Our results demonstrates that a structured three-dimensional co-culture, in which a spherical population of human mesenchymal stem cells (hMSC) is surrounded by a layer of juvenile chondrocytes (JC), promotes a chondrogenic phenotype without the induction of chondrocyte hypertrophy. The amount of proteoglycan produced by BCPs was nearly equivalent to JC pellets even though the BCPs contain only 25% JC cells. Furthermore, the BCPs produce more proteoglycan than hMSC pellets or hMSC pellets treated with TGFβ. Taken together, these data indicate that the structured co-culture of juvenile chondrocytes and hMSC in the BCP are inducing chondrogenic differentiation. Most notably, in the setting of inflammatory cytokines and/or hypoxia, BCPs outperform JC as well as hMSC pellets in the production of proteoglycan. Therefore, the unique combination of physical, spatial, and biochemical cues provided by BCP co-culture overcomes several obstacles that currently limit the utility of hMSC for articular cartilage repair.
Unlike TGFβ-induced chondroinduction of hMSC, chondrogenic differentiation in BCPs occurred with reduced hypertrophy. The observation that BCP co-culture directs a more stable chondrogenic differentiation than does TGFβ is consistent with those of Aung, et al., who found that the co-culture of hydrogel-encapsulated hMSC with chondrocytes enhanced differentiation without hypertrophy. Chondrocyte conditioned media was unable to replicate this effect or that of BCP co-culture (16, data not shown). Therefore, chondroinduction requires an exchange of soluble factors between the two cell populations, and/or a spatial gradient of these factors. Although mass spectrometry studies by Aung, et al. identified many soluble factors present only in co-cultured media, which of the co-culture-specific cues provided by BCP co-culture are responsible for this stable chondrogenic differentiation remains unclear. Nonetheless, several studies suggest factors that may be involved. Specifically, TGFβ is known to play an important role in the induction of chondrocyte lineage selection and in the regulation of chondrogenic hypertrophy (5
). BCPs that were cultured with SB431542, a specific pharmacologic inhibitor of the type I TGFβ receptor, showed a reduced expression of both pro-chondrogenic (collagen 2A1 and aggrecan) and hypertrophic (MMP13 and collagen 10A1) genes (data not shown). Likely, the combination of factors produced by BCP co-culture modulate the hMSC response to TGFβ, such that the anabolic effects are enhanced, while hypertrophy-inducing effects are suppressed. Other factors may include agonists and antagonists of the fibroblast growth factor (FGF), hedgehog, bone morphogenetic protein (BMP) and Wnt pathways (22
While several studies demonstrate the important role of soluble factors in the chondroinductive effects of co-culture (10
), physical and spatial cues may also participate (18
). In development, the spatial juxtaposition of the perichondrium with the undifferentiated mesenchymal cells provides a critical signaling feedback loop that self-promotes chondrogenesis (22
). A computational model predicts that a graded diffusion of soluble factors may impact chondrogenic differentiation (16
). Our observations in BCPs may provide experimental support for this model, suggesting that cellular crosstalk between two cell populations across a boundary or gradient may enhance the inductive effect. The report by Allon, et al., provides further support for this conclusion by demonstrating that the structured BCP configuration is superior to mixed co-cultures of MSCs and chondrocyte-related nucleus pulposus cells (23
). Since the BCP configuration used herein is based on Allon, et al., we anticipate but have not yet verified similar advantages of BCP culture over random mixtures of hMSC and chondrocytes. Finally, physical cues ranging from extracellular matrix stiffness to topographical cues have been shown to direct cell fate and may contribute to the chondroinductive effects of BCPs (32
); important points to consider as cells and engineered biomaterials are combined to develop stem cell-based therapies.
Another key advantage of BCP co-culture is the ability of BCPs to resist the adverse hypoxic and inflammatory conditions anticipated in injured articular cartilage, which are recognized challenges to the success of cartilage tissue engineering for the treatment of osteoarthritis. This study found that BCPs maintain their chondrogenic potential under hypoxic and inflammatory conditions, producing more proteoglycan than pellets composed of either cell type alone. While hMSC are susceptible to hypoxia and JC are susceptible to inflammatory conditions, the combined cell populations in the BCP conferred resistance to these adverse environments, possibly due to the well-documented anti-inflammatory capability of hMSC (36
) and the adaptation of chondrocytes to hypoxic conditions. Though these results are provocative, a limitation of our study is that we used two cytokines and a hypoxia to mimic the complex microenvironment of the injured joint. Additional studies are necessary to evaluate the performance of BCP under in vivo conditions that more effectively model the healthy and injured articular cartilage microenvironment.
Further study is also needed to conclusively demonstrate which of the two cell populations in BCPs are responsible for the increased proteoglycan production and chondrogenic gene expression. Several prior studies have explored whether the MSC, chondrocytes, or both are responsible for enhanced chondrogenesis in co-cultures (12
). The possibility that BCP co-culture induces chondrogenic differentiation of hMSC is supported by several lines of evidence. First, the magnitude of proteoglycan production by BCPs and JC pellets is comparable, even though BCPs contain 75% hMSC. Second, histological analyses of BCPs show aggrecan mRNA and proteoglycan deposition throughout the BCP, not only in the outer shell composed of juvenile chondrocytes. Third, FISH analysis confirmed that aggrecan and safranin-O-positive regions of BCPs contained only hMSC, and that the JC were confined to the pellet surface even after 21 days of culture. Fourth, similar analyses of bilaminar cell pellets composed of 25% bovine nucleus pulposus cells and 75% human MSC showed an induction of human (not bovine) collagen 2A1 and aggrecan expression after 21 days of culture (38
). These results are consistent with those in which co-culture of hMSC with chondrocytes or cartilage matrix, for example, in a transwell, can increase expression of chondrocyte marker genes by hMSC (12
). Alternatively, hMSC may promote increased levels of proteoglycan production by the juvenile chondrocytes (40
). Human MSC are known to generate instructive signals for other cell types, and several other studies demonstrate that MSC increase chondrogenic capacity of chondrocytes in pellet co-cultures of these two cell types (14
). While our data suggest that BCP co-culture promotes chondrogenic differentiation of hMSC, it is does not exclude the possibility that hMSC enhance the chondrogenic activity of the human chondrocytes. Indeed, both mechanisms likely contribute.
In summary, our data indicate that the structured cellular interactions within BCPs promote a robust chondrogenic phenotype, as demonstrated by proteoglycan production and chondrogenic gene expression. Importantly, BCP co-culture addresses two major limitations of cartilage tissue engineering: a limited chondrocyte supply and the generation of chondrocytes without hypertrophy. hMSC are plentiful, therefore BCP formation may be used to expand the limited supply of chondrocytes for cartilage bioengineering. Furthermore, BCP co-culture does not require exogenous growth factors nor does it exhibit a hypertrophic phenotype. Although under chondrogenic conditions JC pellets express genes indicative of a chondrogenic phenotype, chondrocytes are limited in supply and also have impaired production of proteoglycan under inflammatory conditions that are seen in osteoarthritic joints. By contrast, BCPs do not appear to be negatively affected when exposed to inflammatory and hypoxic conditions. Consequently, BCPs may provide significant advantages as a therapeutic approach for cartilage regeneration.