Due to the limited self-repair capacity of articular cartilage and the lack of efficient pharmacologic treatments for chondral defects, cell-based approaches for articular cartilage regeneration have been developed. One of these approaches, autologous chondrocyte transplantation (ACT), has been used with encouraging clinical results (1–5). For ACT, chondrocytes are harvested by biopsy of a non–weight-bearing region of the damaged joint, expanded ex vivo, and then reinjected into the site of the defect. Among the limitations of ACT are a paucity of the cell source and tissue damage at the donor site, with the risk of emerging osteoarthritis. To overcome these problems, adult mesenchymal stem cells (MSCs) have been proposed as an alternative cell source. MSCs can be easily obtained from different sources, such as bone marrow (6, 7) and adipose tissue (8), and possess good proliferation and differentiation potential, including differentiation into a chondrogenic phenotype (8, 9).
Chondrogenesis is a complex and tightly regulated process, the underlying molecular mechanisms of which are not yet fully understood. During early chondrogenesis, progenitor cells condense and differentiate into resting chondrocytes, producing aggregating proteoglycans and types II, IX, and XI collagen. This phenotype is stably maintained in the hyaline cartilage of joints, whereas further differentiation occurs during endochondral ossification in the development and growth of bones. These maturing chondrocytes proliferate and subsequently become hypertrophic, characterized by a marked increase in metabolic activity and cell volume. Hypertrophic cells deposit large quantities of extracellular matrix, including type X collagen, which is a marker of this stage of differentiation (10). Hypertrophic chondrocytes also begin to produce alkaline phosphatase (AP), an enzyme involved in matrix mineralization, marking the late stage of terminal differentiation. Mineralized cartilage is then invaded and replaced by bone cells and bone marrow cells.
To better understand the chondrogenesis of MSCs as well as to produce MSC-derived tissue-engineered cartilage for use in cartilage repair, various protocols for the in vitro chondrogenesis of MSCs have been developed. These include pellet culture of cells in serum-free chondrogenic medium supplemented with dexamethasone and transforming growth factor β (TGFβ) (11–13). This pellet culture results in the up-regulation of type II collagen and proteoglycans, but it also up-regulates markers of hypertrophy, such as type X collagen, AP, and matrix metalloproteinase 13 (MMP-13) (11, 14, 15). Predifferentiated chondrogenic MSC constructs transplanted subcutaneously into immunodeficient mice have been shown to undergo calcification, vascular invasion, and micro-ossicle formation (15), suggesting the formation of undesirable, transient cartilage reminiscent of endochondral ossification, rather than stable articular cartilage. In contrast, articular chondrocytes have been shown to maintain their nonhypertrophic phenotype and to be capable of stable ectopic cartilage formation, unless they are subjected to too extensive dedifferentiation by monolayer culture (15, 16).
Improvement in the differentiation protocols is therefore needed in order to generate MSC-derived chondrocytes that have a stable, nonhypertrophic phenotype. This is particularly challenging because the differentiation of MSCs reflects the natural pathway of endochondral ossification, and the molecular events that trigger terminal differentiation and how this can be suppressed remain largely unclear.
Interestingly, coculture experiments have demonstrated that immature or articular chondrocytes produce soluble factors that are able to suppress the terminal differentiation of maturing growth plate chondrocytes in vitro (17–19). This indicates that resting chondrocytes maintain their stable phenotype by actively inhibiting terminal differentiation and could potentially teach differentiating MSCs to become stable chondrocytes in coculture. It has been suggested that coculture-induced mechanisms may rely on the soluble factors TGFβ2 and fibroblast growth factor 2 (FGF-2) acting in synergy (17), whereas other postulated mechanisms are TGFβ-independent (18).
Three published studies have addressed the coculture of MSCs and articular chondrocytes; however, those studies focused on the promotion of chondrogenesis of MSCs (20–22). Two of the studies found positive effects on the induction of type II collagen messenger RNA (mRNA) and/or protein. However, the animal MSCs that were chosen for study (20, 21) also exhibited chondrocyte marker induction, including type II collagen, in the absence of exogenous TGFβ and thus displayed some degree of autoinduction. While this has not been observed with primary human MSCs, it may apply to the immortalized human MSC line that was used in coculture with immortalized human chondrocytes in the third study (22).
It thus remains unknown whether primary human articular chondrocytes (HACs) can suppress the hypertrophic development of primary human MSCs in coculture and prevent ectopic matrix calcification. Such knowledge is not only important for unraveling the molecular mechanisms and mediators of articular chondrocyte phenotype stability that are relevant to osteoarthritis (23, 24), but may also help to further improve clinical stem cell–based cartilage repair strategies.
In the present study, we therefore sought to determine whether direct or indirect coculture with HACs is able to suppress hypertrophy during TGFβ-driven chondrogenesis of MSCs and whether either technique will reduce the calcification of ectopic transplants in vivo. We further searched for candidate soluble mediators of this action in a novel indirect coculture system in which conditioned medium from 3-dimensional (3-D) chondrocyte pellet cultures was transferred to parallel 3-D MSC-pellet cultures during a followup period of 6 weeks.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Resting chondrocytes in the growth plate and articular chondrocytes maintain their nonhypertrophic phenotype by active inhibition of terminal differentiation, and soluble factors have been discussed as the main mediators of this action (17–19, 30). This encouraged us to evaluate whether articular chondrocytes can teach MSCs to become stable chondrocytes, either by direct or indirect coculture, during TGFβ-driven in vitro chondrogenesis.
Direct coculture was chosen for these experiments since we expected the strongest effects when MSCs and HACs were mixed in a single pellet. Furthermore, we established a novel indirect coculture system in which conditioned medium was generated by 3-D HAC pellets cultivated in parallel with MSC pellets under standard chondrogenic conditions. TGFβ was added to the medium to be conditioned, since a pilot study showed that HACs deposited very little proteoglycan and type II collagen in chondrogenic medium without TGFβ. Serum-free conditions were used, since this is standard for chondrogenic induction and because the inhibitory effects of serum components on chondrogenesis were seen in a previous study (31).
HAC-conditioned medium stimulated COL2A1 mRNA levels—and thus, in vitro chondrogenesis—in most MSC donor cultures, except those which reached very high COL2A1 mRNA levels (>900% β-actin) already under control conditions (data not shown). Most strikingly, however, the HAC-conditioned medium induced significant inhibition of hypertrophy, as evidenced by the in vitro down-regulation of the COL10A1 to COL2A1 mRNA ratio, the relative amount of type X collagen deposition, and the AP enzyme activity. As a consequence, matrix calcification was reduced after ectopic transplantation of pellets in vivo, an effect that was even more pronounced in direct coculture experiments. Our finding of repressed type X collagen production while maintaining type II collagen production is consistent with the results of studies using a rat model, in which MSCs were cocultured with cartilage chips in a Transwell system (21). In that study, vascular endothelial growth factor, MMP-13, and tissue inhibitor of metalloproteinases types 1 and 2 were identified in the conditioned medium and were suggested to be involved in the regulation of type X collagen.
Our objective was to identify the candidate inhibitory factors released by HACs, which may modulate hypertrophy. We therefore performed assays for FGF-2 and PTHrP, both of which are negative regulators of chondrocyte differentiation (32) and MSC chondrogenesis (33). FGF-2 and PTHrP have been shown to severely reduce type X collagen expression, AP activity, and cell enlargement of lower sternal chondrocytes from immature chicken (34, 35), and both molecules are soluble factors produced by articular chondrocytes (36–38).
While we detected FGF-2 secretion by HACs on day 7 only, PTHrP mRNA and protein were produced throughout the culture period, making PTHrP a likely candidate for this action. Indeed, exposure of MSCs to 10 ng/ml of PTHrP in chondrogenic medium from day 21 of culture until week 6 significantly reduced COL10A1 and IHH expression and AP activity. However, the complete suppression seen with HACs was not reached by MSCs exposed to PTHrP. We, therefore, propose that PTHrP is one of the main candidate mediators of coculture-induced reduction of hypertrophy in our model. This is consistent with the established direct suppression of COL10A1 by PTHrP via a PTH/PTHrP-responsive region in the human COL10A1 enhancer (39) and parallels the suggestion that PTHrP may mediate the suppression of AP activity and matrix mineralization in a coculture model of deep-zone with surface-zone bovine cartilage (30).
Most importantly, our study established a causal relationship between the presence of PTHrP during late MSC chondrogenesis and a reduced, but not fully suppressed, hypertrophic development of cells. The final proof that PTHrP is the sole relevant inhibitor of HAC-conditioned medium is not provided by this study, however. This would require the prevention of PTHrP actions in HAC culture supernatants by a specific inhibitor that would block PTHrP. Known inhibitors, such as PTHrP(7–34), target the PTHrP receptor PTHR-1, and our previous results (33) suggest that PTHrP exerts receptor-independent effects during early chondrogenesis, when PTHR-1 mRNA is not yet detected (33, 40).
In light of the improved chondrogenesis in HAC-conditioned medium, as opposed to the down-regulation of COL2A1 in the presence of 10 ng/ml of PTHrP observed in our previous study (33), we believe that PTHrP is certainly not the only factor involved in the attenuation of hypertrophy by HAC-conditioned medium. Most likely, factors that support anabolic differentiation act in synergy with more than one negative regulator; however, PTHrP is a strong candidate negative regulator. Candidate stimulatory factors could further be examined by use of limiting concentrations of TGFβ in our model, an appealing approach for upcoming studies.
One remarkable finding was that MSCs produced PTHrP only during the first 2–3 weeks of chondrogenesis, which was then down-regulated in favor of a strong induction of IHH (Figure 6B). Thus, MSCs differed from phenotypically stable HAC cultures by a loss of endogenous PTHrP expression during culture, while no such autocrine PTHrP/IHH regulation was observed in HACs. Only MSC differentiation was reminiscent of growth plate development, during which PTHrP is expressed by immature chondrocytes, whereas IHH is secreted later on, allowing for further maturation of the cells toward hypertrophy. This implies that we should try to understand why PTHrP is down-regulated during MSC chondrogenesis and that we should try to prevent this step with new protocols that induce a stable chondrogenic phenotype, as is desirable for cartilage repair studies.
In conclusion, HAC-derived soluble factors and direct coculture were potent means of improving TGFβ-driven chondrogenesis by suppressing the development of hypertrophy and ectopic matrix mineralization of MSCs. PTHrP was identified as an important candidate soluble factor involved in this action. Our study suggests that second-generation differentiation protocols for MSCs be developed in which the PTHrP/IHH autoregulation discovered in this study is modulated in favor of the generation of chondrocytes, which display no hypertrophic phenotype and thus seem more suitable as a substitute for articular chondrocytes for use in cartilage repair. The knowledge of how to stop MSC differentiation before and after hypertrophy is important, since calcified hypertrophic cartilage is highly relevant for the intimate anchoring of articular cartilage to the subchondral bone. A functional cartilage repair tissue should therefore always consist of both a stable middle and upper cartilage layer and a hypertrophic mineralized lower region (which only together can form a functional unit) as a prerequisite for a long-lasting success of MSC-based cartilage repair strategies.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Richter had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Fischer, Dickhut, Rickert, Richter.
Acquisition of data. Fischer, Dickhut.
Analysis and interpretation of data. Fischer, Dickhut, Rickert, Richter.