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## **Meet the editor**

Dr. Qian Chen is the Michael G. Ehrlich, MD Endowed Chair in Orthopaedic Research, Professor of Medical Science, and Vice Chair for Research in the Department of Orthopaedics at the Warren Alpert Medical School of Brown University. He is the director of Center of Biomedical Research Excellence in Skeletal Health and Repair in Rhode Island Hospital, a multi-disciplinary

translational research center established by National Institute of Health. Dr. Chen received his PhD degree in Cell, Molecular, and Developmental Biology from Tufts University School of Medicine in Boston, and performed Post-Doctoral Fellowship at Harvard Medical School and Massachusetts General Hospital. Dr. Chen's research interest includes cartilage molecular biology, skeletal development and aging, extracellular matrix, mechanotransduction, osteoarthritis, and nanomedicine.

### Contents



Chapter 7 **Validation of Mechanical Hypothesis of hip Arthritis Development by HIPSTRESS Method 131** Veronika Kralj-Iglič

### Chapter 8 **Specific Proteases for Osteoarthritis Diagnosis and Therapy 159**

Xiao-Yu Yuan, Liping Zhang and Yuqing Wu


### Preface

Chapter 8 **Specific Proteases for Osteoarthritis Diagnosis**

**Section 3 Therapy, Treatment and Management 189**

Rie Kurose and Takashi Sawai

**of the Literature 207**

**Knee Osteoarthritis 249**

Chapter 10 **Cell-Based Therapy for Human Osteoarthritis 191**

Xiao-Yu Yuan, Liping Zhang and Yuqing Wu

Chapter 9 **The Cholinergic System in Relation to Osteoarthritis 179**

Chapter 11 **Local Therapies for Osteoarthritis — An Update and a Review**

Chapter 12 **The Conservative Management of Osteoarthritis — Hyaluronic Acid, Platelet Rich Plasma or the Combination? 225** Michele Abate, Isabel Andia and Vincenzo Salini

Chapter 13 **Superficial Heat and Cold Applications in the Treatment of**

Çalışkan Nurcan and Mevlüde Karadağ

Sarah Karrar and Charles Mackworth-Young

**and Therapy 159**

**VI** Contents

Sture Forsgren

Cartilage and joint disease, or arthritis, is a leading cause of disability worldwide. In United States, ar‐ thritis affects an estimated 46.4 million U.S. Citizens — more than one in every five adults aged 18 or older. The national average is 24% women and 18% men. Because the prevalence of arthritis increases with age, half of adults aged 65 and over are affected. However, nearly two-thirds of the adults report‐ ing doctor-diagnosed arthritis are younger than 65 years. Among them, more than 60% are women. Population projections predict that both the number and the proportion of persons aged 65 and older will rise sharply, thereby increasing the prevalence of arthritis. By 2030, the number of persons with doctor-diagnosed arthritis is projected to increase by 40% over current levels to nearly 67 million, or 25% of the adult population nationally. The most common form of arthritis is osteoarthritis (OA), which most often affects the hip, knee, foot and hand. Also called "degenerative joint disease", the degenera‐ tion of joint cartilage and changes in underlying bone and supporting tissues such as ligament lead to pain, stiffness, movement problems and activity limitations.

The alarming figures of cartilage and joint diseases are compounded by the enormous costs we bear for arthritis treatment, its complications and the resulting disability. The prevalence of the joint disease rates illustrates the significance for research, treatment, and prevention of this debilitating disease. The most effective strategy should be multi-disciplinary, which includes understanding the fundamental mechanisms of skeletal diseases by analyzing not only how bone and joint are degenerated, but also how they are built up. To develop new therapeutic strategies in skeletal medicine, we need to under‐ stand not only how diseases are developed, but also what the repair strategies will be. Because bone and joints ultimately support mechanical functions of the body, we need to design and test the treat‐ ment strategies not only from the biological perspective, but also on biomechanical principles. Because our ultimate goal is to improve patients' life quality, we need to study not only basic mechanisms, but also clinical manifestations. These are the guiding principles of organizing and editing the current book "Osteoarthritis - Progress in Basic Research and Treatment".

This book contains three major sections: I. Physiology, Cell and Molecular Biology; II. Pathology and Biomechanics; and III. Therapy, Treatment and Management. It is an update of the book "Osteoarthritis - Diagnosis, Treatment and Surgery" published by InTech in 2012. The authors are experts in the osteo‐ arthritis field, which include biologists, bioengineers, clinicians, and health professionals. The scientific content of the book will be beneficial to patients, students, researchers, educators, physicians, and health care providers who are interested in the recent progress in osteoarthritis research and therapy. I believe that this book can provide a glimpse of the rapid progress occurring in the osteoarthritis field right now, and truly hope that breakthrough of novel research and treatment in the field will signifi‐ cantly increase the life quality of OA patients in the near future.

> **Qian Chen, Ph.D.** Rhode Island Hospital Alpert Medical School of Brown University Providence, RI, USA

**Physiology, Cell and Molecular Biology**

VIII Preface

### **Classifications and Definitions of Normal Joints**

Xiaoming Zhang, Darryl Blalock and Jinxi Wang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59977

#### **1. Introduction**

The anatomical definition of a joint (articulation) is the location where two or more bones, or any rigid parts of the skeleton, connect with each other. It forms a mechanical support to the skeleton and allows a variety of movements in different ranges between the rigid skeletal elements. Some joints, such as the sutures between cranial bones, allow very little or no movement. Other joints, such as the shoulder and hip joints, allow free movement in a large range [1].

#### **2. Joint classification**

Joints differ from each other by their tissue formation, function, structure, and movement.

#### **2.1. Types of joints by tissue formation**

When the articulating bones are connected, the connection may mainly involve three types of tissues – fibers, cartilages, or synovial membrane. Therefore, joints are generally classified according to these three tissues.

The fibrous joints are united by dense connective fibers such as the sutures between cranial bones, the interosseous membrane in the forearm, and the socket articulation between the root of the tooth and the alveolar processes of the maxilla and the mandible. The interosseous membrane allows movement between the two articulating bones. This type of fibrous joint is classified as "syndesmoses" (discussed below). The root of the tooth and its alveolar process associate in a manner similar to how a cone-shaped peg fits into a socket. There is very little room to move in this type of joint, which is defined as "gomphosis".

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

The cartilaginous joints have cartilage in between articulating bones to form a cartilaginous plate or disc. An example of a primary cartilaginous joint is the epiphyseal plate of a long bone, which disappears after puberty. An example of a secondary cartilaginous joint is the interver‐ tebral disc between each vertebra.

The synovial joint is also called the "diarthrodial joint" and is formed by three basic compo‐ nents – the joint cavity, the joint (articular) cartilage which covers the surfaces of articulating bones, and the joint capsule which is composed of a synovial membrane layer lining up the joint cavity and a fibrous layer outside the synovial membrane. In addition, a synovial joint may have ligaments that form a portion of the fibrous joint capsule or ligaments that exist inside the joint capsule (Figure 1). Cartilaginous discs, such as the menisci in the knee joint, may also be found inside the joint cavity.

**Figure 1.** An illustration of the shoulder joint shows that the synovial membrane (red) lines the inner surface of the fibrous capsule. The joint/synovial cavity is marked in black. The tendon of the long head of the biceps passes through the joint and is enclosed in a tubular sheath of synovial membrane, which is continued around the tendon into the intertubercular sulcus as far as the surgical neck of the humerus.

#### **2.2. Types of joints by tissue formation and mobility**

Synarthroses joint: A fibrous type of joint that allows very little or no movement under normal conditions. If a joint is formed with an intervening tissue (fibrous connective tissue, cartilage, or bone) in between the two joint forming elements, it is a "synarthroses" joint. This type of joint usually has limited mobility. The above mentioned suture joints between cranial bones and the gomphosis are synarthroses joints.

Diarthroses joint: If a joint has a space in between the two joint forming elements, it is a "diarthroses" joint. Synovial joints are diathroses joints and their mobility varies in a large range.

Synostoses joint: This term is used to define when two bones fuse with each other under either normal or abnormal conditions. Normally, cranial bones fuse together when a child reaches adulthood, or when the diaphysis and epiphysis fuses in puberty. Abnormal fusion of bones may occur too early in development such as the early fusion of cranial bones (craniostenosis) or when bones are abnormally joined together such as the synostosis of the cervical vertebrae.

Syndesmoses joint: It refers to fibrous joints containing either an interosseous membrane or a ligament that allows movement. The distal tibiofibular joint is a typical syndesmosis joint.

Synchondroses joint: A cartilaginous joint containing a hyaline cartilage in between the two joined bones. The primary cartilaginous joint with an epiphyseal plate between the epiphysis and the diaphysis of a long bone is a typical synchondrosis joint. The joint between the first rib and the sternum (not including any other ribs) is also a synchondroses joint. However, the intervertebral discs are not considered synchondroses joints because they are composed of fibrous cartilage. They, along with the joint between the pubic bones, are instead referred to as "symphysis" joints.

#### **2.3. Types of joints categorized by structure and movement.**

The cartilaginous joints have cartilage in between articulating bones to form a cartilaginous plate or disc. An example of a primary cartilaginous joint is the epiphyseal plate of a long bone, which disappears after puberty. An example of a secondary cartilaginous joint is the interver‐

The synovial joint is also called the "diarthrodial joint" and is formed by three basic compo‐ nents – the joint cavity, the joint (articular) cartilage which covers the surfaces of articulating bones, and the joint capsule which is composed of a synovial membrane layer lining up the joint cavity and a fibrous layer outside the synovial membrane. In addition, a synovial joint may have ligaments that form a portion of the fibrous joint capsule or ligaments that exist inside the joint capsule (Figure 1). Cartilaginous discs, such as the menisci in the knee joint,

**Figure 1.** An illustration of the shoulder joint shows that the synovial membrane (red) lines the inner surface of the fibrous capsule. The joint/synovial cavity is marked in black. The tendon of the long head of the biceps passes through the joint and is enclosed in a tubular sheath of synovial membrane, which is continued around the tendon into the

Synarthroses joint: A fibrous type of joint that allows very little or no movement under normal conditions. If a joint is formed with an intervening tissue (fibrous connective tissue, cartilage, or bone) in between the two joint forming elements, it is a "synarthroses" joint. This type of

tebral disc between each vertebra.

4 Osteoarthritis - Progress in Basic Research and Treatment

may also be found inside the joint cavity.

intertubercular sulcus as far as the surgical neck of the humerus.

**2.2. Types of joints by tissue formation and mobility**

Joints develop into different physical forms. As a result, the structural formation of a joint determines its movement. There are six types of joints in this category and they are all synovial joints (Figure 2).

Pivot type: A round bony process fits into a bony groove permitting rotation. For example, the atlanto-axial joint (between C1 and the Dens of C2 vertebrae).

Ball and socket type: A ball shaped bony head fits into a concavity allowing movement in several axes. The glenohumeral (shoulder) and hip joints are ball and socket type joints.

Plane type: The joint surfaces of both bones are flat against each other. Gliding action in one direction (uniaxial) can happen in this type of joint, for example, the acromioclavicular joint.

Hinge type: A joint forms like a door hinge allowing only one direction (uniaxial) movement, for example, the elbow joint.

Saddle type: The opposing articular surfaces are both saddle shaped. As a result, this type of joint can move in two directions (biaxial), for example, the carpometacarpal joint of the thumb.

Condyloid type: A rounded bony prominence of one bone articulates with a shallow inden‐ tation of another bone. This type joint allows two direction (biaxial) movement as well as circumduction, for example, the metacarpophalangeal joints.

**Figure 2.** Diagrammatic illustrations show the structural features of six different types of human joints.

#### **3. Cartilage associated with joints**

Cartilage is a semi-rigid type of connective tissue composed of cells called chondrocytes and a large amount (>95%) of specialized extracellular matrix. Chondrocytes are scattered among the matrix and located in spaces called "lacunae".

A primary cartilaginous joint (synchondroses) such as the epiphyseal plate contains hyaline cartilage. The secondary cartilaginous joints (symphyses) such as the intervertebral discs contain fibrous cartilage. In synovial joints, a layer of hyaline cartilage "caps" the articulating surfaces of each joint-forming bone. This particular cartilage is called "articular cartilage". Articular cartilage provides smooth, low-friction, gliding surfaces for free movement. It also absorbs mechanical impacts placed on the articulating bones [1].

Cartilage is usually covered on its external surface by a thin layer of connective tissue named "perichondrium". However, perichondrium does not cover articular cartilage, the epiphyseal plate, and cartilage immediately under the skin such as the cartilage at the ear and nose.

Based on the characteristics of its matrix, cartilage is classified as the following and each type has its own different appearance and mechanical property [2].

#### **3.1. Hyaline cartilage**

Hyaline cartilage is named such because of the glassy (transparent) appearance of its matrix in living state. The matrix contains predominantly water (60-80%), with type II collagen fibers (~15%), chondroitin sulfate proteoglycan aggregates (~9%), and adhesive non-collagen glycoproteins (~5%) [2]. The highly hydrated matrix is due to the high content of proteoglycans, which allows water molecules to bind and stay. The hydrated matrix permits diffusion of small metabolites and nutrients as well as providing resilience to mechanical pressures during weight bearing. The matrix is made by the chondrocytes, which continuously remodel the matrix throughout life in response to mechanical, chemical, and biological signals. However, as the body ages, this remodeling process slows down or stops, resulting in degradation that surpasses synthesis. This is particularly the case in articular cartilage located in synovial joints.

#### **3.2. Fibrocartilage**

Fibrocartilage is a combination of hyaline cartilage and dense regular connective tissue [2]. The chondrocytes in fibrous cartilage appear similar to those in the hyaline cartilage. These cells make various amounts of type I and type II collagen fibers resulting in different propor‐ tions of type I and type II collagen in fibrocartilages at different regions of the body. Fibrocar‐ tilage can be found in intervertebral discs, pubic symphysis, articular discs inside several joint cavities, and the menisci in the knee joint. There is no perichondrium surrounding the fibrocartilages in these regions. Fibrocartilage functions much like a shock absorber for joints.

#### **3.3. Elastic cartilage**

**3. Cartilage associated with joints**

6 Osteoarthritis - Progress in Basic Research and Treatment

the matrix and located in spaces called "lacunae".

absorbs mechanical impacts placed on the articulating bones [1].

Cartilage is a semi-rigid type of connective tissue composed of cells called chondrocytes and a large amount (>95%) of specialized extracellular matrix. Chondrocytes are scattered among

**Figure 2.** Diagrammatic illustrations show the structural features of six different types of human joints.

A primary cartilaginous joint (synchondroses) such as the epiphyseal plate contains hyaline cartilage. The secondary cartilaginous joints (symphyses) such as the intervertebral discs contain fibrous cartilage. In synovial joints, a layer of hyaline cartilage "caps" the articulating surfaces of each joint-forming bone. This particular cartilage is called "articular cartilage". Articular cartilage provides smooth, low-friction, gliding surfaces for free movement. It also Elastic cartilage is characterized by hyaline cartilage containing elastic fibers with the presence of elastin in its matrix. This type of cartilage is found mainly in the external ear, the wall of external acoustic meatus, the auditory (Eustachian) tube, and the epiglottis of the larynx [2].

#### **4. Definition, structure, and function of joint tissues**

#### **4.1. Articular cartilage**

Articular cartilage is a highly specialized viscoelastic hyaline cartilage that is found overlying the bone ends in synovial joints and forming the joint surface [3,4]. Articular cartilage has all the characteristics of hyaline cartilage with some additional features of its own.

Human articular cartilage is normally 2-4 mm thick and does not have a perichondrium on either the surface side that faces the joint cavity or the deep side that connects to the bone [3]. It has a limited intrinsic capacity to heal and repair, and is closely related to joint health.

From the joint surface to subchondral bone, a cross section of articular cartilage can be divided into four zones under the microscope (Figure 3).

**Figure 3.** Illustrations showing the zonal features of human articular cartilage and subchondral bone. **Left panel** (chon‐ drocyte organization)**:** The superficial zone chondrocytes are small and flattened, which are orientated parallel to the joint surface. The middle zone chondrocytes are rounded and oriented randomly. The deep zone chondrocytes are grouped in columns or clusters. **Right panel:** Collagen orientation in articular cartilage. In the superficial (tangential) zone, small and medium-sized collagen fibrils aggregate into bundles that are aligned parallel to the joint surface. In the middle and deep zones, collagen fibrils aggregate into larger bundles and are aligned more perpendicular to the joint surface.

The superficial (tangential) zone contains numerous elongated and flattened chondrocytes. These cells are surrounded by dense type II collagen fibril fascicles arranged parallel to the surface [2]. This zone occupies 10-20% of the articular cartilage's thickness and is in contact with synovial fluid. It bears sheer, tensile, and compressive forces imposed on the articular surface [3].

The intermediate (middle or transitional) zone is deep to the superficial zone and contains randomly distributed round chondrocytes. Collagen fibers are generally organized in an oblique orientation to the surface. It occupies 40-60% of the thickness of articular cartilage and mainly resists compressive forces.

The deep (radial) zone has round chondrocytes arranged in short columns perpendicular to the joint surface. Collagen fibers located between cell columns are thick and are generally perpendicular to the joint surface. The proteoglycan content is the highest among all the zones while water concentration is low. It occupies about 30% of the thickness of articular cartilage and provides additional resistance to compressive forces [5,6].

The calcified zone contains a small amount of chondrocytes and a large amount of calcified matrix. A smooth and heavily calcified line called the "tidemark" separates this zone from the deep zone. The primary role of the calcified zone is to firmly secure articular cartilage to subchondral bone. This highly organized structure is responsible for the unique mechanical properties of articular cartilage.

In addition to the histologically defined zones, the matrix distribution of articular cartilage is distinguished in three regions [3].

The pericellular matrix is a thin layer adjacent to the cell membrane completely surrounding the chondrocyte. It contains mainly proteoglycans, glycoproteins, and other noncollagenous proteins. This matrix region may function to initiate signal transduction within cartilage.

The territorial matrix is thicker and surrounds the pericellular matrix. It is composed mostly of fine collagen fibrils that form a network around the cells. This region may protect the cartilage cells against mechanical stresses when there are substantial forces loaded.

The interterritorial region is the largest and refers to the collagen fibrils arranged parallel to the surface in the superficial zone, obliquely in the middle zone, and perpendicular in the deep zone. It contributes the most to the biomechanical properties of articular cartilage. The extracellular matrix accounts for approximately 95% of the dry weight of articular cartilage [4]. The primary macromolecule found in articular cartilage is type II collagen, which represents 90-95% of the total collagen content. The remaining are collagen types I, IV, V, VI, IX, and XI. The second largest group of macromolecules is proteoglycans which consist of a protein core with covalently linked glycosaminoglycan chains (GAGs). The main proteoglycans found in articular cartilage include aggrecan, decorin, biglycan and fibromodulin with aggrecan being the most abundant. Negatively charged carboxyl and sulfate groups found on these GAGs, namely keratin sulfate and chondroitin sulfate, have a high affinity for water [2,3].

The renewal process of mature articular cartilage is very slow due to the stable type II collagen structure and the long half-life of GAGs. The matrix degrading enzyme metalloproteinase activity is also low [2,3].

#### **4.2. Synovial membrane (synovium)**

Human articular cartilage is normally 2-4 mm thick and does not have a perichondrium on either the surface side that faces the joint cavity or the deep side that connects to the bone [3]. It has a limited intrinsic capacity to heal and repair, and is closely related to joint health.

From the joint surface to subchondral bone, a cross section of articular cartilage can be divided

**Figure 3.** Illustrations showing the zonal features of human articular cartilage and subchondral bone. **Left panel** (chon‐ drocyte organization)**:** The superficial zone chondrocytes are small and flattened, which are orientated parallel to the joint surface. The middle zone chondrocytes are rounded and oriented randomly. The deep zone chondrocytes are grouped in columns or clusters. **Right panel:** Collagen orientation in articular cartilage. In the superficial (tangential) zone, small and medium-sized collagen fibrils aggregate into bundles that are aligned parallel to the joint surface. In the middle and deep zones, collagen fibrils aggregate into larger bundles and are aligned more perpendicular to the

The superficial (tangential) zone contains numerous elongated and flattened chondrocytes. These cells are surrounded by dense type II collagen fibril fascicles arranged parallel to the surface [2]. This zone occupies 10-20% of the articular cartilage's thickness and is in contact with synovial fluid. It bears sheer, tensile, and compressive forces imposed on the articular

The intermediate (middle or transitional) zone is deep to the superficial zone and contains randomly distributed round chondrocytes. Collagen fibers are generally organized in an oblique orientation to the surface. It occupies 40-60% of the thickness of articular cartilage and

The deep (radial) zone has round chondrocytes arranged in short columns perpendicular to the joint surface. Collagen fibers located between cell columns are thick and are generally perpendicular to the joint surface. The proteoglycan content is the highest among all the zones while water concentration is low. It occupies about 30% of the thickness of articular cartilage

The calcified zone contains a small amount of chondrocytes and a large amount of calcified matrix. A smooth and heavily calcified line called the "tidemark" separates this zone from the deep zone. The primary role of the calcified zone is to firmly secure articular cartilage to subchondral bone. This highly organized structure is responsible for the unique mechanical

into four zones under the microscope (Figure 3).

8 Osteoarthritis - Progress in Basic Research and Treatment

joint surface.

surface [3].

mainly resists compressive forces.

properties of articular cartilage.

and provides additional resistance to compressive forces [5,6].

Synovial membrane is the inner layer of the joint capsule facing the joint cavity and synovial fluid. The synovium lines the joint cavity producing synovial fluid that lubricates the joint surfaces and provides nutrition to the articular cartilage [7]. The synovial membrane is composed of two layers; the surface layer consisting of one or two layers of synovial cells and the underlying connective tissue layer.

There are two types of synovial cells. Type A synovial cells are macrophage-like cells whereas type B cells are fibroblast-like. These cells secrete hyaluronic acid and glycoprotein molecules, which are part of the synovial fluid lubricating the joint surfaces. There is no basal lamina separating the synovial cells from the underlying connective tissue. This connective tissue contains a rich network of fenestrated capillaries, which allow plasma to flow out of blood circulation and enter the joint cavity. The filtered plasma content combines with hyaluronic acid, glycoproteins, and leukocytes becoming the synovial fluid [8,9]. Normal synovial fluid appears clear to pale yellow in color, transparent, and contains less than 200 white blood cells/µl [10].

#### **4.3. Joint capsule**

The joint capsule is essential to the proper function of synovial joints. It forms the seal that contains synovial fluid within the joint, imparts passive stability by limiting joint movement, and provides active stability via its proprioceptive nerve endings [11]. It is composed of collagen fibers that are firmly adhered to bone through a fibrocartilaginous attachment. Localized thickenings of the capsule form capsular ligaments that provide strong points of fixation to bone. Tendons commonly attach to the joint capsule and occasionally replace it as is the case with the quadriceps and patellar tendons in the anterior knee. Blood vessels and nerves pass through the joint capsule supplying both the capsule and the underlying synovi‐ um. Nerve endings in the joint capsule are thought to be proprioceptive and play an important role in the active protection of the capsule and associated ligaments by reflex control of the appropriate musculature [11].

#### **4.4. Tendon**

Tendon is a tough band of fibrous connective tissue that usually connects muscle to bone. Tendons are mainly composed of type I collagen fibers arranged in fascicles and bands. Proteoglycans are primarily responsible for holding the collagen fibrils together. Specialized fibroblast cells called "tenocytes" exist in tendons and are responsible for collagen synthesis in tendon. Tendon is surrounded by a loose connective tissue layer called "peritendineum", which blends with the periosteum when the tendon attaches to the bone [12-14].

At the site of tendon insertion, parallel collagen fibers penetrate through the periosteum and insert into the mineralized fibrocartilage zone of the bone. Elastic fibers function to prevent overstretching and cartilage cells function to resist transverse shortening at the tendon insertion site [15].

Tendons contain some, but not many blood vessels. Enhanced physical activity can increase blood flow in the tendon. In regions where the tendons wrap around bony pulleys, blood supply is largely reduced. Tendons also have nerve supply in various degrees [12].

#### **4.5. Ligament**

Ligaments can be defined as dense bands of collagenous fibers that span a joint and are anchored to bone at either end [16]. Like tendon, ligament is made of dense connective tissue consisting mainly of type I collagen. Some ligaments are located outside the joint cavity; others are inside the joint cavity. Some ligaments are discrete structures that stand alone such as the cruciate ligaments in the knee; some are regional thickenings of the joint capsule as a part of the fibrous layer of the joint capsule. The primary function of ligaments is to provide passive stability to a joint through a normal range of motion under an applied load. Bundles of collagen fibrils form the majority of the ligament substance [17]. These fibrils are typically aligned in the direction of tension applied to the ligament during normal joint motion.

The ligament-bone interface is a complex structure that has been described as two distinct insertion types: direct and indirect. Direct insertion involves passage of a ligament directly into cortical bone. The superficial ligament collagen fibers merge with the fibrous layer of the periosteum while the majority of the insertion consists of deeper fibers directly penetrating the cortex [17]. These deep fibers pass through ligament substance, fibrocartilage, mineralized fibrocartilage and finally into bone. This direct insertion typically occurs at a right angle to the bone. In contrast, indirect insertions typically occur more obliquely. This type of insertion is less common and usually involves a wide surface area of insertion along the bone surface as opposed to directly into the cortex [17]. These insertions are believed to allow gradual transmission of force between ligament and bone.

#### **4.6. Subchondral bone**

**4.3. Joint capsule**

10 Osteoarthritis - Progress in Basic Research and Treatment

appropriate musculature [11].

**4.4. Tendon**

insertion site [15].

**4.5. Ligament**

The joint capsule is essential to the proper function of synovial joints. It forms the seal that contains synovial fluid within the joint, imparts passive stability by limiting joint movement, and provides active stability via its proprioceptive nerve endings [11]. It is composed of collagen fibers that are firmly adhered to bone through a fibrocartilaginous attachment. Localized thickenings of the capsule form capsular ligaments that provide strong points of fixation to bone. Tendons commonly attach to the joint capsule and occasionally replace it as is the case with the quadriceps and patellar tendons in the anterior knee. Blood vessels and nerves pass through the joint capsule supplying both the capsule and the underlying synovi‐ um. Nerve endings in the joint capsule are thought to be proprioceptive and play an important role in the active protection of the capsule and associated ligaments by reflex control of the

Tendon is a tough band of fibrous connective tissue that usually connects muscle to bone. Tendons are mainly composed of type I collagen fibers arranged in fascicles and bands. Proteoglycans are primarily responsible for holding the collagen fibrils together. Specialized fibroblast cells called "tenocytes" exist in tendons and are responsible for collagen synthesis in tendon. Tendon is surrounded by a loose connective tissue layer called "peritendineum",

At the site of tendon insertion, parallel collagen fibers penetrate through the periosteum and insert into the mineralized fibrocartilage zone of the bone. Elastic fibers function to prevent overstretching and cartilage cells function to resist transverse shortening at the tendon

Tendons contain some, but not many blood vessels. Enhanced physical activity can increase blood flow in the tendon. In regions where the tendons wrap around bony pulleys, blood

Ligaments can be defined as dense bands of collagenous fibers that span a joint and are anchored to bone at either end [16]. Like tendon, ligament is made of dense connective tissue consisting mainly of type I collagen. Some ligaments are located outside the joint cavity; others are inside the joint cavity. Some ligaments are discrete structures that stand alone such as the cruciate ligaments in the knee; some are regional thickenings of the joint capsule as a part of the fibrous layer of the joint capsule. The primary function of ligaments is to provide passive stability to a joint through a normal range of motion under an applied load. Bundles of collagen fibrils form the majority of the ligament substance [17]. These fibrils are typically aligned in

The ligament-bone interface is a complex structure that has been described as two distinct insertion types: direct and indirect. Direct insertion involves passage of a ligament directly

supply is largely reduced. Tendons also have nerve supply in various degrees [12].

the direction of tension applied to the ligament during normal joint motion.

which blends with the periosteum when the tendon attaches to the bone [12-14].

The bony component lying under (deep to) the calcified zone of the articular cartilage is called subchondral bone, which can be separated into two distinct anatomic entities: subchondral bone plate and subchondral trabecular bone.

The subchondral bone plate is the thin cortical lamella, lying parallel to and immediately under the calcified cartilage. This cortical endplate is a penetrable structure and is invaded by channels that provide a direct link between articular cartilage and subchondral trabecular bone. A number of arterial and venous vessels, as well as nerves penetrate through the channels and send tiny branches into calcified cartilage, communicating between the calcified cartilage and the trabeculae bone [18-20].

Compared to the subchondral bone plate, the sunchondral trabecular bone is more porous and metabolically active, containing blood vessels, sensory nerves, and bone marrow. It exerts important shock-absorbing and supportive functions in normal joints and may also be important for cartilage nutrient supply and metabolism [18,19].

Subchondral bone changes are important features of osteoarthritis (OA), suggesting that subchondral bone plays a vital role in the pathogenesis of OA [21-23]. Bone marrow edemalike lesions (BMELs), which are strongly associated with pain among patients with OA, are frequently identified by magnetic resonance imaging (MRI) in patients with progressive OA. BMELs are also observed in the healthy, asymptomatic population and predict an increased risk of OA [24,25].

#### **5. Conclusion**

This chapter describes the classification of different types of joints based on their structural and functional features. The anatomical and functional features of six different types of human joints are demonstrated. Definitions and structures of specific joint tissues associated with the function of joints such as the joint cartilage, synovial membrane, joint capsule, tendon, ligament, ligament-bone interface, and subchondral bone are described in great detail. The clinical relevance of specific joint structures to joint injury, joint instability, and the develop‐ ment of OA is discussed at both macro- and micro-anatomical levels.

#### **Acknowledgements**

This work was supported in part by the U.S. National Institutes of Health (NIH)/NIAMS grant R01 AR059088, the U.S. Department of Defense medical research grant W81XWH-12-1-0304, and the Harrington Distinguished Professorship Endowment. The authors thank Mr. Brian Egan and Mr. Zhaoyang Liu for their graphic and editorial assistance.

#### **Author details**

Xiaoming Zhang1 , Darryl Blalock2 and Jinxi Wang2

\*Address all correspondence to: jwang@kumc.edu

1 Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, USA

2 Department of Orthopedic Surgery, University of Kansas School of Medicine, Kansas City, USA

#### **References**


[8] Edwards JC. The nature and origins of synovium: experimental approaches to the study of synoviocyte differentiation. J Anat. 1994;184 (Pt 3):493-501.

**Acknowledgements**

12 Osteoarthritis - Progress in Basic Research and Treatment

**Author details**

Xiaoming Zhang1

City, USA

**References**

phia, pp198-217.

Anat. 1977;123(Pt 2):437-457.

PA: Elsevier/Saunders; 2013.

USA

This work was supported in part by the U.S. National Institutes of Health (NIH)/NIAMS grant R01 AR059088, the U.S. Department of Defense medical research grant W81XWH-12-1-0304, and the Harrington Distinguished Professorship Endowment. The authors thank Mr. Brian

and Jinxi Wang2

1 Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas

2 Department of Orthopedic Surgery, University of Kansas School of Medicine, Kansas City,

[1] Moore KL, Dalley AF, Agur AMR.. Introduction to Clinically Oriented Anatomy. In: Moore KL, Dalley AF, Agur AMR (eds.), Clinically Oriented Anatomy, 7th ed. 2014;

[2] Ross MH, Pawlina W. Cartilage. In: Ross MH and Pawlina W. (eds.) Histology: A text and Atlas 6th ed. 2011; Wolters Kluwer|Lippincott Williams & Wilkins, Philadel‐

[3] Sophia Fox AJ, Bedi A, Rodeo SA. The basic science of articular cartilage: structure,

[4] Alford JW, Cole BJ. Cartilage restoration, part 1: basic science, historical perspective, patient evaluation, and treatment options. Am J Sports Med. 2005;33(2):295-306.

[5] Curtin WA, Reville WJ. Ultrastructural observations on fibril profiles in normal and degenerative human articular cartilage. Clin Orthop Relat Res. 1995(313):224-230.

[6] Minns RJ, Steven FS. The collagen fibril organization in human articular cartilage. J

[7] Firestein GS, Kelley WN. Kelley's textbook of rheumatology. 9th ed. Philadelphia,

Wolters Kluwer|Lippincott Williams & Wilkins, Philadelphia, p25-29.

composition, and function. Sports Health. 2009;1(6):461-468.

Egan and Mr. Zhaoyang Liu for their graphic and editorial assistance.

, Darryl Blalock2

\*Address all correspondence to: jwang@kumc.edu


#### **Chapter 2**

### **Epigenetic Mechanisms in Osteoarthritis**

#### Antonio Miranda-Duarte

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60533

#### **1. Introduction**

[24] Link TM, Steinbach LS, Ghosh S, Ries M, Lu Y, Lane N, Majumdar S. Osteoarthritis: MR imaging findings in different stages of disease and correlation with clinical find‐

[25] Wluka AE, Hanna F, Davies-Tuck M, Wang Y, Bell RJ, Davis SR, Adams J, Cicuttini FM. Bone marrow lesions predict increase in knee cartilage defects and loss of carti‐ lage volume in middle-aged women without knee pain over 2 years. Ann Rheum

ings. Radiology. 2003;226(2):373-381.

Dis. 2009;68(6):850-855.

14 Osteoarthritis - Progress in Basic Research and Treatment

The hallmark of osteoarthritis (OA) is the progressive degeneration of articular cartilage, although bone and synovia are also involved in the development of the disease [1]. Chondro‐ cytes are the unique cellular component of articular cartilage and, under physiological conditions, are responsible for a subtle balance between the synthesis of extracellular matrix (ECM) components, mainly type-II collagen and aggrecan [2], and its degradation by proteolyt‐ ic enzymes such as the matrix metalloproteinases (MMP)[3] and A disintegrin and metallopro‐ tease with thrombospondin motifs (ADAMTS) [4]. In OA, there is an imbalance of this process driven by cytokines and the production of inflammatory mediators, resulting in an increase of the degradation process with respect to synthesis, and leading to articular cartilage loss [1, 5].

OA is considered a multifactorial disease in which genetics and environmental factors, such as aging, gender andobesity, among others, are strongly related withitsdevelopment[6].Primary OA possesses an important genetic component, and several genetic association studies have demonstrated that it is associated with different genes that encode molecules involved in a number of pathways, such as inflammation, Wnt signalling, bone morphogenetic proteins (BMPs), proteases and their inhibitors, and extracellular matrix proteins, among others [7, 8]. However, there has not always been consistency in the results, probably due to the low penetranceofthegenepolymorphisms studied,ortodifferentgene-gene interactions andgeneenvironment interactions. In this regard, epigenetics is one mechanism through which geneenvironment interactions occur. Epigenetics refers to heritable changes in gene expression that occur without changes in DNA, and includes DNA methylation, histone modifications, chromatin remodelling and microRNAs (miRNAs), although debate continues concerning whether miRNA can be categorized as an epigenetic phenomenon [9]. Recent evidence has made it apparent that epigenetic changes alter the expression of genes that could participate in the pathogenesis of OA. This chapter does not intend to conduct a deep review of epigenetic modifications, but rather to review the main findings directly related with OA.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. DNA methylation in osteoarthritis**

DNA methylation is the most studied epigenetic mark in humans. DNA methylation involves the addition of a methyl group at CpG dinucleotides to convert cytosine into 5 methylcytosine. These CpG dinucleotides tend to cluster in regions termed 'islands', and approximately 70% of human gene promoters are associated with CpG, which are usual‐ ly unmethylated. CpG-island methylation is associated with gene silencing due to the binding of methyl-CpG-binding proteins, which recruit proteins to the gene promoter, blocking its transcription. DNA methylation can also occur in CpG island shores, regions of lower CpG density that lie in close proximity to CpG islands (~2 kb); their methylation is associated with transcriptional inactivation. DNA methylation is mediated by the DNA methyltransferases (DNMTs) family of enzymes that catalyse the transfer of a methyl group to DNA. In mammals, five members of the DNMT family have been reported: DNMT1, DNMT2, DNMT3a, DNMT3b and DNMT3L, but only DNMT1, DNMT3a and DNMT3b possess methyltransferase activity. DNMTs are classified as *de novo* and maintenance enzymes. DNMT1 is the maintenance DNMT and has a preference for hemimethylated DNA; this is the most abundant DNMT in cells and is transcribed mostly during the S phase of the cell cycle. DNMT3A and DNMT3B are *de novo* DNMTs and are responsible for establishing the pattern of methylation during embryonic development [9-13].

Analysis of the overall methylation level of genomic DNA in the chondrocytes of degenerated OA cartilage shows no difference in comparison with normal chondrocytes. However, an inhibitor of cell proliferation, the cyclin-dependent kinase inhibitor 1 (*p21WAF1/CIP1*) gene, which may mediate the re-initiation of cell proliferation in OA cartilage and which has demonstrated itself to be epigenetically regulated in neoplastic cells [14], is significantly downregulated in OA chondrocytes and does not exhibit hypermethylation in its promoter [15].

In chick embryos' chondrocytes, the *Col2a1* gene shows reduced methylation in comparison with other cells, such as fibroblasts and erythrocytes [16]. In humans, it remains unclear whether the methylation of DNA alters the regulation of cartilage matrix genes in OA. Aggrecan is reduced during aging and in OA cartilage; however, the aggrecan (*ACAN*) gene expression of normal aging and osteoarthritic articular human cartilage does not correlate with increased methylation of the *ACAN*-promoter CpG island [17]. Human articular chondrocytes remain negative for type X collagen, unless they become hypertrophic. In the latter cells, the gene methylation patterns and the expression of *COL10A1* and *COL2A1* have shown that the *COL10A1* promoter is methylated, which correlates with the suppression of hypertrophy observed in articular chondrocytes, and there is no evidence of *COL2A1* regulation at the methylation level, which demonstrates a low methylation rate [18].

The nuclear transcription factor SOX9, along with other SOX family members, is required for the control of the expression of ECM components. SOX9 is an important regulator of the chondrocyte phenotype and controls the expression of the *COL2A1*, *COL9A1*, *COL11A1* and *ACAN* genes. The SOX9 protein binds to its promoter elements and forms transactivating complexes with other proteins, such as SOX5/SOX6. The SOX family maintains the chondro‐ cytic phenotypes, and is vital for chondrogenesis in embryonic development [19, 20]. In human synovium-derived mesenchymal stem cells (MSCs) subjected to chondrogenesis the CpG island of *SOX9* is hypomethylated; as well as in other chondrogenesis related genes such as runt-related transcription factor 2 (*RUNX2)* and fibroblast growth factor receptor 3 (*FGFR3)* [21]. While in OA chondrocytes, *SOX9*-promoter is hypermethylated, which reduces the binding affinity of the transcription factors CCAAT-binding factor/nuclear factor-Y (CBF/NF-Y) and the cAMP response element binding (CREB) [22]. This means that the methylation of the *SOX9* promoter remains low during chondrogenesis, and in OA there is change in the epigenetic status of *SOX9*, including increased DNA methylation.

**2. DNA methylation in osteoarthritis**

16 Osteoarthritis - Progress in Basic Research and Treatment

DNA methylation is the most studied epigenetic mark in humans. DNA methylation involves the addition of a methyl group at CpG dinucleotides to convert cytosine into 5 methylcytosine. These CpG dinucleotides tend to cluster in regions termed 'islands', and approximately 70% of human gene promoters are associated with CpG, which are usual‐ ly unmethylated. CpG-island methylation is associated with gene silencing due to the binding of methyl-CpG-binding proteins, which recruit proteins to the gene promoter, blocking its transcription. DNA methylation can also occur in CpG island shores, regions of lower CpG density that lie in close proximity to CpG islands (~2 kb); their methylation is associated with transcriptional inactivation. DNA methylation is mediated by the DNA methyltransferases (DNMTs) family of enzymes that catalyse the transfer of a methyl group to DNA. In mammals, five members of the DNMT family have been reported: DNMT1, DNMT2, DNMT3a, DNMT3b and DNMT3L, but only DNMT1, DNMT3a and DNMT3b possess methyltransferase activity. DNMTs are classified as *de novo* and maintenance enzymes. DNMT1 is the maintenance DNMT and has a preference for hemimethylated DNA; this is the most abundant DNMT in cells and is transcribed mostly during the S phase of the cell cycle. DNMT3A and DNMT3B are *de novo* DNMTs and are responsible

for establishing the pattern of methylation during embryonic development [9-13].

OA chondrocytes and does not exhibit hypermethylation in its promoter [15].

methylation level, which demonstrates a low methylation rate [18].

Analysis of the overall methylation level of genomic DNA in the chondrocytes of degenerated OA cartilage shows no difference in comparison with normal chondrocytes. However, an inhibitor of cell proliferation, the cyclin-dependent kinase inhibitor 1 (*p21WAF1/CIP1*) gene, which may mediate the re-initiation of cell proliferation in OA cartilage and which has demonstrated itself to be epigenetically regulated in neoplastic cells [14], is significantly downregulated in

In chick embryos' chondrocytes, the *Col2a1* gene shows reduced methylation in comparison with other cells, such as fibroblasts and erythrocytes [16]. In humans, it remains unclear whether the methylation of DNA alters the regulation of cartilage matrix genes in OA. Aggrecan is reduced during aging and in OA cartilage; however, the aggrecan (*ACAN*) gene expression of normal aging and osteoarthritic articular human cartilage does not correlate with increased methylation of the *ACAN*-promoter CpG island [17]. Human articular chondrocytes remain negative for type X collagen, unless they become hypertrophic. In the latter cells, the gene methylation patterns and the expression of *COL10A1* and *COL2A1* have shown that the *COL10A1* promoter is methylated, which correlates with the suppression of hypertrophy observed in articular chondrocytes, and there is no evidence of *COL2A1* regulation at the

The nuclear transcription factor SOX9, along with other SOX family members, is required for the control of the expression of ECM components. SOX9 is an important regulator of the chondrocyte phenotype and controls the expression of the *COL2A1*, *COL9A1*, *COL11A1* and *ACAN* genes. The SOX9 protein binds to its promoter elements and forms transactivating complexes with other proteins, such as SOX5/SOX6. The SOX family maintains the chondro‐ cytic phenotypes, and is vital for chondrogenesis in embryonic development [19, 20]. In human

Metalloproteinase expression in normal cartilage is relatively low but is elevated in OA, resulting in ECM degradation [3, 23]. The altered synthesis of the cartilage-degrading enzymes in OA is the result of changes in the methylation status, as demonstrated by the analysis of the methylation of the promoter region of *MMP3*, *MMP9*, *MMP13* and *ADAMTS4* in the cartilage of patients with OA, in which the overall percentage of non-methylated sites increased in comparison with normal controls. However, not all CpG sites were equally susceptible to loss of methylation, and for each gene there was a specific site where OA demethylation was higher, namely: -635 for *MMP3*, -36 for *MMP9*, -110 for *MMP13*, and -753 for *ADAMTS4* [24]. This is interesting because it was generally thought that the methylation of many CpG sites was required to repress gene expression, and these findings suggest that methylation of a single site may be sufficient to affect gene expression. In agreement with this, demethylation of -110 in *MMP13* promoters and -299 in *IL1B* promoters is correlated with an increased gene expression. In addition, methylation of the -110 CpG site in *MMP13* decreases the hypoxia inducible factor 2α (HIF-2α), binding to the *MMP13* promoter. HIF-2α is a transcription factor that regulates *MMP13* expression [25]. On the other hand, demethylation of the -104 CpG region of the *MMP13* promoter correlates with increased gene expression and avoids the binding of the transcription factor CREB to its promoter [26]. ADAMTS5 is considered to be the major aggrecanase; however, ADAMTS4 also contributes to aggrecan degradation in OA. *ADAMTS4* is epigenetically regulated and, although methylation is lost in several promoter sites, the -753 site is that most consistently demethylated [27].

Nitric oxide (NO) is a multifunctional molecule that suppresses energy production by mitochondrial respiration. In OA, high amounts of NO are produced, a consequence of upregulation in the chondrocyte of the inducible NO synthase (iNOS) induced by inflamma‐ tory cytokines, such as interleukin 1-beta (IL-1β) and tumour necrosis factor alpha (TNFα), among others [28]. NO suppresses the synthesis of the cartilaginous matrix [29]. In culture, under-stimulated chondrocytes produce *iNOS*, and its promoter contains nuclear factor-kappa beta (NF-κB) binding sites; this regulates *iNOS* at the transcriptional level. NF-κB is a signalling factor activated by tissue damage and inflammation; its demethylation in specific enhancer elements favours the activation of *iNOS* in chondrocytes [30]. Interestingly, a study showed that glucosamine and an NF-kB inhibitor inhibit cytokine-induced demethylation at a specific site in the *IL1B* promoter, resulting in decreased gene and protein expression [31].

Leptin (*LEP*) is a cytokine-like peptide hormone secreted by white adipose tissue, which plays a key role in OA [32] because it has been shown that *LEP* exerts a detrimental effect on articular cartilage by promoting NO synthesis in chondrocytes [33]. In normal chondrocytes, *LEP* is highly methylated and, in OA, it is demethylated and highly expressed. Additionally, *LEP* downregulation with small interference RNA (siRNA) decreases *MMP13* expression [34].

To date, it is well recognized that OA has an important inflammatory component in its development mediated by proinflammatory cytokines, such as IL-1β and TNFα [2, 32]. Healthy chondrocytes do not express *IL1B*, however, promoter demethylation increases the expression of the gene [35]. Suppressor of cytokine signalling (SOCS) proteins are inhibitors of cytokine signalling. There are eight SOCS proteins, including SOCS1-SOCS7 and cytokine-inducible SH2-domain-1 (CIS-1), with SOCS1, -2 and -3, and CIS-1 the best characterized. *SOCS2* and *CIS-1* expression is reduced in OA chondrocytes compared with normal chondrocytes, while *SOCS1* and *SOCS3* expression remains unchanged. In addition, the *SOCS2* promoter does not exhibit a change in its methylation status [36].

Bone morphogenetic protein-7 (BMP-7) – or osteogenic protein-1(OP-1) – is one of the most potent growth factors for cartilage maintenance and repair. It possesses a critical role in human cartilage homeostasis, regulating numerous metabolic pathways that are not only limited to its well-documented anabolic function but also to its anti-catabolic activity [37]. There is a positive correlation between age and the methylation of the *BMP7* promoter's status in aged chondrocytes; this age-related promoter methylation may contribute to a decrease in BMP7 production in cartilage, with the decreased expression of the insulin-like growth factor-1 (*IGF-1*) and the IGF-1 receptor (*IGF-1R*) genes, as well as the ECM component gene *ACAN* [38].

The growth differentiation factor 5 (*GDF5*) gene is a member of the transforming growth factor β (TGFβ) superfamily that is involved in chondrogenesis and chondrocyte proliferation [39]. The rs143383 C/T single nucleotide polymorphism (SNP) in the 5´ untranslated region (5´UTR) of the gene is associated with an increased risk of OA [40, 41]. This SNP is itself functional and exerts a joint-wide effect on *GDF5* expression, causing a significant reduction in the expression of the disease-associated T allele relative to the C allele in the cartilage and other joint tissues [42], a phenomenon known as 'differential allelic expression' (DAE). The transcriptional effect of rs143383 SNP is dependent on a second C-to-T SNP in the 5´UTR of *GDF5*, rs143384 C/T, with decreased expression of the T allele of rs143383 only observed in individuals which are compound-heterozygous for both SNPs. When the OA-protective C alleles are present at the rs143383 and rs143384 SNPs, they form CpG dinucleotides, which are potentially amenable to regulation by DNA methylation. In cell lines, *GDF5* is upregulated after demethylation, and methylation decreases transcriptional activity. Interestingly, CpG sites formed by the C alleles of both SNPs are methylated; however, their demethylation is associated with increased expression of the C allele of rs143383 relative to the T allele, which indicates that the OAsusceptibility conferred by rs143383 of the *GDF5* gene is regulated by methylation [43].

Methylation analysis of 23,367 sites (corresponding to 13,463 genes) through a genome-wide methylation profile of bone from patients with OA and osteoporosis (OP) revealed an inverse relationship between methylation and gene expression in both groups, with 271 CpG sites being less methylated in OP than in OA. *In silico* pathway analysis revealed genes associated in glycoprotein metabolism or cell differentiation, particularly the homeobox superfamily of transcription factors such as homeobox A9 (*HOXA9*), Iroquois homeobox 2 (*IRX2*) and msh homeobox 2 (*MSX2*), which are involved in embryonic development [44].

#### **3. Histone modification, chromatin remodelling and osteoarthritis**

highly methylated and, in OA, it is demethylated and highly expressed. Additionally, *LEP* downregulation with small interference RNA (siRNA) decreases *MMP13* expression [34]. To date, it is well recognized that OA has an important inflammatory component in its development mediated by proinflammatory cytokines, such as IL-1β and TNFα [2, 32]. Healthy chondrocytes do not express *IL1B*, however, promoter demethylation increases the expression of the gene [35]. Suppressor of cytokine signalling (SOCS) proteins are inhibitors of cytokine signalling. There are eight SOCS proteins, including SOCS1-SOCS7 and cytokine-inducible SH2-domain-1 (CIS-1), with SOCS1, -2 and -3, and CIS-1 the best characterized. *SOCS2* and *CIS-1* expression is reduced in OA chondrocytes compared with normal chondrocytes, while *SOCS1* and *SOCS3* expression remains unchanged. In addition, the *SOCS2* promoter does not

Bone morphogenetic protein-7 (BMP-7) – or osteogenic protein-1(OP-1) – is one of the most potent growth factors for cartilage maintenance and repair. It possesses a critical role in human cartilage homeostasis, regulating numerous metabolic pathways that are not only limited to its well-documented anabolic function but also to its anti-catabolic activity [37]. There is a positive correlation between age and the methylation of the *BMP7* promoter's status in aged chondrocytes; this age-related promoter methylation may contribute to a decrease in BMP7 production in cartilage, with the decreased expression of the insulin-like growth factor-1 (*IGF-1*) and the IGF-1 receptor (*IGF-1R*) genes, as well as the ECM component gene *ACAN* [38]. The growth differentiation factor 5 (*GDF5*) gene is a member of the transforming growth factor β (TGFβ) superfamily that is involved in chondrogenesis and chondrocyte proliferation [39]. The rs143383 C/T single nucleotide polymorphism (SNP) in the 5´ untranslated region (5´UTR) of the gene is associated with an increased risk of OA [40, 41]. This SNP is itself functional and exerts a joint-wide effect on *GDF5* expression, causing a significant reduction in the expression of the disease-associated T allele relative to the C allele in the cartilage and other joint tissues [42], a phenomenon known as 'differential allelic expression' (DAE). The transcriptional effect of rs143383 SNP is dependent on a second C-to-T SNP in the 5´UTR of *GDF5*, rs143384 C/T, with decreased expression of the T allele of rs143383 only observed in individuals which are compound-heterozygous for both SNPs. When the OA-protective C alleles are present at the rs143383 and rs143384 SNPs, they form CpG dinucleotides, which are potentially amenable to regulation by DNA methylation. In cell lines, *GDF5* is upregulated after demethylation, and methylation decreases transcriptional activity. Interestingly, CpG sites formed by the C alleles of both SNPs are methylated; however, their demethylation is associated with increased expression of the C allele of rs143383 relative to the T allele, which indicates that the OAsusceptibility conferred by rs143383 of the *GDF5* gene is regulated by methylation [43].

Methylation analysis of 23,367 sites (corresponding to 13,463 genes) through a genome-wide methylation profile of bone from patients with OA and osteoporosis (OP) revealed an inverse relationship between methylation and gene expression in both groups, with 271 CpG sites being less methylated in OP than in OA. *In silico* pathway analysis revealed genes associated in glycoprotein metabolism or cell differentiation, particularly the homeobox superfamily of transcription factors such as homeobox A9 (*HOXA9*), Iroquois homeobox 2 (*IRX2*) and msh

homeobox 2 (*MSX2*), which are involved in embryonic development [44].

exhibit a change in its methylation status [36].

18 Osteoarthritis - Progress in Basic Research and Treatment

DNA is compacted by the tight weaving of approximately 147 base pairs around the proteins' denominated histones, forming a DNA-protein complex termed a 'nucleosome', the basic unit of chromatin. Each nucleosome consists of an octamer of two copies of the following four core histones: H2A, H2B, H3 and H4. The nucleosomes comprise a barrier to transcription that blocks the access of activators and transcription factors to their sites in DNA. The histones are subject to post-transcriptional modification; the most common are acetylation and methyla‐ tion, although other modifications have been identified, including phosphorylation, ubiquiti‐ nation, SUMOylation, citrullination, and adenosine diphosphate ribosylation. Histones acetylation occurs at lysine residues and is associated with DNA accessibility and transcrip‐ tional activity, whereas deacetylation is associated with transcriptional repression. Histone acetyltransferases (HATs) are enzymes that transfer the acetyl group onto the ε-amino group of the lysine residues within a histone tail; this is a reversible process, and the enzymes that remove the acetyl groups are known as 'histone deacetylases' (HDACs). Classical isoforms of HDACs comprise a total of 11, and are broadly divided into two classes: HDACs 1, -2, -3 and -8 are Class I HDACs, while Class II encompasses HDAC isoforms 4, -5, -6, -7, -9, -10 and -11. The newly characterized SIR2 family of HDACs (sirtuins), termed 'Class III', operate through a nicotinamide adenine dinucleotide (NAD+)-dependent mechanism. Histone methylation is another major modification that takes place in the ε-amino group of lysine residues; it is mediated by histone H3 N-lysine lysine methyltransferases (HKMTs). The effect of histone lysine methylation on gene regulation is highly complex, mediating either transcription repression or activation. Likewise, the methylation of arginine residues is catalysed by the protein arginine methyltransferase (PRMT) family. Regulation of gene transcription can also occur by chromatin remodelling. The SWItch/sucrose non-fermentable protein complex binds to the nucleosome and disconnects the DNA from the histones, creating a transient DNA loop and resulting in nucleosome repositioning, such that the transcription of targeted genes can be increased or decreased depending upon whether the gene is located in the open-chromatin or compacted chromatin region. Polycomb-group proteins are also involved in gene silencing through chromatin remodelling, repressing transcription by maintaining a heterochromatin state through particular histone modifications and DNA methylation [9-13].

Chondrocyte differentiation is controlled by transcription factors such as SOX9, among others. SOX9 requires other cofactors, such as the CREB binding protein, which activates SOX9-dependent transcription due to its intrinsic histone acetyl-transferase activity [45]. In human chondrocytes inducted by IL-1β and the fibroblast growth factor 2 (FGF2), there is increased expression of MMPs and ADAMTS, responsible for collagen and aggrecan loss, respectively. However, HDAC inhibitors (HDACi) block the induction of these enzymes at the mRNA and protein levels [46, 47]. Thus, HDACi also suppress IL-1β-induced NO and prostaglandin E2 (PGE2) synthesis, which plays an important role in OA as well as in proteoglycan degradation [48].

Specific HDACs appear to be involved in different processes as well as targeting different chondrocyte-specific genes. In a murine model, *HDAC4* demonstrates the regulation of chondrocyte hypertrophy and endochondral bone formation by interacting with Runx2 and inhibiting its activity [49]. In the chondrocytes of patients with OA, HDAC1 and HDAC2 proteins are elevated with the specific downregulation of *COL2A1* and *ACAN*, though not with other cartilage marker genes. This is because the snail transcription factor acts as a mediator of the HDAC1 and HDAC2 repression of *COL2A1* via its interaction with HDACs' carboxyterminal domains [50]. *HDAC7* shows a significant increase in OA cartilage, while its knock‐ down by siRNA in a chondrosarcoma cell line suppress *MMP-13* expression [51].

The role of HDACi has been explored in OA. HDAC activity decreases during chondrocyte dedifferentiation, and the inhibition of HDAC with HDACi trichostatin suppresses type II collagen expression. This is because HDACi promotes the acetylation of Wnt-5a, increasing its expression, which is known to inhibit type II collagen [52]. Human chondrocytes under mechanical stress exhibit the downregulation of *COL2A1* and upregulation of *RUNX2*, *ADAMTS4* and *MMP3*; however, after treatment with HDACi there is an increase of *COL2A1* expression, the downregulation of *RUNX2, ADAMTS4* and *MMP3*, and an inhibition of the mechanical stress-induced phosphorylation of mitogen-activated protein kinase (MAPK) molecules after treatment with HDACi [53]. RUNX family members regulate the gene expression involved in cellular differentiation and cell cycle progression. *RUNX2* plays a key role in bone mineralization by stimulating osteoblast differentiation [54] and it contributes to OA pathogenesis through chondrocyte hypertrophy and matrix breakdown after the initiation of joint instability [55]. MAPK pathways play essential regulatory roles in early osteoblast differentiation in response to mechanical stress via the activation of *RUNX2* [56]. *In vivo,* the effects of HDACi trichostatin on cartilage degradation in a rabbit experimental model showed that HDACi decreases cartilage degradation as well as the expression of IL1 and MMPs, such as *MMP1*, *MMP3* and *MMP13* [57].

Histone H3 lysine-4 (H3K4) methylation is associated with transcriptional activation, whereas H3K9 methylation correlates with transcriptional repression. In human osteoarthritic chon‐ drocytes, the induction of iNOS and cyclooxygenase 2 (COX2) expressions by IL-1β are associated with H3K4 di and trimethylation at the *iNOS* and *COX2* promoters; these changes correlate with the recruitment of SET-1A, a HKMT. Furthermore, HKMT inhibition prevents the IL-1β induction of iNOS and COX2 [58].

*Nfat1* is a nuclear factor of the activated T cells' transcription factor family and is a regulator of cytokine gene-expression. It has been reported that adult *Nfat1*-deficient mice display abnormal chondrocyte differentiation in their articular cartilage and develop several articular cartilage characteristics that resemble these changes in OA in humans. These OA-like changes appear at the adult stage and an increase in *Nfat1* expression in the chondrocytes is associated with increased H3K4 methylation, whereas a decrease of *Nfat1* is associated with an increase in H3K9 methylation, which demonstrates that *Nfat1* specifically regulates the function of adult articular chondrocytes through its age-dependent expression, mediated by dynamic histone methylation [59].

NAD-dependent class III HDACs consist of SIRT1-7. SIRT1 plays a key role in the regulation of metabolism, as well in regulating cell differentiation, proliferation, survival and longevity [60, 61]. SIRT1 increases cartilage-specific gene expression, such as *ACAN*, *COL2A1*, *COL9A1* and *COMP*. SIRT1 deacetylate SOX9 enhances the transcription of *COL2A1* and, at least for the *COL2A1* gene promoter, also enhances the acetylation of critical histone core residues in the promoter through the recruitment of activator/co-activator proteins [62]. In human chondrocytes, SIRT1 inhibits NO-induced apoptosis caused, at least partially, by caspases 3 and 9 [63]. Another mechanism by means of which SIRT1 inhibits apoptosis in human chondrocytes is that of repressing protein tyrosine phosphatase 1B (PTP1B), a potent proa‐ poptotic protein, and there is an inverse relationship in the expression patterns of SIRT1 and PTP1B in normal and OA cartilage. In contrast, SIRT1 levels are high and PTP1B levels are low in normal cartilage, while in OA SIRT1 levels are low and PTP1B levels are high [64]. In OA, the inhibition of SIRT1 induces OA-like gene expression changes with a downregulation of *ACAN* and upregulation of *COL10A1* and *ADAMTS5*, which suggests that SIRT1 expression decreases with the development of OA, favouring chondrocyte hypertrophy and cartilage matrix loss [65].

chondrocyte hypertrophy and endochondral bone formation by interacting with Runx2 and inhibiting its activity [49]. In the chondrocytes of patients with OA, HDAC1 and HDAC2 proteins are elevated with the specific downregulation of *COL2A1* and *ACAN*, though not with other cartilage marker genes. This is because the snail transcription factor acts as a mediator of the HDAC1 and HDAC2 repression of *COL2A1* via its interaction with HDACs' carboxyterminal domains [50]. *HDAC7* shows a significant increase in OA cartilage, while its knock‐

The role of HDACi has been explored in OA. HDAC activity decreases during chondrocyte dedifferentiation, and the inhibition of HDAC with HDACi trichostatin suppresses type II collagen expression. This is because HDACi promotes the acetylation of Wnt-5a, increasing its expression, which is known to inhibit type II collagen [52]. Human chondrocytes under mechanical stress exhibit the downregulation of *COL2A1* and upregulation of *RUNX2*, *ADAMTS4* and *MMP3*; however, after treatment with HDACi there is an increase of *COL2A1* expression, the downregulation of *RUNX2, ADAMTS4* and *MMP3*, and an inhibition of the mechanical stress-induced phosphorylation of mitogen-activated protein kinase (MAPK) molecules after treatment with HDACi [53]. RUNX family members regulate the gene expression involved in cellular differentiation and cell cycle progression. *RUNX2* plays a key role in bone mineralization by stimulating osteoblast differentiation [54] and it contributes to OA pathogenesis through chondrocyte hypertrophy and matrix breakdown after the initiation of joint instability [55]. MAPK pathways play essential regulatory roles in early osteoblast differentiation in response to mechanical stress via the activation of *RUNX2* [56]. *In vivo,* the effects of HDACi trichostatin on cartilage degradation in a rabbit experimental model showed that HDACi decreases cartilage degradation as well as the expression of IL1 and MMPs, such

Histone H3 lysine-4 (H3K4) methylation is associated with transcriptional activation, whereas H3K9 methylation correlates with transcriptional repression. In human osteoarthritic chon‐ drocytes, the induction of iNOS and cyclooxygenase 2 (COX2) expressions by IL-1β are associated with H3K4 di and trimethylation at the *iNOS* and *COX2* promoters; these changes correlate with the recruitment of SET-1A, a HKMT. Furthermore, HKMT inhibition prevents

*Nfat1* is a nuclear factor of the activated T cells' transcription factor family and is a regulator of cytokine gene-expression. It has been reported that adult *Nfat1*-deficient mice display abnormal chondrocyte differentiation in their articular cartilage and develop several articular cartilage characteristics that resemble these changes in OA in humans. These OA-like changes appear at the adult stage and an increase in *Nfat1* expression in the chondrocytes is associated with increased H3K4 methylation, whereas a decrease of *Nfat1* is associated with an increase in H3K9 methylation, which demonstrates that *Nfat1* specifically regulates the function of adult articular chondrocytes through its age-dependent expression, mediated by dynamic histone

NAD-dependent class III HDACs consist of SIRT1-7. SIRT1 plays a key role in the regulation of metabolism, as well in regulating cell differentiation, proliferation, survival and longevity [60, 61]. SIRT1 increases cartilage-specific gene expression, such as *ACAN*, *COL2A1*, *COL9A1*

down by siRNA in a chondrosarcoma cell line suppress *MMP-13* expression [51].

as *MMP1*, *MMP3* and *MMP13* [57].

20 Osteoarthritis - Progress in Basic Research and Treatment

methylation [59].

the IL-1β induction of iNOS and COX2 [58].

To date, OA is well-recognized as an inflammatory disease in which inflammatory cytokines play a central role. In human OA chondrocytes treated with TNFα there is an impaired SIRT1 activity due to cleavage mediated by cathepsin B, resulting in the upregulation of *MMP13* and *ADAMTS4* and reduced cartilage-specific gene expression, such as *COL2A1*, *COL11A1* and *ACAN* [66]. Null mice for SirT1 (SirT1 -/-) do not survive, and heterozygous mice for SirT1 (+/-) are smaller and exhibit a greater increase in OA changes than normal mice. In addition, in heterozygous mice, inflammatory cytokines are upregulated and demonstrate a marked increase in apoptosis, which suggests that SirT1 may prolong the viability of articular chon‐ drocytes in adult mice [67]. In human chondrocytes, the overexpression of SIRT1 significantly inhibits the upregulation of genes caused by the pro-inflammatory cytokines IL-1β and TNFα (*MMP1*, *-2*, *-9*, and -*13*, and *ADAMTS5*), while in the OA cartilage SIRT1 expression decreased while that of *MMP13* and *ADAMTS5* increased [68, 69]. Therefore, SIRT1 exerts an antiinflammatory effect and prevents chondrocyte apoptosis.

#### **4. microRNAs in chondrogenesis and osteoarthritis**

MicroRNA (miRNA) are small noncoding RNAs of ~20–25 nucleotides (nt) in length that are transcribed in the nucleus by RNA polymerase II or III into a long precursor denominated primary-miRNA (pri-miRNA). This pri-miRNA is processed by the microprocessor Drosha-DGCR8 complex, an RNase III-type enzyme, to generate a precursor of ~70–100 nt, known as 'pre-miRNA', which is translocated into the cytoplasm via exportin 5. Pre-miRNA is processed by the ribonuclease dicer, generating a miRNA duplex of ~22 nt. Finally, one of the strands is incorporated into the RNA-induced silencing complex (RISC), where it is guided to its target mRNA. miRNA is involved in post-transcriptional gene-expression regulation, targeting 30% of the encoding genes through complementary base-pairing between the miRNA and the 3´- UTR of the messenger RNA (mRNA) target, resulting in the translational suppression or direct degradation of the mRNA [9-13].

#### **4.1. Cartilage and miRNA**

The influence of miRNAs in cartilage homeostasis and skeletal development has been demonstrated in recent years. Dicer is an essential enzyme for the generation of mature miRNA and for proper skeletal morphogenesis. In mouse models, the loss of *dicer1* leads to embryonic lethality, with animals surviving until embryonic day 7.5 [70] with limbs that are small in size due to the loss of processed miRNAs [71]. Dicer possesses an important function in cartilage as demonstrated in mice in which the gene was specifically deleted in cartilage. These mice exhibited a progressive reduction in the proliferating pool of chondro‐ cytes in growth plates, leading to severe growth defects because of a decrease in proliferat‐ ing chondrocytes and an accelerated differentiation into hypertrophic chondrocytes. The latter results may be explained by dicer loss having distinct functional effects at different stages of chondrocyte development [72].

miR-140 is the most studied miRNA in both cartilage and OA. This miRNA was originally identified as a cartilage-restricted miRNA in developing zebra fish, with its expression in the jaw, head and fins during embryonic development [73]. In mice, miR-140 is also expressed in cartilage during embryonic long- and flat-bone development [74]. In a murine model with a targeted deletion of miR-140, mice are borne with grossly normal skeletal development; however, postnatally they manifest skeletal deformities with short stature and craniofacial deformities, probably as a result of abnormal chondrocyte proliferation [75, 76]. miR-140 is encoded in an intronic region of the ubiquitin E3 ligase gene, *WWP2*, which plays an important role in cartilage biology. miR-140 is highly conserved among vertebrates and it is not present in invertebrates, which suggests that it plays an important role in skeletal development [77]. In mice, miR-140 is exclusively expressed in chondrocyte, is co-expressed with *Wwp2*, and is directly induced by the transcription factor SOX9. Sp1, the activator of the cell cycle regulator p15INK4b, is a target of miR-140, suggesting that it regulates chondrogenic proliferation in part via the inhibition of Sp1 [78].

In the gene expression pattern in human articular chondrocytes and human MSC, miR-140 saw the largest differences in expression. During chondrogenesis, miR-140 increases in parallel with the expression of *SOX9* and *COL2A1*, and treating chondrocytes with IL-1β, suppresses miR-140 expression. On the other hand, transfection of chondrocytes with miR-140 downregulate IL-1β-induced *ADAMTS5* expression and rescue the IL-1β-dependent repression of *ACAN* expression [79]. miR-140 has offered several targets: in mice it potentially suppresses *Hdac4*, a co-repressor of Runx2, the transcription factor essential for chondrocyte hypertrophy and osteoblast differentiation [74]. Other targets include the CXC group of chemokine ligand 12 (*Cxcl12*, also known as 'stromal-derived factor 1' (*SDF-1*)) [80] and *SMAD3* [81], both of which are implicated in chondrocyte differentiation. Interestingly, miR-140 was reported as suppressing *Dnpep*, an aspartyl aminopeptidase that catalyses the sequential removal of amino acids from the unblocked N termini of peptides and proteins, which antagonize BMP signalling downstream of *SMAD* activation [76].

In addition to miR-140, miR-455-3p expression is also restricted to the cartilage and perichon‐ drium of the developing long bones in chicks and to the long bones and joints in mouse embryos, and it contributes to chondrogenesis in humans. miR-455-3p resides in an intron of *COL27A1*, a collagen expressed in cartilage, and its expression is regulated by TGFβ ligands and miRNA-regulated TGFβ signalling. Activin receptor type IIB (*ACVR2B)*, *SMAD2*, and chordin-like protein (*CHRDL1*) are targets of miR-455-3p, and may mediate its functional impact on TGFβ signalling, suppressing the *SMAD2/3* pathway; therefore, its unincreased expression could exacerbate the OA process [82].

**4.1. Cartilage and miRNA**

22 Osteoarthritis - Progress in Basic Research and Treatment

stages of chondrocyte development [72].

via the inhibition of Sp1 [78].

downstream of *SMAD* activation [76].

The influence of miRNAs in cartilage homeostasis and skeletal development has been demonstrated in recent years. Dicer is an essential enzyme for the generation of mature miRNA and for proper skeletal morphogenesis. In mouse models, the loss of *dicer1* leads to embryonic lethality, with animals surviving until embryonic day 7.5 [70] with limbs that are small in size due to the loss of processed miRNAs [71]. Dicer possesses an important function in cartilage as demonstrated in mice in which the gene was specifically deleted in cartilage. These mice exhibited a progressive reduction in the proliferating pool of chondro‐ cytes in growth plates, leading to severe growth defects because of a decrease in proliferat‐ ing chondrocytes and an accelerated differentiation into hypertrophic chondrocytes. The latter results may be explained by dicer loss having distinct functional effects at different

miR-140 is the most studied miRNA in both cartilage and OA. This miRNA was originally identified as a cartilage-restricted miRNA in developing zebra fish, with its expression in the jaw, head and fins during embryonic development [73]. In mice, miR-140 is also expressed in cartilage during embryonic long- and flat-bone development [74]. In a murine model with a targeted deletion of miR-140, mice are borne with grossly normal skeletal development; however, postnatally they manifest skeletal deformities with short stature and craniofacial deformities, probably as a result of abnormal chondrocyte proliferation [75, 76]. miR-140 is encoded in an intronic region of the ubiquitin E3 ligase gene, *WWP2*, which plays an important role in cartilage biology. miR-140 is highly conserved among vertebrates and it is not present in invertebrates, which suggests that it plays an important role in skeletal development [77]. In mice, miR-140 is exclusively expressed in chondrocyte, is co-expressed with *Wwp2*, and is directly induced by the transcription factor SOX9. Sp1, the activator of the cell cycle regulator p15INK4b, is a target of miR-140, suggesting that it regulates chondrogenic proliferation in part

In the gene expression pattern in human articular chondrocytes and human MSC, miR-140 saw the largest differences in expression. During chondrogenesis, miR-140 increases in parallel with the expression of *SOX9* and *COL2A1*, and treating chondrocytes with IL-1β, suppresses miR-140 expression. On the other hand, transfection of chondrocytes with miR-140 downregulate IL-1β-induced *ADAMTS5* expression and rescue the IL-1β-dependent repression of *ACAN* expression [79]. miR-140 has offered several targets: in mice it potentially suppresses *Hdac4*, a co-repressor of Runx2, the transcription factor essential for chondrocyte hypertrophy and osteoblast differentiation [74]. Other targets include the CXC group of chemokine ligand 12 (*Cxcl12*, also known as 'stromal-derived factor 1' (*SDF-1*)) [80] and *SMAD3* [81], both of which are implicated in chondrocyte differentiation. Interestingly, miR-140 was reported as suppressing *Dnpep*, an aspartyl aminopeptidase that catalyses the sequential removal of amino acids from the unblocked N termini of peptides and proteins, which antagonize BMP signalling

In addition to miR-140, miR-455-3p expression is also restricted to the cartilage and perichon‐ drium of the developing long bones in chicks and to the long bones and joints in mouse embryos, and it contributes to chondrogenesis in humans. miR-455-3p resides in an intron of To study the miRNA-mediated regulation of chondrogenesis, the expression of 35 miRNAs in chondroblasts derived from MSC was analysed and it was found that miR-199a and miR-124a were strongly upregulated, while miR-96 was substantially suppressed. The potential targets of the miRNAs are the following transcriptional factors: HIF-1α for miR-199a, regulatory factor X1 (RFX1) for miR-124a, and SOX5 for miR-96, which demonstrate that miRNAs and tran‐ scription factors could fine-tune cellular differentiation [83].

In another miRNA microarray in MSC at four different stages of TGF-β3-induced chondro‐ genesis differentiation, eight significantly upregulated and five downregulated miRNA were observed. Two miRNA clusters, miR-143/145 and miR-132/212, were maintained on downre‐ gulation, while miR-140-3p was the most upregulated. By means of bioinformatics approaches, the following target genes were predicted: *ADAMTS5* for miR-140-3p; activin receptor 1B (*ACVR1B*) for miR143/145; *SOX6* for miR-132/212; and *RUNX2* for miR-30a [84]. Consistent with that finding, miR-145 decreased during TGF-β3-induced chondrogenic differentiation of murine MSC, and targeted the SRY-related high-mobility group-box gene 9 (SOX9), the key transcription factor for chondrogenesis. In cells overexpressing miR-145, the expression of chondrogenic markers was significantly decreased at the mRNA level, including *COL2A1*, *ACAN*, the cartilage oligomeric matrix protein (*COMP*), *COL9A2* and *COL11A1* [85], indicating that miR-145 comprises a key mediator of early chondrogenic differentiation attenuating SOX9 at the post-transcriptional level. In this way, it was reported that miR-145 is a direct regulator of SOX9 in normal human articular chondrocytes through binding to a non-conserved specific site in its human 3´-UTR. In addition, the increased expression of miR-145 in articular chon‐ drocytes greatly reduced the expression *COL2A1* and *ACAN*, and critical cartilage ECM genes, and significantly increased the hypertrophic markers *RUNX2* and *MMP13*, responsible for the changes during the OA process [86].

miR-675 could also regulate *COL2A1* expression. miR-675 is processed from H19, a noncoding RNA, in healthy human chondrocytes. This miRNA is highly expressed and is regulated by SOX9 during chondrocyte differentiation, and upregulates the expression of *COL2A1.* The overexpression of miR-675 rescued COL2A1 levels in H19- or SOX9-depleted chondrocytes, which suggests that the regulation of *COL2A1* by SOX9/H19 is mediated specifically by miR-675. These data suggest that miR-675 may modulate cartilage homeostasis by suppressing a *COL2A1* transcriptional repressor [87].

A comparative miRNA array of approximately 380 miRNAs in C2C12 cells induced by BMP2 found that several miRNAs, including let-7e, miR-221, miR-199a-3p, miR-374 and miR-298 were positively regulated, while miR-125a, miR-210, miR-125b, miR-21, miR-145 and miR-143 were repressed. Among these, miR-199a-3p was the most significantly upregulated at 24 h following BMP2 induction in the C3H10T1/2 cells and in an *in vitro* cell model of chondrogen‐ esis. When these cells were transfected with miRNA-199a-3p, they exhibited a significant decrease in mRNA expression levels of the chondrogenic markers COL2A1 and COMP, suggesting that miR-199a-3p is an inhibitor of the early stages of chondrogenic differentiation. miRNA target-prediction demonstrated that the putative target genes are *SMAD1* and *SMAD5*, which are known downstream mediators of BMP signalling in osteochondroproge‐ nitor cells, which suggests that miR-199a-3p is a BMP2 responsive microRNA that adversely regulates early chondrocyte differentiation via the direct targeting of the *SMAD1* transcription factor [88].

To investigate the miRNA expression pattern involved in the chondrocyte dedifferentiation process, a microarray analysis was performed. Several miRNA were deregulated, including 13 upregulated and 12 downregulated miRNA in differentiated as compared with dediffer‐ entiated chondrocytes. The most notable changes were for miR-491-3p, miR-140-3p, miR-140-5p and let-7d, which were upregulated, and for miR-548e, miR-342-5p, miR-1248 and miR-146a, which were downregulated. Bioinformatics analysis revealed 21 microRNA–gene target pairs potentially involved in chondrocyte dedifferentiation. Among these, miR-548e-*SOX9*, miR-1248-*ACAN*, miR-18a-*IGF1*, miR-193b-*SOX5*, miR-631-*RUNX1*, miR-335-*CRTAP*, miR-153-*MATN2* and miR-26a-*COL1A2* are involved in ECM proteins and homeostasis in chondrocytes, miR-365-*BCL2* in the apoptosis mechanism, and let-7a, d, f- *ITGB3*, miR-320d-*DBN1*, miR-1260-*LAMC2* and miR-222-*ITGB3* are involved in cytoskeleton organization [89]. Human primary articular chondrocytes allow the initiation of proliferation to produce ECM molecules similar to embryological chondroblasts, and are called 'chondroblast-like' cells. The miRNA expression profile in these cells showed five differentially expressed miRNA clusters. Among these, one cluster consisted of miR-451, four miRNA included miR-140-3p, another cluster had five miRNA including miR-221 and miR-222, and the last cluster consisted of 11 miRNA, including miR-143 and miR-145, all of these being upregulated at different differen‐ tiation stages that might inhibit the molecules from participating in the dedifferentiation process [90].

miR-194 decreased during chondrogenic differentiation in adipose-derived stem cells (ASCs) (which are capable of differentiating into cartilage lineages *in vitro*). This downregulation increases its direct target gene, *SOX5*, resulting in the chondrogenic differentiation of ASC; thus, miR-194 may mediate chondrogenic differentiation via the suppression of the transcrip‐ tion factor *SOX5* [91]. In a rat model, miR-337 is associated with chondrogenesis by repressing transforming growth factor-beta type II receptor (TGFBR2) expression and modulating the expression of cartilage-specific genes, such as *ACAN*, in chondrocytes [92].

During the chondrogenesis differentiation of chick-limb mesenchymal cells, miR-221 may be involved in chondrocyte apoptosis; its inhibition reverses the chondro-inhibitory actions of a Jun N-terminal protein kinase (JNK) inhibitor in the proliferation and migration of chondro‐ genic progenitors. A target for miR-221 is Mmd2, an oncoprotein that has been shown to inhibit the activity of the p53 tumour suppressor protein with E3 ubiquitin ligase activity, and downregulation of Mmd2 prevents the degradation of the slug protein, which negatively regulates the proliferation of chondroprogenitors. The slug protein is a snail family member – it controls the developmental process by regulating the genes involved in cell adhesion and migration [93]. In the same model, miR-34a is a negative modulator of chondrogenesis and affects MSC migration but not proliferation. EphA5, a receptor in Eph/ephrin signalling that mediates cell-to-cell interaction, is a miR-34a target [94]. Moreover, miR-34a regulates RhoA/ Rac1 cross-talk and negatively modulates the reorganization of the actin cytoskeleton, which is one of the essential processes in establishing chondrocyte morphology [95]. Table 1 depicts the miRNAs implicated in chondrogenesis.

decrease in mRNA expression levels of the chondrogenic markers COL2A1 and COMP, suggesting that miR-199a-3p is an inhibitor of the early stages of chondrogenic differentiation. miRNA target-prediction demonstrated that the putative target genes are *SMAD1* and *SMAD5*, which are known downstream mediators of BMP signalling in osteochondroproge‐ nitor cells, which suggests that miR-199a-3p is a BMP2 responsive microRNA that adversely regulates early chondrocyte differentiation via the direct targeting of the *SMAD1* transcription

To investigate the miRNA expression pattern involved in the chondrocyte dedifferentiation process, a microarray analysis was performed. Several miRNA were deregulated, including 13 upregulated and 12 downregulated miRNA in differentiated as compared with dediffer‐ entiated chondrocytes. The most notable changes were for miR-491-3p, miR-140-3p, miR-140-5p and let-7d, which were upregulated, and for miR-548e, miR-342-5p, miR-1248 and miR-146a, which were downregulated. Bioinformatics analysis revealed 21 microRNA–gene target pairs potentially involved in chondrocyte dedifferentiation. Among these, miR-548e-*SOX9*, miR-1248-*ACAN*, miR-18a-*IGF1*, miR-193b-*SOX5*, miR-631-*RUNX1*, miR-335-*CRTAP*, miR-153-*MATN2* and miR-26a-*COL1A2* are involved in ECM proteins and homeostasis in chondrocytes, miR-365-*BCL2* in the apoptosis mechanism, and let-7a, d, f- *ITGB3*, miR-320d-*DBN1*, miR-1260-*LAMC2* and miR-222-*ITGB3* are involved in cytoskeleton organization [89]. Human primary articular chondrocytes allow the initiation of proliferation to produce ECM molecules similar to embryological chondroblasts, and are called 'chondroblast-like' cells. The miRNA expression profile in these cells showed five differentially expressed miRNA clusters. Among these, one cluster consisted of miR-451, four miRNA included miR-140-3p, another cluster had five miRNA including miR-221 and miR-222, and the last cluster consisted of 11 miRNA, including miR-143 and miR-145, all of these being upregulated at different differen‐ tiation stages that might inhibit the molecules from participating in the dedifferentiation

miR-194 decreased during chondrogenic differentiation in adipose-derived stem cells (ASCs) (which are capable of differentiating into cartilage lineages *in vitro*). This downregulation increases its direct target gene, *SOX5*, resulting in the chondrogenic differentiation of ASC; thus, miR-194 may mediate chondrogenic differentiation via the suppression of the transcrip‐ tion factor *SOX5* [91]. In a rat model, miR-337 is associated with chondrogenesis by repressing transforming growth factor-beta type II receptor (TGFBR2) expression and modulating the

During the chondrogenesis differentiation of chick-limb mesenchymal cells, miR-221 may be involved in chondrocyte apoptosis; its inhibition reverses the chondro-inhibitory actions of a Jun N-terminal protein kinase (JNK) inhibitor in the proliferation and migration of chondro‐ genic progenitors. A target for miR-221 is Mmd2, an oncoprotein that has been shown to inhibit the activity of the p53 tumour suppressor protein with E3 ubiquitin ligase activity, and downregulation of Mmd2 prevents the degradation of the slug protein, which negatively regulates the proliferation of chondroprogenitors. The slug protein is a snail family member – it controls the developmental process by regulating the genes involved in cell adhesion and migration [93]. In the same model, miR-34a is a negative modulator of chondrogenesis and

expression of cartilage-specific genes, such as *ACAN*, in chondrocytes [92].

factor [88].

24 Osteoarthritis - Progress in Basic Research and Treatment

process [90].



**Table 1.** microRNAs implicated in chondrogenesis

#### **4.2. Osteoarthritis and miRNAs**

The effects of miRNA deregulation on OA are evident in studies comparing the expression of miRNAs between OA tissue specimens and their normal articular counterparts. A study tested 365 miRNA expression in articular cartilage obtained from patients with OA undergoing total joint replacement surgery as well as from normal controls. The study identified 16 miRNA differentially expressed in OA cartilage, of which nine were upregulated and seven downre‐ gulated. Through an *in silico* analysis, miRNA-gene targets were potentially involved in cartilage homeostasis and its structure (*miR-377-CART1, miR-140-ADAMTS5, miR-483-ACAN, miR-23b-CRTAP, miR-16-TPM2, miR-223-GDF5, miR-509-SOX9* and *miR-26a-ASPN*), in biomechanical pathways (*miR-25-ITGA5*), in apoptotic mechanisms (*miR-373-CASP6* and *miR-210-CASP10*), and in lipid metabolism pathways (*miR-22-PPARA, miR-22-BMP7, miR-103- ACOX1, miR-337-RETN* and *miR-29a-LEP*). The comparison of the molecular and clinical data revealed that miR-22 and miR-103 were highly correlated with body mass index (BMI) [97]. In another study, the expression profiling of 157 miRNA in chondrocytes obtained from OA cartilage identified 17 miRNAs that showed differential expression in comparison with normal controls. The most notable changes were observed for miR-9, miR-25 and miR-98, which were upregulated, and for miR-146 and miR-149, which were downregulated. A bioinformatics analysis performed to identify the potential gene targets suggested that a significant number of genes involved in inflammation were related with miR-9, miR-98 and miR-146. In overex‐ pression experiments involving these miRNAs, miR-9, miR-98 and miR-146 were implicated in the control of TNFα, and miR-9 was implicated in MMP13 regulation [98]. To identify and characterize the expression profile of miRNA in the chondrocytes of III and IV grade OA, 723 miRNA were analysed and seven exhibited differential expression, of which one was upre‐ gulated and six were downregulated. In the bioinformatics prediction for knowing potential target genes regulated by these miRNA, it was found that the genes were involved in TGF-β, Wnt, MAPK and the mammalian target of rapamycin (mTOR) signalling pathways, as well in focal adhesion, cytoskeleton regulation, ubiquitin-mediated proteolysis and the cell cycle. Interestingly, TGF-β and Wnt both played a role in OA [99].

microRNA Expression Target Ref. miR-18a ↓ *IGF1* Insulin-Like Growth Factor I 89 miR-153 *MATN2* Matrilin-2 89 miR-1248 ↓ *ACAN* Aggrecan 89 miR-142-3p *ADAM9* A Disintegrin And Metalloproteinase Domain 9 95

Activin a receptor, Type IIB

The effects of miRNA deregulation on OA are evident in studies comparing the expression of miRNAs between OA tissue specimens and their normal articular counterparts. A study tested 365 miRNA expression in articular cartilage obtained from patients with OA undergoing total joint replacement surgery as well as from normal controls. The study identified 16 miRNA differentially expressed in OA cartilage, of which nine were upregulated and seven downre‐ gulated. Through an *in silico* analysis, miRNA-gene targets were potentially involved in cartilage homeostasis and its structure (*miR-377-CART1, miR-140-ADAMTS5, miR-483-ACAN, miR-23b-CRTAP, miR-16-TPM2, miR-223-GDF5, miR-509-SOX9* and *miR-26a-ASPN*), in biomechanical pathways (*miR-25-ITGA5*), in apoptotic mechanisms (*miR-373-CASP6* and *miR-210-CASP10*), and in lipid metabolism pathways (*miR-22-PPARA, miR-22-BMP7, miR-103- ACOX1, miR-337-RETN* and *miR-29a-LEP*). The comparison of the molecular and clinical data revealed that miR-22 and miR-103 were highly correlated with body mass index (BMI) [97]. In another study, the expression profiling of 157 miRNA in chondrocytes obtained from OA cartilage identified 17 miRNAs that showed differential expression in comparison with normal controls. The most notable changes were observed for miR-9, miR-25 and miR-98, which were upregulated, and for miR-146 and miR-149, which were downregulated. A bioinformatics analysis performed to identify the potential gene targets suggested that a significant number of genes involved in inflammation were related with miR-9, miR-98 and miR-146. In overex‐ pression experiments involving these miRNAs, miR-9, miR-98 and miR-146 were implicated in the control of TNFα, and miR-9 was implicated in MMP13 regulation [98]. To identify and characterize the expression profile of miRNA in the chondrocytes of III and IV grade OA, 723 miRNA were analysed and seven exhibited differential expression, of which one was upre‐ gulated and six were downregulated. In the bioinformatics prediction for knowing potential target genes regulated by these miRNA, it was found that the genes were involved in TGF-β, Wnt, MAPK and the mammalian target of rapamycin (mTOR) signalling pathways, as well in

82

Chordin-Like 1

miR-194 ↓ *SOX5* Chondrogenic differentiation 91 miR-337 ↑ *TGFBR2* Transforming Growth Factor-Beta Receptor, Type II 92 miR-181b ↓ *MMP13* Matrix Metalloproteinase 13 96

miR-455-3p ↑

*ACVR2B SMAD2 CHRDL1*

**Table 1.** microRNAs implicated in chondrogenesis

26 Osteoarthritis - Progress in Basic Research and Treatment

**4.2. Osteoarthritis and miRNAs**

Some miRNAs were examined as potential biomarkers of patients with OA of the knee, and miR-16, miR-132, miR-146a and miR-22 were significantly lower in the synovial fluid than in plasma. miR-132 in plasma exhibited a number of miRNAs in plasma, some of which were found at different levels between patients with rheumatoid arthritis (RA) and with OA. Concentrations of miR-16, miR-132, miR-146a and miR-223 are reduced in the synovial fluid of individuals suffering from OA compared with healthy controls [100]. More recently a profiling of 384 miRNAs was developed in the plasma of patients with radiographic OA of the knee, and 12 miRNAs were found to be differentially expressed with a clear differentiation of OA samples from those of healthy controls. *In silico* analysis revealed that potential miRNA targets belonged to OA-related pathways, such as those of chondrocyte maintenance, osteocyte modulation, inflammation, proteases, extracellular matrix (ECM) molecules and signalling pathways. Interestingly, some specific target genes are also involved in OA development, such as fibroblast growth factor receptor 1 (*FGFR1*), histone deacetylase 4 (*HDAC4*), (*FGF2*), vascular endothelial growth factor A (*VEGFA*), the insulin-like growth factor I receptor (*IGF1R*), A disintegrin-like and metalloproteinase with thrombospondin type 1 motif-5 (*ADAMTS5*), tissue inhibitor of metalloproteinase 2 (*TIMP2*), and *WNT1*-inducible signalling pathway protein 1 (*WISP1*) [101].

miR-140 has also been implicated in OA pathogenesis. In mice, the disruption of miR-140 *in vivo* induced the early onset of spontaneous OA-like changes in articular cartilage, in part due to elevated ADAMTS5, characterized by proteoglycan loss and articular cartilage fibrillation in the age-related model, and more severe OA-like changes in the surgical model [75]. In OA cartilage chondrocytes, *COL2A1* expression is low and *ADAMTS5* is increased. In response to IL-1β stimulation, miR-140 expression decreases while that of *ADAMTS5* and *MMP13* increases. These results demonstrate that miR-140 regulates cartilage-specific genes, playing an important role in regulating the balance between ECM synthesis and degradation [79]. Cartilage degradation in OA is due to factors such as MMP and to insulin-like growth factor protein 5 (IGFBP-5). In human OA chondrocytes, miR-140 significantly decreases *IGFBP5* expression, and anti-miR-140 exerts the contrary effect; therefore, these data suggest that *IGFBP5* is a direct target of miR-140 [102]. miR-140 was shown to mediate *MMP13* expression directly *in vitro*. In C28/I2 cells, a model cell of OA, stimulation by IL-1β increases the expres‐ sion of *MMP13* [103].

In human OA chondrocytes, miR-27a expression is decreased in OA and treatment with antimiR-27a increases the expression of *IGFBP5* and *MMP-13*, which suggests that miR-27a may indirectly regulate the levels of both genes by targeting upstream positive effectors of both genes [102]. In agreement with these results, miR-27b is downregulated in IL-1β-stimulated OA chondrocytes stimulated with an inverse correlation of MMP-13 expression [104].

Members of the miR-34 family are induced by p53, leading to apoptosis, cell cycle arrest and senescence through targeting E2F3, cyclin E2 and CDK6, etc. [105]. miR-34a is the most significantly induced miRNA after the activation of p53. In chondrocytes of a rat model of OA, miR-34a is upregulated after IL-1β stimulation and its silencing prevents the IL-1β-induced upregulation of iNOS and the downregulation of Col2a1 [106].

miR-146a/b has been described as a key molecule in the inflammatory response [107] and is expressed in all layers of human articular cartilage, especially in the superficial zone. In grade I OA, the expression of miR-146a and COL2A1 is significantly increased, and is decreased in grades II and III, along with MMP13. Thus, their expression gradually decreased with progressive tissue degeneration [108]. In a rat model with surgically induced OA, miR-146a is upregulated in articular chondrocytes in response to treatment with IL-1β. *SMAD4* is a direct target of miR-146a, and the inhibition of *SMAD4* results in the upregulation of the vascular endothelial growth factor (VEGF) and apoptosis in chondrocytes. This VEGF induction by miR-146a may affect angiogenesis and inflammation during OA pathogenesis [109]. Interest‐ ingly, miR-146a has been implicated in the control of knee joint homeostasis and OA-associated algesia by balancing the inflammatory response in cartilage and synovia with pain-related factors in glial cells [110], because miR-146a is induced by joint stability resulting from medial collateral ligament transaction and medial meniscal tearing in the knee joints of an OA mouse model, suggesting that miR-146a might be a mechano-responsive miRNA in articular cartilage [109]. Previously, miR-365 was described as a mechano-responsive miRNA in chicken primary proliferative chondrocytes under mechanical stimulation. This miRNA stimulates chondro‐ cyte proliferation and differentiation, and increases the expression of the hypertrophic marker COL10A1. Additionally, it targets *HDAC4*, which modulates cell growth and inhibits chon‐ drocyte hypertrophy and endochondral bone formation by inhibiting Runx2 [111]. Table 2 depicts the miRNAs implicated in OA.



**Table 2.** microRNAs implicated in Osteoarthritis

#### **5. Conclusions**

miR-34a is upregulated after IL-1β stimulation and its silencing prevents the IL-1β-induced

miR-146a/b has been described as a key molecule in the inflammatory response [107] and is expressed in all layers of human articular cartilage, especially in the superficial zone. In grade I OA, the expression of miR-146a and COL2A1 is significantly increased, and is decreased in grades II and III, along with MMP13. Thus, their expression gradually decreased with progressive tissue degeneration [108]. In a rat model with surgically induced OA, miR-146a is upregulated in articular chondrocytes in response to treatment with IL-1β. *SMAD4* is a direct target of miR-146a, and the inhibition of *SMAD4* results in the upregulation of the vascular endothelial growth factor (VEGF) and apoptosis in chondrocytes. This VEGF induction by miR-146a may affect angiogenesis and inflammation during OA pathogenesis [109]. Interest‐ ingly, miR-146a has been implicated in the control of knee joint homeostasis and OA-associated algesia by balancing the inflammatory response in cartilage and synovia with pain-related factors in glial cells [110], because miR-146a is induced by joint stability resulting from medial collateral ligament transaction and medial meniscal tearing in the knee joints of an OA mouse model, suggesting that miR-146a might be a mechano-responsive miRNA in articular cartilage [109]. Previously, miR-365 was described as a mechano-responsive miRNA in chicken primary proliferative chondrocytes under mechanical stimulation. This miRNA stimulates chondro‐ cyte proliferation and differentiation, and increases the expression of the hypertrophic marker COL10A1. Additionally, it targets *HDAC4*, which modulates cell growth and inhibits chon‐ drocyte hypertrophy and endochondral bone formation by inhibiting Runx2 [111]. Table 2

**microRNA Expression Target Ref.**

A Disintegrin-Like And Metalloproteinase With

75,79,97,98,102,

103

98

108, 109

97

Insulin-Like Growth Factor-Binding Protein 5

Interleukin 1 Receptor-Associated Kinase 1 TNF Receptor-Associated Factor 6

Mothers against decapentaplegic homolog 4

Peroxisome Proliferator-Activated Receptor

Thrombospondin Type 1 Motif, 5 Matrix Metalloproteinase 13

Matrix Metalloproteinase 13

Bone Morphogenetic Protein 7

miR-103 ↑ *ACOX1* AcyL-CoA Oxidase 1 97 miR-16 ↑ *TIPM2* Tissue Inhibitor Of Metalloproteinase 2 97 miR-210 ↓ *CASP10* Caspase 10 97

miR-223 ↑ *GDF5* Growth/Differentiation Factor 5 97 miR-23b ↑ *CRTAP* Cartilage-Associated Protein 97

upregulation of iNOS and the downregulation of Col2a1 [106].

28 Osteoarthritis - Progress in Basic Research and Treatment

depicts the miRNAs implicated in OA.

*ADAMTS5 MMP13 IGFBP5*

*IRAK1 TRAF6*

*MMP13 SMAD4*

*PPAR BMP7*

miR-140 ↓

miR-146 ↓

miR-146a ↑

miR-22 ↑

At present, there is increasing progress in the description of epigenetic mechanisms under normal conditions, as well in disease, and much of the current knowledge has focused on cancer. Yet epigenetics research has provided new insights in relation to other entities, such as neurological and autoimmune diseases. Epigenetics research into OA continues to be developed, but could shed light on its pathological mechanisms. One promising field is related with OA treatment, such as HDACi or miRNAs. However, although HDACi and miRNAs could inhibit several genes related with its development, they also inhibit ECM genes. To date, there is no appropriate biomarker for OA. Epigenetics marks in OA have been associated with the condition, and even with its progression, and could be biomarkers of disease and progres‐ sion, as miRNAs determined in plasma. However, research into epigenetics continues to be required.

#### **Acknowledgements**

This work was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT), proyecto SALUD-2012-C01-180720.

#### **Author details**

Antonio Miranda-Duarte

Address all correspondence to: fovi01@prodigy.net.mx

Department of Genetics, National Rehabilitation Institute (INR), Mexico City, Mexico

To Ivonne, Ivana, and Romina, with love To my father, Antonio Miranda-Novoa, in loving memory.

#### **References**


**Author details**

**References**

Antonio Miranda-Duarte

Address all correspondence to: fovi01@prodigy.net.mx

To my father, Antonio Miranda-Novoa, in loving memory.

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### **Biomechanics of Cartilage and Osteoarthritis**

Herng-Sheng Lee and Donald M. Salter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60011

#### **1. Introduction**

The etiology of osteoarthritis (OA) is not known with certainty. Undoubtedly, many factors contribute to articular cartilage failure but consistently abnormal biomechanics appear central to the condition. Indeed it is recognized that mechanical loading that is either below or in excess of the physiology range leads cartilage degeneration. There are currently no cures for OA and no effective pharmacological treatments that slow or halt disease progression. Physical activity is one of the most widely prescribed non-pharmacological therapies for OA management, based on its ability to limit pain and improve physical function. The detailed mechanisms underlying these beneficial effects of exercise and physical therapy are largely unknown. Structural integrity is important for joint function and can be lost as a consequence of a range of physical, biomechanical and inflammatory factors. This chapter will overview how joint loading influences cartilage structure and how mechanical loading is perceived by chondro‐ cytes resulting in cellular responses that are either chondroprotective or promoting inflam‐ matory and catabolic responses initiating and progressing OA.

#### **2. Mechanical integrity of joint structure**

The synovial or diarthrodial joint allows movement between bones and permits transmission of mechanical loads. The mechanical integrity of the joint elements including articular cartilage, synovium, subchondral bone, joint capsule, ligaments and periarticular connective tissues cooperates to provide optimal function. Loss of mechanical integrity results in a range of pathological changes within the joint recognized as osteoarthritis.

In a synovial joint the articulating bone ends are covered by a thin, highly hydrated specialized connective tissue, articular cartilage. A high interstitial fluid content distinguishes cartilage from most other connective tissues and contributes to the mechanical properties of the tissue

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

[1,2]. The major components of articular cartilage are type II collagen, proteoglycans, noncol‐ lagenous proteins and glycoproteins. Type II collagen forms the fibrillar meshwork which provides tensile strength [3-6] and entraps aggregating hydrophilic proteoglycans which help maintain the high tissue hydration [7-9]. Disruption of the fibrillar meshwork or loss of proteoglycan results in an in-ability of cartilage to distribute loads and its contribution to near frictionless joint movement leading, in time, to progressive structural damage and pathological features of OA.

#### **3. Articular cartilage responses to mechanical loading** *in vivo*

Mechanical loading within a physiological range is necessary to maintain joints in a healthy state. During normal daily activity articular cartilage is exposed to a range of mechanical forces during joint movement. Peak forces across the human hip and knee joints have been shown to reach 4 and 7 times body weight, respectively, during normal walking [10,11]. In vivo, mechanical loading is applied cyclically and the cells within cartilage, chondrocytes, are exposed to a composite of radial, tangential and shear stresses [12]. The effects of mechanical load bearing on the development and microscopic structure of the articular cartilage have been studied in some detail [13]. Elevated loading increases cartilage thickness, causes hypertrophy of the superficial zone chondrocytes, and increases the amount of intercellular matrix [14-17]. In normal human joints, load-bearing areas of the cartilage are thicker with a higher proteo‐ glycan concentration and are mechanically stronger than non-load-bearing regions of the same joint [18-20]. Increasing weight-bearing of joints, in a variety of animal models, leads to elevation of proteoglycan content within articular cartilage [15,16,21-23]. In contrast, removal of load bearing leads to a reduction in proteoglycan content [13]. In a dog model, immobili‐ zation of a joint by placing a leg in a cast leads to cartilage atrophy, loss of Safranin O staining, and a decrease in its uronic acid content [21]. These changes are reversible on remobilization. Mechanical regulation is also an important factor for chondrogenesis and has been involved in the development of cell-based therapies for cartilage degeneration and disease [24].

#### **3.1. Mechanical stress within articular cartilage**

Articular cartilage is exposed to surprisingly large mechanical loads during joint movement. Using an instrumented hip prosthesis mechanical stresses have been measured in a 74-yearold female [25]. Rising from a chair, pressures in the hip joint cartilage can reach nearly 20 MPa and during walking, pressures cycle between atmospheric and 3-4 MPa at a frequency of around 1 Hz. With walking or running forces at the joint surface may vary from near zero to several times the whole body weight within a period of 1 second [10,11]. Loading of articular cartilage generates a combination of tensile, compressive and shear stress in the material. The tensile modulus of healthy human articular cartilage varies from 5-25 MPa, depending on the site of movement on the joint surface (i.e., high or low weight bearing regions), and the depth and orientation of the test specimen relative to the joint surface [4,26]. The compressive modulus varies from 0.4-2.0 MPa [27,28]. Articular cartilage responds to shearing forces by both stretching and deformation of the solid matrix. The dynamic shear modulus is within the range of 0.2-2.0 MPa for healthy bovine or canine cartilage [29-31]. These physiological stresses are important regulators of cartilage metabolism and integrity as mechanical loading serves to maintain fluid flow and ion phase function within the tissue and act to stimulate chondrocyte metabolism [32].

[1,2]. The major components of articular cartilage are type II collagen, proteoglycans, noncol‐ lagenous proteins and glycoproteins. Type II collagen forms the fibrillar meshwork which provides tensile strength [3-6] and entraps aggregating hydrophilic proteoglycans which help maintain the high tissue hydration [7-9]. Disruption of the fibrillar meshwork or loss of proteoglycan results in an in-ability of cartilage to distribute loads and its contribution to near frictionless joint movement leading, in time, to progressive structural damage and pathological

Mechanical loading within a physiological range is necessary to maintain joints in a healthy state. During normal daily activity articular cartilage is exposed to a range of mechanical forces during joint movement. Peak forces across the human hip and knee joints have been shown to reach 4 and 7 times body weight, respectively, during normal walking [10,11]. In vivo, mechanical loading is applied cyclically and the cells within cartilage, chondrocytes, are exposed to a composite of radial, tangential and shear stresses [12]. The effects of mechanical load bearing on the development and microscopic structure of the articular cartilage have been studied in some detail [13]. Elevated loading increases cartilage thickness, causes hypertrophy of the superficial zone chondrocytes, and increases the amount of intercellular matrix [14-17]. In normal human joints, load-bearing areas of the cartilage are thicker with a higher proteo‐ glycan concentration and are mechanically stronger than non-load-bearing regions of the same joint [18-20]. Increasing weight-bearing of joints, in a variety of animal models, leads to elevation of proteoglycan content within articular cartilage [15,16,21-23]. In contrast, removal of load bearing leads to a reduction in proteoglycan content [13]. In a dog model, immobili‐ zation of a joint by placing a leg in a cast leads to cartilage atrophy, loss of Safranin O staining, and a decrease in its uronic acid content [21]. These changes are reversible on remobilization. Mechanical regulation is also an important factor for chondrogenesis and has been involved in the development of cell-based therapies for cartilage degeneration and disease [24].

Articular cartilage is exposed to surprisingly large mechanical loads during joint movement. Using an instrumented hip prosthesis mechanical stresses have been measured in a 74-yearold female [25]. Rising from a chair, pressures in the hip joint cartilage can reach nearly 20 MPa and during walking, pressures cycle between atmospheric and 3-4 MPa at a frequency of around 1 Hz. With walking or running forces at the joint surface may vary from near zero to several times the whole body weight within a period of 1 second [10,11]. Loading of articular cartilage generates a combination of tensile, compressive and shear stress in the material. The tensile modulus of healthy human articular cartilage varies from 5-25 MPa, depending on the site of movement on the joint surface (i.e., high or low weight bearing regions), and the depth and orientation of the test specimen relative to the joint surface [4,26]. The compressive modulus varies from 0.4-2.0 MPa [27,28]. Articular cartilage responds to shearing forces by both stretching and deformation of the solid matrix. The dynamic shear modulus is within the range of 0.2-2.0 MPa for healthy bovine or canine cartilage [29-31]. These physiological stresses

**3. Articular cartilage responses to mechanical loading** *in vivo*

**3.1. Mechanical stress within articular cartilage**

features of OA.

42 Osteoarthritis - Progress in Basic Research and Treatment

#### **4. Articular cartilage explant and chondrocyte responses to mechanical loading** *in vitro*

Rodan et al. [33] studied the effects of application of compressive forces (80 g/cm2 ) to chick tibial epiphyses (16-day-old embryos) in culture and found that glucose consumption reduced to half of controls. Twenty four hours after the release of pressure, glucose utilization again increased, approaching control levels. The same pressure also stimulated thymidine incorpo‐ ration into DNA. Exposing chick tibial epiphyses to continuous compressive forces (60 g/cm2 , equal to 5.865 kPa) caused a reduction of both cAMP and cGMP [34]. An equivalent hydrostatic pressure applied directly to cells isolated from chick tibial epiphyses also affects cyclic nucleotide accumulation [34]. Veldhuijzen et al. developed a model system that exposed cultured monolayer chondrocytes on the walls of tissue culture tubes to intermittent com‐ pressive forces of 12.8 kPa for 6 hours at a frequency of 0.3 Hz [35]. Contrary to the effect of continuous compressive forces, intermittent compressive forces caused a rise in levels of cAMP and a reduction in DNA synthesis. Palmoski and Brandt [36] studied the effects of both static and intermittent mechanical stress on full-thickness plugs of canine articular cartilage. When the plugs were exposed to compressive force using a regime of 60 sec on/60 sec off, glycosa‐ minoglycan synthesis was reduced to 30-60% of controls. However, when a regime of 4 sec on/ 11 sec off was employed, the glycosaminoglycan synthesis increased by 34%, although protein synthesis and DNA, uronic acid, and water content remained unaltered indicating that different frequencies of cyclical strain produce differences in metabolic activity within chondrocytes.

Some models designed to test the effects of mechanical force on chondrocytes in vitro have focused on the effects of cell stretching. In these models there is usually deformation of a cellladen, flexible membrane which can be regulated according to (1) the method of deformation of the membrane - by control of either the displacement or the force, and (2) the shape and mounting of the deformable membrane - either a circular membrane held at its periphery or a rectangular strip held at the two ends [37]. The devices utilized in the production of the displacement include (a) a vacuum driven diaphragm (silicone elastomer membrane, 2.5 mm in thickness) [38,39], (b) pin shaped displacement (silicone elastomer membrane, 0.254 mm in thickness) [40], (c) glass dome displacement (polytetrafluoroethylene membrane, 0.025 mm in thickness) [41,42], (d) air or fluid displacement (polyurethane membrane, 0.094 mm in thickness) [43-45], and (e) a circular groove displacement (silicone elastomer membrane, 0.076 mm in thickness; polyurethane membrane, 0.094 mm in thickness) [46,47]. The FlexercellTM strain unit [38] consists of a computer-controlled vacuum unit and a baseplate on which are held the culture dishes. These dishes have a flexible base. A vacuum is applied to the dishes via the baseplate. When a precise vacuum level is applied to the system, the bases of the culture plates are deformed by known percentage elongation that is maximal at the edge of the culture dish, but decreases towards the center. Using the system, straining the base of a culture dish leads to strain of the attached cultured cells. When the vacuum is released, the bases of the dishes return to their original conformation.

By stretching a supportive flexible membrane on which chondrocytes were cultured, Lee et al. [48] found that a cyclic 10% mechanical stretch for 8 hours increased glycosaminoglycan synthesis and decreased protein and collagen synthesis. DeWitt et al. [49] showed increased radiosulphate and 14C-glucosamine incorporation into glycosaminoglycans by chick epiphy‐ seal chondrocytes in high density cultures subjected to a 5.5% strain at a frequency of 0.2 Hz. Protein synthesis after 24 hours mechanical strain remained unchanged. Using the Flexer‐ cellTM strain system, Fujisawa et al. [50] investigated the influence of cyclic tension force on the metabolism of cultured chondrocytes. Two levels of force (5 kPa or 15 kPa) and three frequen‐ cies 30 cycles/min (1 sec on/1 sec off), 0.5 cycles/min (1 sec on/119 sec off) and 0.25 cycles/min (1 sec on/239 sec off) were used. Both 5 and 15 kPa of high frequency cyclic mechanical stress for 48 hours significantly inhibited the syntheses of DNA, proteoglycan, collagen, and protein. The expression of interleukin-1, matrix metalloproteinase (MMP)-2 and MMP–9 mRNA were induced by 15 kPa of high frequency force. The production of pro- and active-MMP-9 which would lead to cartilage breakdown in vivo was also increased at this pressure and frequency of stimulation. Reducing the applied frequency decreased the inhibition of proteoglycan synthesis. Mechanical stretch producing 25% maximal elongation at a frequency of 0.05 Hz for 48 hours also induces the expression of high molecular weight heat shock protein (HSP) 105 kDa in the human chondrocytic cell line CS-OKB [51]. These findings suggest that the fre‐ quency of cyclic tension force is one of the key determinants of chondrocyte metabolism.

Using confocal microscopy and fluorescent techniques it is possible to monitor and measure intracellular calcium ion concentration in isolated articular chondrocytes subjected to control‐ led deformation with the edge of a glass micropipette [52]. Intracellular calcium ion concen‐ tration reaches a peak within 5 sec following 25% deformation of the cells and returns to baseline levels in 3-5 minutes. The immediate and transient increase of intracellular calcium waves is abolished by removing Ca2+ from the culture medium and is significantly reduced by the presence of gadolinium and amiloride, agents known to block mechanosensitive ion channels [53-56]. Inhibitors of intracellular Ca2+ release or agents known to cause cytoskeletal disruption including cytochalasin D and colchicine had no significant effect on the Ca2+ waves. The results indicate that mechanosensitive ion channels are upstream in the mechanotrans‐ duction signaling pathway, consistent with results obtained using electrophysiological parameters in the assessment of the cell response to cyclical mechanical strain [56].

The effects of fluid-induced shear stress on articular chondrocyte morphology and metabolism in vitro have also been investigated [57]. Fluid-induced shear stress (1.6 Pa = 16 dynes/cm2 ) was applied by cone viscometer to both normal human and bovine articular chondrocytes. Shear stress for 48 and 72 hours caused individual chondrocytes to elongate and align tangential to the direction of cone rotation. Glycosaminoglycan synthesis was increased 2-fold. After 48 hours of shear stress, the release of prostaglandin E2 (PGE2) was increased 10 to 20 fold. In human articular chondrocytes, mRNA levels for tissue inhibitor of metalloproteinase (TIMP) increased 9-fold in response to shear stress compared to controls. In contrast, mRNA levels for the neutral metalloproteinases, collagenase, stromelysin, and gelatinase, did not show significant changes [57].

#### **5. Chondrocyte mechanoreceptors**

The mechanisms by which chondrocytes recognize and respond to the various mechanical stresses encountered in mechanically loaded cartilage continue to be elaborated. A number of potential mechanoreceptors, sensory receptors that respond to vibration, stretching, pressure, or other mechanical stimuli have been identified in chondrocytes. In cartilage the extracellular matrix (ECM) transmits mechanical signals to the cell interior through changes in tension on the cell membrane. Integrins, stretch-activated ion channels, connexins, and primary cilia have each been identified as candidate mechanoreceptors [58-61].

#### **5.1. Integrins**

leads to strain of the attached cultured cells. When the vacuum is released, the bases of the

By stretching a supportive flexible membrane on which chondrocytes were cultured, Lee et al. [48] found that a cyclic 10% mechanical stretch for 8 hours increased glycosaminoglycan synthesis and decreased protein and collagen synthesis. DeWitt et al. [49] showed increased radiosulphate and 14C-glucosamine incorporation into glycosaminoglycans by chick epiphy‐ seal chondrocytes in high density cultures subjected to a 5.5% strain at a frequency of 0.2 Hz. Protein synthesis after 24 hours mechanical strain remained unchanged. Using the Flexer‐ cellTM strain system, Fujisawa et al. [50] investigated the influence of cyclic tension force on the metabolism of cultured chondrocytes. Two levels of force (5 kPa or 15 kPa) and three frequen‐ cies 30 cycles/min (1 sec on/1 sec off), 0.5 cycles/min (1 sec on/119 sec off) and 0.25 cycles/min (1 sec on/239 sec off) were used. Both 5 and 15 kPa of high frequency cyclic mechanical stress for 48 hours significantly inhibited the syntheses of DNA, proteoglycan, collagen, and protein. The expression of interleukin-1, matrix metalloproteinase (MMP)-2 and MMP–9 mRNA were induced by 15 kPa of high frequency force. The production of pro- and active-MMP-9 which would lead to cartilage breakdown in vivo was also increased at this pressure and frequency of stimulation. Reducing the applied frequency decreased the inhibition of proteoglycan synthesis. Mechanical stretch producing 25% maximal elongation at a frequency of 0.05 Hz for 48 hours also induces the expression of high molecular weight heat shock protein (HSP) 105 kDa in the human chondrocytic cell line CS-OKB [51]. These findings suggest that the fre‐ quency of cyclic tension force is one of the key determinants of chondrocyte metabolism.

Using confocal microscopy and fluorescent techniques it is possible to monitor and measure intracellular calcium ion concentration in isolated articular chondrocytes subjected to control‐ led deformation with the edge of a glass micropipette [52]. Intracellular calcium ion concen‐ tration reaches a peak within 5 sec following 25% deformation of the cells and returns to baseline levels in 3-5 minutes. The immediate and transient increase of intracellular calcium waves is abolished by removing Ca2+ from the culture medium and is significantly reduced by the presence of gadolinium and amiloride, agents known to block mechanosensitive ion channels [53-56]. Inhibitors of intracellular Ca2+ release or agents known to cause cytoskeletal disruption including cytochalasin D and colchicine had no significant effect on the Ca2+ waves. The results indicate that mechanosensitive ion channels are upstream in the mechanotrans‐ duction signaling pathway, consistent with results obtained using electrophysiological

parameters in the assessment of the cell response to cyclical mechanical strain [56].

show significant changes [57].

The effects of fluid-induced shear stress on articular chondrocyte morphology and metabolism in vitro have also been investigated [57]. Fluid-induced shear stress (1.6 Pa = 16 dynes/cm2

was applied by cone viscometer to both normal human and bovine articular chondrocytes. Shear stress for 48 and 72 hours caused individual chondrocytes to elongate and align tangential to the direction of cone rotation. Glycosaminoglycan synthesis was increased 2-fold. After 48 hours of shear stress, the release of prostaglandin E2 (PGE2) was increased 10 to 20 fold. In human articular chondrocytes, mRNA levels for tissue inhibitor of metalloproteinase (TIMP) increased 9-fold in response to shear stress compared to controls. In contrast, mRNA levels for the neutral metalloproteinases, collagenase, stromelysin, and gelatinase, did not

)

dishes return to their original conformation.

44 Osteoarthritis - Progress in Basic Research and Treatment

Integrins were first isolated, characterized, and sequenced from chick embryo fibroblast cDNA clones which encoded one subunit of the complex of membrane glycoproteins [62]. The name 'integrin' was proposed as the consequence of its role as an integral membrane complex involved in the transmembrane association between the ECM and the cytoskeleton. Integrins are a large family of α/β heterodimeric cell surface adhesion receptors that can bind a wide variety of ECM and cell surface ligands [63-69]. Most integrins bind ligands that are compo‐ nents of ECM, e.g. fibronectin, collagen, and vitronectin [70]; certain integrins can bind to soluble ligands (such as fibrinogen) or to counter receptors (such as intercellular adhesion molecules) on adjacent cells, leading to homo- or heterotypic aggregation [71,72]. Some of the integrin recognition sites in the ligands and counter receptors have been defined [73,74]. The first binding site to be defined was the Arg-Gly-Asp (RGD) containing sequence present in fibronectin, vitronectin, and a variety of other adhesive proteins [75,76].

Integrin expression by human chondrocytes has been investigated utilizing several techniques including immunohistochemistry, flow cytometry, immunoprecipitation, and northern blotting [77-79]. Normal adult human articular chondrocytes express α1β1, α3β1, α5β1, αVβ5, αVβ3, α6β1, α10β1, and α2β1, in which the α1β1 (receptor for collagen), α5β1 (receptor for fibronectin) and the αVβ5 (receptor for vitronectin) heterodimers are consistently expressed. In cartilage, integrin-ECM interactions are thought to be important in many aspects, physio‐ logical and pathological, of chondrocyte function, including adhesion, spreading, prolifera‐ tion, signal transduction, biomechanical regulation, chondrogenesis, and gene expression [80]. Chondrocyte β1 integrin-ECM interactions are required for chondrocyte survival, matrix deposition and differentiation in models of chondrocyte development [81]. Integrins mediate chondrocyte adhesion to many ECM proteins including type II collagen, type VI collagen, fibronectin, laminin, and osteopontin [82-85]. Chondrocyte spreading and migration on type II collagen, type VI collagen, or fibronectin substrates in vitro is mediated by interactions with β1 integrins [86,87]. The interaction of the α5β1 integrin with fibronectin is necessary for adhesion, spreading, and proliferation of both chicken and rabbit chondrocytes [88,89]. *In vitro* chondrogenesis (the differentiation of blastemal cells to chondroblasts and the formation of cartilage matrix) is inhibited by the function blocking anti-β1 integrin antibodies [90]. Laminin-α3β1/α6β1 interactions are regulated by the ligand trend (depletion/reconstitution or competition experiments) during early chondrocyte differentiation [89]. Other integrinmediated chondrocyte-matrix interactions include β1-matrix Gla protein, β3-bone sialoprotein II, β3-osteopontin, and α2β1-chondroadherin associations [84,86].

Integrins are involved in regulation of both cartilage matrix synthesis and integrity. Loss of integrin function inhibits type II collagen synthesis by chondrocytes in culture [91]. However, integrins are also involved in cartilage breakdown processes. Fibronectin fragments stimulate chondrolysis and decrease proteoglycan synthesis in cartilage explants through fibronectinintegrin dependent interactions [92]. Ligation of α5β1 integrin with fibronectin in cultured chondrocytes results in the formation of focal adhesion complexes comprising actin, focal adhesion kinase (FAK) and the G protein Rho [93]. Nitric oxide (NO), a potential mediator of events occurring in osteoarthritis, can inhibit the assembly of the intracellular activation complex and the subsequent upregulation of proteoglycan synthesis that occurs following ligation of α5β1 integrin to fibronectin [93].

Integrins also act as mechanoreceptors and transmit mechanical signals from the extracellular environment to the cytoskeleton [94-96]. Integrins can provide a gating function for signal transduction, by either supporting or prohibiting force transmission between ECM and the cytoskeleton [94]. Wang et al. [97], using a magnetic twisting device applied mechanical forces directly to cell surface receptors. They showed that the integrin subunit β1 induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhe‐ sion receptors did not [97]. Maniotis et al. [98] reported that living cells and nuclei are hardwired. When integrins were stimulated by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoske‐ leton and nucleus [98]. Using a similar magnetic drag force device, intracellular Ca2+ concen‐ tration was shown to increase when the α2 or the β1 integrin subunits were stressed, whereas mechanical loading of the transferrin receptor produced a significantly reduced effect [99]. An increase in tyrosine phosphorylation was observed as a reaction to mechanical stress on the β1-subunits of the integrin family, whilst stress to the transferrin or low density lipoprotein receptors which have no connection to the cytoskeleton did not produce this reaction [100,101].

Integrins are also modulated by mechanical stress [102]. When chondrosarcoma cells were exposed to mechanical stimulation, mRNA expression of the α5 integrin subunit was found to increase whilst expression of the β1, α2, and αV did not increase significantly [102]. The effect of mechanical stress on integrin subunit expression has also been investigated in cells cultured on type II collagen-coated dishes with a flexible base. Mechanical stress increased mRNA expression of the α2 integrin subunit whilst the levels of mRNA for integrin subunits β1, α1, α5, and αV showed no or only small changes [102]. It is likely that mechanical induced regulation of integrins is closely regulated and may be dependent on the nature of the mechanical force acting on the cell and specific mechanoreceptor stimulated.

#### **5.2. Stretch-activated Ion channels**

Stretch-activated or stretch-sensitive ion channels (SACs) open as a consequence of mechanical deformation of the cell membrane [103]. SACs are directly activated by mechanical forces applied along the plane of the cell membrane that induce membrane tension and distortion of the lipid bilayer. These result in conformational changes which alter opening or closing rates of the channels permitting ion flux [104]. Application of mechanical forces perpendicular to the cell membrane, as seen with hydrostatic pressure, appears to be less effective in activating SACs [103]. Activation of calcium permeable SACs leads to local increase in intracellular calcium levels and stimulation of downstream calcium-dependent intracellular signal cas‐ cades. SACs sensitive to gadolinium are necessary for load and fluid flow related cellular responses in both chondrocytes and bone cells.

#### **5.3. Connexins**

Integrins are involved in regulation of both cartilage matrix synthesis and integrity. Loss of integrin function inhibits type II collagen synthesis by chondrocytes in culture [91]. However, integrins are also involved in cartilage breakdown processes. Fibronectin fragments stimulate chondrolysis and decrease proteoglycan synthesis in cartilage explants through fibronectinintegrin dependent interactions [92]. Ligation of α5β1 integrin with fibronectin in cultured chondrocytes results in the formation of focal adhesion complexes comprising actin, focal adhesion kinase (FAK) and the G protein Rho [93]. Nitric oxide (NO), a potential mediator of events occurring in osteoarthritis, can inhibit the assembly of the intracellular activation complex and the subsequent upregulation of proteoglycan synthesis that occurs following

Integrins also act as mechanoreceptors and transmit mechanical signals from the extracellular environment to the cytoskeleton [94-96]. Integrins can provide a gating function for signal transduction, by either supporting or prohibiting force transmission between ECM and the cytoskeleton [94]. Wang et al. [97], using a magnetic twisting device applied mechanical forces directly to cell surface receptors. They showed that the integrin subunit β1 induced focal adhesion formation and supported a force-dependent stiffening response, whereas nonadhe‐ sion receptors did not [97]. Maniotis et al. [98] reported that living cells and nuclei are hardwired. When integrins were stimulated by micromanipulating bound microbeads or micropipettes, cytoskeletal filaments reoriented, nuclei distorted, and nucleoli redistributed along the axis of the applied tension field. These effects were specific for integrins, independent of cortical membrane distortion, and were mediated by direct linkages between the cytoske‐ leton and nucleus [98]. Using a similar magnetic drag force device, intracellular Ca2+ concen‐ tration was shown to increase when the α2 or the β1 integrin subunits were stressed, whereas mechanical loading of the transferrin receptor produced a significantly reduced effect [99]. An increase in tyrosine phosphorylation was observed as a reaction to mechanical stress on the β1-subunits of the integrin family, whilst stress to the transferrin or low density lipoprotein receptors which have no connection to the cytoskeleton did not produce this reaction [100,101].

Integrins are also modulated by mechanical stress [102]. When chondrosarcoma cells were exposed to mechanical stimulation, mRNA expression of the α5 integrin subunit was found to increase whilst expression of the β1, α2, and αV did not increase significantly [102]. The effect of mechanical stress on integrin subunit expression has also been investigated in cells cultured on type II collagen-coated dishes with a flexible base. Mechanical stress increased mRNA expression of the α2 integrin subunit whilst the levels of mRNA for integrin subunits β1, α1, α5, and αV showed no or only small changes [102]. It is likely that mechanical induced regulation of integrins is closely regulated and may be dependent on the nature of the

Stretch-activated or stretch-sensitive ion channels (SACs) open as a consequence of mechanical deformation of the cell membrane [103]. SACs are directly activated by mechanical forces applied along the plane of the cell membrane that induce membrane tension and distortion of the lipid bilayer. These result in conformational changes which alter opening or closing rates of the channels permitting ion flux [104]. Application of mechanical forces perpendicular to

mechanical force acting on the cell and specific mechanoreceptor stimulated.

ligation of α5β1 integrin to fibronectin [93].

46 Osteoarthritis - Progress in Basic Research and Treatment

**5.2. Stretch-activated Ion channels**

Connexins are widely expressed in connective tissue where networks of cells are seen such as in bone, tendon and, meniscus. They probably act to allow propagation of a mechanical stimulus through a tissue. They are a superfamily of twenty-one transmembrane proteins that form gap junctions and hemichannels [105]. Gap junctions allow continuity between cells permitting diffusion of ions, metabolites and small signaling molecules such as cyclic nucleo‐ tides and inositol derivatives. Cx43 is the most abundant connexin present in skeletal tissue. Conditional deletion of Cx43 reduces mineral apposition rate to mechanical loading and Cx43 hemichannels are important for fluid shear induced PGE2 and ATP release in osteocytic cells [106]. Connexins and gap junctions are present at the tip of osteocyte dendritic processes and between these processes and osteoblasts indicating their potential importance in permitting cell–cell communication among the osteocytic network although propagation may be only directed from osteocytes to osteoblasts [107]. Cx43 hemichannels are activated and mediate small molecule exchange between cells and matrix under mechanical stimulation in rat temporomandibular joint (TMJ) chondrocytes [108]. Cx32 and Cx43 are important in tenocyte mechanotransduction [109]. Cx32 junctions form a communication network arranged along the line of principal loading and stimulate collagen production in response to strain. Connexin dependent mechanotransducation may be important in adaptation of subchondral bone to mechanical loading of joints rather than having a major role in chondrocyte dependent mechanotransduction. Nevertheless recent studies suggest that primary cilia associated connexins may be functional in responses of chondrocytes to mechanical loading.

#### **5.4. Primary Cilia**

Primary cilia are solitary, immotile cilium present in most cells including chondrocytes and bone cells. They are microtubule-based organelles, growing from the centrosome to extend from the cell surface and contain large concentrations of cell membrane receptors, including integrins [110]. They function both as chemosensors and mechanosensors [111,112]. Bending of the cilium upon matrix deformation or with fluid flow is thought to cause cilium bending, pulling on associated matrix receptors and activation of the mechanoreceptors [113]. In addition to integrins, Cx43 hemichannels are also present on primary cilia and by regulating ATP release cilia and activation of purine receptors cilia-associated connexins may also be involved in mechanotransduction.

#### **6. Chondrocyte mechanotransduction**

Mechanoresponsiveness is a fundamental feature of all living cells [94,114,115]. Studies with cultured cells confirm that mechanical stresses can directly alter many cellular processes, including signal transduction, gene expression, growth, differentiation, and survival [116]. Wright et al. [117] investigated the effects of applied hydrostatic pressure on the transmem‐ brane potentials of articular chondrocytes. These studies have been pivotal in identifying potential mechanotransduction pathways in both normal and osteoarthritic human chondro‐ cytes. In this system cells in monolayer culture were exposed to an increase in hydrostatic pressure by placing culture dishes in a sealed perspex pressure chamber with a gas inlet and outlet. Nitrogen or helium gas was used to pressurize the cultures. A hyperpolarization of the chondrocyte plasma membrane was induced by cyclic pressurization (0.33 Hz, 120 mmHg for 20 min) whilst depolarization was induced by continuous pressure (120 mmHg, 20 min). For the frequencies tested, the maximum values for chondrocyte hyperpolarization occurred at approximately 0.3-0.4 Hz. The mechanical stimulation regime (0.33 Hz, 120 mmHg, 20 min), similar to that used by Veldhuijzen et al. [35], allowed identification of a number of integral components of the electrophysiological response providing insight into molecules and pathways activated in chondrocyte upon mechanical stimulation. By the use of pharmacolog‐ ical inhibitors, it was shown that the hyperpolarization response in cultured human chondro‐ cytes induced by cyclic pressurization involved Ca2+ activated K+ channels and L-type calcium channels. Hyperpolarization was also produced by addition of the calcium ionophore A23187 to the culture medium showing that a rise in intracellular Ca2+ concentration within the cell could induce the response. Plasma membrane histamine H1 and H2 receptors, and β-adre‐ noreceptors did not appear to be involved in the hyperpolarization response. The studies also showed that the actin cytoskeleton, but not microtubules, was involved in the chondrocyte hyperpolarization response [117].

Subsequent studies identified that the electrophysiological response to cyclical pressurization was the result of deformation of the base of the culture dishes to which the chondrocytes were attached and therefore deformation (strain) on the chondrocytes rather than a direct effect of the increased hydrostatic pressure on chondrocytes [56]. The hyperpolarization response was proportional to the microstrain to which cells were subjected and did not occur when chon‐ drocytes were subjected to cyclical pressurization in rigid glass culture dishes or when the plastic dishes were positioned in the pressurization chamber so as to avoid deformation of the base of the culture dish [56].

Experiments undertaken to identify the source of intracellular calcium that activated the SK channels leading to hyperpolarization demonstrated a requirement for extracellular calcium and activity of L-type calcium channels [118-120]. Thapsigargin which raises intracellular Ca2+ by inhibition of Ca2+-ATPase in endoplasmic reticulum [121-123] caused hyperpolariza‐ tion independent of mechanical strain but further hyperpolarization of the cells occurred after cyclical pressurization further supporting the idea that mechanically induced chondrocyte hyperpolarization is dependent on intracellular free Ca2+ levels [56]. In addition, TRPV4 mediated Ca2+ signaling has been demonstrated to play a central role in the transduction of mechanical signals to support cartilage extracellular matrix maintenance [124].

#### **6.1. Intracellular signal cascades activated by mechanical stimulation**

Stimulation of connective tissue cell mechanoreceptors is followed by generation of the secondary messenger molecules and activation of a cascade of downstream signaling events that regulate gene expression and cell function. Many intracellular signaling pathways are known to be activated by mechanical forces applied to tissues and cells including heterotri‐ meric guanine nucleotide binding proteins (G-proteins), protein kinases and transcription factors. These pathways that regulate tissue modelling/remodelling may be activated directly as a consequence of mechanoreceptor signaling or indirectly following production of auto‐ crine/paracrine acting molecules.

including signal transduction, gene expression, growth, differentiation, and survival [116]. Wright et al. [117] investigated the effects of applied hydrostatic pressure on the transmem‐ brane potentials of articular chondrocytes. These studies have been pivotal in identifying potential mechanotransduction pathways in both normal and osteoarthritic human chondro‐ cytes. In this system cells in monolayer culture were exposed to an increase in hydrostatic pressure by placing culture dishes in a sealed perspex pressure chamber with a gas inlet and outlet. Nitrogen or helium gas was used to pressurize the cultures. A hyperpolarization of the chondrocyte plasma membrane was induced by cyclic pressurization (0.33 Hz, 120 mmHg for 20 min) whilst depolarization was induced by continuous pressure (120 mmHg, 20 min). For the frequencies tested, the maximum values for chondrocyte hyperpolarization occurred at approximately 0.3-0.4 Hz. The mechanical stimulation regime (0.33 Hz, 120 mmHg, 20 min), similar to that used by Veldhuijzen et al. [35], allowed identification of a number of integral components of the electrophysiological response providing insight into molecules and pathways activated in chondrocyte upon mechanical stimulation. By the use of pharmacolog‐ ical inhibitors, it was shown that the hyperpolarization response in cultured human chondro‐

channels. Hyperpolarization was also produced by addition of the calcium ionophore A23187 to the culture medium showing that a rise in intracellular Ca2+ concentration within the cell could induce the response. Plasma membrane histamine H1 and H2 receptors, and β-adre‐ noreceptors did not appear to be involved in the hyperpolarization response. The studies also showed that the actin cytoskeleton, but not microtubules, was involved in the chondrocyte

Subsequent studies identified that the electrophysiological response to cyclical pressurization was the result of deformation of the base of the culture dishes to which the chondrocytes were attached and therefore deformation (strain) on the chondrocytes rather than a direct effect of the increased hydrostatic pressure on chondrocytes [56]. The hyperpolarization response was proportional to the microstrain to which cells were subjected and did not occur when chon‐ drocytes were subjected to cyclical pressurization in rigid glass culture dishes or when the plastic dishes were positioned in the pressurization chamber so as to avoid deformation of the

Experiments undertaken to identify the source of intracellular calcium that activated the SK channels leading to hyperpolarization demonstrated a requirement for extracellular calcium and activity of L-type calcium channels [118-120]. Thapsigargin which raises intracellular Ca2+ by inhibition of Ca2+-ATPase in endoplasmic reticulum [121-123] caused hyperpolariza‐ tion independent of mechanical strain but further hyperpolarization of the cells occurred after cyclical pressurization further supporting the idea that mechanically induced chondrocyte hyperpolarization is dependent on intracellular free Ca2+ levels [56]. In addition, TRPV4 mediated Ca2+ signaling has been demonstrated to play a central role in the transduction of

Stimulation of connective tissue cell mechanoreceptors is followed by generation of the secondary messenger molecules and activation of a cascade of downstream signaling events that regulate gene expression and cell function. Many intracellular signaling pathways are

mechanical signals to support cartilage extracellular matrix maintenance [124].

**6.1. Intracellular signal cascades activated by mechanical stimulation**

channels and L-type calcium

cytes induced by cyclic pressurization involved Ca2+ activated K+

hyperpolarization response [117].

48 Osteoarthritis - Progress in Basic Research and Treatment

base of the culture dish [56].

PKB/Akt is a protein family of serine/threonine kinases that have multiple roles including inhibition of apoptosis by phosphorylation and inactivation of pro-apoptotic factors. Integrindependent activation of phosphoinositide3 kinase (PI3 kinase) by mechanical forces regulates PKB activity and can inhibit cell death. Inactivation of the PI3-K/PKB pathway may be important in deleterious effects of mechanical overloading of cartilage and bone loss in response to withdrawal of loading [125]. The activity of mammalian target of rapamycin (mTOR) may be an essential mechanotransduction component modulated by SH2-containing protein tyrosine phosphatase 2 and is required for cartilage development [126].

Mitogen-activated protein kinases (MAPKs) regulate multiple cellular activities, such as gene expression, mitosis, differentiation, and cell survival/apoptosis. ERK1/2, JNK and p38, of critical importance in regulation of matrix protein and protease gene expression have each been shown to be activated in chondrocytes following mechanical stimulation [127]. Mechan‐ ical stimuli may activate different MAPKs and through this mechanism differential cellular responses may occur. MAPK responses may also be cell type dependent. Mechanical stimu‐ lation induced ERK1/2 activation in bone cells requires calcium-dependent ATP release whilst in cartilage activation, under certain circumstances, is dependent on FGF-2 rather than through integrin mechanoreceptors [128]. Tyrosine phosphorylation of focal adhesion kinase (pp125FAK), beta-catenin, and paxillin following mechanical stimulation is also recognized in human articular chondrocytes [129].

In bone cells NF-**κ**B, a protein complex that acts as a transcription factor, is directly stimulated by mechanical stimulation is dependent on intracellular calcium release [130]. In chondrocytes biomechanical signals within the physiological range block NF-**κ**B activity and proinflamma‐ tory chondrocyte responses [131]. Mechanical stimuli that induce catabolic rather than anabolic responses in chondrocytes induce rapid nuclear translocation of NF-**κ**B subunits p65 and p50 in a similar manner to IL-1**β** [132].

#### **6.2. Growth factors and autocrine/paracrine signaling in mechanotransduction**

As part of the cellular response to mechanical stimulation mechanosensitive connective tissue cells release a range of soluble mediators. These may be present in the cell and available for immediate release, or secretion may depend de novo synthesis by enzymatic activity or transcriptional activation and protein production. These mediators, including prostaglandins, nitric oxide, cytokines, growth factors, and neuropeptides are involved in downstream tissue modelling and remodelling responses initiated by the mechanosensitive cells or other effector cells. Production of soluble mediators by connective tissue cells in response to mechanical stimulation however may also be intrinsic to mechanotransduction pathways. Autocrine and paracrine activity allows increased regulation of the cellular response to mechanical stimuli by permitting cross talk between different components of a mechanotransduction cascade. As the cellular responses to mechanical stimuli and soluble mediators activate similar signal cascades inducing either anabolic or catabolic responses, it would be expected that they may be antagonistic, additive or synergistic. Anabolic cytokines and growth factors enhance production of matrix under mechanical loading conditions whilst anabolic mechanical stimuli antagonize the effects of catabolic cytokines such as IL-1**β** [133].

Prostaglandins, predominantly PGE2, NO and ATP are produced when bone cells and chondrocytes are mechanically stimulated. Prostaglandin production is integrin dependent requiring an intact cytoskeleton and activation of SACs, PKC, and PLA2. In cartilage PGE2 induced by mechanical loads is catabolic. Mechanical loading of chondrocytes by physiologi‐ cal stimuli inhibits production of PGE2 and NO whereas damaging loading induces PGE2 release [134].Followingmechanical stimulationbone cells andchondrocytes releaseATPwhich can bind and activate purinergic receptors on these and adjacent cells. Both metabotropic P2Y receptors and ionotropic P2X receptors, have been shown to be involved in mechanical load activatedsignal cascades inchondrocytesandbone cellsandmayhavephysiologicalroles [135].

IL-4 and IL-1**β** autocrine/paracrine activity is seen in the integrin-dependent mechanotrans‐ duction cascade of chondrocytes (IL-4 and IL-1**β**) and bone cells (IL-1**β**) to mechanical stimulation [136,137]. These molecules are secreted within 20 minutes of the onset of mechan‐ ical stimulation, suggesting release from preformed stores. IL-4 release relies on secretion of the neuropeptide substance P which binds to its NK1 receptor. Both IL-4 and substance P are necessary but not sufficient for the increased expression of aggrecan mRNA and decrease in MMP3 mRNA induced by the mechanical stimulus suggesting cross talk with other mecha‐ nosensitive signaling pathways. IL-1**β** is involved in the early mechanotransduction pathway of both osteoarthritic chondrocytes and human trabecular bone derived cells [138]. Mechanical loading may also induce release or activation of sequestered growth factors in extracellular matrix which will then act on near-by resident connective tissue cells. Basic fibroblast growth factor (FGF2) is a possible mediator of mechanical signaling in cartilage through such a mechanism [128]. Dynamic compression of porcine cartilage induces release of FGF2 with activation of ERK MAP kinase, synthesis and secretion of TIMP-1. In contrast FGF2 production by bovine cartilage is inhibited by 1 hour of compressive stress of 20 MPa [139]. This mechanical induced suppression of FGF2 is blocked by IL-4 indicating further roles for this pleiotropic cytokine in the regulation of chondrocyte responses to mechanical stimulation.

#### **7. Mechanical loading and osteoarthritis**

Abnormal mechanical loading is associated with osteoarthritis [140]. Most animal models of OA are mechanically induced, for example, by introducing joint instability by anterior cruciate ligament section [22] or by altering the loading across the joint by menisectomy [141]. These changes in joint loading affect cartilage structure and chondrocyte activity within days of the procedure, and may eventually result in complete loss of cartilage [142]. When cartilage matrix is lost or made deficient as a consequence of direct physical effects or proteolytic digestion the articular cartilage loses its mechanical function. The tensile modulus has been shown to decrease by as much as 90%, reflecting damage to the cartilage matrix network [26]. Animal

studies, for example, have shown that the tensile modulus of canine knee articular cartilage was reduced after one month of immobilization [143]. In the dog, severe OA lesions in the knee joint have been produced by treadmill exercise after the limb was immobilized for several weeks [144]. The compressive modulus also decreases with increasing severity of degeneration [27]. Other joint tissues (e.g., anterior cruciate ligament) also undergo similar changes of tensile and compressive modulus in an experimental OA model [145].

the cellular responses to mechanical stimuli and soluble mediators activate similar signal cascades inducing either anabolic or catabolic responses, it would be expected that they may be antagonistic, additive or synergistic. Anabolic cytokines and growth factors enhance production of matrix under mechanical loading conditions whilst anabolic mechanical stimuli

Prostaglandins, predominantly PGE2, NO and ATP are produced when bone cells and chondrocytes are mechanically stimulated. Prostaglandin production is integrin dependent requiring an intact cytoskeleton and activation of SACs, PKC, and PLA2. In cartilage PGE2 induced by mechanical loads is catabolic. Mechanical loading of chondrocytes by physiologi‐ cal stimuli inhibits production of PGE2 and NO whereas damaging loading induces PGE2 release [134].Followingmechanical stimulationbone cells andchondrocytes releaseATPwhich can bind and activate purinergic receptors on these and adjacent cells. Both metabotropic P2Y receptors and ionotropic P2X receptors, have been shown to be involved in mechanical load activatedsignal cascades inchondrocytesandbone cellsandmayhavephysiologicalroles [135].

IL-4 and IL-1**β** autocrine/paracrine activity is seen in the integrin-dependent mechanotrans‐ duction cascade of chondrocytes (IL-4 and IL-1**β**) and bone cells (IL-1**β**) to mechanical stimulation [136,137]. These molecules are secreted within 20 minutes of the onset of mechan‐ ical stimulation, suggesting release from preformed stores. IL-4 release relies on secretion of the neuropeptide substance P which binds to its NK1 receptor. Both IL-4 and substance P are necessary but not sufficient for the increased expression of aggrecan mRNA and decrease in MMP3 mRNA induced by the mechanical stimulus suggesting cross talk with other mecha‐ nosensitive signaling pathways. IL-1**β** is involved in the early mechanotransduction pathway of both osteoarthritic chondrocytes and human trabecular bone derived cells [138]. Mechanical loading may also induce release or activation of sequestered growth factors in extracellular matrix which will then act on near-by resident connective tissue cells. Basic fibroblast growth factor (FGF2) is a possible mediator of mechanical signaling in cartilage through such a mechanism [128]. Dynamic compression of porcine cartilage induces release of FGF2 with activation of ERK MAP kinase, synthesis and secretion of TIMP-1. In contrast FGF2 production by bovine cartilage is inhibited by 1 hour of compressive stress of 20 MPa [139]. This mechanical induced suppression of FGF2 is blocked by IL-4 indicating further roles for this pleiotropic

cytokine in the regulation of chondrocyte responses to mechanical stimulation.

Abnormal mechanical loading is associated with osteoarthritis [140]. Most animal models of OA are mechanically induced, for example, by introducing joint instability by anterior cruciate ligament section [22] or by altering the loading across the joint by menisectomy [141]. These changes in joint loading affect cartilage structure and chondrocyte activity within days of the procedure, and may eventually result in complete loss of cartilage [142]. When cartilage matrix is lost or made deficient as a consequence of direct physical effects or proteolytic digestion the articular cartilage loses its mechanical function. The tensile modulus has been shown to decrease by as much as 90%, reflecting damage to the cartilage matrix network [26]. Animal

**7. Mechanical loading and osteoarthritis**

antagonize the effects of catabolic cytokines such as IL-1**β** [133].

50 Osteoarthritis - Progress in Basic Research and Treatment

Mechanical loading out with that which joint tissues can normally withstand or are required to maintain a healthy state is central to the development of OA. OA arises when there is an imbalance between the mechanical forces within a joint and the ability of the cartilage to withstand these forces. This arises in two situations. In the first normal articular cartilage is exposed to abnormal mechanical loads whereas in the other the articular cartilage is funda‐ mentally defective with biomaterial properties that are insufficient to withstand normal load bearing. Risk factors associated with development of OA may have influences in either one or both of these scenarios. The accumulation of advanced glycation end products (AGEs) in ECM with age results in a more brittle collagen network that is less able to withstand normal loads, again leading to cartilage degeneration.

The mechanisms by which abnormal mechanical loading may influence chondrocyte function to promote cartilage breakdown are beginning to be understood. Chondrocytes from osteo‐ arthritic cartilage share mechanoreceptors with chondrocytes from normal chondrocytes [146,147]. Whilst activation of these receptors and downstream signaling cascades such as FAK, PKC, JAK/STAT and MAP kinases[148,149] result in pro-anabolic activity in normal chondrocytes activity through release of locally acting mediators that include the antiinflammatory cytokine IL-4 and the neuropeptide substance P [136,150]. However the response of chondrocytes is aberrant with production of proinflammatory cytokines such as IL-1 and TNF-**α** which increase production of MMPs and aggrecanases, further accelerating disease progression and attenuating cartilage repair [151-156].

#### **7.1. Altered responses to mechanical stimulation in osteoarthritic chondrocytes**

Chondrocytes from osteoarthritic cartilage show a membrane depolarization response to IL-4 that is inhibited by functional receptor antibodies. It is unclear why chondrocytes from osteoarthritic cartilage should show differences in their response to 0.33 Hz mechanical stimulation and recombinant IL-4. This may be result of a general phenotypic change seen in OA chondrocytes in which the cells are resident in a pro-inflammatory, catabolic environment. Indeed the observation that mechanical stimulation in osteoarthritis may result in production of proinflammatory mediators is supported by the findings that α5β1 integrin ligation increases production of IL-1**β** by osteoarthritic human chondrocytes with subsequent induc‐ tion of nitric oxide, PGE2, IL-6, and IL-8 [157]. These cytokines will inhibit anabolic responses and increase cartilage matrix breakdown by MMPs. This may be through direct mechanisms or by interfering with integrin signaling. Expression and function of molecules such as members of the SOCS (suppressors of cytokine signaling) which regulate cytokine signaling pathways [158] may also be implicated. These molecules modulate intracellular signals stimulated by IL-4 including JAK/STAT activation. SOCS-1 has been shown to bind to and inhibit kinase activity of JAK family members and inhibit IL-4 induced activation of JAK1 and STAT6. SOCS-3 has been shown mediate IL-1**β** inhibition of STAT5 activity. These and other regulators of STAT transcription factor signaling may be responsible for modulation of IL-4 dependent responses of chondrocytes in osteoarthritic cartilage to mechanical stimulation.

#### **8. Summary**

A healthy synovial joint requires exposure to mechanical loads within a physiological range. Osteoarthritis develops when joints are subjected to mechanical loads which they are not biomechanically conditioned to withstand. This may be because the loads are excessive due to obesity or joint malalignment or a consequence of intrinsic or acquired biomechanical weakness of joint tissues such as seen when cartilage proteoglycans are depleted secondary to synovial inflammation. Abnormal mechanical loads may have direct physical effects on joint tissues including cartilage but increasingly knowledge of the pathological process within the osteoarthritic joint indicate that chondrocytes regulated catabolic processes are of prime importance in cartilage degradation. The mechanisms by which chondrocytes recognize mechanical loads and how these mechanical stimuli are transduced into biochemical responses which subsequently lead to altered gene expression and cell function is being increasing understood (Figure 1). Knowledge of how anabolic and catabolic signaling cascades are differentially regulated in response to physiological and pathological mechanical stimuli will enable future strategies to be developed to prevent and treat the progression of cartilage pathology in osteoarthritis.

**Figure 1.** The major mechanotransduction components in chondrocytes. The integrin, connexin, and stretch-activated ion channel mechanoreceptors are stimulated by the mechanical forces transduced via the extracellular matrix (ECM). Downstream transduction pathways involve the cytoskeleton and signaling molecules, including FAK, PKC, PI3K,

PKB, NF-kB, and MAPK, which act to regulate gene expression, cell function and survival/apoptosis. The release and paracrine/autocrine activity of the anti-inflammatory cytokine IL-4 has beneficial effects in regulating an anabolic re‐ sponse with enhanced expression of aggrecan and inhibition of MMP expression. In contrast production of IL-1β, as seen in OA, has a catabolic outcome with activation of pathways resulting in increased expression of COX2 and MMPs.

#### **Author details**

regulators of STAT transcription factor signaling may be responsible for modulation of IL-4 dependent responses of chondrocytes in osteoarthritic cartilage to mechanical stimulation.

A healthy synovial joint requires exposure to mechanical loads within a physiological range. Osteoarthritis develops when joints are subjected to mechanical loads which they are not biomechanically conditioned to withstand. This may be because the loads are excessive due to obesity or joint malalignment or a consequence of intrinsic or acquired biomechanical weakness of joint tissues such as seen when cartilage proteoglycans are depleted secondary to synovial inflammation. Abnormal mechanical loads may have direct physical effects on joint tissues including cartilage but increasingly knowledge of the pathological process within the osteoarthritic joint indicate that chondrocytes regulated catabolic processes are of prime importance in cartilage degradation. The mechanisms by which chondrocytes recognize mechanical loads and how these mechanical stimuli are transduced into biochemical responses which subsequently lead to altered gene expression and cell function is being increasing understood (Figure 1). Knowledge of how anabolic and catabolic signaling cascades are differentially regulated in response to physiological and pathological mechanical stimuli will enable future strategies to be developed to prevent and treat the progression of cartilage

**Figure 1.** The major mechanotransduction components in chondrocytes. The integrin, connexin, and stretch-activated ion channel mechanoreceptors are stimulated by the mechanical forces transduced via the extracellular matrix (ECM). Downstream transduction pathways involve the cytoskeleton and signaling molecules, including FAK, PKC, PI3K,

**8. Summary**

52 Osteoarthritis - Progress in Basic Research and Treatment

pathology in osteoarthritis.

Herng-Sheng Lee1 and Donald M. Salter2

1 Department of Pathology and Laboratory Medicine, Kaohsiung Veterans General Hospital, Zuoying Dist., Kaohsiung City, Taiwan

2 Center for Genomics and Experimental Medicine, MRC IGMM, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, United Kingdom

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### **Structural and Functional features of Major Synovial Joints and Their Relevance to Osteoarthritis**

Xiaoming Zhang, Brian Egan and Jinxi Wang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59978

#### **1. Introduction**

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[156] Millward-Sadler S J, Wright M O, Lee H S, Caldwell H, Nuki G, Salter D M. Altered electrophysiological responses to mechanical stimulation and abnormal signalling through alpha5beta1 integrin in chondrocytes from osteoarthritic cartilage. Osteoar‐ thritis and cartilage / OARS, Osteoarthritis Research Society 2000;8(4): 272-278. [157] Attur M G, Dave M N, Clancy R M, Patel I R, Abramson S B, Amin A R. Functional genomic analysis in arthritis-affected cartilage: yin-yang regulation of inflammatory mediators by alpha 5 beta 1 and alpha V beta 3 integrins. Journal of immunology

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66 Osteoarthritis - Progress in Basic Research and Treatment

Osteoarthritis (OA) is considered to be an organ disease that may affect all of the articular and peri-articular tissues such as articular cartilage, synovium, ligament, capsule, subchondral bone, and peri-articular muscles [1-3]. Understanding the structural and functional features of the joint is of great significance for the diagnosis and treatment of OA. Although OA can occur in any synovial joint in the body, it mainly attacks the joints responsible for weight/load bearing such as the knee, hip, hand, and ankle joints. In this chapter, we will focus only on the structural and functional features of the major synovial joints and their relevance to osteoar‐ thritis.

### **2. Shoulder (glenohumeral) joint**

The glenohumeral joint is a ball-and-socket joint formed by the shallow glenoid cavity of the scapula and the head of the humerus. The joint cavity is slightly deepened by a ring-shaped fibrocartilage structure called the "glenoidal labrum", which attaches to the edge of the glenoid cavity. Because of its structure, the joint has a wide range of movement in all directions. However, its wide range of mobility is accompanied by instability. Only about 1/3 of the humeral head surface area attaches to the glenoid cavity. The humeral head is held onto the glenoid cavity by the rotator cuff muscles, namely, the supraspinatus, infraspinatus, teres minor, and subscapularis. These four muscles are located superior, posterior, and anterior on three sides around the joint cavity.

The fibrous joint capsule originates from the margin of the glenoid cavity and attaches to the anatomical neck of the humerus. The arrangement of the joint capsule is remarkably loose,

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particularly inferiorly when the arm is fully adducted (in anatomical position), allowing great separation between the bones of the joint and freedom of motion [4]. There are two apertures: one opens toward the intertubercular groove (sulcus) of the humerus to allow the long head of the biceps tendon to travel into the joint cavity, the other opens to the subscapularis bursa located anterior and inferior to the coracoid process of the scapula. The synovial membrane is lined up the inner surface of the joint capsule. In addition, it forms a tubular sheath wrapping the long head of the biceps tendon and extends into the intertubercular groove.

There are two intrinsic ligaments that are part of the joint capsule, the glenohumeral ligament that strengthens the anterior portion of the capsule and the coracohumeral ligament that is located superiorly. In addition, the transverse humeral ligament holds the long head of the biceps brachii muscle tendon inside the intertuberbular groove, and the coraco-acromial ligament stabilizes the glenohumeral joint from above.

The supraspinatus muscle tendon travels laterally in between the acromion of the scapula and the superior aspect of the joint capsule and attaches to the greater tubercle of the humerus. A synovial bursa called the "subacromial/subdeltoid bursa" is located in between the acromion and the muscle tendon to prevent friction of the latter against the bone. A second bursa associated with the glenohumeral joint is located anterior-inferiorly to the coracoid process at the neck of the scapula. It protects the tendon of the subscapularis muscle from friction against the neck of scapula. This bursa communicates with the joint cavity.

The movement of the glenohumeral joint is in three axes plus circumduction. The extensive range of movement of the joint is due to its structural feature, a large humeral head articulating with a small glenoid cavity and a loose joint capsule. Many muscles move the glenohumeral joint including the thoracoappendicular muscles (muscles that originate from the thoracic wall and attach to the humerus) and scapulohumeral muscles (muscles that originate from the scapula and attach to the humerus).

The glenohumeral joint is supplied by the anterior and posterior circumflex humeral arteries and the suprascapular artery. The joint is innervated by the axillary and lateral pectoral nerves[5].

Commonly seen injuries to the glenohumeral joint and its associated bursae are the following:


Young, active individuals can experience dislocation or partial-dislocation of the shoulder during exercise, practice, or competitive events. In prospective cohort studies of young military populations, 3% to 6% sustained shoulder dislocations or partial-dislocations were observed [8,9].


OA in the glenohumeral joint. Primary OA in the glenohumeral joint is relatively uncommon, and occurs more often in women and patients over the age of 60 [14,15]. In younger patients, it is usually caused by injuries to the joint that occurred several years earlier such as joint dislocation, fracture, rotator cuff tear, and glenoid labrum injury.

#### **3. Elbow joint**

particularly inferiorly when the arm is fully adducted (in anatomical position), allowing great separation between the bones of the joint and freedom of motion [4]. There are two apertures: one opens toward the intertubercular groove (sulcus) of the humerus to allow the long head of the biceps tendon to travel into the joint cavity, the other opens to the subscapularis bursa located anterior and inferior to the coracoid process of the scapula. The synovial membrane is lined up the inner surface of the joint capsule. In addition, it forms a tubular sheath wrapping

There are two intrinsic ligaments that are part of the joint capsule, the glenohumeral ligament that strengthens the anterior portion of the capsule and the coracohumeral ligament that is located superiorly. In addition, the transverse humeral ligament holds the long head of the biceps brachii muscle tendon inside the intertuberbular groove, and the coraco-acromial

The supraspinatus muscle tendon travels laterally in between the acromion of the scapula and the superior aspect of the joint capsule and attaches to the greater tubercle of the humerus. A synovial bursa called the "subacromial/subdeltoid bursa" is located in between the acromion and the muscle tendon to prevent friction of the latter against the bone. A second bursa associated with the glenohumeral joint is located anterior-inferiorly to the coracoid process at the neck of the scapula. It protects the tendon of the subscapularis muscle from friction against

The movement of the glenohumeral joint is in three axes plus circumduction. The extensive range of movement of the joint is due to its structural feature, a large humeral head articulating with a small glenoid cavity and a loose joint capsule. Many muscles move the glenohumeral joint including the thoracoappendicular muscles (muscles that originate from the thoracic wall and attach to the humerus) and scapulohumeral muscles (muscles that originate from the

The glenohumeral joint is supplied by the anterior and posterior circumflex humeral arteries and the suprascapular artery. The joint is innervated by the axillary and lateral pectoral

Commonly seen injuries to the glenohumeral joint and its associated bursae are the following:

**c.** Bicipital tendonitis, an inflammatory process of the long head of the biceps tendon inside the intertubercular groove. This process can be accompanied by tendon rupture, and/or

**e.** Dislocation of the glenohumeral joint, which often happens with the humeral head dislocating inferiorly. If the dislocated humeral head is positioned anterior to the long head of the triceps brachii muscle tendon, it is called an "anterior dislocation". Risk factors for shoulder injury include athletic participation, male gender, and young or old age [6,7].

**b.** Supraspinatus tendonitis, usually as a further development of subacromial bursitis.

the long head of the biceps tendon and extends into the intertubercular groove.

ligament stabilizes the glenohumeral joint from above.

68 Osteoarthritis - Progress in Basic Research and Treatment

scapula and attach to the humerus).

transverse humeral ligament tear.

**d.** Subscapular bursa inflammation (bursitis).

nerves[5].

the neck of scapula. This bursa communicates with the joint cavity.

**a.** Subacromial/subdeltoid bursitis due to wear-and-tear.

The elbow joint is a complex structure involving three bones, the humerus, ulna, and radius, articulating together. There are three joints wrapped within one joint capsule: the humeroulnar joint, the humeroradial joint, and the proximal radioulnar joint.

The humeroulnar joint is formed between the trochlear of the humerus and the trochlear notch of the ulna. It is a typical hinge joint capable of flexion and extension. The humeroradial joint is formed between the capitulum of the humerus and the head of the radius. The capitulum is a ball shaped structure that allows the head of the radius, which is a disc-shaped structure articulating with the capitulum on its flat surface, to move in two directions: flexion and extension, plus axial rotation against the capitulum. The proximal radioulnar joint is formed between the head of the radius (round surface) and the radial notch of the ulna. The radial structure rotates against the ulnar structure when the forearm carries out pronation and supination actions.

The fibrous joint capsule surrounds the elbow joint on four sides with the anterior and posterior sides weaker than those on the medial and lateral sides. Therefore, elbow joint dislocation often happens anteriorly or posteriorly. Synovial membrane lines the inner surface of the fibrous joint capsule.

The fibrous joint capsule thickens on the medial and lateral sides to become medial (ulnar) or lateral (radial) collateral ligaments. The ulnar collateral ligament is a triangular shaped ligament containing three components: the anterior cord-like band (strongest), the posterior fan-like band, and the oblique band. The radial collateral ligament is fan-shaped and connects the lateral epicondyle of the humerus with the annular ligament of the radial head. The annular ligament is a ring-shaped ligament that surrounds the circumference of the disc-shaped radial head and fixes it to the radial notch of the ulna.

The movement of the elbow joint includes flexion/extension and pronation/supination. There are more than a dozen muscles across the elbow joint that participate in moving the joint. Blood is supplied to the elbow joint by anastomosing branches from the humeral artery, radial artery, and ulna artery. Musculocutaneous, radial, and ulnar nerves innervate this joint.

OA in the elbow joint: The elbow is one of the least affected joints by osteoarthritis because of its well matched joint surfaces and strong stabilizing ligaments. As a result, the elbow joint can tolerate large forces across it without becoming unstable. Development of osteoarthritis in elbow joint is usually due to previous injuries to the joint.

#### **4. Wrist (radiocarpal) joint**

The wrist joint is a condyloid joint. The proximal joint surface is the distal end of the radius and the articular disc. The distal joint surface is formed by three of the proximal row of carpal bones (scaphoid, lunate, and triquetrum). The ulna and pisiform are not involved in wrist joint formation. The articular disc is a triangular-shaped fibrocartilage structure that connects the styloid process of the ulna to the distal end of the radius. The distal end of the ulna is located proximal to the articular disc and thus does not contact the carpal bones.

The fibrous joint capsule is strengthened by several ligaments which are all part of the fibrous joint capsule. Anteriorly, there is the palmar radiocarpal ligament. Posteriorly, there is the dorsal radiocarpal ligament. On the medial side, there is the ulnar collateral ligament which attaches to the ulnar styloid process. On the lateral side, there is the radial collateral ligament which attaches to the radial styloid process. The synovial membrane lines the internal surface of the fibrous joint capsule and forms numerous synovial folds.

The movement of the wrist joint involves flexion/extension, abduction/adduction, and circumduction. Many muscles from the forearm to the hand move this joint. The wrist joint is supplied by the palmar and dorsal carpal arches which are branches of the radial and ulnar arteries. The innervation of this joint is by median, radial, and ulnar nerves.

OA in the wrist joint: There are different causes, both idiopathic and traumatic, of wrist osteoarthritis. Traumatic causes of wrist OA include injuries to ligament, articular carti‐ lage, and bone. Although injuries to many wrist ligaments can lead to progressive wrist arthrosis, a chronic scapholunate ligament tear in particular is known to produce intercar‐ pal instability, altered wrist kinematics and joint loading, and degeneration of the radiocar‐ pal joint. Fracture and subsequent nonunion of the scaphoid also leads to a series of predictable degenerative changes. Wrist OA can also occur secondary to an intra-articular fracture of the distal radius or ulna or from an extra-articular fracture resulting in maluni‐ on and abnormal joint loading [16].

### **5. Hand joints**

ligament containing three components: the anterior cord-like band (strongest), the posterior fan-like band, and the oblique band. The radial collateral ligament is fan-shaped and connects the lateral epicondyle of the humerus with the annular ligament of the radial head. The annular ligament is a ring-shaped ligament that surrounds the circumference of the disc-shaped radial

The movement of the elbow joint includes flexion/extension and pronation/supination. There are more than a dozen muscles across the elbow joint that participate in moving the joint. Blood is supplied to the elbow joint by anastomosing branches from the humeral artery, radial artery,

OA in the elbow joint: The elbow is one of the least affected joints by osteoarthritis because of its well matched joint surfaces and strong stabilizing ligaments. As a result, the elbow joint can tolerate large forces across it without becoming unstable. Development of osteoarthritis

The wrist joint is a condyloid joint. The proximal joint surface is the distal end of the radius and the articular disc. The distal joint surface is formed by three of the proximal row of carpal bones (scaphoid, lunate, and triquetrum). The ulna and pisiform are not involved in wrist joint formation. The articular disc is a triangular-shaped fibrocartilage structure that connects the styloid process of the ulna to the distal end of the radius. The distal end of the ulna is located

The fibrous joint capsule is strengthened by several ligaments which are all part of the fibrous joint capsule. Anteriorly, there is the palmar radiocarpal ligament. Posteriorly, there is the dorsal radiocarpal ligament. On the medial side, there is the ulnar collateral ligament which attaches to the ulnar styloid process. On the lateral side, there is the radial collateral ligament which attaches to the radial styloid process. The synovial membrane lines the internal surface

The movement of the wrist joint involves flexion/extension, abduction/adduction, and circumduction. Many muscles from the forearm to the hand move this joint. The wrist joint is supplied by the palmar and dorsal carpal arches which are branches of the radial and ulnar

OA in the wrist joint: There are different causes, both idiopathic and traumatic, of wrist osteoarthritis. Traumatic causes of wrist OA include injuries to ligament, articular carti‐ lage, and bone. Although injuries to many wrist ligaments can lead to progressive wrist arthrosis, a chronic scapholunate ligament tear in particular is known to produce intercar‐ pal instability, altered wrist kinematics and joint loading, and degeneration of the radiocar‐ pal joint. Fracture and subsequent nonunion of the scaphoid also leads to a series of predictable degenerative changes. Wrist OA can also occur secondary to an intra-articular

and ulna artery. Musculocutaneous, radial, and ulnar nerves innervate this joint.

head and fixes it to the radial notch of the ulna.

70 Osteoarthritis - Progress in Basic Research and Treatment

**4. Wrist (radiocarpal) joint**

in elbow joint is usually due to previous injuries to the joint.

proximal to the articular disc and thus does not contact the carpal bones.

arteries. The innervation of this joint is by median, radial, and ulnar nerves.

of the fibrous joint capsule and forms numerous synovial folds.

There are several groups of joints in the hand. From proximal to distal, the groups are:


OA in hand: Hand OA is a prevalent disorder. It is not one single disease, but a heterogeneous group of disorders. It may appear as osteophyte or joint space narrowing, interphalangeal nodal, or thumb base erosion [17-19].

#### **6. Hip joint**

The hip joint is a ball-and-socket joint formed by the head of the femur (ball) and the acetab‐ ulum of the pelvis (socket). It is a very stable joint that bears all the weight of the upper body yet maintains a wide range of movement.

The head of the femur is covered by articular cartilage except for the center where a depression called the "fovea" allows for the attachment of the ligament to the femoral head.

The acetabulum is formed by the fusion of three pelvic bones: pubis, ischium, and ilium. It is a hemispherical hollow socket facing anteriolaterally. The edge of the acetabulum is called the "acetabular rim", which is covered by semilunar-shaped articular cartilage called the "lunate surface of the acetabulum". It is an incomplete circle with the inferior part missing. The missing inferior segment is called the "acetabular notch". This notch is bridged by the "transverse acetabular ligament", which is part of a fibrocartilaginous ring that attaches to the margin of the acetabulum. This lip-shaped ring structure is called the "acetabular labrum". It increases the articular surface of the acetabulum by 10%. The central region of the acetabulum is not covered by any articular cartilage; rather, it is filled with a fat pad. This region is called the "acetabular fossa", which has a thin wall from the ischium and communicates with the acetabular notch (Figure 1).

**Figure 1.** An illustration showing the lateral view of the hip joint. The ligament of the head of the femur has been transected and the femoral head has been dislocated to show the internal structure of the acetabulum.

More than half of the femoral head fits into the acetabulum making the joint the most stable for weight bearing.

The capsule of the hip joint is strong in its fibrous layer. It attaches just outside the acetabular rim proximally and the femoral neck, intertrochanteric line, and greater trochanter distally. Most of the fibers of this joint capsule run in a spiral direction between its two ends. This is particularly true when the hip joint is extended at a standing position (anatomical position). At this position, the joint capsule is tightened, pushing the head of the femur against the acetabulum firmly. When the hip joint is flexed, such as when one is in a sitting position, the spiral joint capsule fibers are "unwound" becoming straight. The straightened joint capsule fibers are longer than their spiral state making the joint capsule loosen for more mobility. The synovial membrane lines up the inner surface of the fibrous joint capsule and forms synovial folds at the femoral neck.

There are three intrinsic joint ligaments that are part of the joint capsule.

the articular surface of the acetabulum by 10%. The central region of the acetabulum is not covered by any articular cartilage; rather, it is filled with a fat pad. This region is called the "acetabular fossa", which has a thin wall from the ischium and communicates with the

**Figure 1.** An illustration showing the lateral view of the hip joint. The ligament of the head of the femur has been

More than half of the femoral head fits into the acetabulum making the joint the most stable

The capsule of the hip joint is strong in its fibrous layer. It attaches just outside the acetabular rim proximally and the femoral neck, intertrochanteric line, and greater trochanter distally. Most of the fibers of this joint capsule run in a spiral direction between its two ends. This is particularly true when the hip joint is extended at a standing position (anatomical position). At this position, the joint capsule is tightened, pushing the head of the femur against the acetabulum firmly. When the hip joint is flexed, such as when one is in a sitting position, the spiral joint capsule fibers are "unwound" becoming straight. The straightened joint capsule

transected and the femoral head has been dislocated to show the internal structure of the acetabulum.

acetabular notch (Figure 1).

72 Osteoarthritis - Progress in Basic Research and Treatment

for weight bearing.


The ligament of the head of the femur is actually a synovial fold located inside the joint cavity. It attaches to the fovea of the head of the femur at one end and the transverse acetabular ligament at the other. There is a small artery running inside this ligament. It is a weak ligament of little importance for the stability of the joint.

The movement of the hip joint is extensive in all three axes (flexion/extension, abduction/ adduction, and medial/lateral rotation) plus circumduction. Its movement is also affected by the positions of the knee and the vertebral column. Muscles in the gluteal region, lumbar region, anterior thigh, medial thigh, and posterior thigh are involved when moving the hip joint. Some muscles move the joint in more than one direction.

The major blood supply to the hip joint is the retinacular arteries arising from the medial and lateral circumflex femoral arteries. Both are branches of the profunda femoris artery or the femoral artery. The medial and lateral circumflex arteries travel along the intertrochanteric ridge and the intertrochanteric line of the femur and anastomose with each other. The retinacular arteries branch off from the circumflex arteries and travel along the neck of the femur to reach the femoral head and the hip joint. When fracture happens to the neck of the femur, retinacular arteries are injured resulting in reduced blood supply to the femoral head and the hip joint.

"Hilton's Law" states that the nerve that innervates the muscles moving the joint also inner‐ vates the joint. The following nerves innervate the muscles that move the hip joint: femoral nerve, obturator nerve, and superior and inferior gluteal nerves.

OA in the hip joint: In addition to idiopathic OA, acetabular fracture is a known cause of posttraumatic OA of the hip joint [20]. Acetabular dysplasia is predictive of hip OA and subsequent hip arthroplasty [21]. An increased prevalence of radiographic hip OA and osteophytosis is observed in high bone mass (HBM) cases compared with controls [22]. In addition, the development of knee OA is related to variations in hip and pelvic anatomy [23].

#### **7. Knee joint**

The knee joint is formed by three bones: femur, tibia, and patella. It is basically a hinge joint for flexion and extension with additional motions such as gliding (between the femur and patella), rolling (between the femur and tibia), and rotation (between the femur and tibia). There are three articulations in this joint: medial femorotibial (between the medial condyles of the femur and the tibia), lateral femorotibial (between the lateral condyles of the femur and the tibia), and femoropatellar (between the femur and the patella). The articulating surfaces of the femur are ball-shaped, whereas the articulating surfaces of the tibia are flat. When they articulate with each other, it is like two balls placed on a warp‐ ed table top, making the articulation very unstable. Ligaments, menisci, and muscles strengthen the knee joint (Figure 2).

**Figure 2.** An illustration showing the anterior view of the knee joint with major intra-articular and peri-articular tis‐ sues. The patellar ligament has been reflected downward with the attached patella.

The fibrous capsule of the knee joint thickens in some areas to become the intrinsic joint ligaments. Anteriorly, the fibrous capsule merges with the quadriceps tendon, the patella, and the patellar ligament so that these structures become part of the anterior fibrous joint capsule. Posteriorly, the fibrous joint capsule has an opening at the medial condyle of the tibia. This opening allows the tendon of the popliteus muscle to exit the joint capsule and attach to the tibia.

The synovial membrane lines the inside surface of the fibrous joint capsule. In the center of the joint where the intercondylar fossa houses the anterior and posterior cruciate ligaments, the synovial membrane leaves the posterior fibrous capsule and reflects anteriorly into the intercodyle fossa area forming the "infrapatella synovial fold". This synovial fold excludes the cruciate ligaments and the infrapatella fat pad from the joint cavity and almost sub-divides the knee joint cavity into medial and lateral halves. This unique anatomical feature allows surgeons to approach the cruciate ligaments through the posterior fibrous capsule without entering the joint cavity. However, the synovial membrane does not cover the following joint structures: articular cartilages on femur and tibia, the posterior surface of the patella, and the menisci.

**7. Knee joint**

tibia.

strengthen the knee joint (Figure 2).

74 Osteoarthritis - Progress in Basic Research and Treatment

The knee joint is formed by three bones: femur, tibia, and patella. It is basically a hinge joint for flexion and extension with additional motions such as gliding (between the femur and patella), rolling (between the femur and tibia), and rotation (between the femur and tibia). There are three articulations in this joint: medial femorotibial (between the medial condyles of the femur and the tibia), lateral femorotibial (between the lateral condyles of the femur and the tibia), and femoropatellar (between the femur and the patella). The articulating surfaces of the femur are ball-shaped, whereas the articulating surfaces of the tibia are flat. When they articulate with each other, it is like two balls placed on a warp‐ ed table top, making the articulation very unstable. Ligaments, menisci, and muscles

**Figure 2.** An illustration showing the anterior view of the knee joint with major intra-articular and peri-articular tis‐

The fibrous capsule of the knee joint thickens in some areas to become the intrinsic joint ligaments. Anteriorly, the fibrous capsule merges with the quadriceps tendon, the patella, and the patellar ligament so that these structures become part of the anterior fibrous joint capsule. Posteriorly, the fibrous joint capsule has an opening at the medial condyle of the tibia. This opening allows the tendon of the popliteus muscle to exit the joint capsule and attach to the

sues. The patellar ligament has been reflected downward with the attached patella.

There are about 12 bursae around the knee joint; some of them communicate with the joint cavity.

Anteriorly, there are 5 bursae. The suprapatellar bursa is a large, deep bursa located above the patella and under the quadriceps tendon. It communicates with the joint cavity. The synovial membrane of the knee joint becomes the lining of this bursa. There are 2 prepatella bursae: the subtendinous prepatellar bursa is located between the patellar tendon and the patella and the subcutaneous prepatellar bursa is located between the skin and the patellar tendon. There are also 2 infrapatellar bursae: the deep infrapatellar bursa is located between the patellar tendon and the tibia and the subcutaneous infrapatellar bursa is located between the skin and the patellar tendon.

Posteriorly, there are several bursae associated with the muscle attachments around the knee joint such as the gastrocnemius bursae, the semimembranosus bursa, and the popliteus bursa. These bursae are less clinically significant than those located in the anterior aspect of the knee.

The knee joint is strengthened by two groups of ligaments, external ligaments and internal ligaments. There are five external knee joint ligaments, and most of them are part of the fibrous joint capsule (intrinsic ligaments).

The patellar ligament is the distal portion of the quadriceps tendon when it wraps the patella and goes on to insert into the tibial tuberosity. On each side of the patellar ligament extending from the aponeurosis of the vastus medialis and vastus lateralis, are the medial and lateral "patellar retinacula", which help to maintain the position of the patella.

There are two collateral ligaments on each side of the knee joint. The medial (tibial) collateral ligament (MCL or TCL) is a flat, broad band of the fibrous joint capsule. Its fibers continue into the medial meniscus connecting the two. When the MCL is injured, the medial meniscus is mostly involved. The lateral (fibular) collateral ligament (LCL or FCL) is a cord-like strong extracapsular ligament. It attaches to the fibular head splitting the tendon of the biceps femoris muscle. It is separated from the joint capsule by the tendon of the popliteus muscle, and therefore is not connected to the lateral meniscus.

The oblique and arcuate popliteal ligaments are located posteriorly to the knee joint and strengthen the joint capsule posteriorly.

The internal or intra-articular ligaments include the cruciate ligaments and the meniscal ligaments. The cruciate ligaments are located inside the fibrous joint capsule in the intercon‐ dylar fossa but outside the synovial membrane, and therefore outside the joint cavity. They cross each other and play the most important role in maintaining the contact between the femur and the tibia when the knee is flexed. Whatever position the knee joint is at, one of the cruciate ligaments is maintained in tension.

The anterior cruciate ligament (ACL) arises from the anterior intercondylar area of the tibia posterior to the attachment of the medial meniscus, travels posterior-laterally, and attaches to the medial surface of the lateral condyle of the femur. When the ACL travels across the posterior cruciate ligament (PCL), it is on the lateral side of the PCL. The ACL prevents the posterior movement of the femur from the tibial plateau when the knee is extended. When the knee joint is flexed, the ACL prevents the anterior movement of the tibia from the femur [24,25].

The posterior cruciate ligament (PCL) arises from the posterior intercondylar area of the tibia, travels anteriorly on the medial side of the ACL, and attaches to the lateral surface of the medial condyle of the femur. It is stronger than the ACL. When the knee joint is extended, the PCL prevents the anterior movement of the femur from the tibial plateau. When the knee is flexed, the PCL prevents the posterior movement of the tibia from the femur.

Because of the anatomical relationship between the two cruciate ligaments, the medial rotation of the tibia is limited to about 10° when the knee is flexed. This is because the ACL is pushed against the PCL and the latter blocks the ACL from moving medially during the rotation. Under the same situation but reversing direction, the lateral rotation of the tibia is about 60° because the two cruciate ligaments are moving away from each other.

The menisci are crescent-shaped fibrocartilage structures located on the articular surface of the tibia. They are thicker at the external margins and thin in the central edges, thereby deepening the surface of the tibial articular surface. They attach to the intercondylar area of the tibia with their ends and to the fibrous joint capsule on each side. Other than these attachments, the menisci are free of attachment to other joint structures. Therefore, they are mobile along with the knee joint movement. The medial meniscus is C-shaped, attaches to the medial collateral ligament and is less mobile. The lateral meniscus is almost O-shaped and is more movable.

The movement of the knee joint is essentially flexion and extension. During these actions, the patella glides against the femur and the femur rolls against the tibial plateau. When the knee joint is in the fully extended position with the foot on the ground, the femur may rotate 5° medially along its longitudinal axis on the tibial plateau. This is the locking of the knee. When the knee is "locked", the knee joint is stable for weight bearing and the thigh and leg muscles can briefly relax. To "unlock" the knee, the popliteus muscle rotates the femur laterally about 5° [26-28].

When the knee joint is extended, the contacting area between the femur and the tibia moves anteriorly; when the knee is flexed, this contacting area moves posteriorly. As a result, the menisci, particularly the lateral meniscus, moves anteriorly during extension, and posteriorly during flexion.

The blood supply to the knee joint is from the genicular arteries branched from the popliteal artery. Extensive anastomoses form around the knee joint. The nerve innervation of the knee joint follows Hilton's law by femoral, obturator, and sciatic nerves.

The knee joint is the most vulnerable joint for injury. Structures that are most frequently injured are the ACL, MCL, and the medial meniscus. Because of its weight bearing feature, the knee joint is also the most affected joint for OA [29-31].

#### **8. Ankle joint**

The internal or intra-articular ligaments include the cruciate ligaments and the meniscal ligaments. The cruciate ligaments are located inside the fibrous joint capsule in the intercon‐ dylar fossa but outside the synovial membrane, and therefore outside the joint cavity. They cross each other and play the most important role in maintaining the contact between the femur and the tibia when the knee is flexed. Whatever position the knee joint is at, one of the cruciate

The anterior cruciate ligament (ACL) arises from the anterior intercondylar area of the tibia posterior to the attachment of the medial meniscus, travels posterior-laterally, and attaches to the medial surface of the lateral condyle of the femur. When the ACL travels across the posterior cruciate ligament (PCL), it is on the lateral side of the PCL. The ACL prevents the posterior movement of the femur from the tibial plateau when the knee is extended. When the knee joint is flexed, the ACL prevents the anterior movement of the tibia from the femur [24,25]. The posterior cruciate ligament (PCL) arises from the posterior intercondylar area of the tibia, travels anteriorly on the medial side of the ACL, and attaches to the lateral surface of the medial condyle of the femur. It is stronger than the ACL. When the knee joint is extended, the PCL prevents the anterior movement of the femur from the tibial plateau. When the knee is flexed,

Because of the anatomical relationship between the two cruciate ligaments, the medial rotation of the tibia is limited to about 10° when the knee is flexed. This is because the ACL is pushed against the PCL and the latter blocks the ACL from moving medially during the rotation. Under the same situation but reversing direction, the lateral rotation of the tibia is about 60°

The menisci are crescent-shaped fibrocartilage structures located on the articular surface of the tibia. They are thicker at the external margins and thin in the central edges, thereby deepening the surface of the tibial articular surface. They attach to the intercondylar area of the tibia with their ends and to the fibrous joint capsule on each side. Other than these attachments, the menisci are free of attachment to other joint structures. Therefore, they are mobile along with the knee joint movement. The medial meniscus is C-shaped, attaches to the medial collateral ligament and is less mobile. The lateral meniscus is almost O-shaped and is

The movement of the knee joint is essentially flexion and extension. During these actions, the patella glides against the femur and the femur rolls against the tibial plateau. When the knee joint is in the fully extended position with the foot on the ground, the femur may rotate

medially along its longitudinal axis on the tibial plateau. This is the locking of the knee. When the knee is "locked", the knee joint is stable for weight bearing and the thigh and leg muscles can briefly relax. To "unlock" the knee, the popliteus muscle rotates the femur laterally about

When the knee joint is extended, the contacting area between the femur and the tibia moves anteriorly; when the knee is flexed, this contacting area moves posteriorly. As a result, the menisci, particularly the lateral meniscus, moves anteriorly during extension, and posteriorly

the PCL prevents the posterior movement of the tibia from the femur.

because the two cruciate ligaments are moving away from each other.

ligaments is maintained in tension.

76 Osteoarthritis - Progress in Basic Research and Treatment

more movable.

5°

5° [26-28].

during flexion.

The ankle joint is a hinge joint involving three bones: distal tibia, distal fibula, and superior surface of talus. The distal end of the tibia forms an L-shaped joint surface with its horizontal aspect articulating with the talus from above and its vertical aspect articulating with the talus on the medial side. The distal end of the tibia forms the medial malleolus. The fibula articulates with the talus on the lateral side and forms the lateral malleolus. The distal tibia and distal fibula are connected together by ligaments forming an open rectangular recess like a mortise facing inferiorly. The superior surface of the talus sits inside the mortise like a trochlea to form the ankle joint with three articular surfaces, superior and medially by tibia and laterally by fibula.

The superior articular surface of the talus is not rectangular in shape, but rather trapezoidal with a wider anterior measure and a narrower posterior measure. When the ankle joint is dorsiflexed, the wider anterior portion of the talus sits in the mortise formed by the tibia and fibula. In this situation, there is little room for the talus to move inside the joint cavity. Therefore, the ankle joint is most stable when the foot is dorsiflexed. On the contrary, when the ankle joint is plantarflexed, the narrower posterior portion of the talus sits inside the mortise and there is more room laterally for the talus to move. In this situation, the ankle joint is unstable and is vulnerable to injuries.

The joint capsule of the ankle joint is loose anteriorly and posteriorly but strengthened on each side by collateral ligaments. Synovial membrane lines the internal surface of the fibrous capsule.

The ligaments of the ankle joint can be grouped into those that stabilize the tibia and the fibula and those that are located on each side of the joint.

There is an interosseous ligament located deep between the tibia and the fibula. In addition, there are the anterior superior tibiofibular ligament, anterior inferior tibiofibular ligament in the front, and posterior tibiofibular ligament at the back. All of these ligaments strengthen the bond between tibia and fibula and stabilize the ankle joint.

On the lateral side of the ankle, the fibrous joint capsule is reinforced by the lateral ligaments of the ankle. They are intrinsic joint ligaments (being part of the fibrous joint capsule) and are actually three separate structures (Figure 3A).

**a.** Anterior talofibular ligament – from the lateral malleolus to talus.


The medial ligament of the ankle is also referred to as the deltoid ligament of the ankle. It is a fan-shaped ligament that originates from the medial malleolus and attaches to several bones distally. From anterior to posterior in sequence, the portions of the medial ligament of the ankle are the anterior tibiotalar part, the tibionavicular part, the tibiocalcaneal part, and the posterior tibiotalar part (Figure 3C).

The major movements of the ankle joint are dorsiflexion and plantarflexion. The ankle joint can slightly abduct and adduct. When the foot is in plantarflexion in combination with adduction, the movement is inversion (Figure 3B). When the foot is in dorsiflexion in combi‐ nation with abduction, the ankle joint is carrying out eversion (Figure 3D).

**Figure 3.** (A) A representation of major lateral ligaments of the ankle and the tibiofibular ligaments. (B) A typical inver‐ sion injury of the ankle that leads to damage of the lateral ankle ligaments. (C) The deltoid ligament which is the pri‐ mary medial ankle ligament complex. (D) A typical eversion injury of the ankle that results in damage to the medial ligaments of the ankle.

The blood supply to the ankle joint is via the anterior tibial artery, the posterior tibial artery, and the fibular artery which is a branch of the posterior tibial artery. The nerve innervation is by the tibial nerve and the deep fibular nerve.

Ankle joint injury: The ankle is a second joint that demonstrates a high susceptibility to injury. A severe injury of major ligaments of the ankle may cause instability of the joint.

Like knee injuries, ankle injuries often occur during participation in sports or exercise; consequently, populations of athletes are often used in incidence studies. For example, ankle injuries are estimated to account for 14% of all athletic injuries, with sprains to ankle ligaments accounting for over 75% of ankle injuries [32-34]. The anterior talofibular ligament is the most commonly injured ankle ligament, involved in an estimated 85% of sprains sustained during United States high school sports [35]. A major problem accompanying ankle injury is the high rate of recurrence associated with chronic ankle instability. Approximately 15% of all ankle sprains occur in ankles with previous ligament injury[35]. Current models of chronic ankle instability (CAI) identify sufferers as experiencing—individually or in combination—mechan‐ ical instability, perceived instability, and recurrent sprains. Further characterizing patients with CAI by specific impairment, activity limitations, and participation restrictions, could help in the design of targeted treatments and injury reduction programs[36].

Ankle joint osteoarthritis: Idiopathic OA is common in the hand, foot, knee, spine, and hip joints, but rarely occurs in the ankle joint mainly due to its stable anatomical structure. However, the risk of post-traumatic OA in the ankle appears to be at least as great as the risk in the other joints. Differences among joints in congruity, articular cartilage thickness, force transmission across the joint surfaces, joint stability, and the presence of menisci could make some joints more vulnerable to OA. For example, the knee has thick menisci but the ankle does not. In addition, the ankle joint has a smaller bearing surface and is more constrained. The distal tibial articular surface has much thinner cartilage than the proximal tibial articular surface. Mechanical loading on the articular surface of the distal tibia after chondral damage causes higher subchondral bone strains than the loading on the proximal tibial articular surface. These differences may make the distal tibial articular surface more vulnerable to degradation of cartilage and development of OA [37-42].

#### **9. Conclusion**

**b.** Posterior talofibular ligament – from the lateral malleolus to talus at the back.

nation with abduction, the ankle joint is carrying out eversion (Figure 3D).

tibiotalar part (Figure 3C).

78 Osteoarthritis - Progress in Basic Research and Treatment

ligaments of the ankle.

**c.** Calcaneofibular ligament – from lateral malleolus to the lateral surface of the calcaneus. The medial ligament of the ankle is also referred to as the deltoid ligament of the ankle. It is a fan-shaped ligament that originates from the medial malleolus and attaches to several bones distally. From anterior to posterior in sequence, the portions of the medial ligament of the ankle are the anterior tibiotalar part, the tibionavicular part, the tibiocalcaneal part, and the posterior

The major movements of the ankle joint are dorsiflexion and plantarflexion. The ankle joint can slightly abduct and adduct. When the foot is in plantarflexion in combination with adduction, the movement is inversion (Figure 3B). When the foot is in dorsiflexion in combi‐

**Figure 3.** (A) A representation of major lateral ligaments of the ankle and the tibiofibular ligaments. (B) A typical inver‐ sion injury of the ankle that leads to damage of the lateral ankle ligaments. (C) The deltoid ligament which is the pri‐ mary medial ankle ligament complex. (D) A typical eversion injury of the ankle that results in damage to the medial This chapter summarizes the structural and functional features of major synovial joints of the human body and their relevance to joint injury and the development of OA. Although OA can affect any synovial joint, the prevalence of OA in specific joints is closely related to their structural and functional features. Idiopathic OA rarely occurs in the ankle, wrist, elbow, and shoulder, but it is common in the hand, foot, knee, spine, and hip joints. The risk of posttraumatic OA in the ankle, wrist, elbow, and shoulder appears to be as great as the risk in the hand, foot, knee, and hip. Differences among joints in articular surface congruity, articular cartilage thickness, mechanical force transmission, ligament structure-related joint stability, and the presence of menisci could make some joints more vulnerable to the development of OA. A better understanding of the structural and functional features of major synovial joints of the human body may help us develop more effective strategies for the prevention and treatment of OA.

#### **Acknowledgements**

This work was supported in part by the U.S. National Institutes of Health (NIH)/NIAMS grant R01 AR059088, the U.S. Department of Defense medical research grant W81XWH-12-1-0304, and the Harrington Distinguished Professorship Endowment. The authors thank Mr. Zhaoyang Liu for editorial assistance.

#### **Author details**

Xiaoming Zhang1 , Brian Egan2 and Jinxi Wang2\*

\*Address all correspondence to: jwang@kumc.edu

1 Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, USA

2 Department of Orthopedic Surgery, University of Kansas School of Medicine, Kansas City, USA

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OA. A better understanding of the structural and functional features of major synovial joints of the human body may help us develop more effective strategies for the prevention and

This work was supported in part by the U.S. National Institutes of Health (NIH)/NIAMS grant R01 AR059088, the U.S. Department of Defense medical research grant W81XWH-12-1-0304, and the Harrington Distinguished Professorship Endowment. The authors thank Mr.

1 Department of Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas

2 Department of Orthopedic Surgery, University of Kansas School of Medicine, Kansas City,

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82 Osteoarthritis - Progress in Basic Research and Treatment


## **Pathology and Biomechanics**

**Chapter 5**

**Hand Osteoarthritis — Clinical Presentation, Phenotypes and Management**

Nidhi Sofat and Sharenja Jeyabaladevan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60540

#### **1. Introduction**

Osteoarthritis (OA) is the most common arthritic disorder, affecting increasing numbers of people in an ageing population [1]. Nearly 27 million people are estimated to have OA among US adults [2]. OA of the hand is known to cause significant morbidity and can have a severe impact on patients' functional capacity. For example, the US Framingham study found that 27% of adults aged over 26 have hand OA (HOA) [2]. In addition, a large European study of 7983 people demonstrated that 25% of participants with hand pain showed significant hand disability [3]. Hand OA can lead to the development of chronic pain, causing significant emotional and financial burden to those affected, impacting on carers and on society as a whole [4]. Treatment of HOA currently comprises analgesia with analgesic drugs including topical or oral nonsteroidal anti-inflammatory agents (NSAIDs), opioid analgesics and rehabilitative hand physiotherapy [5]. However, large numbers of people continue to experience impaired hand function and pain.

Pathologically, OA is typified by cartilage degradation, osteophyte formation and underlying subchondral sclerosis. More recently, imaging-detected synovitis and bone marrow lesions have also been found to correlate with OA inflammation and pain. In this chapter we discuss the recognised clinical phenotypes of hand OA, typical features of hand OA, including Heberden's nodes, radiographic correlates of the clinical features observed and new insights into treatments for this chronic painful arthritic condition.

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2. Clinical phenotypes of hand OA**

#### **2.1. Nodal hand OA**

Nodal hand OA characteristically involves the distal interphalangeal (DIP) and proximal interphalangeal (PIP) joints of both hands. A typical swelling of the interphalangeal joints evolves, which can enlarge to a maximum size during its development (Figure 1). At the early to mid-stages when enlargement occurs, DIP joints can become painful, erythematous and difficult to mobilise. The underlying pathogenesis of this process includes bony enlargement of the underlying interphalangeal joints, synovitis and soft tissue swelling of the region affected [6]. When involving the DIP joints, the enlargement may give rise to an often typical feature described as a 'Heberden's node'. The historical context of the description of Heber‐ den's nodes is given further below. The PIP joints are also a recognised feature of nodal hand OA. They can be associated with a very similar pathological process to the joints described in DIPs as above and are sometimes called 'Bouchard's nodes' [7]. The radiographic features of nodal hand OA are demonstrated in Figure 2. Interestingly, the presence of Heberden's and Bouchard's nodes can occur with or sometimes without associated symptoms of pain, stiffness and disability.

**Figure 1.** Distal interphalangeal (DIP) joint swelling of the index, middle and ring fingers demonstrating Heberden's nodes in a patient with hand OA. The patient also has involvement of the proximal interphalangeal joints (PIP) of the index, middle and ring fingers (Bouchard's nodes).

Hand Osteoarthritis — Clinical Presentation, Phenotypes and Management http://dx.doi.org/10.5772/60540 89

**Figure 2.** Radiographic features of nodal hand OA demonstrating DIP joint involvement (index and ring fingers espe‐ cially) with joint space narrowing and osteophyte formation. This patient also has involvement of the first carpometa‐ carpal (CMC) joint of the thumb. The metacarpophalangeal joints are spared, which is often a characteristic finding.

#### **2.2. First carpometacarpal joint OA**

The first carpometacarpal (CMC) joint is a distinct recognised feature of hand OA. It is often seen bilaterally in people affected and is often a major cause of symptomatic joint pain. Risk factors for first CMC joint hand OA include mechanical factors and manual occupations [8]. The typical radiographic features of CMC joint OA are demonstrated in Figure 2. They often give rise to a 'square-shaped' hand in people affected by this condition. Interestingly nodal hand OA can co-exist with first CMC joint OA.

#### **2.3. Erosive hand OA**

**2. Clinical phenotypes of hand OA**

88 Osteoarthritis - Progress in Basic Research and Treatment

Nodal hand OA characteristically involves the distal interphalangeal (DIP) and proximal interphalangeal (PIP) joints of both hands. A typical swelling of the interphalangeal joints evolves, which can enlarge to a maximum size during its development (Figure 1). At the early to mid-stages when enlargement occurs, DIP joints can become painful, erythematous and difficult to mobilise. The underlying pathogenesis of this process includes bony enlargement of the underlying interphalangeal joints, synovitis and soft tissue swelling of the region affected [6]. When involving the DIP joints, the enlargement may give rise to an often typical feature described as a 'Heberden's node'. The historical context of the description of Heber‐ den's nodes is given further below. The PIP joints are also a recognised feature of nodal hand OA. They can be associated with a very similar pathological process to the joints described in DIPs as above and are sometimes called 'Bouchard's nodes' [7]. The radiographic features of nodal hand OA are demonstrated in Figure 2. Interestingly, the presence of Heberden's and Bouchard's nodes can occur with or sometimes without associated symptoms of pain, stiffness

**Figure 1.** Distal interphalangeal (DIP) joint swelling of the index, middle and ring fingers demonstrating Heberden's nodes in a patient with hand OA. The patient also has involvement of the proximal interphalangeal joints (PIP) of the

index, middle and ring fingers (Bouchard's nodes).

**2.1. Nodal hand OA**

and disability.

The erosive phenotype of hand OA is a particularly aggressive form of hand involvement. It is associated with erosions particularly in the DIP and PIP joints [9]. It is important to exclude other forms of inflammatory arthritis in its management. Typical radiographic features of this condition are shown in Figure 3. Historically, clinicians have considered the use of diseasemodifying anti-rheumatic drugs (DMARDs) in this variant to avert the progression of this erosive form of the disease [10].

**Figure 3.** Typical radiographic features of erosive hand OA are shown. There has been aggressive erosive damage to the proximal and distal interphalangeal joints (PIPs) and (DIPs) respectively. A central erosion is observed in several of these joints with a corresponding characteristic 'gull-wing' deformity and osteophytes observed laterally on either side. The interphalangeal (IP) joint of the thumb is also involved and characteristic joint space narrowing at the first carpometacarpal (CMC) joint is observed. The metacarpophalangeal joints are characteristically spared.

Other rarer forms of hand OA are also observed in the context of underlying conditions e.g. inflammatory arthritides, where hand OA can be a secondary phenomenon. The may include rheumatoid arthritis or the crystal arthropathies including gout and pseudogout. In the crystal arthropathies, clinical and radiographic appearances can be very similar and may also respond to similar therapies [11], giving prolonged episodes of joint inflammation and pain. Other underlying conditions which may give rise to chronic OA of the hands include genetic conditions such as Stickler's syndrome, and others such as haemochromatosis, hyperpara‐ thyroidism and acromegaly.

#### **3. Risk factors for hand OA development**

condition are shown in Figure 3. Historically, clinicians have considered the use of diseasemodifying anti-rheumatic drugs (DMARDs) in this variant to avert the progression of this

**Figure 3.** Typical radiographic features of erosive hand OA are shown. There has been aggressive erosive damage to the proximal and distal interphalangeal joints (PIPs) and (DIPs) respectively. A central erosion is observed in several of these joints with a corresponding characteristic 'gull-wing' deformity and osteophytes observed laterally on either side. The interphalangeal (IP) joint of the thumb is also involved and characteristic joint space narrowing at the first

Other rarer forms of hand OA are also observed in the context of underlying conditions e.g. inflammatory arthritides, where hand OA can be a secondary phenomenon. The may include rheumatoid arthritis or the crystal arthropathies including gout and pseudogout. In the crystal arthropathies, clinical and radiographic appearances can be very similar and may also respond to similar therapies [11], giving prolonged episodes of joint inflammation and pain. Other underlying conditions which may give rise to chronic OA of the hands include genetic conditions such as Stickler's syndrome, and others such as haemochromatosis, hyperpara‐

carpometacarpal (CMC) joint is observed. The metacarpophalangeal joints are characteristically spared.

erosive form of the disease [10].

90 Osteoarthritis - Progress in Basic Research and Treatment

thyroidism and acromegaly.

The risk of OA rises with increasing age, with the prevalence of OA increasing over the age of 50 [4]. With respect to the question of whether heavy physical work is associated with hand OA, some researchers have suggested that heavy occupational work was not associated with the presence of OA [12] and other studies have favoured a positive correlation of manual work with OA [13]. Obesity has also been investigated as an independent risk factor for hand OA with occasionally conflicting results. Hochberg *et al.* previously suggested that age and not obesity, were the main risk factor for hand OA [14]. In contrast, Denisov *et al.* suggested that obesity was associated with the progression of knee and hand OA in a cohort of almost 300 patients [15]. Cicutinni *et al.* have also suggested that there is a 9 to 13% increase in knee and hand OA for every kilogram increase in body weight [16].

Since the range of presentation of hand OA is varied, with several clinical phenotypes recognised, it is perhaps not surprising that a number of genes have been identified as risk factors for hand OA. These have been summarised in a recent review [17]. The main themes which have arisen from genetic studies include the observation that the genetic associations for hand OA cover a broad nature of genes, perhaps reflecting the multifactorial risk factors in this condition. Reported genetic associations include a female preponderance carried in many family cohorts, OA susceptibility loci mapping to chromosome 6 for hip and knee OA. Recently a group reported a significant association between hand OA and susceptibility loci on chromosome 6 [18]. In a UK cohort, Zhang *et al.* have investigated four putatively functional genetic variants in the KLOTHO gene, which is a strong ageing-related gene [19]. The group suggested that one variant in the KLOTHO gene was associated with hand OA susceptibility and especially with osteophyte formation rather than cartilage damage. Further studies have reported four SNPs in the IL-1R1 gene suggesting an association between the gene encoding the IL-1R1 and hand OA. Since IL-1 is a cytokine that has catabolic effects on cartilage, this may be particularly relevant in human disease [20]. Recent work has also focused on the extracellular matrix protein found in cartilage: aggrecan. Kamareinen *et al.* [21] showed that patients homozygous for the most common aggrecan VTNR (variable number of tandem repeats) allele, A27, had a significantly lower risk of hand OA. People who carried 2 copies of the aggrecan alleles with less than 27 repeats or more than 27 repeats demonstrated a higher risk of hand OA. The link between hand OA and extracellular matrix proteins found in cartilage has received further attention with the recent reports that single nucleotide polymorphisms (SNPs) in the asporin (ASPN) gene are associated with hand OA progression [22]. A recent genome-wide association study in an Icelandic population has shown that variants within the ALDH1A2 gene was associated with hand OA. The variants within the ALDH1A2 gene were confirmed in replication sets from The Netherlands and UK [23].

Although the studies above suggest a strong familial association with hand OA, the broad nature of clinical presentation and phenotypes suggests that strong genetic associations with hand OA are difficult to identify. In future it would be useful to accumulate larger populations of specific phenotypes to establish genetic associations in greater detail.

#### **4. Mechanisms of pain in hand osteoarthritis**

Osteophytes, which are a pathognomonic feature of OA, are often observed to create a physical barrier to optimal range of movement and can give rise to severe joint pain as well. Recently, inflammatory changes have been found to relate to higher risk of structural damage in a study which looked at 2 years follow-up [6]. Kortekaas and colleagues showed that inflammatory features, defined by synovial thickening, effusion and increased power Doppler signal on ultrasound scan (demonstrated in Figure 4), when they persisted, were related to increased radiographic progression of OA after 2 years. The presence of synovitis by ultrasound may therefore guide treatment decisions such as corticosteroid-guided intra-articular injection in hand joints affected by OA.

**Figure 4.** Ultrasound image of the right first carpometacarpal joint, showing evidence of synovial thickening and in‐ creased vascularity on power Doppler imaging.

Recent work has demonstrated the evidence of bone marrow lesions (BML), which are defined as high density signal lesions on MRI with T2-weighted imaging, especially in the knee [24], have also been observed in people with hand OA [25]. In their study, Haugen *et al.* showed that bone marrow lesions, synovitis and erosions were associated with joint tenderness. It is therefore possible that synovitis and bone marrow lesions could be future targets for thera‐ peutic interventions in hand OA.

#### **5. Heberden's nodes: A historical perspective**

"What are those little hard knobs, about the size of a small pea, which are frequently seen upon the fingers, particularly a little below the top, near the joint? They have no connection with the gout, being found in persons who never had it; they continue for life; and being hardly ever attended with pain, or disposed to become sores, are rather unsightly, than inconvenient, though they must be some little hindrance to the free use of the fingers." [26]. These were the observations of William Heberden whose philosophy in medicine was that one must always be guided by their own direct observations. Heberden's nodes, which are classical lesions of osteoarthritis, were initially described as 'digitorum nodi' in Latin by Heberden himself, which led him to make the important distinction between gout and osteoarthritis [27]. OA remains a common disabling condition worldwide and the most common form of arthritis [28]. However, there are few diagnostic tests and unfortunately current treatments for OA have not been able to successfully eliminate pain from the clinical manifestations of the disease [5].

**4. Mechanisms of pain in hand osteoarthritis**

92 Osteoarthritis - Progress in Basic Research and Treatment

hand joints affected by OA.

creased vascularity on power Doppler imaging.

peutic interventions in hand OA.

**5. Heberden's nodes: A historical perspective**

Osteophytes, which are a pathognomonic feature of OA, are often observed to create a physical barrier to optimal range of movement and can give rise to severe joint pain as well. Recently, inflammatory changes have been found to relate to higher risk of structural damage in a study which looked at 2 years follow-up [6]. Kortekaas and colleagues showed that inflammatory features, defined by synovial thickening, effusion and increased power Doppler signal on ultrasound scan (demonstrated in Figure 4), when they persisted, were related to increased radiographic progression of OA after 2 years. The presence of synovitis by ultrasound may therefore guide treatment decisions such as corticosteroid-guided intra-articular injection in

**Figure 4.** Ultrasound image of the right first carpometacarpal joint, showing evidence of synovial thickening and in‐

Recent work has demonstrated the evidence of bone marrow lesions (BML), which are defined as high density signal lesions on MRI with T2-weighted imaging, especially in the knee [24], have also been observed in people with hand OA [25]. In their study, Haugen *et al.* showed that bone marrow lesions, synovitis and erosions were associated with joint tenderness. It is therefore possible that synovitis and bone marrow lesions could be future targets for thera‐

"What are those little hard knobs, about the size of a small pea, which are frequently seen upon the fingers, particularly a little below the top, near the joint? They have no connection with the In addition to Heberden's description further observation of the nodes have led them to be classified according to the location (Figure 1). Nodes either appear on the lateral aspect on the dorsolateral margins or in the central midline where occasionally they fuse with the lateral nodes to form a ridge. They can also be classified as idiopathic or traumatic, with the latter most commonly resulting in a solitary Heberden node [29]. Idiopathic nodes are easily identifiable from the clinical history, which usually consists of a node, which slowly and gradually increases in size on one finger and then spreads to other fingers. Reports from patients about pain associated with the nodes are inconsistent, with some reporting painful growth of the nodes whereas others report the growth as painless [30]. Stecher [29] described the progression of Heberden's nodes having observed around 7,000 individuals with Heber‐ den's nodes. He noted that the progression could be divided into three stages. The initial stage consists of a visible enlargement of the joint; this enlargement is also palpable at the proximal end of the distal phalanx as two spherical nodules or as prominent ridge. In some people it was noted that the enlargement was so severe that the nodules were palpable at the sides of the joint and on the palmar surface. The second stage consists of palmar flexion of the distal phalanx in addition to the enlargement. In addition to the previous two stages the third stage consists of lateral deviation of the distal phalanx from the midline (see Figure 1) [29].

It is well established that Heberden's nodes are associated with underlying radiographic changes [30]. More recent studies have shown that Heberden's nodes, which are more developed and affecting both sides of the joint show joint space narrowing as opposed to Heberden's nodes in their initial stages. This can be attributed to the slow growth of Heberden's nodes and that joint space narrowing is possibly a late manifestation of the disease [31]. With this knowledge it can be assumed that Heberden's nodes affecting both aspects of the joint can be used as a clinical marker for radiographic change [31].

OA is a process of cartilage and bone damage in which changes are eventually irreversible. However it seems that Heberden's nodes have a different process governing their formation and they are histologically different depending on the location on the joint. Midline nodes are traction spurs growing in the extensor tendon, this growth is usually found in athletes as a physiological response to excessive tension. It is important to note that this spur is not a true osteophyte and that it can be identified by its location and the lack of a cartilage cap. Con‐ versely, the lateral node has shown to have a constant presence of osteophyte, which arises from one or both of the phalanges and is located lateral to the extensor tendon. These histo‐ logical findings are supported by radiological observation. Radiography of Heberden's nodes shows that there is evidence of osteophyte formation in the lateral node and a traction spur in the middle node [31]. Osteophytes consist of both new bone and cartilage formation, which arise from progenitor cells, indicating that the sequelae of joint destruction induces a pluri‐ potent cell response [32]. The exact function of osteophytes in osteoarthritis is yet be under‐ stood but can been seen as an adaptive mechanism against joint injury to stabilize the joint [33, 34]. Analysis of osteophytes at different developmental stages has shown a sequential process of differentiation and the presence of the anabolic factor transforming growth factor-beta (TGFβ) [32]. Osteophyte growth usually occurs in the direction of least resistance; in joints such as the shoulder and ankle the strong capsules restrict osteophyte growth. At the distal interpha‐ langeal joint the only obstruction is a thin capsule, which holds in synovial fluid. This ob‐ struction is not sufficient to restrict osteophyte growth hence the presence of such prominent Heberden's nodes. To what extent the osteophyte causes deviation of the distal phalanx is dependent on the strength of the collateral ligament, which is genetically determined [31]. Extensive research by Stecher has shown that there may be a single autosomal gene responsible for the inheritance of Heberden's nodes however it seems to exhibit a sex-linked pattern as it is dominant in females and recessive in males [29].

Although Heberden's nodes were first described over 150 years ago and despite extensive research their exact mechanism and purpose has not been fully understood. They are part of the clinical picture of osteoarthritis but it seems that their pathophysiology and genetic inheritance somewhat differs to our current understanding of generalised osteoarthritis.

#### **6. Treatments for hand osteoarthritis**

A range of treatments have been used in hand OA, including physical techniques such as taping and splinting to reduce pain and improve function [35, 36]. Such treatment is often administered through physiotherapy and/or occupational therapy units and can be repeated during flares.

Pharmacological treatments are often focused around symptomatic pain relief including paracetamol, topical and oral non-steroidal anti-inflammatory drugs such as ibuprofen, naproxen and others, which have been shown to be efficacious over and above other analgesic treatments in large scale meta-analyses [4]. Such treatments are now recommend‐ ed within several international guidelines for the treatment of OA [36]. Recent imaging studies which have shown synovitis and bone marrow lesions to correlate with painful progressive hand OA have led to renewed interest in considering disease-modifying antirheumatic agents for hand OA.

Previous studies have shown that intra-articular injections of corticosteroid can be particularly beneficial in hand OA, particularly at the first CM joint [37]. However, repeated injections are not without significant side-effects, including local tissue damage and skin atrophy. The general consensus is that, if not straightforward, local injections may best be performed under ultrasound guidance. Some reports have suggested intra-articular hyaluronic acid may also be a potential therapeutic option, but such studies have not yet been subjected to large scale clinical trials [38]. With respect to systemic steroid, a recent study showed improvement in synovitis following intramuscular depomedrone injection of steroid for hand OA, which was sustained for 4 weeks [39]. However, the effects of systemic corticosteroid treatment were relatively short-lived and synovitis returned by ultrasound measures after 12 weeks of treatment.

With respect to the use of other disease-modifying agents, a recent trial of hydroxychloroquine has been conducted for the treatment of hand OA [40] and the results of this study are now awaited. Other groups have investigated the use of bisphosphonates e.g. intravenous clodro‐ nate in the treatment of hand OA with beneficial outcome for pain in their study [41]. However, a recent meta-analysis by our group did not show overall significant benefit overall for pain and function after use of bisphosphonates across several phenotype of OA, including hip, knee and hand [42]. Future studies targeted at specific stages of disease with proven synovitis and bone marrow lesions may be more helpful in establishing the potential future therapeutic use of bone-modulating agents in the clinic.

#### **Author details**

logical findings are supported by radiological observation. Radiography of Heberden's nodes shows that there is evidence of osteophyte formation in the lateral node and a traction spur in the middle node [31]. Osteophytes consist of both new bone and cartilage formation, which arise from progenitor cells, indicating that the sequelae of joint destruction induces a pluri‐ potent cell response [32]. The exact function of osteophytes in osteoarthritis is yet be under‐ stood but can been seen as an adaptive mechanism against joint injury to stabilize the joint [33, 34]. Analysis of osteophytes at different developmental stages has shown a sequential process of differentiation and the presence of the anabolic factor transforming growth factor-beta (TGFβ) [32]. Osteophyte growth usually occurs in the direction of least resistance; in joints such as the shoulder and ankle the strong capsules restrict osteophyte growth. At the distal interpha‐ langeal joint the only obstruction is a thin capsule, which holds in synovial fluid. This ob‐ struction is not sufficient to restrict osteophyte growth hence the presence of such prominent Heberden's nodes. To what extent the osteophyte causes deviation of the distal phalanx is dependent on the strength of the collateral ligament, which is genetically determined [31]. Extensive research by Stecher has shown that there may be a single autosomal gene responsible for the inheritance of Heberden's nodes however it seems to exhibit a sex-linked pattern as it

Although Heberden's nodes were first described over 150 years ago and despite extensive research their exact mechanism and purpose has not been fully understood. They are part of the clinical picture of osteoarthritis but it seems that their pathophysiology and genetic inheritance somewhat differs to our current understanding of generalised osteoarthritis.

A range of treatments have been used in hand OA, including physical techniques such as taping and splinting to reduce pain and improve function [35, 36]. Such treatment is often administered through physiotherapy and/or occupational therapy units and can be repeated

Pharmacological treatments are often focused around symptomatic pain relief including paracetamol, topical and oral non-steroidal anti-inflammatory drugs such as ibuprofen, naproxen and others, which have been shown to be efficacious over and above other analgesic treatments in large scale meta-analyses [4]. Such treatments are now recommend‐ ed within several international guidelines for the treatment of OA [36]. Recent imaging studies which have shown synovitis and bone marrow lesions to correlate with painful progressive hand OA have led to renewed interest in considering disease-modifying anti-

Previous studies have shown that intra-articular injections of corticosteroid can be particularly beneficial in hand OA, particularly at the first CM joint [37]. However, repeated injections are not without significant side-effects, including local tissue damage and skin atrophy. The general consensus is that, if not straightforward, local injections may best be performed under

is dominant in females and recessive in males [29].

94 Osteoarthritis - Progress in Basic Research and Treatment

**6. Treatments for hand osteoarthritis**

during flares.

rheumatic agents for hand OA.

Nidhi Sofat\* and Sharenja Jeyabaladevan

\*Address all correspondence to: nsofat@sgul.ac.uk

Institute for Infection and Immunity, St George's, University of London, Cranmer Terrace, London, UK

#### **References**


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[5] Sofat, N., Ejindu, V. and Kiely, P. (2011) 'What makes osteoarthritis painful? The evi‐ dence for local and central pain processing', *Rheumatology (Oxford, England),* 50 (12),

[6] Kortekaas, M.C., et al. (2014) 'Inflammatory ultrasound features show indepdendent associations with progression of structural damage after 2 years of follow-up in pa‐ tients with hand osteoarthritis'. *Ann Rheum Dis*. Doi: 10.1136/annrheum‐

[7] Rees, F. et al. (2012). 'Distribution of finger nodes and their association with underly‐ ing radiographic features of osteoarthritis. *Arthritis Care Res (Hoboken)* 64(4): 533-8 [8] Jensen J.C., Sherson, D. (2007) 'Work-related bilateral osteoarthritis of the first carpo‐

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[10] Bryant LR, et al. (1995) 'Hydroxychloroquine in the treatment of erosive osteoarthri‐

[11] Rothschild, B.M. (2013) 'Distinguishing erosive osteoarthritis and calcium pyrophos‐

[12] Goekoop, R.J et al. (2011) 'Determinants of absence of osteoarthritis in old age'. *Scan J*

[13] Snodgrass SJ, Rivett DA, Chiarelli P, Bates AM, Rowe LJ, Aust J Physio 2003; 49(4):

[14] Hochberg, M.C. et al. (1993) 'Obesity and osteoarthritis of the hands in women'. *Os‐*

[15] Denisov, L.N. et al. (2011) 'Role of obesity in the development of osteoarthrosis and

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[36] Hochberg, M.C. et al. (2012). 'American College of Rheumatology 2012 recommenda‐ tions for the use of nonpharmacologic and pharmacologic therapies in osteoarthritis

[37] Maarse, W. et al. (2009) 'Medium-term outcomes following intra-articular corticoste‐ roid injection in first CMC joint arthritis using fluoroscopy'. *Hand Surg* 14(2-3):

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[39] Wenham, C.Y. (2012). 'A randomized, double-blind, placebo-controlled trial of lowdose oral prednisolone for treating painful hand osteoarthritis. *Rheumatology (Oxford)*

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al'. *Trials* 14:64

Elizabeth Pérez-Hernández, Nury Pérez-Hernández and Ariel Fuerte-Hernández

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60940

#### **1. Introduction**

Osteoarthritis (OA) is a widespread disease and is considered the most common form of arthritis. The current OA prevalence is estimated at 15% of the population [1], but this rate is predicted to double by 2020 [2] and this increase is related with some lifestyle diseases like obesity [2]. OA affects the joints of the hand and the lower extremities such as knee and hip. OA of the spine occurs in approximately 40–85% of the adult population, but it is often omitted in prevalence studies. The rate disparities are due to the differences on the definition of the disease and to the variability between demographic groups studied [3]. Generally, the costs for OA care are high, as well as the economic implications for prolonged work disability [4, 5]. Symptomatically, it is considered that 80% of Americans suffer an episode of low back pain (LBP) in their lifetime [6, 7], thus care costs for LBP are estimated to be more than 100 billion dollars per year in the US [8], with a loss of 149 million workdays per year [9, 10].

OA of the lumbar spine is related with the degeneration of the intervertebral disc (ID) and bone formation, which is called spondylosis [11]. There has been a lack consensus on whether or not the combination of decreased disc space and osteophyte formation is a characteristic of OA or is a separate phenomenon. Clinically, the association between OA of the hand, knee and facet joint has been described, but no relationship was found between disc degeneration (DD) and OA of the hip, knee, or hand, or between the formation of osteophytes and OA of the hip and hand [12].

OA is defined as a disease resulting in structural and functional failure of synovial joints, which usually is characterized by progressive articular cartilage damage, involvement of the synovium and subchondral bone hypertrophy. OA affects the spinal zygapophyseal joints and is closely related to degenerative disc disease (DDD) despite the pathophysiological differen‐

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ces between the two disorders [13]. This degenerative cycle has mechanical impact with significant and progressive changes in the functional anatomy and mechanobiology, mani‐ festing pain syndromes, destabilization, and impaired quality of life.

#### **2. Anatomy and mechanics of the spinal joints**

#### **2.1. Normal facet joints**

The vertebral joints are complex structures made up of posterior and anterior elements. The posterolateral spine consists of facet joints that are considered true synovial joint. One ID and two facet joints comprises a "three-joint complex." This complex binds two adjacent vertebrae, and the superior articular process of the inferior vertebra joins with the inferior articular processes of the overlying vertebra (Figure 1). The joint surfaces in the cervical and thoracic spinal regions are convex and concave, and the lumbar region of the facet joints shows a devastated form [14]. The orientation of the articular surfaces has a basic biomechanical role. The articular surfaces of the cervical and thoracic spinal segments are arranged horizontally [14, 15], favoring the axial rotation and lateral flexion [16, 17]. Comparatively, lower thoracic and lumbar spinal regions tend to adopt a more vertical orientation [18] that limits lateral flexion and rotation, protecting the IDs and spinal cord. Generally, the inclination angles of cervical facet joints in the sagittal plane ranges from 20° to 78° and in the axial plane from 70° to 96°, while the angle between the thoracic facet joints range from 55° to 80° and 85° to 120°, and the lumbar region range from 82° to 86° and 15° to 70° in both planes, respectively [14, 18].

Typically, facet synovial joints have a hyaline cartilage cover, subchondral bone, synovium, and a ligament system that envelops the entire joint [19, 20]. The cartilage layer is thinner at the edges of the joint surface and gradually thicker in the central portion thereof [21]. The composition of the articular cartilage does not differ from that observed in other diarthrodial surfaces, a cellular component, chondrocytes, and an abundant extracellular matrix (EM) composed of water, fibrillar proteins, glycosaminoglycans, and proteoglycans. The mechanical properties, such as load distribution and low-friction movement are dependent on the articular cartilage integrity [22, 23].

The subchondral bone has been considered as a morphological unit that provides a link between the articular cartilage and cancellous bone, which plays a key role in mitigate the impact of axial forces during dynamic joint load [24, 25]. It has been reported that the sub‐ chondral bone thickness is greater in asymptomatic males and increases with each successive lower spinal level, suggesting its association with the increased load [25].

Additionally, synovium and the ligament system facilitate movement with minimal friction and provide mechanical resistance, while synovial fluid lubricates and nourishes the joint surfaces [26]. The meniscoides or intraarticular synovial folds also protect the articular cartilage during movement [27], compensate the irregularities of the joint surface and increase the contact surface with the facets [28]. The meniscoides are formed by fatty, fibrous connective tissues and a lining of synovium [29, 28].

**Figure 1.** Normal anatomy of the facet joint and ID. A. Sagittal view of segments L1–L5; B. Sagittal view (L3); C–D. Coronal views (L3); E–F. Axial view (L3); G. Lumbar disc-facet unit (L3–L4); H. ID (L3–L4).

Moreover, the capsule comprises ligament fibroblasts, dense collagen fibers, elastic fibers, and proteoglycans [30, 31], and one of its main functions is to allow movement without provide mechanical resistance.

#### **2.2. Normal intervertebral disc**

ces between the two disorders [13]. This degenerative cycle has mechanical impact with significant and progressive changes in the functional anatomy and mechanobiology, mani‐

The vertebral joints are complex structures made up of posterior and anterior elements. The posterolateral spine consists of facet joints that are considered true synovial joint. One ID and two facet joints comprises a "three-joint complex." This complex binds two adjacent vertebrae, and the superior articular process of the inferior vertebra joins with the inferior articular processes of the overlying vertebra (Figure 1). The joint surfaces in the cervical and thoracic spinal regions are convex and concave, and the lumbar region of the facet joints shows a devastated form [14]. The orientation of the articular surfaces has a basic biomechanical role. The articular surfaces of the cervical and thoracic spinal segments are arranged horizontally [14, 15], favoring the axial rotation and lateral flexion [16, 17]. Comparatively, lower thoracic and lumbar spinal regions tend to adopt a more vertical orientation [18] that limits lateral flexion and rotation, protecting the IDs and spinal cord. Generally, the inclination angles of cervical facet joints in the sagittal plane ranges from 20° to 78° and in the axial plane from 70° to 96°, while the angle between the thoracic facet joints range from 55° to 80° and 85° to 120°, and the lumbar region range from 82° to 86° and 15° to 70° in both planes, respectively [14, 18].

Typically, facet synovial joints have a hyaline cartilage cover, subchondral bone, synovium, and a ligament system that envelops the entire joint [19, 20]. The cartilage layer is thinner at the edges of the joint surface and gradually thicker in the central portion thereof [21]. The composition of the articular cartilage does not differ from that observed in other diarthrodial surfaces, a cellular component, chondrocytes, and an abundant extracellular matrix (EM) composed of water, fibrillar proteins, glycosaminoglycans, and proteoglycans. The mechanical properties, such as load distribution and low-friction movement are dependent on the articular

The subchondral bone has been considered as a morphological unit that provides a link between the articular cartilage and cancellous bone, which plays a key role in mitigate the impact of axial forces during dynamic joint load [24, 25]. It has been reported that the sub‐ chondral bone thickness is greater in asymptomatic males and increases with each successive

Additionally, synovium and the ligament system facilitate movement with minimal friction and provide mechanical resistance, while synovial fluid lubricates and nourishes the joint surfaces [26]. The meniscoides or intraarticular synovial folds also protect the articular cartilage during movement [27], compensate the irregularities of the joint surface and increase the contact surface with the facets [28]. The meniscoides are formed by fatty, fibrous connective

lower spinal level, suggesting its association with the increased load [25].

festing pain syndromes, destabilization, and impaired quality of life.

**2. Anatomy and mechanics of the spinal joints**

**2.1. Normal facet joints**

100 Osteoarthritis - Progress in Basic Research and Treatment

cartilage integrity [22, 23].

tissues and a lining of synovium [29, 28].

In the anterior spine, the vertebral bodies are attached through the IDs. These structures provide support for load and flexibility during mechanical exposure; they also facilitate the movements of flexion, extension, and rotation. The typical composition of the ID consists of a central nucleus pulposus, which is contained in an outer annulus fibrous at the periphery, and the inferior and superior cartilage endplates [32].

Annulus fibers are agreed in concentric lamellae and consist predominantly of type I collagen [33]. In the early stages of life, these lamellae are arranged regularly, these are divide and interdigitate, and during aging they form an intricate and complex network in response to the load. Adult lumbar discs may contain up to 25 lamellae, thus lead to an increase in thickness toward the center portion thereof [34]. Annulus cells are small, elongated, disposed parallel to lamellae, and synthesize types I and II collagen [35], elastin [36], proteoglycans [37], and types III, IV, and VI collagens in various proportions [38, 39, 40, 41]. Other proteins with leucine-rich repeat, such as fibromodulin, decorin, and lumican, regulate the assembly of collagen fibers; similarly, the cartilage oligomeric matrix protein (COMP) is involved in regulation of the assembly of fibrillar proteins. Furthermore, chondroadherin, other protein with leucine-rich repeat without carbohydrate substituents and without the N-terminal binds to collagen participates in the maintenance of chondrocyte phenotype [41].

The center of the ID is the nucleus pulposus, which becomes gelatinous and more fibrous with aging. The nucleus pulposus is surrounded by a fibrous capsule and consists of round or oval chondrocyte-like cell with abundant cytoplasm and prominent cytoskeleton. These cells are called "physaliphorus" cells, which present large vacuoles. In this region, these cells are responsible in the synthesis of type II collagen [35]. The nucleus pulposus is rich in proteo‐ glycan aggrecan, which consists of approximately chondroitin sulfate chains hundred, and each polysaccharide chain has about hundred negatively charged groups. Furthermore, the keratan sulfate chains are disposed in clusters located in a different area to that of chondroitin sulfate chains. Moreover, hundred molecules bind aggrecan and hyaluronate, as well as fibulin and tenascin proteins [41, 42]. These negatively charged macromolecular structures promote osmotic water retention.

The interface between the disc and the vertebral body consists of a thin layer of hyaline cartilage called endplate. This is extended across most of the vertebral body except at the outer rim, where the fibers of annulus fibrosus are inserted. In adults, this tissue is avascular, so the metabolites diffuse through it to the cells of the endplate and the center of the disc. During adulthood, the endplate thickness is reduced to approximately 0.6 mm [43]. Biochemically, the endplate contains type X collagen, which is involved in calcification processes [44].

It is well known that the main source of energy disc cells is derived from glycolysis [45]. Due to the low oxygen tension, protein synthesis and macromolecules, such as sulfated glycosa‐ minoglycans is inhibited. Stimuli such as growth factors also come from the extracellular fluid [46], while the ATP production in disc cells depends on the local pH and nutrient availability. Most studies have reported that an acidic pH level significantly reduces the glycolytic metabolism and the rate of oxygen consumption, with concomitant decrease in ATP produc‐ tion [47, 48]. However, the effect of oxygen concentration in disc cells remains controversial, as some studies have described a positive effect [45, 46]. Protein synthesis is also a process affected by local oxygen and pH levels, which significant decrease if the low oxygen tension to less than 5% [46]. Similarly, extracellular pH affects protein synthesis, so an abrupt decrease in acidic environments [48]. Contrary to this event, the activity of matrix metalloproteinases is generally not inhibited at low pH, which may enhance the rate of matrix breakdown [49].

Dependent glucose supply (primary energy source), disc cells can die within 24 hours if glucose concentrations fall below 0.2 mM. Under these conditions, the intracellular glucose transport is also significantly reduced [50]. In this respect, it has been reported that the rate of cell death of the disc increases in acidic conditions (pH 6.0) despite of an adequate glucose intake [51, 52].

The avascular nature of the adult human ID is well known, with minimal penetration of capillaries and nerve endings in the outer regions of the annulus. This capillary network comes from the vertebral arteries which across the subchondral bone forming the loops of the interface between cartilage endplate and the bone [53, 54]. Thus, vascularity is protected by the cartilage endplate and promotes selective transport of molecules through the disc [55, 56].

Nutrients diffuse from capillary vascularity of the disc, through cartilage endplate, EM until the cells [57, 58, 59]. This solute movement is associated with load patterns to which it is usually subjected the disc. Apparently, the mechanical load on the disc is inversely proportional to nutrient transport. For example, during the redistribution of the load, the disc thickness is decreased, favoring the transport of nutrients from the cartilage endplate; however, if the proportion of fluid in the tissue matrix is decreased, diffusion is reduced, and this could affect the metabolic levels [60, 61]. This charge-nutrients ratio in the ID is still under investigation. Diffusion gradient, which is dependent on passive transport, leads to differences in the metabolic activity of the disc cells. Generally, the center of the disc contains lower concentra‐ tions of glucose and oxygen, and higher concentrations of lactic acid [45].

#### **2.3. Mechanobiology**

with leucine-rich repeat without carbohydrate substituents and without the N-terminal binds

The center of the ID is the nucleus pulposus, which becomes gelatinous and more fibrous with aging. The nucleus pulposus is surrounded by a fibrous capsule and consists of round or oval chondrocyte-like cell with abundant cytoplasm and prominent cytoskeleton. These cells are called "physaliphorus" cells, which present large vacuoles. In this region, these cells are responsible in the synthesis of type II collagen [35]. The nucleus pulposus is rich in proteo‐ glycan aggrecan, which consists of approximately chondroitin sulfate chains hundred, and each polysaccharide chain has about hundred negatively charged groups. Furthermore, the keratan sulfate chains are disposed in clusters located in a different area to that of chondroitin sulfate chains. Moreover, hundred molecules bind aggrecan and hyaluronate, as well as fibulin and tenascin proteins [41, 42]. These negatively charged macromolecular structures promote

The interface between the disc and the vertebral body consists of a thin layer of hyaline cartilage called endplate. This is extended across most of the vertebral body except at the outer rim, where the fibers of annulus fibrosus are inserted. In adults, this tissue is avascular, so the metabolites diffuse through it to the cells of the endplate and the center of the disc. During adulthood, the endplate thickness is reduced to approximately 0.6 mm [43]. Biochemically, the

It is well known that the main source of energy disc cells is derived from glycolysis [45]. Due to the low oxygen tension, protein synthesis and macromolecules, such as sulfated glycosa‐ minoglycans is inhibited. Stimuli such as growth factors also come from the extracellular fluid [46], while the ATP production in disc cells depends on the local pH and nutrient availability. Most studies have reported that an acidic pH level significantly reduces the glycolytic metabolism and the rate of oxygen consumption, with concomitant decrease in ATP produc‐ tion [47, 48]. However, the effect of oxygen concentration in disc cells remains controversial, as some studies have described a positive effect [45, 46]. Protein synthesis is also a process affected by local oxygen and pH levels, which significant decrease if the low oxygen tension to less than 5% [46]. Similarly, extracellular pH affects protein synthesis, so an abrupt decrease in acidic environments [48]. Contrary to this event, the activity of matrix metalloproteinases is generally not inhibited at low pH, which may enhance the rate of matrix breakdown [49].

Dependent glucose supply (primary energy source), disc cells can die within 24 hours if glucose concentrations fall below 0.2 mM. Under these conditions, the intracellular glucose transport is also significantly reduced [50]. In this respect, it has been reported that the rate of cell death of the disc increases in acidic conditions (pH 6.0) despite of an adequate glucose intake [51, 52].

The avascular nature of the adult human ID is well known, with minimal penetration of capillaries and nerve endings in the outer regions of the annulus. This capillary network comes from the vertebral arteries which across the subchondral bone forming the loops of the interface between cartilage endplate and the bone [53, 54]. Thus, vascularity is protected by the cartilage

endplate and promotes selective transport of molecules through the disc [55, 56].

endplate contains type X collagen, which is involved in calcification processes [44].

to collagen participates in the maintenance of chondrocyte phenotype [41].

osmotic water retention.

102 Osteoarthritis - Progress in Basic Research and Treatment

The mechanical load on the ID can cause multiple physical changes and mechanobiological effects. Volumetric changes, fluid flows, pressure changes, electrokinetic activity, and changes in cell shape are events that occur secondarily to tension, compression, or shear. Previous studies in vitro and in vivo on animal models have shown that the static compressive load or strain (0.2 to 0.4 MPa) [62] can induce anabolic cellular responses in the disc, with increased gene expression and synthesis of EM components such as proteoglycans and types I and II collagens [63].

Comparatively, during dynamic compression at low frequency (0.01 Hz, 1 MPa) in cells of rodent disc, induce an increase in the gene expression of macromolecules such as aggrecan and types I and II collagens. At higher frequencies of load an increase in expression of mRNA of proteases like MMP-3, MMP-13, ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs) has been observed [64, 65]. Additionally, disc cell death occurred after exposure to high magnitudes (> 0.4 MPa) and low frequency (0.01 Hz) of dynamic load.

In response to moderate hydrostatic pressure (<3.0 MPa), the cells cultured or tissue explants of disc may increase the synthesis collagen, proteoglycans, and tissue inhibitor of metallopro‐ teinase 1 (TIMP1), which applies to cells of the nucleus pulposus and annulus inner regions [66, 67, 68]. The inhibition of protein synthesis, the increase in the nitric oxide and the synthesis of MMP-3 have been shown in disc cells in extreme downward or high pressures [69].

Similarly, the metabolic activity of the disc cells has been influenced by changes in osmotic pressure. In vitro tests have shown high rates of proteoglycan synthesis at in situ extracellular osmolarity (~430 mOsm); however, when this concentration is increased or decreased, protein synthesis declines [70]. Another manifestation of compressive deformation of disc cells is the reorganization of the cytoskeleton, including the increase of early polymerization of vimentin [63]. Also depolymerization of actin filament calcium-dependent and the volume change on disc cells grown in hyper or hypo-osmotic media has also been shown [71, 72].

Moreover, disc cells exposed to stress in vitro undergo changes in the membrane potential associated with apoptosis. These cells increase nitric oxide production and decrease the proteoglycans synthesis [73, 74].

### **3. Epidemiology**

#### **3.1. Facet arthrosis**

LBP is considered epidemic, and its prevalence varies in developed countries from 60% to 90% of patients undergoing orthopedic consultation [75, 76]. The cost of care for these patients varies from \$100 to \$200 billion annually [8]. One of the main causes of LBP is facet arthrosis. Since 1930, it has been called as facet syndrome [77, 78]; many studies on cadavers have described the presentation of facet arthrosis around the third decade of life [79, 80]. According to epidemiological studies based on imaging, the cervical facet OA has been reported in 19% of adults between 45 and 64 years of age and in 57% of adults over 65 years [81]. On severe lumbar facet OA diagnosed by computed tomography (CT) images, prevalence rates are estimated to be 36% in adults under 45 years of age, 67% in adults between 45 and 64 years, and 89% in individuals over 65 years [82]. Thus, it can be concluded that the prevalence of facet OA and its progression are dependent on age [81, 83].

The literature reports higher prevalence and degree of arthrosis at L4–L5 facet joints [79, 84, 85]. This was more prevalent at L4–L5 (from 45.1% to 79%), followed by L5–S1 (from 38.2% to 59%), and finally L3–L4 (from 30.6% to 72%) [79, 84]. These reports support the fact that the degenerative lumbar stenosis is related to the more mobile segments (L3–L4, L4–L5) of the lumbar spine [86, 87]. On the frequency of occurrence on the right or left side, an equal distribution has been reported [88].

With respect to gender, higher prevalence has been reported in males, and apparently, there is no significant difference in relation to race [79, 80]. However, in image studies using CT scans and planar radiography, women have been shown to have a higher prevalence of lumbar facet OA than men [12].

Moreover, the body mass index (BMI) has been found to be associated with an increased prevalence of cervical facet OA [83] and, even more, of the lumbar region [12, 82]. In this respect, the risk of lumbar facet OA is almost three times higher in overweight individuals (BMI 25–30 kg/m2 ) and five times more associated with obesity (BMI 30–35 kg/m2 ), in com‐ parison with the normal-weight reference group (BMI ≤ 25 kg/m<sup>2</sup> ) [12].

Another risk factor for facet OA is the anatomy of the spine; for example, the changes in the orientation of the articular facet and facet joint asymmetry or "tropism" [89, 90, 91, 92] and simultaneously the disc-height narrowing represents the risk of contracting the disease [82]. Similarly, other factors such as the poor quality of the extensor muscles have been associated with facet OA of L4–L5 [93].

#### **3.2. Disc degeneration**

Although there is no standard definition of disc degeneration (DD), it is considered the product of the degradation and remodeling of the ID and adjacent vertebrae, with adaptive changes and/or consequential damage induced by physical load. Radiographic studies, particularly magnetic resonance imaging (MRI), have allowed qualitative assessment of disc degeneration (DD). Particular emphasis has been given on disc space narrowing, the disc-vertebra remod‐ eling with formation of osteophytes and disc bulge peripherals, changes or loss of signal intensity with development of annular tears, herniations, Schmorl nodes, and endplate sclerosis [94, 95]. In this regard, the prevalence of disc-bulging has been described from 10% to over 80% in asymptomatic patients, and the prevalence of annular tears varies from 6% to 56% [3].

Age has been widely related to disc degeneration (DD). Degenerative changes have been described since childhood and young adulthood, such as the presence of annular tears between 3 and 10 years old [96, 97, 98]. Moreover, the water content of the nucleus pulposus shown by the disc signal intensity has been reported from 35 years of age [99].

It has also been proposed that mechanical load and nutritional states could contribute to the early development of DD, and it has found more severe in men than in women [100]. The association of heavy physical load and DD is still controversial and inconsistent. Similarly, relation to smoking has not been found [99].

Studies on the genetic influence in monozygotic male twins have shown a substantial familial influence on disc degenerative changes such as lumbar disc-height narrowing, bulging or herniation, and disc desiccation [99, 101, 102]. It seems likely that the DD is a multifactorial genetic condition, oligogenic. Some of the human gene forms reported as TaqI and FokI of the vitamin-D receptor gene have been associated with low intensity of magnetic resonance signal of thoracic and lumbar discs [103]. Two genotypes of MMP-3 gene have been related with degenerative disc changes in the elderly, and type IX collagen, alpha 2 (COL9A2), and 3 (COL9A3) gene forms have been linked with symptomatic disc pathology [104]. Nevertheless, research in this is still lacking.

#### **4. Pathophysiology**

**3. Epidemiology**

104 Osteoarthritis - Progress in Basic Research and Treatment

**3.1. Facet arthrosis**

LBP is considered epidemic, and its prevalence varies in developed countries from 60% to 90% of patients undergoing orthopedic consultation [75, 76]. The cost of care for these patients varies from \$100 to \$200 billion annually [8]. One of the main causes of LBP is facet arthrosis. Since 1930, it has been called as facet syndrome [77, 78]; many studies on cadavers have described the presentation of facet arthrosis around the third decade of life [79, 80]. According to epidemiological studies based on imaging, the cervical facet OA has been reported in 19% of adults between 45 and 64 years of age and in 57% of adults over 65 years [81]. On severe lumbar facet OA diagnosed by computed tomography (CT) images, prevalence rates are estimated to be 36% in adults under 45 years of age, 67% in adults between 45 and 64 years, and 89% in individuals over 65 years [82]. Thus, it can be concluded that the prevalence of

The literature reports higher prevalence and degree of arthrosis at L4–L5 facet joints [79, 84, 85]. This was more prevalent at L4–L5 (from 45.1% to 79%), followed by L5–S1 (from 38.2% to 59%), and finally L3–L4 (from 30.6% to 72%) [79, 84]. These reports support the fact that the degenerative lumbar stenosis is related to the more mobile segments (L3–L4, L4–L5) of the lumbar spine [86, 87]. On the frequency of occurrence on the right or left side, an equal

With respect to gender, higher prevalence has been reported in males, and apparently, there is no significant difference in relation to race [79, 80]. However, in image studies using CT scans and planar radiography, women have been shown to have a higher prevalence of lumbar

Moreover, the body mass index (BMI) has been found to be associated with an increased prevalence of cervical facet OA [83] and, even more, of the lumbar region [12, 82]. In this respect, the risk of lumbar facet OA is almost three times higher in overweight individuals

Another risk factor for facet OA is the anatomy of the spine; for example, the changes in the orientation of the articular facet and facet joint asymmetry or "tropism" [89, 90, 91, 92] and simultaneously the disc-height narrowing represents the risk of contracting the disease [82]. Similarly, other factors such as the poor quality of the extensor muscles have been associated

Although there is no standard definition of disc degeneration (DD), it is considered the product of the degradation and remodeling of the ID and adjacent vertebrae, with adaptive changes and/or consequential damage induced by physical load. Radiographic studies, particularly

) and five times more associated with obesity (BMI 30–35 kg/m2

) [12].

), in com‐

facet OA and its progression are dependent on age [81, 83].

parison with the normal-weight reference group (BMI ≤ 25 kg/m<sup>2</sup>

distribution has been reported [88].

facet OA than men [12].

(BMI 25–30 kg/m2

with facet OA of L4–L5 [93].

**3.2. Disc degeneration**

#### **4.1. Mechanical response and degeneration**

DD is the manifestation of damage set caused by heavy physical load, posture or improper movement, and vibration. Therefore, it is extremely important to know the mechanical consequences of spinal motion segments under conditions of cyclic load, load magnitude, and frequency [105]. The investigations on degenerative mechanisms have been greatly supported in numerical models of the disc in animals, such as a finite element model [106]. One of the main advantages of the finite element model is the ability to parametrically manipulate one input factor and evaluate the resulting effects. These models have improved gradually, with the inclusion of poroelastic material properties of the motion segment facilitating the evalua‐ tion of physiological parameters related to cyclic load [107].

The application of the models in the evaluation of DD include analysis of disc geometry and mechanical properties of the nucleus, changes in permeability, porosity, and water content. The decreased content of fluid that occurs in degenerative processes is known to affect not only the nucleus pulposus but also the annulus matrix resulting in disc stiffness [106, 108, 109]. Poroelastic finite models have allowed evaluation of the effect on the strain-dependent permeability and osmotic potential in cyclic compression and expansion [110, 111]. These studies have shown time-dependent deformation of a lumbar motion segment subjected to multiple creep-compression-expansion loads. Another study that used an asymmetric discbody-disc poroelastic finite element model has shown that sustained compression maintains tensile stresses in the outer portion of the annulus but not in the middle and inner regions [108]. This correlates with the progressive disruption of the annulus fibrosus observed in vivo as well as the increase in apoptosis and the consequent decrease of cellularity. Similarly, other authors have demonstrated changes in the density and distribution of electric charges in healthy versus degenerated discs, which induced stress, water loss, and nutritional implica‐ tions [59, 112].

A poroelastic finite element model determines the interaction of fluid with the proteoglycans in the nucleus. Using this model, it has been shown that normal discs are much more deform‐ able that discs degenerate in response to cyclic load. The loss of healthy disc height in load cycle at a maximum load of 2000 N varied between 2.5 mm and 4.5 mm as opposed to between 1.0 mm and 1.8 mm in the degenerated disc [107, 113]. Similarly, stiffness of the disc was shown to be inversely proportional to the load cycles, while under higher compression loads (3000 N), loss of healthy disc height was demonstrated in 48% in comparison with 40% in degener‐ ated discs [108].

Additionally, the poroelastic finite element model can predict the evolution of disc failure. A previous study showed that disc failure is propagated when the elastic modulus is decreased and the rate of disc failure associated with increase load was greater than that due to the decrease in elastic modulus [107].

#### **4.2. Intervertebral disc aging and degeneration**

DD is a process related to physiological conditions, such as aging in most asymptomatic individuals, and is associated with pathological processes involving pain and disability. The definition of DD has not been fully established; two possibilities have emerged: one in which degenerative disc changes correspond to premature aging, and the other in which there is similarity between DD: and age changes but at an accelerated rate [114, 115]. It is possible that changes in the spine associated with aging are genetically predetermined and/or are associated with exposure to heavy mechanical forces throughout life. Independent of the trigger mech‐ anism, degenerative changes begin with biochemical alterations, followed by structural changes of the spinal functional units [116].

The notochordal cells constitute the primordium of the nucleus during the development of the ID and generally decrease in number rapidly after birth [117, 118]. Gradually, cellularity of the nucleus pulposus is replaced by chondrocyte-like cells, which may originate and migrate from the cartilaginous endplate and inner annulus [119]. Apparently, the Fas-mediated apoptosis plays an important role in this process [120]. The notochordal cells synthesize more proteo‐ glycans than chondrocytes and might be responsible in maintaining the fluid gelatinous nucleus pulposus [121]. Due to the reduction of these cells, the nucleus pulposus becomes more solid cartilage, which also decreases the signal intensity on MRI. On cell density, some studies have suggested an increase in the proportion of cells in the inner annulus fibrosus and the nucleus pulposus [122, 123].

The normal ID maintains a balance between synthesis and degradation of EM components, but it is well known that the age-related early degenerative changes are loss of aggrecan, collagen, and water in the nucleus pulposus. In addition, the release of molecules, including proinflammatory cytokines such as interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α) [124, 125], increases the synthesis of metalloproteinases [126] contributes also to this degen‐ erative process. However, it is noteworthy that annulus cells in the early stages of DD synthesize a larger amount of proteoglycans and collagen, probably in response to a repair process [127]. The progress of degeneration involves the reduction of production of most of the molecules of the EM, except for biglycan and fibronectin [127].

Generally, degenerative disc changes are of multifactorial origin. One of the most important determinants is the nutritional deficit secondary to decreased blood supply to the endplate. Apparently, this process could start early in the second decade of life [97]. Vascularization and innervation of the disc are also associated with aging and degeneration. Similarly, inflamma‐ tory cells and macrophages have been identified in degenerated discs and have been found responsible in the synthesis of cytokines and proteases by endogenous cells and by the vascular cells of the invading vessels [128]. The painful sensation that accompanies DD associated with aging is due to the presence of nociceptive nerve fibers in the annulus and inner nucleus [129].

Additionally, macroscopic changes can be observed, as well as concentric fissures and radial tears in the annulus from the third and fourth decades of life [130]. These modifications are due to increased synthesis of metalloproteinases that occurs as a result of the ad‐ vance of age [130].

Besides, cell viability is also affected in aging due to thinning and calcification of the endplates, which impair the nutritional contribution of the disc [131]. Also, other factors like stress induced by overload or nonphysiological static compression and cyclic stretching are involved in cell death and DD [65, 74, 108, 132, 133, 134]. Furthermore, there are reports of cell prolif‐ eration in human degenerative discs especially in areas where cell clusters are integrated [135].

#### **4.3. Facet articular OA and aging**

The decreased content of fluid that occurs in degenerative processes is known to affect not only the nucleus pulposus but also the annulus matrix resulting in disc stiffness [106, 108, 109]. Poroelastic finite models have allowed evaluation of the effect on the strain-dependent permeability and osmotic potential in cyclic compression and expansion [110, 111]. These studies have shown time-dependent deformation of a lumbar motion segment subjected to multiple creep-compression-expansion loads. Another study that used an asymmetric discbody-disc poroelastic finite element model has shown that sustained compression maintains tensile stresses in the outer portion of the annulus but not in the middle and inner regions [108]. This correlates with the progressive disruption of the annulus fibrosus observed in vivo as well as the increase in apoptosis and the consequent decrease of cellularity. Similarly, other authors have demonstrated changes in the density and distribution of electric charges in healthy versus degenerated discs, which induced stress, water loss, and nutritional implica‐

A poroelastic finite element model determines the interaction of fluid with the proteoglycans in the nucleus. Using this model, it has been shown that normal discs are much more deform‐ able that discs degenerate in response to cyclic load. The loss of healthy disc height in load cycle at a maximum load of 2000 N varied between 2.5 mm and 4.5 mm as opposed to between 1.0 mm and 1.8 mm in the degenerated disc [107, 113]. Similarly, stiffness of the disc was shown to be inversely proportional to the load cycles, while under higher compression loads (3000 N), loss of healthy disc height was demonstrated in 48% in comparison with 40% in degener‐

Additionally, the poroelastic finite element model can predict the evolution of disc failure. A previous study showed that disc failure is propagated when the elastic modulus is decreased and the rate of disc failure associated with increase load was greater than that due to the

DD is a process related to physiological conditions, such as aging in most asymptomatic individuals, and is associated with pathological processes involving pain and disability. The definition of DD has not been fully established; two possibilities have emerged: one in which degenerative disc changes correspond to premature aging, and the other in which there is similarity between DD: and age changes but at an accelerated rate [114, 115]. It is possible that changes in the spine associated with aging are genetically predetermined and/or are associated with exposure to heavy mechanical forces throughout life. Independent of the trigger mech‐ anism, degenerative changes begin with biochemical alterations, followed by structural

The notochordal cells constitute the primordium of the nucleus during the development of the ID and generally decrease in number rapidly after birth [117, 118]. Gradually, cellularity of the nucleus pulposus is replaced by chondrocyte-like cells, which may originate and migrate from the cartilaginous endplate and inner annulus [119]. Apparently, the Fas-mediated apoptosis

tions [59, 112].

ated discs [108].

decrease in elastic modulus [107].

106 Osteoarthritis - Progress in Basic Research and Treatment

**4.2. Intervertebral disc aging and degeneration**

changes of the spinal functional units [116].

The degenerative processes associated with age or other factors may also affect the facet joint indirectly. These changes are usually associated with variations in the load surfaces of the joint [136]. This can manifest macroscopically with osteophytes and bone overgrowth with stenosis of the foramen, lateral and central spinal canal [85, 88, 90, 137, 138, 139]. Previous studies reported that the subchondral cortex shows no significant morphological variation in different spinal levels as a result of aging, suggesting that it may be due to a slower rate of remodeling. Moreover, the fraction of bone volume and trabecular thickness decrease is more frequent in women than in men during aging [140].

It is generally accepted that degenerative facet changes are preceded by DD [137, 141]. We already mentioned that the consequences of DD include segmental instability and increase in facet load, which could induce joint subluxation and damage the cartilage surface. The changes in the cartilage are characterized by progressive erosion and subchondral bone sclerosis. Degenerative changes of facet articular are identical to OA seen in other synovial articulations. In addition to facet hypertrophy, apophyseal misalignment and osteophyte formation may narrow the spinal canal. Also, involvement of the triple articulation can influence degenerative spondylolisthesis and scoliosis [116].

Bone also undergoes sclerosis with consequent redistribution of loads, which may prog‐ ress and induce bone remodeling and subsequent rotatory deformities of the posterior elements [142].

#### **5. Spinal pain**

#### **5.1. Lumbar facet syndrome and cervical facet pain**

The facet joints are often associated with neck pain and LBP. The mechanical painful stimuli have been detected in sensory fibers, nociceptive endings, sensory afferent nerve endings, and types III/A and IV/C fibers located in the joint capsule, ligaments, periosteum, and subchondral bone [143, 144].

Neurophysiological studies have shown the involvement of small-diameter sensory neurons of the capsule, facet sensory neurons during inflammation and the effect of substance P in lumbar facet pain [145, 146, 147]. Furthermore, it has been demonstrated that substance P is also contained in nerve endings of subchondral bone in patients with facet OA [148].

The prevalence of cervical facet pain has been reported in about 55% of patients with chronic nonspecific pain [149]. Previous studies have suggested that the cervical facet pain signals are derived from the capsule, where the immunoreactivity of substance P and calcitonin generelated peptides has been demonstrated [150]. Several mechanisms have been proposed in facet joint injury including facet-joint impingement, synovial pinching, and strain injury to the capsule [151, 152, 153, 154, 155]. In this regard, it was deduced that noxious and trigger nociceptive discharges from the capsule are transmitted to the central nervous system for pain sensation [155]. This response was seen not only as a result of the injury but also secondary to high-magnitude mechanical stimuli such as tension, compression, and rotation. This persistent discharge was related to nerve or capsular injury with the consequent release of inflammatory mediators, which could stimulate signaling pathways of pain in the spinal cord by central sensitization [156, 157].

#### **5.2. Disc pain**

Moreover, the fraction of bone volume and trabecular thickness decrease is more frequent in

It is generally accepted that degenerative facet changes are preceded by DD [137, 141]. We already mentioned that the consequences of DD include segmental instability and increase in facet load, which could induce joint subluxation and damage the cartilage surface. The changes in the cartilage are characterized by progressive erosion and subchondral bone sclerosis. Degenerative changes of facet articular are identical to OA seen in other synovial articulations. In addition to facet hypertrophy, apophyseal misalignment and osteophyte formation may narrow the spinal canal. Also, involvement of the triple articulation can influence degenerative

Bone also undergoes sclerosis with consequent redistribution of loads, which may prog‐ ress and induce bone remodeling and subsequent rotatory deformities of the posterior

The facet joints are often associated with neck pain and LBP. The mechanical painful stimuli have been detected in sensory fibers, nociceptive endings, sensory afferent nerve endings, and types III/A and IV/C fibers located in the joint capsule, ligaments, periosteum, and subchondral

Neurophysiological studies have shown the involvement of small-diameter sensory neurons of the capsule, facet sensory neurons during inflammation and the effect of substance P in lumbar facet pain [145, 146, 147]. Furthermore, it has been demonstrated that substance P is

The prevalence of cervical facet pain has been reported in about 55% of patients with chronic nonspecific pain [149]. Previous studies have suggested that the cervical facet pain signals are derived from the capsule, where the immunoreactivity of substance P and calcitonin generelated peptides has been demonstrated [150]. Several mechanisms have been proposed in facet joint injury including facet-joint impingement, synovial pinching, and strain injury to the capsule [151, 152, 153, 154, 155]. In this regard, it was deduced that noxious and trigger nociceptive discharges from the capsule are transmitted to the central nervous system for pain sensation [155]. This response was seen not only as a result of the injury but also secondary to high-magnitude mechanical stimuli such as tension, compression, and rotation. This persistent discharge was related to nerve or capsular injury with the consequent release of inflammatory mediators, which could stimulate signaling pathways of pain in the spinal cord by central

also contained in nerve endings of subchondral bone in patients with facet OA [148].

women than in men during aging [140].

108 Osteoarthritis - Progress in Basic Research and Treatment

spondylolisthesis and scoliosis [116].

**5.1. Lumbar facet syndrome and cervical facet pain**

elements [142].

**5. Spinal pain**

bone [143, 144].

sensitization [156, 157].

Degenerative spinal disease is the condition most frequently associated with chronic LBP, particularly in older adults. As a definition, degenerative spinal disease includes DDD and degenerative facet disease or facet OA [158]. A study for the purpose analyze and compare the radiographic severity of DDD and facet degeneration of the lumbosacral spine in adult subjects with and without chronic LBP showed no association between them. This was despite the fact that the highest radiographic severity scores were associated with the presence of pain [159]. Usually, DD may result in radicular pain secondary to stenosis and nerve-root or cauda equina irritation, and discogenic pain derived from disc lesion [160].

Animal studies of healthy IDs have demonstrated the presence of mechanoreceptors in the outer portion of the annulus fibrosus. These nerve fibers correspond to small myelinated (group-III or A-delta fibers) and unmyelinated (group-IV or C fibers) fibers [161, 162, 163]. These fibers are classified into those containing neuropeptides, which express substance P and calcitonin gene-related peptides [164], and nociceptors fibers related to inflammatory pain. These fibers are also dependent on nerve growth factor and have high affinity with the tyrosine kinase A (TrkA) receptor [165]. Discal nerve fibers generally exhibit afferent axons, and cell bodies are located in the dorsal root ganglia [166].

#### **6. System grading in DD and facet joint degeneration**

Currently, MRI is considered as the gold standard in imaging of the spine; however, the diagnosis of facet OA remains a challenge for clinicians. For this purpose, different methods have been used such as the planar X-ray, CT and MRI scans, dynamic bending films, and planar radionuclide bone scanning [167].

Usually, the degree of DD is determined by macroscopic observation on MRI. Comparatively, facet OA may not be evaluated with precision by MRI as with CT scans [168, 169]. Commonly, conventional radiography (X-ray films) is used in the evaluation of arthritic changes of the spine, although CT shows joints with better resolution [170, 171]. It has been reported that CT can show the axial plane of the facet joint and the osteoarthritic changes with precision [171]. However, MRI provides axial and sagittal images of the facet, which are useful in assessing degenerative spinal joint disease [168]. Several studies have reported accuracy in the evalua‐ tion of the facet OA with MRI at the rate of between 93% and 95% [85, 172]. So far, it has been accepted that MRI is a useful method in the assessment of OA of the lumbar facet joints.

Different scoring systems have been described in evaluating the disc and facet degeneration. A previous study recommends intraobserver and interobserver reliability tests in the evalua‐ tion of lumbar degenerative changes [173]. One of these systems used lateral radiographic projections and was easy to apply [174]. However, one that used MRI showed high feasibility [175]. Comparatively, other systems cannot be applied to patients and have been used to evaluate DD in vitro based on detailed morphological studies [97, 176]. In cervical DD, a system based on lateral radiographs and easy to implement was the only one recommended [177].

Regarding grading systems for lumbar facet joint degeneration, recommendations were based solely on CT [170] or CT and MRI systems [178]. Differentially in cervical facet joint degeneration, a system based on lateral radiographs was recommended [177].

Lumbar DD is classified into five grades according to macroscopic characteristics such as fibrosis, mucinous degeneration, erosion of cartilage endplate, and osteophyte formation on sagittal sections [176]. Histologically, lumbar DD on sagittal paraffin sections contains parameters such as cell proliferation, mucinous degeneration, cell death, tear and cleft formation, and disc granular changes. Additional features include disorganization and cracks of cartilage, microfractures, bone neoformation, and endplate sclerosis [97].

Radiographically, a method for assessing the presence and severity of lumbar DD is based on joint space narrowing, anterior and posterior osteophyte formation, and subchondral sclerosis [174]. However, the best accepted grading system is based on the characteristics of the degenerative lumbar disc on MRI, such as the distinction of nucleus and annulus, the signal intensity, and height of ID [175]. Moreover, grading of lumbar facet joint degeneration appreciates the joint space narrowing, sclerosis, hypertrophy, and osteophyte formation on oblique conventional radiographs and CT scans [170] or CT and MRI scans [178].

Categorization of cervical DD is based on plain radiography and includes parameters such as osteophytosis, disc space narrowing, and sclerosis of vertebral plates [177]. Furthermore, grading of cervical facet joint degeneration on lateral radiographs determines the presence of osteophytes on the articular margins of facets of apophyseal joints and sclerosis [177].

Additionally, radionuclide bone scintigraphy with single photon emission CT (SPECT) has been used to detect microcalcification due to increased osteoblastic activity [179, 180]. More recently, it was reported that the hybrid SPECT/CT imaging identifies potential chronic spinal pain generators in 92% of cervical spine scans and 86% of lumbar spine scans [181].

Figures 2 and 3 show the MRI results of patients with cervical and lumbar OA.

#### **7. Inflammatory cytokines and degenerative lumbar spinal disease**

It is known that OA is associated with facet joint pain. The generation mechanisms of pain could be due to mechanical stress and joint instability or misalignment that often accompany DD and aging. In this regard, the presence of inflammatory mediators such as prostaglandins in facet joints of patients with lumbar spinal degenerative disorders were found [182]. These findings suggest that chemical factors besides mechanical factors arising from the facet joint could be related to pain in OA [183, 184, 185].

Another study demonstrated increase in the concentration of IL-6 in the synovium and cartilage of the facet joint by CLEIA method (Chemiluminescent Enzyme Immunoassay). The tissues analyzed in this study were obtained from patients with disc herniation and lumbar spinal stenosis [186]. The role of IL-6 in the spinal joint disease is controversial; it can facilitate the inflammation together with IL-1β and TNF-α in the early stages of the immune reaction, or may be involved in autoimmune states producing antibodies or act as an anti-inflammatory cytokine. According to these assertions, the authors proposed that IL-6 induces continuous local inflammation caused by mechanical stress on the facet joint [186]. Similarly, a significant increase was detected in IL-1β in patients with lumbar spinal canal stenosis than the lumbar herniated disc, which correlated with higher scores on scales of leg pain [187]. More recently, the overexpression of MMP-1 induced by IL-1β was revealed, suggesting an important role in the inflammation associated with lumbar facet joint degeneration [188].

Regarding grading systems for lumbar facet joint degeneration, recommendations were based solely on CT [170] or CT and MRI systems [178]. Differentially in cervical facet joint

Lumbar DD is classified into five grades according to macroscopic characteristics such as fibrosis, mucinous degeneration, erosion of cartilage endplate, and osteophyte formation on sagittal sections [176]. Histologically, lumbar DD on sagittal paraffin sections contains parameters such as cell proliferation, mucinous degeneration, cell death, tear and cleft formation, and disc granular changes. Additional features include disorganization and cracks

Radiographically, a method for assessing the presence and severity of lumbar DD is based on joint space narrowing, anterior and posterior osteophyte formation, and subchondral sclerosis [174]. However, the best accepted grading system is based on the characteristics of the degenerative lumbar disc on MRI, such as the distinction of nucleus and annulus, the signal intensity, and height of ID [175]. Moreover, grading of lumbar facet joint degeneration appreciates the joint space narrowing, sclerosis, hypertrophy, and osteophyte formation on

Categorization of cervical DD is based on plain radiography and includes parameters such as osteophytosis, disc space narrowing, and sclerosis of vertebral plates [177]. Furthermore, grading of cervical facet joint degeneration on lateral radiographs determines the presence of osteophytes on the articular margins of facets of apophyseal joints and sclerosis [177].

Additionally, radionuclide bone scintigraphy with single photon emission CT (SPECT) has been used to detect microcalcification due to increased osteoblastic activity [179, 180]. More recently, it was reported that the hybrid SPECT/CT imaging identifies potential chronic spinal

pain generators in 92% of cervical spine scans and 86% of lumbar spine scans [181].

**7. Inflammatory cytokines and degenerative lumbar spinal disease**

It is known that OA is associated with facet joint pain. The generation mechanisms of pain could be due to mechanical stress and joint instability or misalignment that often accompany DD and aging. In this regard, the presence of inflammatory mediators such as prostaglandins in facet joints of patients with lumbar spinal degenerative disorders were found [182]. These findings suggest that chemical factors besides mechanical factors arising from the facet joint

Another study demonstrated increase in the concentration of IL-6 in the synovium and cartilage of the facet joint by CLEIA method (Chemiluminescent Enzyme Immunoassay). The tissues analyzed in this study were obtained from patients with disc herniation and lumbar spinal stenosis [186]. The role of IL-6 in the spinal joint disease is controversial; it can facilitate the inflammation together with IL-1β and TNF-α in the early stages of the immune reaction,

Figures 2 and 3 show the MRI results of patients with cervical and lumbar OA.

could be related to pain in OA [183, 184, 185].

degeneration, a system based on lateral radiographs was recommended [177].

110 Osteoarthritis - Progress in Basic Research and Treatment

of cartilage, microfractures, bone neoformation, and endplate sclerosis [97].

oblique conventional radiographs and CT scans [170] or CT and MRI scans [178].

Also were reported inflammatory chemical mediators such as prostaglandins and leukotrienes in facet cartilage and subchondral bone obtained from patients with degenerative lumbar spinal disorders. Here, it was suggested that these chemical mediators may be involved in inflammation and pain generation at the local lumbar facet joints [182].

**Figure 2.** A. Lateral cervical spine radiograph with decreased general bone density; correction of the cervical lordosis; vertebral platforms sclerosis; decrease in intervertebral spaces at C3–C4, C4–C5, and C5–C6; syndesmophytes; reduc‐ tion in diameter of intervertebral foramina at C2–C3, C3–C4, and C4–C5; decreased facet interface at C2–C3, C3–C4, and C4–C5; and spondylolisthesis at C4–C5. B. Anterior-posterior cervical spine radiograph with loss facet interface at C2–C3, C3–C4, and C4–C5; decreased vertebral space at C2–C3, C3–C4, and C4–C5. C. Sagittal MRI of cervical spine at stage T1 with vertebral platforms sclerosis, osteophyte formation, decreased height of IDs, and disc extrusion mainly in the intervertebral spaces at C4 and at C3–C4–C5. D. Sagittal MRI in T2 phase with decrease caliber medullary canal by the presence of posterior osteophytes, hypertrophy of posterior longitudinal ligament, and ID extrusion. E–F. MRI axial slices in T2 phase with reduced caliber of the cervical canal and compression of the spinal cord.

**Figure 3.** A. Antero-posterior radiograph with left lumbar scoliosis, plastic deformation of the last three lumbar verte‐ brae, vertebral platforms sclerosis, osteophytes, and decreased height of intervertebral spaces. B. Lateral radiograph of lumbar spine with spinal platform sclerosis, osteophytes, and reduced height of the L3–L4, L4–L5, and L5–S1 IDs. C. Sagittal MRI of lumbar spine in T1 phase with sclerosis of the vertebral platforms, Modic changes, osteophytes, de‐ creased height of IDs, and disc protrusion at L3–L4, L4–L5, and L5–S1 with narrow lumbar canal. D. Sagittal MRI of lumbar spine in T2 phase with vertebral platform sclerosis; osteophytes; decreased height of IDs; disc protrusion at L3– L4, L4–L5, and L5–S1 with lumbar canal narrowing; decreased spaces at L3–L4 and L4–L5; and bulging of IDs at L2–L3 and L5–S1. E–F. MRI axial slices of lumbar spine facet degenerative changes and hypertrophy of the ligamentum fla‐ vum and lumbar stenosis at different levels.

#### **8. Angiogenesis, calcification, and programmed cell death in DD**

As described above, degenerative changes of the ID involve processes such as neovasculari‐ zation, calcification, and cell death. Angiogenesis has been described in degenerated and herniated discs [189]. A degenerative disc is defined as a disc protruding into the spinal canal or neural foramens resulting in compression of the nerve roots [189]. The herniated nucleus pulposus develops fibrotic and angiogenic reactions [190, 191]. This process involves factors such as TGF-β, TNF-α, VEGF, MMP-1, and MMP-3 [191, 192].

Similarly, the intradiscal calcification has been significantly correlated with DD [193]. Disc calcification occurs in the annulus, fibrocartilaginous plate, and nucleus pulposus that appears as amorphous deposits of calcium salts [194] asymptomatic in most cases. The frequency of degenerative disc calcification varies from 3.1% to 65% as assessed by microscopy and MRI [195, 196, 197]. Another study reported that microscopic calcification was significantly higher in degenerative discs than in those obtained from normal cadavers (54.4% vs. 6.7%), and it is also higher in Modic type III than in type I (95.0% and 13.0%, respectively). The same study also refers angiogenesis in degenerative discs (41.0%) and in calcified discs (59.2%) [198].

The etiology of disc calcification remains uncertain. Two possible mechanisms of calcification disc have been proposed: one in which inflammatory cytokines such as VEGF and MMPs released into the degenerative disc promote expression of osteopontin, induce differentiation of osteoprogenitor cells, and allow calcification; and the other through indirect mechanisms in which these molecular mediators promote angiogenesis, and this, in turn, stimulates macrophage infiltration, the formation of new osteoprogenitor cells, and finally the progres‐ sive calcification [198].

As we have mentioned in this review, in addition to mechanical and genetic factors, apoptotic cell death is another event type that contributes to the development of disc degeneration (DD) [74, 132, 134, 199, 200, 201, 202, 203]. Apoptosis in degenerative discs is described as that occurring through activation of the mitochondrial [74], death receptor [204], and the endo‐ plasmic reticulum pathway [205, 206].

The static axial compressive load [74, 108, 134], the static bending compressive load [133], the dynamic axial compressive load [65, 207], and the imbalance of dynamic and/or static forces of the spine [199] have been considered in programmed cell death (PCD) on degenerative disc. Load effects increase lactate concentration, decline oxygen tension, decrease nutrient level, reduces tissue permeability and secondarily the water content [208, 209]. Biomechanical stimuli such as serum deprivation [210], nitric oxide [201], lipid peroxidation [132], hypoxiainducible factor-1α [211], and even normal oxygen concentrations [212] have also been involved in the induction and increase of PCD.

The death of disc cells has been reported to be significant as age increases [97]. Elsewhere in the body, it is well established that apoptotic cells are removed by phagocytosis; however, macrophages or phagocytes are not cells that are normally present on the disc. In vitro studies have shown that nucleus pulposus cells are able to perform, as well as competent phagocytes and stimulate phagocytosis [213].

#### **9. Conclusion**

**Figure 3.** A. Antero-posterior radiograph with left lumbar scoliosis, plastic deformation of the last three lumbar verte‐ brae, vertebral platforms sclerosis, osteophytes, and decreased height of intervertebral spaces. B. Lateral radiograph of lumbar spine with spinal platform sclerosis, osteophytes, and reduced height of the L3–L4, L4–L5, and L5–S1 IDs. C. Sagittal MRI of lumbar spine in T1 phase with sclerosis of the vertebral platforms, Modic changes, osteophytes, de‐ creased height of IDs, and disc protrusion at L3–L4, L4–L5, and L5–S1 with narrow lumbar canal. D. Sagittal MRI of lumbar spine in T2 phase with vertebral platform sclerosis; osteophytes; decreased height of IDs; disc protrusion at L3– L4, L4–L5, and L5–S1 with lumbar canal narrowing; decreased spaces at L3–L4 and L4–L5; and bulging of IDs at L2–L3 and L5–S1. E–F. MRI axial slices of lumbar spine facet degenerative changes and hypertrophy of the ligamentum fla‐

**8. Angiogenesis, calcification, and programmed cell death in DD**

such as TGF-β, TNF-α, VEGF, MMP-1, and MMP-3 [191, 192].

As described above, degenerative changes of the ID involve processes such as neovasculari‐ zation, calcification, and cell death. Angiogenesis has been described in degenerated and herniated discs [189]. A degenerative disc is defined as a disc protruding into the spinal canal or neural foramens resulting in compression of the nerve roots [189]. The herniated nucleus pulposus develops fibrotic and angiogenic reactions [190, 191]. This process involves factors

Similarly, the intradiscal calcification has been significantly correlated with DD [193]. Disc calcification occurs in the annulus, fibrocartilaginous plate, and nucleus pulposus that appears as amorphous deposits of calcium salts [194] asymptomatic in most cases. The frequency of degenerative disc calcification varies from 3.1% to 65% as assessed by microscopy and MRI

vum and lumbar stenosis at different levels.

112 Osteoarthritis - Progress in Basic Research and Treatment

Spinal OA is a condition characterized by failure in motion segments, usually as a result of exposure to heavy physical load and aging. By definition, this disease induces degenerative changes in the facet joints and the IDs. One of the predominant symptoms of spinal OA is neck pain and LBP syndrome, which involves prolonged disability and high care costs. However there is controversy whether the prevalence, severity, and imaging findings are related to the pain sensation. The fields of molecular biology and mechanobiology of the degenerative process also require research to understand the pathophysiological mechanisms that lead to it and, thus, be able to contribute in the development of regenerative medicine and techno‐ logical innovation with the improvement of prototypes for design of orthopedic components.

#### **Acknowledgements**

We thank M.D. Eulalio Elizalde Martínez, Chief of Orthopaedic Spine Surgery; M.D. Armando Fabio Ramos Guerrero, Spine Surgery Training of Hospital "Dr. Victorio de la Fuente Narváez", IMSS, Distrito Federal, Mexico; and M.D. Misael Vargas López of the Escuela Nacional de Medicina y Homeopatía-IPN for their valuable support in the preparation of this chapter. This work was supported by the SIP 20141449 project.

#### **Author details**

Elizabeth Pérez-Hernández1\*, Nury Pérez-Hernández2 and Ariel Fuerte-Hernández3

\*Address all correspondence to: perezheliza@aol.com

1 División de Educación e Investigación en Salud, UMAE "Dr. Victorio de la Fuente Narváez", Hospital de Ortopedia, Instituto Mexicano del Seguro Social, Distrito Federal, México

2 Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional, Distrito Federal, México

3 Escuela Superior de Ingeniería Mecánica y Eléctrica, Instituto Politécnico Nacional, Distrito Federal, México

#### **References**


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**Acknowledgements**

114 Osteoarthritis - Progress in Basic Research and Treatment

**Author details**

Federal, México

Federal, México

**References**

S230-5.

work was supported by the SIP 20141449 project.

Elizabeth Pérez-Hernández1\*, Nury Pérez-Hernández2

\*Address all correspondence to: perezheliza@aol.com

We thank M.D. Eulalio Elizalde Martínez, Chief of Orthopaedic Spine Surgery; M.D. Armando Fabio Ramos Guerrero, Spine Surgery Training of Hospital "Dr. Victorio de la Fuente Narváez", IMSS, Distrito Federal, Mexico; and M.D. Misael Vargas López of the Escuela Nacional de Medicina y Homeopatía-IPN for their valuable support in the preparation of this chapter. This

1 División de Educación e Investigación en Salud, UMAE "Dr. Victorio de la Fuente Narváez",

3 Escuela Superior de Ingeniería Mecánica y Eléctrica, Instituto Politécnico Nacional, Distrito

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### **Validation of Mechanical Hypothesis of hip Arthritis Development by HIPSTRESS Method**

Veronika Kralj-Iglič

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/59976

#### **1. Introduction**

Hip joint connects the upper part of the body to the lower limb. As in human (a bipodal) the motion derives from periodical extension of lower limbs, the one-limb support is a common body position attained in everyday life. Keeping the body in balance and performing the required activities by means of attaining particular body positions or motions and by activating particular muscles is the main function of the hip joint. When the load is transmitted to the supporting leg, the hip bears the body weight (aside from the weight of the supporting leg). Besides the weight the joint is affected also by forces exerted by the surrounding tissues (e.g. muscles, tendons, ligaments and fluids). As the human body is subject to laws of physics, it is therefore indicated that mechanical parameters such as forces and stresses can be connected to physiological and patophysiological processes in the hip joint.

Understanding of causes of the effects on development of the body was dramatically acceler‐ ated by the discovery of X-rays in 1895, which enabled imaging of inner body structures without cutting them. It was found that the lateral coverage of the femoral head with the acetabulum is an important parameter in predicting the development of hip cartilage degen‐ eration and hip osteoarthritis [1]. Besides providing new diagnostic technologies, physical methods contributed also to revealing mechanisms of disease development. According to the mechanical hypothesis, too high load of the hip was empirically considered as a cause for deterioration of the hip joint.

#### **2. Hip stress as a relevant biomechanical parameter**

Poor lateral coverage of the femoral head by the acetabulum was connected to smaller load bearing area and therefore larger contact stress on the hip cartilage and bones. To increase the

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

load bearing area and prevent early hip osteoarthritis, various operative techniques were suggested [2-9]. In these operations the load bearing area was increased by increasing the lateral coverage of the femoral head by the acetabular roof. However, proof for the mechanical hypothesis stating that unfavorable distribution of stress in the hip is connected to early hip osteoarthritis, requires a method for assessment of hip stress.

Hip stress was measured in vitro and in vivo by using different techniques (e.g. pressure sensitive film and instrumented prothesis) [10]. After a thorough study involving development of a special Austin Moore partial endoprothesis and its validation in vitro, a specimen was implanted into a patient [11]. Contact hip stress was recorded by electromagnetic signal deriving from piezoelectric transducers on the head of the prothesis. The signal was recorded by a coil placed arround the patient's thigh. The location of the particular transducer was distinguished by the frequency of the signal. The patient was followed during the rehabilita‐ tion and in different activities for several years. Measurements recorded nonuniform distri‐ bution of hip stress over the load bearing area. Peak stresses as high as 15 MPa were recorded in everyday activities (e.g. standing up from a cca 25 cm chair).

To assess biomechanical parameters, theoretical models were developed. Finite element method was used to predict stresses within hip bones [12]. According to this method the hip is imagined as composed of small elements which act one upon another according to laws of elastomechanics. Two dimensional and three dimensional models were elaborated. Taking into account the materials elastic constants, the relevant constraints and the load on the hip the values of stress subject to each element can be calculated. Calculation requires solving large systems of equations which became possible by development of powerful computers. Impor‐ tant general knowledge was obtained by this method as for example the effect of the bone stiffening and cartilage elastic modulus on the stress values and distribution. Dynamic effects were studied by measurements of the effect of the movement on the piezoelectric force plate and by recording the motion of the subject by the video camera in combination with mathe‐ matical model [13]. In the model, the body was divided into segments connected by joints. Muscle and tendon mechanics was taken into account. Resultant joint forces were calculated from intersegmental forces by solving the inverse dynamics problem.

However, these methods were not appropriate for clinical studies where large number of hips should be assessed within reasonable time and possibilities. Also, the elastic constants of the material composing hip and pelvis for a particular person are largely unknown. For clinical studies of disease specificity, analytically or almost analytically solvable models based on the individual hip geometry were found more appropriate [14-16]. The strength of these methods lies in appropriate abstractions and choices of a small number of relevant features that render the model simple enough to be transparent with respect to the effect of the parameters. Development of such methods enabled analysis of large populations of hips with different pathologies. The mechanical hypothesis claiming that elevated contact hip stress is a possible cause of hip osteoarthritis was tested on a large cohort of hips with idiopathic osteoarthrosis and compared to a population of »normal« hips [16]. It was found that »compressive stress is of minor importance with respect to the etiology of idiopathic osteoarthrosis of the hip joint«. The results of this thorough study seemed decisive and discouraging for further questioning the mechanical hypotehsis as regards osteoarthritis, but the effect of stress was further investigated by determination of stress in patients with congenital dislocation of te hip [17,18]. It was found that an integral of stress over time statistically significantly correlated with clinical status [18] and that the values of stress beyond a threshold of 2MPa and integrated stress beyond threshold of 10MPa-years were connected to poor clinical outcome [17].

#### **3. Method HIPSTRESS**

load bearing area and prevent early hip osteoarthritis, various operative techniques were suggested [2-9]. In these operations the load bearing area was increased by increasing the lateral coverage of the femoral head by the acetabular roof. However, proof for the mechanical hypothesis stating that unfavorable distribution of stress in the hip is connected to early hip

Hip stress was measured in vitro and in vivo by using different techniques (e.g. pressure sensitive film and instrumented prothesis) [10]. After a thorough study involving development of a special Austin Moore partial endoprothesis and its validation in vitro, a specimen was implanted into a patient [11]. Contact hip stress was recorded by electromagnetic signal deriving from piezoelectric transducers on the head of the prothesis. The signal was recorded by a coil placed arround the patient's thigh. The location of the particular transducer was distinguished by the frequency of the signal. The patient was followed during the rehabilita‐ tion and in different activities for several years. Measurements recorded nonuniform distri‐ bution of hip stress over the load bearing area. Peak stresses as high as 15 MPa were recorded

To assess biomechanical parameters, theoretical models were developed. Finite element method was used to predict stresses within hip bones [12]. According to this method the hip is imagined as composed of small elements which act one upon another according to laws of elastomechanics. Two dimensional and three dimensional models were elaborated. Taking into account the materials elastic constants, the relevant constraints and the load on the hip the values of stress subject to each element can be calculated. Calculation requires solving large systems of equations which became possible by development of powerful computers. Impor‐ tant general knowledge was obtained by this method as for example the effect of the bone stiffening and cartilage elastic modulus on the stress values and distribution. Dynamic effects were studied by measurements of the effect of the movement on the piezoelectric force plate and by recording the motion of the subject by the video camera in combination with mathe‐ matical model [13]. In the model, the body was divided into segments connected by joints. Muscle and tendon mechanics was taken into account. Resultant joint forces were calculated

However, these methods were not appropriate for clinical studies where large number of hips should be assessed within reasonable time and possibilities. Also, the elastic constants of the material composing hip and pelvis for a particular person are largely unknown. For clinical studies of disease specificity, analytically or almost analytically solvable models based on the individual hip geometry were found more appropriate [14-16]. The strength of these methods lies in appropriate abstractions and choices of a small number of relevant features that render the model simple enough to be transparent with respect to the effect of the parameters. Development of such methods enabled analysis of large populations of hips with different pathologies. The mechanical hypothesis claiming that elevated contact hip stress is a possible cause of hip osteoarthritis was tested on a large cohort of hips with idiopathic osteoarthrosis and compared to a population of »normal« hips [16]. It was found that »compressive stress is of minor importance with respect to the etiology of idiopathic osteoarthrosis of the hip joint«. The results of this thorough study seemed decisive and discouraging for further questioning

osteoarthritis, requires a method for assessment of hip stress.

132 Osteoarthritis - Progress in Basic Research and Treatment

in everyday activities (e.g. standing up from a cca 25 cm chair).

from intersegmental forces by solving the inverse dynamics problem.

Method HIPSTRESS was intended for analyses of large populations of hips. Its use is simple enough to be used by medical doctors and it requires some minutes to assess biomechanical parameters if the geometrical parameters of hips are known. The method consists of two mathematical models, one for determination of the resultant hip force in one legged stance [19] and the other for determination of contact hip stress distribution [20]. Both methods introduced some improvements with respect to previously developed models.

#### **3.1. Model HIPSTRESS for resultant hip force**

The model for the resultant hip force [19] considers the body to be composed of two segments: the lower segment (the loaded leg) and the upper segment (the rest of the body). The static equilibrium requires that the resultant of all external forces acting on each segment is zero and that the resultant of all external torques acting on each segment is zero. For the upper segment this requirement is

$$\left(\mathbf{W\_B} - \mathbf{W\_L}\right) + \sum \mathbf{F\_i} - \mathbf{R} = 0,\tag{1}$$

$$\mathbf{a} \times \left(\mathbf{W\_B} - \mathbf{W\_L}\right) + \sum \mathbf{r}\_i \times \mathbf{F\_i} = 0,\tag{2}$$

where **W**L is the weight of the loaded leg, **W**B is the body weight, **F***<sup>i</sup>* are forces of muscles that are active in the one-legged stance, **R** is the resultant hip force, **a** is the vector to the center of the mass of the body without the loaded leg and **r***<sup>i</sup>* are vectors to the origins of the muscle forces. The coordinate system for the upper body segment was chosen at the origin of the resultant hip force (the center of the feomral head) and therefore the torque due to this force is 0. The index *i* runs over all included muscles.

For the upper body segment, forces of 9 effective muscles are taken into account. Muscle forces are considered to act in straight lines between the muscle attachment points. The muscles that attach in larger areas were represented by several effective muscles. The effective muscles included in the model are gluteus minimus anterior, gluteus minimus middle, gluteus minimus posterior, gluteus medius anterior, gluteus medius middle, gluteus medius posterior, tensor fasciae lateae, piriformis and rectus femoris. The force of each muscle **F***<sup>i</sup>* was considered proportional to the muscle cross section area A*<sup>i</sup>* and average tension in the muscle f*<sup>i</sup>* ,

$$\mathbf{F}\_{i} = \mathbf{f}\_{i} \mathbf{A}\_{i} \left(\mathbf{r}\_{i} - \mathbf{r}\_{i}^{\prime}\right) \bigwedge \left(\mathbf{r}\_{i} - \mathbf{r}\_{i}^{\prime}\right)\big|\_{\prime}, i = 1, 2, 3, \ldots, 9. \tag{3}$$

**Figure 1.** Geometrical parameters of the hip and pelvis for the HIPSTRESS method, resultant hip force and hip stress distribution. From [23].

The forces and the torques have three dimensions, therefore the model consists of six equations (3 for equilibrium of forces and 3 for equilibrium of torques). For known origin and insertion points of the muscles, and known cross section areas the unknown quantities are the muscle tensions and three components of the resultant hip force **R**. Since there are 9 effective muscles and 3 components of the force **R,** there are 12 unknowns and 6 equations. To solve this problem, a simplification was introduced by dividing the muscles into three groups (anterior, middle, posterior) with respect to the position. It was assumed that the muscles in the same group have the same tension. This reduced the number of unknowns to 6 as required for solution of the complex of 6 equations. The muscle origin and insertion points and the muscle cross-section were taken from [21] and [22], respectively. The geometry of the individual patient was taken into account by correction of muscle attachment points according to the geometrical parame‐ ters obtained from the standard anteroposterior radiograph (the interhip distance (*l*), the height (*H*) and the width (*C*) of the pelvis, and the position on the greater trochanter relative to the centre of the femoral head (*x,z*)).

Results obtained with the HIPSTRESS model for resultant hip force showed that the force lies almost in the frontal plane of the body through both femoral heads. To further simplify the calculations it was assumed in most studies that the force lies in the frontal plane and is represented by its magnitude *R* and its inclination with respect to the vertical *ϑR*.

#### **3.2. Model HIPSTRESS for contact hip stress**

=f A – ' ' , 1,2,3,...,9. ( )( ) *<sup>i</sup> <sup>i</sup> <sup>i</sup> <sup>i</sup> ii <sup>i</sup>* **r r r** - = **rF** *i* (3)

**Figure 1.** Geometrical parameters of the hip and pelvis for the HIPSTRESS method, resultant hip force and hip stress

The forces and the torques have three dimensions, therefore the model consists of six equations (3 for equilibrium of forces and 3 for equilibrium of torques). For known origin and insertion points of the muscles, and known cross section areas the unknown quantities are the muscle tensions and three components of the resultant hip force **R**. Since there are 9 effective muscles and 3 components of the force **R,** there are 12 unknowns and 6 equations. To solve this problem, a simplification was introduced by dividing the muscles into three groups (anterior, middle, posterior) with respect to the position. It was assumed that the muscles in the same group have the same tension. This reduced the number of unknowns to 6 as required for solution of the complex of 6 equations. The muscle origin and insertion points and the muscle cross-section were taken from [21] and [22], respectively. The geometry of the individual patient was taken into account by correction of muscle attachment points according to the geometrical parame‐ ters obtained from the standard anteroposterior radiograph (the interhip distance (*l*), the height (*H*) and the width (*C*) of the pelvis, and the position on the greater trochanter relative to the

Results obtained with the HIPSTRESS model for resultant hip force showed that the force lies almost in the frontal plane of the body through both femoral heads. To further simplify the

distribution. From [23].

134 Osteoarthritis - Progress in Basic Research and Treatment

centre of the femoral head (*x,z*)).

Model for contact hip stress was thorougly described in a previous contribution [24] and will be only briefly described here. Femoral head is represented by a part of the sphere and acetabulum is represented by a part of the spherical shell. Articular spehere represents both, the acetabular sphere and the femoral head sphere. When unloaded, both representative spheres have the same origin. Between the spheres there is an elastic continuum representing cartilage. The cartilage is subject to Hooke's law. When loaded, the origin of the femoral head sphere is slightly displaced with respect to the acetabular sphere and the cartilage is squeezed. It is assumed that stress is proportional to displacement. Some points on the femoral head are moved closer to the acetabulum and some points are moved away from the acetabulum. The stress pole is the point on the articular sphere that corresponds to the closest approach of the femoral head and the acetabulum spheres. It is assumed that there is no friction in the tangential direction to the spherical surface, so the normal stress is the only relevant stress acting in the hip.

The base of the mathematical model is the cosine dependence of contact stress on the space angle between the position of the stress pole and the chosen point on the articular surface *γ* [16],

$$p = p\_0 \cos \chi \quad \text{ } \tag{4}$$

where *p*<sup>0</sup> is the value of stress at the pole. The essential contribution of the HIPSTRESS model for stress is the elaboration of the load bearing area. The choice of the coordinate system and the definition of the borders of the load bearing area renders the elaborated load bearing area always symmetric, regardless of the direction of the resultant hip force. The lateral boundary of the load bearing area is defined by intersection of the articular sphere and the plane which is inclined with respect to the sagittal plane for *ϑ*CE (the centre-edge angle of Wiberg). On the medial side the border is defined by the condition that the stress vanishes, i.e. the medial border is for the angle π/2 allienated from the stress pole. The connection between the resultant hip force and the hip stress distribution

<sup>=</sup> *<sup>p</sup>* , ò **R dA** (5)

where the integration is performed over the load bearing area, yields three equations for three unknowns: the angles defining the position of the hip stress pole (Θ and Φ) and the value of stress at the pole *p*0 [20,24]

$$
\Phi = \begin{array}{c} 0, \ \pi \end{array} \tag{6}
$$

$$\tan\left(\mathcal{G}\_{\mathbb{R}} + \Theta\right) = \cos^2\left(\mathcal{G}\_{\mathbb{CE}} \cdot \Theta\right) \left(\pi/2 + \mathcal{G}\_{\mathbb{CE}} - \Theta + \sin\left(2\left(\mathcal{G}\_{\mathbb{CE}} - \Theta\right)\right)/2\right),\tag{7}$$

$$p\_0 = \ \mathfrak{R}\text{sinc}\left(\mathfrak{G}\_{\mathbb{R}} + \Theta\right) \Big/ 2r^2 \cos^2\left(\mathfrak{G}\_{\mathbb{C}\mathbb{E}} - \Theta\right) \,,\tag{8}$$

where Φ is the azimuth coordinate of the stress pole on the articular surface. To determine stress at any point of the load bearing area, the solution of the nonlinear equation for the coordinate of the pole Θ Eq. (4) should be found. Re-arranging Eq.(7) by substitution [25]

$$\left\|\mathfrak{P}\mathbf{=x} - \left(\mathfrak{G}\_{\mathbb{R}} - \mathfrak{G}\_{\text{CE}}\right)\right\|\mathfrak{I} \tag{9}$$

transforms the nonlinear equation (7) into

$$\begin{aligned} &\tan\left(\left(\mathcal{S}\_{\mathbb{R}} + \mathcal{S}\_{\mathrm{CE}}\right) / 2 \cdot \mathbf{x}\right) = \\ &= \cos^2\left(\left(\mathcal{S}\_{\mathbb{R}} + \mathcal{S}\_{\mathrm{CE}}\right) / 2 \cdot \mathbf{x}\right) \left\langle \left(\pi/2 + \left(\mathcal{S}\_{\mathbb{R}} + \mathcal{S}\_{\mathrm{CE}}\right) / 2 \cdot \mathbf{x} + \sin\left(2\left(\left(\mathcal{S}\_{\mathbb{R}} + \mathcal{S}\_{\mathrm{CE}}\right)/2 \cdot \mathbf{x}\right)\right) / 2\right) \cdot \mathbf{x} \right\rangle \end{aligned} \tag{10}$$

It follows from Eq. (10) that the solution of Eq. (7) (i.e. the position of the stress pole depends solely on (*ϑ*R + *ϑ*CE)).

The integration of Eq.(5) is performed over the load bearing area. Stress is unevenly distributed over the load bearing area. It decreases towards the medial border while on the lateral side there are two possibilities, depending on the position of the hip stress pole. If the pole lies within the load bearing area, stress increases in the medial direction, reaches maximum and then decreases. The pole, being an abstract quantity that reflects the extent and the direction of the relative movement of the femoral head and the acetabulum upon loading, may however lie outside the load bearing area. In this case, stress monotonously decreases towards the medial border. The contact hip stress distribution is represented by the peak value of hip stress on the load bearing area (*p*max). If the pole lies in the weight bearing area the peak stress is equal to its value at the pole (*p*max = *p*0). If the stress pole lies outside the load bearing area, the peak stress is equal to the value of stress at the point of the load bearing area which is closest to the pole. Other parameters that represent stress distribution are the index of the stress gradient at the lateral acetabular rim (*G*p)

$$G\_{\rm p} = \cdot p\_0 / r \cdot \cos \mathcal{S}\_{\rm F} \tag{11}$$

where *ϑ*F is the functional angle of the load bearing,

$$
\Delta \mathcal{G}\_{\rm F} = \pi \Big/ 2 + \mathcal{G}\_{\rm CE} - \Theta \tag{12}
$$

and the load bearing *A*<sup>F</sup>

( ) ( ) ( ( ( )) ) <sup>2</sup>

<sup>0</sup> 3 sin 2 cos , *<sup>R</sup> CE pR r* =

= – 2 *x* (J J

(( ) ) ( ( ) ( (( ) )) )

It follows from Eq. (10) that the solution of Eq. (7) (i.e. the position of the stress pole depends

The integration of Eq.(5) is performed over the load bearing area. Stress is unevenly distributed over the load bearing area. It decreases towards the medial border while on the lateral side there are two possibilities, depending on the position of the hip stress pole. If the pole lies within the load bearing area, stress increases in the medial direction, reaches maximum and then decreases. The pole, being an abstract quantity that reflects the extent and the direction of the relative movement of the femoral head and the acetabulum upon loading, may however lie outside the load bearing area. In this case, stress monotonously decreases towards the medial border. The contact hip stress distribution is represented by the peak value of hip stress on the load bearing area (*p*max). If the pole lies in the weight bearing area the peak stress is equal to its value at the pole (*p*max = *p*0). If the stress pole lies outside the load bearing area, the peak stress is equal to the value of stress at the point of the load bearing area which is closest to the pole. Other parameters that represent stress distribution are the index of the stress gradient at

*x xx*

Jp

=cos 2 - /2 + + 2 - sin 2 /2 - 2 .

p0 F *G pr* = - cos

<sup>F</sup> CE = 2 + p JJ

J

(11)


R CE R CE R CE

+ ++

J J J

( ) ( ) 2 2

where Φ is the azimuth coordinate of the stress pole on the articular surface. To determine stress at any point of the load bearing area, the solution of the nonlinear equation for the coordinate of the pole Θ Eq. (4) should be found. Re-arranging Eq.(7) by substitution [25]

Jp

 J


+ Q - Q (8)

R CE -Q ) (9)

J

(10)

<sup>R</sup> CE CE CE tan = cos - 2 + sin 2

J


JQ

JQ

136 Osteoarthritis - Progress in Basic Research and Treatment

transforms the nonlinear equation (7) into

*x*

(( ) )

J

tan 2 + =

R CE

+

J

the lateral acetabular rim (*G*p)

where *ϑ*F is the functional angle of the load bearing,

2

solely on (*ϑ*R + *ϑ*CE)).

J J

$$A\_{\rm F} = 2r^2 \mathcal{G}\_{\rm F} \ . \tag{13}$$

An example of the stress distribution calculated by HIPSTRESS method is shown in Figure 2. The green line denotes the magnitude of the contact hip stress *p* in the frontal plane through centres of both articular spheres. The left hip has normal shape. The right hip is considerably deformed as the patient underwent in the childhood the Perthes disease. In the normal (left) hip the stress increases in the medial direction, reaches maximum and then decreases. The functional angle and the load bearing area are large. The pole lies within the load bearing area and the index of hip stress gradient is negative. In the deformed (right) hip the stress monot‐ onously decreases in the medial direction. The functional angle is small. The pole lies outside of the load bearing area and the index of hip stress gradient is positive. However, the load bearing area is not small in the deformed hip as the smaller functional angle is compensated by the larger radius of the articular sphere (Eq. (13)). Consequnetly, the peak stress is almost equal in both hips.

**Figure 2.** Distribution of hip stress on the load bearing area of a normal hip (left) and a hip after Legg-Calve-Perthes disease (right). The green line represents the magnitude of stress in the frontal plane through the centers of the femoral heads. The red line indicates the functional angle determining the load bearing area. From [26].

Figure 3 shows the peak hip stress (A) and the coordinate of the stress pole (B) in dependence on the sum of the angles (*ϑ*CE + *ϑR*). As this sum implies the solution of the system of equations representing the vector equation (5), the two angles can compensate each other. Smaller centre-

**Figure 3.** A: Peak contact stress as a function of the parameter (*ϑ*CE + *ϑR*). B: position of the stress pole as a function of the parameter (*ϑ*CE + *ϑR*). Lines were for a hypothetical hip with *R/W*B = 2.6 and *r* = 1.6 cm. Adapted from [25].

edge angle can be complemented by larger inclination of the resultant hip force to assure favorably large enough functional angle and load bearing area. It is expected that large centreedge angle means larger load bearing area and lower stress, but Figure 3 shows that for very large centre-edge angles combined with large inclinations of the resultant hip force, stress increases. Such situation would take place for large centre-edge angles (larger than 70 degrees) since the resultant hip force in the one-legged stance is usually smaller than 20 degrees. The above described model of resultant hip force describes the one-legged stance. The model for stress is general with respect to body position and enables calculation of hip stress distribution if the resultant hip force and some additional geometrical parameters of the hip are known. To obtain stress with the HIPSTRESS model for stress it is therefore not necessary that the resultant hip force is calculated by the HIPSTRESS model for the resultant hip force. The force can also be determined experimentally. However, it is necessary to know additional geomet‐ rical parameters such as the centre-edge angle *ϑ*CE and the radius of the articular sphere *r* (Figure 1).

#### **4. Computer program and nomograms for determination of the resultant hip force and peak contact stress in the HIPSTRESS method**

Computer program was developed to calculate the force **R** (its magnitude and inclination with respect to the vertical direction), the peak hip stress *p*max and the coordinate Θ of the pole. The program is available at http://physics.fe.uni-lj.si/projects/orthopaedic.htm Also, the nomograms were elaborated [27] for those who do not have a possibility to use the computer. They prove useful also as the rapid development of computer science requires compatibility of the program with hardware and other software which is not always available. The input data of the program HIPSTRESS are the geometrical parameters of the hip and pelvis that can be assessed from images of the hip and pelvis geometry (e.g. Xrays or magnetic resonance) (Figure 1): the interhip distance (*l*), the pelvic width (*C*), the pelvic height (*H*), the coordinates of the effective muscle attachment point on the greater trochanter in the coordinate system of the femur (*z* and *x*), the femoral head radius (*r*) and the centre-edge angle *ϑ*CE. To determine the resultant hip force and the contact hip stress distribution also the magnitude of the body weight *W*B should be known. However, besides the force and the stress, also the parameters normalized with respect to body weight are of interest, *R/W*B, *p*max/*W*B and *G*p/*W*B. These normalized parameters reflect the effect of the hip and pelvis geometry on the force and the stress.

Below we present nomograms for determination of resultant hip force and peak hip stress. The nomograms were calculated by using the computer programs. As there are many parameters that define the model it was not appropriate to consider all possible combina‐ tions of parameters but only those that yield the largest effect. The vertical position of the effective muscle attachment point on the greater trochanter was therefore not taken into account. Determination of the force is performed in two steps: determination of the inclination of the resultant hip force and determination of the magnitude of the resultant hip force. In determination of the magnitude, the effect of the pelvic width and height was disregarded as these parameters proved less important than the lateral extension of the greater trochanter and the interhip distance. To assess stress from the nomograms, we choose the *R/W*B(*l*/2) curve (Figure 4) pertaining to the combination of *z* and *l*/2 closest to the measured values and determine *R/W*B. Then we choose the *ϑR*(*l*/2) diagram (Figure 5) pertaining to the combination of *z, H* and *C* closest to the measured values and deter‐ mine *ϑR*. Using thus determined *R/W*B and *ϑR*, and the measured values of *ϑ*CE and *r* we choose the *p*max*r*<sup>2</sup> /*W*B(*ϑ*CE+*ϑR*) diagram pertaining to the relevant interval of (*ϑ*CE+*ϑR*) (Figure 6) and determine *p*max*r*<sup>2</sup> /*W*B. To assess *p*max/*W*B we divide the obtained value by *r*<sup>2</sup> . To obtain *p*max we multiply the obtained value with body weight *W*B.

edge angle can be complemented by larger inclination of the resultant hip force to assure favorably large enough functional angle and load bearing area. It is expected that large centreedge angle means larger load bearing area and lower stress, but Figure 3 shows that for very large centre-edge angles combined with large inclinations of the resultant hip force, stress increases. Such situation would take place for large centre-edge angles (larger than 70 degrees) since the resultant hip force in the one-legged stance is usually smaller than 20 degrees. The above described model of resultant hip force describes the one-legged stance. The model for stress is general with respect to body position and enables calculation of hip stress distribution if the resultant hip force and some additional geometrical parameters of the hip are known. To obtain stress with the HIPSTRESS model for stress it is therefore not necessary that the resultant hip force is calculated by the HIPSTRESS model for the resultant hip force. The force can also be determined experimentally. However, it is necessary to know additional geomet‐ rical parameters such as the centre-edge angle *ϑ*CE and the radius of the articular sphere *r*

**Figure 3.** A: Peak contact stress as a function of the parameter (*ϑ*CE + *ϑR*). B: position of the stress pole as a function of the parameter (*ϑ*CE + *ϑR*). Lines were for a hypothetical hip with *R/W*B = 2.6 and *r* = 1.6 cm. Adapted from [25].

(Figure 1).

138 Osteoarthritis - Progress in Basic Research and Treatment

For example, let us determine the resultant hip force and peak hip stress in a hip with parame‐ ters *l*/2=9.8 cm, *C*=5.6 cm, *H* =15.0 cm*, x* = 1.0 cm, *z* = 6.1 cm*, r* = 2.3 cm, *ϑ*CE = 32 degrees and *W*B=750 N. To assess *R/W*B we chose in Figure 4 the curve pertaining to *z* = 6 cm and obtain for *l*/2 = 10 cm the value *R/W*B = 2.7 (see dotted lines in Figure 4). Then we assess *ϑR*. The closest values of the parameters *l*/2, *H* and *z* are *l*/2 = 10cm, *H* =16cm and *z* = 6 cm while the value of *C* is between 5

**Figure 4.** Nomograms for determination of the magnitude of the resultant hip force. The dependence of the magnitude of the force *R* on half of the interhip distance *l*/2 for different lateral extensions of the greater trochanter *z*. Adapted from [27].

and 6 cm, therefore we can assess ϑR from panels h and k (Figure 5). The value of *ϑR* obtained from panel h (pertaining to *C* = 5 cm) is 8 degrees and the value of *ϑR* obtained from panel k (pertaining to *C* = 6cm) is 7 degrees (dotted lines in Figure 5). For *C* = 5.6 cm we can estimate that *ϑR* = 7.5 cm. Using the obtained values *ϑR* = 7.5 degrees, *R/W*B = 2.7 and the geometrical parame‐ ters *r* = 2.3 cm and *ϑ*CE = 32 degrees we assess *p*max*r*<sup>2</sup> /*W*B from Figure 6 (see dotted lines in Figure 6). The sum (*ϑ*CE+*ϑR*) is (32+7.5 ≅ 40) degrees, therefore we chose panel c and the curve pertain‐ ing to *z* = 6 cm. The obtained value of *p*max*r*<sup>2</sup> /*W*B is 1.75. Dividing this value by *r* = 2.3 cm yields *p*max/ *W*B =3300 m-2. For *W*B=750N the peak stress is finally *p*max=2.47 MPa.

It can be seen from the nomograms that smaller *C* and smaller *H* are biomechanically favorable as they yield larger *ϑR*. As regards *ϑR*, for large *z* smaller interhip distance is favorable and for smaller *z* larger interhip distance is more favorable, however, the dependencies are weak. Smaller lateral extension of the greater trochanter *z* and larger interhip distance *l* increase the magnitude of resultant hip force *R/W*B. Peak hip stress decreases with decreasing (*ϑ*CE + *ϑR*) up to (*ϑ*CE + *ϑR* = π/2) and increases with increasing *R/W*B [25]. For low hip stress (*ϑ*CE + *ϑR*) is large enough that the curve *p*max*r*<sup>2</sup> /*W*B(*ϑ*CE+*ϑR*) is almost flat, mostly on the account of large *ϑ*CE. Thus, the most favorable hip geometry has small interhip distance, large lateral extension of the grater trochanter, small pelvic width and height, large radius of the femoral head and large (but within limits) centre edge angle.

and 6 cm, therefore we can assess ϑR from panels h and k (Figure 5). The value of *ϑR* obtained from panel h (pertaining to *C* = 5 cm) is 8 degrees and the value of *ϑR* obtained from panel k (pertaining to *C* = 6cm) is 7 degrees (dotted lines in Figure 5). For *C* = 5.6 cm we can estimate that *ϑR* = 7.5 cm. Using the obtained values *ϑR* = 7.5 degrees, *R/W*B = 2.7 and the geometrical parame‐

**Figure 4.** Nomograms for determination of the magnitude of the resultant hip force. The dependence of the magnitude of the force *R* on half of the interhip distance *l*/2 for different lateral extensions of the greater trochanter *z*. Adapted

6). The sum (*ϑ*CE+*ϑR*) is (32+7.5 ≅ 40) degrees, therefore we chose panel c and the curve pertain‐

It can be seen from the nomograms that smaller *C* and smaller *H* are biomechanically favorable as they yield larger *ϑR*. As regards *ϑR*, for large *z* smaller interhip distance is favorable and for smaller *z* larger interhip distance is more favorable, however, the dependencies are weak. Smaller lateral extension of the greater trochanter *z* and larger interhip distance *l* increase the magnitude of resultant hip force *R/W*B. Peak hip stress decreases with decreasing (*ϑ*CE + *ϑR*) up to (*ϑ*CE + *ϑR* = π/2) and increases with increasing *R/W*B [25]. For low hip stress (*ϑ*CE + *ϑR*) is large

the most favorable hip geometry has small interhip distance, large lateral extension of the grater trochanter, small pelvic width and height, large radius of the femoral head and large

/*W*B from Figure 6 (see dotted lines in Figure

/*W*B is 1.75. Dividing this value by *r* = 2.3 cm yields *p*max/

/*W*B(*ϑ*CE+*ϑR*) is almost flat, mostly on the account of large *ϑ*CE. Thus,

ters *r* = 2.3 cm and *ϑ*CE = 32 degrees we assess *p*max*r*<sup>2</sup>

*W*B =3300 m-2. For *W*B=750N the peak stress is finally *p*max=2.47 MPa.

ing to *z* = 6 cm. The obtained value of *p*max*r*<sup>2</sup>

140 Osteoarthritis - Progress in Basic Research and Treatment

enough that the curve *p*max*r*<sup>2</sup>

from [27].

(but within limits) centre edge angle.

**Figure 5.** Nomograms for determination of the inclination of the resultant hip force. The dependence of the incli‐ nation of the force *ϑR* on half of the interhip distance *l*/2 for different lateral extensions of the greater trochanter *z*. a: *C*=3cm, *H*=12 cm, b: *C*=3cm, *H*=16cm, c: *C*=3cm, *H*=20 cm, d: *C*=4cm, *H*=12 cm, e: *C*=4 cm, *H*=16 cm, f: *C*=4 cm, *H*=20 cm, g: *C*=5 cm, *H*=12 cm, h: *C*=5 cm, *H*=16 cm, i: *C*=5cm, *H*=20 cm, j: *C*=6 cm, *H*=12 cm, k: *C*=6 cm, *H*=16 cm, l: *C*=6 cm, *H*=20 cm. Adapted from [27].

**Figure 6.** Nomograms for determination of the peak hip stress. The dependence of *p*max*r*<sup>2</sup> /*W*B on the sum of the angles (*ϑ*CE+*ϑR*) for different values of the resultant hip force *R/W*B. Due to large variation of the values with (*ϑ*CE+*ϑR*) panel a pertains to the range of (*ϑ*CE+*ϑR*) between 10 and 20 degrees, panel b pertains to the range between 20 and 30 degrees and panel c pertains to the range between 30 and 60 degrees. Adapted from [27].

#### **5. Biomechanical parameters in normal and dysplastic hips**

#### **5.1. Comparison between »normal« female and male hips**

The early population studies by the HIPSTRESS method considered »normal« hips. Geomet‐ rical parameters were assessed from standard anteroposterior radiograms retrieved from the archives. The pictures that showed no abnormalities in the hip region were included in the analysis; the patients had the pictures taken due to back pain. The exclusion criteria for participation in the study were clinical or radiographic signs of hip pathology, insufficient technical quality of the radiograph and incomplete presentation of pelvis on the radiograph. The first clinical study addressed differences between female and male hips [28]. Study of relevant geometrical parameters showed differences between 79 female and 21 male hips (Table 1). Female subjects had considerable and statistically significantly larger interhip distance and smaller femoral heads than male subjects, which is biomechanically unfavorable as it increases the magnitude of the peak stress. It was suggested that less favorable hip and pelvis geometry as regards hip stress »could be one of the reasons for the increased incidence of arthritis in women« [28]. However, stress was not actually calculated in that study.


**Table 1.** Median values of the geometrical parameters of 79 female and 21 male »normal« hips as determined in the first HIPSTRESS population study. Parameter *w* is the distance between the medial acetabular rims.

The differences between parameters (e.g., *x*) were calculated with respect to the mean value (*x*female + *x*male)/2. The statistical significance of the difference (the probability p) was calculated by using Mann-Whitney test. Instead of the interhip distance *l* the study considered the distance between the medial acetabular rims *w* as to avoid the effect of the femoral head size on the interhip distance. Adapted from [28].

#### **5.2. Comparison between »normal« and dysplastic hips**

**Figure 6.** Nomograms for determination of the peak hip stress. The dependence of *p*max*r*<sup>2</sup>

**5. Biomechanical parameters in normal and dysplastic hips**

and panel c pertains to the range between 30 and 60 degrees. Adapted from [27].

142 Osteoarthritis - Progress in Basic Research and Treatment

**5.1. Comparison between »normal« female and male hips**

(*ϑ*CE+*ϑR*) for different values of the resultant hip force *R/W*B. Due to large variation of the values with (*ϑ*CE+*ϑR*) panel a pertains to the range of (*ϑ*CE+*ϑR*) between 10 and 20 degrees, panel b pertains to the range between 20 and 30 degrees

The early population studies by the HIPSTRESS method considered »normal« hips. Geomet‐ rical parameters were assessed from standard anteroposterior radiograms retrieved from the archives. The pictures that showed no abnormalities in the hip region were included in the analysis; the patients had the pictures taken due to back pain. The exclusion criteria for participation in the study were clinical or radiographic signs of hip pathology, insufficient technical quality of the radiograph and incomplete presentation of pelvis on the radiograph. The first clinical study addressed differences between female and male hips [28]. Study of relevant geometrical parameters showed differences between 79 female and 21 male hips (Table 1). Female subjects had considerable and statistically significantly larger interhip

/*W*B on the sum of the angles

Stress was assessed in a population of dysplastic hips to test the hypothesis that it is higher than in »normal« hips and at the same time test the models [29]. The diagnosis of dysplasia was made on the basis of standard clinical and radiographic evaluation [29]. As it was found in the previous study [28] that female and male hips have considerably different geometrical and biomechanical parameters, the groups of female and male hips were considered sepa‐ rately, however the group of male hips was too small to allow for gender-matched comparison with »normal« hips, therefore the male hips were excluded from the analysis. 47 dysplastic female hips were included in the final analysis. The sample consisted of 20 right and 27 left hips, and the age of the subjects ranged from 18 to 52 years with a median of 33 years. The gender- and age-matched control group consisted of subjects who had had a radiograph taken of the pelvic region for reasons other than degenerative hip disease and in whom the pelvic radiograph had shown no signs of hip pathology. This group consisted of 36 hips, 18 right and 18 left, and the age of the subjects ranged from 18 to 41 years with a median age of 33 years. The results showed considerable and statistically very significant differences in most of geometrical parameters relevant for the HIPSTRESS model, in particular in resultant hip force and in peak contact stress (Table 2). The largest difference (80%) was in the centre-edge angle (Table 2). There was a 65% difference in contact hip stress; small centre-edge angle in dysplastic hips was to some extent compensated by larger radius of the femoral head and more favorable shape of the pelvis (smaller width and height). This study [28] was the first one that clearly showed on a relatively large cohort that contact hip stress is considerably higher (for about twice) in dysplastic female hips than in »normal« female hips. Also, it provided the first estimate of »normal« stress, i.e. the average value 3100 m-2. It was therefore suggested that the peak contact stress is a suitable parameter to assess risk for development of early arthritis of the hip.


SD: standard deviation. Adapted from [29].

**Table 2.** Average values of geometrical and biomechanical parameters of 47 dysplastic and 36 »normal« female hips.

It seems reasonable that in hips that were assigned dysplastic, mostly due to poor coverage of the femoral head by the acetabulum the area that bears resultant hip force is smaller. So it could be concluded that in these hips the reasons for development of arthritis are mechanical in a sense that too high stress causes degeneration of the tissues and inflammation of hip joint. In other words, hip arthritis in these cases is secondary to increased contact hip stress that reflects unfavorable geometry of the hips and pelvis.

It can be seen from Table 2 that the parameters for the above example assessed from nomo‐ grams were the average parameters of the »normal« hips. The peak stress obtained by using the nomograms (3300 m-2) differs from the average hip stress calculated by the computer program 3500±900 m-2 (Table 2) for about 6%. Yet it should be considered that the value 3500 m-2 was not obtained by calculating stress from the average parameters presented in Table 2 but by averag‐ ing stresses of hips included in the study.

#### **5.3. »Normal« hips**

Secondary arthritis caused by hip dysplasia represents a minor part in the population of hip arthritis, so the question was posed whether the hips with diagnosis »dysplasia coxae« are in fact the extreme subpopulation of hips with too high hip stress and that a considerable number of hips with diagnosis »idiopathic hip arthritis« are in fact poorly described hips with too high hip stress. This question has already been addressed previously and the negative answer given by the thorough study of large cohort [16] brought evidence against the mechanical hypothesis. However, decisive and transparent description of dysplastic hips by the HIPSTRESS method was an indication to reconsider the validity of the mechanical hypothesis also in idiopathic osteoarthritis.

showed on a relatively large cohort that contact hip stress is considerably higher (for about twice) in dysplastic female hips than in »normal« female hips. Also, it provided the first estimate of »normal« stress, i.e. the average value 3100 m-2. It was therefore suggested that the peak contact stress is a suitable parameter to assess risk for development of early arthritis of

(*l* ± SD) (cm) 20.8±1.2 19.5±0.9 6 <0.001 (*C* ± SD) (cm) 4.7±1.0 5.6±1.1 -17 <0.001 (*H* ± SD) (cm) 14.4±1.3 15.0±1.0 4 0.024 (*x* ± SD) (cm) 1.4±0.6 1.0±0.5 33 <0.001 (*z* ± SD) (cm) 5.6±0.6 6.1±0.6 8 <0.001 (*r* ± SD) (cm) 2.6±0.2 2.3±0.1 12 <0.001 (*ϑ*CE±SD) (degrees) 13±8 31±6 80 <0.001 *R/W*B± SD 3.1±0.3 2.7±0.1 14 <0.001 (*ϑR* ± SD) (degrees) 8±2 8±1 0 0.60 (*p*max/*W*B ± SD) (m-2) 7100±3500 3500±900 65 <0.001

**Table 2.** Average values of geometrical and biomechanical parameters of 47 dysplastic and 36 »normal« female hips.

It seems reasonable that in hips that were assigned dysplastic, mostly due to poor coverage of the femoral head by the acetabulum the area that bears resultant hip force is smaller. So it could be concluded that in these hips the reasons for development of arthritis are mechanical in a sense that too high stress causes degeneration of the tissues and inflammation of hip joint. In other words, hip arthritis in these cases is secondary to increased contact hip stress that reflects

It can be seen from Table 2 that the parameters for the above example assessed from nomo‐ grams were the average parameters of the »normal« hips. The peak stress obtained by using the nomograms (3300 m-2) differs from the average hip stress calculated by the computer program 3500±900 m-2 (Table 2) for about 6%. Yet it should be considered that the value 3500 m-2 was not obtained by calculating stress from the average parameters presented in Table 2 but by averag‐

Secondary arthritis caused by hip dysplasia represents a minor part in the population of hip arthritis, so the question was posed whether the hips with diagnosis »dysplasia coxae« are in fact the extreme subpopulation of hips with too high hip stress and that a considerable number of hips with diagnosis »idiopathic hip arthritis« are in fact poorly described hips with too high hip stress. This question has already been addressed previously and the negative answer given by the thorough study of large cohort [16] brought evidence against the mechanical hypothesis.

**Dysplastic (47) Normal (36) Difference(%) p**

the hip.

SD: standard deviation. Adapted from [29].

144 Osteoarthritis - Progress in Basic Research and Treatment

unfavorable geometry of the hips and pelvis.

ing stresses of hips included in the study.

**5.3. »Normal« hips**

As it was expected that the differences between the diseased and »normal« hips would in the population considering hips with idiopathic osteoarthritis be smaller, another question was rised, i.e. which hips can be considered »normal«. To better define the »normal« hips, a more thorough study was performed considering asymptomatic hips [30]. The population consid‐ ered in the previous study [27] was expanded to 164 female and 42 male »normal« hips. In the female group the subjects' age ranged from 18 to 86, median 54. In the male group the subjects' age ranged from 23 to 82, median 54.

Figure 7 shows the dependence of the peak hip stress on the age of the subject. It can be seen that in the female and in the male population the values of peak stress were scattered over a large interval (between 2000 and 6000 m-2 in the female population and between 1500 and 4000 m-2 in the male population). With increasing age, the lower bound of the peak stress values remained more or less the same while the upper bound diminished. There were no »normal« old subjects with high hip stress. The average value of peak hip stress decreased with age. It was interpreted that hips that seem »normal« at young age (are asymptomatic) but have high peak stress are removed from the population of »normal« hips in the middle or old age due to development of early hip arthritis, thereby leaving in the »normal« population only hips with low peak stress. With aims at healthy ageing and higher lifespan it would be more appropri‐ ate to consider hip as »normal« only if it is asymptomatic at old age. According to the results presented in Figure 7,the appropriate value for »healthy« hips asymptomatic at 80 years would be about 2000 m-2 in both sexes. Most importantly, it was concluded that when comparing populations, special care should be taken with regard to the age of the subjects. In the study of Brinckmann et al. (1981) [16] the subjects in the group of »normal« hips were on the average younger than the subjects in the group of hips with arthritis, so some hips that were regarded as »normal« couldhave atthemathching agepertainedto the groupofhips with arthritis.These arguments encouraged reconsideration of validation ofthe mechanical hypotehsis also for hips with idiopathic arthritis.

**Figure 7.** Peak contact hip stress in the population of »normal« hips in dependence on subject's age. Adapted from [30].

#### **6. Comparison of »normal« hips and hips with idiopathic arthritis**

The mechanical hypothesis for the primary hip arthritis was validated by considering a group of 431 female patients who underwent total hip replacement [31]. Patients for whom secondary causes for hip arthritis were known were excluded (90 patients with rheumatoid or psoriatic arthritis, avascular necrosis, slipped capital femoral epiphysis, dysplasia of the hip or lower extremity fracture). Radiograms of hips and pelvis of 92 of the patients that were taken years before the operation for various reasons (back pain, discrete pain in the hips or minor injury to the pelvis) were retrieved from the archives, 65 of these radiograms were of required quality and showed hips without considerable joint space narrowing (mean width 3 mm), large osteophytes, subchondral cysts or acetabular protrusion. Three of these patients could not be located and one did not consent to participate in the study while two patients reported a fracture of the lower extremity during childhood. The final analysis was performed on 59 radiograms of hips with no or only initial stage of hip arthritis. The side of arthritis that developed later was determined from medical records on arthroplasty. Geometrical and biomechanical parameters were assessed. There were 22 female patients with unilateral hip arthritis (aged 45 to 79 years, median 69 years) and 37 female patients with bilateral disease (aged 50 to 80 years, median 68 years). In the population with unilateral disease, the parameters of the hips with arthritis were compared to the respective parameters of the contralateral hips with no sign of degenerative process. In the population with bilateral disease, the parameters of hips with earlier implantation of hip endoprosthesis were compared to the respective parameters of contralateral hips with later implementation of hip endoprosthesis.


SD: standard deviation. Adapted from [31].

**Table 3.** Average values of geometrical and biomechanical parameters of 22 female hips with arthritis and 22 contralateral »normal« hips.


**6. Comparison of »normal« hips and hips with idiopathic arthritis**

146 Osteoarthritis - Progress in Basic Research and Treatment

parameters of contralateral hips with later implementation of hip endoprosthesis.

(*r* ± SD) (cm) 2.6±1.5 2.6±1.4 0

SD: standard deviation. Adapted from [31].

contralateral »normal« hips.

(*C* ± SD) (cm) 6.4±1.1 6.5±1.0 -2 0.50 (*H* ± SD) (cm) 15.7±0.8 15.8±0.8 -1 0.18 (*x* ± SD) (cm) 1.1±0.9 1.2±0.9 -9 0.41 (*z* ± SD) (cm) 6.7±0.6 6.8±0.5 -1 0.07

(*ϑ*CE±SD) (degrees) 33.5±7.1 35.2±7.3 5 0.03 *R/W*B± SD 2.61±0.21 2.58±0.21 1 0.21 (*ϑR* ± SD) (degrees) 7.8±1.3 7.9±1.1 -1 0.47 (*p*max/*W*B ± SD) (m-2) 2440±490 2320±210 5 <0.001

**Table 3.** Average values of geometrical and biomechanical parameters of 22 female hips with arthritis and 22

**Hips with arthritis (22) Normal hips (36) Difference (%) p**

The mechanical hypothesis for the primary hip arthritis was validated by considering a group of 431 female patients who underwent total hip replacement [31]. Patients for whom secondary causes for hip arthritis were known were excluded (90 patients with rheumatoid or psoriatic arthritis, avascular necrosis, slipped capital femoral epiphysis, dysplasia of the hip or lower extremity fracture). Radiograms of hips and pelvis of 92 of the patients that were taken years before the operation for various reasons (back pain, discrete pain in the hips or minor injury to the pelvis) were retrieved from the archives, 65 of these radiograms were of required quality and showed hips without considerable joint space narrowing (mean width 3 mm), large osteophytes, subchondral cysts or acetabular protrusion. Three of these patients could not be located and one did not consent to participate in the study while two patients reported a fracture of the lower extremity during childhood. The final analysis was performed on 59 radiograms of hips with no or only initial stage of hip arthritis. The side of arthritis that developed later was determined from medical records on arthroplasty. Geometrical and biomechanical parameters were assessed. There were 22 female patients with unilateral hip arthritis (aged 45 to 79 years, median 69 years) and 37 female patients with bilateral disease (aged 50 to 80 years, median 68 years). In the population with unilateral disease, the parameters of the hips with arthritis were compared to the respective parameters of the contralateral hips with no sign of degenerative process. In the population with bilateral disease, the parameters of hips with earlier implantation of hip endoprosthesis were compared to the respective

**Table 4.** Average values of geometrical and biomechanical parameters of 37 female hips with bilateral arthritis. The hips in which arthritis developed earlier were compared with the hips in which arthritis developed later.

These results provided evidence in favor of the mechanical hypothesis by showing that hips with idiopathic arthritis had statistically significantly higher peak hip stress than contralateral asymptomatic hips (Table 3) and that higher peak hip stress meant clinically worse result (Table 4). As it is clear that hips with small centre-edge angle (smaller than 20 degrees) have very high peak stress and that hips with rather large centre-edge angle (larger than 35 degrees) have low hip stress, in hips in between these values the other geometrical parameters can importantly influence the stress. In standard procedures which are often based on the centreedge angle and the shape of the femoral head, these hips are not recognized as dysplastic, however, a combination of high and wide pelvis, laterally small extension of the greater trochanter and small radius of the femoral head may result in high hip stress. It is therefore indicated that rough estimation of stress on the basis of the visual experience should be supported by actually calculating stress.

#### **7. Hip stress gradient index as a relevant biomechanical parameter**

It was suggested [23] that hip stress gradient index is an appropriate parameter to assess hip dysplasia. As described above, the hip stress gradient index describes the derivative of the hip stress with respect to medial direction, at the lateral acetabular rim. If the hip stress increases in the medial direction at the lateral rim, *G*p is negative, while if it decreases, it is positive.

A population of hips diagnosed with hip dysplasia according to standard criteria and a group of »normal« hips were examined for the hip stress gradient index [23]. The respective popu‐ lations consisted of 56 dysplastic hips (9 male and 47 female) and 146 »normal« hips. Figure 3 shows a dependence of hip stress gradient index on the centre-edge angle for both popula‐ tions of hips. It can be seen that for small centre edge angles *G*<sup>p</sup> is positive, but it diminishes with increasing centre-edge angle. The parameter *G*p changes sign at the centre-edge angle approximately equal 20 degrees. The scattering of *G*p shows that parameters besides the centreedge angle are also important; the scattering is larger for smaller centre-edge angles. The difference between the average values of *G*p pertaining to the group of dysplastic hips (1.48.105 m-3) and to the group of normal hips (-0.44. 105 m-3) as assessed by the t-test was statistically signifficant (p<0.001).

**Figure 8.** Hip stress gradient index in dependence on centre-edge angle for dysplastic and for »normal« hips. Adapted from [23].

An independent group of 45 dysplastic and »normal« hips was assessed by the Harris hip score forpain,performanceandmobility[23].Astatisticallysignificantcorrelationwasfoundbetween the Harris hip score and the hip stress gradient index (ρ = -0.426, p<0.01). Hip stress gradient index was tested as a criterion for hip dysplasia in these hips. The hips were divided into two groups. The group with positive *G*p (16 hips) and the group with negative *G*p (29 hips). For comparison, the method for estimating hip dysplasia based on the centre-edge angle was used; thehips withcentre-edge angle smallerthan20degrees were considereddysplastic.There were 10 such hips and 35 hips with centre-edge angle largerthan 20 degrees. The difference in Harris hip score between the corresponding groups of dysplastic and »normal« hips were estimated by a non-parametrical statistical test (Kolmogorov-Smirnov test). By considerin*g G*p as the criterion, the difference between the two groups was found statistically significant (p = 0.031) while by considering the centre-edge angle as a criterion, the difference was not statistically significant(p=0.233).Inthisgroupofhipsthehipstressgradientindexprovedabetterparameter to predict the Harris hip score than the centre-edge angle [23]. As the hip stress gradient index reflects the distribution of stress over the weight bearing area, it was suggested due to these results that *G*p may prove complementary or even more important than he peak stress in assessment of the risk for hip arthritis development.

### **8. Hip stress gradient index as a relevant biomechanical parameter in hips that were in childhood subjected to Legg-Calve-Perthes disease**

3 shows a dependence of hip stress gradient index on the centre-edge angle for both popula‐ tions of hips. It can be seen that for small centre edge angles *G*<sup>p</sup> is positive, but it diminishes with increasing centre-edge angle. The parameter *G*p changes sign at the centre-edge angle approximately equal 20 degrees. The scattering of *G*p shows that parameters besides the centreedge angle are also important; the scattering is larger for smaller centre-edge angles. The difference between the average values of *G*p pertaining to the group of dysplastic hips (1.48.105 m-3) and to the group of normal hips (-0.44. 105 m-3) as assessed by the t-test was

**Figure 8.** Hip stress gradient index in dependence on centre-edge angle for dysplastic and for »normal« hips. Adapted

An independent group of 45 dysplastic and »normal« hips was assessed by the Harris hip score forpain,performanceandmobility[23].Astatisticallysignificantcorrelationwasfoundbetween the Harris hip score and the hip stress gradient index (ρ = -0.426, p<0.01). Hip stress gradient index was tested as a criterion for hip dysplasia in these hips. The hips were divided into two groups. The group with positive *G*p (16 hips) and the group with negative *G*p (29 hips). For comparison, the method for estimating hip dysplasia based on the centre-edge angle was used; thehips withcentre-edge angle smallerthan20degrees were considereddysplastic.There were 10 such hips and 35 hips with centre-edge angle largerthan 20 degrees. The difference in Harris hip score between the corresponding groups of dysplastic and »normal« hips were estimated by a non-parametrical statistical test (Kolmogorov-Smirnov test). By considerin*g G*p as the criterion, the difference between the two groups was found statistically significant (p = 0.031) while by considering the centre-edge angle as a criterion, the difference was not statistically significant(p=0.233).Inthisgroupofhipsthehipstressgradientindexprovedabetterparameter to predict the Harris hip score than the centre-edge angle [23]. As the hip stress gradient index reflects the distribution of stress over the weight bearing area, it was suggested due to these results that *G*p may prove complementary or even more important than he peak stress in

assessment of the risk for hip arthritis development.

statistically signifficant (p<0.001).

148 Osteoarthritis - Progress in Basic Research and Treatment

from [23].

Legg-Calve-Perthes disease may considerably affect the development of the hip resulting in deformed femur and acetabulum (Figure 1, right hip). As the risk for arthritis development is increased in hips that were in the childhood subjected to Legg-Calve-Perthes disease, it was of interest to determine biomechanical parameters in these hips and compare them with the corresponding parameters of »normal« hips. The group of contralateral asymptomatic hips with no aparent deformities were considered as the group of »normal« hips. 259 patients were initially considered in the study [26]. 167 patients (64.5%) attended a control examination which included measurement of height and weight of the patient. 3 patients were omitted for missing X-ray pictures from the time of the disease, 19 patients were omitted due to bilateral disease, 3 patients were omitted due to absence of radiograms at the follow-up, 5 patients were omitted due to inadequate X-ray pictures and 2 patients were omitted as they already had a hip prosthesis implanted due to hip arthritis. The final cohort included 135 patients. 24 hips were female (17.8%) and 111 hips were male (82.2%). The mean age at follow-up was 32.5 (20.6 – 47.6) years and the mean body mass index (BMI) at follow-up was 26.7 (18 – 38) kg/m2 .The mean time interval between treatment and follow up was 25.6 (14.5 – 34.5) years. As the body weight was measured at the control examination it was possible to determine both, the normalized biomechanical parameters and the »whole« parameters.


**Table 5.** Average values of geometrical and biomechanical parameters of hips that were in the childhood subjected to Legg-Calve-Perthes disease and "normal" (contralateral) hips. The probabilities (p-value) were calculated with the two-tailed paired t-test and post-hoc statistical power (1-β) calculated for α = 0.05 and sample size N = 135. Statistical power was 1 also for α = 10-8 for the variables that yielded differences with statistical significance p <10-8. Adapted from [26].

It can be seen in Table 5 that there were no statistically significant differences between the two groups in resultant hip force and peak contact hip stres (normalized and »whole«). The centreedge angle was considerably and statistically significantly more favorable in »normal« hips, however, the hips that were in the childhood subjected to the disease had developed a considerably and statistically significantly larger femoral head which compensated the effect of the smaller centre-edge angle. Figure 9 shows that in the group of »normal« hips (red dots) the radii were smaller and uniformly distributed over the interval of centre-edge angles. The lower bound of this interval was approximately 20 degrees as previously acknowledged to be a criterion for hip dysplasia. The test group extended also below this interval, but here the radii were considerably larger. This effect overcompensated the load bearing area which was (statistically significantly) larger in the test group although the centre-edge angle was smaller (Table 5).

**Figure 9.** Interdependence between the radius of the femoral head and the centre-edge angle in hips that were in child‐ hood subjected to Legg-Calve-Perthes disease (test group) and contralateral "normal" hips (control group). From [26].

In the hips that were in childhood subjected to Legg-Calve-Perthes disease the resultant hip force and the peak stress did not show differences while the load bearing area was more favorable, however, the stress pole was located considerably and statistically significantly more laterally than in the control group which was reflected also in the difference in the functional angle (Table 5). Most importantly, the biomechanical parameter that showed the difference between the two groups in favor of »normal« hips was the hip stress gradient index. The respective differences in the normalized and the »whole« parameter were considerable (larger than 100%) and statistically very significant (Table 5). The cohort was large enough (with high statistical power at very small probabilities) to render the above results decisive.

Figure 10 shows dependence of hip stress gradient index on the centre-edge angle (A) and on the radius of the femoral head (B). Almost all »normal« hips are confined within the boundaries of radii smaller than 3 cm, centre-edge angles larger than 20 degrees and negative hip stress gradient indexes while the hips that were in childhood subjected to Legg-Calve-Perthes disease extended beyond these boundaries (to larger radii, smaller centre-edge angles and positive hip stress gradient indexes). But there were also many hips from the test group that fitted well within the group of »normal« hips showing successful recovery from the disease in the childhood.

It can be seen in Table 5 that there were no statistically significant differences between the two groups in resultant hip force and peak contact hip stres (normalized and »whole«). The centreedge angle was considerably and statistically significantly more favorable in »normal« hips, however, the hips that were in the childhood subjected to the disease had developed a considerably and statistically significantly larger femoral head which compensated the effect of the smaller centre-edge angle. Figure 9 shows that in the group of »normal« hips (red dots) the radii were smaller and uniformly distributed over the interval of centre-edge angles. The lower bound of this interval was approximately 20 degrees as previously acknowledged to be a criterion for hip dysplasia. The test group extended also below this interval, but here the radii were considerably larger. This effect overcompensated the load bearing area which was (statistically significantly) larger in the test group although the centre-edge angle was smaller

**Figure 9.** Interdependence between the radius of the femoral head and the centre-edge angle in hips that were in child‐ hood subjected to Legg-Calve-Perthes disease (test group) and contralateral "normal" hips (control group). From [26].

In the hips that were in childhood subjected to Legg-Calve-Perthes disease the resultant hip force and the peak stress did not show differences while the load bearing area was more favorable, however, the stress pole was located considerably and statistically significantly more laterally than in the control group which was reflected also in the difference in the functional angle (Table 5). Most importantly, the biomechanical parameter that showed the difference between the two groups in favor of »normal« hips was the hip stress gradient index. The respective differences in the normalized and the »whole« parameter were considerable (larger than 100%) and statistically very significant (Table 5). The cohort was large enough (with high statistical power at very small probabilities) to render the above results decisive.

(Table 5).

150 Osteoarthritis - Progress in Basic Research and Treatment

**Figure 10.** Dependence of the hip stress gradient index on the centre-edge angle (A) and on the radius of the femoral head (B) in hips that were in childhood subjected to Legg-Calve-Perthes disease (test group) and contralateral "nor‐ mal" hips (control group). From [26].

#### **9. Conclusion**

Method HIPSTRESS proved useful in contributing evidence in favor of mechanical hypothesis stating that long lasting unfavorable stress distribution is an etiological factor in development of hip arthritis. The mathematical model for resultant hip force contains the relevant choice of muscles and appropriate scaling of their attachment points that emphasize the individual geometry. The mathematical model is not simple in its derivation, however, it is expressed by transparent and almost analytical solution. The computer program and nomograms enable medical doctors and students to use the mathematical models without extensive mathematical skills. Knowing the geometrical parameters the resultant hip force and the peak stress can be determined within minutes. It is shown above that peak hip stress showed differences between dysplastic and normal hips and between hips with idiopathic arthritis and normal hips. However in dysplastic hips also the hip stress gradient index was less favorable (larger), so it is unclear which of these parameters is the most relevant to estimate the risk for development of hip arthritis. The role of hip stress gradient index is emphasized also by the study of hips that were in childhood subjected to Legg-Calve-Perthes disease since in these hips the resultant hip force and the peak stress were not elevated. Further studies are needed to obtain answer to this question. The method could be supported by using improved imaging of hips (three dimensional data on muscle attachment points) and refined by considering particularities of diseases (such as nonsphericity of femoral head after the Legg-Calve-Perthes disease). Most importantly, the hypothesis involving macroscopic hip stress distribution should be connected to molecular mechanisms underlaying cartilage deterioration and onset and spreading of inflammation.

The criteria for biomechanical measures suggested by R.A. Brand [32] are that they (a) should be accurate and reproducible, (b) the measuring technique must not significantly alter the function it is measuring, (c) it should exhibit reasonable stability, (d) the measure should not be directly observable by the skilled clinician, (e) it should be independent of mood, motivation or pain, (f) it must clearly distinguish between normal and abnormal, (g) it should be reported in a form analogous to some accepted clinical concept, (h) it should be cost-effective and (i) it must be appropriately validated. As a method based on physical laws the HIPSTRESS method completely fulfills the criteria (b), (c), (d), (e), (g) and (h). As for criterion (a) it is reproducible, but its accuarcy is limited by the model assumptions and by the accuracy of measurement of geometrical parameters. Further improvements should be made in these directions. Criterion (f) adresses »normal« and »abnormal«. A clear criterion can be given within the HIPSTRESS method (i.e. threshold values of biomechanical parameters such as Gp=0), however, these are based on correspondence of the parameters with clinical assessment which is also not always clear. The criterion (*i*) was implemented by validation of the HIPSTRESS method by studies of the effect of different operations on biomechanical and clinical outcome and related problems [32-57]. The strongest point of the method is low invasiveness (it uses data that were obtained for therapeutic purposes). Also, the development of the method and its use required no experiments on laboratory animals or any other material that would require burden on patients, volunteers or animals.

#### **Author details**

**9. Conclusion**

152 Osteoarthritis - Progress in Basic Research and Treatment

inflammation.

patients, volunteers or animals.

Method HIPSTRESS proved useful in contributing evidence in favor of mechanical hypothesis stating that long lasting unfavorable stress distribution is an etiological factor in development of hip arthritis. The mathematical model for resultant hip force contains the relevant choice of muscles and appropriate scaling of their attachment points that emphasize the individual geometry. The mathematical model is not simple in its derivation, however, it is expressed by transparent and almost analytical solution. The computer program and nomograms enable medical doctors and students to use the mathematical models without extensive mathematical skills. Knowing the geometrical parameters the resultant hip force and the peak stress can be determined within minutes. It is shown above that peak hip stress showed differences between dysplastic and normal hips and between hips with idiopathic arthritis and normal hips. However in dysplastic hips also the hip stress gradient index was less favorable (larger), so it is unclear which of these parameters is the most relevant to estimate the risk for development of hip arthritis. The role of hip stress gradient index is emphasized also by the study of hips that were in childhood subjected to Legg-Calve-Perthes disease since in these hips the resultant hip force and the peak stress were not elevated. Further studies are needed to obtain answer to this question. The method could be supported by using improved imaging of hips (three dimensional data on muscle attachment points) and refined by considering particularities of diseases (such as nonsphericity of femoral head after the Legg-Calve-Perthes disease). Most importantly, the hypothesis involving macroscopic hip stress distribution should be connected to molecular mechanisms underlaying cartilage deterioration and onset and spreading of

The criteria for biomechanical measures suggested by R.A. Brand [32] are that they (a) should be accurate and reproducible, (b) the measuring technique must not significantly alter the function it is measuring, (c) it should exhibit reasonable stability, (d) the measure should not be directly observable by the skilled clinician, (e) it should be independent of mood, motivation or pain, (f) it must clearly distinguish between normal and abnormal, (g) it should be reported in a form analogous to some accepted clinical concept, (h) it should be cost-effective and (i) it must be appropriately validated. As a method based on physical laws the HIPSTRESS method completely fulfills the criteria (b), (c), (d), (e), (g) and (h). As for criterion (a) it is reproducible, but its accuarcy is limited by the model assumptions and by the accuracy of measurement of geometrical parameters. Further improvements should be made in these directions. Criterion (f) adresses »normal« and »abnormal«. A clear criterion can be given within the HIPSTRESS method (i.e. threshold values of biomechanical parameters such as Gp=0), however, these are based on correspondence of the parameters with clinical assessment which is also not always clear. The criterion (*i*) was implemented by validation of the HIPSTRESS method by studies of the effect of different operations on biomechanical and clinical outcome and related problems [32-57]. The strongest point of the method is low invasiveness (it uses data that were obtained for therapeutic purposes). Also, the development of the method and its use required no experiments on laboratory animals or any other material that would require burden on

Veronika Kralj-Iglič\*

Address all correspondence to: veronika.kralj-iglic@fe.uni-lj.si

Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia

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## **Specific Proteases for Osteoarthritis Diagnosis and Therapy**

Xiao-Yu Yuan, Liping Zhang and Yuqing Wu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60570

#### **1. 1. Introduction**

#### **1.1. Osteoarthritis (OA)**

#### *1.1.1. Causes and Symptoms of Osteoarthritis*

Osteoarthritis is the most common style of arthritis, and this complicated and chronic degen‐ erative joint disease is extremely found in adults and especially in old people. It mostly affects the whole joint structures associated with progressive changes in cartilage, menisci, ligaments and subchondral bone.[1-3] The cartilage covers the end of joint bones and provides slippery touch during movement, so it is obvious that the degradation of the cartilage extracellular matrix is a central feature of this disease. Normal articular cartilage makes bones frictionless with each other, and additionally it can also reduce the damage caused by shock of movement. [4] However, in osteoarthritis the top layer of articular cartilage wears out and even breaks down, which initiates the bone rubbing against each other, and therefore causes pain, sclerosis, swelling and loss of organ function in joint. As time going on, the symptoms are increasing in frequency and severity, finally the shape of joint changes with deformity, bone spurs may also occur at the edges of joints, bits of bones even fractures and floats among the joint space.

According to the pre-existing investigations, osteoarthritis has affected the health of a growing number of people world-widely.[5] Though osteoarthritis will not endanger the life safety of sufferers, its occurrence and development may not only seriously threaten people's physical fitness, but also directly reduce their quality of life.

It is well known that the loss of cartilage is concerned with the etiology of osteoarthritis. [1] The articular cartilage failure is triggered by several correlate factors, such as genetic, metabolic, and biomechanical factors with secondary components inflammation which react

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mutually. Until now the pathogenesis has not been wholly revealed due to the multifactorial pathological mechanism of osteoarthritis, though many groups have researched for a long time.[6-10] Moreover, other risk factors including obesity, older age, joint injury, family history, over using, bone density, defect in joint cartilage contribute to osteoarthri‐ tis progression.[11-14]

Based on the above description, it is not hard to see that there must be osteoarthritis syndrome with a series of heterogeneous presentations,[15] such as **a)** joint pain, also the major clinical manifestation; **b)** joint stiffness, especially morning stiffness; **c)** functional disorder, like joint instability and activity limitation. These symptoms of osteoarthritis develop slowly and get serious increasingly with time.

Osteoarthritis is short of the physical and biochemical integrity of a joint, and also presents as a mono-arthritis, oligo-arthritis or a poly-arthritis, with several distinct patterns which exist in most ethnic and racial groups.[16] The common feature of osteoarthritis is characterized by the early inflammation followed by degeneration of chondrocytes including irreversible biodegradation. Then osteoarthritis appears as transformation of whole joint structures including degradation of the articular cartilage, menisci and ligaments, and these are also accompanied by other performance, such as joint space narrowing (JSN), bone marrow lesions, synovial inflammation, changes of subchondral bone and generation of osteophytes at the joint edge (See Figure 1).[2, 10, 13, 17] The degradative process of cartilage is widely considered to be regulated by the protease involved in osteoarthritis.[4, 6, 10, 18]

**Figure 1.** Diagrammatic presentation of normal and osteoarthritic joint.[10]

As the major mediators of collagen and proteoglycan cleavage, two classes of proteases are thought to be responsible for the degradation of cartilage components in osteoarthritis. It was thought that the collagen degradation is majorly due to the action of matrix metalloproteinase (MMP) collagenases. Mort *et al.* have indicated that members of both MMP and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) families are important mediators of the degradation of proteoglycans.[19] At the same time there is evidence for the role of the cysteine protease cathepsin K in collagen degradation in articular cartilage as revealed by Konttinen *et al.*.[20] The cleavage of cartilage proteins often occurs at specific sites on these molecules, resulting in the generation of characteristic N- and C-terminal epitopes which can be used for the production of antibodies specific for these cleavage products (antineoepitope antibodies).[21] Recent years, Chan *et al.* have also reported that the increased chondrocyte sclerostin may protect against cartilage degradation in osteoarthritis.[22]

#### **1.2. Importance of osteoarthritis diagnosis and therapy**

mutually. Until now the pathogenesis has not been wholly revealed due to the multifactorial pathological mechanism of osteoarthritis, though many groups have researched for a long time.[6-10] Moreover, other risk factors including obesity, older age, joint injury, family history, over using, bone density, defect in joint cartilage contribute to osteoarthri‐

Based on the above description, it is not hard to see that there must be osteoarthritis syndrome with a series of heterogeneous presentations,[15] such as **a)** joint pain, also the major clinical manifestation; **b)** joint stiffness, especially morning stiffness; **c)** functional disorder, like joint instability and activity limitation. These symptoms of osteoarthritis develop slowly and get

Osteoarthritis is short of the physical and biochemical integrity of a joint, and also presents as a mono-arthritis, oligo-arthritis or a poly-arthritis, with several distinct patterns which exist in most ethnic and racial groups.[16] The common feature of osteoarthritis is characterized by the early inflammation followed by degeneration of chondrocytes including irreversible biodegradation. Then osteoarthritis appears as transformation of whole joint structures including degradation of the articular cartilage, menisci and ligaments, and these are also accompanied by other performance, such as joint space narrowing (JSN), bone marrow lesions, synovial inflammation, changes of subchondral bone and generation of osteophytes at the joint edge (See Figure 1).[2, 10, 13, 17] The degradative process of cartilage is widely considered to

As the major mediators of collagen and proteoglycan cleavage, two classes of proteases are thought to be responsible for the degradation of cartilage components in osteoarthritis. It was thought that the collagen degradation is majorly due to the action of matrix metalloproteinase (MMP) collagenases. Mort *et al.* have indicated that members of both MMP and ADAMTS (a

be regulated by the protease involved in osteoarthritis.[4, 6, 10, 18]

**Figure 1.** Diagrammatic presentation of normal and osteoarthritic joint.[10]

tis progression.[11-14]

serious increasingly with time.

160 Osteoarthritis - Progress in Basic Research and Treatment

Currently the total number of osteoarthritis patients in world-wide is more than 600 million. There are about 1/6 of people in Asia suffering from osteoarthritis at some stage of their life, among which patients account for 10% of the total population in China, and are increasing in recent years. It is even estimated that the number may reach to 150 million by 2015. In addition, it has been reported that a lifetime risk of OA-specific morbidity of about 45% for the knee and 25% for the hip.[23] The National Healthy and Nutrition Examination Survey of USA points out that the symptoms and signs of clinical osteoarthritis are only in 12% of 6913 people, aged among from 25 to 74 years, and X-ray results of osteoarthritis appeared in at least one site occupy 33%.[24] It will be worse that 53% of osteoarthritis patients may lead to disability, loss of joint function and the ability to work.

Unfortunately, the disease-modifying drugs for osteoarthritis are not available currently, the drugs have the role of analgesic effect and symptom improvement, but they do not involve the OA pathology and change the abnormal structure. Even though some styles of drugs can slow down and reverse the degradation of cartilage, they produce the desired result tardily, and the curative effect only can maintain for a short period. Osteoarthritis has become the largest disease causing disability and is known as "no-lethal cancer", it is so harmful to human health, and the related research about it has been carried out.[25-27]

While there is no complete cure for OA until now, the early detection followed by efficient therapy may slow down its detrimental effects. Apparently, a diagnostic system that enables early and reliable diagnosis of this degenerative joint disease is necessary,[28] such as bio‐ chemical test and imageological diagnosis (X-ray or/and NMR examination). **a)** biochemical test: recent years, the special biochemical marker have been drawn attention, hydroxylysyl‐ pyridinoline, heoxylysylpyridinoline, C-reactive protein, Serum amyloid A and so on are all used to calculate the OA; **b)** imageological diagnosis: the diagnosis base on radiology mani‐ festation of OA through X-ray or/and NMR examination. However, the imaging technique can not accurately, selectively evaluate quantitative property of articular cartilage. In addition, once it is diagnosed as osteoarthritis, the disease has proceeded into the mid- or late- period, and the opportune moment and prompt treatment to patients are all missed, so the new technology and strategy need to be further developed. Treatments are also limited to relieve the symptoms and surgically replace the affected joints. Therefore, to minimize the damage, understanding the early symptoms of bone disease will help to detect and treat the disease. The development of bone-related diagnostic and therapeutic programs will be essential,[29] and a wide range of work in the past years has proved that several proteases might related directly to osteoarthritis.

#### **2. Diagnosis of osteoarthritis in targeting specific proteases**

#### **2.1. Cathepsin B (Cath B)**

As a cysteine proteinase, cathepsin B is a lysosomal cysteine protease, which belongs to the papain surper-family and has been implicated in the pathology of a number of important human disease, including cancer and arthritis.[30] It has been shown to be up-regulated in patients with rheumatoid arthritis,[31] and components of the estracellular matrix are shown to be substrates for this protease.[32] Cathepsin B is active in aggrecan and cartilage,[33-34] cleaves the aggrecan G1-G2 domain fragment, and engenders two fragments from the cleavage at Gly-Val bond to the metalloproteinase cleavage site. For example, Fosang *et al.* have revealed that cathepsin B degraded the proteoglycan extensively producing several bands of faster migration and therefore it can be used as a biomarker of diagnosis of OA.[35]

Then Lai's group took twelve male nude mice to investigate early diagnosis of osteoarthritis on a molecular basis, by using the developed cathepsin B sensitive near-infrared (NIR) fluorescent probe.[36] Firstly, they injected collagenase (1.0%, w/v) into the right knee joint to induce osteoarthritis and the left knee joint served as a comparison. Secondly, the cathepsin B sensitive near-infrared fluorescence probe was activated, which could radiate an intensive NIR fluorescence signal. Finally, the NIR fluorescence signals were caught by an optical imaging system which could receive an emission wavelength of 680–720 nm. Using this mechanism, they discovered that 3-fold difference in signal intensity between osteoarthritic and normal joints can be detected after 24h intravenous injection (see Figure 2). Therefore, it is believed that cathepsin B activatable NIR fluorescence probe can offer a potential new imaging technology for early osteoarthritis diagnosis.

**Figure 2.** Near infrared fluorescence reflectance imaging, which were taken 24 h after intravenous injection of the cath‐ epsin B sensitive auto-quenched probe in a representative animal; (A and B) white light images, (C and D) NIRF im‐ ages.[36]

#### **2.2. Lactate Dehydrogenase (LDH)**

**2. Diagnosis of osteoarthritis in targeting specific proteases**

migration and therefore it can be used as a biomarker of diagnosis of OA.[35]

As a cysteine proteinase, cathepsin B is a lysosomal cysteine protease, which belongs to the papain surper-family and has been implicated in the pathology of a number of important human disease, including cancer and arthritis.[30] It has been shown to be up-regulated in patients with rheumatoid arthritis,[31] and components of the estracellular matrix are shown to be substrates for this protease.[32] Cathepsin B is active in aggrecan and cartilage,[33-34] cleaves the aggrecan G1-G2 domain fragment, and engenders two fragments from the cleavage at Gly-Val bond to the metalloproteinase cleavage site. For example, Fosang *et al.* have revealed that cathepsin B degraded the proteoglycan extensively producing several bands of faster

Then Lai's group took twelve male nude mice to investigate early diagnosis of osteoarthritis on a molecular basis, by using the developed cathepsin B sensitive near-infrared (NIR) fluorescent probe.[36] Firstly, they injected collagenase (1.0%, w/v) into the right knee joint to induce osteoarthritis and the left knee joint served as a comparison. Secondly, the cathepsin B sensitive near-infrared fluorescence probe was activated, which could radiate an intensive NIR fluorescence signal. Finally, the NIR fluorescence signals were caught by an optical imaging system which could receive an emission wavelength of 680–720 nm. Using this mechanism, they discovered that 3-fold difference in signal intensity between osteoarthritic and normal joints can be detected after 24h intravenous injection (see Figure 2). Therefore, it is believed that cathepsin B activatable NIR fluorescence probe can offer a potential new imaging

**Figure 2.** Near infrared fluorescence reflectance imaging, which were taken 24 h after intravenous injection of the cath‐ epsin B sensitive auto-quenched probe in a representative animal; (A and B) white light images, (C and D) NIRF im‐

**2.1. Cathepsin B (Cath B)**

162 Osteoarthritis - Progress in Basic Research and Treatment

technology for early osteoarthritis diagnosis.

ages.[36]

LDH-4 and LDH-5 play an important role in anaerobic metabolism of articular cartilage. In early 1975, Weseloh and Fiegelmann began to realize the importance of LDH-isoenzym patterns in human cartilage, and attempted to evaluate it. The result of experience was that LDH-5 dominates with an average of 75.3%, whereas the LDH-4 (21.7%) and the LDH-3 (3.2%) were considerably lower, which was significant for the later study. [37]

As we know from previous literatures, degenerative joint diseases were deemed to associate with increased LDH activity in the synovial fluid. In order to verify the distribution of LDH, Eveline *et al.* have made a study to examine healthy and degenerative stifle joints for the goal of clarifying the origin of LDA in synovial fluid through many technical means, such as transmission electron microscopy (TEM), immunolabeling and enzyme cytochemistry. And then all techniques corroborated that the presence of LDH in chondrocytes and in the inter‐ territorial matrix of degenerative stifle joints. Whereas LDH is retained in healthy cartilage due to permeability limitations, it is released into synovial fluid through abrasion as well as through unrestricted diffusion as a result of degradation of collagen and increased water content in degenerative joints.[38]

In addition, the spectrophotometric technique using the pyruvate to lactate conversion was taken to measure the total LDH activity, while agar gel electrophoresis followed by a tetrazo‐ lium enzymatic staining reaction was used to establish the LDH isoenzyme patterns. Veys *et al.* have found long before that the cases of arthritis had high LDH activity both in cellular material and in cell-free fluid. Moreover, these cases also had an increased percentage of LDH-5 in the cellular extract. It was concluded that the LDH could be a symbol reflecting the degree of arthritis and used to the diagnosis of early OA.[39]

#### **3. Therapeutics of osteoarthritis in targeting specific proteases**

#### **3.1. Sclerostin (SOST)**

Sclerostin is a kind of extracellular protease (see Figure 3). As it is well known, the SOST gene encodes for the secreted protein sclerostin.[40] The expression of SOST in the adult body exclusively is produced by osteocytes located in bones. Therefore, sclerostin is considered as negatively modulating osteoblast development and bone formation.[41-42]

At first it was thought that sclerostin might implement its regulatory function *via* acting as a modulator of bone morphogenetic proteins (BMPs).[43] Afterwards the accumulating evi‐ dence showed that sclerostin interferes with the Wnt signaling pathway due to binding to the Wnt co-receptor LRP5 and consequently regulating bone growth.[44-45] SOST restrained the express of Wnt signaling, which was important in the skeletal development[41, 46] and could regulate the activity of β-catenin. It was also reported that excess SOST could lead to chon‐ drocyte apoptosis and cartilage damage, namely arthritis, through suppressing the normal function of β-catenin.[47] At the same time, the study performed by Blom *et al.* have shown that superabundant β-catenin would produce a procatabolic effect in the cartilage and promote

**Figure 3.** "Sausage" plot of the averaged minimized structure of murine sclerostin showing the highly flexible regions of mSOST. Regions colored in blue structurally marked the highly defined areas, regions marked in red are highly dis‐ ordered.[51]

chondrocyte hypertrophy directly related with osteoarthritic pathology. Therefore, moderate sclerostin would be necessary to keep β-catenin at an appropriate level, and it would be a crucial indicator in the treatment or diagnosis of osteoarthritis.[48-50] Of note is that, the importance of Wnt/β-catenin signaling in the pathogenesis of osteoarthritis in humans has not been well understood. For example, the findings in preclinical studies using anti-sclerostin therapy in animal models of osteoarthritis have been disappointing, with no reported benefit on cartilage remodeling during aging or mechanical injury.[50] In addition, the role of sclerostin in the pathogenesis of osteoarthritis in humans has not yet been well defined, and the potential utility of treating osteoarthritis with interventions that alter sclerostin is not known. Still, we are surely pleased to see that summary of SOST in therapeutics of OA will be performed.

#### **3.2. Matrix Metalloproteinases (MMPs) and Adamalysin with Thrombospondin Motifs (ADAMTS)**

A significant characteristic of osteoarthritis is the degradation of the extracellular cartilage tissue. According to the previous reports it is widely known that the structural component of the matrix is mainly composed of collagen and aggrecan, which are regulated by the proteolytic enzymes, MMPs and ADAMTs.

#### *3.2.1. MMPs*

The collagen found primarily in the cartilage ECM is type II collagen, which appears as the fibrillar network (see Figure 4) and offers strong elasticity to the cartilage matrix. It will be difficult to be repaired for cartilage once the collagen was lost (see Figure 5).[52-53] Matrix metalloproteinases (MMP) comprises a family of zinc-dependent enzymes, they are called collagenases which possess the collagenolytic abilities that degrade extracellular matrix components.[54] These proteases regulate the initial cleavage of the collagen triple helix, occurring at 3/4 of the distance from the amino-terminal end of each chain, forming collagen fragments of 3/4 and 1/4 length.[55] To be directly related to these processes there are three kinds of collagenases: collagenase-1 or interstitial collagenase (MMP-1); collagenase-2 or neutrophil collagenase (MMP-8); and collagenase-3 (MMP-13). In addition, MMP-13 is considered as the primary collagenase in collagen degradation.[6-57] Neuhold *et al.* showed that MMP-13 transgenic animals exhibited joint pathology which strongly resembled osteo‐ arthritis. Such a result provided direct evidence in support of a role for this proteinase in the pathology of this disease.[18]

#### *3.2.2. ADAMTS*

chondrocyte hypertrophy directly related with osteoarthritic pathology. Therefore, moderate sclerostin would be necessary to keep β-catenin at an appropriate level, and it would be a crucial indicator in the treatment or diagnosis of osteoarthritis.[48-50] Of note is that, the importance of Wnt/β-catenin signaling in the pathogenesis of osteoarthritis in humans has not been well understood. For example, the findings in preclinical studies using anti-sclerostin therapy in animal models of osteoarthritis have been disappointing, with no reported benefit on cartilage remodeling during aging or mechanical injury.[50] In addition, the role of sclerostin in the pathogenesis of osteoarthritis in humans has not yet been well defined, and the potential utility of treating osteoarthritis with interventions that alter sclerostin is not known. Still, we are surely pleased to see that summary of SOST in therapeutics of OA will be

**Figure 3.** "Sausage" plot of the averaged minimized structure of murine sclerostin showing the highly flexible regions of mSOST. Regions colored in blue structurally marked the highly defined areas, regions marked in red are highly dis‐

**3.2. Matrix Metalloproteinases (MMPs) and Adamalysin with Thrombospondin Motifs**

A significant characteristic of osteoarthritis is the degradation of the extracellular cartilage tissue. According to the previous reports it is widely known that the structural component of the matrix is mainly composed of collagen and aggrecan, which are regulated by the proteolytic

The collagen found primarily in the cartilage ECM is type II collagen, which appears as the fibrillar network (see Figure 4) and offers strong elasticity to the cartilage matrix. It will be difficult to be repaired for cartilage once the collagen was lost (see Figure 5).[52-53] Matrix

performed.

ordered.[51]

164 Osteoarthritis - Progress in Basic Research and Treatment

**(ADAMTS)**

*3.2.1. MMPs*

enzymes, MMPs and ADAMTs.

Aggrecan is a large proteoglycan including chondroitin sulphate and keratan sulphate glycosaminoglycan moieties, and is crucial for bringing water to the cartilage matrix which gives joints the ability to bear the heavy load. Aggrecan plays such a good role, short of it can lead to the articular cartilage softening and loss of fixed charges, then the joint function will be reduced and even forfeited.[58] Aggrecan molecules possess two major cleavage sites in the interglobular domain (IGD) region of the core protein. Without the G1 domain, aggrecan molecules can be free in and out of the cartilage matrix, leading to the lack of cartilage function. [59] The first cleavage site at the Asn341-Phe342 bond, creating the neoepitope VDIPEN, is found to be generated by MMPs.[60] The second site at the Glu373-Ala374 bond, creating the NITEGE neoepitope, is found to be very important and results from aggrecan cleavage, which is associated with lots of pathologies.

**Figure 4.** Electron micrographs of healthy cartilage. **a**) A dense superficial network of collagen fibers running in paral‐ lel with the articular surface is present. Chondrocytes contain numerous organelles and vesicles. *Inset* An amorphous layer (*double arrow*) extends at the surface. Underneath, densely packed collagen fibers run in parallel with the articular surface. **b**) Territorial (A) and interterritorial (B) zones of extracellular matrix are clearly demarcated. Note the large nucleus and abundance of organelles in the chondrocyte.[38]

Related studies have shown that ADAMTS-5 is a pivotal enzyme for cutting the Glu373~Ala374 bond, and the inhibitors of ADAMTS-5 can debase the aggrecan decomposition effectively. So ADAMTS-5 is important in osteoarthritis of individuals and responsible for aggrecan degra‐

**Figure 5.** Electron micrograph of degenerative cartilage. The amorphous layer is missing, and the articular surface is uneven. Collagen Wbers are loosely packed and arranged at random. Note amorphous foci in the extracellular matrix (arrows). (A) Territorial extracellular matrix.[38]

dation in normal and diseased cartilage.[61] A new drug, AGG523, in targeting ADAMTS-5 and ADAMTS-4 for therapeutics has entered clinical trials phase I. Studies by Glasson *et al.* and Majumdar *et al.* carried out with ADAMTS-5 knockout and ADAMTS-4/-5 double knockout mice showed that these animals were more resistant to cartilage degradation after destabilizing knee surgery.[62-63] However, in human, assumed damaging polymorphisms in the ADAMTS-5 gene does not show any modification in selectivity to osteoarthritis.[64] The search for the most important aggrecanase in human osteoarthritis is still going.[65]

#### **3.3. Cathepsin K**

Cathepsin K (Cath K) is a cysteine proteinase of papain family. It has been implicated in the resorption of the bone matrix. Like most of the proteinases, cathepsin K is synthesized and secreted from the cell as an inactive proenzyme, it should be noted that cathepsin K is secreted from macrophage and synovial fibroblasts. Cathepsin K cleaves the triple-helical type II collagen[66], and the special distribution of cathepsin K in osteoarthritic cartilage suggests an important role of this protease in the etiopathogenesis of osteoarthritis.

Early in 2004, Morko *et al.* took transgenic mice which were predisposed to early osteoarthritis because of harboring a short deletion mutation and their non-transgenic litter mates as controls for study.[53] They used the immunohistochemistry and morphometry to investigate the distribution of cathepsin K in the knee joints. In the knee joints of transgenic mice, Cathepsin K was found near sites of matrix destruction in articular chondrocytes, particularly in calcified cartilaginous matrix and proliferating cells. It indicted that cathepsin K played an important role in the pathogenesis of osteoarthritis. They also gave an explain that cathepsin K could digest cartilage matrix components, therefore, it was considered to contribute to the progres‐ sion of osteoarthritic damage. Such studies have provided new clew for the development of treatments aimed at holding back cartilage degeneration.[53]

#### **4. Inhibition of proteases related to OA**

There is currently no disease-modifying OA drug available, and treatment is limited to symptomatic relief or surgical replacement of affected joints. There is thus considerable interest in developing effective treatments that can halt or reverse the progression of the disease.

#### **4.1. Inhibition of sclerostin**

dation in normal and diseased cartilage.[61] A new drug, AGG523, in targeting ADAMTS-5 and ADAMTS-4 for therapeutics has entered clinical trials phase I. Studies by Glasson *et al.* and Majumdar *et al.* carried out with ADAMTS-5 knockout and ADAMTS-4/-5 double knockout mice showed that these animals were more resistant to cartilage degradation after destabilizing knee surgery.[62-63] However, in human, assumed damaging polymorphisms in the ADAMTS-5 gene does not show any modification in selectivity to osteoarthritis.[64] The

**Figure 5.** Electron micrograph of degenerative cartilage. The amorphous layer is missing, and the articular surface is uneven. Collagen Wbers are loosely packed and arranged at random. Note amorphous foci in the extracellular matrix

Cathepsin K (Cath K) is a cysteine proteinase of papain family. It has been implicated in the resorption of the bone matrix. Like most of the proteinases, cathepsin K is synthesized and secreted from the cell as an inactive proenzyme, it should be noted that cathepsin K is secreted from macrophage and synovial fibroblasts. Cathepsin K cleaves the triple-helical type II collagen[66], and the special distribution of cathepsin K in osteoarthritic cartilage suggests an

Early in 2004, Morko *et al.* took transgenic mice which were predisposed to early osteoarthritis because of harboring a short deletion mutation and their non-transgenic litter mates as controls for study.[53] They used the immunohistochemistry and morphometry to investigate the distribution of cathepsin K in the knee joints. In the knee joints of transgenic mice, Cathepsin K was found near sites of matrix destruction in articular chondrocytes, particularly in calcified cartilaginous matrix and proliferating cells. It indicted that cathepsin K played an important role in the pathogenesis of osteoarthritis. They also gave an explain that cathepsin K could digest cartilage matrix components, therefore, it was considered to contribute to the progres‐ sion of osteoarthritic damage. Such studies have provided new clew for the development of

search for the most important aggrecanase in human osteoarthritis is still going.[65]

important role of this protease in the etiopathogenesis of osteoarthritis.

treatments aimed at holding back cartilage degeneration.[53]

**3.3. Cathepsin K**

(arrows). (A) Territorial extracellular matrix.[38]

166 Osteoarthritis - Progress in Basic Research and Treatment

Till date, it is well documented that SOST inhibition is effective for treatment of osteoporo‐ sis. Tanners group have performed the DNA aptamer selectively against sclerostin, and characterized DNA aptamer-sclerostin binding affinity.[67] Aptamers can be used for therapeutic purposes and have been investigated in major disease such as osteoarthritis and osteoporosis.[68]

There are several potential advantages of using aptamers for osteoarthritis and osteoporosis. Nucleic acids show good pharmacokinetic parameters in cartilage and joints, and many therapeutic targets tend to be extracellular so that the challenge of cross-membrane delivery of the nucleic acid can be avoided. Furthermore, aptamers also hold particular promise in conjunction with other technologies such as fluorescent nanoclusters which open up new possibilities for diagnostic imaging. At last the stable aptamers display effective and specific dose-dependent inhibition of sclerostin's antagonistic effect on Wnt activity. Their studies have provided an alternative approach to inhibit sclerostin function with therapeutic potential.

However, there is no pre-clinical or clinical evidence to show its efficacy for the treatment of osteoarthritis. Its role in OA treatment is still under premature. É. Abed *et al.* explored the role played by Sirtuin 1 and SOST on the abnormal mineralization and cWnt signaling in human osteoarthritic subchondral osteoblast (OA Ob). The results indicated that high level of SOST was responsible, in part for the reduced cWnt and mineralization of human OA Ob, which in turn is linked with abnormal SIRT1 levels in these pathological cells.[69] A recent study found that absence of sclerostin in mice with genetic knockout of sclerostin did not alter development of age-dependent osteoarthritis, and that anti-sclerostin therapy with a monoclonal antibody in rats with post-traumatic osteoarthritis had no effect on articular cartilage remodeling.[50] Anyway, antisclerostin therapy has appeared be a promising approach to the treatment of osteoporosis, while Wnt/β-catenin signaling has also been implicated in the pathogenesis of osteoarthritis, with the potential for therapeutic intervention yet to be determined.[70]

#### **4.2. Inhibition of MMPs and ADAMTs**

According to the reports, it is known that the MMPs can be effectively inhibited by TIMP-1, -2, -3, -4.[71] Similar to MMPs, ADAMTS family members can also be inhibited effectively by TIMPs.[72-73] It is illustrated that TIMPs is a significant candidate of blocking cartilage degradation. And of particular note is that TIMP-3 can inhibit several ADAM/ADAMTS proteinases, as reported in lots of existed literatures, while TIMP-1 shows ability to inhibit ADAMTS-10.[72-77] In addition, it is also reported that TIMP-3 can be endocytosed and degraded by chondrocytes,[78] suggesting that its activity in cartilage may be regulated posttranslationally rather than transcriptionally.[10]

Wayne *et al*. have demonstrated that inhibition of full-length ADAMTS-4 by TIMP-3 was enhanced in the presence of aggrecan, and this interaction was mediated largely through the binding of aggrecan and the spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. Therefore, the results also indicated that the cartilage environment could modulate the function of protease-inhibitor systems and have relevance for therapeutic approaches to aggrecanase modulation.[79]

ADAMTS-2 is an activity necessary for the formation of extracellular matrix and responsible for cleaving the N-propeptides of procollagens I–III. Wang *et al.* have shown that TIMP-3 inhibited ADAMTS-2 *in vitro* with apparent Ki values of 160 and 602 nM, in the presence of heparin or without respectively. In a word, TIMP-3 was shown to inhibit procollagen proc‐ essing by cells.[80]

#### **4.3. Inhibition of cathepsin K**

Cathepsin K contains a highly conserved catalytic triad Cys25, His159, and Asn175 within its active site. And in design of the cathepsin K inhibitors, there are several key elements that need to be considered, its inhibitors must be reversible so as to prevent antigenicity arising from covalent modification of proteins *via* irreversible, such as aldehydes, ketones and nitriles. These reversible inhibitors have become more crucial in recent years because they combine strongly to the cathepsin K with low reactivity towards cellular nucleophiles.[81] In addition, the selectivity of inhibitors towards cathepsin K and other cysteine are also desirable to avoid side effects.[82]

#### *4.3.1. Aldehydes and ketones*

Aldehydes and ketones modulate cathepsin K activity *via* hemiacetal/thiohemiacetal and bond formation. Some representatives of aldehyde- and ketone-based reversible covalent cathepsin K inhibitors have reached stages of clinical trials.[83-84]

Since selectivity is important when designing potential drugs to avoid undesired toxicities, Boros *et al*. have obtained several of selective aldehyde-based inhibitors[85] and some of them presented at least 500-fold more potent for cathepsin K than cathepsin B or L. These results gave the encouragement that the aldehyde-based inhibitors could be developed as an anti-OA drug in targeting Cath K. In addition, cyclic ketone was also indicated as the inhibitors of the cysteine protease cathepsin K.

Crystallographic and structure-activity (SA) studies on acyclic ketone-based inhibitors of cathepsin K have guided the design and identification of two series of cyclic ketone inhibitors. Marquis *et al.* have found that the 3-amidopyrrolidin-4-one inhibitors were bound into the active site of the cathepsin K with two alternate directions. Epimerization issues associated with the labile alpha-amino ketone diastereomeric center contained within these inhibitor classes has proven to limit their utility.[86] The results showed that the heterogeneous ketone inhibitors have different influence on the practical application.

#### *4.3.2. Nitriles*

Wayne *et al*. have demonstrated that inhibition of full-length ADAMTS-4 by TIMP-3 was enhanced in the presence of aggrecan, and this interaction was mediated largely through the binding of aggrecan and the spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. Therefore, the results also indicated that the cartilage environment could modulate the function of protease-inhibitor systems and have relevance for therapeutic approaches to aggrecanase modulation.[79]

ADAMTS-2 is an activity necessary for the formation of extracellular matrix and responsible for cleaving the N-propeptides of procollagens I–III. Wang *et al.* have shown that TIMP-3

heparin or without respectively. In a word, TIMP-3 was shown to inhibit procollagen proc‐

Cathepsin K contains a highly conserved catalytic triad Cys25, His159, and Asn175 within its active site. And in design of the cathepsin K inhibitors, there are several key elements that need to be considered, its inhibitors must be reversible so as to prevent antigenicity arising from covalent modification of proteins *via* irreversible, such as aldehydes, ketones and nitriles. These reversible inhibitors have become more crucial in recent years because they combine strongly to the cathepsin K with low reactivity towards cellular nucleophiles.[81] In addition, the selectivity of inhibitors towards cathepsin K and other cysteine are also desirable to avoid

Aldehydes and ketones modulate cathepsin K activity *via* hemiacetal/thiohemiacetal and bond formation. Some representatives of aldehyde- and ketone-based reversible covalent cathepsin

Since selectivity is important when designing potential drugs to avoid undesired toxicities, Boros *et al*. have obtained several of selective aldehyde-based inhibitors[85] and some of them presented at least 500-fold more potent for cathepsin K than cathepsin B or L. These results gave the encouragement that the aldehyde-based inhibitors could be developed as an anti-OA drug in targeting Cath K. In addition, cyclic ketone was also indicated as the inhibitors of the

Crystallographic and structure-activity (SA) studies on acyclic ketone-based inhibitors of cathepsin K have guided the design and identification of two series of cyclic ketone inhibitors. Marquis *et al.* have found that the 3-amidopyrrolidin-4-one inhibitors were bound into the active site of the cathepsin K with two alternate directions. Epimerization issues associated with the labile alpha-amino ketone diastereomeric center contained within these inhibitor classes has proven to limit their utility.[86] The results showed that the heterogeneous ketone

values of 160 and 602 nM, in the presence of

inhibited ADAMTS-2 *in vitro* with apparent Ki

168 Osteoarthritis - Progress in Basic Research and Treatment

K inhibitors have reached stages of clinical trials.[83-84]

inhibitors have different influence on the practical application.

essing by cells.[80]

side effects.[82]

*4.3.1. Aldehydes and ketones*

cysteine protease cathepsin K.

**4.3. Inhibition of cathepsin K**

Inhibitors for papain-like cysteine are derived from peptides, they contains electrophilic group and have been shown to covalently interact with the thiol group at the active site of cathepsin K under formation a thioimidate adduct. Among many types of inhibitors, nitriles have received much attention in recent study.[87-88]

Three chemical classes of nitrile-containing inhibitors of cysteine proteases are known as: **a**) cyanamides,[89] **b**) aromatic nitriles,[90] and **c**) aza-nitrile derivatives, which includes a P1 aza-amino nitrile.[91-93] Among these inhibitors the aza-nitrile derivatives are the optimal ones due to the unique properties such as proteolytical stabilization, reversible binding and excellent inhibitory activity.[94]

In 2013, Ren and Yuan *et al*. in our research group has synthesized two new series of cathepsin K inhibitors. The first series with P2-P3 amide linker have both high selectivity and potency, especially the *meta*-triaryl compound **13** is significantly more potent to cathepsin K (Ki = 0.0031 nM). The second series without the P2-P3 amide linkage have showed a remarkable improvements, the triaryl *meta*-product **13'** possessed a favorable balance between potency (Ki = 0.29 nM) and selectivity of cathepsin K over L, S, B (320-, 1784-, 8566-fold). Such a selective improvement would be useful to avoid harmful side effects in practical applications of the inhibitors.[94-95]

#### **5. Conclusion and perspective**

A significant improvement about scientific cognition of osteoarthritis have been achieved in the past decades, both the aggrecan and collagen play a supporting role in cartilage. It is indisputable that there is a close relationship between the component lost and cartilage degradation, and people are becoming increasingly aware that osteoarthritis is a serious joint disease which is adjusted by proteases and is performed as the degradation of the cartilage extracellular matrix. In addition, we also summarized the profoundly reorganizations that risk factors for OA contain obesity, sports injury, joint instability, particular muscle weakness, genetic, occupational factors and so on. These are related with mechanical, genetic, metabolic factors launch and hold the biochemical changes result the joint dysfunction.

As stated above, a wide range of work in the past years has proved that several proteases are intertwined with osteoarthritis and focused upon in clinical trails. Sclerostin, MMPs, ADAMTS, cathepsins and LDH have become key targets in the development of the diagnosis and treatment of osteoarthritis, and significant progress has been made over the decades. It is no doubt that international co-work in this area will made great progress in the near future and lead to some effective treatment methods in order to alleviate the symptoms and hamper the progression of osteoarthritis.

#### **Acknowledgements**

This work was supported by the National Natural Science Foundation of China (21373101, 20973073, and 91027027) and the Innovation Program of the State Key Laboratory of Supra‐ molecular Structure and Materials, Jilin University.

#### **Author details**

Xiao-Yu Yuan1 , Liping Zhang2 and Yuqing Wu1\*

\*Address all correspondence to: yqwu@jlu.edu.cn

1 State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, China

2 Grain and Oil Food Processing Key Laboratory of Jilin Province, Jilin Business and Technology College, Changchun, China

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170 Osteoarthritis - Progress in Basic Research and Treatment

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### **The Cholinergic System in Relation to Osteoarthritis**

#### Sture Forsgren

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60537

#### **1. Introduction**

The cholinergic system is of interest for the synovial tissue of joints and for arthritic processes, including in osteoarthritis (OA). One aspect is that stimulation via the vagal nerve leads to hampering of arthritic processes. Another is that there is evidence of local acetylcholine (ACh) production within the synovial tissue of human joints, including joints in OA and rheumatoid arthritis. There is furthermore a marked presence of the nicotinic acetylcholine receptor AChRα7 (α7nAChR) in the synovial tissue. Influences on this receptor are known to have antiinflammatory and healing effects. Overall, the concept of a "cholinergic anti-inflammatory pathway" has emerged for various parts of the body. That includes the situation in arthritis. This means that released ACh can have anti-inflammatory effects, in parallel with other favourable effects including wound-healing effects, implying that increased ACh effects might be of value in situations with arthritis. However, focus should further be made on the fact that there is not only evidence of ACh production but also ACh degradation within the synovial tissue. This is related to expressions of acetylcholinesterase (AChE). Of interest in this respect is that reductions of AChE activity via use of AChE inhibitor drugs are used in other situations (e.g. Alzheimer´s disease). The aspects concerning ACh production/degradation in the synovial tissue, the fact that vagal stimulation decreases arthritic processes and the known presence of the potent AChRα7 receptor in synovial tissue should be further considered concerning arthritis in the future. That includes the situation in OA.

#### **2. Why focus on the cholinergic system when discussing osteoarthritis**

It can seem far-fetched to consider aspects related to the cholinergic system when discus‐ sing arthritis, including osteoarthritis. It is thus namely well-known that there is no cholinergic innervation of the joints. They are on the other hand well-equipped with sensory

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and sympathetic innervations. Nevertheless, as will be discussed below, cholinergic stimulations (via the vagal nerve) have been shown to have effects for joints. It has actually also been shown that the bones of mice are functionally innervated by the vagus nerve [1]. On the whole, it is well-known that the vagus nerve plays an anti-inflammatory role in various other parts of the body [2].

Another aspect concerning joint synovial tissue and the cholinergic system has evolved. That is related to the existence of a so-called non-neuronal cholinergic system. Such a system is nowadays well-known for different parts of the body [3]. New information on this system is gradually attained. We have noticed the existence of a non-neuronal cholinergic system in the synovial tissue of the knee joint of humans [4]. We have thus observed that immunoactive cells as well as fibroblasts in the synovial tissue of patients suffering from severe arthritis [rheu‐ matoid arthritis (RA) as well as osteoarthritis (OA)] show expressions of the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) at both mRNA and protein levels [4]. This observation suggests that there is a local ACh production in the synovial tissue. There is only little information on the other aspect of ACh metabolism, namely ACh degra‐ dation. This will be discussed below.

Another noteworthy aspect is the finding that there are marked expressions of the nicotinic alpha7nACh receptor (α7nAChR) in the synovial tissue of arthritic patients. These observa‐ tions have been made in studies in our laboratory [5] and in studies by other researchers [6,7]. That includes patients with osteoarthritis and is related to α7nAChR expressions for the fibroblast-like and inflammatory cells within the synovial tissue as well as for the synovial lining [5,8]. This receptor is known to be involved in inflammatory and remodulation proc‐ esses, most notably having anti-inflammatory effects [9,10]. The findings have lead to sugges‐ tions that increased functions of ACh via effects on the α7nAChR can be positive for the arthritic processes [8,11].

Based on the aspects described above, further considerations on the cholinergic system for arthritis are here focused on. These relate to considerations on vagal effects for joint function, possible interference of the ACh-degrading enzyme acetylcholinesterase (AChE) and the possibility that increased ACh influences on the α7nAChR might be a promising strategy.

#### **3. The vagus nerve in relation to arthritis**

It has since several years been considered that effects via the vagal nerve can be of functional importance for the synovial tissue, not least in situations with arthritis. Thus, stimulatory effects on the vagal nerve have been show to be hampering for experimentally-induced arthritic processes [6] and paw inflammation [12]. There is on the other hand an exacerbation of the experimental arthritis after vagotomy [6,13]. The findings concerning the vagus nerve and vagotomy are unexpected as there is no vagal, nor other cholinergic, innervation in synovial tissue. One possibility that is discussed is that the effects are indirect, namely via vagal effects on other sites such as the region of the spleen [14] (see also [6]). It is well known that there are communications between the vagal nerve and the splenic nerve via the celiac and superior mesenteric ganglia (for a review, see [15,16]).

It is known that signaling via the vagus nerve that is leading to anti-inflammatory effects is initiated in the brainstem nuclei of the vagus nerve, secondarily leading to effects on the peripheral ganglia referred to above. This is part of the so-called inflammatory reflex whereby peripheral afferent nerves are primarily sensed, secondarily leading to efferent effects [17], in this case via the efferent part of the vagus nerve. It is well-known that the vagus nerve is a main component in the neuro-endocrine homeostasis via effects through its afferent and efferent neurons (for a review, see [2]).

From a functional point of view, it is of interest to note that ACh released from cholinergic nerves like the vagus nerve has immunomodulatory effects. These effects are considered to be anti-inflammatory [9,12]. The concept of a "cholinergic anti-inflammatory pathway" has hereby emerged [10,18], including for the synovial tissue [11]. In accordance with this, it is shown that electrical stimulation of the vagus nerve leads to an attenuation in macrophage activation [19].

It has been shown that subdiaphragmatic vagotomy in mice leads to reduced bone mass, bearing in mind that the mouse skeleton normally has a vagal innervation [1]. In a recent sudy it was shown that the severity of collagen-induced arthritis was reduced by electrical vagus nerve stimulation using a cuff electrode [20]. The cuff electrode that was used is analogous to the one used in treatment of drug-resistent epilepsy [21]. It was suggested that electrical neurostimulation via use of implanted vagus nerve stimulation cuff electrodes can be useful in treatment tests for various immune-mediated inflammatory disorders in man [20].

#### **4. The α7nAChR in relation to arthritis**

and sympathetic innervations. Nevertheless, as will be discussed below, cholinergic stimulations (via the vagal nerve) have been shown to have effects for joints. It has actually also been shown that the bones of mice are functionally innervated by the vagus nerve [1]. On the whole, it is well-known that the vagus nerve plays an anti-inflammatory role in

Another aspect concerning joint synovial tissue and the cholinergic system has evolved. That is related to the existence of a so-called non-neuronal cholinergic system. Such a system is nowadays well-known for different parts of the body [3]. New information on this system is gradually attained. We have noticed the existence of a non-neuronal cholinergic system in the synovial tissue of the knee joint of humans [4]. We have thus observed that immunoactive cells as well as fibroblasts in the synovial tissue of patients suffering from severe arthritis [rheu‐ matoid arthritis (RA) as well as osteoarthritis (OA)] show expressions of the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) at both mRNA and protein levels [4]. This observation suggests that there is a local ACh production in the synovial tissue. There is only little information on the other aspect of ACh metabolism, namely ACh degra‐

Another noteworthy aspect is the finding that there are marked expressions of the nicotinic alpha7nACh receptor (α7nAChR) in the synovial tissue of arthritic patients. These observa‐ tions have been made in studies in our laboratory [5] and in studies by other researchers [6,7]. That includes patients with osteoarthritis and is related to α7nAChR expressions for the fibroblast-like and inflammatory cells within the synovial tissue as well as for the synovial lining [5,8]. This receptor is known to be involved in inflammatory and remodulation proc‐ esses, most notably having anti-inflammatory effects [9,10]. The findings have lead to sugges‐ tions that increased functions of ACh via effects on the α7nAChR can be positive for the

Based on the aspects described above, further considerations on the cholinergic system for arthritis are here focused on. These relate to considerations on vagal effects for joint function, possible interference of the ACh-degrading enzyme acetylcholinesterase (AChE) and the possibility that increased ACh influences on the α7nAChR might be a promising strategy.

It has since several years been considered that effects via the vagal nerve can be of functional importance for the synovial tissue, not least in situations with arthritis. Thus, stimulatory effects on the vagal nerve have been show to be hampering for experimentally-induced arthritic processes [6] and paw inflammation [12]. There is on the other hand an exacerbation of the experimental arthritis after vagotomy [6,13]. The findings concerning the vagus nerve and vagotomy are unexpected as there is no vagal, nor other cholinergic, innervation in synovial tissue. One possibility that is discussed is that the effects are indirect, namely via vagal effects on other sites such as the region of the spleen [14] (see also [6]). It is well known that

various other parts of the body [2].

180 Osteoarthritis - Progress in Basic Research and Treatment

dation. This will be discussed below.

**3. The vagus nerve in relation to arthritis**

arthritic processes [8,11].

The α7nAChR is considered to be much involved in the obtaining of anti-inflammatory effects of ACh in various situations [3,22]. It is namely shown that this receptor contributes to antiinflammatory effects of ACh in several models [12,23]. α7nAChR agonists are shown to suppress the production of various cytokines such as TNF alpha [12,22].

Based on what is described above, it is of great interest to note that the α7nAChR is present in the synovial tissue. That has been shown for the synovial tissue of patients with OA [5,8], RA [7,11] and psoriatic arthritis [7]. The findings concerning the α7nAChR have led to suggestions that interference with this receptor in clinical situations with arthritis might be useful [24,25]. α7nAChR agonists are not least suggested to be candidates as treatments for RA [26]. In accordance with such a proposal are the findings that synovial fibroblasts respond in vitro to cholinergic stimulation, via the α7nAChR, leading to a potent inhibition of proinflammatory cytokines [27]. Studies on the healing of skin wounds do also suggest that the α7nAChR is involved in the repair processes that occur for these wounds [28]. It is also shown that the α7nAChR is involved in the repair of wounds of respiratory epithelium [29].

#### **5. AChE in relation to arthritis**

The main ACh-degrading enzyme is acetylcholinesterase (AChE). The bulk of AChE of the neurons is in the axons and AChE is known to be associated with the membrane of this [30]. AChE is shown to be functional in embryonic muscle before it is accumulated at the sites of nerve-muscle contact [31]. AChE activity is also shown for a large number of non-neuronal cell types. That includes T-cells [32], fibroblasts of various locations [33], cells in lung tissue [34], cells of human gingival and esophageal epithelia [35] and embryonic stem cells [36]. AChE is also typically confined to the membranes of red blood cells [37]. Other components of the cholinergic system are also present in these cell types.

It is of relevance to notice that interference with AChE activity can be performed and that treatments for which this is done are used clinically. That includes the situations in myastenia gravis and Alzheimer´s disease. In the case of myastenia gravis, where there is an occurrence of few receptors, the treatment is of value in order to extend the effects of ACh [38]. The AChE inhibitor drugs donepezil, galantamine and rivastigmine are being tried for patients with Alzheimer´s disease [39]. In this case, where there is a reduced concentration of ACh, the point with the AChE inhibitors is to increase the concentration of the transmitter. A cholinergic deficiency is a feature that can be important for the development of the cognitive decline that occurs in Alzheimer´s disease. There are also other fields of usage of AChE inhibitors; they are e.g. used in insecticides and nerve gases.

There is very little information on the patterns of AChE activity for synovial tissue. Neverthe‐ less, AChE activity, in parallel with other components of cholinergic function, has been clearly detected in the knee joint synovial tissue of patients with RA and OA in a study using RT-PCR methods [40]. In our laboratory, existence of AChE activity in human knee joint synovial tissue has also been observed histochemically (unpublished observations).

The most well known function of AChE is to terminate neurotransmission at the cholinergic synapses via splitting of ACh. ACh is hereby hydrolyzed into choline and acetate. The degradation is rapid. However, AChE is also known to exhibit several non-classical roles, features that are of importance when considering both the neuronal and non-neuronal cholinergic systems [41]. That includes effects on cell differentiation and synaptogenesis along the nervous system, hydrolysis of neuropeptides, and effects in heart morphogenesis (for a review, see [42]). One cell type for which AChE is highly expressed is re-epithelialising epidermal keratinocytes during in vivo healing of mouse skin [43].

The exact functions of AChE in relation to the regulations of the non-neuronal cholinergic system at its various locations in the body are somewhat unclear [3]. It may be that the magnitude of ACh degrading activity is low in tissues like airway epithelium [44,45], and in cells of the placenta [33]. How the situation is for synovial tissue remains to be defined. Nevertheless, as there indeed is an occurrence of AChE in the synovial tissue it is likely that the function of the ACh that is produced in synovial tissue is limited to the precise area where it is produced. It may well be so that up- and down-regulations of production and release of ACh in the synovial tissue are parallelled by up- and down-regulations of AChE activity. In line with such a proposal is the finding that the immunological stimulation that leads to T-cell activation and upregulation of ACh synthesis and ACh receptor expression also leads to a marked ACh degradation [46].

Further studies on the importance and function of AChE for synovial tissue are needed in order to reveal the possible usefulness of interference with the effects of the enzyme in arthritis.

#### **6. Concluding remarks**

**5. AChE in relation to arthritis**

182 Osteoarthritis - Progress in Basic Research and Treatment

cholinergic system are also present in these cell types.

e.g. used in insecticides and nerve gases.

The main ACh-degrading enzyme is acetylcholinesterase (AChE). The bulk of AChE of the neurons is in the axons and AChE is known to be associated with the membrane of this [30]. AChE is shown to be functional in embryonic muscle before it is accumulated at the sites of nerve-muscle contact [31]. AChE activity is also shown for a large number of non-neuronal cell types. That includes T-cells [32], fibroblasts of various locations [33], cells in lung tissue [34], cells of human gingival and esophageal epithelia [35] and embryonic stem cells [36]. AChE is also typically confined to the membranes of red blood cells [37]. Other components of the

It is of relevance to notice that interference with AChE activity can be performed and that treatments for which this is done are used clinically. That includes the situations in myastenia gravis and Alzheimer´s disease. In the case of myastenia gravis, where there is an occurrence of few receptors, the treatment is of value in order to extend the effects of ACh [38]. The AChE inhibitor drugs donepezil, galantamine and rivastigmine are being tried for patients with Alzheimer´s disease [39]. In this case, where there is a reduced concentration of ACh, the point with the AChE inhibitors is to increase the concentration of the transmitter. A cholinergic deficiency is a feature that can be important for the development of the cognitive decline that occurs in Alzheimer´s disease. There are also other fields of usage of AChE inhibitors; they are

There is very little information on the patterns of AChE activity for synovial tissue. Neverthe‐ less, AChE activity, in parallel with other components of cholinergic function, has been clearly detected in the knee joint synovial tissue of patients with RA and OA in a study using RT-PCR methods [40]. In our laboratory, existence of AChE activity in human knee joint synovial tissue

The most well known function of AChE is to terminate neurotransmission at the cholinergic synapses via splitting of ACh. ACh is hereby hydrolyzed into choline and acetate. The degradation is rapid. However, AChE is also known to exhibit several non-classical roles, features that are of importance when considering both the neuronal and non-neuronal cholinergic systems [41]. That includes effects on cell differentiation and synaptogenesis along the nervous system, hydrolysis of neuropeptides, and effects in heart morphogenesis (for a review, see [42]). One cell type for which AChE is highly expressed is re-epithelialising

The exact functions of AChE in relation to the regulations of the non-neuronal cholinergic system at its various locations in the body are somewhat unclear [3]. It may be that the magnitude of ACh degrading activity is low in tissues like airway epithelium [44,45], and in cells of the placenta [33]. How the situation is for synovial tissue remains to be defined. Nevertheless, as there indeed is an occurrence of AChE in the synovial tissue it is likely that the function of the ACh that is produced in synovial tissue is limited to the precise area where it is produced. It may well be so that up- and down-regulations of production and release of ACh in the synovial tissue are parallelled by up- and down-regulations of AChE activity. In

has also been observed histochemically (unpublished observations).

epidermal keratinocytes during in vivo healing of mouse skin [43].

This review shows three aspects of the cholinergic system in relation to arthritis. It is obvious that stimulation of the vagal nerve has effects, that there is a non-neuronal cholinergic system in the synovial tissue and that there in parallel with expressions favouring ACh production also are expressions favouring ACh degradation in the synovial tissue. Although a lot of the information is related to the situation in RA, the various features of the cholinergic system are also related to the situation in OA. All these features concerning the cholinergic system highlight the relevance of further studies on the functional importance of this system for joint function, including the situation in OA.

#### **Acknowledgements**

Financial support for the studies performed at Department has been provided by the Faculty of Medicine.

#### **Author details**

Sture Forsgren\*

Address all correspondence to: Sture.Forsgren@anatomy.umu.se

Department of Integrative Medical Biology Anatomy Section Umeå University Umeå, Sweden

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**Therapy, Treatment and Management**

### **Cell-Based Therapy for Human Osteoarthritis**

Rie Kurose and Takashi Sawai

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/60754

#### **1. Introduction**

Articular cartilage has a function to smooth the movement of the joints and to decrease the coefficient of friction. Recently, it has been reported that "lubricin," a mucinous glycoprotein encoded by the *PRG4* gene, provides boundary lubrication in the articular joints [1]. Also, the articular cartilage has a role of shock absorber against an external force. The articular cartilage is hyaline cartilage composed of water (approximately 70%), cell (less than 3%), and abundant extracellular matrix (approximately 20%) such as type II collagen and proteoglycan. The articular cartilage is highly differentiated avascular tissue, and blood vessels, nerves, and lymphatic vessels are not present in the articular cartilage of adults. Based on the above, it is well known that damaged articular cartilage has a very limited capacity for self-repair. Even minor injuries may lead to progressive damage and result in osteoarthritic joint with significant pain and disability.

In 1989, it was reported that cartilage defect could be repaired with cultured chondrocytes in animal experiments in rabbits [2]. Based on this result, Brittberg et al. [3] performed clinical application for humans of autologous chondrocyte transplantation in 1994. However, some problems have been pointed out in this surgery. One was the possibility of dedifferentiated of cultured cells, which decreased matrix production ability because of monolayer culture. Another was the uneven distribution of injected chondrocytes caused in part by leak of the cell suspension from the periosteum covering the cartilage defect. To solve these problems, Ochi et al. [4] devised the use of atelocollagen as a scaffold: implantation of three-dimensional cartilage-like tissues using cultured autologous chondrocytes embedded in atelocollagen gel. This method has been currently used as a clinical application for osteochondritis dissecans since 2012 [5]. However, there is yet no clinically approved cell-based strategy for treatment of osteoarthritis (OA)-based cartilage lesions. Basic researchers and clinicians are focusing on alternative methods for cartilage repair, aiming to regenerate OA cartilage tissues. Cell-based

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

therapy is an attractive biological method, and its studies have progressed in accordance with the development of tissue engineering.

#### **2. Cell-based therapy for OA**

Cell-based therapies using various cell types such as the chondrocytes or the bone marrow have been researched conventionally. Autologous chondrocytes are actually in clinical application for cartilage defects, and the surgical procedures such as marrow stimulation have also been performed for the purpose of cartilage repair. Although short-term results of these methods are good, there remains in doubt about long-term results. Mesenchymal stem cells (MSCs) are harvested from different sources such as the bone marrow, synovial tissue, and adipose tissue and have multilineage potentials. Recently, research in cartilage tissue engi‐ neering focuses on the use of MSCs as an alternative to autologous chondrocytes. Furthermore, induced pluripotent stem (iPS) cells or Muse cells might overcome the disadvantages of MSCs: insufficient number of cells, cell harvesting procedures with pain, and unstable differentiation potential of cells.

The regeneration of hyaline cartilage provides to improve the symptoms and ultimately prevent or delay progression to osteoarthritic joints. Cell-based therapies have been increas‐ ingly applied because they have the potential to regenerate the cartilage tissues.

#### **2.1. Chondrocyte**

#### *2.1.1. Autologous chondrocyte implantation*

Autologous chondrocyte implantation (ACI) was first reported in 1994 for treatment of focal cartilage defects in the tibiofemoral and patellofemoral compartments [3]. Since this report, chondrocyte-based therapy has become to be expected; a periosteal cover (first-generation ACI), a collagen-membrane cover (second -generation ACI), and a variety of three-dimensional scaffolds (third-generation ACI) are used for the methods of fixation. In these methods, arthrotomy and a two-stage surgical procedure are used. Long-term durability and success as long as 11 years of follow-up periods have been reported [6–13]. ACI is the first articular cartilage repair method using tissue engineering, but there is a problem that a sufficient number of cells cannot be secured by the case. Its usefulness is still under discussion.

#### *2.1.2. Matrix-induced autologous chondrocyte implantation*

A variation of the original periosteum membrane technique is matrix-induced autologous chondrocyte implantation (MACI). MACI membrane consists of a porcine type I/III collagen bilayer seeded with chondrocytes and MACI can promote hyaline-like cartilage repair. The technique of MACI procedure can eliminate many problems of first- or second-generation ACI, and the cell-seeded membrane can be implanted over a less inaccessible area or at osteochon‐ dral junctions because of its adhesive property [14]. Meyerkort et al. reported that both MACI and tibial tubercle transfer (TTT) using the Fulkerson technique were used to treat cartilage defects in the patellofemoral joints and provided a durable graft on 5-years resultant with clinical improvement [15–17].

#### **2.2. Bone marrow**

therapy is an attractive biological method, and its studies have progressed in accordance with

Cell-based therapies using various cell types such as the chondrocytes or the bone marrow have been researched conventionally. Autologous chondrocytes are actually in clinical application for cartilage defects, and the surgical procedures such as marrow stimulation have also been performed for the purpose of cartilage repair. Although short-term results of these methods are good, there remains in doubt about long-term results. Mesenchymal stem cells (MSCs) are harvested from different sources such as the bone marrow, synovial tissue, and adipose tissue and have multilineage potentials. Recently, research in cartilage tissue engi‐ neering focuses on the use of MSCs as an alternative to autologous chondrocytes. Furthermore, induced pluripotent stem (iPS) cells or Muse cells might overcome the disadvantages of MSCs: insufficient number of cells, cell harvesting procedures with pain, and unstable differentiation

The regeneration of hyaline cartilage provides to improve the symptoms and ultimately prevent or delay progression to osteoarthritic joints. Cell-based therapies have been increas‐

Autologous chondrocyte implantation (ACI) was first reported in 1994 for treatment of focal cartilage defects in the tibiofemoral and patellofemoral compartments [3]. Since this report, chondrocyte-based therapy has become to be expected; a periosteal cover (first-generation ACI), a collagen-membrane cover (second -generation ACI), and a variety of three-dimensional scaffolds (third-generation ACI) are used for the methods of fixation. In these methods, arthrotomy and a two-stage surgical procedure are used. Long-term durability and success as long as 11 years of follow-up periods have been reported [6–13]. ACI is the first articular cartilage repair method using tissue engineering, but there is a problem that a sufficient

number of cells cannot be secured by the case. Its usefulness is still under discussion.

A variation of the original periosteum membrane technique is matrix-induced autologous chondrocyte implantation (MACI). MACI membrane consists of a porcine type I/III collagen bilayer seeded with chondrocytes and MACI can promote hyaline-like cartilage repair. The technique of MACI procedure can eliminate many problems of first- or second-generation ACI, and the cell-seeded membrane can be implanted over a less inaccessible area or at osteochon‐ dral junctions because of its adhesive property [14]. Meyerkort et al. reported that both MACI and tibial tubercle transfer (TTT) using the Fulkerson technique were used to treat cartilage

ingly applied because they have the potential to regenerate the cartilage tissues.

the development of tissue engineering.

192 Osteoarthritis - Progress in Basic Research and Treatment

**2. Cell-based therapy for OA**

potential of cells.

**2.1. Chondrocyte**

*2.1.1. Autologous chondrocyte implantation*

*2.1.2. Matrix-induced autologous chondrocyte implantation*

Marrow stimulation techniques such as abrasion arthroplasty, drilling, and microfracture penetrate the subchondral bone and induce the formation of fibrocartilage repair tissues [18]. Although these methods were performed traditionally, it has been shown recently that bleeding from the bone marrow resulted in the supply of cytokines, osteoprogenitor cells, and chondroprogenitor cells. Also, there have been many reports related to induction of MSCs by bone marrow stimulation techniques. Clinically, although excellent short-term outcomes have been reported after bone marrow stimulation, the durability of marrow-stimulated repair tissues has shown the tendency to functional decline with further follow-up.

#### *2.2.1. Abrasion arthroplasty*

In 1986, Johnson [19] reported about achievement of abrasion arthroplasty, removing dead bone superficially and providing vascularity tissues for blood clot attachment. According to this method, subsequent fibrocartilage formation was maintained integrity for up to 6 years. Sansone et al. [20] reported a 20-year follow-up of abrasion arthroplasty, with a positive functional outcome of 67.9%.

#### *2.2.2. Multiple perforation (drilling)*

In 1959, Pridie [21] reported about a method for multiple perforation to subchondral bone using a drill with a 6-mm diameter. After partial weight bearing for 6 weeks, holes drilled were filled with fibrocartilage. However, recent reports related to medial opening-wedge high tibial osteotomy suggest that subchondral drilling is not necessary because there is no significant difference in the formation of fibrocartilage with or without subchondral drilling [22].

#### *2.2.3. Microfracture*

Microfracture is common procedure for cartilage repair, which produces a small fracture of the subchondral bone using awls to penetrate eburnated bone to promote blood flow to the bony surface. There is a report that short-term results were good, but 38.1% proceeded to total knee arthroplasty (TKA) in a 6.8-year follow-up [23]. In other papers, the survival rate was 88.8% at a 5-year follow-up and decreased 67.9% at a 10-year follow-up [24, 25].

#### **2.3. Mesenchymal stem cell**

Nucleated cells in the bone marrow are mostly hematological cells, which float when they are cultured. However, some of the cells in the bone marrow adhere to culture dishes *in vitro*, proliferate itself, and form colonies. Thus, adherent cells are regarded as bone marrow mesenchymal cells (BMMCs). In 1974, Friedenstein et al. [26] reported that osteochondral progenitor cells were present in BMMCs, and in 1999, Pittenger et al. [27] reported about the pluripotency of BMMCs, named MSCs. Currently, it is well known that MSCs are adult stem cells and have the possibility of differentiating into multiple cell types, including adipocytes, chondrocytes, osteocytes, and cardiomyocytes [28].

#### *2.3.1. Bone marrow MSC*

Articular cartilage is insufficient for the capacity of cartilage repair, and the damaged cartilage tissues are not restored in complete hyaline cartilage in adults. We reasoned that the chon‐ droprogenitor cells supplied to the cartilage defects are not sufficient. Then we have focused on the BMMCs, which might include MSCs, to supply sufficient chondroprogenitor cells to cartilage defects [29]. In our study in rabbits, BMMCs, which had a fibroblastic morphology and pluripotency for differentiation, were isolated from the bone marrow of the tibiae of rabbits, grown in monolayer culture. The autologous cells were then implanted into fullthickness articular cartilage defects in the knee joints of each rabbit. Advantages of this method included the use of autologous cells and absence of immunoreactivity. Furthermore, we investigated the efficiency of cartilage-derived morphogenetic protein 1 (CDMP1) genetransfected autologous BMMCs for cartilage repair in a rabbit cartilage defect model [30]. CDMP1, a member of the transforming growth factor-β superfamily, is an essential molecule for the aggregation of mesenchymal cells and acceleration of chondrogenic differentiation. BMMCs were isolated from the bone marrow of the tibiae of rabbits, grown in monolayer culture, and transfected with the CDMP1 gene or a control gene (GFP) by a lipofection method. During *in vivo* repair of full-thickness articular cartilage defects, cartilage regeneration was enhanced by the implantation of CDMP1-transfected autologous BMMCs (Figure 1). The defects were filled with hyaline cartilage, and the deeper zone showed remodeling to sub‐ chondral bone over time. The repair and the reconstitution of zones of hyaline articular cartilage were superior to simple BMMC implantation. The histological score of the CDMP1 transfected BMMC group was significantly better than those of both control BMMC group and empty control group (Tables 1 and 2). Our studies suggest that the modulation of BMMCs by factors such as CDMP1 allows enhanced repair and remodeling compatible with hyaline articular cartilage.

#### *2.3.2. Synovial MSC*

Sekiya et al. [31] previously reported that MSCs in synovial fluid from anterior cruciate ligament injury, meniscus injury, or patients with OA were much more than those from healthy volunteers and increased according to postinjury period or severity. The MSCs in synovial fluid are considered to be derived from synovial tissue and are positive for CD44, CD73, and CD90, which are markers of MSCs, and negative for CD34 and CD45, which is a marker of hematopoietic stem cells and leukocyte progenitor cells, respectively. Intra-articular injection of the synovial MSCs promoted meniscus regeneration and protected articular cartilage by arthroscopic and histological observations in pig [32], rat [33], or porcine [34] massive meniscal defect models. We research for synovial fluid cells that are not accompanied by pain in the cell harvest and describe about its advantage in the latter part of this manuscript.

**Figure 1.** Representative histological appearance of the defects after 4 weeks. (A–J) Safranin-O/fast green staining. (A– C) Empty control group. (D–F) Left knees of GFP-transfected BMMC group. (G–I) Right knees of CDMP1-transfected BMMC group. (D and G, E and H, F and I) Bilateral knee specimens from the same rabbits. (J) Higher magnification of I. (K) Immunohistochemical staining specific for type II collagen. (L) Immunohistochemical staining specific for type I collagen. (A–L) Scale bar is 500 µm.

#### *2.3.3. Adipose-derived MSC*

pluripotency of BMMCs, named MSCs. Currently, it is well known that MSCs are adult stem cells and have the possibility of differentiating into multiple cell types, including adipocytes,

Articular cartilage is insufficient for the capacity of cartilage repair, and the damaged cartilage tissues are not restored in complete hyaline cartilage in adults. We reasoned that the chon‐ droprogenitor cells supplied to the cartilage defects are not sufficient. Then we have focused on the BMMCs, which might include MSCs, to supply sufficient chondroprogenitor cells to cartilage defects [29]. In our study in rabbits, BMMCs, which had a fibroblastic morphology and pluripotency for differentiation, were isolated from the bone marrow of the tibiae of rabbits, grown in monolayer culture. The autologous cells were then implanted into fullthickness articular cartilage defects in the knee joints of each rabbit. Advantages of this method included the use of autologous cells and absence of immunoreactivity. Furthermore, we investigated the efficiency of cartilage-derived morphogenetic protein 1 (CDMP1) genetransfected autologous BMMCs for cartilage repair in a rabbit cartilage defect model [30]. CDMP1, a member of the transforming growth factor-β superfamily, is an essential molecule for the aggregation of mesenchymal cells and acceleration of chondrogenic differentiation. BMMCs were isolated from the bone marrow of the tibiae of rabbits, grown in monolayer culture, and transfected with the CDMP1 gene or a control gene (GFP) by a lipofection method. During *in vivo* repair of full-thickness articular cartilage defects, cartilage regeneration was enhanced by the implantation of CDMP1-transfected autologous BMMCs (Figure 1). The defects were filled with hyaline cartilage, and the deeper zone showed remodeling to sub‐ chondral bone over time. The repair and the reconstitution of zones of hyaline articular cartilage were superior to simple BMMC implantation. The histological score of the CDMP1 transfected BMMC group was significantly better than those of both control BMMC group and empty control group (Tables 1 and 2). Our studies suggest that the modulation of BMMCs by factors such as CDMP1 allows enhanced repair and remodeling compatible with hyaline

Sekiya et al. [31] previously reported that MSCs in synovial fluid from anterior cruciate ligament injury, meniscus injury, or patients with OA were much more than those from healthy volunteers and increased according to postinjury period or severity. The MSCs in synovial fluid are considered to be derived from synovial tissue and are positive for CD44, CD73, and CD90, which are markers of MSCs, and negative for CD34 and CD45, which is a marker of hematopoietic stem cells and leukocyte progenitor cells, respectively. Intra-articular injection of the synovial MSCs promoted meniscus regeneration and protected articular cartilage by arthroscopic and histological observations in pig [32], rat [33], or porcine [34] massive meniscal defect models. We research for synovial fluid cells that are not accompanied by pain in the cell

harvest and describe about its advantage in the latter part of this manuscript.

chondrocytes, osteocytes, and cardiomyocytes [28].

194 Osteoarthritis - Progress in Basic Research and Treatment

*2.3.1. Bone marrow MSC*

articular cartilage.

*2.3.2. Synovial MSC*

Adipose tissues contain various cells such as blood cells, endothelial cells, and smooth muscle cells, in addition to adipocytes. Adipose tissues are also rich in microvasculature which adjoins with MSCs. Adipose-derived MSCs (ASCs) can be established by following method. Subcu‐ taneous or visceral adipose tissues are minced and treated with type I collagenase. Then infranatant cells are centrifuged at low speed, and the cell pellet is placed in a flask. ASCs propagate themselves rapidly. Currently, two clinical trials for humans, which are the intraarticular injection for OA in France and the intravenous administration to rheumatoid arthritis in Spain, have been undergoing.

#### **2.4. Induced pluripotent stem cells**

iPS cells have pluripotency and the potential for self-renewal similar to ES cells. Recent study has made it possible to generate integration-free iPS cells and to differentiate iPS cells toward chondrocytes [35]. As an alternative approach, chondrocytic cells can be induced directly from dermal fibroblasts without going through the iPS cell stage. In 2011, Hiramatsu et al. [35] generated *in vitro* polygonal chondrogenic cells from adult dermal fibroblast cultures by ectopic expression of reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9). Namely, this approach could lead to the preparation of hyaline cartilage directly from skin without generating iPS cells. Recently, Yamashita et al. [36] reported that hyaline cartilage was generated from human iPS cells in immunodeficiency rats and immunosuppressed minipigs.

Table 1. Histological grading scale for cartilage defect\*


Total maximum 20

\*Modified from the scale described by Pineda et al. [46] and Wakitani et al. [47]. † Total smooth area of the reparative cartilage compared with the entire area of \*Modified from the scale described by Pineda et al. [46] and Wakitani et al. [47].

the cartilage defect. † Total smooth area of the reparative cartilage compared with the entire area of the cartilage defect.

1

**Table 1.** Histological grading scale for cartilage defect\*


Table 2. Results of the histological grading scale

\**P* < 0.05, when compared to the GFP group at corresponding time (Mann–Whitney *<sup>U</sup>*-test). # *P* < 0.05, when compared to the empty control at corresponding time (Scheffe test for multiple comparison). \**P* < 0.05, when compared to the GFP group at corresponding time (Mann–Whitney *U*-test).
