Preface

Chapter 6 **Chondral Lesion in the Hip Joint and Current Chondral Repair**

Adrian J. Cassar-Gheiti, Neil G. Burke, Theresa M. Cassar-Gheiti and

**Techniques 103**

Chapter 7 **Osteochondritis Dissecans of the Knee 123** Anthony C. Egger and Paul Saluan

Chapter 8 **Autologous Chondrocyte Implantation: Scaffold-Based**

Michael E. Hantes and Apostolos H. Fyllos

Chapter 10 **MRI Mapping for Cartilage Repair Follow-up 177**

Chapter 11 **Applied Basic Science of the Auricular Cartilage 203** Mohamed Khamis Tolba Mahmoud Abdalla

David C. Flanigan, Joshua S. Everhart and Nicholas A. Early

**Matrix-Induced Chondrogenesis (AMIC) Technique 163**

Chapter 9 **Management of Knee Cartilage Defects with the Autologous**

Kevin J. Mulhall

**VI** Contents

**Solutions 143**

Mars Mokhtar

**Section 3 Head and Neck 201**

This work is the result of a partnership that began in 2011, when I received for the first time the invitation to be the scientific editor of a book on bone grafting, by the still little publisher known as InTech. I remember very well the publisher's proposal to make the knowledge more accessible through the open access system. At that time, I decided to accept the invita‐ tion of the still young publisher, founded in 2004. The reason was the enthusiasm of Ms. Ana Pantar, editorial consultant, and Ms. Jana Sertic, publishing process manager, also, be‐ cause I agreed to the need for a new and must fair publication system.

Now six years later, InTech has grown and thrived. This is the fourth book in which I am the scientific editor. I can say that my respect and warm approval for the quality of the publish‐ er's work only increased.

In this book, entitled *Cartilage Repair and Regeneration*, I am pleased to work with a subject that has gained much notoriety. The hyaline cartilage is a tissue that challenges tissue engi‐ neering and regenerative medicine because of its avascular nature. The chondrocyte, the cell responsible for producing the extracellular matrix that confers the unique properties of the hyaline cartilage, is one of the most difficult cells to be cultured, as well as neurons and en‐ dothelial cells, because of their high degree of differentiation and specialization.

At the same time, the advancement of the life span of the population and the increase in the practice of sports activities have led to an increasing incidence of pains caused by problems associated with cartilage lesions.

In the eleven chapters of this book, the reader will find texts written by researchers working on advanced topics related to basic laboratory research, as well as excellent reviews on the clinical use of currently available therapies.

> **Alessandro R. Zorzi, MD, MSc, PhD and João Batista de Miranda, Prof** Department of Orthopedic Surgery State University of Campinas, Brazil

**Section 1**

**Basic Science**

**Section 1**

## **Basic Science**

**Chapter 1**

**Provisional chapter**

**Viruses: Friends and Foes**

**Viruses: Friends and Foes**

Penny A. Rudd and Lara J. Herrero

Penny A. Rudd and Lara J. Herrero

http://dx.doi.org/10.5772/intechopen.71071

**Abstract**

**repair**

Additional information is available at the end of the chapter

**Keywords:** viral gene therapy, cartilage and bone healing

Additional information is available at the end of the chapter

DOI: 10.5772/intechopen.71071

In this chapter, we will review how viruses can be used to positively affect joints and cartilage of their hosts. Many viruses are arthrogenic, and cause persistent and debilitating arthritis. Even those viruses that are not typically arthrogenic can also cause bone lesions as secondary pathogenesis. Some of these foes include members of the alphaviruses, like chikungunya and Ross River viruses, the rubiviruses, such as rubella, and erythoparvoviruses, like parvovirus B19. Some more uncommon viruses, which can occasionally have detrimental effects on their hosts' joints, include herpes simplex virus, varicella zoster, mumps, human cytomegalovirus, avian orthoreovirus, and caprine arthritis-encephalitis virus. Despite some viruses having negative impacts on cartilage and joints, others have been used as an effective means of gene therapy for bone and cartilage repair. We will take an in-depth look at the current therapeutic strategies for treating arthritis using various viral vectors.

**1. Introduction: viral peptides/vectors used as gene therapy for joint** 

ment of viral vectors to treat the musculoskeletal system, including the joints [6].

Viruses have long been used as vectors for gene therapy. Some of the more popular viral vectors include retroviruses, oncolytic viruses, lentiviruses, adenoviruses, and adeno-associated viruses to name just a few. They are used in a wide variety of fields and are able to treat a diverse range of diseases, including Parkinson's disease, many cancers, amyotrophic lateral sclerosis, genetic disorders, cardiovascular diseases, hemophilia, and central nervous system CNS diseases and disorders [1–5]. In recent years, there has been an increase in the develop-

> © 2016 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,

© 2018 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 reproduction in any medium, provided the original work is properly cited.

Articular cartilage damage can result from a variety of insults, either from over usage, diseases and disorders, or accidents, and often leads to different types of arthritis including

## **Chapter 1**

**Provisional chapter**

## **Viruses: Friends and Foes**

**Viruses: Friends and Foes**

Penny A. Rudd and Lara J. Herrero Penny A. Rudd and Lara J. Herrero Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71071

#### **Abstract**

In this chapter, we will review how viruses can be used to positively affect joints and cartilage of their hosts. Many viruses are arthrogenic, and cause persistent and debilitating arthritis. Even those viruses that are not typically arthrogenic can also cause bone lesions as secondary pathogenesis. Some of these foes include members of the alphaviruses, like chikungunya and Ross River viruses, the rubiviruses, such as rubella, and erythoparvoviruses, like parvovirus B19. Some more uncommon viruses, which can occasionally have detrimental effects on their hosts' joints, include herpes simplex virus, varicella zoster, mumps, human cytomegalovirus, avian orthoreovirus, and caprine arthritis-encephalitis virus. Despite some viruses having negative impacts on cartilage and joints, others have been used as an effective means of gene therapy for bone and cartilage repair. We will take an in-depth look at the current therapeutic strategies for treating arthritis using various viral vectors.

DOI: 10.5772/intechopen.71071

**Keywords:** viral gene therapy, cartilage and bone healing

## **1. Introduction: viral peptides/vectors used as gene therapy for joint repair**

Viruses have long been used as vectors for gene therapy. Some of the more popular viral vectors include retroviruses, oncolytic viruses, lentiviruses, adenoviruses, and adeno-associated viruses to name just a few. They are used in a wide variety of fields and are able to treat a diverse range of diseases, including Parkinson's disease, many cancers, amyotrophic lateral sclerosis, genetic disorders, cardiovascular diseases, hemophilia, and central nervous system CNS diseases and disorders [1–5]. In recent years, there has been an increase in the development of viral vectors to treat the musculoskeletal system, including the joints [6].

Articular cartilage damage can result from a variety of insults, either from over usage, diseases and disorders, or accidents, and often leads to different types of arthritis including

osteoarthritis (OA) [7]. Articular cartilage damage can cause swelling, pain, and subsequent loss of joint function. Due to its structure, cartilage does not usually regenerate after injury or disease, thus leading to loss of tissue and formation of a defect [8]. Cartilage is devoid of nerves, lymph, and blood supply, thereby explaining the limitations to self-repair. Current therapies targeted at treating articular damage have demonstrated variable results. These therapies include oral administration of a variety of components of the extracellular matrix, such as glucosamine or intra-articular injections of corticosteroids, biological agents (e.g., infliximab, etanercept), analgesics, and autologous blood products [9, 10]. Many approaches have also been investigated to help heal cartilage damage, including the use of viral peptides/ vectors as a means of gene therapy for joint repair. These strategies mostly rely on overexpressing therapeutic factors or suppressing genes involved in joint destruction. In this chapter, we will examine the use of the severe acute respiratory syndrome (SARS)-coronaviruses (CoV), recombinant adeno-associated, and adenovirus vectors as well as retroviruses and lentiviral vectors for the treatment of joint repair.

**2.1. Recombinant adeno-associated virus vectors (rAAV)**

with proposed usage for melanoma cancers.

and finally a combination of these strategies.

target tissue.

Adeno-associated vectors (AAV) are frequently used as viral vectors for gene therapy. They are small nonpathogenic members of the *Parvoviridae* family and the genus *Dependovirus*. These members are nonenveloped viruses with a single-stranded DNA genome (≈ 4.7 kb) [20] and only about 20–25 nm in size [21]. They are safe to use as viral replication (the lytic stage) can only occur in the presence of a helper virus, either adenoviruses or herpesviruses. AAVs were first isolated from stocks of human and simian adenoviruses and thought to be contaminants [22]. AAVs are of interest since they have the ability to specifically integrate into host genomes and establish latent infections. Furthermore, more great advantages are that the preparations are stable and can be produced at titers of more than 1012 particles per ml [23]. Many clinical trials have commenced looking at the use of rAAV for treating a variety of conditions including but not limited to Pompe disease, cystic fibrosis, Parkinson's disease, muscular dystrophy, α-1 antitrypsin deficiency, and hemophilia [24–28]. Europe has even approved a rAAV drug manufactured under the name Glybera, which is the first gene therapy, to treat a very rare disease called lipoprotein lipase deficiency [29]. Despite the efficacy, the staggering cost of such a treatment has hindered the commercial success and use of this drug. There are currently a few other gene therapy drugs in the pipeline, including Amgen's FDA-approved drug IMLYGIC, which is a genetically modified oncolytic virus (Herpes simplex virus type 1)

Viruses: Friends and Foes

5

http://dx.doi.org/10.5772/intechopen.71071

Previous work had shown that cell proliferation actually increases rAAV transduction, thereby making arthritis a candidate disease to be treated by rAAV [30]. Arthritis is not only accompanied by local influx of immune cells but also proliferation of cells in the synovial lining. The first *in vivo* experiment to examine the use of rAAV for the treatment of arthritis was done in the late 1990s. The authors chose to use a rat model of acute arthritis by intra-articular injection of lipopolysaccharide (LPS). The rAAV vector contained the *Escherichia coli* β-galactosidase gene regulated by the cytomegalovirus (CMV). The paper goes on to show efficient and stable gene delivery by rAAV and similar to previous *in vitro* findings, inflammation or disease state seems necessary to facilitate gene delivery. There is a clear enhancement of gene expression during the inflammatory process and the severity of the arthritis. At peak disease, 95% of the synoviocytes expressed high levels of the transgene, whereas when the arthritis subsided at 30 days post-LPS treatment, only basal levels of expression was seen. Interestingly, the study supports the feasibility of a preventative treatment approach, since rAAV responds to the disease state of the

Since that study, rAAV vectors have demonstrated a great efficiency at transducing a variety of joint/articular cells, both *in vitro* and *in vivo,* including chondrocytes [31–33]. In the hopes of treating osteoarthritis, not only has the transduction of chondrocytes been investigated but also other important cells, including osteocytes, meniscal fibrochondrocytes, tendon/ligament cells, muscle cells, cells of the synovial lining and progenitor cells that may differentiate to form joint tissues [6]. The gene therapy approach is aimed at targeting a variety of mechanisms involved in the development of osteoarthritis, including cell proliferation and survival, the stimulation of anabolic pathways, the inhibition of inflammatory or catabolic pathways,

## **2. SARS-coronavirus peptides**

Coronaviruses (CoV) are potentially lethal viruses of the *Coronaviridae* family. They are positive-sense enveloped RNA viruses, which infect humans and animals. Two virulent strains, HCoV-229E and HCoV-OC43, were first identified in the 1960s from patients who presented with coryzal symptoms. Due to increased surveillance of CoV disease prevalence, other strains circulating in the population have recently been identified, including HCoV-NL63 and HCoV-HKU1 [11, 12]. Also, since 2003, more pathogenic strains of coronaviruses have been discovered, including severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS), which predominantly infect the lower respiratory track and cause lethal pneumonia [13, 14].

Despite being pathogenic, coronaviruses have been used as both viral vaccine vectors and gene therapy vectors [15–18]. The uses of CoV as vectors range from delivering immunostimulatory cytokines and antigens to treatment of feline infectious peritonitis. A recent publication has shown the potential of using CoV vectors for the treatment of arthritis [19]. In this study, the authors demonstrated that the use of a small synthetic peptide (MG11, 11 amino acids in length) derived from SARS-CoV fusion protein was able to reduce inflammation in a collagen-induced arthritis (CIA) mouse model. Furthermore, MG11 also was shown to protect mice against bone and cartilage damage. A 14-day treatment regimen with a dose of 25 mg/kg was considered efficient at reducing arthritis in this common autoimmune animal model of rheumatoid arthritis. Histological analysis showed treated mice had no or very minimal inflammation and minimal cartilage damage in the joints of the paws. Knees and ankles also had limited inflammation, or no inflammation, and synovial membrane thickening did not differ from normal limits. The findings suggested that the decreased pathogenesis is due to the ability of MG11 to inhibit cytokine and growth factor production mediated by inflammatory T cells. This study is interesting and paves the way for potential usage of CoV peptides as a novel therapeutic to alleviate rheumatoid arthritis.

#### **2.1. Recombinant adeno-associated virus vectors (rAAV)**

osteoarthritis (OA) [7]. Articular cartilage damage can cause swelling, pain, and subsequent loss of joint function. Due to its structure, cartilage does not usually regenerate after injury or disease, thus leading to loss of tissue and formation of a defect [8]. Cartilage is devoid of nerves, lymph, and blood supply, thereby explaining the limitations to self-repair. Current therapies targeted at treating articular damage have demonstrated variable results. These therapies include oral administration of a variety of components of the extracellular matrix, such as glucosamine or intra-articular injections of corticosteroids, biological agents (e.g., infliximab, etanercept), analgesics, and autologous blood products [9, 10]. Many approaches have also been investigated to help heal cartilage damage, including the use of viral peptides/ vectors as a means of gene therapy for joint repair. These strategies mostly rely on overexpressing therapeutic factors or suppressing genes involved in joint destruction. In this chapter, we will examine the use of the severe acute respiratory syndrome (SARS)-coronaviruses (CoV), recombinant adeno-associated, and adenovirus vectors as well as retroviruses and

Coronaviruses (CoV) are potentially lethal viruses of the *Coronaviridae* family. They are positive-sense enveloped RNA viruses, which infect humans and animals. Two virulent strains, HCoV-229E and HCoV-OC43, were first identified in the 1960s from patients who presented with coryzal symptoms. Due to increased surveillance of CoV disease prevalence, other strains circulating in the population have recently been identified, including HCoV-NL63 and HCoV-HKU1 [11, 12]. Also, since 2003, more pathogenic strains of coronaviruses have been discovered, including severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS), which predominantly infect the lower respiratory track and cause lethal

Despite being pathogenic, coronaviruses have been used as both viral vaccine vectors and gene therapy vectors [15–18]. The uses of CoV as vectors range from delivering immunostimulatory cytokines and antigens to treatment of feline infectious peritonitis. A recent publication has shown the potential of using CoV vectors for the treatment of arthritis [19]. In this study, the authors demonstrated that the use of a small synthetic peptide (MG11, 11 amino acids in length) derived from SARS-CoV fusion protein was able to reduce inflammation in a collagen-induced arthritis (CIA) mouse model. Furthermore, MG11 also was shown to protect mice against bone and cartilage damage. A 14-day treatment regimen with a dose of 25 mg/kg was considered efficient at reducing arthritis in this common autoimmune animal model of rheumatoid arthritis. Histological analysis showed treated mice had no or very minimal inflammation and minimal cartilage damage in the joints of the paws. Knees and ankles also had limited inflammation, or no inflammation, and synovial membrane thickening did not differ from normal limits. The findings suggested that the decreased pathogenesis is due to the ability of MG11 to inhibit cytokine and growth factor production mediated by inflammatory T cells. This study is interesting and paves the way for potential usage of CoV peptides as a novel therapeutic to alleviate

lentiviral vectors for the treatment of joint repair.

**2. SARS-coronavirus peptides**

4 Cartilage Repair and Regeneration

pneumonia [13, 14].

rheumatoid arthritis.

Adeno-associated vectors (AAV) are frequently used as viral vectors for gene therapy. They are small nonpathogenic members of the *Parvoviridae* family and the genus *Dependovirus*. These members are nonenveloped viruses with a single-stranded DNA genome (≈ 4.7 kb) [20] and only about 20–25 nm in size [21]. They are safe to use as viral replication (the lytic stage) can only occur in the presence of a helper virus, either adenoviruses or herpesviruses. AAVs were first isolated from stocks of human and simian adenoviruses and thought to be contaminants [22]. AAVs are of interest since they have the ability to specifically integrate into host genomes and establish latent infections. Furthermore, more great advantages are that the preparations are stable and can be produced at titers of more than 1012 particles per ml [23].

Many clinical trials have commenced looking at the use of rAAV for treating a variety of conditions including but not limited to Pompe disease, cystic fibrosis, Parkinson's disease, muscular dystrophy, α-1 antitrypsin deficiency, and hemophilia [24–28]. Europe has even approved a rAAV drug manufactured under the name Glybera, which is the first gene therapy, to treat a very rare disease called lipoprotein lipase deficiency [29]. Despite the efficacy, the staggering cost of such a treatment has hindered the commercial success and use of this drug. There are currently a few other gene therapy drugs in the pipeline, including Amgen's FDA-approved drug IMLYGIC, which is a genetically modified oncolytic virus (Herpes simplex virus type 1) with proposed usage for melanoma cancers.

Previous work had shown that cell proliferation actually increases rAAV transduction, thereby making arthritis a candidate disease to be treated by rAAV [30]. Arthritis is not only accompanied by local influx of immune cells but also proliferation of cells in the synovial lining. The first *in vivo* experiment to examine the use of rAAV for the treatment of arthritis was done in the late 1990s. The authors chose to use a rat model of acute arthritis by intra-articular injection of lipopolysaccharide (LPS). The rAAV vector contained the *Escherichia coli* β-galactosidase gene regulated by the cytomegalovirus (CMV). The paper goes on to show efficient and stable gene delivery by rAAV and similar to previous *in vitro* findings, inflammation or disease state seems necessary to facilitate gene delivery. There is a clear enhancement of gene expression during the inflammatory process and the severity of the arthritis. At peak disease, 95% of the synoviocytes expressed high levels of the transgene, whereas when the arthritis subsided at 30 days post-LPS treatment, only basal levels of expression was seen. Interestingly, the study supports the feasibility of a preventative treatment approach, since rAAV responds to the disease state of the target tissue.

Since that study, rAAV vectors have demonstrated a great efficiency at transducing a variety of joint/articular cells, both *in vitro* and *in vivo,* including chondrocytes [31–33]. In the hopes of treating osteoarthritis, not only has the transduction of chondrocytes been investigated but also other important cells, including osteocytes, meniscal fibrochondrocytes, tendon/ligament cells, muscle cells, cells of the synovial lining and progenitor cells that may differentiate to form joint tissues [6]. The gene therapy approach is aimed at targeting a variety of mechanisms involved in the development of osteoarthritis, including cell proliferation and survival, the stimulation of anabolic pathways, the inhibition of inflammatory or catabolic pathways, and finally a combination of these strategies.

Approaches looking at stimulating growth and regeneration focus primarily on expressing known growth and cell survival factors, such as fibroblast growth factor-2, bone morphogenetic proteins (BMPs), telomerase, and antiapoptotic molecules like Bcl-2. Stimulating anabolic pathways involves building new molecules out of the products of catabolism. It is thought to aid in restoring function/production to the extracellular matrix (ECM), using growth and transcription factors or signaling molecules, for example, insulin-like growth factor I (IGF-I), parathyroid hormone-related peptide, Indian Hedgehog, SOX factors, etc. Whereas the inhibition of catabolic pathways uses inhibitors of matrix-degrading enzymes, inflammatory cytokines, as well as that of chondroprotective cytokines like IL-4 and IL-10.

cortical surface with 5 × 107

vascularized, remodeling.

*2.1.2. Using rAAV for cartilage repair*

and stimulated osteoclastogenesis.

particles of rAAV, expressing caALK2. caALK2 can potently induce

Viruses: Friends and Foes

7

http://dx.doi.org/10.5772/intechopen.71071

mesenchymal cell differentiation *in vitro* and *in vivo*, and its signals cannot be blocked by noggin or chordin, endogenous BMP antagonists. The results showed endochondral bone formation on the allograft. Interestingly, this procedure also prevented the formation of fibrotic tissue around the allograft, promoted blood vessel ingrowth, live bone marrow within the allograft,

The group that opted to use rAAV expressing VEGF and RANKL did so because studies have shown that these factors significantly decrease during allograft healing [44]. Structural musculoskeletal grafts (i.e.*,* bone, ligament), unlike other grafts, are often derived from allogenic cadavers. However, a significant drawback is that these transplants lack viability due to the absence of vascularization. This study aimed to examine that this rAAV could stimulate allograft vascularization and remodeling. The overarching hypothesis is that resorption of the graft through angiogenesis and osteoclast formation/activation leading to bone remodeling is a superior method to improve graft incorporation. VEGF/RANKL is known to regulate angiogenesis [45] and bone resorption [46] during skeletal repair. VEGF is secreted by hypertrophic chondrocytes and the perichondrium thereby recruiting endothelial cells and favor vascularization [47]. The data showed that if you block RANKL and VEGF signaling, there is indeed diminished bone formation on the autograft. A gain-of-function assay was also performed. RANKL and VEGF are sufficient to significantly improve healing by leading to a live,

Despite these positive results, more work is needed before this method can be used in humans. The connectivity between new and old bone needs to be ameliorated. In addition, technology allowing large animal, *in vivo,* 3D imaging of new bone formation and vascular ingrowth of allografts needs to be developed and biomechanical properties of rAAV-coated allografts

Cartilage is formed of connective tissue and found in many parts of the body, including joints. It is composed of chondrocytes surrounded by extracellular matrix, which contains glycoproteins, glycoaminoglycans, and structural and functional proteins. Articular cartilage is strong and flexible and protects the bones where they articulate to insure smooth movement and also absorbs shocks during weight-bearing activities. People with cartilage damage suffer from stiffness, pain, and swelling. Strategies for cartilage regeneration aim at modifying a variety of target cells including chondrocytes, synovial lining, osteocytes, meniscal fibrochondrocytes, tendon/ligament cells, muscle cells, and progenitor cells that may differentiate to form joint

Several papers have reported the ability to modulate cartilage both *in vitro* and *in vivo*. These studies aimed at over-expressing a variety of molecules like insulin-like growth factor-I (IGF-I), transforming growth factor-β (TGFβ), SOX-9, fibroblast growth factor-2 (FGF-2), antioxidant protein heme oxygenase-1 (HO-1), CTLA4-FasL fusion gene, bone morphogenetic protein-7 (BMP-7), dominant negative to Ikappaβ kinase β (IKKβdn), interleukin 38 (IL-38), interleukin-1-receptor antagonist (IL-1Ra), and osteoprotegerin (OPG) [32, 48–57]. These molecules can

must be determined and correlated with micro-CT parameters.

tissues [6]. Many rAAVs have been designed to target these cells.

Caution needs to be taken when trying to implement the use of rAAV vectors in humans as a large proportion of the population have antibodies against AAV, which would greatly hinder its therapeutic efficacy. However, most of these antibodies are against the serotype AAV2 [34]. With several different serotypes, often therapeutic strategies aim to engineer variants to generate vectors with improved tissue specificity and transduction efficiency, while also avoiding the effects of preexisting neutralizing antibodies [35].

#### *2.1.1. Using rAAV to treat bone regeneration*

Bone loss occurs in a wide spectrum of inflammatory diseases including rheumatoid arthritis (RA), coeliac disease, Crohn's disease, asthma, psoriatic arthritis, nephritis and myositis [36, 37]. Bone loss and associated sequelae greatly reduce the quality of life of many patients. Bone remodeling/regeneration is a dynamic and highly complex process involving a delicate interplay between osteoclasts and osteoblasts. Each year, our bodies regenerate about a quarter of trabecular and 3% of cortical bone [38].

Several studies have shown the ability of rAAV vectors to efficiently express bone morphogenic proteins into myoblast C2C12. Skeletal myoblasts, fibroblasts, and bone marrow-derived cells are pluripotent and can be stimulated with various BMPs (or other factors) to become osteoblast lineage cells [39–41]. These studies even showed relatively good success *in vivo*, where new bone formation was detected in rats between 3 and 8 weeks post injection [41]. More recently, rAAV was also examined to repair bone in a cranioplasty model [42]. Calvarial autografts and allografts were coated with 109 particles/mm2 of rAAV2 vector expressing BMP-2 and transplanted into osteocalcin/luciferase (Oc/Luc) transgenic female mice. Microcomputed tomography (μCT) was used to measure the extent of bone formation, and findings showed that rAAV allografts resulted in significantly better bone repair. Furthermore, histological analysis also showed a variety of bone cells, as well as revitalization factors present in the grafts strengthening the conclusions of significant bone growth. However, the mechanisms involved in this AAV bone repair system still need to be elucidated.

Other studies have focused on expressing vascular endothelial growth factor (VEGF), receptor activator of nuclear factor κB ligand (RANKL), and constitutively active form of the activin receptor-like kinase-2 (caALK2) in rAAV vectors. Koefoed et al*.* also used AAV-coated allografts in a murine femur model [43]. This model is fairly popular where a mid-diaphyseal femoral segment is removed and replaced by an autograft, isograft, or allograft, which is secured by an intramedullary pin. In this report, authors used a frozen allograft that was coated on the cortical surface with 5 × 107 particles of rAAV, expressing caALK2. caALK2 can potently induce mesenchymal cell differentiation *in vitro* and *in vivo*, and its signals cannot be blocked by noggin or chordin, endogenous BMP antagonists. The results showed endochondral bone formation on the allograft. Interestingly, this procedure also prevented the formation of fibrotic tissue around the allograft, promoted blood vessel ingrowth, live bone marrow within the allograft, and stimulated osteoclastogenesis.

The group that opted to use rAAV expressing VEGF and RANKL did so because studies have shown that these factors significantly decrease during allograft healing [44]. Structural musculoskeletal grafts (i.e.*,* bone, ligament), unlike other grafts, are often derived from allogenic cadavers. However, a significant drawback is that these transplants lack viability due to the absence of vascularization. This study aimed to examine that this rAAV could stimulate allograft vascularization and remodeling. The overarching hypothesis is that resorption of the graft through angiogenesis and osteoclast formation/activation leading to bone remodeling is a superior method to improve graft incorporation. VEGF/RANKL is known to regulate angiogenesis [45] and bone resorption [46] during skeletal repair. VEGF is secreted by hypertrophic chondrocytes and the perichondrium thereby recruiting endothelial cells and favor vascularization [47]. The data showed that if you block RANKL and VEGF signaling, there is indeed diminished bone formation on the autograft. A gain-of-function assay was also performed. RANKL and VEGF are sufficient to significantly improve healing by leading to a live, vascularized, remodeling.

Despite these positive results, more work is needed before this method can be used in humans. The connectivity between new and old bone needs to be ameliorated. In addition, technology allowing large animal, *in vivo,* 3D imaging of new bone formation and vascular ingrowth of allografts needs to be developed and biomechanical properties of rAAV-coated allografts must be determined and correlated with micro-CT parameters.

## *2.1.2. Using rAAV for cartilage repair*

Approaches looking at stimulating growth and regeneration focus primarily on expressing known growth and cell survival factors, such as fibroblast growth factor-2, bone morphogenetic proteins (BMPs), telomerase, and antiapoptotic molecules like Bcl-2. Stimulating anabolic pathways involves building new molecules out of the products of catabolism. It is thought to aid in restoring function/production to the extracellular matrix (ECM), using growth and transcription factors or signaling molecules, for example, insulin-like growth factor I (IGF-I), parathyroid hormone-related peptide, Indian Hedgehog, SOX factors, etc. Whereas the inhibition of catabolic pathways uses inhibitors of matrix-degrading enzymes, inflammatory cytokines, as well as that of chondroprotective cytokines like IL-4 and IL-10.

Caution needs to be taken when trying to implement the use of rAAV vectors in humans as a large proportion of the population have antibodies against AAV, which would greatly hinder its therapeutic efficacy. However, most of these antibodies are against the serotype AAV2 [34]. With several different serotypes, often therapeutic strategies aim to engineer variants to generate vectors with improved tissue specificity and transduction efficiency, while also avoiding

Bone loss occurs in a wide spectrum of inflammatory diseases including rheumatoid arthritis (RA), coeliac disease, Crohn's disease, asthma, psoriatic arthritis, nephritis and myositis [36, 37]. Bone loss and associated sequelae greatly reduce the quality of life of many patients. Bone remodeling/regeneration is a dynamic and highly complex process involving a delicate interplay between osteoclasts and osteoblasts. Each year, our bodies regenerate about a quarter of

Several studies have shown the ability of rAAV vectors to efficiently express bone morphogenic proteins into myoblast C2C12. Skeletal myoblasts, fibroblasts, and bone marrow-derived cells are pluripotent and can be stimulated with various BMPs (or other factors) to become osteoblast lineage cells [39–41]. These studies even showed relatively good success *in vivo*, where new bone formation was detected in rats between 3 and 8 weeks post injection [41]. More recently, rAAV was also examined to repair bone in a cranioplasty model [42]. Calvarial auto-

particles/mm2

and transplanted into osteocalcin/luciferase (Oc/Luc) transgenic female mice. Microcomputed tomography (μCT) was used to measure the extent of bone formation, and findings showed that rAAV allografts resulted in significantly better bone repair. Furthermore, histological analysis also showed a variety of bone cells, as well as revitalization factors present in the grafts strengthening the conclusions of significant bone growth. However, the mechanisms

Other studies have focused on expressing vascular endothelial growth factor (VEGF), receptor activator of nuclear factor κB ligand (RANKL), and constitutively active form of the activin receptor-like kinase-2 (caALK2) in rAAV vectors. Koefoed et al*.* also used AAV-coated allografts in a murine femur model [43]. This model is fairly popular where a mid-diaphyseal femoral segment is removed and replaced by an autograft, isograft, or allograft, which is secured by an intramedullary pin. In this report, authors used a frozen allograft that was coated on the

involved in this AAV bone repair system still need to be elucidated.

of rAAV2 vector expressing BMP-2

the effects of preexisting neutralizing antibodies [35].

*2.1.1. Using rAAV to treat bone regeneration*

6 Cartilage Repair and Regeneration

trabecular and 3% of cortical bone [38].

grafts and allografts were coated with 109

Cartilage is formed of connective tissue and found in many parts of the body, including joints. It is composed of chondrocytes surrounded by extracellular matrix, which contains glycoproteins, glycoaminoglycans, and structural and functional proteins. Articular cartilage is strong and flexible and protects the bones where they articulate to insure smooth movement and also absorbs shocks during weight-bearing activities. People with cartilage damage suffer from stiffness, pain, and swelling. Strategies for cartilage regeneration aim at modifying a variety of target cells including chondrocytes, synovial lining, osteocytes, meniscal fibrochondrocytes, tendon/ligament cells, muscle cells, and progenitor cells that may differentiate to form joint tissues [6]. Many rAAVs have been designed to target these cells.

Several papers have reported the ability to modulate cartilage both *in vitro* and *in vivo*. These studies aimed at over-expressing a variety of molecules like insulin-like growth factor-I (IGF-I), transforming growth factor-β (TGFβ), SOX-9, fibroblast growth factor-2 (FGF-2), antioxidant protein heme oxygenase-1 (HO-1), CTLA4-FasL fusion gene, bone morphogenetic protein-7 (BMP-7), dominant negative to Ikappaβ kinase β (IKKβdn), interleukin 38 (IL-38), interleukin-1-receptor antagonist (IL-1Ra), and osteoprotegerin (OPG) [32, 48–57]. These molecules can act on a plethora of functions, including enhancing cartilage anabolism (IGF-1, FGF-2, TGFβ, BMP-7), stimulating cartilage formation (SOX-9), exhibiting anti-inflammatory properties (CTLA4-FasL, HO-1, IKKβdn, IL-38, IL-1Ra), reducing oxidative stresses shown to exist is certain forms of arthritis (HO-1), and by blocking osteoclastogenesis (OPG). One paper examined using cystatin C (cysC) to inhibit cathepsin activity in the synovium of rabbit model of osteoarthritis. Unfortunately, this approach was unsuccessful. Despite completely blocking cathepsin activity in the synovium, synovitis, bone sclerosis and cartilage degradation remained [58].

repair may require expression of several therapeutic factors. Toward this, 3D cultures and cartilage explants were used. rAAV-FGF-2 showed greater transduction efficiency and effective expression of FGF-2. While the addition of SOX-9 was equally efficient, it did not add to the overall effectiveness of the expression. The authors did not test but did suggest that repeated administration of the combination might improve the outcomes of cartilage

The most recent report in the literature examined the use of polymer micelles in aiding rAAV as gene therapy. The polymer micelles enhanced the stability and bioactivity of rAAV, leading to higher levels of transgene expression in human OA chondrocytes *in vitro.* It was also found to aid in human osteochondral defect cultures to mimic a more natural environment. In addition, the micelles protected the viral vector against neutralization of the viral capsid. No detrimental effect on cell viability was observed when delivering rAAV/micelles to the cells at

An investigation looking at the use of adjuvants for *in vivo* rAAV articular cartilage gene therapy has also been done. One group showed that light-activated gene transduction (LAGT) could be one such method. UV light accelerates the formation of the double-stranded transducing rAAV vector episome by activation of a host DNA polymerase. The use of UV expo-

transduced gene eGFP in cultures of immortalized and primary human articular chondrocytes, as well as articular cartilage explants. Importantly, this amount of light was noted as insufficient to cause harm to cells [63]. A follow-up study looking at the ability of UV lightactivated gene transduction (LAGT) in chondrocytes *in vivo* showed that in rabbit chondrocyte cultures, as well as in intra-articular transduction of rabbit knees, LAGT treatment resulted in higher efficiencies compared to nonirradiated samples [64]. However, after 3 weeks, the mean fluorescence intensity of positive cells of the non-LAGT group had increased to the same level as the LAGT group, despite the proportion of transgene-expressing chondrocytes were still higher in the LAGT group. Overall results showed that LAGT probably does not benefit healthy cartilage. However, in diseased tissue, more chondrocytes were transduced in general and especially those close to the irradiated surface respond to the treatment. Importantly, further investigation needs to be done to assess if the biological effect is sufficient to provide

Despite being a promising avenue for gene therapy, consistency among findings and systems using rAAV appears to be difficult and unpredictable. Further experimentation and stringent conditions will need to be done to establish if this treatment strategy is a viable and promising

Adenoviruses (AdV) are medium-sized nonenveloped viruses, composed of a nucleocapsid and a double-stranded, linear DNA genome of approximately 36 kb. Over 50 different human serotypes can be found and they cause 5–10% of all childhood upper respiratory infections. Adults can also suffer from illness caused by adenoviruses, but disease is generally mild and

actively increases transduction efficiency and expression of the

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9

http://dx.doi.org/10.5772/intechopen.71071

repair over time.

any time point of the analysis.

sure at doses of up to 200 J/m2

a desired metabolic response toward repair.

avenue to promote cartilage restoration.

resembles that of a common cold.

**2.2. Recombinant adenovirus vectors (rAdV)**

Due to the large scope of these studies, for this review, we will summarize some of the main findings of rAAV and chondrocytes. The first attempts to transduce chondrocytes were in 2000. One group transduced primary human chondrocytes as well as human cartilage organ cultures with a rAAV-GFP. Their results were encouraging with GFP expression seen in more than 90% of monocultures after 7 days and over 45% of the cells in the organ cultures fluoresced for up to 28 days [59]. Around that same time, another group was looking at the ability of rAAV to be used *in vivo.* They used a rAAV-expressing bacterial beta-galactosidase (beta-gal) gene in an arthritis mouse model overexpressing tumor necrosis factor-alpha (hTNFalpha-Tg).

Another group also looking at transduction of a variety of primary human cells including tissues of mesenchymal, endodermal, neuroectodermal origin, and cartilage showed very different results. Chondrocytes appeared to have the lowest transduction rates along with dermal papilla follicles, epithelial cells, and fibroblasts. Transduction levels between 4.3 and 19.5% were seen [60]. Only melanocytes, G-CSF mobilized CD34+ and CD19+ cells fared worse, with no visible transduction seen.

Using genes, which are responsible for producing growth factors or molecules involved in cartilage repair, is a preferred method for viral therapy. Fibroblast growth factor-2 (FGF-2) is a member of the multifunctional fibroblast growth factor family and has broad mitogenic and angiogenic activities. One study examined whether rAAV is capable of delivering a functional FGF-2 gene cassette to isolated articular chondrocytes and to sites of articular cartilage damage *in vitro* and *in vivo* [61]*.* After encouraging results *in vitro*, the authors applied rAAV-hFGF-2 to osteochondral defects created in the patellar groove of knee joints in rabbits. Repair was seen at day 10 post infection and by day 20, the initial repair had progressed further, and integration into surrounding cartilage was seen. A follow-up at 4 months showed that the "new" cartilage now closely resembled the original tissue, but margins of new cartilage were barely visible. Results were even more encouraging as there were no apparent secondary effects such as synovitis or adverse reactions. Further histological analysis showed the absence of infiltrating cells at all time points observed. Earlier studies showed that rAAV could also transduce bone marrow-derived mesenchymal stem cells that migrate to injury sites [62]. Therefore, the mechanism of action is thought to be on two fronts: (1) rAAV can stimulate long-term FGF-2 transduction in damaged areas of cartilage, as well as in chondrocytes found in surrounding healthy areas and (2) rAAV also transduces the mesenchymal stem cells that will be recruited to damage areas and commence tissue repair. In 2008, *in vitro* studies using a combination of FGF-2 and SOX-9, a transcription factor that activates the expression of major cartilage matrix components, were also undertaken [53]. The premise behind this is that due to the complex nature of osteoarthritis and the plethora of processes involved in this pathology, efficient cartilage repair may require expression of several therapeutic factors. Toward this, 3D cultures and cartilage explants were used. rAAV-FGF-2 showed greater transduction efficiency and effective expression of FGF-2. While the addition of SOX-9 was equally efficient, it did not add to the overall effectiveness of the expression. The authors did not test but did suggest that repeated administration of the combination might improve the outcomes of cartilage repair over time.

The most recent report in the literature examined the use of polymer micelles in aiding rAAV as gene therapy. The polymer micelles enhanced the stability and bioactivity of rAAV, leading to higher levels of transgene expression in human OA chondrocytes *in vitro.* It was also found to aid in human osteochondral defect cultures to mimic a more natural environment. In addition, the micelles protected the viral vector against neutralization of the viral capsid. No detrimental effect on cell viability was observed when delivering rAAV/micelles to the cells at any time point of the analysis.

An investigation looking at the use of adjuvants for *in vivo* rAAV articular cartilage gene therapy has also been done. One group showed that light-activated gene transduction (LAGT) could be one such method. UV light accelerates the formation of the double-stranded transducing rAAV vector episome by activation of a host DNA polymerase. The use of UV exposure at doses of up to 200 J/m2 actively increases transduction efficiency and expression of the transduced gene eGFP in cultures of immortalized and primary human articular chondrocytes, as well as articular cartilage explants. Importantly, this amount of light was noted as insufficient to cause harm to cells [63]. A follow-up study looking at the ability of UV lightactivated gene transduction (LAGT) in chondrocytes *in vivo* showed that in rabbit chondrocyte cultures, as well as in intra-articular transduction of rabbit knees, LAGT treatment resulted in higher efficiencies compared to nonirradiated samples [64]. However, after 3 weeks, the mean fluorescence intensity of positive cells of the non-LAGT group had increased to the same level as the LAGT group, despite the proportion of transgene-expressing chondrocytes were still higher in the LAGT group. Overall results showed that LAGT probably does not benefit healthy cartilage. However, in diseased tissue, more chondrocytes were transduced in general and especially those close to the irradiated surface respond to the treatment. Importantly, further investigation needs to be done to assess if the biological effect is sufficient to provide a desired metabolic response toward repair.

Despite being a promising avenue for gene therapy, consistency among findings and systems using rAAV appears to be difficult and unpredictable. Further experimentation and stringent conditions will need to be done to establish if this treatment strategy is a viable and promising avenue to promote cartilage restoration.

## **2.2. Recombinant adenovirus vectors (rAdV)**

act on a plethora of functions, including enhancing cartilage anabolism (IGF-1, FGF-2, TGFβ, BMP-7), stimulating cartilage formation (SOX-9), exhibiting anti-inflammatory properties (CTLA4-FasL, HO-1, IKKβdn, IL-38, IL-1Ra), reducing oxidative stresses shown to exist is certain forms of arthritis (HO-1), and by blocking osteoclastogenesis (OPG). One paper examined using cystatin C (cysC) to inhibit cathepsin activity in the synovium of rabbit model of osteoarthritis. Unfortunately, this approach was unsuccessful. Despite completely blocking cathepsin activity in the synovium, synovitis, bone sclerosis and cartilage degradation remained [58].

Due to the large scope of these studies, for this review, we will summarize some of the main findings of rAAV and chondrocytes. The first attempts to transduce chondrocytes were in 2000. One group transduced primary human chondrocytes as well as human cartilage organ cultures with a rAAV-GFP. Their results were encouraging with GFP expression seen in more than 90% of monocultures after 7 days and over 45% of the cells in the organ cultures fluoresced for up to 28 days [59]. Around that same time, another group was looking at the ability of rAAV to be used *in vivo.* They used a rAAV-expressing bacterial beta-galactosidase (beta-gal) gene in an

Another group also looking at transduction of a variety of primary human cells including tissues of mesenchymal, endodermal, neuroectodermal origin, and cartilage showed very different results. Chondrocytes appeared to have the lowest transduction rates along with dermal papilla follicles, epithelial cells, and fibroblasts. Transduction levels between 4.3 and 19.5% were seen [60]. Only melanocytes, G-CSF mobilized CD34+ and CD19+ cells fared

Using genes, which are responsible for producing growth factors or molecules involved in cartilage repair, is a preferred method for viral therapy. Fibroblast growth factor-2 (FGF-2) is a member of the multifunctional fibroblast growth factor family and has broad mitogenic and angiogenic activities. One study examined whether rAAV is capable of delivering a functional FGF-2 gene cassette to isolated articular chondrocytes and to sites of articular cartilage damage *in vitro* and *in vivo* [61]*.* After encouraging results *in vitro*, the authors applied rAAV-hFGF-2 to osteochondral defects created in the patellar groove of knee joints in rabbits. Repair was seen at day 10 post infection and by day 20, the initial repair had progressed further, and integration into surrounding cartilage was seen. A follow-up at 4 months showed that the "new" cartilage now closely resembled the original tissue, but margins of new cartilage were barely visible. Results were even more encouraging as there were no apparent secondary effects such as synovitis or adverse reactions. Further histological analysis showed the absence of infiltrating cells at all time points observed. Earlier studies showed that rAAV could also transduce bone marrow-derived mesenchymal stem cells that migrate to injury sites [62]. Therefore, the mechanism of action is thought to be on two fronts: (1) rAAV can stimulate long-term FGF-2 transduction in damaged areas of cartilage, as well as in chondrocytes found in surrounding healthy areas and (2) rAAV also transduces the mesenchymal stem cells that will be recruited to damage areas and commence tissue repair. In 2008, *in vitro* studies using a combination of FGF-2 and SOX-9, a transcription factor that activates the expression of major cartilage matrix components, were also undertaken [53]. The premise behind this is that due to the complex nature of osteoarthritis and the plethora of processes involved in this pathology, efficient cartilage

arthritis mouse model overexpressing tumor necrosis factor-alpha (hTNFalpha-Tg).

worse, with no visible transduction seen.

8 Cartilage Repair and Regeneration

Adenoviruses (AdV) are medium-sized nonenveloped viruses, composed of a nucleocapsid and a double-stranded, linear DNA genome of approximately 36 kb. Over 50 different human serotypes can be found and they cause 5–10% of all childhood upper respiratory infections. Adults can also suffer from illness caused by adenoviruses, but disease is generally mild and resembles that of a common cold.

AdV are interesting because they can infect a broad range of human cells and tend to yield high levels of gene transfer compared to levels achieved with other currently available vectors. This also includes high *in vitro* gene transfer efficiencies in chondrocytes and mesenchymal stem cells [65, 66]. These viruses can accommodate large genomic insertions up to 14 kb and have the ability to transduce these genes in both proliferating and quiescent cells. At least three regions of the viral genome can accept insertions or substitutions of DNA to generate therapeutic vectors. Also, the viral genome is relatively stable and undergoes limited rearrangements and inserted foreign genes are very well maintained through successive rounds of viral replication. Genetic manipulation of these vectors is easy by using standard recombinant DNA techniques, and they are easily grown, reaching titers of up to high up to 1013 particles/ml. Taken together, these factors make adenoviruses excellent candidates for viral gene therapy.

inflammation and plays an important role in the pathogenesis of rheumatoid arthritis. IL-1ra is a natural receptor antagonist that competes with IL-1 for binding to type I IL-1 receptors and as a result blocks the effects of IL-1 [71]. Again, authors used New Zealand white rabbits as an *in vivo* model. After verifying *in vitro* that the expression of IL-1ra is biologically active, they found that direct intra-articular injection of rAdV into the synovium of rabbits led to the expression of high levels of IL-1ra within 1 week, as determined by Southern blot. However, like in their previous work, within 4 weeks, the levels of IL-1ra expression within synoviocytes decreased a major limitation to the approach. However, it is noteworthy to mention that these studies were undertaken using first generation vectors, which as mentioned above, are

Viruses: Friends and Foes

11

http://dx.doi.org/10.5772/intechopen.71071

Since the first studies in the 1990s, a plethora of publications regarding AdV have been published. Similar to rAAV, the focus of these studies seems to be targeted mainly on either bone or cartilage repair. Many studies have been interested in using rAdV to transduce bone morphogenic proteins including BMP-2, BMP-4, BMP-7, and BMP-13. In addition, soluble growth factors like PDGF, FGF and IGF, anabolic factors like growth factor and PTH, systemic angiogenic factors like VEGF as well as transcription factors associated with bone- and cartilage-related gene expression like Runx2, SOX9, osterix, and extracellular matrix molecules associated with induction or repression of mineralization like Gla protein, osteopontin,

Most of the studies using rAdV focus on the transduction of the various BMPs. One study by an American group based in Chicago investigated the feasibility of using a recombinant AdV to express 14 different bone morphogenic proteins (BMPs) [72]. It is known that bone demineralization can induce *de novo* synthesis of bone formation [73]. BMPs have been demonstrated to be the factors involved in bone regeneration. They belong to the TGFβ superfamily and are important in embryogenesis as well as in bone modeling. There are at least 15 different BMPs in humans, and this study attempted to establish which BMPs were the most effective at bone regeneration. The authors first examined the ability of the rAdVs to express ALP (an osteogenic marker) in the C2C12 cell line that is a precursor of osteoblasts. Four days after transduction, five BMPs were able to express ALP. These were BMP-2, BMP-4 BMP-6, BMP-7, and BMP-9. Findings were similar when looking *in vivo* at athymic nude mice. AdV were used to transduce C2C12 *in vitro,* and cells were then injected into the quadriceps muscle. Ossification was seen in animals that received AdBMP-2, 6, 7, and 9. However, BMP-7 was less robust than the other BMPs, and interestingly, BMP-6 and 9 were the most efficient. Since this study, numerous others have investigated the use of these bone-regenerating BMPs

Two studies of interest showed the ability of AdBMP-7 and AdBMP-2 to form bone intramuscularly and subdermally in immunocompetent rodents. A key factor in the success of these studies was to reduce immune responses to the adenoviral vector. Strong immune responses can decrease or inhibit therapeutic transgene expression. It was found that when the vector is delivered in conjunction with a collagen carrier, the vector becomes more effective in decreasing

associated with major drawbacks.

and bone sialoprotein.

*2.2.1. Using rAdV to treat bone regeneration*

for *in vitro*, *in vivo,* and clinical studies [74].

Human serotypes AdV2 and AdV5 from group C are the classic adenoviruses used as therapeutic vectors. Early versions of adenovirus vectors were unsuccessful due to the deletion of E1 region to accommodate the therapeutic transgene and to prohibit viral replication [67]. This deletion led to a strong innate immune response followed by adaptive responses, which destroyed the transduced cells, thereby defeating the purpose of gene therapy. Second generation vectors were generated by deleting several areas of the genome and allowing a larger amount of DNA to be inserted. Unfortunately, these vectors still triggered immunogenicity and led to cell death. Third generation vectors were known as "gutted" vectors. All viral coding regions were removed to prevent an immunological trigger. However, they need a helper vector that codes for the viral genome to allow for replication. These third-generation vectors facilitate insertion of up to 35 kb of genetic material and are therefore deemed high capacity. Gutless AdV have been delivered to different tissues in rodents, dogs, and nonhuman primates. These third-generation vectors have been shown to be nonimmunogenic for the life of a mouse, whereas the first generation induced a response within 3 months [68].

Along with rAAV, AdV is a very popular choice for gene therapy delivery. Much work has been done in a variety of fields including cancer, metabolic diseases, motoneuronal injuries/ diseases, and cerebrovascular diseases. One of the first reports in the early 1990s examined the ability of AdV vectors to be useful tools in overexpressing anti-inflammatory molecules in rabbit synoviocytes to alleviate rheumatoid arthritis. Synoviocytes were chosen due to the ease of access via the intra-articular space and their longevity (type A, macrophage-like synoviocytes are estimated to live for 3–6 months) making them ideal candidates for viral transduction [69]. This study showed the ability of rAdV vectors to express lacZ via different techniques including *in situ* staining, immunohistochemistry, and transmission electron microscopy. The transduction remained detectable for over 8 weeks; however, efficiency did wane over time. Clinically, the rabbits fared well with no signs of arthritis, synovitis, or adverse effects for up to 8 weeks post-transduction, despite having preexisting antibodies to either human or rabbit adenoviruses. The authors were unable to identify exactly to which one the animals were previously exposed to, human or rabbit adenoviruses, since antibodies against rAdV are crossreactive against many species including humans, rabbits, and cattle.

A follow-up study by the same group looked at replacing lacZ expression with that of human interleukin-1 receptor antagonist protein (IL-1ra) [70]. IL-1 is an important mediator of inflammation and plays an important role in the pathogenesis of rheumatoid arthritis. IL-1ra is a natural receptor antagonist that competes with IL-1 for binding to type I IL-1 receptors and as a result blocks the effects of IL-1 [71]. Again, authors used New Zealand white rabbits as an *in vivo* model. After verifying *in vitro* that the expression of IL-1ra is biologically active, they found that direct intra-articular injection of rAdV into the synovium of rabbits led to the expression of high levels of IL-1ra within 1 week, as determined by Southern blot. However, like in their previous work, within 4 weeks, the levels of IL-1ra expression within synoviocytes decreased a major limitation to the approach. However, it is noteworthy to mention that these studies were undertaken using first generation vectors, which as mentioned above, are associated with major drawbacks.

Since the first studies in the 1990s, a plethora of publications regarding AdV have been published. Similar to rAAV, the focus of these studies seems to be targeted mainly on either bone or cartilage repair. Many studies have been interested in using rAdV to transduce bone morphogenic proteins including BMP-2, BMP-4, BMP-7, and BMP-13. In addition, soluble growth factors like PDGF, FGF and IGF, anabolic factors like growth factor and PTH, systemic angiogenic factors like VEGF as well as transcription factors associated with bone- and cartilage-related gene expression like Runx2, SOX9, osterix, and extracellular matrix molecules associated with induction or repression of mineralization like Gla protein, osteopontin, and bone sialoprotein.

#### *2.2.1. Using rAdV to treat bone regeneration*

AdV are interesting because they can infect a broad range of human cells and tend to yield high levels of gene transfer compared to levels achieved with other currently available vectors. This also includes high *in vitro* gene transfer efficiencies in chondrocytes and mesenchymal stem cells [65, 66]. These viruses can accommodate large genomic insertions up to 14 kb and have the ability to transduce these genes in both proliferating and quiescent cells. At least three regions of the viral genome can accept insertions or substitutions of DNA to generate therapeutic vectors. Also, the viral genome is relatively stable and undergoes limited rearrangements and inserted foreign genes are very well maintained through successive rounds of viral replication. Genetic manipulation of these vectors is easy by using standard recombinant DNA techniques, and they are easily grown, reaching titers of up to high up to 1013 particles/ml. Taken together,

Human serotypes AdV2 and AdV5 from group C are the classic adenoviruses used as therapeutic vectors. Early versions of adenovirus vectors were unsuccessful due to the deletion of E1 region to accommodate the therapeutic transgene and to prohibit viral replication [67]. This deletion led to a strong innate immune response followed by adaptive responses, which destroyed the transduced cells, thereby defeating the purpose of gene therapy. Second generation vectors were generated by deleting several areas of the genome and allowing a larger amount of DNA to be inserted. Unfortunately, these vectors still triggered immunogenicity and led to cell death. Third generation vectors were known as "gutted" vectors. All viral coding regions were removed to prevent an immunological trigger. However, they need a helper vector that codes for the viral genome to allow for replication. These third-generation vectors facilitate insertion of up to 35 kb of genetic material and are therefore deemed high capacity. Gutless AdV have been delivered to different tissues in rodents, dogs, and nonhuman primates. These third-generation vectors have been shown to be nonimmunogenic for the life of

these factors make adenoviruses excellent candidates for viral gene therapy.

10 Cartilage Repair and Regeneration

a mouse, whereas the first generation induced a response within 3 months [68].

crossreactive against many species including humans, rabbits, and cattle.

Along with rAAV, AdV is a very popular choice for gene therapy delivery. Much work has been done in a variety of fields including cancer, metabolic diseases, motoneuronal injuries/ diseases, and cerebrovascular diseases. One of the first reports in the early 1990s examined the ability of AdV vectors to be useful tools in overexpressing anti-inflammatory molecules in rabbit synoviocytes to alleviate rheumatoid arthritis. Synoviocytes were chosen due to the ease of access via the intra-articular space and their longevity (type A, macrophage-like synoviocytes are estimated to live for 3–6 months) making them ideal candidates for viral transduction [69]. This study showed the ability of rAdV vectors to express lacZ via different techniques including *in situ* staining, immunohistochemistry, and transmission electron microscopy. The transduction remained detectable for over 8 weeks; however, efficiency did wane over time. Clinically, the rabbits fared well with no signs of arthritis, synovitis, or adverse effects for up to 8 weeks post-transduction, despite having preexisting antibodies to either human or rabbit adenoviruses. The authors were unable to identify exactly to which one the animals were previously exposed to, human or rabbit adenoviruses, since antibodies against rAdV are

A follow-up study by the same group looked at replacing lacZ expression with that of human interleukin-1 receptor antagonist protein (IL-1ra) [70]. IL-1 is an important mediator of Most of the studies using rAdV focus on the transduction of the various BMPs. One study by an American group based in Chicago investigated the feasibility of using a recombinant AdV to express 14 different bone morphogenic proteins (BMPs) [72]. It is known that bone demineralization can induce *de novo* synthesis of bone formation [73]. BMPs have been demonstrated to be the factors involved in bone regeneration. They belong to the TGFβ superfamily and are important in embryogenesis as well as in bone modeling. There are at least 15 different BMPs in humans, and this study attempted to establish which BMPs were the most effective at bone regeneration. The authors first examined the ability of the rAdVs to express ALP (an osteogenic marker) in the C2C12 cell line that is a precursor of osteoblasts. Four days after transduction, five BMPs were able to express ALP. These were BMP-2, BMP-4 BMP-6, BMP-7, and BMP-9. Findings were similar when looking *in vivo* at athymic nude mice. AdV were used to transduce C2C12 *in vitro,* and cells were then injected into the quadriceps muscle. Ossification was seen in animals that received AdBMP-2, 6, 7, and 9. However, BMP-7 was less robust than the other BMPs, and interestingly, BMP-6 and 9 were the most efficient. Since this study, numerous others have investigated the use of these bone-regenerating BMPs for *in vitro*, *in vivo,* and clinical studies [74].

Two studies of interest showed the ability of AdBMP-7 and AdBMP-2 to form bone intramuscularly and subdermally in immunocompetent rodents. A key factor in the success of these studies was to reduce immune responses to the adenoviral vector. Strong immune responses can decrease or inhibit therapeutic transgene expression. It was found that when the vector is delivered in conjunction with a collagen carrier, the vector becomes more effective in decreasing immunogenicity [75, 76]. Another method of prolonging transgene expression is by administering anti-T cell receptor monoclonal antibody following adenovirus-mediated *in vivo* gene transfer [77].

seen between naive chondrocyte-implanted or AdIGF-1-transduced repair tissues. These data were determined by examining inflammatory markers (including MMP-1, MMP-3, MMP-13, and aggrecanase-1) by qPCR. In addition, it was shown that IGF-1-enhanced repair also involved an increase in tissue thickness. It appears that there was a greater defect filling, and upon examination, these cells morphologically resembled chondrocytes rather than a fibrocartilaginous-like phenotype seen within the control tissues. Another study in humans looked at the effects of AdV gene transduction FGF-2, FGF-2 combined with interleukin-1 receptor antagonist protein (IL-Ra), and/or insulin-like growth factor-1 (IGF-1). This was determined in both human osteoarthritis (OA) chondrocytes as well as in a leporine OA model [83]. FGF-2 expression protected human OA chondrocytes and decreased cartilage degradation *in vivo* (rabbit model). *In vitro,* FGF-2 induced collagen type II and an increased production of GAG. Furthermore, combining all three factors FGF-2, IL-1Ra, and IGF-1 leads to significantly lower levels of ADAMTS-5, MMP-13, and MMP-3, and increased amounts of TIMP-1. This was also true as seen in the rabbit model. The combined therapy seems to have a synergistic effect to achieve optimal results. The trigene expression system appears to promote GAG synthesis of chondrocyte, increases TIMP-1 expression, and reduces ADAMTS-5, MMP-13 and aggrecanase expression. Haupt et al*.* also found that an adenovirus-mediated gene therapy combining several factors was more efficient. In this study, IGF-1 and IL-1Ra were shown to promote the healing of cartilage injury in degenerative joint diseases, suggesting combination

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therapy could be beneficial for cartilage repair in degenerative joint diseases [84].

GDF-5 has been shown to be essential for normal appendicular skeletal and joint development in humans and mice. It positively regulates differentiation of chondrogenic tissue through its binding with bone morphogenetic protein receptor type 1 A and B (BMPR1A and BMPR1B). It also negatively regulates chondrogenic differentiation through its interaction with noggin (NOG). One study conducted by Luo et al*.* investigated the effects of adenovirus-mediated GDF-5 (AdGDF-5) on ECM expression in human degenerative disc nucleus pulposus (NP) cells in order to determine if AdGDF-5 is a viable therapy to treat intervertebral disc degeneration (IDD) [85]. Like many other studies, they began by determining the expression of GDF-5 *in vitro* after treating HEK293 cells with AdGDF-5 and then determined the optimal amount of viral vector needed for efficient transduction of NP cells. Following this, they investigated the effects of expression of GDF-5 had on the ECM. It was noted that GDF-5 promotes the synthesis of sulfated glycoaminoglycans and hydroxyproline, two major structures forming the ECM network. In addition, immunohistochemistry showed an increase in proteoglycans in the AdGDF-5-treated NP cells, stimulated NP proliferation, and increased the expression of collagen II and aggrecan genes. The outcome of this study indicates that NP cells within degraded discs would be ideal targets for the transduction of transgenic proteins and that AdV therapy could be a promising new avenue for the treatment of disc degeneration.

As like for rAAV, the effects of Sox9 on MSCs have been examined as a novel treatment of cartilage repair. This is of no surprise, considering that Sox9 is considered a master regulator of chondrocyte phenotype [86]. Like that of rAAV, Sox9 has been shown to be able to modulate cartilage both *in vitro* and *in vivo*. In the study led by Cao et al*.,* Sox9 expression successfully promoted a chondrocyte morphology after AdV transduction of rabbit bone marrow mesenchymal stem cells (BMSCs) [87]. Overexpression of Sox9 resulted in the upregulation

One of the most recent publications examined the effect of AdBMP-2 on the osteogenic ability of human mesenchymal stem cells (hMSCs) [78]. MSCs are multipotent somatic stem cells that are able to differentiate into a variety of cell types, including chondrocytes, myocytes, osteoblasts, and adipocytes. Targeting these cells with BMP-2 could potentially lead to their osteogenic differentiation and promote bone healing. *In vitro* experiments showed that when treated with AdBMP-2, hMSCs change phenotypes and resemble osteoblast-like cells. Further analysis showed that these changed cells also expressed ALP, an enzyme present in osteoblasts and critical for bone mineralization and calcification. Immunohistochemistry using a von Kossa stain (used for the quantification of mineralization in cell culture and tissue sections) showed increased positive staining at d14 post-treatment. Taken together, this study showed the potential of AdBMP-2 to skew the differentiation of hMSCs toward osteoblast-like cells, thereby potentially becoming a novel treatment for delayed or nonunion fractures.

In addition to BMPs, several other factors have been investigated to determine if their expression via an adenoviral vector leads to bone healing. Nell-1 is a novel direct transcriptional target of runt homology domain transcription factor-2 (Runx2). Nel-like molecule-1 (Nell-1) is osteoinductive on cells of the osteochondral lineage. Adenovirus vectors containing Nell-1 was shown to promote osteoblastic differentiation in calvarial cells (from the skull cap) [79]. An *in vivo* study demonstrated that Null-1 could be as efficient as BMP-2, one of the most potent BMPs, to induce rat calvarial bone formation [80]. VEGF, Sox9, Core binding factor alpha 1 (Cbfa1), Runx2, and noggin have all been investigated with varying degrees of success.

#### *2.2.2. Using rAdV to treat cartilage regeneration*

A recent publication showed that AdBMP-2 stimulates chondrogenesis of equine synovial membrane-derived progenitor cells. Chondrogenesis was determined by the up-regulation of collagen II, X and aggrecan, as well as the secretion of sulfated glycosaminoglycans and production of alkaline phosphatase [81]. Two other growth factors, Insulin-like growth factor-I (IGF1) and human growth and differentiation factor-5 (GDF-5), have also been examined for cartilage regeneration using rAdVs. IGF-1 is the major anabolic mediator for articular cartilage and plays an important role in maintaining cartilage homeostasis. IGF-1 enhances cartilage matrix metabolism by increasing the production of aggrecan, hyaluronan, and proteoglycan link protein-1 and by preventing degradation of proteoglycans. It also protects cartilage from the harmful effects of interleukin-1 or TNF following assault or injury. In one study, the ability of adenovirus vector encoding equine IGF-1 (AdIGF-1) to heal cartilage in an equine femoropatellar joint model was examined [82]. Then, 2 × 10<sup>7</sup> AdIGF-1-modified chondrocytes were injected into the joint and the animals were monitored for repair over the course of 8 months. The results showed that the AdIGF-1-modified chondrocytes were able to induce high levels of IGF-1, which persists for up to 9 weeks post-transplant. The increase in IGF-1 also led to an increase in collagen II expression. Histological analysis of tissue repair showed significant amelioration over control joints. Furthermore, no difference in inflammation was seen between naive chondrocyte-implanted or AdIGF-1-transduced repair tissues. These data were determined by examining inflammatory markers (including MMP-1, MMP-3, MMP-13, and aggrecanase-1) by qPCR. In addition, it was shown that IGF-1-enhanced repair also involved an increase in tissue thickness. It appears that there was a greater defect filling, and upon examination, these cells morphologically resembled chondrocytes rather than a fibrocartilaginous-like phenotype seen within the control tissues. Another study in humans looked at the effects of AdV gene transduction FGF-2, FGF-2 combined with interleukin-1 receptor antagonist protein (IL-Ra), and/or insulin-like growth factor-1 (IGF-1). This was determined in both human osteoarthritis (OA) chondrocytes as well as in a leporine OA model [83]. FGF-2 expression protected human OA chondrocytes and decreased cartilage degradation *in vivo* (rabbit model). *In vitro,* FGF-2 induced collagen type II and an increased production of GAG. Furthermore, combining all three factors FGF-2, IL-1Ra, and IGF-1 leads to significantly lower levels of ADAMTS-5, MMP-13, and MMP-3, and increased amounts of TIMP-1. This was also true as seen in the rabbit model. The combined therapy seems to have a synergistic effect to achieve optimal results. The trigene expression system appears to promote GAG synthesis of chondrocyte, increases TIMP-1 expression, and reduces ADAMTS-5, MMP-13 and aggrecanase expression. Haupt et al*.* also found that an adenovirus-mediated gene therapy combining several factors was more efficient. In this study, IGF-1 and IL-1Ra were shown to promote the healing of cartilage injury in degenerative joint diseases, suggesting combination therapy could be beneficial for cartilage repair in degenerative joint diseases [84].

immunogenicity [75, 76]. Another method of prolonging transgene expression is by administering anti-T cell receptor monoclonal antibody following adenovirus-mediated *in vivo* gene

One of the most recent publications examined the effect of AdBMP-2 on the osteogenic ability of human mesenchymal stem cells (hMSCs) [78]. MSCs are multipotent somatic stem cells that are able to differentiate into a variety of cell types, including chondrocytes, myocytes, osteoblasts, and adipocytes. Targeting these cells with BMP-2 could potentially lead to their osteogenic differentiation and promote bone healing. *In vitro* experiments showed that when treated with AdBMP-2, hMSCs change phenotypes and resemble osteoblast-like cells. Further analysis showed that these changed cells also expressed ALP, an enzyme present in osteoblasts and critical for bone mineralization and calcification. Immunohistochemistry using a von Kossa stain (used for the quantification of mineralization in cell culture and tissue sections) showed increased positive staining at d14 post-treatment. Taken together, this study showed the potential of AdBMP-2 to skew the differentiation of hMSCs toward osteoblast-like cells, thereby potentially becoming a novel treatment for delayed or nonunion fractures.

In addition to BMPs, several other factors have been investigated to determine if their expression via an adenoviral vector leads to bone healing. Nell-1 is a novel direct transcriptional target of runt homology domain transcription factor-2 (Runx2). Nel-like molecule-1 (Nell-1) is osteoinductive on cells of the osteochondral lineage. Adenovirus vectors containing Nell-1 was shown to promote osteoblastic differentiation in calvarial cells (from the skull cap) [79]. An *in vivo* study demonstrated that Null-1 could be as efficient as BMP-2, one of the most potent BMPs, to induce rat calvarial bone formation [80]. VEGF, Sox9, Core binding factor alpha 1 (Cbfa1),

A recent publication showed that AdBMP-2 stimulates chondrogenesis of equine synovial membrane-derived progenitor cells. Chondrogenesis was determined by the up-regulation of collagen II, X and aggrecan, as well as the secretion of sulfated glycosaminoglycans and production of alkaline phosphatase [81]. Two other growth factors, Insulin-like growth factor-I (IGF1) and human growth and differentiation factor-5 (GDF-5), have also been examined for cartilage regeneration using rAdVs. IGF-1 is the major anabolic mediator for articular cartilage and plays an important role in maintaining cartilage homeostasis. IGF-1 enhances cartilage matrix metabolism by increasing the production of aggrecan, hyaluronan, and proteoglycan link protein-1 and by preventing degradation of proteoglycans. It also protects cartilage from the harmful effects of interleukin-1 or TNF following assault or injury. In one study, the ability of adenovirus vector encoding equine IGF-1 (AdIGF-1) to heal cartilage in an equine

were injected into the joint and the animals were monitored for repair over the course of 8 months. The results showed that the AdIGF-1-modified chondrocytes were able to induce high levels of IGF-1, which persists for up to 9 weeks post-transplant. The increase in IGF-1 also led to an increase in collagen II expression. Histological analysis of tissue repair showed significant amelioration over control joints. Furthermore, no difference in inflammation was

AdIGF-1-modified chondrocytes

Runx2, and noggin have all been investigated with varying degrees of success.

*2.2.2. Using rAdV to treat cartilage regeneration*

femoropatellar joint model was examined [82]. Then, 2 × 10<sup>7</sup>

transfer [77].

12 Cartilage Repair and Regeneration

GDF-5 has been shown to be essential for normal appendicular skeletal and joint development in humans and mice. It positively regulates differentiation of chondrogenic tissue through its binding with bone morphogenetic protein receptor type 1 A and B (BMPR1A and BMPR1B). It also negatively regulates chondrogenic differentiation through its interaction with noggin (NOG). One study conducted by Luo et al*.* investigated the effects of adenovirus-mediated GDF-5 (AdGDF-5) on ECM expression in human degenerative disc nucleus pulposus (NP) cells in order to determine if AdGDF-5 is a viable therapy to treat intervertebral disc degeneration (IDD) [85]. Like many other studies, they began by determining the expression of GDF-5 *in vitro* after treating HEK293 cells with AdGDF-5 and then determined the optimal amount of viral vector needed for efficient transduction of NP cells. Following this, they investigated the effects of expression of GDF-5 had on the ECM. It was noted that GDF-5 promotes the synthesis of sulfated glycoaminoglycans and hydroxyproline, two major structures forming the ECM network. In addition, immunohistochemistry showed an increase in proteoglycans in the AdGDF-5-treated NP cells, stimulated NP proliferation, and increased the expression of collagen II and aggrecan genes. The outcome of this study indicates that NP cells within degraded discs would be ideal targets for the transduction of transgenic proteins and that AdV therapy could be a promising new avenue for the treatment of disc degeneration.

As like for rAAV, the effects of Sox9 on MSCs have been examined as a novel treatment of cartilage repair. This is of no surprise, considering that Sox9 is considered a master regulator of chondrocyte phenotype [86]. Like that of rAAV, Sox9 has been shown to be able to modulate cartilage both *in vitro* and *in vivo*. In the study led by Cao et al*.,* Sox9 expression successfully promoted a chondrocyte morphology after AdV transduction of rabbit bone marrow mesenchymal stem cells (BMSCs) [87]. Overexpression of Sox9 resulted in the upregulation of collagen II and aggrecan, while inhibiting osteogenic differentiation. The latter was shown by a decrease in ALP staining and reduced expression of Runx2, Col I, and osteopontin. In rabbits, the AdVSox9 group had a better outcome regarding cartilage repair. This was seen by integration of *de novo* cartilage tissue repair, cells in the repaired tissue had distinctive morphology resembling chondrocytes that were surrounded by matrix that stained positive for safranin O and type II collagen. Finally, overexpression of Sox9 led to suppressing makers of hypertrophic chondrocytes (ColX and osteocalcin), thereby avoiding cartilage calcification.

plasmid containing gag and pol structural genes are needed to supply reverse transcriptase and integration functions for the therapeutic vector particles. Finally, the last part is composed of plasmids encoding envelope proteins for the therapeutic viral particles and perhaps Rev. protein. Typically, envelope gene used is that of the glycoprotein G from vesicular stomatitis virus (VSV-G). The addition of this foreign viral envelope is called pseudotyping, and it alters

Viruses: Friends and Foes

15

http://dx.doi.org/10.5772/intechopen.71071

Retroviruses and lentiviruses have been used to transfer genetic material since the 1980s. In the early 1990s, γ-retrovirus gene transfer was shown to be possible in hematopoietic stem cells [89]. This era also saw the first clinical trial that aimed at treating severe combined immunodeficiency (SCID) [90]. A major accomplishment in this field happened in the early 2000s, when 11 children were successfully treated for X-SCID by introducing the common interleukin receptor γ-chain in bone marrow using a retrovirus vector based on mouse leukemia virus (MLV) [91].

One of the first reports of using a lentivirus for the treatment of joints occurred in 2008. Ricchetti et al*.* overexpressed IL-10 in the patellar tendons of mice. IL-10 is known for its potent anti-inflammatory properties that limit host response to pathogens, but also can inhibit scar formation in fetal wound healing. In this study, a murine model of patellar tendon injury was used to investigate the effect of IL-10 overexpression on the properties of adult healing tendon. Findings showed successful transfer of IL-10 into patellar tendons with more than six times greater expression in comparison with endogenous IL-10 levels. IL-10 expression peaked at 10 days after injury. Furthermore, treated tendons showed improved maximum stress and percent relaxation was increased in the treated group. However, there were significant limitations regarding the study. The empty vector control also showed improved tendon properties compared to the sham control group, which could indicate that injection of the vector itself, rather than IL-10, as a beneficial effect. The authors hypothesize that injection of the viral vector may actually lead to more robust immune responses that subsequently drive

Many attempts have been made to use retroviruses and lentiviruses for a long-term transgene expression in chondrocytes. Toward this, many different animal cells have been used, including human, rat, rabbit, goat, and cattle [92–95]. One group showed that transduction of chondrocytes with GFP was associated with an approximate 60% success rate [92]. After 6 weeks, only 21% of the cells remained GFP positive, whereas other studies showed greater efficiency rates with up to 85% of osteoarthritic chondrocytes being transduced [94]. Human articular chondrocytes have been shown to be highly susceptible to lentiviral infection, with 74% being

Like for the other viral vectors described in this chapter, studies have focused on inserting factors, which could help cartilage or bone repair, either by incorporating molecules stimulating the ECM, chondrogenesis, or immunomodulatory molecules. One such study examined the

GFP positive and expression was maintained *in vitro* for up to 22 weeks [93].

the viral tropism to specifically target certain cell types.

*2.3.1. Using lentiviruses for joint repair*

better scar formation and wound healing.

*2.3.2. Lentiviruses toward cartilage regeneration*

In summary, like for rAAVs, rAdVs show a promising future for gene therapy to treat, or limit, joint damage. They have the advantage of growing to high titers, allowing high transduction efficiencies in a variety of cells and have shown promise in animal experiments as well as in explants. However, the main drawbacks for AdVs remain a long-term efficiency and overall safety. Prior exposure to various strains results in robust host immune responses against the vectors, greatly hindering long-term transgene expression in targeted patients. Moreover, the first patient death associated with gene therapy trials was that of an 18-year-old boy receiving a rAdV [88]. This vector contained ornithine transcarbamoylase (OTC), an enzyme needed to eliminate ammonia, and essential to treat the patient's partial OTC deficiency, which was present since birth. Unfortunately, the boy died 4 days after receiving the infusion and this adverse effect sparked controversy and ended in a lawsuit and formal investigation. Despite being the only death in nearly 4000 gene-therapy patients (over 400 trials), this hindered progress and saw extra measures for monitoring, reporting, and obtaining informed consent. The FDA and participants will probably still err on the side of caution when it comes to these types of clinical trials.

## **2.3. Retroviruses and lentiviral vectors**

Lentiviral vectors are members of the *Retroviridae* family. These vectors can deliver a substantial amount of genetic information by spontaneously penetrating the intact nuclear membrane and inserting the "carried" DNA into the host's DNA. Due to this unique property, they are among the most efficient methods for gene delivery. Furthermore, they can integrate into either actively replicating or quiescent cells. For these reasons, they are commonly used for *in vivo* delivery of genome editing therapies. However, this ability to integrate into the host's DNA also raises a number of safety and ethical concerns. Another drawback of this class of vector is the possibility to activate tumor genes and to provoke insertional mutagenesis events upon integration. Examples of most frequently used lentiviruses include human, simian, and feline immunodeficiency viruses (HIV, SIV, and FIV).

Compared to other forms of viral gene therapy, the main advantages of using lentiviruses include low or absence of preexisting immunity, ability to transport one or more transgenes, delivery of genetic material to replicating and nonreplicating cells, as well as prolonged transgene expression (upward of 6 months). In order to make a lentivirus vector, a split component system is needed, where each part is in itself nonpathogenic and only the sum of it is parts can actively infect cells. Target cells are usually transfected with the viral vector, which is flanked with long terminal repeats (LTRs). It is this feature that allows the carried transgene to integrate into the genome of the target cell. The vector could also contain the Rev-responsive element (RRE) for most efficient vector production and, of course, the gene of interest. In parallel, a plasmid containing gag and pol structural genes are needed to supply reverse transcriptase and integration functions for the therapeutic vector particles. Finally, the last part is composed of plasmids encoding envelope proteins for the therapeutic viral particles and perhaps Rev. protein. Typically, envelope gene used is that of the glycoprotein G from vesicular stomatitis virus (VSV-G). The addition of this foreign viral envelope is called pseudotyping, and it alters the viral tropism to specifically target certain cell types.

Retroviruses and lentiviruses have been used to transfer genetic material since the 1980s. In the early 1990s, γ-retrovirus gene transfer was shown to be possible in hematopoietic stem cells [89]. This era also saw the first clinical trial that aimed at treating severe combined immunodeficiency (SCID) [90]. A major accomplishment in this field happened in the early 2000s, when 11 children were successfully treated for X-SCID by introducing the common interleukin receptor γ-chain in bone marrow using a retrovirus vector based on mouse leukemia virus (MLV) [91].

## *2.3.1. Using lentiviruses for joint repair*

of collagen II and aggrecan, while inhibiting osteogenic differentiation. The latter was shown by a decrease in ALP staining and reduced expression of Runx2, Col I, and osteopontin. In rabbits, the AdVSox9 group had a better outcome regarding cartilage repair. This was seen by integration of *de novo* cartilage tissue repair, cells in the repaired tissue had distinctive morphology resembling chondrocytes that were surrounded by matrix that stained positive for safranin O and type II collagen. Finally, overexpression of Sox9 led to suppressing makers of hypertrophic chondrocytes (ColX and osteocalcin), thereby avoiding cartilage calcification.

In summary, like for rAAVs, rAdVs show a promising future for gene therapy to treat, or limit, joint damage. They have the advantage of growing to high titers, allowing high transduction efficiencies in a variety of cells and have shown promise in animal experiments as well as in explants. However, the main drawbacks for AdVs remain a long-term efficiency and overall safety. Prior exposure to various strains results in robust host immune responses against the vectors, greatly hindering long-term transgene expression in targeted patients. Moreover, the first patient death associated with gene therapy trials was that of an 18-year-old boy receiving a rAdV [88]. This vector contained ornithine transcarbamoylase (OTC), an enzyme needed to eliminate ammonia, and essential to treat the patient's partial OTC deficiency, which was present since birth. Unfortunately, the boy died 4 days after receiving the infusion and this adverse effect sparked controversy and ended in a lawsuit and formal investigation. Despite being the only death in nearly 4000 gene-therapy patients (over 400 trials), this hindered progress and saw extra measures for monitoring, reporting, and obtaining informed consent. The FDA and participants will probably still err on the side of caution when it comes to these types of clinical trials.

Lentiviral vectors are members of the *Retroviridae* family. These vectors can deliver a substantial amount of genetic information by spontaneously penetrating the intact nuclear membrane and inserting the "carried" DNA into the host's DNA. Due to this unique property, they are among the most efficient methods for gene delivery. Furthermore, they can integrate into either actively replicating or quiescent cells. For these reasons, they are commonly used for *in vivo* delivery of genome editing therapies. However, this ability to integrate into the host's DNA also raises a number of safety and ethical concerns. Another drawback of this class of vector is the possibility to activate tumor genes and to provoke insertional mutagenesis events upon integration. Examples of most frequently used lentiviruses include human, simian, and

Compared to other forms of viral gene therapy, the main advantages of using lentiviruses include low or absence of preexisting immunity, ability to transport one or more transgenes, delivery of genetic material to replicating and nonreplicating cells, as well as prolonged transgene expression (upward of 6 months). In order to make a lentivirus vector, a split component system is needed, where each part is in itself nonpathogenic and only the sum of it is parts can actively infect cells. Target cells are usually transfected with the viral vector, which is flanked with long terminal repeats (LTRs). It is this feature that allows the carried transgene to integrate into the genome of the target cell. The vector could also contain the Rev-responsive element (RRE) for most efficient vector production and, of course, the gene of interest. In parallel, a

**2.3. Retroviruses and lentiviral vectors**

14 Cartilage Repair and Regeneration

feline immunodeficiency viruses (HIV, SIV, and FIV).

One of the first reports of using a lentivirus for the treatment of joints occurred in 2008. Ricchetti et al*.* overexpressed IL-10 in the patellar tendons of mice. IL-10 is known for its potent anti-inflammatory properties that limit host response to pathogens, but also can inhibit scar formation in fetal wound healing. In this study, a murine model of patellar tendon injury was used to investigate the effect of IL-10 overexpression on the properties of adult healing tendon. Findings showed successful transfer of IL-10 into patellar tendons with more than six times greater expression in comparison with endogenous IL-10 levels. IL-10 expression peaked at 10 days after injury. Furthermore, treated tendons showed improved maximum stress and percent relaxation was increased in the treated group. However, there were significant limitations regarding the study. The empty vector control also showed improved tendon properties compared to the sham control group, which could indicate that injection of the vector itself, rather than IL-10, as a beneficial effect. The authors hypothesize that injection of the viral vector may actually lead to more robust immune responses that subsequently drive better scar formation and wound healing.

#### *2.3.2. Lentiviruses toward cartilage regeneration*

Many attempts have been made to use retroviruses and lentiviruses for a long-term transgene expression in chondrocytes. Toward this, many different animal cells have been used, including human, rat, rabbit, goat, and cattle [92–95]. One group showed that transduction of chondrocytes with GFP was associated with an approximate 60% success rate [92]. After 6 weeks, only 21% of the cells remained GFP positive, whereas other studies showed greater efficiency rates with up to 85% of osteoarthritic chondrocytes being transduced [94]. Human articular chondrocytes have been shown to be highly susceptible to lentiviral infection, with 74% being GFP positive and expression was maintained *in vitro* for up to 22 weeks [93].

Like for the other viral vectors described in this chapter, studies have focused on inserting factors, which could help cartilage or bone repair, either by incorporating molecules stimulating the ECM, chondrogenesis, or immunomodulatory molecules. One such study examined the possibility of expressing a member of the nuclear factor of activated T-cells (NFAT) as a means to treat osteoarthritis [96]. NFAT was initially identified as a regulator of gene transcription in response to T-cell receptor-mediated signals in lymphocytes. However, it is also involved in regulating bone formation and osteoclastic bone resorption [97, 98]. Interestingly, NFAT knockout mice have normal skeletal development, but with age, display loss of type II collagen, and aggrecan. They also show overexpression of specific matrix-degrading proteinases including MMPs and ADAMTS in addition to proinflammatory cytokines. The authors then used a lentiviral vector to express NFAT1 in cultured primary *Nfat1*−/− articular chondrocytes. This rescue of NFAT partially or completely rescued the abnormal catabolic and anabolic activities of *Nfat1*−/− articular chondrocytes.

Despite some promising results, the use of lentiviruses will probably always raise concerns about safety due to the ability to integrate into the host genome. Clinical trials will be challenging due to the unknown risks associated with their administration. Thorough justification for their use will be warranted especially with so many other types of viral vectors currently

Viruses: Friends and Foes

17

http://dx.doi.org/10.5772/intechopen.71071

Overall, this chapter examined some of the most recent literature surrounding the use of viral vectors for bone and cartilage repair. This is a vast field with many exciting studies and promising developments. There has been a huge amount of progress since the early development of viral gene therapy, and it is only a matter of time before joint disorders and injuries will be

[1] Kirik D, Cederfjall E, Halliday G, Petersen A. Gene therapy for Parkinson's disease. Disease modification by GDNF family of ligands. Neurobiology of disease. 2017;**97**(Pt B):179-188

[2] Joshi CR, Labhasetwar V, Ghorpade A. Destination brain: The past, present, and future of therapeutic gene delivery. Journal of Neuroimmune Pharmacology: The Official Journal of

[3] Kumar SR, Markusic DM, Biswas M, High KA, Herzog RW. Clinical development of gene therapy: Results and lessons from recent successes. Molecular Therapy Methods & Clinical

[4] Simonato M, Bennett J, Boulis NM, Castro MG, Fink DJ, Goins WF, et al. Progress in gene therapy for neurological disorders. Nature Reviews Neurology. 2013;**9**(5):277-291

[5] Yla-Herttuala S, Baker AH.Cardiovascular gene therapy: Past, present, and future. Molecular Therapy: The Journal of the American Society of Gene Therapy. 2017;**25**(5):1095-1106 [6] Evans CH, Huard J. Gene therapy approaches to regenerating the musculoskeletal sys-

[7] Loeser RF. Molecular mechanisms of cartilage destruction: Mechanics, inflammatory mediators, and aging collide. Arthritis and Rheumatism. 2006;**54**(5):1357-1360

[8] Buckwalter JA, Brown TD. Joint injury, repair, and remodeling: Roles in post-traumatic

osteoarthritis. Clinical orthopaedics and related research. 2004;**423**:7-16

available, although it is possible to see successful joint repair using such a system.

treated using these approaches.

Penny A. Rudd and Lara J. Herrero\*

Development. 2016;**3**:16034

\*Address all correspondence to: l.herrero@griffith.edu.au

Institute for Glycomics, Griffith University, Southport, Qld, Australia

the Society on Neuroimmune Pharmacology. 2017;**12**(1):51-83

tem. Nature Reviews Rheumatology. 2015;**11**(4):234-242

**Author details**

**References**

Another study looked at using the lentivirus vector to knock down aggrecanase activity [99]. RNAi was used to specifically target both aggrecanase-1 and -2 in primary rat chondrocytes. This approach was relatively successful *in vitro* with increased amounts of glycosaminoglycans and total collagen being produced as well as an increase in chondrocyte proliferation. This data provided the proof-of-principle that it is feasible to use this vector system to modulate chondrocyte phenotype and may be useful for future studies.

Several reports examined the ability of lentivirus vectors to be used to target MSCs in order to ameliorate the ECM surrounding the joints. One interesting example is the use of these vectors to help create a bioactive scaffold where sustained transgene expression and ECM formation are accomplished by human MSCs (hMSCs) [100]. The lentivirus vectors were used to express transforming growth factor β3 (TGF-β3) under the control of a constitutive EF-1α promoter. TGF-β3 was chosen as it was previously shown to be the most potent driver for chondrogenesis in hMSCs. After transduction, hMSCs developed a spherical shape comparable to chondrocyte-like morphology. Also, there was a substantial increase in col. II and glycosaminoglycan. Bioactive scaffolds with immobilized TGF-β3 expressed in lentivirus vectors showed a production of 17 ng/mL TGF-β3 and 12.87 μg sGAG/μg DNA at 1–3 weeks after seeding scaffolds. The results of this study indicate that the scaffold-mediated transduction technique could eventually be used *in vivo* to direct cell lineage commitment and ECM development in a controlled and persistent manner. The field of bioengineering is rapidly growing and the possibility of creating alternative methods for tissue replacement is not so far away.

One of the most recent publications examining the use of lentiviruses for cartilage repair used ovine perivascular stem cells (oPSCs). These cells are said to be natural ancestors of mesenchymal stem cells. The goal of this study was to develop an autologous large animal model for PSC transplantation and determine if implanted cells are retained in articular cartilage defects. oPSCs could be sourced from various locations including bone marrow, subcutaneous fat, and the infrapatellar fat pad. The lentivirus was used to transduce the cells with eGFP to allow tracking when implanted into the animals. The transduced cells were implanted into articular cartilage defects on the medial femoral condyle using hydrogel and collagen membranes. Results showed that GFP-emitting cells could be found at the base of the articular cartilage defect up to 4 weeks after transplantation. However, no repair tissue was seen by immunohistochemistry. Overall, more work needs to be done for this model to be a robust example of cartilage repair, but it could be an alternative replacement to the current canine model.

Despite some promising results, the use of lentiviruses will probably always raise concerns about safety due to the ability to integrate into the host genome. Clinical trials will be challenging due to the unknown risks associated with their administration. Thorough justification for their use will be warranted especially with so many other types of viral vectors currently available, although it is possible to see successful joint repair using such a system.

Overall, this chapter examined some of the most recent literature surrounding the use of viral vectors for bone and cartilage repair. This is a vast field with many exciting studies and promising developments. There has been a huge amount of progress since the early development of viral gene therapy, and it is only a matter of time before joint disorders and injuries will be treated using these approaches.

## **Author details**

possibility of expressing a member of the nuclear factor of activated T-cells (NFAT) as a means to treat osteoarthritis [96]. NFAT was initially identified as a regulator of gene transcription in response to T-cell receptor-mediated signals in lymphocytes. However, it is also involved in regulating bone formation and osteoclastic bone resorption [97, 98]. Interestingly, NFAT knockout mice have normal skeletal development, but with age, display loss of type II collagen, and aggrecan. They also show overexpression of specific matrix-degrading proteinases including MMPs and ADAMTS in addition to proinflammatory cytokines. The authors then used a lentiviral vector to express NFAT1 in cultured primary *Nfat1*−/− articular chondrocytes. This rescue of NFAT partially or completely rescued the abnormal catabolic and anabolic

Another study looked at using the lentivirus vector to knock down aggrecanase activity [99]. RNAi was used to specifically target both aggrecanase-1 and -2 in primary rat chondrocytes. This approach was relatively successful *in vitro* with increased amounts of glycosaminoglycans and total collagen being produced as well as an increase in chondrocyte proliferation. This data provided the proof-of-principle that it is feasible to use this vector system to modu-

Several reports examined the ability of lentivirus vectors to be used to target MSCs in order to ameliorate the ECM surrounding the joints. One interesting example is the use of these vectors to help create a bioactive scaffold where sustained transgene expression and ECM formation are accomplished by human MSCs (hMSCs) [100]. The lentivirus vectors were used to express transforming growth factor β3 (TGF-β3) under the control of a constitutive EF-1α promoter. TGF-β3 was chosen as it was previously shown to be the most potent driver for chondrogenesis in hMSCs. After transduction, hMSCs developed a spherical shape comparable to chondrocyte-like morphology. Also, there was a substantial increase in col. II and glycosaminoglycan. Bioactive scaffolds with immobilized TGF-β3 expressed in lentivirus vectors showed a production of 17 ng/mL TGF-β3 and 12.87 μg sGAG/μg DNA at 1–3 weeks after seeding scaffolds. The results of this study indicate that the scaffold-mediated transduction technique could eventually be used *in vivo* to direct cell lineage commitment and ECM development in a controlled and persistent manner. The field of bioengineering is rapidly growing and the possibility of creating alternative methods for tissue replacement is

One of the most recent publications examining the use of lentiviruses for cartilage repair used ovine perivascular stem cells (oPSCs). These cells are said to be natural ancestors of mesenchymal stem cells. The goal of this study was to develop an autologous large animal model for PSC transplantation and determine if implanted cells are retained in articular cartilage defects. oPSCs could be sourced from various locations including bone marrow, subcutaneous fat, and the infrapatellar fat pad. The lentivirus was used to transduce the cells with eGFP to allow tracking when implanted into the animals. The transduced cells were implanted into articular cartilage defects on the medial femoral condyle using hydrogel and collagen membranes. Results showed that GFP-emitting cells could be found at the base of the articular cartilage defect up to 4 weeks after transplantation. However, no repair tissue was seen by immunohistochemistry. Overall, more work needs to be done for this model to be a robust example of

cartilage repair, but it could be an alternative replacement to the current canine model.

activities of *Nfat1*−/− articular chondrocytes.

16 Cartilage Repair and Regeneration

not so far away.

late chondrocyte phenotype and may be useful for future studies.

Penny A. Rudd and Lara J. Herrero\*

\*Address all correspondence to: l.herrero@griffith.edu.au

Institute for Glycomics, Griffith University, Southport, Qld, Australia

## **References**


[9] Evans CH, Kraus VB, Setton LA. Progress in intra-articular therapy. Nature Reviews Rheumatology. 2014;**10**(1):11-22

[24] Tenenbaum L, Humbert-Claude M.Glial cell line-derived Neurotrophic factor gene delivery in Parkinson's disease: A delicate balance between neuroprotection, trophic effects, and unwanted compensatory mechanisms. Frontiers in Neuroanatomy. 2017;**11**:29 [25] Loring HS, Flotte TR. Current status of gene therapy for alpha-1 antitrypsin deficiency.

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19

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[26] Ward P, Walsh CE. Current and future prospects for hemophilia gene therapy. Expert

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[28] Mingozzi F, High KA. Therapeutic in vivo gene transfer for genetic disease using AAV:

[29] Bryant LM, Christopher DM, Giles AR, Hinderer C, Rodriguez JL, Smith JB, et al. Lessons learned from the clinical development and market authorization of Glybera. Human

[30] Russell DW, Miller AD, Alexander IE. Adeno-associated virus vectors preferentially transduce cells in S phase. Proceedings of the National Academy of Sciences of the United

[31] Santangelo KS, Bertone AL. Effective reduction of the interleukin-1beta transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis.

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**Chapter 2**

**Provisional chapter**

**Chondrocyte Turnover in Lung Cartilage**

**Chondrocyte Turnover in Lung Cartilage**

DOI: 10.5772/intechopen.70860

Yareth Gopar-Cuevas, Alberto Niderhauser-García,

Yareth Gopar-Cuevas, Alberto Niderhauser-García, Adriana Ancer-Arellano, Ivett C. Miranda-Maldonado, María-de-Lourdes Chávez-Briones, Laura E. Rodríguez-Flores,

Adriana Ancer-Arellano, Ivett C. Miranda-Maldonado,

Laura E. Rodríguez-Flores, Marta Ortega-Martínez and Gilberto Jaramillo-Rangel

Jaramillo-Rangel

**Abstract**

**1. Introduction**

María-de-Lourdes Chávez-Briones,

Marta Ortega-Martínez and Gilberto

http://dx.doi.org/10.5772/intechopen.70860

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

© 2016 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,

© 2018 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 reproduction in any medium, provided the original work is properly cited.

Cartilage is a connective tissue consisting of cells and extracellular matrix (ECM). These cells are called chondrocytes and reside within spaces called lacunae. The ECM is a three-dimensional

Cartilage is a highly differentiated connective tissue that forms mechanical support to soft tissues and is important for bone development from fetal period to puberty. It is conformed by chondrocytes and extracellular matrix. It is generally believed that adult cartilage has no capacity to renewal. A delicate balance between cell proliferation and cell death ensures the maintenance of normal tissue morphology and function. Stem cells play essential roles in this process. Mesenchymal stem cells (MSCs) can give rise to multiple lineages including bone, adipose and cartilage. Nestin protein was initially identified as a marker for neural stem cells, but its expression has also been detected in many types of cells, including MSCs. *In vivo*, chondrocyte turnover has been almost exclusively studied in articular cartilage. In this chapter we will review the findings about the chondrocyte turnover in lung cartilage. We have presented evidence that there exist nestinpositive MSCs in healthy adulthood that participates in the turnover of lung cartilage and in lung airway epithelium renewal. These findings may improve our knowledge about the biology of the cartilage and of the stem cells, and could provide new cell candidates for cartilage tissue engineering and for therapy for devastating pulmonary diseases. **Keywords:** lung, cartilage, chondrocyte, turnover, apoptosis, proliferation, stem cells


## **Chapter 2**

**Provisional chapter**

## **Chondrocyte Turnover in Lung Cartilage Chondrocyte Turnover in Lung Cartilage**

DOI: 10.5772/intechopen.70860

Yareth Gopar-Cuevas, Alberto Niderhauser-García, Adriana Ancer-Arellano, Ivett C. Miranda-Maldonado, María-de-Lourdes Chávez-Briones, Laura E. Rodríguez-Flores, Marta Ortega-Martínez and Gilberto Jaramillo-Rangel Yareth Gopar-Cuevas, Alberto Niderhauser-García, Adriana Ancer-Arellano, Ivett C. Miranda-Maldonado, María-de-Lourdes Chávez-Briones, Laura E. Rodríguez-Flores, Marta Ortega-Martínez and Gilberto Jaramillo-Rangel Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70860

#### **Abstract**

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Cartilage is a highly differentiated connective tissue that forms mechanical support to soft tissues and is important for bone development from fetal period to puberty. It is conformed by chondrocytes and extracellular matrix. It is generally believed that adult cartilage has no capacity to renewal. A delicate balance between cell proliferation and cell death ensures the maintenance of normal tissue morphology and function. Stem cells play essential roles in this process. Mesenchymal stem cells (MSCs) can give rise to multiple lineages including bone, adipose and cartilage. Nestin protein was initially identified as a marker for neural stem cells, but its expression has also been detected in many types of cells, including MSCs. *In vivo*, chondrocyte turnover has been almost exclusively studied in articular cartilage. In this chapter we will review the findings about the chondrocyte turnover in lung cartilage. We have presented evidence that there exist nestinpositive MSCs in healthy adulthood that participates in the turnover of lung cartilage and in lung airway epithelium renewal. These findings may improve our knowledge about the biology of the cartilage and of the stem cells, and could provide new cell candidates for cartilage tissue engineering and for therapy for devastating pulmonary diseases.

**Keywords:** lung, cartilage, chondrocyte, turnover, apoptosis, proliferation, stem cells

## **1. Introduction**

Cartilage is a connective tissue consisting of cells and extracellular matrix (ECM). These cells are called chondrocytes and reside within spaces called lacunae. The ECM is a three-dimensional

© 2016 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. © 2018 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.

macromolecular network composed of fibers and ground substance. In the mammals, much of the skeleton is first laid down in cartilage, and in the adult body it covers the articular surfaces of bones and forms the sole skeletal support of several structures [1].

glycoproteins. Cartilage is subdivided into three varieties depending on their molecular composition: hyaline, elastic, and fibrous [11]. Of these, hyaline cartilage is the most widely dis-

Chondrocyte Turnover in Lung Cartilage http://dx.doi.org/10.5772/intechopen.70860 27

With the exception of the free surfaces of articular cartilages, hyaline cartilage is surrounded by a membrane of fibrous connective tissue, the perichondrium. Cartilage is usually devoid of blood vessels, so its cells must obtain their oxygen and nutrients by long-range diffusion

The upper respiratory tract includes the nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal cords. The lower respiratory tract includes the trachea and within the lungs, the bronchi, bronchioles, and alveoli. This system performs or participates in several functions: air conduction, gas exchange, olfaction, and phonation [13–15]. Although the air passages take on their mature appearance well before a fetus is viable, they undergo significant maturational changes in late gestation. Thereafter, the lungs undergo a phase of growth and maturation during the first two decades of live and achieve maximal lung function approximately at the age of 20 years old for women and 25 years old for men. Lung function remains steady from age 20 to 35 years and starts declining thereafter. It has been suggested that airway cartilage plays an important role in determining airway compressibility and distensibility. Age-related differences in airway mechanical function may reflect an increase in stiffness of both airway muscle and cartilage that occurs with

Cartilage (hyaline type) has the function of maintaining airway patency and it also serves for the attachment of local muscle and connective tissue. It exists in the form of plates of cartilage

In the trachea and right and left main bronchi, cartilage is present in the anterior and lateral walls as C-shaped plates. Approximately 15–20 cartilaginous rings support the trachea. The cartilage in the wall of intrapulmonary bronchi is in the form of irregular cartilage plates that form a complete but not continuous circumferential support. The smallest bronchi have only widely scattered cartilaginous plates in their walls. Terminal and respiratory bronchioles lack

Physiological cell turnover plays an important role in maintaining normal tissue function and morphology. During this process, older differentiated cells are typically eliminated by programmed cell death (apoptosis) and replaced by the division progeny of adult stem cells

A delicate balance among all factors influencing cell turnover is needed to maintain the normal volume and function of tissues in healthy people. The key points of this homoeostatic

which have characteristics shapes and arrangements at different airway levels [19].

tributed type.

from the perichondrium [12].

**2.3. Pulmonary cartilage**

increasing age [16–18].

**3. Cell turnover**

(ASC) [20, 21].

supporting cartilaginous plates [13, 19].

Normal chondrocytes maintain a functional ECM that replaces itself very slowly and provides a shock absorber [2]. However, in mature cartilage, metabolic activity is low, and has been thought that adult chondrocytes resist proliferation throughout life. As a result, the mechanical properties of cartilage deteriorate with age [3–5].

Cell death and cell proliferation must be balanced in adult organisms in order to maintain homeostasis. Programmed cell death or apoptosis is important in mature organisms for deleting unwanted cells (e.g. aged cells). Most tissues contain stem cells that are able of proliferate and differentiate to replace cells that have been lost. A defective cell turnover process may have serious consequences to the tissues and the entire organism [6].

The role of chondrocyte turnover in cartilage aging and disease has been poorly analyzed and most of the related studies have been carried out in articular cartilage. In this chapter we will review the findings about the chondrocyte turnover in lung cartilage.

## **2. Chondrocyte, cartilage, and pulmonary cartilage**

## **2.1. The chondrocyte**

There are two forms of cells in cartilage: chondroblasts and chondrocytes. Chondroblasts are actively dividing immature cells which form ECM. They are oval or spindle-shaped cells with a spherical nucleus. The cytoplasm is basophilic, rich in ribosomes, rough endoplasmic reticulum, and Golgi saccules [7].

When chondroblasts are completely surrounded by ECM, they are called chondrocytes. They reside in spaces within the cartilage matrix known as lacunae. However, the cells fill the lacunae *in vivo*, as verified by electron microscopic studies. Chondrocytes vary from elongate to spherical in shape in relation to their position within the cartilage. They have a spherical nucleus with one or more nucleoli. Chondrocyte cytoplasm contains, in addition to glycogen and lipid, the usual characteristics of a secretory cell: abundant rough endoplasmic reticulum and prominent Golgi complex [8, 9].

The main function of the chondrocyte is to produce, maintain, and remodel the ECM of the cartilage. Chondrocytes receive mechanical, electrical, and physicochemical signals transmitted by the ECM and respond by regulating their metabolic activity [3, 9].

## **2.2. Cartilage**

Cartilage is flexible and strong, and is resilient to compression. It forms mechanical support to soft tissues and is important for bone development from fetal period to puberty [1, 10].

Cartilage consists of cells (chondroblasts and chondrocytes) and ECM. The ECM is primarily composed of tissue fluid and macromolecules, including collagens, proteoglycans, and glycoproteins. Cartilage is subdivided into three varieties depending on their molecular composition: hyaline, elastic, and fibrous [11]. Of these, hyaline cartilage is the most widely distributed type.

With the exception of the free surfaces of articular cartilages, hyaline cartilage is surrounded by a membrane of fibrous connective tissue, the perichondrium. Cartilage is usually devoid of blood vessels, so its cells must obtain their oxygen and nutrients by long-range diffusion from the perichondrium [12].

## **2.3. Pulmonary cartilage**

macromolecular network composed of fibers and ground substance. In the mammals, much of the skeleton is first laid down in cartilage, and in the adult body it covers the articular sur-

Normal chondrocytes maintain a functional ECM that replaces itself very slowly and provides a shock absorber [2]. However, in mature cartilage, metabolic activity is low, and has been thought that adult chondrocytes resist proliferation throughout life. As a result, the mechani-

Cell death and cell proliferation must be balanced in adult organisms in order to maintain homeostasis. Programmed cell death or apoptosis is important in mature organisms for deleting unwanted cells (e.g. aged cells). Most tissues contain stem cells that are able of proliferate and differentiate to replace cells that have been lost. A defective cell turnover process may

The role of chondrocyte turnover in cartilage aging and disease has been poorly analyzed and most of the related studies have been carried out in articular cartilage. In this chapter we will

There are two forms of cells in cartilage: chondroblasts and chondrocytes. Chondroblasts are actively dividing immature cells which form ECM. They are oval or spindle-shaped cells with a spherical nucleus. The cytoplasm is basophilic, rich in ribosomes, rough endoplasmic reticu-

When chondroblasts are completely surrounded by ECM, they are called chondrocytes. They reside in spaces within the cartilage matrix known as lacunae. However, the cells fill the lacunae *in vivo*, as verified by electron microscopic studies. Chondrocytes vary from elongate to spherical in shape in relation to their position within the cartilage. They have a spherical nucleus with one or more nucleoli. Chondrocyte cytoplasm contains, in addition to glycogen and lipid, the usual characteristics of a secretory cell: abundant rough endoplasmic reticulum

The main function of the chondrocyte is to produce, maintain, and remodel the ECM of the cartilage. Chondrocytes receive mechanical, electrical, and physicochemical signals transmit-

Cartilage is flexible and strong, and is resilient to compression. It forms mechanical support to soft tissues and is important for bone development from fetal period to puberty [1, 10].

Cartilage consists of cells (chondroblasts and chondrocytes) and ECM. The ECM is primarily composed of tissue fluid and macromolecules, including collagens, proteoglycans, and

ted by the ECM and respond by regulating their metabolic activity [3, 9].

faces of bones and forms the sole skeletal support of several structures [1].

have serious consequences to the tissues and the entire organism [6].

review the findings about the chondrocyte turnover in lung cartilage.

**2. Chondrocyte, cartilage, and pulmonary cartilage**

**2.1. The chondrocyte**

26 Cartilage Repair and Regeneration

lum, and Golgi saccules [7].

and prominent Golgi complex [8, 9].

**2.2. Cartilage**

cal properties of cartilage deteriorate with age [3–5].

The upper respiratory tract includes the nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal cords. The lower respiratory tract includes the trachea and within the lungs, the bronchi, bronchioles, and alveoli. This system performs or participates in several functions: air conduction, gas exchange, olfaction, and phonation [13–15].

Although the air passages take on their mature appearance well before a fetus is viable, they undergo significant maturational changes in late gestation. Thereafter, the lungs undergo a phase of growth and maturation during the first two decades of live and achieve maximal lung function approximately at the age of 20 years old for women and 25 years old for men. Lung function remains steady from age 20 to 35 years and starts declining thereafter. It has been suggested that airway cartilage plays an important role in determining airway compressibility and distensibility. Age-related differences in airway mechanical function may reflect an increase in stiffness of both airway muscle and cartilage that occurs with increasing age [16–18].

Cartilage (hyaline type) has the function of maintaining airway patency and it also serves for the attachment of local muscle and connective tissue. It exists in the form of plates of cartilage which have characteristics shapes and arrangements at different airway levels [19].

In the trachea and right and left main bronchi, cartilage is present in the anterior and lateral walls as C-shaped plates. Approximately 15–20 cartilaginous rings support the trachea. The cartilage in the wall of intrapulmonary bronchi is in the form of irregular cartilage plates that form a complete but not continuous circumferential support. The smallest bronchi have only widely scattered cartilaginous plates in their walls. Terminal and respiratory bronchioles lack supporting cartilaginous plates [13, 19].

## **3. Cell turnover**

Physiological cell turnover plays an important role in maintaining normal tissue function and morphology. During this process, older differentiated cells are typically eliminated by programmed cell death (apoptosis) and replaced by the division progeny of adult stem cells (ASC) [20, 21].

A delicate balance among all factors influencing cell turnover is needed to maintain the normal volume and function of tissues in healthy people. The key points of this homoeostatic process are apoptosis and cell proliferation. Cell turnover is precisely regulated by the interplay of various factors, which modulate tissue and cell-specific responses on apoptosis and proliferation, either directly, or by altering expression and function of key death and/or cell proliferative genes [6, 20, 22].

There are two main apoptotic pathways: the extrinsic or death receptor pathway, which is triggered from outside of the cell by death ligands, and the intrinsic or mitochondrial pathway, which is triggered from inside the cell as a response to various stress signals. Both intrinsic as well as extrinsic pathways of apoptosis are associated and influence each other [33]. Another pathway of apoptosis as also been recognized that involves T- and NK-cell mediated

Chondrocyte Turnover in Lung Cartilage http://dx.doi.org/10.5772/intechopen.70860 29

The three pathways converge on the same execution pathway: the activation of cysteine proteases of the caspase family, which selectively digest the cell from within. The perforin/granzyme pathway also activates another cell death pathway via single stranded DNA damage

Since the pathways of apoptosis are very complicated, there are a lot of features of it than can be evaluated. A great number of methods have been developed to detect apoptosis, such as morphological techniques, proteomic and genomic approaches, spectroscopic methods, flow cytometry, caspase activity assays, microfluidic applications, and electrochemical methods [35]. Each assay has advantages and disadvantages. Understanding the strengths and limitations of the assays would allow investigators to select the best methods for their needs [28, 36]. A description of all assays for detecting apoptosis is beyond the scope of this chapter. We will

Detection of apoptotic cells in hematoxylin and eosin-stained tissue sections with light microscopy is possible because of characteristic morphological features of apoptosis. They include condensation of the chromatin in granular masses along the nuclear envelope, cell shrinkage, convolution of the cellular and nuclear outlines, and fragmentation of the nucleus. The apoptotic cell breaks into membrane bound bodies that are quickly removed by neighboring macrophages. The condensed or fragmented nucleus can be detected with DNA dyes such as propidium iodide, Hoechst dye, or DAPI (4′,6-diamidino-2-phenylindole). Light microscopy detects the later events of apoptosis and confirmation with other methods may

A more definitive method of morphologic identification of apoptotic cells is TEM, because apoptosis is confirmed by several of its ultrastructural characteristics. TEM detects chromatin condensation and convulsions in and around the nuclear envelope that precedes nuclear fragmentation, the condensation of cytoplasm with the disappearance of the microvilli, blebs on the cell surface, and the loss of cell junctions. If immunochemical staining is employed, then chemical information can be also obtained. However, there are limitations in TEM as an apoptosis detection method, including that apoptotic cells detected by TEM are in the last stage of apoptosis, and that much time and a high skill are required for preparation of ultra-

cytotoxicity and perforin-granzyme-dependent killing of the cell [34].

briefly describe the assays to detect apoptosis most employed by our group.

[29, 34].

*3.1.3. Methods of apoptosis detection*

*3.1.3.1. Light microscopy*

be necessary [37, 38].

*3.1.3.2. Transmission electron microscopy (TEM)*

thin sections used in TEM [35, 39].

Age-specific changes in tissue regeneration and repair lead to cell loss and compromise of tissue homeostasis, structure, and function. These phenomena parallel changes in resident stem cell function [23, 24].

## **3.1. Apoptosis**

Apoptosis is a process of controlled cellular death whereby the activation of specific deathsignaling pathways leads to deletion of cells from tissue [25]. The term apoptosis was first used in a paper by Kerr, Wyllie, and Currie in 1972 to describe a morphologically distinct form of cell death [26], discriminating it from necrosis.

Apoptosis plays an essential role in survival of the organisms and is responsible for many biological processes such as normal cell turnover, embryonic and brain development, proper development and functioning of the immune system, and hormone-dependent atrophy [27, 28].

## *3.1.1. Apoptosis versus necrosis. Other forms of cell death*

Cell death has been broadly classified in two categories: apoptosis and necrosis. Apoptosis is a synchronized and energy-requiring process than involves altered expression of key cell proliferation and death-inducing genes, and the activation of a group of cysteine proteases (caspases) in a complex cascade of events that link the initiating stimuli to the final demise of the cell, while necrosis does not involve gene expression and is a passive externally driven event resulting from acute cellular injury [20, 29]. However, increasing evidence has been accumulating that necrosis can occur in a regulated manner, and that necrosis has a prominent role in multiple physiological and pathological settings [30].

Apoptosis is morphologically characterized by cell shrinkage, detachment from the substrate, chromatin condensation, nuclear and DNA fragmentation, cytoplasmic membrane blebbing, package of the cell debris into apoptotic bodies, and engulfment by resident phagocytes. Necrosis involves increase in cell volume, swelling of organelles, rupture of the plasma membrane, and the subsequent release of the cytoplasmic contents into the surrounding tissue, leading to inflammatory reaction [31].

Recently, new forms of cell death have been progressively described, which can be more precisely distinguished based on molecular pathways. A functional classification of cell death forms have been proposed that includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death, and mitotic catastrophe [30, 31].

## *3.1.2. Apoptosis mechanisms*

Apoptosis can be initiated by exogenous stimuli such as ionizing radiation and chemotherapeutic drugs, as well as by endogenous stimuli such as the absence of oxygen, nutrients or growth/survival factors, the presence of DNA damage, or the action of cytokines [32].

There are two main apoptotic pathways: the extrinsic or death receptor pathway, which is triggered from outside of the cell by death ligands, and the intrinsic or mitochondrial pathway, which is triggered from inside the cell as a response to various stress signals. Both intrinsic as well as extrinsic pathways of apoptosis are associated and influence each other [33]. Another pathway of apoptosis as also been recognized that involves T- and NK-cell mediated cytotoxicity and perforin-granzyme-dependent killing of the cell [34].

The three pathways converge on the same execution pathway: the activation of cysteine proteases of the caspase family, which selectively digest the cell from within. The perforin/granzyme pathway also activates another cell death pathway via single stranded DNA damage [29, 34].

## *3.1.3. Methods of apoptosis detection*

process are apoptosis and cell proliferation. Cell turnover is precisely regulated by the interplay of various factors, which modulate tissue and cell-specific responses on apoptosis and proliferation, either directly, or by altering expression and function of key death and/or cell

Age-specific changes in tissue regeneration and repair lead to cell loss and compromise of tissue homeostasis, structure, and function. These phenomena parallel changes in resident stem

Apoptosis is a process of controlled cellular death whereby the activation of specific deathsignaling pathways leads to deletion of cells from tissue [25]. The term apoptosis was first used in a paper by Kerr, Wyllie, and Currie in 1972 to describe a morphologically distinct

Apoptosis plays an essential role in survival of the organisms and is responsible for many biological processes such as normal cell turnover, embryonic and brain development, proper development and functioning of the immune system, and hormone-dependent atrophy [27, 28].

Cell death has been broadly classified in two categories: apoptosis and necrosis. Apoptosis is a synchronized and energy-requiring process than involves altered expression of key cell proliferation and death-inducing genes, and the activation of a group of cysteine proteases (caspases) in a complex cascade of events that link the initiating stimuli to the final demise of the cell, while necrosis does not involve gene expression and is a passive externally driven event resulting from acute cellular injury [20, 29]. However, increasing evidence has been accumulating that necrosis can occur in a regulated manner, and that necrosis has a promi-

Apoptosis is morphologically characterized by cell shrinkage, detachment from the substrate, chromatin condensation, nuclear and DNA fragmentation, cytoplasmic membrane blebbing, package of the cell debris into apoptotic bodies, and engulfment by resident phagocytes. Necrosis involves increase in cell volume, swelling of organelles, rupture of the plasma membrane, and the subsequent release of the cytoplasmic contents into the surrounding tissue,

Recently, new forms of cell death have been progressively described, which can be more precisely distinguished based on molecular pathways. A functional classification of cell death forms have been proposed that includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death, and mitotic catastrophe [30, 31].

Apoptosis can be initiated by exogenous stimuli such as ionizing radiation and chemotherapeutic drugs, as well as by endogenous stimuli such as the absence of oxygen, nutrients or

growth/survival factors, the presence of DNA damage, or the action of cytokines [32].

proliferative genes [6, 20, 22].

28 Cartilage Repair and Regeneration

form of cell death [26], discriminating it from necrosis.

*3.1.1. Apoptosis versus necrosis. Other forms of cell death*

leading to inflammatory reaction [31].

*3.1.2. Apoptosis mechanisms*

nent role in multiple physiological and pathological settings [30].

cell function [23, 24].

**3.1. Apoptosis**

Since the pathways of apoptosis are very complicated, there are a lot of features of it than can be evaluated. A great number of methods have been developed to detect apoptosis, such as morphological techniques, proteomic and genomic approaches, spectroscopic methods, flow cytometry, caspase activity assays, microfluidic applications, and electrochemical methods [35]. Each assay has advantages and disadvantages. Understanding the strengths and limitations of the assays would allow investigators to select the best methods for their needs [28, 36]. A description of all assays for detecting apoptosis is beyond the scope of this chapter. We will briefly describe the assays to detect apoptosis most employed by our group.

#### *3.1.3.1. Light microscopy*

Detection of apoptotic cells in hematoxylin and eosin-stained tissue sections with light microscopy is possible because of characteristic morphological features of apoptosis. They include condensation of the chromatin in granular masses along the nuclear envelope, cell shrinkage, convolution of the cellular and nuclear outlines, and fragmentation of the nucleus. The apoptotic cell breaks into membrane bound bodies that are quickly removed by neighboring macrophages. The condensed or fragmented nucleus can be detected with DNA dyes such as propidium iodide, Hoechst dye, or DAPI (4′,6-diamidino-2-phenylindole). Light microscopy detects the later events of apoptosis and confirmation with other methods may be necessary [37, 38].

#### *3.1.3.2. Transmission electron microscopy (TEM)*

A more definitive method of morphologic identification of apoptotic cells is TEM, because apoptosis is confirmed by several of its ultrastructural characteristics. TEM detects chromatin condensation and convulsions in and around the nuclear envelope that precedes nuclear fragmentation, the condensation of cytoplasm with the disappearance of the microvilli, blebs on the cell surface, and the loss of cell junctions. If immunochemical staining is employed, then chemical information can be also obtained. However, there are limitations in TEM as an apoptosis detection method, including that apoptotic cells detected by TEM are in the last stage of apoptosis, and that much time and a high skill are required for preparation of ultrathin sections used in TEM [35, 39].

## *3.1.3.3. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)*

TUNEL method is based on the assumption that genomic DNA is fragmented in a dying cell, producing fragments of consistent length in apoptotic cell death, as opposed to necrotic cell death where DNA is believed to be randomly degraded [40, 41]. The method consists of the labeling of DNA nick ends by terminal deoxynucleotidyl transferase (TdT) which incorporates the labeled nucleotide (most often dUTP) in the places of DNA strain breaks. The dUTP can then be labeled with a variety of probes to allow detection by light microscopy, fluorescence microscopy, or flow cytometry [42].

to ensure that they occur in the correct order with respect to each other and that they occur

At least two types of cell cycle regulation mechanisms have been recognized: cell cycle checkpoints, which are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events of the cell cycle [54], and a cascade of activation and deactivation of a series

Cell cycle checkpoints are a series of control systems enabling proliferation only in the presence of stimulatory signals (e.g. growth factors). They also arrest the cell cycle in response to DNA damage in order to provide time for DNA repair. After damage repair, progression through the cell cycle resumes. If the damage cannot be repaired, the cell is eliminated by

times known as the "point of no return" in the cell cycle with respect to S phase entry [56].

Additional checkpoints exist in S phase to activate DNA repair mechanisms when necessary. Furthermore, incomplete DNA replication or DNA damage triggers checkpoint pathways

Finally, the spindle assembly checkpoint acts during mitosis to maintain genome stability by delaying cell division (cytokinesis) until accurate chromosome segregation can be guar-

The main families of proteins that play key roles in controlling cell cycle progression are the Cdks, the cyclins, the Cdk inhibitors (CKIs), and the tumor-suppressor gene products—the

Progression of the cell through the cell cycle is mediated by sequential activation and inactivation of Cdks. The Cdks are a family of serine/threonine protein kinases that are activated at specific points of the cell cycle by the cyclins. Cdks activity can be counteracted by cell cycle

Activated Cdks induce downstream processes by phosphorylating selected proteins. pRb is a downstream target of Cdks-cyclins complexes [62]. Full pRb hyperphosphorylation releases pRb from E2F relieving repression of E2F target genes and allowing for activated

p53 is stabilized in response to DNA damage, oncogenic stress, and various other stress conditions and activates transcription of a number of genes (including *p21*, *Mdm2*, and *Bax*)

E2F-dependent transcriptional induction and cell cycle progression [63].

. Once the cell has entered S phase, it is bound to con-

Chondrocyte Turnover in Lung Cartilage http://dx.doi.org/10.5772/intechopen.70860 31

/S checkpoint (see above), cell cycle arrest

, and M and thus produce two daughter cells. This checkpoint is some-

/M transition to ensure that cells have completely replicated their DNA and

only once per cell cycle [53].

*3.2.1.1.1. Cell cycle checkpoints*

The primary checkpoint acts late in G<sup>1</sup>

that it is intact before they enter mitosis [57].

*3.2.1.1.2. Cyclin-dependent kinases (Cdks) regulation*

that induce cell cycle arrest or apoptosis. At the G<sup>1</sup>

induced by DNA damage is p53-dependent [64].

retinoblastoma protein (pRb) and p53 [59].

inhibitory proteins, the CKIs [60, 61].

apoptosis [55].

tinue through S, G<sup>2</sup>

that block the G<sup>2</sup>

anteed [58].

of proteins that relay a cell from one stage to the next [47].

TUNEL method is suitable for analysis of apoptosis in individual cells applicable to all kinds of material: cultured cells, tissues, and blood samples, even if a material contains only a few apoptotic cells. Another advantage of the TUNEL staining is that detects cells at a relatively early stage of apoptosis [39, 43]. However, this method also has drawbacks. Notably, it has been reported that the TUNEL assay also detect necrotic and autolytic cells in addition to apoptotic cells [44, 45].

## **3.2. Cell proliferation**

Cell proliferation is the process whereby cells reproduce themselves by growing and then dividing into two equal copies [46]. This process is a fundamental requirement for normal development and homeostasis.

Cell division consists of two consecutive processes, mainly characterized by DNA replication and segregation of replicated chromosomes into two separate cells. The process of replicating DNA and dividing a cell can be described as a series of coordinated events that compose a cell cycle [47, 48].

## *3.2.1. The cell cycle*

The cell cycle can be subdivided into two stages: interphase and mitosis. Genome replication occurs during the interphase, and its segregation to the daughter cells during the mitosis. The interphase includes G<sup>1</sup> , S, and G<sup>2</sup> phases. Cells in G<sup>0</sup> are not actively cycling and have to be stimulated by growth factors in order to enter the cell cycle in G<sup>1</sup> [49]. Mitosis includes prophase, prometaphase, metaphase, anaphase, and telophase, and also cell division (cytokinesis), which overlaps the final stages of mitosis [50, 51]. In this chapter we will further analyze only the interphase.

DNA synthesis and doubling of the genome take place during the synthetic or S phase. This is preceded by a period or gap of variable duration called G<sup>1</sup> during which the cell is preparing for DNA synthesis, and is followed by a period known as the second gap or G<sup>2</sup> , during which the cell prepares for mitosis [48, 52].

## *3.2.1.1. Cell cycle regulation*

Cell proliferation is a process fundamental to development, growth, homeostasis, adaptation to disease, and neoplasia. For this reason, cell cycle events must be tightly regulated to ensure that they occur in the correct order with respect to each other and that they occur only once per cell cycle [53].

At least two types of cell cycle regulation mechanisms have been recognized: cell cycle checkpoints, which are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events of the cell cycle [54], and a cascade of activation and deactivation of a series of proteins that relay a cell from one stage to the next [47].

## *3.2.1.1.1. Cell cycle checkpoints*

*3.1.3.3. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)*

cence microscopy, or flow cytometry [42].

apoptotic cells [44, 45].

30 Cartilage Repair and Regeneration

**3.2. Cell proliferation**

cell cycle [47, 48].

*3.2.1. The cell cycle*

interphase includes G<sup>1</sup>

the cell prepares for mitosis [48, 52].

*3.2.1.1. Cell cycle regulation*

the interphase.

, S, and G<sup>2</sup>

preceded by a period or gap of variable duration called G<sup>1</sup>

stimulated by growth factors in order to enter the cell cycle in G<sup>1</sup>

development and homeostasis.

TUNEL method is based on the assumption that genomic DNA is fragmented in a dying cell, producing fragments of consistent length in apoptotic cell death, as opposed to necrotic cell death where DNA is believed to be randomly degraded [40, 41]. The method consists of the labeling of DNA nick ends by terminal deoxynucleotidyl transferase (TdT) which incorporates the labeled nucleotide (most often dUTP) in the places of DNA strain breaks. The dUTP can then be labeled with a variety of probes to allow detection by light microscopy, fluores-

TUNEL method is suitable for analysis of apoptosis in individual cells applicable to all kinds of material: cultured cells, tissues, and blood samples, even if a material contains only a few apoptotic cells. Another advantage of the TUNEL staining is that detects cells at a relatively early stage of apoptosis [39, 43]. However, this method also has drawbacks. Notably, it has been reported that the TUNEL assay also detect necrotic and autolytic cells in addition to

Cell proliferation is the process whereby cells reproduce themselves by growing and then dividing into two equal copies [46]. This process is a fundamental requirement for normal

Cell division consists of two consecutive processes, mainly characterized by DNA replication and segregation of replicated chromosomes into two separate cells. The process of replicating DNA and dividing a cell can be described as a series of coordinated events that compose a

The cell cycle can be subdivided into two stages: interphase and mitosis. Genome replication occurs during the interphase, and its segregation to the daughter cells during the mitosis. The

phase, prometaphase, metaphase, anaphase, and telophase, and also cell division (cytokinesis), which overlaps the final stages of mitosis [50, 51]. In this chapter we will further analyze only

DNA synthesis and doubling of the genome take place during the synthetic or S phase. This is

Cell proliferation is a process fundamental to development, growth, homeostasis, adaptation to disease, and neoplasia. For this reason, cell cycle events must be tightly regulated

are not actively cycling and have to be

during which the cell is preparing

[49]. Mitosis includes pro-

, during which

phases. Cells in G<sup>0</sup>

for DNA synthesis, and is followed by a period known as the second gap or G<sup>2</sup>

Cell cycle checkpoints are a series of control systems enabling proliferation only in the presence of stimulatory signals (e.g. growth factors). They also arrest the cell cycle in response to DNA damage in order to provide time for DNA repair. After damage repair, progression through the cell cycle resumes. If the damage cannot be repaired, the cell is eliminated by apoptosis [55].

The primary checkpoint acts late in G<sup>1</sup> . Once the cell has entered S phase, it is bound to continue through S, G<sup>2</sup> , and M and thus produce two daughter cells. This checkpoint is sometimes known as the "point of no return" in the cell cycle with respect to S phase entry [56].

Additional checkpoints exist in S phase to activate DNA repair mechanisms when necessary. Furthermore, incomplete DNA replication or DNA damage triggers checkpoint pathways that block the G<sup>2</sup> /M transition to ensure that cells have completely replicated their DNA and that it is intact before they enter mitosis [57].

Finally, the spindle assembly checkpoint acts during mitosis to maintain genome stability by delaying cell division (cytokinesis) until accurate chromosome segregation can be guaranteed [58].

## *3.2.1.1.2. Cyclin-dependent kinases (Cdks) regulation*

The main families of proteins that play key roles in controlling cell cycle progression are the Cdks, the cyclins, the Cdk inhibitors (CKIs), and the tumor-suppressor gene products—the retinoblastoma protein (pRb) and p53 [59].

Progression of the cell through the cell cycle is mediated by sequential activation and inactivation of Cdks. The Cdks are a family of serine/threonine protein kinases that are activated at specific points of the cell cycle by the cyclins. Cdks activity can be counteracted by cell cycle inhibitory proteins, the CKIs [60, 61].

Activated Cdks induce downstream processes by phosphorylating selected proteins. pRb is a downstream target of Cdks-cyclins complexes [62]. Full pRb hyperphosphorylation releases pRb from E2F relieving repression of E2F target genes and allowing for activated E2F-dependent transcriptional induction and cell cycle progression [63].

p53 is stabilized in response to DNA damage, oncogenic stress, and various other stress conditions and activates transcription of a number of genes (including *p21*, *Mdm2*, and *Bax*) that induce cell cycle arrest or apoptosis. At the G<sup>1</sup> /S checkpoint (see above), cell cycle arrest induced by DNA damage is p53-dependent [64].

## *3.2.2. Identification and measurement of cell proliferation*

Assessment of cell proliferation is often of relevance in biomedical science, and a range of techniques have evolved to identify and quantify the process, generally by recognition and calculation of the number of cells in S or M phase [65].

asymmetrical cell division, producing an exact multipotent replica cell, and an additional

Chondrocyte Turnover in Lung Cartilage http://dx.doi.org/10.5772/intechopen.70860 33

Stem cells are classified according to their origin and developmental status in embryonic stem cells (ESC) and adult stem cells. Embryonic stem cells (ESC) can be derived from the inner cell mass of a blastocyst during gastrulation. They are totipotent cells giving rise to the germ line during development and virtually to all tissues of the organism [78, 79]. Adult stem cells (ASC) are tissue-resident stem cells that, based on their differentiation potency, can be classified as multipotent, oligopotent, or even unipotent [80]. In their tissue of residency, ASC function as lineage-committed progenitors to cells capable of more highly specialized tasks [78]. They are involved in tissue homeostasis and repair after wounding over

Among the tissues and organs harboring ASC, there are bone marrow, vascular walls, adipose tissues, skeletal muscles, heart, and brain, as well as epithelium of lung, liver, pancreas, digestive tract, skin, retina, breast, ovaries, prostate, and testis [81]. The bone marrow stem cell niche includes the hematopoietic stem cell population, which provides continuous renewal of blood cell lineages and the foundation of the immune system, and the mesenchymal stem cell population, responsible for osteogenic, adipogenic, and chondrogenic dif-

The minimal criteria for defining MSCs include: (a) remain plastic-adherent under standard culture conditions; (b) express CD73, CD90, and CD105, and lack expression of CD34, CD45, CD14 or CD11b, CD79a or CD19, and HLA-DR surface molecules; and (c) differentiate into

Originally isolated from bone marrow, MSCs have being isolated from other sites including spleen, thymus, muscle, adipose tissue, endometrium, placenta, umbilical cord, umbilical cord blood, peripheral blood, periosteum, periodontal ligament, dental pulp, synovium, synovial fluid, tendons, and cartilage [84, 85]. A perivascular location for MSCs has been suggested, correlating these cells with pericytes. This would explain why MSCs can be virtually

MSCs have demonstrated significant potential for clinical use due to their convenient isolation, their lack of significant immunogenicity permitting allogenic transplantation, their lack of ethical controversy, and their potential to differentiate into tissue-specific cell types [87]. MSCs may have therapeutic applications in several clinical disorders including myocardial infarction, diabetes, sepsis, hepatic failure, acute renal failure, several kinds of lung disease,

The human nestin protein consists of 1621 amino acids and displays a predicted molecular weight of 177.4 kDa. It is a class VI intermediate filament protein. Intermediate filaments represent, along with microtubules and actin filaments, one of the main components of cyto-

as well as in spinal cord injuries, and bone and cartilage diseases [88, 89].

progeny cell than can perform a more specialized function [78].

the lifetime [79].

ferentiation [82].

**4.2. Mesenchymal stem cells (MSCs)**

isolated from all tissues [79, 86].

**4.3. Nestin-positive MSCs**

skeleton in animal cells [90].

osteoblasts, adipocytes, and chondrocytes *in vitro* [83, 84].

A variety of markers have been used to determine cell cycle status and quantify cell proliferation, including the identification of mitotic figures, tritiated thymidine incorporation, bromodeoxyuridine incorporation, expression of proteins such as the proliferative cell nuclear antigen (PCNA), Ki-67, cyclins and Cdks, and the analysis of Cdks phosphorylation status [62, 66].

Of importance for this chapter are the immunohistochemical methods that detect proliferation-associated antigens. Ideally, such methods should be applicable to routinely processed tissues, they should be relatively inexpensive and the results easily quantified and interpreted [67]. The best known markers employed to recognize proliferating cells are Ki-67 and PCNA.

Cells express Ki-67 during G<sup>1</sup> , S, G<sup>2</sup> , and M phases, but not during the resting phase G<sup>0</sup> . Its levels are low in the G<sup>1</sup> and S phases and rise to their peak level in M. Later in the M phase, a sharp decrease in Ki-67 levels occurs [68]. Ki-67 is required to maintain individual mitotic chromosomes dispersed in the cytoplasm after their release from the nuclear envelope, through a surfactant mechanism [69].

Ki-67 is widely used as a proliferation marker because it provides a rapid and relatively inexpensive method of measuring dividing cells [65, 70]. However, the short half-life of Ki-67 (1–1.5 h, regardless of the cell position in the cell cycle [71, 72]) makes its detection difficult. Furthermore, some healthy tissues can express low levels of Ki-67 [68].

PCNA was first shown to act as a cofactor/auxiliary protein for DNA polymerase δ, which is required for DNA synthesis during replication. However, besides DNA replication, PCNA functions have been associated with other cellular processes such as chromatin remodeling, DNA repair, sister-chromatid cohesion, and cell cycle control [73]. During DNA replication, presence of PCNA is necessary for synthesis of the leading strand. Levels of PCNA expression are therefore highest during S phase, with little to no expression during G<sup>1</sup> and intermediate levels in G<sup>2</sup> and M phases [62, 74].

PCNA detection has been widely used in immunohistochemical studies of cell proliferation. However, some authors claim that PCNA is not a reliable marker of this process because it is a pleiotropic protein involved in several aspects of cell control and not only in proliferation [66]. On the opposite, other authors affirm that PCNA is the most reliable and versatile of all markers used to analyze cell proliferation [75]. In the past, we have successfully used the immunohistochemical detection of PCNA in studies of cell turnover in lung [76].

## **4. Stem cells**

## **4.1. Definition and classification**

Stem cells are generally defined as clonogenic cells capable of both self-renewal and multilineage differentiation [77]. For a cell to be considered a stem cell, it must be capable of asymmetrical cell division, producing an exact multipotent replica cell, and an additional progeny cell than can perform a more specialized function [78].

Stem cells are classified according to their origin and developmental status in embryonic stem cells (ESC) and adult stem cells. Embryonic stem cells (ESC) can be derived from the inner cell mass of a blastocyst during gastrulation. They are totipotent cells giving rise to the germ line during development and virtually to all tissues of the organism [78, 79]. Adult stem cells (ASC) are tissue-resident stem cells that, based on their differentiation potency, can be classified as multipotent, oligopotent, or even unipotent [80]. In their tissue of residency, ASC function as lineage-committed progenitors to cells capable of more highly specialized tasks [78]. They are involved in tissue homeostasis and repair after wounding over the lifetime [79].

Among the tissues and organs harboring ASC, there are bone marrow, vascular walls, adipose tissues, skeletal muscles, heart, and brain, as well as epithelium of lung, liver, pancreas, digestive tract, skin, retina, breast, ovaries, prostate, and testis [81]. The bone marrow stem cell niche includes the hematopoietic stem cell population, which provides continuous renewal of blood cell lineages and the foundation of the immune system, and the mesenchymal stem cell population, responsible for osteogenic, adipogenic, and chondrogenic differentiation [82].

## **4.2. Mesenchymal stem cells (MSCs)**

*3.2.2. Identification and measurement of cell proliferation*

calculation of the number of cells in S or M phase [65].

, S, G<sup>2</sup>

Furthermore, some healthy tissues can express low levels of Ki-67 [68].

are therefore highest during S phase, with little to no expression during G<sup>1</sup>

immunohistochemical detection of PCNA in studies of cell turnover in lung [76].

Cells express Ki-67 during G<sup>1</sup>

through a surfactant mechanism [69].

and M phases [62, 74].

levels are low in the G<sup>1</sup>

32 Cartilage Repair and Regeneration

levels in G<sup>2</sup>

**4. Stem cells**

**4.1. Definition and classification**

Assessment of cell proliferation is often of relevance in biomedical science, and a range of techniques have evolved to identify and quantify the process, generally by recognition and

A variety of markers have been used to determine cell cycle status and quantify cell proliferation, including the identification of mitotic figures, tritiated thymidine incorporation, bromodeoxyuridine incorporation, expression of proteins such as the proliferative cell nuclear antigen (PCNA), Ki-67, cyclins and Cdks, and the analysis of Cdks phosphorylation status [62, 66].

Of importance for this chapter are the immunohistochemical methods that detect proliferation-associated antigens. Ideally, such methods should be applicable to routinely processed tissues, they should be relatively inexpensive and the results easily quantified and interpreted [67]. The best known markers employed to recognize proliferating cells are Ki-67 and PCNA.

a sharp decrease in Ki-67 levels occurs [68]. Ki-67 is required to maintain individual mitotic chromosomes dispersed in the cytoplasm after their release from the nuclear envelope,

Ki-67 is widely used as a proliferation marker because it provides a rapid and relatively inexpensive method of measuring dividing cells [65, 70]. However, the short half-life of Ki-67 (1–1.5 h, regardless of the cell position in the cell cycle [71, 72]) makes its detection difficult.

PCNA was first shown to act as a cofactor/auxiliary protein for DNA polymerase δ, which is required for DNA synthesis during replication. However, besides DNA replication, PCNA functions have been associated with other cellular processes such as chromatin remodeling, DNA repair, sister-chromatid cohesion, and cell cycle control [73]. During DNA replication, presence of PCNA is necessary for synthesis of the leading strand. Levels of PCNA expression

PCNA detection has been widely used in immunohistochemical studies of cell proliferation. However, some authors claim that PCNA is not a reliable marker of this process because it is a pleiotropic protein involved in several aspects of cell control and not only in proliferation [66]. On the opposite, other authors affirm that PCNA is the most reliable and versatile of all markers used to analyze cell proliferation [75]. In the past, we have successfully used the

Stem cells are generally defined as clonogenic cells capable of both self-renewal and multilineage differentiation [77]. For a cell to be considered a stem cell, it must be capable of

, and M phases, but not during the resting phase G<sup>0</sup>

and S phases and rise to their peak level in M. Later in the M phase,

. Its

and intermediate

The minimal criteria for defining MSCs include: (a) remain plastic-adherent under standard culture conditions; (b) express CD73, CD90, and CD105, and lack expression of CD34, CD45, CD14 or CD11b, CD79a or CD19, and HLA-DR surface molecules; and (c) differentiate into osteoblasts, adipocytes, and chondrocytes *in vitro* [83, 84].

Originally isolated from bone marrow, MSCs have being isolated from other sites including spleen, thymus, muscle, adipose tissue, endometrium, placenta, umbilical cord, umbilical cord blood, peripheral blood, periosteum, periodontal ligament, dental pulp, synovium, synovial fluid, tendons, and cartilage [84, 85]. A perivascular location for MSCs has been suggested, correlating these cells with pericytes. This would explain why MSCs can be virtually isolated from all tissues [79, 86].

MSCs have demonstrated significant potential for clinical use due to their convenient isolation, their lack of significant immunogenicity permitting allogenic transplantation, their lack of ethical controversy, and their potential to differentiate into tissue-specific cell types [87]. MSCs may have therapeutic applications in several clinical disorders including myocardial infarction, diabetes, sepsis, hepatic failure, acute renal failure, several kinds of lung disease, as well as in spinal cord injuries, and bone and cartilage diseases [88, 89].

## **4.3. Nestin-positive MSCs**

The human nestin protein consists of 1621 amino acids and displays a predicted molecular weight of 177.4 kDa. It is a class VI intermediate filament protein. Intermediate filaments represent, along with microtubules and actin filaments, one of the main components of cytoskeleton in animal cells [90].

Although nestin was first described as a marker of neural stem cells [91], its expression has also been shown in various prenatal and adult cells and tissues. Nestin-expressing cell types in embryonic and fetal tissues includes developing skeletal muscle cells, developing cardiomyocytes, endothelial cells of developing blood vessels, pancreatic epithelial progenitor cells, and hepatic oval cells. In adult, nestin expression has been found in, for example, satellite cells in dorsal root ganglia, retina, pancreatic stellate and endothelial cells, interstitial cells of Cajal, muscularis propria, Sertolli cells, and odontoblasts. Nestin has also been found to be expressed in injured tissues and in cancer cells [92].

For a long time it has been considered that cartilage contains a unique type of cell: the chondrocyte. However, nestin-positive MSCs has been found in cultured human adult lung cells, which underwent chondrogenic differentiation [100], and evidence from our investigations [98, 99] indicates that besides chondrocytes there exist nestin-positive MSCs in the adult lung cartilage.

Chondrocyte Turnover in Lung Cartilage http://dx.doi.org/10.5772/intechopen.70860 35

The nestin-positive MSCs might be circulating in the blood stream or remain located in local blood vessels and be able to populate the cartilage when necessary, and/or might reside inside it. Other authors have shown that murine MSCs embolised within pulmonary blood vessels following systemic injection, and then transmigrated and differentiated into cartilage [101].

Finally, in another work, we found nestin-positive cells in perivascular areas and in connective tissue that were in close proximity of the bronchial airway epithelium. Nestin-positive cells were also found among the cells lining the airway epithelium, perhaps in order to participate in epithelial renewal [102]. Thus, stem cell reported in our works might be a pluripotent cell, which are able to generate several types of lung tissues. Other researchers presented evidence that a pluripotent stem cell exists in the lung that can generate lung-like tissue *in vitro* [103, 104].

Most of cells, tissues, and organs show continuous turnover. A delicate balance between cell proliferation and cell death ensures the maintenance of normal tissue morphology and function. Stem cells play essential roles in the growth, homeostasis and repair of many tissues. MSCs can give rise to multiple lineages including bone, adipose, and cartilage. The intermediate filament protein nestin was initially identified as a marker for neural stem cells, but its

It is generally believed that adult cartilage has no capacity to renewal. Taken together, our findings indicate that there exist nestin-positive MSCs in healthy adulthood that participates in the turnover of lung cartilage and in lung airway epithelium renewal. These findings may improve our knowledge about the biology of the cartilage and of the stem cells, and could provide new cell candidates for cartilage tissue engineering and for therapy for devastating

Ivett C. Miranda-Maldonado, María-de-Lourdes Chávez-Briones, Laura E. Rodríguez-Flores,

Department of Pathology, School of Medicine, Autonomous University of Nuevo Leon,

expression has also been detected in many types of cells, including MSCs.

Yareth Gopar-Cuevas, Alberto Niderhauser-García, Adriana Ancer-Arellano,

Marta Ortega-Martínez and Gilberto Jaramillo-Rangel\*

\*Address all correspondence to: gjaramillorangel@yahoo.com.mx

**6. Conclusion**

pulmonary diseases.

**Author details**

Monterrey, Mexico

In most of the studies, nestin has been detected by immunohistochemistry [92]. The principal advantage of immunohistochemistry over other techniques is that it enables the observation of processes in the context of intact tissue [93].

Normally, nestin becomes up-regulated in tissues during embryogenesis and down-regulated during maturation. During tissue injury in the adult, nestin is expressed in cells with progenitor cell properties. Furthermore, observational and interventional studies in animals and humans have shown that nestin may be an important marker for MSCs. These cells seem to act as a tissue reserve and to participate in tissue repair, regeneration, and growth [94, 95].

## **5. Cell renewal in lung cartilage**

Cartilage grows by two methods: appositional growth and interstitial growth. In the former, chondroblasts in the perichondrium are transformed into chondrocytes. Interstitial growth result from mitotic division of pre-existing chondrocytes within the matrix. These two mechanisms occur early in life [96].

In the past, it has been believed that healthy adult chondrocytes maintain a stable resting phenotype and resist proliferation and differentiation throughout life [5]. Most cell types reach cell cycle arrest after a characteristic number of population doublings. The limit for human chondrocytes has been estimated at ~35 population doublings [4]. Their decreasing proliferative potential has been attributed to replicative senescence associated with erosion of telomere length [97].

We analyzed lung specimens from adult mice embedded in paraffin. Apoptosis was analyzed by TUNEL assay. PCNA and nestin were examined by immunohistochemistry. Apoptosis and PCNA were detected in lung chondrocytes. Serial section analysis demonstrated that cells in apoptosis were different from PCNA-positive cells, indicating that turnover was occurring. Chondrocytes were negative for nestin. However, nestin-positive cells were found in connective tissue associated with cartilage, in some specimens in close proximity of it and in perivascular cells. Thus, the findings of this work indicated that cell turnover in adult lung cartilage is possible, and that it may be mediated by nestin-positive cells [98].

In another related work, we found nestin-positive cells inside of lung cartilage and cells in division very close from them. This finding indicated that there exist nestin-positive MSCs in the adult that are able to differentiate into lung chondrocytes, perhaps to maintain homeostasis and/or repair damaged tissue [99].

For a long time it has been considered that cartilage contains a unique type of cell: the chondrocyte. However, nestin-positive MSCs has been found in cultured human adult lung cells, which underwent chondrogenic differentiation [100], and evidence from our investigations [98, 99] indicates that besides chondrocytes there exist nestin-positive MSCs in the adult lung cartilage.

The nestin-positive MSCs might be circulating in the blood stream or remain located in local blood vessels and be able to populate the cartilage when necessary, and/or might reside inside it. Other authors have shown that murine MSCs embolised within pulmonary blood vessels following systemic injection, and then transmigrated and differentiated into cartilage [101].

Finally, in another work, we found nestin-positive cells in perivascular areas and in connective tissue that were in close proximity of the bronchial airway epithelium. Nestin-positive cells were also found among the cells lining the airway epithelium, perhaps in order to participate in epithelial renewal [102]. Thus, stem cell reported in our works might be a pluripotent cell, which are able to generate several types of lung tissues. Other researchers presented evidence that a pluripotent stem cell exists in the lung that can generate lung-like tissue *in vitro* [103, 104].

## **6. Conclusion**

Although nestin was first described as a marker of neural stem cells [91], its expression has also been shown in various prenatal and adult cells and tissues. Nestin-expressing cell types in embryonic and fetal tissues includes developing skeletal muscle cells, developing cardiomyocytes, endothelial cells of developing blood vessels, pancreatic epithelial progenitor cells, and hepatic oval cells. In adult, nestin expression has been found in, for example, satellite cells in dorsal root ganglia, retina, pancreatic stellate and endothelial cells, interstitial cells of Cajal, muscularis propria, Sertolli cells, and odontoblasts. Nestin has also been found to be

In most of the studies, nestin has been detected by immunohistochemistry [92]. The principal advantage of immunohistochemistry over other techniques is that it enables the observation

Normally, nestin becomes up-regulated in tissues during embryogenesis and down-regulated during maturation. During tissue injury in the adult, nestin is expressed in cells with progenitor cell properties. Furthermore, observational and interventional studies in animals and humans have shown that nestin may be an important marker for MSCs. These cells seem to act as a tissue reserve and to participate in tissue repair, regeneration, and growth [94, 95].

Cartilage grows by two methods: appositional growth and interstitial growth. In the former, chondroblasts in the perichondrium are transformed into chondrocytes. Interstitial growth result from mitotic division of pre-existing chondrocytes within the matrix. These two mecha-

In the past, it has been believed that healthy adult chondrocytes maintain a stable resting phenotype and resist proliferation and differentiation throughout life [5]. Most cell types reach cell cycle arrest after a characteristic number of population doublings. The limit for human chondrocytes has been estimated at ~35 population doublings [4]. Their decreasing proliferative potential has been attributed to replicative senescence associated with erosion of telomere length [97]. We analyzed lung specimens from adult mice embedded in paraffin. Apoptosis was analyzed by TUNEL assay. PCNA and nestin were examined by immunohistochemistry. Apoptosis and PCNA were detected in lung chondrocytes. Serial section analysis demonstrated that cells in apoptosis were different from PCNA-positive cells, indicating that turnover was occurring. Chondrocytes were negative for nestin. However, nestin-positive cells were found in connective tissue associated with cartilage, in some specimens in close proximity of it and in perivascular cells. Thus, the findings of this work indicated that cell turnover in adult lung

In another related work, we found nestin-positive cells inside of lung cartilage and cells in division very close from them. This finding indicated that there exist nestin-positive MSCs in the adult that are able to differentiate into lung chondrocytes, perhaps to maintain homeosta-

cartilage is possible, and that it may be mediated by nestin-positive cells [98].

expressed in injured tissues and in cancer cells [92].

of processes in the context of intact tissue [93].

**5. Cell renewal in lung cartilage**

nisms occur early in life [96].

34 Cartilage Repair and Regeneration

sis and/or repair damaged tissue [99].

Most of cells, tissues, and organs show continuous turnover. A delicate balance between cell proliferation and cell death ensures the maintenance of normal tissue morphology and function. Stem cells play essential roles in the growth, homeostasis and repair of many tissues. MSCs can give rise to multiple lineages including bone, adipose, and cartilage. The intermediate filament protein nestin was initially identified as a marker for neural stem cells, but its expression has also been detected in many types of cells, including MSCs.

It is generally believed that adult cartilage has no capacity to renewal. Taken together, our findings indicate that there exist nestin-positive MSCs in healthy adulthood that participates in the turnover of lung cartilage and in lung airway epithelium renewal. These findings may improve our knowledge about the biology of the cartilage and of the stem cells, and could provide new cell candidates for cartilage tissue engineering and for therapy for devastating pulmonary diseases.

## **Author details**

Yareth Gopar-Cuevas, Alberto Niderhauser-García, Adriana Ancer-Arellano, Ivett C. Miranda-Maldonado, María-de-Lourdes Chávez-Briones, Laura E. Rodríguez-Flores, Marta Ortega-Martínez and Gilberto Jaramillo-Rangel\*

\*Address all correspondence to: gjaramillorangel@yahoo.com.mx

Department of Pathology, School of Medicine, Autonomous University of Nuevo Leon, Monterrey, Mexico

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Practical Approaches to Applied Research and Education. Badajoz: Formatex Research

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**Chapter 3**

Provisional chapter

**Alternative Therapeutic Approach for Cartilage Repair**

DOI: 10.5772/intechopen.72478

Alternative Therapeutic Approach for Cartilage Repair

The cartilage is a flexible tissue, which supports the adjacent soft tissues. The damages that cause degenerative articular diseases are marked by the increase of cytokines such as tumor necrosis factor-α (TNF-α), IL-1β, IL-6, IL-18, and IL-17, which cause intense inflammatory process and release of metalloproteinases and disintegrin enzymes that lead to cartilage degradation. The Curcuma longa possesses bioactive compounds designated as curcuminoids that display therapeutic potential in several pathologies. Curcumin is one of these compounds that may exhibit anti-inflammatory, antioxidant, antiviral, antibacterial, and antitumor effects. It may promote decrease of IL-1β, IL-6, IL-8, TNF-α, COX-2, and reactive oxygen species. Furthermore, curcumin inhibits the activity of several kinases related to the degradation of the cartilage, including tyrosine kinase, p21-activated kinase, mitogenactivated protein kinase, protein kinase C, the activator protein 1 pathway, and NF-κB leading to the suppression of the production of metalloproteinases and inflammatory cytokines. Curcumin has also been related to the stimulation of the production of type II collagen and glycosaminoglycan by chondrocytes. Studies have shown that this compound may alleviate joint pain and crepitation, reduce the use of other drugs for pain relief, stimulate the production of type II collagen and glycosaminoglycan resulting in a protective and antiinflammatory action of cartilage and bones, and improve the quality of life of the patients.

The articular cartilage is a flexible tissue, which supports the adjacent soft tissues and possesses the extracellular matrix (ECM), collagen, chondrocyte, proteoglycans, and water [1]. This tissue is alymphatic, avascular, and aneural, and for these reasons, when a severe damage

> © 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.

© 2018 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.

Marina Cristina Akuri, Mariana Ricci Barion,

Marina Cristina Akuri, Mariana Ricci Barion,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72478

Sandra Maria Barbalho and

Élen Landgraf Guiguer

Abstract

1. Introduction

Sandra Maria Barbalho and Élen Landgraf Guiguer

Keywords: cartilage, inflammation, Curcuma longa, curcumin

occurs, the self-repair is a highly difficult process [2–4].


## **Alternative Therapeutic Approach for Cartilage Repair** Alternative Therapeutic Approach for Cartilage Repair

DOI: 10.5772/intechopen.72478

Marina Cristina Akuri, Mariana Ricci Barion, Sandra Maria Barbalho and Élen Landgraf Guiguer Marina Cristina Akuri, Mariana Ricci Barion, Sandra Maria Barbalho and

Additional information is available at the end of the chapter Élen Landgraf Guiguer

http://dx.doi.org/10.5772/intechopen.72478 Additional information is available at the end of the chapter

#### Abstract

[96] Martini FH. Anatomy & Physiology. 1st ed. Jurong: Pearson Education, Inc.; 2005. 845 p [97] Martin JA, Brown TD, Heiner AD, Buckwalter JA. Chondrocyte senescence, joint loading and osteoarthritis. Clinical Orthopaedics and Related Research. 2004;**427**(Suppl):S96-103

[98] Ortega-Martínez M, Romero-Núñez E, Niderhauser-García A, De-la-Garza-González C, Ancer-Rodríguez J, Jaramillo-Rangel G. Evidence of chondrocyte turnover in lung cartilage, with the probable participation of nestin-positive cells. Cell Biology

[99] Ortega-Martínez M, De-la-Garza-González C, Ancer-Rodríguez J, Jaramillo-Rangel G. Nestin-positive stem cells participate in chondrocyte renewal in healthy adult lung car-

[100] Sabatini F, Petecchia L, Tavian M, Jodon de Villeroché V, Rossi GA, Brouty-Boyé D. Human bronchial fibroblasts exhibit a mesenchymal stem cell phenotype and multilin-

[101] Aguilar S, Nye E, Chan J, Loebinger M, Spencer-Dene B, Fisk N, Stamp G, Bonnet D, Janes SM. Murine but not human mesenchymal stem cells generate osteosarcoma-like

[102] Ortega-Martínez M, Rodríguez-Flores LE, De-la-Garza-González C, Ancer-Rodríguez J, Jaramillo-Rangel G. Detection of a novel stem cell probably involved in normal turnover of the lung airway epithelium. Journal of Cellular and Molecular Medicine.

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42 Cartilage Repair and Regeneration

2015;**19**:2679-2681

2001;**80**:455-460

The cartilage is a flexible tissue, which supports the adjacent soft tissues. The damages that cause degenerative articular diseases are marked by the increase of cytokines such as tumor necrosis factor-α (TNF-α), IL-1β, IL-6, IL-18, and IL-17, which cause intense inflammatory process and release of metalloproteinases and disintegrin enzymes that lead to cartilage degradation. The Curcuma longa possesses bioactive compounds designated as curcuminoids that display therapeutic potential in several pathologies. Curcumin is one of these compounds that may exhibit anti-inflammatory, antioxidant, antiviral, antibacterial, and antitumor effects. It may promote decrease of IL-1β, IL-6, IL-8, TNF-α, COX-2, and reactive oxygen species. Furthermore, curcumin inhibits the activity of several kinases related to the degradation of the cartilage, including tyrosine kinase, p21-activated kinase, mitogenactivated protein kinase, protein kinase C, the activator protein 1 pathway, and NF-κB leading to the suppression of the production of metalloproteinases and inflammatory cytokines. Curcumin has also been related to the stimulation of the production of type II collagen and glycosaminoglycan by chondrocytes. Studies have shown that this compound may alleviate joint pain and crepitation, reduce the use of other drugs for pain relief, stimulate the production of type II collagen and glycosaminoglycan resulting in a protective and antiinflammatory action of cartilage and bones, and improve the quality of life of the patients.

Keywords: cartilage, inflammation, Curcuma longa, curcumin

## 1. Introduction

The articular cartilage is a flexible tissue, which supports the adjacent soft tissues and possesses the extracellular matrix (ECM), collagen, chondrocyte, proteoglycans, and water [1]. This tissue is alymphatic, avascular, and aneural, and for these reasons, when a severe damage occurs, the self-repair is a highly difficult process [2–4].

© 2018 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.

© 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 damages that cause degenerative articular diseases are marked by the increase of cytokines that cause intense inflammatory process and enzymes that cause cartilage degradation [5, 6].

Activated synoviocytes also produce inflammatory cytokines such as IL-1, IL-6, and TNF-α, which act by amplifying the inflammatory process and cartilaginous destruction. There is increased release of reactive oxygen species (ROS), mainly nitric oxide (NO), peroxynitrite (ONOO), and superoxide anion radicals (O2). Other inflammatory mediators such as cyclooxygenase-2 (COX-2), produced by synovial monocytes, and prostaglandins 2 (PGE2) are also

Alternative Therapeutic Approach for Cartilage Repair http://dx.doi.org/10.5772/intechopen.72478 45

The nuclear factor-kappa B (NF-κβ) pathway is responsible for the production of various cytokines and induction of inflammation. When stimulated by interleukins IL-1β and TNF-α, there is activation of I kappa beta kinase (IKK), which promotes the phosphorylation of IKB-α. Thus, IKB-α is degraded by ubiquitination, and the dimers compounded by p50 and p65 reach the nucleus and can stimulate the expression of more than 400 genes, of which some are proinflammatory and pro-apoptotic genes [8, 18]. Therefore, there is production of various inter-

Besides the destruction of cartilage in OA, an intense process of bone resorption occurs. This process is a result of osteoclast activation known as osteoclastogenesis [19]. The receptor activator NF-kappa ligand (RANKL) is produced by some cells as the osteoblast and has an affinity for RANK, which is present in the membrane of osteoclast precursor cells [20]. When RANKL binds to RANK, a phosphorylation process occurs, culminating in the activation of NF-kB [5, 21, 22]. The osteoprotegerin also has an affinity for RANK, thus competing with RANKL, inducing apoptosis of mature osteoclasts [5]. In the OA, the increase of RANKL and the decrease of OPG

Figure 1. The activation of NF-kβ is related to the release of TGFβ, IL-1, IL-6, and IL-8 and further activation of TH17 that leads to the stimulation of several cells and expression of other inflammatory cytokines and metalloproteinases (MMP), and further development of features characteristic of inflammation and degradation of cartilage and bone. NF-kβ: nuclear factor kβ; IL: interleukin; TH17: T-helper 17; MMP: matrix metalloproteinase; RANKL: receptor activator of nuclear factor κB ligand; TH17: type 17 T-helper; TNF-α: tumor necrosis factor-α; TGF-β, transforming growth factor-β; ROS: reactive

are observed [23, 24]. Figure 1 summarizes the inflammatory process in the cartilage.

involved in the pathophysiology of the disease [6, 15].

leukins, including IL-1, IL-6, IL-8, and IL-10 [8, 15, 16].

oxygen species; and NO: nitric oxide.

The osteoarthritis (OA) is an example of a progressive degenerative disease characterized by a chronic inflammatory process, joint pain, and loss of function and injury of adjacent tissue. The great destruction of the articular cartilage is the main characteristic of this disease [7–9], and therefore, it is used, in this chapter, as a prototype of cartilage destruction and regeneration.

Drugs such as nonsteroidal anti-inflammatory drugs (NSAIDS) and acetaminophen are the therapeutic approaches for the pharmacological treatment of degenerative diseases. However, this kind of medications is associated with gastrointestinal, cardiovascular, and renal adverse effects and do not effectively inhibit the disease progression and destruction of cartilage [7, 8, 10, 11]. Furthermore, corticosteroids, another therapeutic option due to their potent antiinflammatory action and ability to reduce symptoms, should also not be used for an extended period because they can lead to a more rapid progression of OA [12].

The development of therapeutic alternatives that do not cause adverse effects and inhibit the progression of the disease is urgent and, therefore, has been widely studied. Curcuma longa, herbal medicine, has been shown to be one of these possible alternatives because it presents significant benefits in degenerative diseases such as OA, and this plant may play a crucial role in the reduction of the inflammatory pathways [13].

## 2. Physiopathology and cartilage destruction

In healthy cartilage, chondrocytes can form ECM components and enzymes that degrade cartilage in equilibrium. Although the pathophysiology of OA and its triggers have not yet been fully elucidated, it is known that inflammation, joint destruction, synovitis, and osteoclastogenesis are involved [9].

In OA, there is an increase in the enzymes involved in the cartilage degradation such as disintegrin and metalloproteinase (MMP) with a thrombospondin motif (ADAMTS). This enzymatic increase occurs due to stimulation by interleukins (IL) and inflammatory mediators such as tumor necrosis factor-α (TNF-α), IL-1β, IL-6, IL-18, and IL-17 [6, 14].

The most widely studied and related to the destruction of cartilage are ADAMTS4 and ADAMTS5 that are released after stimulation of inflammatory cytokines such as IL-1β. They are aggrecanases and aggrecan aggregation of proteoglycans and one of the components of ECM. After degradation of aggrecans by these enzymes, MMP-3 acts in synergism in the degradation of proteoglycans [8, 15–17].

MMPs are enzymes implicated primarily in the destruction of type II collagen and therefore play a fundamental role in the destruction of cartilage. MMP-1, MMP-3, MMP-9, and MMP-13 are the most involved enzymes in this process, and the last one is not found in adult cartilage without OA. Fragments from cleavage of collagen type 2 by MMPs amplify the destruction of ECM and amplify the release of more MMPs [5–9].

Activated synoviocytes also produce inflammatory cytokines such as IL-1, IL-6, and TNF-α, which act by amplifying the inflammatory process and cartilaginous destruction. There is increased release of reactive oxygen species (ROS), mainly nitric oxide (NO), peroxynitrite (ONOO), and superoxide anion radicals (O2). Other inflammatory mediators such as cyclooxygenase-2 (COX-2), produced by synovial monocytes, and prostaglandins 2 (PGE2) are also involved in the pathophysiology of the disease [6, 15].

The damages that cause degenerative articular diseases are marked by the increase of cytokines that cause intense inflammatory process and enzymes that cause cartilage degradation [5, 6].

The osteoarthritis (OA) is an example of a progressive degenerative disease characterized by a chronic inflammatory process, joint pain, and loss of function and injury of adjacent tissue. The great destruction of the articular cartilage is the main characteristic of this disease [7–9], and therefore, it is used, in this chapter, as a prototype of cartilage destruc-

Drugs such as nonsteroidal anti-inflammatory drugs (NSAIDS) and acetaminophen are the therapeutic approaches for the pharmacological treatment of degenerative diseases. However, this kind of medications is associated with gastrointestinal, cardiovascular, and renal adverse effects and do not effectively inhibit the disease progression and destruction of cartilage [7, 8, 10, 11]. Furthermore, corticosteroids, another therapeutic option due to their potent antiinflammatory action and ability to reduce symptoms, should also not be used for an extended

The development of therapeutic alternatives that do not cause adverse effects and inhibit the progression of the disease is urgent and, therefore, has been widely studied. Curcuma longa, herbal medicine, has been shown to be one of these possible alternatives because it presents significant benefits in degenerative diseases such as OA, and this plant may play a crucial role

In healthy cartilage, chondrocytes can form ECM components and enzymes that degrade cartilage in equilibrium. Although the pathophysiology of OA and its triggers have not yet been fully elucidated, it is known that inflammation, joint destruction, synovitis, and osteoclas-

In OA, there is an increase in the enzymes involved in the cartilage degradation such as disintegrin and metalloproteinase (MMP) with a thrombospondin motif (ADAMTS). This enzymatic increase occurs due to stimulation by interleukins (IL) and inflammatory mediators

The most widely studied and related to the destruction of cartilage are ADAMTS4 and ADAMTS5 that are released after stimulation of inflammatory cytokines such as IL-1β. They are aggrecanases and aggrecan aggregation of proteoglycans and one of the components of ECM. After degradation of aggrecans by these enzymes, MMP-3 acts in synergism in the

MMPs are enzymes implicated primarily in the destruction of type II collagen and therefore play a fundamental role in the destruction of cartilage. MMP-1, MMP-3, MMP-9, and MMP-13 are the most involved enzymes in this process, and the last one is not found in adult cartilage without OA. Fragments from cleavage of collagen type 2 by MMPs amplify the destruction of

such as tumor necrosis factor-α (TNF-α), IL-1β, IL-6, IL-18, and IL-17 [6, 14].

period because they can lead to a more rapid progression of OA [12].

in the reduction of the inflammatory pathways [13].

2. Physiopathology and cartilage destruction

tion and regeneration.

44 Cartilage Repair and Regeneration

togenesis are involved [9].

degradation of proteoglycans [8, 15–17].

ECM and amplify the release of more MMPs [5–9].

The nuclear factor-kappa B (NF-κβ) pathway is responsible for the production of various cytokines and induction of inflammation. When stimulated by interleukins IL-1β and TNF-α, there is activation of I kappa beta kinase (IKK), which promotes the phosphorylation of IKB-α. Thus, IKB-α is degraded by ubiquitination, and the dimers compounded by p50 and p65 reach the nucleus and can stimulate the expression of more than 400 genes, of which some are proinflammatory and pro-apoptotic genes [8, 18]. Therefore, there is production of various interleukins, including IL-1, IL-6, IL-8, and IL-10 [8, 15, 16].

Besides the destruction of cartilage in OA, an intense process of bone resorption occurs. This process is a result of osteoclast activation known as osteoclastogenesis [19]. The receptor activator NF-kappa ligand (RANKL) is produced by some cells as the osteoblast and has an affinity for RANK, which is present in the membrane of osteoclast precursor cells [20]. When RANKL binds to RANK, a phosphorylation process occurs, culminating in the activation of NF-kB [5, 21, 22]. The osteoprotegerin also has an affinity for RANK, thus competing with RANKL, inducing apoptosis of mature osteoclasts [5]. In the OA, the increase of RANKL and the decrease of OPG are observed [23, 24]. Figure 1 summarizes the inflammatory process in the cartilage.

Figure 1. The activation of NF-kβ is related to the release of TGFβ, IL-1, IL-6, and IL-8 and further activation of TH17 that leads to the stimulation of several cells and expression of other inflammatory cytokines and metalloproteinases (MMP), and further development of features characteristic of inflammation and degradation of cartilage and bone. NF-kβ: nuclear factor kβ; IL: interleukin; TH17: T-helper 17; MMP: matrix metalloproteinase; RANKL: receptor activator of nuclear factor κB ligand; TH17: type 17 T-helper; TNF-α: tumor necrosis factor-α; TGF-β, transforming growth factor-β; ROS: reactive oxygen species; and NO: nitric oxide.

## 3. Curcuma longa

C. longa, or turmeric or saffron, is native to Asia and India and belongs to the Zingiberaceae family, and its rhizome has been used as a seasoning and in the traditional medicine since ancient times [18, 25].

kinase, p21-activated kinase 1 (PAK1), mitogen-activated protein kinase (MAPK), and protein kinase C (PKC). Figure 3 shows the process of cartilage inflammation and the effects of

Alternative Therapeutic Approach for Cartilage Repair http://dx.doi.org/10.5772/intechopen.72478 47

Many studies have shown that curcumin has potent effects on the induction of apoptosis and decreased tumor cell proliferation and may promote the inhibition of important angiogenesis regulators, signal transducers and activators of transcription 3 (STAT3), and vascular endothelial growth factor (VEGF). Besides, it downregulates the expression of differentiated embryochondrocyte expressed gene 1 (DEC1) and hypoxia-inducible factor-1-α (HIF-1α) [33–36].

Furthermore, several authors have shown that the supplementation with curcumin may bring a plethora of benefits in the treatment and prevention of the osteopenia [37]. This compound has been demonstrated to be able to avert the suppression of osteoblasts proliferation and to enhance the index of osteoprotegerin and RANKL, which indicates osteoblastogenesis [38].

As mentioned earlier, the actions of curcumin vary from potent anti-inflammatory and antiapoptotic to antioxidant [39]. The wide variety of sites of actions and consequently decrease in the inflammation markers make this compound and its analogs extremely promising in chronic inflammatory diseases such as OA [5, 28]. Also, this herbal medicine inhibits the phosphoryla-

Conventional OA therapies are restricted to the reduction of symptoms in patients, but they do not decrease the degradation of cartilage and, consequently, do not alter the progression of the disease. For these reasons, the need for new therapies is striking, and curcumin and its analogs

Figure 3. The inflammation of the cartilage may occur due to several processes such as an increase in the expression of enzymes, increase in the formation of ROS, and release of cytokines. The consequence is the loss of type II collagen and glycosaminoglycan resulting in the degradation of the cartilage. Curcumin interferes in this scenario and may help in the healing process. ROS: reactive oxygen species; IL: interleukin; TNF-α: tumor necrosis factor-α; PGE2: prostaglandin E2.

tion of IKB-α and thereby reduces cartilage degradation, as shown in Figure 4.

have become extremely promising in this context [13, 28].

curcumin in the healing process [29, 31, 32].

The bioactive compounds derived from turmeric are called curcuminoids and have shown therapeutic potential in various pathologies. The three most important compounds originated from this rhizome are curcumin (diferuloylmethane), bisdemethoxycurcumin, and demethoxycurcumin, which are present, respectively, in concentrations of 77, 17, and 3%. Curcumin gives the typical yellowish coloration of the rhizome, and this part of the plant is the most widely studied [18, 26, 27].

Several studies have been conducted in order to show their actions in vitro and in vivo. Curcumin acts with different mechanisms and in different cell types and pathways. It shows antiinflammatory, antioxidant, antiviral, antibacterial, and antitumor effects. Its therapeutic potential covers diseases such as cancer, Alzheimer's disease, osteoporosis, inflammatory bowel disease, depression, arthritis, diabetes, vitiligo, endometriosis, and several others. Figure 2 shows some effects of curcumin [5, 28–30].

Studies on the action of curcumin and its analogs show that it can act directly or indirectly in the decrease of the formation of inflammatory molecules and pro-inflammatory transcription factors. Under its action, there is a reduction of IL-1β, IL-6, IL-8, TNF-α, NF-kB, COX-2, and reactive oxygen species (ROS). Apart from that, curcumin has been shown to inhibit the activity of several kinases related to the degradation of the cartilage, including a tyrosine

Figure 2. Some benefits of curcumin on human health.

kinase, p21-activated kinase 1 (PAK1), mitogen-activated protein kinase (MAPK), and protein kinase C (PKC). Figure 3 shows the process of cartilage inflammation and the effects of curcumin in the healing process [29, 31, 32].

3. Curcuma longa

46 Cartilage Repair and Regeneration

ancient times [18, 25].

widely studied [18, 26, 27].

effects of curcumin [5, 28–30].

Figure 2. Some benefits of curcumin on human health.

C. longa, or turmeric or saffron, is native to Asia and India and belongs to the Zingiberaceae family, and its rhizome has been used as a seasoning and in the traditional medicine since

The bioactive compounds derived from turmeric are called curcuminoids and have shown therapeutic potential in various pathologies. The three most important compounds originated from this rhizome are curcumin (diferuloylmethane), bisdemethoxycurcumin, and demethoxycurcumin, which are present, respectively, in concentrations of 77, 17, and 3%. Curcumin gives the typical yellowish coloration of the rhizome, and this part of the plant is the most

Several studies have been conducted in order to show their actions in vitro and in vivo. Curcumin acts with different mechanisms and in different cell types and pathways. It shows antiinflammatory, antioxidant, antiviral, antibacterial, and antitumor effects. Its therapeutic potential covers diseases such as cancer, Alzheimer's disease, osteoporosis, inflammatory bowel disease, depression, arthritis, diabetes, vitiligo, endometriosis, and several others. Figure 2 shows some

Studies on the action of curcumin and its analogs show that it can act directly or indirectly in the decrease of the formation of inflammatory molecules and pro-inflammatory transcription factors. Under its action, there is a reduction of IL-1β, IL-6, IL-8, TNF-α, NF-kB, COX-2, and reactive oxygen species (ROS). Apart from that, curcumin has been shown to inhibit the activity of several kinases related to the degradation of the cartilage, including a tyrosine Many studies have shown that curcumin has potent effects on the induction of apoptosis and decreased tumor cell proliferation and may promote the inhibition of important angiogenesis regulators, signal transducers and activators of transcription 3 (STAT3), and vascular endothelial growth factor (VEGF). Besides, it downregulates the expression of differentiated embryochondrocyte expressed gene 1 (DEC1) and hypoxia-inducible factor-1-α (HIF-1α) [33–36].

Furthermore, several authors have shown that the supplementation with curcumin may bring a plethora of benefits in the treatment and prevention of the osteopenia [37]. This compound has been demonstrated to be able to avert the suppression of osteoblasts proliferation and to enhance the index of osteoprotegerin and RANKL, which indicates osteoblastogenesis [38].

As mentioned earlier, the actions of curcumin vary from potent anti-inflammatory and antiapoptotic to antioxidant [39]. The wide variety of sites of actions and consequently decrease in the inflammation markers make this compound and its analogs extremely promising in chronic inflammatory diseases such as OA [5, 28]. Also, this herbal medicine inhibits the phosphorylation of IKB-α and thereby reduces cartilage degradation, as shown in Figure 4.

Conventional OA therapies are restricted to the reduction of symptoms in patients, but they do not decrease the degradation of cartilage and, consequently, do not alter the progression of the disease. For these reasons, the need for new therapies is striking, and curcumin and its analogs have become extremely promising in this context [13, 28].

Figure 3. The inflammation of the cartilage may occur due to several processes such as an increase in the expression of enzymes, increase in the formation of ROS, and release of cytokines. The consequence is the loss of type II collagen and glycosaminoglycan resulting in the degradation of the cartilage. Curcumin interferes in this scenario and may help in the healing process. ROS: reactive oxygen species; IL: interleukin; TNF-α: tumor necrosis factor-α; PGE2: prostaglandin E2.

many medications that, in most cases, do not show effective actions resulting in the damage of the synovial tissue. For these reasons, new pharmacotherapies and therapies for this illness are

Alternative Therapeutic Approach for Cartilage Repair http://dx.doi.org/10.5772/intechopen.72478 49

As pointed earlier, curcumin may act in many different locals of inflammation resulting, directly or indirectly, in the reduction of the production of inflammatory mediators and interleukins, resulting in less destruction of cartilage. Besides that, patients treated with

Moreover, curcumin has been shown to inhibit the activator protein 1 (AP-1) pathway and NFκB leading to the suppression of the production of MMP-3, MMP-9, and MMP-13 [15, 19]. Zhang et al. [9] demonstrated in a mouse model that the production of MMP-1, MMP-3, MMP-13, IL-1β, TNF-α, and ADAMTS5 was decreased when the animals were treated with curcumin. They also showed an increase in the expression of the chondroprotective gene CITED 2 (Cbp/P300 interacting transactivator with Glu/Asp rich carboxy terminal domain 2), which seems to be involved in the suppression of NF-κB activity [9, 19]. Curcumin has also been related to the stimulation of the production of type II collagen and glycosaminoglycan by chondrocytes [5].

Curcumin inhibits the activation of I kappa B kinase (IKK) in chondrocytes, osteoblasts, and synovial cells [15, 48]. By inhibiting the phosphorylation of this kinase, curcumin prevents the activation of NF-kB. Consequently, it inhibits the expression of pro-apoptotic genes in chondrocytes (caspase-3) and the formation of inflammatory mediators [18]. Thus, it is responsible for the downregulation of lipoxygenases, COX-2, phospholipase A2, prostaglandin E2 (PGE2), IL-1β, IL-6, and IL-8 [19, 30]. Wherefore, curcumin blocks the signaling by NF-kB, leading to the inhibition of this factor resulting in the decrease of the degradation of collagen. This pathway is induced by the activation of the chondrocytes stimulated by IL-1 [15, 16].

Curcumin inhibits TNF-α, which is associated with increased cartilage reabsorption. This

Studies have shown that compounds from Curcuma sp. can alleviate joint pain and crepitation, which lead to improved scores on WOMAC (Western Ontario and McMaster Universities Osteoarthritis Index), improve function, reduce the use of other drugs for pain relief, and is as

Therefore, curcumin acts on the NF-kB system, in addition to the stimulation of the production of type II collagen and glycosaminoglycan resulting in a protective and anti-inflammatory action of cartilage and bones, reducing pain and improving the quality of life of patients with

The major problem of curcumin is that it is extremely hydrophobic and thus has low oral bioavailability, thus decreasing their beneficial effects. Another problem is the rapid metabolism of curcuminoids considering the extensive biotransformation and consequent reduction in

cytokine associated with IL-6 and IL-1 inhibits the proteoglycan synthesis [5, 49, 50].

effective as the use of ibuprofen [51–59].

5. Disadvantages of Curcuma longa

degenerative diseases [18].

the plasmatic levels [25, 60].

curcumin have decreased C-reactive protein, a marker of inflammation [46, 47].

essential [28].

Figure 4. Effects of curcumin on the inhibition of the process involving the degradation of cartilage. IKK: I kappa B kinase; IKBα: inhibitor of kappa B; NF-κβ: nuclear factor κβ.

## 4. The potential effects of Curcuma longa

In articular cartilage, the ECM is composed of different compounds such as collagen, proteoglycans (glycosaminoglycan and proteins) mainly aggrecan and non-collagenous proteins [28, 40].

In the intra-articular space, there is the synovial fluid, which is enveloped by the synovial membrane and is also responsible for the nutrition of articular cartilage cells. The main cells in the synovial membrane are synoviocytes that have phagocytic functions and are responsible for the production of synovial fluid [41] and contribute to the inflammatory process when it releases several cytokines and proteases which contribute to joint destruction [42, 43].

Some degenerative diseases are involved with synovial inflammation and destruction of ECM of articular cartilage [44, 45]. According to the World Health Organization (WHO), musculoskeletal or rheumatic conditions consist over 150 syndromes and diseases. These ailments are liable for chronic pain, disability, and dysfunction. Among these diseases, rheumatoid arthritis, osteoarthritis, spinal disorders, and severe limb trauma deserve special mention because of the greatest impact on society such as healthcare expenditures. In developed countries, OA is one of the most disabling diseases [45].

OA is a degenerative disorder involving synovial inflammation and destruction of ECM leading to several symptoms such as pain, disability, and significant morbidity, requiring many medications that, in most cases, do not show effective actions resulting in the damage of the synovial tissue. For these reasons, new pharmacotherapies and therapies for this illness are essential [28].

As pointed earlier, curcumin may act in many different locals of inflammation resulting, directly or indirectly, in the reduction of the production of inflammatory mediators and interleukins, resulting in less destruction of cartilage. Besides that, patients treated with curcumin have decreased C-reactive protein, a marker of inflammation [46, 47].

Moreover, curcumin has been shown to inhibit the activator protein 1 (AP-1) pathway and NFκB leading to the suppression of the production of MMP-3, MMP-9, and MMP-13 [15, 19]. Zhang et al. [9] demonstrated in a mouse model that the production of MMP-1, MMP-3, MMP-13, IL-1β, TNF-α, and ADAMTS5 was decreased when the animals were treated with curcumin. They also showed an increase in the expression of the chondroprotective gene CITED 2 (Cbp/P300 interacting transactivator with Glu/Asp rich carboxy terminal domain 2), which seems to be involved in the suppression of NF-κB activity [9, 19]. Curcumin has also been related to the stimulation of the production of type II collagen and glycosaminoglycan by chondrocytes [5].

Curcumin inhibits the activation of I kappa B kinase (IKK) in chondrocytes, osteoblasts, and synovial cells [15, 48]. By inhibiting the phosphorylation of this kinase, curcumin prevents the activation of NF-kB. Consequently, it inhibits the expression of pro-apoptotic genes in chondrocytes (caspase-3) and the formation of inflammatory mediators [18]. Thus, it is responsible for the downregulation of lipoxygenases, COX-2, phospholipase A2, prostaglandin E2 (PGE2), IL-1β, IL-6, and IL-8 [19, 30]. Wherefore, curcumin blocks the signaling by NF-kB, leading to the inhibition of this factor resulting in the decrease of the degradation of collagen. This pathway is induced by the activation of the chondrocytes stimulated by IL-1 [15, 16].

Curcumin inhibits TNF-α, which is associated with increased cartilage reabsorption. This cytokine associated with IL-6 and IL-1 inhibits the proteoglycan synthesis [5, 49, 50].

Studies have shown that compounds from Curcuma sp. can alleviate joint pain and crepitation, which lead to improved scores on WOMAC (Western Ontario and McMaster Universities Osteoarthritis Index), improve function, reduce the use of other drugs for pain relief, and is as effective as the use of ibuprofen [51–59].

Therefore, curcumin acts on the NF-kB system, in addition to the stimulation of the production of type II collagen and glycosaminoglycan resulting in a protective and anti-inflammatory action of cartilage and bones, reducing pain and improving the quality of life of patients with degenerative diseases [18].

## 5. Disadvantages of Curcuma longa

4. The potential effects of Curcuma longa

kinase; IKBα: inhibitor of kappa B; NF-κβ: nuclear factor κβ.

48 Cartilage Repair and Regeneration

of the most disabling diseases [45].

In articular cartilage, the ECM is composed of different compounds such as collagen, proteoglycans (glycosaminoglycan and proteins) mainly aggrecan and non-collagenous proteins [28, 40]. In the intra-articular space, there is the synovial fluid, which is enveloped by the synovial membrane and is also responsible for the nutrition of articular cartilage cells. The main cells in the synovial membrane are synoviocytes that have phagocytic functions and are responsible for the production of synovial fluid [41] and contribute to the inflammatory process when it

Figure 4. Effects of curcumin on the inhibition of the process involving the degradation of cartilage. IKK: I kappa B

Some degenerative diseases are involved with synovial inflammation and destruction of ECM of articular cartilage [44, 45]. According to the World Health Organization (WHO), musculoskeletal or rheumatic conditions consist over 150 syndromes and diseases. These ailments are liable for chronic pain, disability, and dysfunction. Among these diseases, rheumatoid arthritis, osteoarthritis, spinal disorders, and severe limb trauma deserve special mention because of the greatest impact on society such as healthcare expenditures. In developed countries, OA is one

OA is a degenerative disorder involving synovial inflammation and destruction of ECM leading to several symptoms such as pain, disability, and significant morbidity, requiring

releases several cytokines and proteases which contribute to joint destruction [42, 43].

The major problem of curcumin is that it is extremely hydrophobic and thus has low oral bioavailability, thus decreasing their beneficial effects. Another problem is the rapid metabolism of curcuminoids considering the extensive biotransformation and consequent reduction in the plasmatic levels [25, 60].

Some techniques, such as nanoparticles, phospholipid complexes, and liposomes, have been used as drug delivery systems to improve the bioavailability of these substances [61, 62]. Some compounds, such as folic acid, piperine, phosphatidylcholine, galactose, and the complex arginine-glycine-aspartic acid, are also used to improve this bioavailability and effects. Green tea and collagen associated with curcumin extracts may also enhance its effects [8, 30, 63, 64].

[5] Akuri MC, Barbalho SM, Val RM, Guiguer EL. Reflections about osteoarthritis and Curcuma longa. Pharmacognosy Reviews. 2017;11:8-12. DOI: 10.4103/phrev.phrev\_54\_16

Alternative Therapeutic Approach for Cartilage Repair http://dx.doi.org/10.5772/intechopen.72478 51

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[7] Xie XW, Wan RZ, Liu ZP. Recent research advances in selective matrix metalloproteinase-13 inhibitors as anti-osteoarthritis agents. ChemMedChem. 2017;12:1157-1168. DOI:

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[9] Zhang Z, Leong DJ, Xu L, He Z, Wang A, Navati M, Kim SJ, Hirsh DM, Hardin JA, Cobelli NJ, Friedman JM, Sun HB. Curcumin slows osteoarthritis progression and relieves osteoarthritis-associated pain symptoms in a post-traumatic osteoarthritis mouse model. Arthritis Research & Therapy. 2016;18:128. DOI: 10.1186/s13075-016-1025-y [10] Rannou F, Pelletier JP, Martel-Pelletier J. Efficacy and safety of topical NSAIDs in the management of osteoarthritis: Evidence from real-life setting trials and surveys. Seminars

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## 6. Conclusions

The curcumin has been used as an alternative therapy in the control of cartilage healing once it may interfere with the inflammatory pathways reducing the release of pro-inflammatory cytokines. Nevertheless, the use of curcumin and its analogs need to be more extensively studied and tested to determine the bioavailability, the therapeutic properties, adequate delivery formulations, doses, and possible risks of use.

## Author details

Marina Cristina Akuri<sup>1</sup> , Mariana Ricci Barion<sup>1</sup> , Sandra Maria Barbalho1,2\* and Élen Landgraf Guiguer1,2

\*Address all correspondence to: smbarbalho@gmail.com


## References


[5] Akuri MC, Barbalho SM, Val RM, Guiguer EL. Reflections about osteoarthritis and Curcuma longa. Pharmacognosy Reviews. 2017;11:8-12. DOI: 10.4103/phrev.phrev\_54\_16

Some techniques, such as nanoparticles, phospholipid complexes, and liposomes, have been used as drug delivery systems to improve the bioavailability of these substances [61, 62]. Some compounds, such as folic acid, piperine, phosphatidylcholine, galactose, and the complex arginine-glycine-aspartic acid, are also used to improve this bioavailability and effects. Green tea and collagen associated with curcumin extracts may also enhance its

The curcumin has been used as an alternative therapy in the control of cartilage healing once it may interfere with the inflammatory pathways reducing the release of pro-inflammatory cytokines. Nevertheless, the use of curcumin and its analogs need to be more extensively studied and tested to determine the bioavailability, the therapeutic properties, adequate delivery formula-

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50 Cartilage Repair and Regeneration

6. Conclusions

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[42] Hao L, Wan Y, Xiao J, Tang Q, Deng H, Chen L. A study of Sirt1 regulation and the effect of resveratrol on synoviocyte invasion and associated joint destruction in rheumatoid arthritis. Molecular Medicine Reports. 2017;16:5099-5106. DOI: 10.3892/mmr.2017.7299

[43] Benedetti G, Bonaventura P, Lavocat F, Miossec P. IL-17A and TNF-α increase the expression of the antiapoptotic adhesion molecule amigo-2 in arthritis synoviocytes.

[44] Buckwalter JA, Mankin HJ. Articular cartilage: Degeneration and osteoarthritis, repair, regeneration, and transplantation. Instructional Course Lectures. 1998;47:487-504

[45] Mobasheri A, Airley R, Foster CS, Schulze-Tanzil G, Shakibaei M. Post-genomic applications of tissue microarrays: Basic research, prognostic oncology, clinical genomics and drug discovery. Histology and Histopathology. 2004;19:325-335. DOI: 10.14670/HH-19.325. [46] Mollazadeh H, Cicero AFG, Blesso CN, Pirro M, Majeed M, Sahebkar A. Immune modulation by curcumin: The role of interleukin-10. Critical Reviews in Food Science and

[47] Jagetia GC, Aggarwal BB. "spicing up" of the immune system by curcumin. Journal of

[48] Buhrmann C, Mobasheri A, Matis U, Shakibaei M. Curcumin mediated suppression of nuclear factor-kB promotes chondrogenic differentiation of mesenchymal stem cells in a high-density co-culture microenvironment. Arthritis Research & Therapy. 2010;12:R127.

[49] Saklatvala J. Tumour necrosis factor alpha stimulates resorption and inhibits synthesis of

[50] Séguin CA, Bernier SM. TNFalpha suppresses link protein and type II collagen expression in chondrocytes: Role of MEK1/2 and NF-kappaB signaling pathways. Journal of

[51] Perkins K, Sahy W, Beckett RD. Efficacy of Curcuma for treatment of osteoarthritis. Journal of Evidence-Based Complementary & Alternative Medicine. 2017;22:156-165.

[52] Panahi Y, Alishiri GH, Parvin S, Sahebkar A. Mitigation of systemic oxidative stress by curcuminoids in osteoarthritis: Results of a randomized controlled trial. Journal of Die-

[53] Kuptniratsaikul V, Dajpratham P, Taechaarpornkul W, Buntragulpoontawee M, Lukkanapichonchut P, Chootip C, et al. Efficacy and safety of Curcuma domestica extracts compared with ibuprofen in patients with knee osteoarthritis: A multicenter study. Clinical

[54] Appelboom T, Maes N, Albert A. A new Curcuma extract (flexofytol®) in osteoarthritis: Results from a Belgian real-life experience. Open Rheumatology Journal. 2014;8:77-81.

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54 Cartilage Repair and Regeneration

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**Chapter 4**

**Provisional chapter**

**Cell Therapy and Tissue Engineering for Cartilage**

**Cell Therapy and Tissue Engineering for Cartilage** 

DOI: 10.5772/intechopen.70406

© 2016 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,

© 2018 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 reproduction in any medium, provided the original work is properly cited.

The integrity of the articular cartilage is necessary for the proper functioning of the diarthrodial joint. The self-repair capacity of this tissue is very limited and, currently, there is no effective treatment capable of restoring it. The degradation of the articular cartilage leads to osteoarthritis (OA), a leading cause of pain and disability mainly among older

Different cell treatments have been developed with the aim of forming a repair tissue with the characteristics of native articular cartilage, including cellular therapy and tissue engineering. Cell therapy-based approaches include bone marrow-stimulating techniques, implants of periosteum and perichondrium, ostechondral grafting and implantation of chondrogenic cells as chondrocytes, mesenchymal stem cells or induced pluripotent stem cells. In tissue engineering-based approaches cell-free scaffolds capable of recruiting endogenous cells or chondrogenic cell-loaded scaffolds may

However, despite the numerous treatments available nowadays, no technique has been able to consistently regenerate native articular cartilage in clinical trials. Although many cell therapy and tissue engineering studies have shown promising results and clinical improvement, these treatments generate a fibrocartilaginous tissue different from native articular cartilage. More research is needed to improve cell-based approaches and prove

**Keywords:** regenerative medicine, chondrogenic cells, mesenchymal stem cells (MSCs),

María Piñeiro-Ramil, Rocío Castro-Viñuelas,

María Piñeiro-Ramil, Rocío Castro-Viñuelas, Clara Sanjurjo-Rodríguez, Tamara Hermida-Gómez, Isaac Fuentes-Boquete, Francisco J. de Toro-Santos, Francisco J. Blanco-García

Additional information is available at the end of the chapter

induced pluripotent stem cells (iPS), scaffolds

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.70406

and Silvia M. Díaz-Prado

**Abstract**

people.

be used.

its efficacy

Clara Sanjurjo-Rodríguez, Tamara Hermida-Gómez, Isaac Fuentes-Boquete, Francisco J. de Toro-Santos, Francisco J. Blanco-García and Silvia M. Díaz-Prado

**Repair**

**Repair**

**Provisional chapter**

## **Cell Therapy and Tissue Engineering for Cartilage Repair Repair**

**Cell Therapy and Tissue Engineering for Cartilage** 

DOI: 10.5772/intechopen.70406

María Piñeiro-Ramil, Rocío Castro-Viñuelas, Clara Sanjurjo-Rodríguez, Tamara Hermida-Gómez, Isaac Fuentes-Boquete, Francisco J. de Toro-Santos, Francisco J. Blanco-García and Silvia M. Díaz-Prado Clara Sanjurjo-Rodríguez, Tamara Hermida-Gómez, Isaac Fuentes-Boquete, Francisco J. de Toro-Santos, Francisco J. Blanco-García and Silvia M. Díaz-Prado

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

María Piñeiro-Ramil, Rocío Castro-Viñuelas,

http://dx.doi.org/10.5772/intechopen.70406

#### **Abstract**

The integrity of the articular cartilage is necessary for the proper functioning of the diarthrodial joint. The self-repair capacity of this tissue is very limited and, currently, there is no effective treatment capable of restoring it. The degradation of the articular cartilage leads to osteoarthritis (OA), a leading cause of pain and disability mainly among older people.

Different cell treatments have been developed with the aim of forming a repair tissue with the characteristics of native articular cartilage, including cellular therapy and tissue engineering. Cell therapy-based approaches include bone marrow-stimulating techniques, implants of periosteum and perichondrium, ostechondral grafting and implantation of chondrogenic cells as chondrocytes, mesenchymal stem cells or induced pluripotent stem cells. In tissue engineering-based approaches cell-free scaffolds capable of recruiting endogenous cells or chondrogenic cell-loaded scaffolds may be used.

However, despite the numerous treatments available nowadays, no technique has been able to consistently regenerate native articular cartilage in clinical trials. Although many cell therapy and tissue engineering studies have shown promising results and clinical improvement, these treatments generate a fibrocartilaginous tissue different from native articular cartilage. More research is needed to improve cell-based approaches and prove its efficacy

**Keywords:** regenerative medicine, chondrogenic cells, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPS), scaffolds

© 2016 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. © 2018 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. Introduction**

The integrity of the structure of the articular cartilage is necessary for the proper functioning of the diarthrodial joint. Articular hyaline cartilage provides a resistant, smooth, and lubricated surface, which avoids friction between bones. Thus, hyaline cartilage absorbs and minimizes the pressures produced in the movement of the joint, allows bones to glide over one another with minimal friction, and facilitates the coupling between articular surfaces. Due to its elasticity, articular cartilage absorbs an important part of the compression force, reducing the load supported by the underlying bone structure [1–3].

Traditionally, osteoarthritis (OA) was defined as a degenerative joint disease, characterized by the alteration in the integrity of the articular cartilage [1]. Nowadays, it is known that although the degradation of articular cartilage is the central event in the pathogenesis of OA, synovial tissue and subchondral bone also participate in the onset and development of this disease [4]. The degree of compromise of these components of the joint leads not only to variability between the clinical profiles of patients, but also between different joints of the same patient [5]. On this basis, the Osteoarthritis Research Society International (OARSI) has defined OA as a heterogeneous disorder of movable joints, manifested as genetic, metabolic, and inflammatory changes in the joint, as well as anatomic and/or physiological conditions that may lead to the symptoms associated with the disease. OA is characterized by cell stress and extracellular matrix degradation initiated by micro- and macro-injury that activates maladaptive repair responses including pro-inflammatory pathways of innate immunity [6]. OA is one of the most common chronic health conditions and a leading cause of pain and disability among adults [2, 7]. OA is one of the most prevalent diseases in older people and its incidence, which increases with age, is expected to rise along with the median age of the population [3, 8].

The self-repair capacity of articular cartilage is very limited as it is an avascular and aneural tissue. Due to this absence of vascularity, progenitor cells present in blood and marrow cannot enter into the damaged region to influence or contribute to the reparative process [9, 10]. In addition, because of aneurality, chondral lesions are not detected, and thus patients are not medically treated until more severe lesions are formed [11, 12].

Currently, there is no effective treatment capable of restoring the physiological properties of the osteochondral unit (**Figure 1A**) [13, 14] and the prosthetic replacement is necessary at the final clinical stage (**Figure 1B**) [6]. Different cell treatments have been developed with the aim of forming a repair tissue with structural, biochemical, and functional characteristics equivalent to those of native articular cartilage (**Figure 2**). Scientists have sought several different ways to repair articular cartilage after traumatic damage, which can lead to secondary OA or degeneration of the cartilage [13, 15–17].

**2. Cell therapy**

tissue using autologous or allogenic cells.

**Figure 1.** Images showing (A) healthy knee joint and (B) prosthetic joint replacement.

**2.1. Marrow stimulating techniques**

with less of 15 mm of diameter [19].

Cell therapy is a relatively new approach based on the regeneration or repair of a damaged

Cell Therapy and Tissue Engineering for Cartilage Repair http://dx.doi.org/10.5772/intechopen.70406 59

**Figure 2.** Diagram showing an overview of the alternative treatments for osteochondral damage.

Bone marrow stimulating techniques (MSTs) are based on the use of endogenous mesenchymal stromal cells (MSCs). This type of technique is used in the treatment of chondral lesions

Penetration of subchondral bone is among the oldest and still the most commonly used method to stimulate regeneration of neocartilage [16, 20]. Arthroscopic techniques like drilling, abrasion arthroplasty or microfracture are different tools to perforate the subchondral

It is necessary to highlight that "repair" refers to the restoration of a damaged articular surface with the formation of a neocartilage tissue, which resembles to the native cartilage and "regeneration" refers to the formation of a tissue indistinguishable from the native articular cartilage [16]. Cellular therapy (using cells) and tissue engineering (combining cells, scaffolds, and bioactive factors) have emerged as alternative clinical approaches. However, despite the numerous treatments available nowadays, no technique has been able to consistently regenerate normal hyaline cartilage in clinical trials [3, 18]. Long-term follow-up studies are expected to be performed in the coming years to confirm safety and effectiveness of these new approaches [3].

**Figure 1.** Images showing (A) healthy knee joint and (B) prosthetic joint replacement.

**Figure 2.** Diagram showing an overview of the alternative treatments for osteochondral damage.

## **2. Cell therapy**

**1. Introduction**

58 Cartilage Repair and Regeneration

The integrity of the structure of the articular cartilage is necessary for the proper functioning of the diarthrodial joint. Articular hyaline cartilage provides a resistant, smooth, and lubricated surface, which avoids friction between bones. Thus, hyaline cartilage absorbs and minimizes the pressures produced in the movement of the joint, allows bones to glide over one another with minimal friction, and facilitates the coupling between articular surfaces. Due to its elasticity, articular cartilage absorbs an important part of the compression force, reducing

Traditionally, osteoarthritis (OA) was defined as a degenerative joint disease, characterized by the alteration in the integrity of the articular cartilage [1]. Nowadays, it is known that although the degradation of articular cartilage is the central event in the pathogenesis of OA, synovial tissue and subchondral bone also participate in the onset and development of this disease [4]. The degree of compromise of these components of the joint leads not only to variability between the clinical profiles of patients, but also between different joints of the same patient [5]. On this basis, the Osteoarthritis Research Society International (OARSI) has defined OA as a heterogeneous disorder of movable joints, manifested as genetic, metabolic, and inflammatory changes in the joint, as well as anatomic and/or physiological conditions that may lead to the symptoms associated with the disease. OA is characterized by cell stress and extracellular matrix degradation initiated by micro- and macro-injury that activates maladaptive repair responses including pro-inflammatory pathways of innate immunity [6]. OA is one of the most common chronic health conditions and a leading cause of pain and disability among adults [2, 7]. OA is one of the most prevalent diseases in older people and its incidence, which increases with age, is expected to rise along with the median age of the population [3, 8].

The self-repair capacity of articular cartilage is very limited as it is an avascular and aneural tissue. Due to this absence of vascularity, progenitor cells present in blood and marrow cannot enter into the damaged region to influence or contribute to the reparative process [9, 10]. In addition, because of aneurality, chondral lesions are not detected, and thus patients are not

Currently, there is no effective treatment capable of restoring the physiological properties of the osteochondral unit (**Figure 1A**) [13, 14] and the prosthetic replacement is necessary at the final clinical stage (**Figure 1B**) [6]. Different cell treatments have been developed with the aim of forming a repair tissue with structural, biochemical, and functional characteristics equivalent to those of native articular cartilage (**Figure 2**). Scientists have sought several different ways to repair articular cartilage after traumatic damage, which can lead to secondary OA or

It is necessary to highlight that "repair" refers to the restoration of a damaged articular surface with the formation of a neocartilage tissue, which resembles to the native cartilage and "regeneration" refers to the formation of a tissue indistinguishable from the native articular cartilage [16]. Cellular therapy (using cells) and tissue engineering (combining cells, scaffolds, and bioactive factors) have emerged as alternative clinical approaches. However, despite the numerous treatments available nowadays, no technique has been able to consistently regenerate normal hyaline cartilage in clinical trials [3, 18]. Long-term follow-up studies are expected to be performed in the coming years to confirm safety and effectiveness of these new approaches [3].

the load supported by the underlying bone structure [1–3].

medically treated until more severe lesions are formed [11, 12].

degeneration of the cartilage [13, 15–17].

Cell therapy is a relatively new approach based on the regeneration or repair of a damaged tissue using autologous or allogenic cells.

#### **2.1. Marrow stimulating techniques**

Bone marrow stimulating techniques (MSTs) are based on the use of endogenous mesenchymal stromal cells (MSCs). This type of technique is used in the treatment of chondral lesions with less of 15 mm of diameter [19].

Penetration of subchondral bone is among the oldest and still the most commonly used method to stimulate regeneration of neocartilage [16, 20]. Arthroscopic techniques like drilling, abrasion arthroplasty or microfracture are different tools to perforate the subchondral bone [12], allowing MSCs and growth factors from the bone marrow to infiltrate the lesion [15]. A blood clot is formed in the defect, acting as a scaffold and mediating the inflammatory response (through cytokines) [19].

In addition to fresh osteochondral grafts, particulated cartilage grafts, which are formed by combining fragments of cartilage with fibrin glue, may also be used. Superficial chondrocytes, released from the extracellular matrix as a consequence of the fragmentation of the cartilage, produce additional extracellular matrix that integrates the particulate graft with native carti-

Chondrogenic potential of different cell types (**Figure 3**) was tested for hyaline cartilage repair.

The autologous chondrocyte implantation (ACI) was firstly described by Peterson et al. [30]. This technique consists of harvesting a cartilage piece from a low-weight-bearing area of the joint and culture-expanding the chondrocytes to implant into the lesion. The lesion is sealed

[15, 28]. Other limitations are dedifferentiation of chondrocytes during culture expansion, the low amount of chondrocytes obtained and multiple surgical procedures involved [31, 32]. Further, donor-site morbidity of cartilage and bone for chondrocyte and periosteum obtain-

ACI is considered superior to MSTs regarding the quality of the repaired tissue, although

**Figure 3.** Diagram showing the different cell sources, most commonly used in cartilage treatment using cell therapy:

chondrocytes (left), mesenchymal stromal cells (middle), and induced pluripotent stem cells (right).

) focal lesions surrounded by healthy cartilage

Cell Therapy and Tissue Engineering for Cartilage Repair http://dx.doi.org/10.5772/intechopen.70406 61

lage and fills the defect [29].

**2.3. Implantation of cells with chondrogenic capacity**

*2.3.1. Autologous chondrocyte implantation*

with autologous periosteum to avoid cell loss.

ACI is only applicable to small size (3–4 cm<sup>2</sup>

ing was observed [15, 33, 34].

there are conflicting results [28].

However, it was described that endogen bone marrow angiogenic factors favor osteogenesis, instead of chondrogenesis, of bone marrow MSCs [11]. Generated repair tissue frequently ends up degenerating [21] and usually presents type I collagen (fibrocartilage phenotype) and lacks hyaline cartilage viscoelastic properties [22].

## **2.2. Tissue grafts**

Tissue grafts have potential benefits in cartilage repair since they contain cell populations with chondrogenic capacity.

#### *2.2.1. Implants of periosteum and perichondrium*

In the 90s, autologous strips of perichondrium were used to treat chondral defects [23, 24]. Periosteum and perichondrium contain MSCs that are capable of chondrogenesis and act as a biological membrane [16]. However, the ability of periosteum MSCs to proliferate and differentiate into chondrocytes decreases with age [25].

The clinical outcomes of perichondrium implants are similar to those of subchondral perforation [26]. Calcification of the periosteum grafts had been mentioned as a problem in the long term [16].

## *2.2.2. Mosaicplasty*

Autologous mosaicplasty is widely used for treating chondral and osteochondral defects. The most used technique is the osteochondral autologous transplantation (OAT), which consists in the translocation of osteochondral cylinders from not loading areas to the affected areas of the joint [15].

Even though good to excellent short-term subjective results were obtained, clinical and radiological midterms to long-term outcomes of mosaicplasty were moderate. Further limitations are donor-site morbidity, technical difficulty, special equipment, lesion size, and fibrocartilaginous repair [16, 27]. OAT might be more appropriate for lesions smaller than 2–3 cm<sup>2</sup> [28].

Another problem is the lack of congruence between the osteochondral cylinders implanted and the lesion area, and the differences in cartilage height of the defect and surrounding native cartilage, altering the distribution of stress and compression forces [16, 27].

Allogenic mosaicplasty has shown successful outcomes and its main advantage over autograft transplantation is the lack of donor-site morbidity. Nevertheless, the amount of transplanted bone has to be minimum because the allograft failure is mostly due to collapse of the subchondral bone [22].

Nowadays, synthetic cylindrical plugs for implant similar to OAT exist but studies have shown universal failure to incorporate these plugs into the subchondral bone, with formation of cysts [22].

In addition to fresh osteochondral grafts, particulated cartilage grafts, which are formed by combining fragments of cartilage with fibrin glue, may also be used. Superficial chondrocytes, released from the extracellular matrix as a consequence of the fragmentation of the cartilage, produce additional extracellular matrix that integrates the particulate graft with native cartilage and fills the defect [29].

## **2.3. Implantation of cells with chondrogenic capacity**

Chondrogenic potential of different cell types (**Figure 3**) was tested for hyaline cartilage repair.

#### *2.3.1. Autologous chondrocyte implantation*

bone [12], allowing MSCs and growth factors from the bone marrow to infiltrate the lesion [15]. A blood clot is formed in the defect, acting as a scaffold and mediating the inflammatory

However, it was described that endogen bone marrow angiogenic factors favor osteogenesis, instead of chondrogenesis, of bone marrow MSCs [11]. Generated repair tissue frequently ends up degenerating [21] and usually presents type I collagen (fibrocartilage phenotype) and

Tissue grafts have potential benefits in cartilage repair since they contain cell populations

In the 90s, autologous strips of perichondrium were used to treat chondral defects [23, 24]. Periosteum and perichondrium contain MSCs that are capable of chondrogenesis and act as a biological membrane [16]. However, the ability of periosteum MSCs to proliferate and dif-

The clinical outcomes of perichondrium implants are similar to those of subchondral perforation [26]. Calcification of the periosteum grafts had been mentioned as a problem in the long

Autologous mosaicplasty is widely used for treating chondral and osteochondral defects. The most used technique is the osteochondral autologous transplantation (OAT), which consists in the translocation of osteochondral cylinders from not loading areas to the affected areas of

Even though good to excellent short-term subjective results were obtained, clinical and radiological midterms to long-term outcomes of mosaicplasty were moderate. Further limitations are donor-site morbidity, technical difficulty, special equipment, lesion size, and fibrocartilaginous repair [16, 27]. OAT might be more appropriate for lesions smaller than 2–3 cm<sup>2</sup>

Another problem is the lack of congruence between the osteochondral cylinders implanted and the lesion area, and the differences in cartilage height of the defect and surrounding

Allogenic mosaicplasty has shown successful outcomes and its main advantage over autograft transplantation is the lack of donor-site morbidity. Nevertheless, the amount of transplanted bone has to be minimum because the allograft failure is mostly due to collapse of the

Nowadays, synthetic cylindrical plugs for implant similar to OAT exist but studies have shown universal failure to incorporate these plugs into the subchondral bone, with formation

native cartilage, altering the distribution of stress and compression forces [16, 27].

[28].

response (through cytokines) [19].

60 Cartilage Repair and Regeneration

**2.2. Tissue grafts**

term [16].

*2.2.2. Mosaicplasty*

the joint [15].

subchondral bone [22].

of cysts [22].

with chondrogenic capacity.

lacks hyaline cartilage viscoelastic properties [22].

*2.2.1. Implants of periosteum and perichondrium*

ferentiate into chondrocytes decreases with age [25].

The autologous chondrocyte implantation (ACI) was firstly described by Peterson et al. [30]. This technique consists of harvesting a cartilage piece from a low-weight-bearing area of the joint and culture-expanding the chondrocytes to implant into the lesion. The lesion is sealed with autologous periosteum to avoid cell loss.

ACI is only applicable to small size (3–4 cm<sup>2</sup> ) focal lesions surrounded by healthy cartilage [15, 28]. Other limitations are dedifferentiation of chondrocytes during culture expansion, the low amount of chondrocytes obtained and multiple surgical procedures involved [31, 32]. Further, donor-site morbidity of cartilage and bone for chondrocyte and periosteum obtaining was observed [15, 33, 34].

ACI is considered superior to MSTs regarding the quality of the repaired tissue, although there are conflicting results [28].

**Figure 3.** Diagram showing the different cell sources, most commonly used in cartilage treatment using cell therapy: chondrocytes (left), mesenchymal stromal cells (middle), and induced pluripotent stem cells (right).

## *2.3.2. Chondrospheres*

The technique of chondrospheres consists of the generation and implantation of spheroids of autologous or allogenic articular chondrocytes [29]. Autologous chondrocytes are obtained from undamaged articular cartilage, expanded *in vitro*, and condensed in order to form spheroids, which then are coalesced. Chondrospheres have shown to be able to adhere, integrate into hyaline cartilage defect and produce cartilaginous extracellular matrix in mouse, mini pig, and horse cartilage defect models, as well as in artificial defects in human cartilage explants [35–37]. A phase III clinical trial is currently ongoing in Germany and Poland to investigate the efficacy of this technology compared to microfracture in the treatment of cartilage defects of knee joints [38].

was performed by Wakitani et al*.* [57]. In this study, bone marrow-derived MSCs were transplanted into the articular cartilage defect and covered with autologous periosteum. Although the arthroscopic and histological grading score was better in the cell-transplanted group than in the control one, the clinical improvement was not very clear. Since then, several clinical studies have been performed, mainly using intra-articular injection of autologous bone marrow-derived MSCs, showing some degree of improvement in terms of clinical outcomes and repaired cartilage tissue quality [58–60]. However, several studies described a lack of engraftment into cartilage defects [61] and it is important to highlight that most of the clinical trials are I and I/II phases, indicating the immaturity of MSC clinical applications in OA [49].

Cell Therapy and Tissue Engineering for Cartilage Repair http://dx.doi.org/10.5772/intechopen.70406 63

Limitations of this approach are that culture expansion is not avoided, cell yield is often low and MSCs differentiation capacity decreases with age of the donor [21]. This is a problem in regenerative therapies for degenerative diseases such as OA, where most of patients are aged [61]. Given that the age of patient and the size of the lesion affect the outcome, the cut-off points for the risk of failure have been suggested at age greater than 60 years and lesion size larger than

A novel cell therapy approach is based on combining autologous chondrocytes in their pericellular matrix (chondrons) and allogenic MSCs, which was called Instant MSC Product accompanying Autologous Chondron Transplantation (IMPACT) and performed by De Windt et al*.* [62]. In this phase I clinical trial, patients with focal cartilage defects were treated using a mix of 80–90% allogenic MSCs and 10–20% autologous chondrons combined with fibrin glue. In this approach, chondrons are "recycled" from debrided cartilage instead of being harvested from a low-weight-bearing area of the joint, as occurring in ACI. The combination of this recycled chondrons with allogenic human bone marrow MSCs stimulates cartilage regeneration and provides clinical improvement. Surprisingly, although the co-implantation of chondrons and MSCs provides better results in comparison with implantation of chondrons or MSCs alone [63], no allogenic cells were detected in the repaired cartilage after 1 year, suggesting that MSCs have trophic effects that stimulate chondrons to regenerate cartilage. The quality of the repaired tissue and the clinical outcome using the IMPACT technique was similar or even superior in comparison with ACI. Furthermore, IMPACT technique presents the advantage of allowing to perform both surgeries on the same day (the extraction of cartilage and the

Pluripotent cells could provide an unlimited and renewable cell source that can be induced to differentiate into any cell type. In fact, pluripotent cells of embryonic origin [61, 64], embryonic human stem cells (hESCs), or induced to pluripotency [65], induced pluripotent stem cells (iPSCs), have shown to produce cartilage under specific conditions. iPSCs have been generated from adult cells (**Figure 4A**) using defined factors (**Figure 4B**) [66]. These cells present similar morphology (**Figure 4C**), proliferation capacity, genetic expression and epigenetic

*2.3.4. Mesenchymal stromal cells combined with autologous chondrons*

6.0 cm2

[28].

implantation of cells) [62].

*2.3.5. Induced pluripotent stem cells*

pattern, and pluripotency characteristics to hESCs [66, 67].

#### *2.3.3. Mesenchymal stromal cells*

Human MSCs are nonhematopoietic multipotent progenitor cells with long-term self-renewal ability and the capacity to differentiate along multiple cell lineages, including cartilage, as well as immunomodulatory features [39–41]. MSCs are responsible for normal tissue renewal and for response to injury and may be an alternative to chondrocytes for the development of new therapeutic approaches for the treatment of cartilage defects.

*In vitro* and *in vivo* studies of clonally derived MSCs demonstrated that these cells consist of subsets that present different surface markers expression and different capacities for cellular differentiation [42]. These cells are considered a potential cell source for cell therapy since they can be easily collected from various tissues such as bone marrow [43], adipose tissue [44], synovial membrane [42], and amniotic membrane [45], among others. However, the equivalence of chondrogenic differentiation potential of MSCs derived from different tissues is a matter of considerable debate [46].

For cell therapy approaches, either autologous or allogenic MSCs can be used. MSCs do not express major histocompatibility complex class II (MHC II) and its co-stimulatory molecules, and barely express major histocompatibility complex class I (MHC I), so that they do not produce alloreactivity, avoiding rejection problems. This feature turns MSCs into a feasible cell source for allogenic transplantation [40, 47].

The therapeutic potential of autologous MSCs derived from different tissues to stimulate the regeneration of cartilage in OA has been reported in several preclinical studies [48, 49]. Bone marrow-derived MSCs suspended in hyaluronic acid and administrated by intra-articular injection have been used to promote cartilage repair in animal models such as guinea pig, mini pig, goat and donkey, leading to improvement in cartilage regeneration, less cartilage destruction and reduced osteophyte formation [50–53]. MSCs derived from other sources have also been used; for example, transplantation of synovial MSCs was used to repair osteochondral defects in rabbits [54], and intra-articular injection of adipose-derived MSCs was used to treat chronic osteoarthritis in dogs, showing significant improvement in MSCs-treated joints [55].

One of the MSCs transplantation techniques for cartilage focal lesions is a variation of ACI in which bone marrow MSCs are injected into defects and closed with periosteal membrane to be differentiated toward chondrocytes [56]. The first clinical study using MSCs to treat OA was performed by Wakitani et al*.* [57]. In this study, bone marrow-derived MSCs were transplanted into the articular cartilage defect and covered with autologous periosteum. Although the arthroscopic and histological grading score was better in the cell-transplanted group than in the control one, the clinical improvement was not very clear. Since then, several clinical studies have been performed, mainly using intra-articular injection of autologous bone marrow-derived MSCs, showing some degree of improvement in terms of clinical outcomes and repaired cartilage tissue quality [58–60]. However, several studies described a lack of engraftment into cartilage defects [61] and it is important to highlight that most of the clinical trials are I and I/II phases, indicating the immaturity of MSC clinical applications in OA [49].

Limitations of this approach are that culture expansion is not avoided, cell yield is often low and MSCs differentiation capacity decreases with age of the donor [21]. This is a problem in regenerative therapies for degenerative diseases such as OA, where most of patients are aged [61]. Given that the age of patient and the size of the lesion affect the outcome, the cut-off points for the risk of failure have been suggested at age greater than 60 years and lesion size larger than 6.0 cm2 [28].

#### *2.3.4. Mesenchymal stromal cells combined with autologous chondrons*

A novel cell therapy approach is based on combining autologous chondrocytes in their pericellular matrix (chondrons) and allogenic MSCs, which was called Instant MSC Product accompanying Autologous Chondron Transplantation (IMPACT) and performed by De Windt et al*.* [62]. In this phase I clinical trial, patients with focal cartilage defects were treated using a mix of 80–90% allogenic MSCs and 10–20% autologous chondrons combined with fibrin glue. In this approach, chondrons are "recycled" from debrided cartilage instead of being harvested from a low-weight-bearing area of the joint, as occurring in ACI. The combination of this recycled chondrons with allogenic human bone marrow MSCs stimulates cartilage regeneration and provides clinical improvement. Surprisingly, although the co-implantation of chondrons and MSCs provides better results in comparison with implantation of chondrons or MSCs alone [63], no allogenic cells were detected in the repaired cartilage after 1 year, suggesting that MSCs have trophic effects that stimulate chondrons to regenerate cartilage. The quality of the repaired tissue and the clinical outcome using the IMPACT technique was similar or even superior in comparison with ACI. Furthermore, IMPACT technique presents the advantage of allowing to perform both surgeries on the same day (the extraction of cartilage and the implantation of cells) [62].

#### *2.3.5. Induced pluripotent stem cells*

*2.3.2. Chondrospheres*

62 Cartilage Repair and Regeneration

tilage defects of knee joints [38].

*2.3.3. Mesenchymal stromal cells*

matter of considerable debate [46].

source for allogenic transplantation [40, 47].

The technique of chondrospheres consists of the generation and implantation of spheroids of autologous or allogenic articular chondrocytes [29]. Autologous chondrocytes are obtained from undamaged articular cartilage, expanded *in vitro*, and condensed in order to form spheroids, which then are coalesced. Chondrospheres have shown to be able to adhere, integrate into hyaline cartilage defect and produce cartilaginous extracellular matrix in mouse, mini pig, and horse cartilage defect models, as well as in artificial defects in human cartilage explants [35–37]. A phase III clinical trial is currently ongoing in Germany and Poland to investigate the efficacy of this technology compared to microfracture in the treatment of car-

Human MSCs are nonhematopoietic multipotent progenitor cells with long-term self-renewal ability and the capacity to differentiate along multiple cell lineages, including cartilage, as well as immunomodulatory features [39–41]. MSCs are responsible for normal tissue renewal and for response to injury and may be an alternative to chondrocytes for the development of

*In vitro* and *in vivo* studies of clonally derived MSCs demonstrated that these cells consist of subsets that present different surface markers expression and different capacities for cellular differentiation [42]. These cells are considered a potential cell source for cell therapy since they can be easily collected from various tissues such as bone marrow [43], adipose tissue [44], synovial membrane [42], and amniotic membrane [45], among others. However, the equivalence of chondrogenic differentiation potential of MSCs derived from different tissues is a

For cell therapy approaches, either autologous or allogenic MSCs can be used. MSCs do not express major histocompatibility complex class II (MHC II) and its co-stimulatory molecules, and barely express major histocompatibility complex class I (MHC I), so that they do not produce alloreactivity, avoiding rejection problems. This feature turns MSCs into a feasible cell

The therapeutic potential of autologous MSCs derived from different tissues to stimulate the regeneration of cartilage in OA has been reported in several preclinical studies [48, 49]. Bone marrow-derived MSCs suspended in hyaluronic acid and administrated by intra-articular injection have been used to promote cartilage repair in animal models such as guinea pig, mini pig, goat and donkey, leading to improvement in cartilage regeneration, less cartilage destruction and reduced osteophyte formation [50–53]. MSCs derived from other sources have also been used; for example, transplantation of synovial MSCs was used to repair osteochondral defects in rabbits [54], and intra-articular injection of adipose-derived MSCs was used to treat chronic osteoarthritis in dogs, showing significant improvement in MSCs-treated joints [55].

One of the MSCs transplantation techniques for cartilage focal lesions is a variation of ACI in which bone marrow MSCs are injected into defects and closed with periosteal membrane to be differentiated toward chondrocytes [56]. The first clinical study using MSCs to treat OA

new therapeutic approaches for the treatment of cartilage defects.

Pluripotent cells could provide an unlimited and renewable cell source that can be induced to differentiate into any cell type. In fact, pluripotent cells of embryonic origin [61, 64], embryonic human stem cells (hESCs), or induced to pluripotency [65], induced pluripotent stem cells (iPSCs), have shown to produce cartilage under specific conditions. iPSCs have been generated from adult cells (**Figure 4A**) using defined factors (**Figure 4B**) [66]. These cells present similar morphology (**Figure 4C**), proliferation capacity, genetic expression and epigenetic pattern, and pluripotency characteristics to hESCs [66, 67].

particles into joint surface defects in immunodeficient rats and immunosuppressed mini pigs, they observed cartilaginous neotissue with potential for integration into native cartilage.

Cell Therapy and Tissue Engineering for Cartilage Repair http://dx.doi.org/10.5772/intechopen.70406 65

Nowadays, there are no clinical studies published about cartilage cell therapy using iPSCs. Although cell therapy or tissue engineering using iPSCs are promising tools, their clinical use is not legalized either by the scientific community or by existing international legislation yet,

The lack of efficient treatments for cartilage repair motivates the researchers to develop, by tissue engineering, biological tissue substitutes that can be implanted to replace the affected area of the joint [74]. Tissue engineering is not widespread yet in surgical procedures, although there are many combinations of different cells and supports being tested

In this way, different strategies were developed for cartilage regeneration, based on the use of scaffolds and endogenous or exogenous cells. Whereas in *in vitro* studies scaffolds are usually combined with cells and bioactive factors, in most *in vivo* studies the scaffolds are used only combined with cells because those factors are present in the joint (e.g., AMIC

*In vitro* administration of growth factors (transforming growth factor 1 or 3, bone morphogenetic proteins 2 or 7, and insulin growth factor 1, among others) have been used to induce chondrogenic differentiation of MSCs and iPSCs. However, the effect of application of these molecules is dose, timing of administration and cell type-dependent [75]. That is why, in recent years, scaffolds were functionalized with bioactive factors or other molecules for *in vivo* cartilage therapies, as a delivery system [76] or stimulation for MSCs. For example, the addition of proteoglycans to collagen biomaterials had improved bone marrow MSCs chondrogenic differentiation [43, 77].

A broad variety of biomaterials have been successfully developed to support proliferation, infiltration, or differentiation of allogeneic transplanted or endogenous MSCs to achieve functional tissue restoration [78]. Scaffolds/biomaterials should be a porous three-dimensional matrix that allow cell migration, adhesion and growth, and support the organization of the

However, despite the diffusion of new tissue-engineering techniques and the high number of scaffolds that have been developed and investigated for cartilage regeneration, the ideal matrix material has not been identified yet. Cartilage-engineering strategies have produced promising *in vitro* data, seeding chondrogenic cells on biomaterials with growth factors. However, thus far, no approach has led to the generation of long-term *in vivo* replacement tissue identical to native hyaline cartilage. There are different factors for the lack of stable

functional tissue as inflammatory stress or biophysical stimuli [80].

except in Japan.

**3. Tissue engineering**

both *in vitro* and *in vivo*.

described below).

growing tissue [79].

**Figure 4.** Scheme representing the role of iPSCs in tissue engineering. (A) Harvesting somatic cells from the patient. (B) Reprogramming the cells using the factors Oct4, Sox2, Klf4, and c-Myc. (C) iPSc colony obtained after reprogramming. (D) Embryoid bodies (EB) formation. (E) Differentiation of the iPSc toward chondrocytes with (W/) or without (W/O) scaffold.

iPSCs seem to be an alternative tool to chondrocytes for cartilage repair as they can be expanded before starting their differentiation (using or not embryoid bodies formation) toward chondrocytes (**Figure 4D**). Then, iPSC-derived chondrocytes can be cultured in three-dimensional culture with scaffold (**Figure 4E**, w/Scaffold), or cultured without a scaffold (**Figure 4E**, w/o Scaffold), to create cartilaginous tissues *in vitro* before transplantation to repair large defects [68].

In addition, iPSCs seem to be an alternative tool to MSCs for cartilage repair. After *in vitro* chondrogenesis, iPSCs showed lower hypertrophic markers than MSCs [69]. The risk of iPSCs teratoma formation in cell therapy or tissue engineering can be avoided using pre-differentiated cells before implantation [70, 71]. Also, the use of iPSCs avoids the problem of *in vivo*age-dependent and *in vitro*-passage-dependent MSC senescence [72].

Yamashita et al. [73] optimized a protocol of chondrogenic differentiation using human iPSCs to form homogenous cartilaginous particles. After the transplantation of these chondrogenic particles into joint surface defects in immunodeficient rats and immunosuppressed mini pigs, they observed cartilaginous neotissue with potential for integration into native cartilage.

Nowadays, there are no clinical studies published about cartilage cell therapy using iPSCs. Although cell therapy or tissue engineering using iPSCs are promising tools, their clinical use is not legalized either by the scientific community or by existing international legislation yet, except in Japan.

## **3. Tissue engineering**

iPSCs seem to be an alternative tool to chondrocytes for cartilage repair as they can be expanded before starting their differentiation (using or not embryoid bodies formation) toward chondrocytes (**Figure 4D**). Then, iPSC-derived chondrocytes can be cultured in three-dimensional culture with scaffold (**Figure 4E**, w/Scaffold), or cultured without a scaffold (**Figure 4E**, w/o Scaffold), to create cartilaginous tissues *in vitro* before transplantation to repair large defects

**Figure 4.** Scheme representing the role of iPSCs in tissue engineering. (A) Harvesting somatic cells from the patient. (B) Reprogramming the cells using the factors Oct4, Sox2, Klf4, and c-Myc. (C) iPSc colony obtained after reprogramming. (D) Embryoid bodies (EB) formation. (E) Differentiation of the iPSc toward chondrocytes with (W/) or without (W/O)

In addition, iPSCs seem to be an alternative tool to MSCs for cartilage repair. After *in vitro* chondrogenesis, iPSCs showed lower hypertrophic markers than MSCs [69]. The risk of iPSCs teratoma formation in cell therapy or tissue engineering can be avoided using pre-differentiated cells before implantation [70, 71]. Also, the use of iPSCs avoids the problem of *in vivo-*

Yamashita et al. [73] optimized a protocol of chondrogenic differentiation using human iPSCs to form homogenous cartilaginous particles. After the transplantation of these chondrogenic

age-dependent and *in vitro*-passage-dependent MSC senescence [72].

[68].

scaffold.

64 Cartilage Repair and Regeneration

The lack of efficient treatments for cartilage repair motivates the researchers to develop, by tissue engineering, biological tissue substitutes that can be implanted to replace the affected area of the joint [74]. Tissue engineering is not widespread yet in surgical procedures, although there are many combinations of different cells and supports being tested both *in vitro* and *in vivo*.

In this way, different strategies were developed for cartilage regeneration, based on the use of scaffolds and endogenous or exogenous cells. Whereas in *in vitro* studies scaffolds are usually combined with cells and bioactive factors, in most *in vivo* studies the scaffolds are used only combined with cells because those factors are present in the joint (e.g., AMIC described below).

*In vitro* administration of growth factors (transforming growth factor 1 or 3, bone morphogenetic proteins 2 or 7, and insulin growth factor 1, among others) have been used to induce chondrogenic differentiation of MSCs and iPSCs. However, the effect of application of these molecules is dose, timing of administration and cell type-dependent [75]. That is why, in recent years, scaffolds were functionalized with bioactive factors or other molecules for *in vivo* cartilage therapies, as a delivery system [76] or stimulation for MSCs. For example, the addition of proteoglycans to collagen biomaterials had improved bone marrow MSCs chondrogenic differentiation [43, 77].

A broad variety of biomaterials have been successfully developed to support proliferation, infiltration, or differentiation of allogeneic transplanted or endogenous MSCs to achieve functional tissue restoration [78]. Scaffolds/biomaterials should be a porous three-dimensional matrix that allow cell migration, adhesion and growth, and support the organization of the growing tissue [79].

However, despite the diffusion of new tissue-engineering techniques and the high number of scaffolds that have been developed and investigated for cartilage regeneration, the ideal matrix material has not been identified yet. Cartilage-engineering strategies have produced promising *in vitro* data, seeding chondrogenic cells on biomaterials with growth factors. However, thus far, no approach has led to the generation of long-term *in vivo* replacement tissue identical to native hyaline cartilage. There are different factors for the lack of stable functional tissue as inflammatory stress or biophysical stimuli [80].

## **3.1. Cell-free scaffolds and endogenous cells**

Cell-free scaffolds are developed for one stage procedure techniques, since they can be implanted alone to attract the endogenous cells. In this case, the aim of using scaffolds is to obtain a suitable microenvironment to recruit and mobilize the host cells, from either the blood or a tissue specific (bone marrow, synovial fluid…) niche for self-repair. Several studies have detected the recruitment of endogenous synovial cells [81, 82] or exogenous-injected MSCs [50] in injured areas after the implantation of empty scaffolds.

bone and cartilage phases [87]. Beside the tissue decellularization, extracellular matrix scaf-

Cell Therapy and Tissue Engineering for Cartilage Repair http://dx.doi.org/10.5772/intechopen.70406 67

The matrix-associated chondrocyte implantation/transplantation (MACI or MACT) is a second generation ACI, which includes the employment of a bilayer collagen membrane [91]. Essentially, the concept is based on the use of biodegradable polymers as temporary scaffolds for *in vitro* growth of cells and their subsequent transplantation into the defect site. In this case, autologous chondrocytes are previously seeded in the scaffold before implantation into the lesion [12, 83]. Other types of scaffolds (hydrogels, fibrous scaffolds, decellularized ECM,

Wakitani et al. [93] observed that MSCs embedded in a collagen gel could differentiate in *in vivo* animal models. Since these first studies, thousands of works were carried out using different types of scaffolds (hydrogels, sponges…), cells, and approaches for chondrogenic

Several *in vivo* studies tried to replicate the distinct osteochondral zones using tri- or bi-layered scaffolds of different composition and/or bioactive factors combined with MSCs. MSCs combined with scaffolds appear to engraft and contribute to cartilage repair, while MSCs injected as a free suspension into the joint do not engraft into the cartilage [61]. This happens because scaffolds can transport cells into the lesion and provide the proper environment for

It was described that cartilage tissue engineering from differentiation-induced *in vitro* MSCs has an inferior quality to that engineered from chondrocytes [95]. However, human amniotic MSCs with human amniotic membrane (as scaffold) showed better reparation in an *in vitro* repair model when compared with bone marrow MSCs and chondrocytes, and demonstrated good adhering capacity to the native cartilage [45]. Also, our group obtained good results using bone marrow MSCs and collagen/heparan sulfate scaffolds in an *in vitro* repair model (**Figure 5**) [96].

Although tissue-engineering studies using iPSCs are scarce, several studies have shown their potential in chondral repair [21]. Liu et al*.* [48] have tested the chondrogenesis of murine cells derived from single embryoid bodies. After seeding these cells on polycaprolactone/gelatin

Nowadays, 3D bioprinting into cartilage using iPSCs and bioinks (that act as scaffolds) is

MACI presents lower rates of graft hypertrophy than first-generation ACI [92].

folds can also be obtained from cultured cells [90].

*3.2.1. Matrix-associated chondrocyte transplantation*

**3.2. Cell-loaded scaffolds**

or composites) were later used [85].

scaffolding.

cell differentiation [75, 94].

being developed [97].

*3.2.2. Mesenchymal stromal cells on scaffolds*

*3.2.3. Induced pluripotent stem cells on scaffolds*

scaffolds, they showed a good chondrogenic capacity.

Implantation of cell-free scaffolds avoids the issues around the *in vitro* cell culture process, as exogenous cell transplantation is not required. However, clinical results after implantation of cell-free scaffolds for OA treatment are few [3].

## *3.1.1. Autologous matrix-induced chondrogenesis*

The autologous matrix induced chondrogenesis (AMIC) is a second generation MSTs. This is a one-step procedure combining subchondral microfracture with the attachment of a collagen scaffold to the lesion. The initially formed blood clot as produced by microfracturing is protected by the collagen scaffold [83]. The collagen scaffold is thought to stabilize the blood clot, helping to promote early mechanical stability and cartilage regeneration [29]. More complex scaffolds have also been tested in AMIC studies, for example, a biphasic scaffold consisting of calcium triphosphate in the osseous region and poly(lactic-co-glycolic acid) in the cartilaginous region [84].

Even though donor-site morbidity due to removal of periosteum from tibia is avoided, AMIC has similar clinical outcomes to ACI [85].

## *3.1.2. Scaffold-based autografts*

Another approach is the use of scaffold-based autografts, in which harvested cartilage is mechanically minced and uniformly affixed to a biodegradable scaffold, using fibrin glue; then, the scaffold with the cartilage fragments is transferred to the lesion. When compared to microfracture, this scaffold-based autograft procedure resulted in an improvement of functional outcomes and cartilage development [86].

## *3.1.3. Decellularized extracellular matrix scaffolds*

Decellularized extracellular matrix may be used as a scaffold with the potential to retain the bioactive factors needed to support specific tissue formation at the implantation site [87]. Cartilage matrix can be harvested from allogenic sources, then decellularized and used as a scaffold. This approach leads to the improvement of neocartilage formation in preclinical models, in comparison with the living-cartilage implantation [88]. One of the drawbacks of this technique is that the protocols required to decellularization of cartilage also imply some degree of destruction of extracellular matrix components [89]. Decellularized cartilage matrix has been used to treat osteochondral defects in a horse model, obtaining repair of both the bone and cartilage phases [87]. Beside the tissue decellularization, extracellular matrix scaffolds can also be obtained from cultured cells [90].

## **3.2. Cell-loaded scaffolds**

**3.1. Cell-free scaffolds and endogenous cells**

66 Cartilage Repair and Regeneration

cell-free scaffolds for OA treatment are few [3].

*3.1.1. Autologous matrix-induced chondrogenesis*

has similar clinical outcomes to ACI [85].

tional outcomes and cartilage development [86].

*3.1.3. Decellularized extracellular matrix scaffolds*

*3.1.2. Scaffold-based autografts*

region [84].

MSCs [50] in injured areas after the implantation of empty scaffolds.

Cell-free scaffolds are developed for one stage procedure techniques, since they can be implanted alone to attract the endogenous cells. In this case, the aim of using scaffolds is to obtain a suitable microenvironment to recruit and mobilize the host cells, from either the blood or a tissue specific (bone marrow, synovial fluid…) niche for self-repair. Several studies have detected the recruitment of endogenous synovial cells [81, 82] or exogenous-injected

Implantation of cell-free scaffolds avoids the issues around the *in vitro* cell culture process, as exogenous cell transplantation is not required. However, clinical results after implantation of

The autologous matrix induced chondrogenesis (AMIC) is a second generation MSTs. This is a one-step procedure combining subchondral microfracture with the attachment of a collagen scaffold to the lesion. The initially formed blood clot as produced by microfracturing is protected by the collagen scaffold [83]. The collagen scaffold is thought to stabilize the blood clot, helping to promote early mechanical stability and cartilage regeneration [29]. More complex scaffolds have also been tested in AMIC studies, for example, a biphasic scaffold consisting of calcium triphosphate in the osseous region and poly(lactic-co-glycolic acid) in the cartilaginous

Even though donor-site morbidity due to removal of periosteum from tibia is avoided, AMIC

Another approach is the use of scaffold-based autografts, in which harvested cartilage is mechanically minced and uniformly affixed to a biodegradable scaffold, using fibrin glue; then, the scaffold with the cartilage fragments is transferred to the lesion. When compared to microfracture, this scaffold-based autograft procedure resulted in an improvement of func-

Decellularized extracellular matrix may be used as a scaffold with the potential to retain the bioactive factors needed to support specific tissue formation at the implantation site [87]. Cartilage matrix can be harvested from allogenic sources, then decellularized and used as a scaffold. This approach leads to the improvement of neocartilage formation in preclinical models, in comparison with the living-cartilage implantation [88]. One of the drawbacks of this technique is that the protocols required to decellularization of cartilage also imply some degree of destruction of extracellular matrix components [89]. Decellularized cartilage matrix has been used to treat osteochondral defects in a horse model, obtaining repair of both the

## *3.2.1. Matrix-associated chondrocyte transplantation*

The matrix-associated chondrocyte implantation/transplantation (MACI or MACT) is a second generation ACI, which includes the employment of a bilayer collagen membrane [91]. Essentially, the concept is based on the use of biodegradable polymers as temporary scaffolds for *in vitro* growth of cells and their subsequent transplantation into the defect site. In this case, autologous chondrocytes are previously seeded in the scaffold before implantation into the lesion [12, 83]. Other types of scaffolds (hydrogels, fibrous scaffolds, decellularized ECM, or composites) were later used [85].

MACI presents lower rates of graft hypertrophy than first-generation ACI [92].

## *3.2.2. Mesenchymal stromal cells on scaffolds*

Wakitani et al. [93] observed that MSCs embedded in a collagen gel could differentiate in *in vivo* animal models. Since these first studies, thousands of works were carried out using different types of scaffolds (hydrogels, sponges…), cells, and approaches for chondrogenic scaffolding.

Several *in vivo* studies tried to replicate the distinct osteochondral zones using tri- or bi-layered scaffolds of different composition and/or bioactive factors combined with MSCs. MSCs combined with scaffolds appear to engraft and contribute to cartilage repair, while MSCs injected as a free suspension into the joint do not engraft into the cartilage [61]. This happens because scaffolds can transport cells into the lesion and provide the proper environment for cell differentiation [75, 94].

It was described that cartilage tissue engineering from differentiation-induced *in vitro* MSCs has an inferior quality to that engineered from chondrocytes [95]. However, human amniotic MSCs with human amniotic membrane (as scaffold) showed better reparation in an *in vitro* repair model when compared with bone marrow MSCs and chondrocytes, and demonstrated good adhering capacity to the native cartilage [45]. Also, our group obtained good results using bone marrow MSCs and collagen/heparan sulfate scaffolds in an *in vitro* repair model (**Figure 5**) [96].

## *3.2.3. Induced pluripotent stem cells on scaffolds*

Although tissue-engineering studies using iPSCs are scarce, several studies have shown their potential in chondral repair [21]. Liu et al*.* [48] have tested the chondrogenesis of murine cells derived from single embryoid bodies. After seeding these cells on polycaprolactone/gelatin scaffolds, they showed a good chondrogenic capacity.

Nowadays, 3D bioprinting into cartilage using iPSCs and bioinks (that act as scaffolds) is being developed [97].

de Reumatología (2014 grant); Universidade da Coruña; Fundación Profesor Novoa Santos;

Cell Therapy and Tissue Engineering for Cartilage Repair http://dx.doi.org/10.5772/intechopen.70406 69

María Piñeiro-Ramil1,2, Rocío Castro-Viñuelas1,2, Clara Sanjurjo-Rodríguez1,2,3, Tamara Hermida-Gómez2,3, Isaac Fuentes-Boquete1,2,3, Francisco J. de Toro-Santos1,3,

1 Cell Therapy and Regenerative Medicine Group, Department of Biomedical Sciences, Physiotherapy and Medicine, Faculty of Health Sciences, University of A Coruña, Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña

2 Tisular Bioengineering and Cell Therapy Unit (GBTTC-CHUAC), Rheumatology

[1] Blanco-García FJ. Aspectos básicos. Chap. I. In: Batlle-Gualda E, Benito Ruiz P, et al.,

[2] Bomer N et al. Translating genomics into mechanisms of disease: Osteoarthritis. Best

[3] Zhang W et al. Current research on pharmacologic and regenerative therapies for osteo-

[4] Lories RJ, Luyten FP. The bone-cartilage unit in osteoarthritis. Nature Reviews.

[5] Blanco-García FJ, Tornero-Molina J. Artrosis. Chap. 112. In: Farreras P, Rozman C.

[6] Kraus VB et al. Call for standardized definitions of osteoarthritis and risk stratification for clinical trials and clinical use. Osteoarthritis and Cartilage. 2015;**23**(8):1233-1241

[7] Allen KD, Golightly YM. Epidemiology of osteoarthritis: State of the evidence. Current

[8] Ross MH, Pawlina W. Histología. Texto y atlas en color con biología celular y molecular.

3 Centro de Investigación Biomédica En Red de Bioingeniería, Biomateriales y

editors. Manual SER de la artrosis. 1st Ed. Madrid: IM&C; 2002

Practice & Research. Clinical Rheumatology. 2015;**29**(6):683-691

Medicina Interna. 18th Ed. Barcelona: Elsevier, v.I; 2016.

Deputación da Coruña; Opocrin S.P.A. (Bruna Parma).

Francisco J. Blanco-García2,3\* and Silvia M. Díaz-Prado1,2,3

(CHUAC), Galician Health Service (SERGAS), A Coruña, Spain

group, INIBIC, CHUAC, SERGAS, A Coruña, Spain

arthritis. Bone Research. 2016;**4**:15040-15040

Opinion in Rheumatology. 2015;**27**(3):276-283

5th ed. Madrid: Editorial médica panamericana; 2007

Rheumatology. 2011;**7**(1):43-49

Nanomedicina (CIBER-BBN), A Coruña, Spain

\*Address all correspondence to: fblagar@sergas.es

**Author details**

**References**

**Figure 5.** Scheme representing different steps during the development of an *in vitro* cartilage repair model. These steps are on one hand (1) to harvest cartilage explants from the joint (hip), (2) make cartilage punches, and (3) generate the lesion with a driller. On the other hand, (4) to seed the cells on the scaffold and (5) introduce the construct inside the lesion. (6) Safranin O staining showing the final result of the repair model after culture in chondrogenic medium during 2 months.

## **4. Gene therapy**

Gene therapy involves the over-expression of the appropriate gene (anabolic factors, chondroinductor, or anti-inflammatory molecules) and cell type (chondrocytes or chondrogenic cells) for their use in cell therapy and tissue engineering.

Nowadays, no gene products have been approved for OA treatment and few clinical trials have been conducted. At present, only TGF-β gene therapy has been clinically investigated in USA and Korea [3].

## **5. Conclusions**

Although many studies of cell therapy and tissue engineering have shown clinical and functional improvement in joints, these treatments generate a fibrocartilaginous tissue that is different from hyaline articular cartilage. The ability to regenerate articular cartilage that resists the degeneration process still remains elusive.

## **Acknowledgements**

The authors would like to acknowledge CIBER-BBN; Rede Galega de Terapia Celular, Xunta de Galicia (R2014/050); Grupos con Potencial de Cremento, Xunta de Galicia (RTC-2016-5386-1); Unión Europea y Fondo Social Europeo; MINECO-FEDER (RTC-2016-5386-1); Fundación Española de Reumatología (2014 grant); Universidade da Coruña; Fundación Profesor Novoa Santos; Deputación da Coruña; Opocrin S.P.A. (Bruna Parma).

## **Author details**

María Piñeiro-Ramil1,2, Rocío Castro-Viñuelas1,2, Clara Sanjurjo-Rodríguez1,2,3, Tamara Hermida-Gómez2,3, Isaac Fuentes-Boquete1,2,3, Francisco J. de Toro-Santos1,3, Francisco J. Blanco-García2,3\* and Silvia M. Díaz-Prado1,2,3

\*Address all correspondence to: fblagar@sergas.es

1 Cell Therapy and Regenerative Medicine Group, Department of Biomedical Sciences, Physiotherapy and Medicine, Faculty of Health Sciences, University of A Coruña, Institute of Biomedical Research of A Coruña (INIBIC), University Hospital Complex A Coruña (CHUAC), Galician Health Service (SERGAS), A Coruña, Spain

2 Tisular Bioengineering and Cell Therapy Unit (GBTTC-CHUAC), Rheumatology group, INIBIC, CHUAC, SERGAS, A Coruña, Spain

3 Centro de Investigación Biomédica En Red de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), A Coruña, Spain

## **References**

**4. Gene therapy**

68 Cartilage Repair and Regeneration

2 months.

USA and Korea [3].

**5. Conclusions**

**Acknowledgements**

Gene therapy involves the over-expression of the appropriate gene (anabolic factors, chondroinductor, or anti-inflammatory molecules) and cell type (chondrocytes or chondrogenic

**Figure 5.** Scheme representing different steps during the development of an *in vitro* cartilage repair model. These steps are on one hand (1) to harvest cartilage explants from the joint (hip), (2) make cartilage punches, and (3) generate the lesion with a driller. On the other hand, (4) to seed the cells on the scaffold and (5) introduce the construct inside the lesion. (6) Safranin O staining showing the final result of the repair model after culture in chondrogenic medium during

Nowadays, no gene products have been approved for OA treatment and few clinical trials have been conducted. At present, only TGF-β gene therapy has been clinically investigated in

Although many studies of cell therapy and tissue engineering have shown clinical and functional improvement in joints, these treatments generate a fibrocartilaginous tissue that is different from hyaline articular cartilage. The ability to regenerate articular cartilage that resists

The authors would like to acknowledge CIBER-BBN; Rede Galega de Terapia Celular, Xunta de Galicia (R2014/050); Grupos con Potencial de Cremento, Xunta de Galicia (RTC-2016-5386-1); Unión Europea y Fondo Social Europeo; MINECO-FEDER (RTC-2016-5386-1); Fundación Española

cells) for their use in cell therapy and tissue engineering.

the degeneration process still remains elusive.


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[31] Demoor M et al. Cartilage tissue engineering: Molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction. Biochimica et Biophysica Acta-

[32] Rackwitz L et al. Functional cartilage repair capacity of dedifferentiated, chondrocyteand mesenchymal stem cell-laden hydrogels in vitro. Osteoarthritis and Cartilage.

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74 Cartilage Repair and Regeneration


**Section 2**

**Orthopedics**

**Section 2**
