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Keywords:

  • Chondrogenesis;
  • Chondrocytes;
  • Stem cells;
  • Differentiation;
  • In vitro

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adult Stem Cells versus Embryonic Stem Cells for Cartilage Repair and Tissue Engineering
  5. Development of Defined Culture Milieu for Directing the Chondrogenic Differentiation of Stem Cells In Vitro
  6. Coculture and Cell-Conditioned Media
  7. Directing Chondrogenic Differentiation through Genetic Modulation
  8. Future Perspectives
  9. Concluding Remarks
  10. References

A major area in regenerative medicine is the application of stem cells in cartilage tissue engineering and reconstructive surgery. This requires well-defined and efficient protocols for directing the differentiation of stem cells into the chondrogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying chondrogenesis and cartilaginous tissue biology. The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for cartilage-related biomaterials and drugs could also utilize protocols developed for the chondrogenic differentiation of stem cells. Hence, this review critically examines the various strategies that could be used to direct the differentiation of stem cells into the chondrogenic lineage in vitro.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adult Stem Cells versus Embryonic Stem Cells for Cartilage Repair and Tissue Engineering
  5. Development of Defined Culture Milieu for Directing the Chondrogenic Differentiation of Stem Cells In Vitro
  6. Coculture and Cell-Conditioned Media
  7. Directing Chondrogenic Differentiation through Genetic Modulation
  8. Future Perspectives
  9. Concluding Remarks
  10. References

Traumatic injury and age-related degenerative diseases associated with cartilage are major health problems worldwide [1, 2]. Despite recent advances in surgical and nonsurgical interventions, the treatment of cartilage lesions remains an intractable problem [3, 4]. This is attributable in large part to the intrinsic biology of cartilaginous tissue, which limits its capacity to self-regenerate. Because cartilage is nonvascularized and noninnervated, the normal mechanism of tissue repair involving humoral factors and recruitment of stem/progenitor cells to the site of damage does not apply [5]. Moreover, the low cell density [6] within cartilaginous tissue reduces the likelihood of local chondrocytes contributing to self-regeneration [7, 8].

Cell transplantation therapy has shown much promise in augmenting the inadequate repair mechanisms within damaged cartilage [913]. At present, the only U.S. Food and Drug Administration–approved cell-based therapy (Carticel™ [Genzyme, Cambridge, MA]) for cartilage repair in the human clinical model uses mature autologous chondrocytes. However, the limited proliferative capacity of differentiated chondrocytes could pose a major problem in providing adequate cell numbers for transplantation therapy [14]. Furthermore, it was also reported that the proliferative potential of autologous chondrocytes decreases with patient age [14]. This would in turn pose a significant challenge in the treatment of age-related chondrodegenerative diseases (i.e., osteoarthritis). Hence, this review examines adult and embryonic stem (ES) cells as alternative sources of donor cells [15] for the treatment of cartilage lesions.

Stem cells can be distinguished from progenitor cells by their capacity for both self-renewal and multilineage differentiation, whereas progenitor cells are capable only of multi-lineage differentiation without self-renewal [16]. It is this capacity for self-renewal that makes stem cells particularly useful for transplantation medicine, because this in theory could provide an unlimited supply of donor material. Moreover, stem cells and their differentiated derivatives possess much higher proliferative and regenerative potential compared with mature differentiated somatic cells. This in turn is more likely to guarantee adequate regeneration and cell turnover at the transplantation site for an extended period of time, possibly a lifetime.

In attempting to use stem cells for cartilage repair [17], it is imperative to develop well-defined and efficient protocols for directing stem cell differentiation into the chondrogenic lineage in vitro. This is followed by selective purification and proliferation of the differentiated subpopulation of stem cells before transplantation within the recipient. This will reduce the likelihood of spontaneous differentiation of stem cells into multiple divergent lineages at the transplantation site [18] other than the chondrogenic lineage. In the case of ES cells, this is particularly important for avoiding teratoma formation [19] within the transplant recipient. The transplantation of stem cells that are well-differentiated into the chondrogenic lineage is also likely to result in higher engraftment efficiency and better integration within recipient cartilaginous tissues. Moreover, cartilage tissue engineering requires differentiated chondrogenic progenitors or chondrocytes, rather than undifferentiated stem cells, for seeding and attachment onto artificially synthesized matrices.

Besides human clinical therapy, the development of such protocols would also provide useful in vitro models for studying chondrogenesis and cartilage development. It is extremely difficult to elucidate the molecular mechanisms and signaling pathways that regulate chondrogenesis in vivo within live animal models. Efficient protocols for directing the chondrogenic differentiation of stem cells in vitro will therefore provide a model that is more amenable to molecular characterization and genetic manipulation. An added advantage is that such protocols may also facilitate the genetic manipulation of stem cells for the delivery of recombinant genes/proteins in cartilage regeneration therapy [20]. The development of pharmacokinetic and cytoxicity/genotoxicity screening tests for cartilage-related biomaterials/drugs may also utilize protocols for the chondrogenic differentiation of human stem cells in vitro. Compared with laboratory testing on animal cell/tissue cultures or live animals, such screening tests based directly on human-derived cells and tissues will be much more clinically relevant, timely, and accurate, as well as more cost-effective. Moreover, the ethical issues surrounding the use of live animals for pharmacokinetic and cytotoxicity/genotoxicity testing will be circumvented through the development of such screening tests.

Hence, the purpose of this review is to critically examine the various strategies that could be used to direct the differentiation of stem cells into the chondrogenic lineage in vitro.

Adult Stem Cells versus Embryonic Stem Cells for Cartilage Repair and Tissue Engineering

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adult Stem Cells versus Embryonic Stem Cells for Cartilage Repair and Tissue Engineering
  5. Development of Defined Culture Milieu for Directing the Chondrogenic Differentiation of Stem Cells In Vitro
  6. Coculture and Cell-Conditioned Media
  7. Directing Chondrogenic Differentiation through Genetic Modulation
  8. Future Perspectives
  9. Concluding Remarks
  10. References

Stem cells for cartilage repair can be derived from two major sources: ES cells derived from the inner cell mass of blasto-cysts-stage embryos [21, 22] and mesenchymal stem cells (MSCs) derived from the bone marrow [17, 23]. Additionally, adult stem cells derived from other tissue types, such as the endothelial [24, 25], epithelial [26], adipogenic [2729], myogenic [30], and hematopoietic [31] lineages, have also been reported to be capable of transdifferentiating into the chondrogenic lineage. However, at this point in time, adult stem cell plasticity and transdifferentiation are highly controversial issues [32] due to evidence of cell fusion and heterokaryon formation [33, 34]. In any case, evidence from the scientific literature suggests that adult stem cell plasticity and transdifferentiation are relatively rare and sporadic events [35]. Hence, this review will focus only on ES cells [3639] and bone marrow–derived MSCs [17, 40, 41] for chondrogenic differentiation.

The most obvious advantage of using ES cells instead of MSCs for cartilage regeneration is that ES cells are immortal and could potentially provide an unlimited supply of differentiated chondrocytes and chondroprogenitor cells for transplantation. In contrast, the self-renewal and proliferative capacity of MSCs is very much limited and seems to decrease with age [4244]. This would obviously limit their usefulness in autologous cell transplantation therapy for the treatment of age-related degenerative diseases of cartilage such as osteoarthritis. The putative MSCs from bone marrow is in fact a highly heterogenous population, with only a limited proportion of cells being capable of differentiation into the chondrogenic lineage. If the capacity for continuous self-renewal is taken strictly as the defining criteria for stem cells, then an extremely low proportion (perhaps approximately 1 in 100,000) of bone marrow stromal cells can be considered true stem cells [44, 45]. This would pose a major challenge in the development of isolation and purification protocols. Additionally, MSCs may also contain more genetic abnormalities than ES cells, caused by exposure to metabolic toxins and errors in DNA replication accumulated during the course of a lifetime [46].

In attempting to use ES cells for cartilage repair, the major challenge is to overcome immunological rejection from the transplant recipient unless an isogenic source of ES cells is derived from therapeutic cloning. Although this has recently been achieved in the human model [47], this is still a remote option, given the technical difficulties of such an approach [48]. A more viable alternative is to create a bank of ES cells with different major histocompatibilty complex (MHC) genotypes for matching with the transplant recipient. It could also be possible to downregulate the antigenicity of ES cells through suppression of MHC gene expression. In contrast, autologous MSCs derived from the patient's bone marrow would not face any immunological barriers in transplantation. Another major problem is the risk of teratoma formation by ES cells upon transplantation in situ [19], which is not the case for MSCs. Moreover, it is much easier to direct MSCs into the chondrogenic lineage compared with ES cells. At present, all reported studies on the chondrogenic differentiation of ES cells were based solely on animal models [3639], with no successful differentiation yet being accomplished with human-derived ES cells.

Development of Defined Culture Milieu for Directing the Chondrogenic Differentiation of Stem Cells In Vitro

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adult Stem Cells versus Embryonic Stem Cells for Cartilage Repair and Tissue Engineering
  5. Development of Defined Culture Milieu for Directing the Chondrogenic Differentiation of Stem Cells In Vitro
  6. Coculture and Cell-Conditioned Media
  7. Directing Chondrogenic Differentiation through Genetic Modulation
  8. Future Perspectives
  9. Concluding Remarks
  10. References

Advantages of Defined Culture Milieu

For clinical applications of stem cell transplantation therapy, it is imperative that in vitro culture protocols should be devoid of animal or human products to avoid potential contamination with pathogens. The avoidance of products of animal or human origin would also reduce variability within the culture milieu and provide a more stringent level of quality control. Moreover, supplemented animal or human proteins may adhere onto the surface of cultured stem cells, which could possibly enhance their antigenicity upon transplantation. Hence, the ideal culture milieu for promoting the chondrogenic differentiation of stem cells in vitro should be chemically defined and either be serum-free or use synthetic serum replacements [49, 50], with the possible supplementation of specific recombinant cytokines and growth factors (Table 1) if so required.

Table Table 1.. Cytokines and growth factors that promote chondrogenesis
  1. a

    Abbreviation: TGF, transforming growth factor.

Cytokines/growth factorsKey references
Various isoforms of TGF-βIwasaki et al. [59],
 Chimal-Monroy et al. [60], Awad et al. [61], Tuli et al. [41]
Various isoforms of bone morphogenetic proteinChen et al. [62],
 Kramer et al. [36], Shea et al. [57]
ActivinJiang et al. [63]
Osteogenic protein-1Asahina et al. [64]
GDF-5Spiro et al. [65]
Fibroblast growth factor-2Quarto et al. [45], Martin et al. [223]
Insulin-like growth factor-1Fortier et al. [76], French et al. [77]
ProlactinOgueta et al. [82]
Interleukin-1βTonon et al. [83]
Cyr61Wong et al. [84]
HB-GAMDreyfus et al. [85]
Growth hormoneTsukazaki et al. [86]

Serum-Free Culture Conditions for Promoting Chondrogenic Differentiation

The major challenge in attempting to culture stem cells under serum-free conditions is that cells generally tend to have a lower mitotic index, become apoptotic, and display poor adhesion in the absence of serum [49]. The removal of serum has been reported to slow down the proliferation rate of MSCs [51, 52]. Nevertheless, the osteochondral potential of MSCs can still be maintained in a chemically defined serum-free medium [50, 53]. Presently, most studies with MSCs use serum-free in vitro culture conditions so as to avoid the arte-factual and pleiotropic effects of serum on the experimental data. However, for optimal ex vivo proliferation of MSCs, the presence of serum is still preferred [54]. With primary chondrocytes, the absence of serum has also been reported to be detrimental to the proliferation rate [55]. In the case of ES cells, serum-free in vitro culture condition for differentiation into the chondrogenic lineage was recently reported by Nakayama et al. [38].

To prevent transmission of pathogens, the patient's own serum could be used for the in vitro culture of stem cells. However, for clinical applications, there are several reasons that make it preferable to eliminate serum from the in vitro culture milieu. First and foremost, the composition of serum is poorly defined, with a considerable degree of interbatch variation, even when obtained from the same patient or manufacturer. This impedes good quality control in the laboratory. Serum is also not completely physiological, because it is essentially a pathological fluid formed in response to blood clotting. Additionally, it may also contain uncharacterized growth and differentiation factors, which may result in the uncontrolled spontaneous differentiation of stem cells into divergent multiple lineages other than the chondrogenic lineage.

A step toward serum-free culture conditions is the development of chemically defined synthetic serum substitutes. At present, there are several such commercially available synthetic serum substitutes [49, 50]. Most notable of these is knockout serum replacement (KSR), which was specifically developed for the maintenance of ES cells in an undifferentiated state within in vitro culture [50]. The exact chemical composition of KSR is not available, because it is a protected trade secret. However, it has been reported to be completely devoid of any undefined growth factors or differentiation-promoting factors [50]. This would be extremely useful for achieving controlled differentiation of stem cells into the chondrogenic lineage in vitro.

Use of Exogenous Cytokines and Growth Factors to Promote Chondrogenic Differentiation

The use of exogenous cytokines and growth factors is another step forward in the development of a defined culture milieu for directing the chondrogenic differentiation of stem cells. Indeed, numerous cytokines and growth factors have been implicated in chondrogenesis (Table 1), and many of these display a high degree of functional overlap. Because the process of chondrogenesis is so closely intertwined with osteogenesis, many of the cytokines and growth factors that promote chondrogenic differentiation are also somewhat implicated in osteogenic differentiation [56, 57]. Hence, the challenge is to find an optimized subtle combination of these various cytokines and growth factors that would bias differentiation specifically toward the chondrogenic lineage. An added complication is that many of these cytokines and growth factors exert nonspecific pleiotropic effects on stem cell differentiation, which is most likely attributable to the activation of multiple intracellular signaling pathways by each individual cytokine or growth factor.

Among the most potent inducers of chondrogenic differentiation are members of the transforming growth factor beta (TGF-β) family. These consist of more than 40 polypeptide growth factors that share a high degree of homology, in particular the seven conserved residues in their C-terminal region [58]. Besides the various isoforms of TGF-β [41, 5961] and bone morphogenetic protein (BMP) [36, 57, 62], other members of the TGF-β superfamily include activin [63], osteogenic protein-1 [64], and GDF-5 [65]. The effects of cytokines of the TGF-β superfamily on chondrogenic differentiation are transduced through two major intracellular signaling pathways. The first of these involves the SMAD family of signaling molecules [66], whereas the second pathway involves mitogen-activated protein kinase (MAPK) signaling [67]. Interestingly, both signaling cascades are activated by the same TGF-β receptor complex. This comprises two distinct transmembrane protein components (type I and II receptor) that undergo heterodimerization upon binding to cytokines of the TGF-β superfamily. More recently, there is also evidence of regulatory cross-talk between TGF-β–activated signaling cascades and the Wnt pathway during chondrogenic differentiation [41, 68]. An interesting aspect of the TGF-β superfamily is the modulation of its biological activity through binding interactions with extracellular matrix proteins such as β-glycan and endoglien [69, 70]. These are thought to alter the presentation of these cytokines to their corresponding receptors on the cell surface.

Another family of cytokines that plays an important role in chondrogenesis is comprised of the various isoforms of fibroblast growth factor (FGF). Numerous studies have implicated the FGF family of cytokines in limb bud cartilage formation [71]. The effects of FGF on chondrogenic differentiation are transduced primarily through MAPK signaling [72]. This leads to increased expression of the transcription factor Sox9, which is the master regulator of chondrogenesis. Additionally, there is also evidence of regulatory cross-talk between FGF-activated signaling cascades and the signaling pathways initiated by parathyroid hormone–related peptides and Indian hedgehog [73, 74].

Besides the TGF-β and FGF family of cytokines, insulin-like growth factor-1 (IGF-1) [7577] has also been shown to have potent stimulatory effects on chondrogenic differentiation. It is believed that the chondrogenic-promoting activity of IGF-1 is primarily transduced through a phosphatidyl inositol 3-kinase signaling pathway [78, 79]. Because the combination of TGF-β1 with either IGF-1 [80] or FGF-2 [81] was reported to have a synergistic effect on chondrogenic differentiation in vitro, it is likely that the IGF-1–activated signaling cascade could have regulatory cross-talk with the signaling pathways initiated by the TGF-β and FGF family of cytokines. Other proteins that have also been shown to have a stimulatory effect on chondrogenesis include prolactin [82], interleukin-1β [83], Cyr61 [84], HB-GAM [85], and growth hormone [86]. All of these are summarized in Table 1.

Use of Nonproteinaceous Chemical Compounds to Promote Chondrogenic Differentiation

In addition to protein-based cytokines and growth factors, several nonproteinaceous chemical compounds have also been shown to promote chondrogenic differentiation in vitro (Table 2). Such chemicals tend to be less labile, with a longer active half-life in solution, compared with protein-based cytokines and growth factors. This is advantageous for prolonged in vitro cell culture over several days or even weeks. Moreover, unlike proteins that have to be synthesized in living cells and subjected to complex post-translational modifications (i.e., glycosylation, peptide splicing, conformational folding), non–protein-based chemical compounds can be manufactured by chemical reactions in the laboratory and hence are more structurally and chemically defined compared with proteins.

Table Table 2.. Nonproteinaceous chemical factors that promote chondrogenesis
Chemical factorsKey references
Prostaglandin E2Miyamoto et al. [96], Biddulph et al. [95]
Thyroid hormoneSiebler et al. [90]
1,25-dihydroxy vitamin DTsonis [92], Harmand et al. [93]
Ascorbic acidFarquharson et al. [97]
DexamethasoneJohnstone et al. [87], Mackay et al. [88]
EthanolKulyk et al. [98], Hoffman et al. [99]
StaurosporineKulyk [100], Kulyk et al. [101]
Dibutryl cAMPRevillion-Carette et al. [102]
Concanavalin AMikhailov et al. [103]
VanadateKato et al. [104]
FK506Nishigaki et al. [105]

Among the nonproteinaceous chemical compounds that are known to promote chondrogenic differentiation in vitro (Table 2) are dexamethasone, thyroid hormone, 1,25-dihydroxy vitamin D3, prostaglandin E2, ascorbic acid, ethanol, staurosporine, dibutryl cAMP, concavalin A, vanadate, and FK506. Dexamethasone is a synthetic glucocorticoid that is a potent inducer of chondrogenic differentiation in human-derived mesenchymal stem cells [87, 88]. Thyroid hormones are steroid derivatives of cholesterol metabolism that have also been implicated in chondrogenic differentiation [89, 90]. In an interesting study by Locker et al. [91], it was reported that the sequential addition of dexamethasone and thyroid hormone (triiodothyronine) under serum-free conditions permitted full chondrogenic differentiation of the pluripotent mesoblastic C1 cell line. By contrast, the addition of exogenous cytokines (IGF-1 and TGF-β), as well as the cell-intrinsic activation of the BMP autocrine signaling pathway, failed to elicit full chondrogenic differentiation [91]. 1,25-dihydroxy vitamin D3, also known as calcitriol, is the active form of vitamin D. It has been reported to stimulate chondrogenesis of embryonic limb bud mesenchymal cells [92] and promote expression of differentiated chondrocyte function within in vitro culture [93]. Prostaglandin E2 (PE2) is a naturally occurring eicosanoid that is derived from arachidonic acid metabolism. Numerous studies have implicated PE2 in limb cartilage formation [94, 95] and chondrocyte differentiation [96]. Ascorbic acid, better known as vitamin C, has been shown to stimulate chondrogenic differentiation by promoting 1,25-dihydroxy vitamin D3 synthesis and cartilage matrix production [97]. The teratogenic effects of ethanol on vertebrate development are well-known. It has been demonstrated to have potent stimulatory effects on the chondrogenic differentiation of both embryonic limb [98] and embryonic facial [99] mesenchyme cells. Staurosporine, a protein kinase C inhibitor, has also been demonstrated to have stimulatory effects on the chondrogenic differentiation of both embryonic limb [100] and embryonic facial [101] mesenchyme cells. Dibutryl cAMP, the cell-permeable form of cAMP, was reported to have a stimulatory effect on embryonic limb bud cartilage formation [102]. Concanavalin A is a member of the lectin family of carbohydrate-based compounds. It has been reported to induce both neural and cartilage tissue formation in amphibian early gastrula ectoderm [103]. Vanadate, an inorganic vanadium salt, has been demonstrated to promote cartilage-matrix proteoglycan synthesis in rabbit coastal chondrocyte cultures [104]. FK506 is an immunosuppressive drug that has been shown to stimulate chondrogenic differentiation of a clonal murine embryonic carcinoma cell line, ATDC5 [105].

Use of Naturally Occurring and Artificially Synthesized Extracellular Matrix Substratum to Promote Chondrogenic Differentiation

The in vitro culture milieu for directing chondrogenic differentiation should also incorporate naturally occurring and artificially synthesized extracellular matrix (ECM) substratum to optimize cell attachment, growth, and differentiation. Indeed, the presence of ECM components would more closely replicate the physiological environment that supports chondrogenesis. In situ, cartilage tissue consists of chondrocytes embedded within an avascular ECM that serves to maintain its structural integrity. This is primarily comprised of collagenous proteins [106] and proteoglycans [107] that account for the bulk of its dry weight (60%–90%), together with smaller quantities of noncollagenous proteins [108, 109] and hyaluronan [110]. Within cartilaginous tissue, the major collagen isoform is collagen type II [111], whereas the predominant proteoglycan is aggrecan [112]. In addition to its structural role, cartilage ECM plays a physiological role by influencing the immediate microenvironment of the chondrocytes embedded within it. Histological studies have reported extensive remodeling of cartilage ECM during growth and development [113] as well as under various pathological conditions [114, 115].

Hence, the introduction of appropriate ECM substratum within in vitro culture would certainly enhance the directed differentiation of stem cells into the chondrogenic lineage [116]. These can either be based on naturally occurring components of cartilage ECM or may use synthetic materials. Additionally, composite matrix scaffolds of both natural and synthetic materials have also been fabricated.

It is important to note the various properties of ECM substratum that would be favorable for chondrogenesis. For tissue engineering applications, it is imperative that the synthesized matrix substratum is biocompatible and has no cytotoxic properties. Biodegradability is also another preferred characteristic [117], although this is not absolutely critical. Culture on 3D matrix scaffolds has been reported to be superior to conventional 2D monolayer culture for maintaining the differentiated phenotype of chondrocytes [118]. Porosity of the ECM substratum is essential for 3D tissue growth. Hence, matrix scaffolds for cartilage tissue engineering are often fabricated as porous foams or granules. At the present moment, there are as yet no reported studies on the use of ECM substratum to promote the chondrogenic differentiation of ES cells. Hence, the following discussion will focus solely on MSC and primary chondrocytes.

The attachment, proliferation, and subsequent chondrogenic differentiation of MSC and chondrocytes on the ECM substratum are dependent on several interrelated properties, namely chemical composition, electrostatic charge, surface texture/roughness, and geometrical configuration [119]. Obviously, the chemical composition of the ECM substratum is the most critical factor in determining its ability to influence chondrogenesis. These should preferably incorporate naturally occurring constituents of cartilage ECM, so as to provide a more physiological environment for chondrogenic differentiation. Indeed, matrix scaffolds fabricated for cartilage tissue engineering are often based on either collagen [120122], hyaluronan [123, 124], chondroitin sulfate [125, 126], mineralized calcium [127, 128], fibrin [129, 130], or composites of these materials [131, 132], all of which are constituents of cartilage ECM. Other naturally occurring materials that are not found in cartilage ECM have also been used. These include gelatin [133], chitosan [134], and alginate [135138]. Additionally, matrix scaffolds based completely on synthetic materials such as poly (lactic-co-glycolic acid), poly (ethylene glycol), poly (epsilon-caprolactone) have also been fabricated [139144]. Nevertheless, it would be preferable to use these as composites with naturally occurring materials. The electrostatic charge on the surface of the ECM substratum is another important factor. The presences of negative charges have been reported to be advantageous for the adhesion and spreading of MSCs on ECM substratum [145, 146]. Furthermore, there is evidence that variations in the electrostatic charge content of the peri-cellular matrix synthesized by chondrocytes have a profound influence on the biomechanical properties of cartilaginous tissue [147]. Surface texture/roughness seems to have differential effects on chondrocyte proliferation, differentiation, and matrix production, depending on the maturational state of the chondrocyte itself [148, 149]. Bhardwaj et al. [150] reported that geometric configuration of the matrix scaffold, as defined by average pore size, had profound effects on chondrocyte proliferation and matrix synthesis.

As mentioned earlier, collagen type II is the most abundant collagen isoform within cartilage ECM. Indeed, it has been reported that cartilage-specific collagen type II is superior to collagen type I for primary culture of chondrocytes [119, 120]. Although chondrocytes appeared to maintain high proliferative capacity when cultured in collagen type I, there was a gradual loss of differentiated phenotype [151]. To enhance their structural and chondroinductive properties, collagen-based matrices are often fabricated as composites that may incorporate a variety of naturally occurring and synthetic materials such as mineralized calcium phosphate [152], agarose [153], proteoglycan aggregates [154], demineralized bone powder [155], fibronectin [154], chondroitin sulfate [125, 126], hyaluronan [156], poly-L-lactic acid [157], and polylactide-coglycolid [158].

Another class of bone ECM molecules that is known to play an integral role in chondrogenic differentiation is the glycosaminoglycan (GAG), which is essentially a long-chain sugar molecule. With the exception of hyaluronan, all of GAGs (heparin sulfate, chondroitin sulfate, keratin sulfate, and dermatin sulfate) are sulfated branch-chained molecules that are conjugated to proteins in the form of proteoglycans. Chondroitin sulfate is the major sulfated glycosaminogly can found in cartilage (predominantly conjugated to aggrecan). Indeed, chondroitin sulfate–based matrix scaffolds have been reported to enhance chondrocyte proliferation and maturation [121, 131]. Additionally, hyaluronan-based matrix scaffolds are also widely utilized in cartilage tissue engineering [123, 124, 159, 160].

At present, the development of ECM substratum for application in cartilage tissue engineering is progressing rapidly. It is anticipated that more novel types of composite matrix scaffolds incorporating a variety of natural and synthetic materials will be developed in the near future for the controlled differentiation of MSC and even ES cells into the chondrogenic lineage.

Coculture and Cell-Conditioned Media

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adult Stem Cells versus Embryonic Stem Cells for Cartilage Repair and Tissue Engineering
  5. Development of Defined Culture Milieu for Directing the Chondrogenic Differentiation of Stem Cells In Vitro
  6. Coculture and Cell-Conditioned Media
  7. Directing Chondrogenic Differentiation through Genetic Modulation
  8. Future Perspectives
  9. Concluding Remarks
  10. References

Another strategy to direct the chondrogenic differentiation of stem cells is to coculture the stem cells with a different cell population. Differentiation of ES cells into the chondrogenic lineage was enhanced by coculture with limb bud progenitor cells [39]. With mesenchymal cells, coculture with synovial-lining macrophages [161] and embryonic calvarial cells [162] was reported to stimulate chondrogenic differentiation. The main advantage of coculture systems is that this allows intimate contact between different cell types, which may lead to a more efficient transduction of molecular signals that induce chondrogenic differentiation. The surface receptors of cocultured cells come into direct physical contact, and the autocrine and paracrine factors secreted by one cell type readily interact with the other cell type.

More recently, there has been evidence that intimate physical contact may lead to fusion of different cell types in vivo, resulting in the formation of heterokaryons [33, 34]. In fact, cell-fusion phenomenon has been used to explain the ability of adult stem cells to transdifferentiate into cell types that are radically different from their tissue of origin when transplanted in vivo [32, 163]. Nevertheless, there is as yet no evidence that the stimulatory effect of coculture on chondrogenic differentiation [39, 161, 162] is the result of cell fusion. Coculture of two or more distinct cell populations also carries a strong risk of transmission of pathogens, in particular viruses. This would constitute a major obstacle to the clinical application of coculture for chondrogenic differentiation. In the clinical situation, it would simply not be practical to stimulate chondrogenic differentiation of stem cells through coculture with either an autogenic or donated cell source. Another major shortcoming of coculture is the difficultly in the separation of cocultured cell populations. The highest degree of purity upon separation could be achieved by fluorescence-activated cell sorting (FACS) [164]. However, FACS is skill intensive and requires expensive instrumentation. Magnetic-affinity cell sorting [165] is much cheaper compared with FACS, but the degree of purity upon separation is much lower. The problem of separating distinct cell populations, as well as the potential problem of cell fusion, may be overcome by keeping cocultured cell populations physically separated through the use of commercially available Transwell inserts [161]. The other alternative is to use filtered cell-conditioned media instead.

Indeed, culture media conditioned by embryonic calvarial cells [162], embryonic limb bud [166], articular perichondrial cells [167], and primary chondrocytes [168170] have all been reported to have stimulatory effects on chondrogenesis. This could therefore suggest that intimate cellular contact within coculture might not actually be necessary to achieve stimulation of chondrogenic differentiation. Nevertheless, it is important to note that the use of filtered conditioned media does not alleviate the risk of viral transmission. Also, secreted factors within conditioned media may be labile and hence may not be suitable for prolonged durations of in vitro culture.

Directing Chondrogenic Differentiation through Genetic Modulation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adult Stem Cells versus Embryonic Stem Cells for Cartilage Repair and Tissue Engineering
  5. Development of Defined Culture Milieu for Directing the Chondrogenic Differentiation of Stem Cells In Vitro
  6. Coculture and Cell-Conditioned Media
  7. Directing Chondrogenic Differentiation through Genetic Modulation
  8. Future Perspectives
  9. Concluding Remarks
  10. References

Directing chondrogenic differentiation with exogenous cytokines, growth factors, and ECM substratum or even with coculture and conditioned media would require prolonged durations of in vitro culture. This has two major disadvantages if autologous adult stem cells are to be used for cartilage regeneration. First, this would obviously delay treatment to the patient. Second, there is evidence that prolonged durations of ex vivo culture could somehow alter the immunogenicity of cultured autologous cells, which could in turn lead to immunorejection upon transplantation [171173].

A novel alternative for directing and controlling the chondrogenic differentiation of stem cells is through genetic modulation, which would obviate prolonged durations of in vitro culture. This could be achieved by transfecting stem cells with recombinant DNA constructs encoding for the expression of certain proteins or growth factors that promote chondrogenesis. Of particular interest are transcription factors implicated in the pathway of chondrogenic differentiation. These control the expression of the entire array of proteins specific to the chondrogenic lineage and include members of the Sox family, Sox5 [174], Sox6 [174, 175], Sox8 [176], and Sox9 [177, 178], as well as a variety of other cytosolic proteins such as Lc-Maf [179], CREB-binding protein [180], Nkx3.2 [181, 182], AP-2, SP-1 [62], Pax1, Pax9 [183], DEC1 [184], Brachyury [185], and the Smad family of signaling molecules [186]. Of these various transcription factors, Sox-8 and Sox-9 are thought to be the most downstream regulators of chondrogenesis [176].

Indeed, recombinant overexpression of Sox9 [187], DEC1 [179], and Brachyury [185] have all been reported to accelerate chondrogenesis. Besides transcription factors, the recombinant expression of several other proteins has also been reported to promote chondrogenic differentiation. These include IGF-1 [188, 189], TGF-β2 [190], Midkine [191], and NCAM [192]. Additionally, it was also reported that recombinant expression of Wnt-3a enhances BMP-2–induced chondrogenesis of murine mesenchymal cells [193].

The disadvantage of directing chondrogenic differentiation through genetic modulation is the potential risk associated with using recombinant DNA technology in human clinical therapy. For example, the constitutive overexpression of any one particular protein or growth factor within transfected stem cells would certainly have unpredictable physiological effects on transplantation in vivo. This problem may be overcome by placing the recombinant expression of the particular protein under the control of switchable promoters, several of which have been developed for expression in eukaryotic systems. Such switchable promoters could be responsive to exogenous chemicals, heat shock, or even light. Of particular interest are light-responsive promoters [194], because these would avoid the potentially toxic or pleiotropic effects of exogenous chemicals and heat treatment. At present, there are as yet no reported studies on the coupling of chondro-specific genes to light-responsive promoters. Indeed, the creation of such recombinant constructs and their subsequent transfection within stem cells would certainly make an interesting study with potentially useful clinical applications.

The cellular signaling pathway for chondrogenic differentiation could be given a kick start through temporary expression of chondro-specific transcription factors coupled with light-inducible promoters. After that, it is possible that the pathway for chondrogenic differentiation could carry on independently of the recombinant expression of these transcription factors, because the entire array of chondro-specific genes would have already been activated. The advantage of this approach is that there is no constitutive overexpression of any one particular transcription factor. Also, the natural cellular pathway for chondrogenic differentiation could carry on physiologically switching off the recombinant expression of these transcription factors through removal of light stimulus. Upon transplantation in vivo, it is extremely unlikely that light-inducible promoters would again be activated, because light stimulus would be completely absent in situ.

Genetically modified stem cells may also run the risk of becoming malignant within the transplanted recipient. Moreover, there are overriding safety concerns with regards to the use of recombinant viral-based vectors in the genetic manipulation of stem cells [195]. It remains uncertain as to whether legislation would ultimately permit the use of genetically modified stem cells for human clinical therapy. At present, the potential detrimental effects of transplanting genetically modified stem cells in vivo are not well-studied. More research needs to be carried out on animal models to address the safety aspects of such an approach.

Future Perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adult Stem Cells versus Embryonic Stem Cells for Cartilage Repair and Tissue Engineering
  5. Development of Defined Culture Milieu for Directing the Chondrogenic Differentiation of Stem Cells In Vitro
  6. Coculture and Cell-Conditioned Media
  7. Directing Chondrogenic Differentiation through Genetic Modulation
  8. Future Perspectives
  9. Concluding Remarks
  10. References

For therapeutic applications, there seem to be two major strategies for directing stem cell differentiation into the chondrogenic lineage in vitro: through the development of a defined serum-free culture milieu and through genetic modulation with recombinant DNA technology. The use of coculture and conditioned media to promote chondrogenic differentiation would most likely not be useful for clinical applications. However, these could still provide useful in vitro experimental models for further studies in chondrogenesis.

As mentioned earlier, there are overwhelming safety concerns with regards to the use of genetic modulation to direct stem cells into the chondrogenic lineage. Unless such safety concerns can be allayed, the method of choice to direct the chondrogenic differentiation of stem cells for therapeutic applications would most probably use a defined serum-free culture milieu incorporating various cytokines, growth factors, chemicals, and ECM substratum. Figure 1 summarizes the major components of such a defined culture milieu.

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Figure Figure 1.. Schematic diagram representing the major components of a defined culture milieu for directing the differentiation of stem cells into the chondrogenic lineage. (A): Protein-based cytokines and growth factors; (B): extracellular matrix; (C): nonproteinaceous chemicals; (D): biophysical parameters such as O2 tension and temperature; (E): cell density, which determines the degree of cell-to-cell contact and gap junction–mediated intercellular coupling. Abbreviation: TGF-β, transforming growth factor β.

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To further optimize the culture milieu for chondrogenic differentiation, it may be worthwhile to look at other biophysical parameters in addition to the culture media composition. This would include varying oxygen tension [196, 197] and temperature within the incubator, as well as the application of physical stimuli in the form of electrical [198] and electromagnetic [199] fields, mechanical forces [200], heat shock [201], ultrasound [202], and even laser irradiation [203]. Indeed, evidence from the scientific literature suggests that the pathway of chondrogenic differentiation can be profoundly influenced by variations in such biophysical parameters.

Because cellular contact and gap junction–mediated intercellular coupling play an integral role in mesenchymal condensation during the initial stages of chondrogenesis [204206], it is likely that variations in cell density within in vitro culture could profoundly affect the pathway of chondrogenic differentiation. Additionally, it may also be useful to look at cytokines that encourage gap junction formation during chondrogenesis [83, 207, 208]. Nevertheless, it is important to note that at more advanced stages of chondrogenic differentiation, the maturing chondrocytes gradually lose their gap junctional communication [205] and become surrounded by a layer of pericellular matrix [209]. This would preclude any direct cell-to-cell contact, as well as gap junction–mediated intercellular coupling.

Other possible techniques to direct chondrogenic differentiation include exposure to cytoplasmic extracts and cybridization. Håkelien and colleagues [210, 211] managed to reprogram 293 T fibroblasts to express T-cell and neuronal function by exposing permeabilized cells (i.e., with streptolysin-O) to concentrated cytoplasmic extracts of T cells and neuronal progenitors, respectively. However, to date, there are as yet no reported studies on the use of cytoplasmic extracts of chondrocytes or chondroblasts to direct the differentiation of either fibroblasts or stem cells into the chondrogenic lineage. Another novel approach is to fuse stem cells with enucleated cytoplasts derived from chondrocytes or chondroblasts to form cytoplasmic hybrids, or cybrids. Techniques for generating enucleated cytoplasts from differentiated somatic cells have been developed and refined over the course of the past three decades [212214]. Briefly, this usually involves treatment of the somatic cells with a microtubulin inhibitor (i.e., cytochalasin), followed by high-speed centrifugation within a layered density gradient (comprised of either Ficoll or Percoll). The enucleated cytoplasts can then be fused with nucleated cells through a variety of different techniques using electrical pulse [215] or polyethylene glycol [216] or with Sendai virus [217]. To date, there are no studies that have yet been reported on the use of cybridization to direct chondrogenic differentiation. Nevertheless, previous studies have shown that cybridization could be used to induce teratocarcinoma cells to express myoblast function [218, 219] as well as direct erythroid [220, 221] and myeloid [222] differentiation.

Concluding Remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adult Stem Cells versus Embryonic Stem Cells for Cartilage Repair and Tissue Engineering
  5. Development of Defined Culture Milieu for Directing the Chondrogenic Differentiation of Stem Cells In Vitro
  6. Coculture and Cell-Conditioned Media
  7. Directing Chondrogenic Differentiation through Genetic Modulation
  8. Future Perspectives
  9. Concluding Remarks
  10. References

Despite the large number of studies that have recently been carried out on the chondrogenic differentiation of stem cells in vitro, this particular area of research is still in its relative infancy. Chondrogenic differentiation may be additionally enhanced if the various techniques that have so far been discussed are used in combination rather than exclusively by themselves. In the natural milieu, the chondrogenic differentiation of stem cells probably involves multiple signaling pathways. This may be mimicked in vitro by using a combination of these various techniques to achieve a synergistic effect on the differentiation of stem cells into the chondrogenic lineage.

However, it must be kept in mind that for clinical applications, it is imperative to develop well-defined and efficient in vitro protocols for the chondrogenic differentiation of stem cells that would use chemically defined culture media supplemented with recombinant cytokines and growth factors. This will then provide the stringent levels of safety and quality control that would make the clinical applications of stem cell transplantation therapy realizable. The hope is that this will be achieved in the near future.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Adult Stem Cells versus Embryonic Stem Cells for Cartilage Repair and Tissue Engineering
  5. Development of Defined Culture Milieu for Directing the Chondrogenic Differentiation of Stem Cells In Vitro
  6. Coculture and Cell-Conditioned Media
  7. Directing Chondrogenic Differentiation through Genetic Modulation
  8. Future Perspectives
  9. Concluding Remarks
  10. References