Trophic Effects of Mesenchymal Stem Cells in Chondrocyte Co-Cultures are Independent of Culture Conditions and Cell Sources

Abstract

Earlier, we have shown that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow-derived mesenchymal stem cells (BM-MSCs) is due to a trophic role of the MSC in stimulating chondrocyte proliferation and matrix production rather than MSCs actively undergoing chondrogenic differentiation. These studies were performed in a culture medium that was not compatible with the chondrogenic differentiation of MSCs. In this study, we tested whether the trophic role of the MSCs is dependent on culturing co-culture pellets in a medium that is compatible with the chondrogenic differentiation of MSCs. In addition, we investigated whether the trophic role of the MSCs is dependent on their origins or is a more general characteristic of MSCs. Human BM-MSCs and bovine primary chondrocytes were co-cultured in a medium that was compatible with the chondrogenic differentiation of MSCs. Enhanced matrix production was confirmed by glycosaminoglycans (GAG) quantification. A species-specific quantitative polymerase chain reaction demonstrated that the cartilage matrix was mainly of bovine origin, indicative of a lack of the chondrogenic differentiation of MSCs. In addition, pellet co-cultures were overgrown by bovine cells over time. To test the influence of origin on MSCs' trophic effects, the MSCs isolated from adipose tissue and the synovial membrane were co-cultured with human primary chondrocytes, and their activity was compared with BM-MSCs, which served as control. GAG quantification again confirmed increased cartilage matrix production, irrespective of the source of the MSCs. EdU staining combined with cell tracking revealed an increased proliferation of chondrocytes in each condition. Irrespective of the MSC source, a short tandem repeat analysis of genomic DNA showed a decrease in MSCs in the co-culture over time. Our results clearly demonstrate that in co-culture pellets, the MSCs stimulate cartilage formation due to a trophic effect on the chondrocytes rather than differentiating into chondrocytes, irrespective of culture condition or origin. This implies that the trophic effect of MSCs in co-cultures is a general phenomenon with potential implications for use in cartilage repair strategies. Introduction Despite the success of autologous chondrocyte implantation in treating large-size cartilage defects, there are some disadvantages of this treatment that limit its broader clinical application. One major issue is the requirement of relatively large quantities of chondrocytes from the patient, 1 which are obtained by in vitro expansion. The partial replacement of the chondrocytes with mesenchymal stem cells (MSC) has been proposed to tackle this problem. Many studies have evaluated the feasibility of this idea by the coculturing of MSCs and chondrocytes.2 Indeed, cartilage matrix deposition was found to be improved in the cocultures of MSCs and chondrocytes compared with the cultures of pure chondrocytes or MSCs. 3,4 In our previous report, 5 we have shown that pellet co-cultures of bovine pri...

Cell-Derived Polymer/Extracellular Matrix Composite Scaffolds for Cartilage Regeneration, Part 1: Investigation of Cocultures and Seeding Densities for Improved Extracellular Matrix Deposition

“…Previous efforts to investigate the utilization of cocultures containing chondrocytes and MSCs have primarily focused on studying the differentiation state of either cell type as opposed to focusing on the development of cartilage-like ECM within a scaffold. [45][46][47] The purpose of this study was to investigate and optimize the use of different coculture ratios of chondrocytes and MSCs in an effort to produce PCL/ECM constructs containing cartilage-like ECM, while reducing the total number of chondrocytes necessary to do so. Further, the influence of seeding density was also examined as it was hypothesized that a higher cell 35K'') or 70,000 cells/scaffold (''70K'') cultured for 7, 14, and 21 days.…”

“…This is consistent with the findings from previous micromass investigations, which indicated that upon coculture, articular chondrocytes were responsible for matrix production as opposed to MSCs in cocultures of adult human bone marrow MSCs with articular chondrocytes from both human and bovine sources. 46,48,58 Among the coculture constructs, a pronounced difference in the cellularity or ECM content was never observed between the 1:3 and 1:5 coculture groups, regardless of culture duration or initial seeding density. As the original objective was to produce a construct laden with cartilage-like ECM, while reducing the number of chondrocytes necessary to do so, these results suggest that ultimately, constructs with 1:5 coculture ratio, and thus, fewer chondrocytes might result in a similar ability to direct cartilage repair as constructs generated with 1:3 cocultures.…”

“…46,48,49 In the case of micromasses, the frequency of cell-cell interactions is increased because the cell density is greatly exaggerated, approximating that of cartilage within the condensation stage of development. 50 Whereas micromasses are useful for studying basic cell interactions and tissue development, they are impractical for tissue engineering applications due to the large cell numbers required to achieve an implantable mass of appreciable dimensions.…”

“…In fact, previous efforts suggest that in cocultures, MSCs elicit a stimulatory trophic effect on chondrocytes, which leads to the observed improved chondrogenic phenotype as opposed to MSC differentiation. 32,46 Possible mechanisms for this communication include paracrine signaling through secreted factors, as well as direct cell-cell contacts. To select the truly optimal culture ratio of MSCs to chondrocytes for the purpose of producing PCL/ECM scaffolds for cartilage regeneration, the effectiveness of the matrix produced must be analyzed for its potential to direct chondrogenesis.…”

Engineering Cartilage Tissue by Pellet Coculture of Chondrocytes and Mesenchymal Stromal Cells

“…Our data showed that the benefi cial effect of coculture is largely due to increased chondrocyte proliferation and matrix formation, while chondrogenic differentiation of MSCs only marginally contributed to cartilage formation. We also demonstrated that these observations present in coculture pellets of chondrocytes and MSCs are independent of donor variation and culture conditions [ 8 ]. Subsequent experiments indicated that increased secretion of fi broblast growth factor 1 (FGF1) in coculture of MSCs and chondrocytes is responsible for increased chondrocyte proliferation in pellet cocultures [ 9 ].…”