**5. Animal models of cartilage for arthritis research**

**Figure 2.** Biomimetic skeletal muscle constructs can be engineered from biological scaffolds. Example model of the typical macroscopic contraction of a type-1 collagen matrix, following 14 days in culture. Note the custom made an‐ chors at either end, which provide predictable lines of longitudinal tension (illustrated by dashed arrows) to promote

A further advantage of using naturally derived polymers is the ability to easily stimulate, both mechanically and electrically, due to the innate mechanical compliance of the polymerised tissue. To this end, numerous investigations have employed such techniques to promote myogenesis and maturation of seeded MPCs [65, 68, 69]. In contrast, the effect of a combined mechanical stimulation protocol, contributed to a reduction in maturation of 3-D seeded myogenic cells compared to monolayer controls [66]. Nevertheless, the effect of increased mechanical signals for the promotion of MPC alignment and fusion, satisfies the desire for a

Skeletal muscle *in vivo* is comprised of myogenic progenitors, non-myogeninc progenitors and fibroblasts. The presence and influence of these non-myogenic populations has not been widely investigated, however, there is evidence to suggest a potential synergistic role in the development and maturation of skeletal muscle *in vitro*. The non-myogenic fibroblast popu‐ lation of cells has been shown to enhance skeletal muscle cell alignment, through contact dependent and independent mechanisms [70]. Data from 3-D skeletal muscle constructs also demonstrates an increased myogenin mRNA expression (marker of terminal differentiation) and increased peak force in a heterogeneous mixture of myogenic and non-myogenic cells, compared to myogenic only cultures [43]. Furthermore the expression of MMP-2 mRNA was greater in the heterogeneous cell population cultures, suggestive of greater matrix remodelling and fusion potential [43]. Together, these data illustrate the requirement of the non-myogenic cell population to recapitulate the *in vivo* skeletal muscle cell niche and associated biological processes. As such the incorporation of a heterogeneous population of cells should be utilised

Consideration of the body of data discussed above, indicates that an argument can be made that the ideal solution for a tissue engineered skeletal muscle construct would embrace the

cell alignment and fusion in a single plane. Scale bar = 10 mm.

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when developing skeletal muscle models *in vitro*.

**1.** Appropriate matrix signals.

**3.** Presence of other cell types.

**2.** Induction of alignment.

**4.** Mechanical stimulation.

biomimetic.

following:

Experimental models of degenerative joint diseases such as osteoarthritis (OA) and rheuma‐ toid arthritis (RA) have been in existence for several decades [72]. Animal models of arthritis have been used to understand elements of the arthritic disease process in human patients [73]. They are powerful tools for studying pathologic changes in articular cartilage and bone in great detail, and can be used to evaluate mechanisms of erosive processes [73]. Animal models of arthritis are also used to evaluate potential anti-arthritic drugs for clinical use in human patients [74-76]. The capacity for predicting efficacy in human disease is one of the most important criteria in the selection of animal models [74]. The use of animals has been indis‐ pensable to the investigation of the aetiology, pathophysiology, and treatment of various forms of juvenile arthritis [77]. Animal models of rheumatoid arthritis (RA) are also well established and have a proven track record of predictability [74, 78]. These include rat adjuvant arthritis [79], rat and mouse type II collagen arthritis [80-82], and antigen-induced arthritis in several species. Many animal species are currently used in OA research [83]. Figure 3 summarises the main procedures and disadvantages of animal models of OA.

**Figure 3.** Procedures and major disadvantages of animal models of arthritis.

The animal models of OA include laboratory animals: mice, rats, guinea pigs, rabbits; farm animals: sheep, goats; and companion animals: dogs, cats, horses. Animal models of OA roughly fall into five categories; firstly, spontaneous OA, which naturally occurs in the knee joints of animals, such as guinea pigs and dogs, and has a similar pathogenesis to human OA [84, 85]. Secondly, the surgical creation of joint instability, for example anterior cruciate ligament transection (ACLT) in dogs [86], meniscal tear model in rats [87], and collateral ligament transection in horses [88]. Thirdly, the surgical replication of joint trauma, for example the canine groove model [89], and carpal chip fragmentation in horses [90]. Fourthly, injection into the joint, for example papain [91], sodium mono-iodoacetate [92] and collagenase [93]. The final category is the knockout model, which deletes certain genes in mice resulting in the development of OA-like degenerative joint disease. For example the deletion of genes that code for type IX collagen [94], or the double deletion of biglycan and fibromodulin [95].

#### **6. Biomimetic models of cartilage**

Biomimetic models of articular cartilage were developed specifically for use in preclinical and clinical research long before the advent of tissue engineering and regenerative medicine, and the realisation that the development of these models is an elementary form of tissue engineer‐ ing. One of the major advantages of articular cartilage is the fact that it is a relatively simple tissue consisting of a fairly homogeneous extracellular matrix and a single cell type. Also, cartilage is avascular, aneural and alymphatic [96, 97], a fact that was disputed for centuries until modern histological techniques were applied to study the tissue [97]. This unique property overcomes many of the obstacles that are involved in culturing other vascularised and innervated tissues. The following sections will discuss the most popular 2- and 3 dimensional models of cartilage.

#### **6.1. 2-Dimensional monolayer models of chondrocyes**

pensable to the investigation of the aetiology, pathophysiology, and treatment of various forms of juvenile arthritis [77]. Animal models of rheumatoid arthritis (RA) are also well established and have a proven track record of predictability [74, 78]. These include rat adjuvant arthritis [79], rat and mouse type II collagen arthritis [80-82], and antigen-induced arthritis in several species. Many animal species are currently used in OA research [83]. Figure 3 summarises the

The animal models of OA include laboratory animals: mice, rats, guinea pigs, rabbits; farm animals: sheep, goats; and companion animals: dogs, cats, horses. Animal models of OA roughly fall into five categories; firstly, spontaneous OA, which naturally occurs in the knee joints of animals, such as guinea pigs and dogs, and has a similar pathogenesis to human OA [84, 85]. Secondly, the surgical creation of joint instability, for example anterior cruciate ligament transection (ACLT) in dogs [86], meniscal tear model in rats [87], and collateral ligament transection in horses [88]. Thirdly, the surgical replication of joint trauma, for example the canine groove model [89], and carpal chip fragmentation in horses [90]. Fourthly, injection into the joint, for example papain [91], sodium mono-iodoacetate [92] and collagenase [93]. The final category is the knockout model, which deletes certain genes in mice resulting in the development of OA-like degenerative joint disease. For example the deletion of genes that code for type IX collagen [94], or the double deletion of biglycan and fibromodulin [95].

Biomimetic models of articular cartilage were developed specifically for use in preclinical and clinical research long before the advent of tissue engineering and regenerative medicine, and

main procedures and disadvantages of animal models of OA.

518 Regenerative Medicine and Tissue Engineering

**Figure 3.** Procedures and major disadvantages of animal models of arthritis.

**6. Biomimetic models of cartilage**

Two-dimensional *in vitro* models of chondrogenesis [98, 99] and methods for isolating and cultivating primary bovine chondrocytes were established in the early 1960's [100]. Methods have been published for human [101] and rabbit [102] cartilage. Primary chondrocytes [103-105] and SV-40 transformed chondrocyte-like cell lines [106] in monolayer culture are routinely used for basic research [107]. These models have been useful for studying the cell biology of cartilage and responses to drugs, pro-inflammatory cytokines, growth factors, nutrients and nutraceuticals. Chondrocytes are also able to survive freezing and cryopreserved cells retain the capacity to proliferate and synthesise ECM [108-111]. Although 2-D culture of chondrocytes is widely used (see Figure 4), this method suffers from several major weaknesses: 1). The cells de-differentiate to fibroblast-like cells after 4-5 passages; 2] They do not synthesise a genuine ECM. Early studies carried out in the 1960's showed that high-density culture preserves the chondrocyte phenotype; when cultured chondrocytes attain a certain cell density they re-acquire their rounded shape, stop dividing, and re-synthesize collagens and proteo‐ glycans. Clusters of chondrocytes synthesize more proteoglycans than isolated chondrocytes [112]. If chondrocytes are actively engaged in ECM synthesis they do not concurrently synthesize DNA and so cannot divide and proliferate [112]. Interaction between chondrocytes is important for maintaining ECM synthesis and failure of interaction between chondrocytes leads to cell proliferation, a fact that has been exploited for expanding small numbers of isolated chondrocytes from limited amounts of clinical material.

Although chondrocyte survival and phenotype are regulated by culture conditions, the expression of the chondrogenic transcription factor Sox9 is of crucial importance [113]. The unique phenotype of the chondrocyte requires sustained expression of Sox9. This transcription factor plays an important role in the normal skeletal development and regulate the expression of other genes involved in chondrogenesis [114, 115].

#### **6.2. 3-Dimensional cartilage culture systems — Alginate beads**

Alginic acid, also called algin or alginate, is an anionic polysaccharide that is widely distributed in the cell walls of brown algae. When extracted in granular or powdered forms, alginate is capable of absorbing 200-300 times its own weight in water. Due to its biocompatibility and simple gelation with divalent cations such as Ca2+, alginate is widely used for cell immobili‐ zation and encapsulation. Therefore, alginate beads offer an ideal substrate for developing support matrices for 3-dimensional chondrocyte culture (see Figure 5). Immobilization of cells

**Figure 4.** Dimensional cultures of primary chondrocytes. This culture system is widely used for basic research on chon‐ drocyte biology and for drug toxicity testing.

along with macromolecules and biomaterials in alginate gels has become a well-established technology. Alginate beads are used in many biomedical and industrial applications. Cells immobilized in alginate gels maintain their differentiated phenotype during long-term culture due to the 3-dimensional environment of the gel network. In tissue engineering applications immobilized cells or tissue explants can be used as bioartificial organs as the alginate gel may function as a protective barrier towards physical stress and to avoid immunological reactions with the host. Chondrocytes can be encapsulated and maintained in calcium alginate beads or gels in 3-dimensional culture [116]. Avian and mammalian chondrocytes cultured in "semisolid" and "hollow" alginate beads exhibit a spherical shape as opposed to the fibroblastic morphology that is observed in monolayer culture [116]. The encapsulation methodology is suitable for the culture of chondrocytes in single beads, in multiwell dishes, or mass culture. Human and bovine adult articular chondrocytes have also been cultured in alginate beads and studies have shown that they retain their spherical shape and typical chondrocyte-like appearance for at least 5 weeks [117]. Alginate culture has also been used for cultivating intervertebral disc cells [118], nucleus pulposus and annulus fibrosus cells [119] and chondro‐ cyte cell lines [120]. Aggrecan appears to be a major ECM molecule produced by alginate cultured chondrocytes. Sensitive assays have been developed for the quantification of glycosaminoglycans (GAGs) and nitric oxide (NO) produced by alginate cultures [121, 122]. Decorin is also synthesized in small amounts but it is rapidly lost from the agarose or alginate gel [117]. Alginate culture can be used to induce the re-expression of cartilage-specific genes (aggrecan and collagen II) by dedifferentiated human articular chondrocytes cultured in alginate beads. However, alginate is unable to restore the chondrocyte phenotype in SV-40 transformed cells [123]. Thus, articular chondrocytes embedded in alginate gel can produce *de novo* a matrix rich in collagens and proteoglycans [124]. The alginate culture system appears to represent a relevant model for maintaining primary populations of chondrocytes and inducing the redifferentiation of dedifferentiated human chondrocytes, especially when they have been expanded several times by passaging in monolayer culture. This approach is particularly useful when small cartilage biopsies are available. New approaches to cartilage tissue-engineering have combined isolated cells with polymer scaffolds (polyglycolic acid fibre meshes and alginate gels) for the purpose of generating new cartilage (neo-cartilage) [125].

**Figure 5.** Chondrocyte culture in alginate beads.

along with macromolecules and biomaterials in alginate gels has become a well-established technology. Alginate beads are used in many biomedical and industrial applications. Cells immobilized in alginate gels maintain their differentiated phenotype during long-term culture due to the 3-dimensional environment of the gel network. In tissue engineering applications immobilized cells or tissue explants can be used as bioartificial organs as the alginate gel may function as a protective barrier towards physical stress and to avoid immunological reactions with the host. Chondrocytes can be encapsulated and maintained in calcium alginate beads or gels in 3-dimensional culture [116]. Avian and mammalian chondrocytes cultured in "semisolid" and "hollow" alginate beads exhibit a spherical shape as opposed to the fibroblastic morphology that is observed in monolayer culture [116]. The encapsulation methodology is suitable for the culture of chondrocytes in single beads, in multiwell dishes, or mass culture. Human and bovine adult articular chondrocytes have also been cultured in alginate beads and studies have shown that they retain their spherical shape and typical chondrocyte-like appearance for at least 5 weeks [117]. Alginate culture has also been used for cultivating intervertebral disc cells [118], nucleus pulposus and annulus fibrosus cells [119] and chondro‐ cyte cell lines [120]. Aggrecan appears to be a major ECM molecule produced by alginate cultured chondrocytes. Sensitive assays have been developed for the quantification of glycosaminoglycans (GAGs) and nitric oxide (NO) produced by alginate cultures [121, 122]. Decorin is also synthesized in small amounts but it is rapidly lost from the agarose or alginate gel [117]. Alginate culture can be used to induce the re-expression of cartilage-specific genes (aggrecan and collagen II) by dedifferentiated human articular chondrocytes cultured in

**Figure 4.** Dimensional cultures of primary chondrocytes. This culture system is widely used for basic research on chon‐

drocyte biology and for drug toxicity testing.

520 Regenerative Medicine and Tissue Engineering

#### **6.3. 3–Dimensional cartilage culture systems — Agarose gels**

Agar is a gelatinous substance derived from algae. It is a mixture of two components: the linear polysaccharide agarose, and a heterogeneous mixture of smaller molecules called agaropectin. Agar and agarose have been used extensively for cell culture. Agar gels are have been used throughout the world to provide a solid surface containing medium for the growth of bacteria and fungi. As a gel, an agarose medium is porous and therefore can be used to re-create the 3 dimensional environment that chondrocytes are accustomed to in articular cartilage. Culturing chondrocytes in agarose gels is actually very similar to alginate beads. Agarose gels proved to be particularly useful for studies on proteoglycans produced by avian [126], porcine [127] and bovine [128] chondrocytes. Agarose gels re-create a biomimetic 3-dimensional environment and stimulate ECM production by chondrocytes [129]. One of the most important studies on chondrocyte differentiation and redifferentiation was done using agarose gels by Benya and Shaffer [130]. As described earlier serial monolayer culture results in chondrocyte dedifferen‐ tiation and loss of phenotype. When chondrocytes dedifferentiate in monolayer culture they stop producing proteoglycans and type II collagen and instead secrete a different ECM consisting predominately of type I collagen and a low level of proteoglycan synthesis. Benya and Shaffer used agarose gels to demonstrate that dedifferentiated chondrocytes re-express the differentiated phenotype, producing proteoglycans and cartilage specific collagens [130]. The same outcome was achieved years later using alginate beads [131]. The original work of Benya and Shaffer demonstrated that a complete return to the differentiated collagen and proteoglycan producing chondrocyte phenotype is possible in agarose gels. Their results also emphasized the essential role of the spherical cell shape in the modulation of the chondrocyte phenotype and demonstrate a reversible system for the study of gene expression [130].

It is important to bear in mind that articular cartilage is subjected to dynamic compressive loading during normal activity and this influences chondrocyte metabolism. Mechanical forces are key determinants of connective tissue differentiation. Agarose gels, alginate beads and other 3-D gel systems are well established for studying the effects of dynamic compression on chondrocytes [132, 133]. These techniques have gained significant popularity over the last two decades [134] and are still in use today to study patterns of gene expression in response to dynamic compression [135] and chondrocyte mechanotransduction pathways [136]. The preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in agarose and alginate gels are promising methods for creating and engineering tissue implants for cartilage repair. These techniques can also be used to create new cartilage tissue from the joints of food producing animals (i.e. cattle, sheep, pigs) without having to sacrifice many smaller laboratory animals. This is an important area of cartilage tissue engineering with important consequences for animal research.

#### **6.4. 3-Dimensional cartilage explant culture**

Explant culture is a technique used for the isolation of cells from a piece of tissue. Tissue harvested in this manner is called an explant. The tissue is harvested under sterile conditions and explants are placed in a cell culture dish containing growth media. In some explant cultures (i.e. skeletal muscle) progenitor cells migrate out of the tissue and grow on the surface of the dish. These primary cells can then be further expanded and exploited. In cartilage explant culture cells remain in their surrounding extracellular matrix and this accurately mimics the *in vivo* environment (see Figure 6). The method became established in the mid 1970's [137, 138] and was used to study the effects of prostaglandins [139-141] and rheumatoid synovium [142] on cartilage degeneration. The establishment of cartilage explant culture later became an important prelude to the development of monolayer culture systems of chondrocytes [143].

**Figure 6.** Overview of cartilage explant culture and its unique advantages.

and fungi. As a gel, an agarose medium is porous and therefore can be used to re-create the 3 dimensional environment that chondrocytes are accustomed to in articular cartilage. Culturing chondrocytes in agarose gels is actually very similar to alginate beads. Agarose gels proved to be particularly useful for studies on proteoglycans produced by avian [126], porcine [127] and bovine [128] chondrocytes. Agarose gels re-create a biomimetic 3-dimensional environment and stimulate ECM production by chondrocytes [129]. One of the most important studies on chondrocyte differentiation and redifferentiation was done using agarose gels by Benya and Shaffer [130]. As described earlier serial monolayer culture results in chondrocyte dedifferen‐ tiation and loss of phenotype. When chondrocytes dedifferentiate in monolayer culture they stop producing proteoglycans and type II collagen and instead secrete a different ECM consisting predominately of type I collagen and a low level of proteoglycan synthesis. Benya and Shaffer used agarose gels to demonstrate that dedifferentiated chondrocytes re-express the differentiated phenotype, producing proteoglycans and cartilage specific collagens [130]. The same outcome was achieved years later using alginate beads [131]. The original work of Benya and Shaffer demonstrated that a complete return to the differentiated collagen and proteoglycan producing chondrocyte phenotype is possible in agarose gels. Their results also emphasized the essential role of the spherical cell shape in the modulation of the chondrocyte phenotype and demonstrate a reversible system for the study of gene expression [130].

It is important to bear in mind that articular cartilage is subjected to dynamic compressive loading during normal activity and this influences chondrocyte metabolism. Mechanical forces are key determinants of connective tissue differentiation. Agarose gels, alginate beads and other 3-D gel systems are well established for studying the effects of dynamic compression on chondrocytes [132, 133]. These techniques have gained significant popularity over the last two decades [134] and are still in use today to study patterns of gene expression in response to dynamic compression [135] and chondrocyte mechanotransduction pathways [136]. The preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in agarose and alginate gels are promising methods for creating and engineering tissue implants for cartilage repair. These techniques can also be used to create new cartilage tissue from the joints of food producing animals (i.e. cattle, sheep, pigs) without having to sacrifice many smaller laboratory animals. This is an important area of cartilage tissue

Explant culture is a technique used for the isolation of cells from a piece of tissue. Tissue harvested in this manner is called an explant. The tissue is harvested under sterile conditions and explants are placed in a cell culture dish containing growth media. In some explant cultures (i.e. skeletal muscle) progenitor cells migrate out of the tissue and grow on the surface of the dish. These primary cells can then be further expanded and exploited. In cartilage explant culture cells remain in their surrounding extracellular matrix and this accurately mimics the *in vivo* environment (see Figure 6). The method became established in the mid 1970's [137, 138] and was used to study the effects of prostaglandins [139-141] and rheumatoid synovium [142] on cartilage degeneration. The establishment of cartilage explant culture later became an important prelude to the development of monolayer culture systems of chondrocytes [143].

engineering with important consequences for animal research.

**6.4. 3-Dimensional cartilage explant culture**

522 Regenerative Medicine and Tissue Engineering

#### *6.4.1. 3-Dimensional High-Density and Pellet Cultures of Chondrocytes*

High-density culture is a 3-dimensional system that has been in the literature since the early 1970's. High-density suspension cultures of chondrocytes were initially used to study cartilage matrix protein synthesis by mammalian chondrocytes [144, 145], specifically mucoprotein [146] and proteoglycan [147] biosynthesis. In an important study published in 1977 the method was refined by von der Mark and von der Mark who used tissue culture plastic dishes on an agar base to monitor chondrogenesis of stage-24 chick limb mesodermal cells *in vitro* by analyzing the onset of type II collagen synthesis [148]. This work demonstrated that highdensity cultures may be used for the analysis of cell specific types of collagen and provide a useful model for detailing the specific events in the differentiation of mesenchymal cells *in vitro*. Several subsequent studies used chick limb bud mesenchymal cells and chick sternal chondrocytes cultured in high-density to study chondrogenesis and proteoglycan synthesis [149] and fibronectin expression [150]. We have successfully used this model as a model for chondrogenic, osteogenic and tenogenic differentiation of mesenchymal stem cells and redifferentiation of dedifferentiated chondrocytes [151-154].

The high-density model exhibits a number of characteristics that make it particularly suitable for studies on chondrogenesis. In the first 24 hours of the high-density chondrocytes culture, cells form prechondrogenic areas composed of densely packed cells with intercellular inter‐ actions (gap junctions); these are surrounded by a perichondrium of flat fibroblast-like cells [155] resembling the situation during the early stage of chondrogenesis *in vivo*. The extensive cell-cell interactions during the first step of chondrogenesis are crucial for cell aggregation [156, 157]. The high-density culture system allows investigations during chondrogenesis starting from early blastema/mesenchymal stem cell condensations until cartilage maturation. This chondrogenic development is accompanied by enlargement of cartilage nodules, which is not due to cell proliferation but rather is due to appositional growth, which includes the transition of perichondral cells into chondrocytes. These conclusions are based on the almost complete absence of mitotic figures in cartilage and perichondrium and on the continuous increase in nodule size during cultivation [156].

#### *6.4.2. Co–cultures of chondrocytes and synoviocytes*

The synovial joint contains several important tissue components. These are articular cartilage, synovium, subchondral bone and fat pads (adipose tissue). Developing *in vitro* models that include all of these tissues is a major challenge. Co-culture models involve growing distinct cell types in a combined culture environment. The mixing of different cell types in culture is normally avoided, although it is becoming increasingly apparent that this approach can be used to model the cellular interactions that occur *in vivo*. Some studies of cellular interactions may actually require direct cell-cell contact in a 2-dimensional model where two different cell types are grown in mixture. Other studies may require proximity between the two cell types without any actual cell-cell contact. In this scenario keeping the two types apart can allow investigators to study the effect of one cell type on another. In a synoviocyte-chondrocyte coculture model synoviocytes grown on a filter insert may be stimulated with pro-inflammatory cytokines or other inflammatory mediators (i.e. phorbol esters such as phorbol myristate acetate (PMA), reactive oxygen species such as hydrogen peroxide or a combination of iron and ascorbic acid to simulate inflammation-like radical attacks) before bringing the "activated" cells in contact with chondrocytes. The use of cell culture inserts controls the physical contact and also the duration of that contact. It is also possible to grow different cell types on either side of the membrane, allowing a simulation of tissue behaviour to be built and studied. A coculture model of rat chondrocytes and a rabbit synoviocyte-like cell line (HIG-82, [158]), has been used to show that chondrocytes establish protective mechanisms against reactive oxygen species by interacting with synoviocytes [159]. This co-culture system presents a model to study mechanisms of inflammation in articular joints under well-defined conditions [159]. With the financial support of the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) we have established a novel co-culture model of primary equine synoviocytes and primary equine chondrocytes for using equine joints derived from the abattoir9 . We have not fully exploited this equine model system. However, since chondrocytes and synoviocytes are the main cell types present in articular joints, such coculture models have been used for metabolic studies [160] and have the capacity to recapitulate the inflammatory and catabolic events that occur in an arthritic joint.
