Mesenchymal stem cells (MSC) are promising for the treatment of articular cartilage defects; however, common protocols for in vitro chondrogenesis induce typical features of hypertrophic chondrocytes reminiscent of endochondral bone formation. This may implicate a risk for graft stability. We here analysed the early healing response in experimental full-thickness cartilage defects, asking whether and how MSC can differentiate to chondrocytes in an orthotopic environment. Cartilage defects in knees of minipigs were covered with a collagen-type I/III membrane, and half of them received transplantation of expanded autologous MSC. Integration into surrounding cartilage tissue was poor to moderate after 1 and 3 weeks and no sign of cartilaginous matrix production as indicated by negative safranin-O staining was visible for both groups. At 8 weeks regenerative tissue was integrated into the surrounding tissue and a safranin-O positively stained neocartilage was detectable in 4 tissue regenerates out of 6 in the MSC group compared to 2 out of 6 in the MSC-free group. At 1 and 3 weeks after surgery only marginal Col2A1 and no AGC expression were detectable in both groups. At 8 weeks Col2A1 and AGC levels had significantly increased. Hypertrophic maker induction (Col10A1 and MMP13) was similar in both groups 8 weeks after surgery. Immunostaining for collagen type X, however, was restricted to the regenerative tissue close to the subchondral bone in both groups, while collagen type II staining was detected from below the superficial to the deep zone. Our data provide molecular evidence for spontaneous differentiation of MSC in cartilage and the development of a collagen type II positive, collagen type X negative neocartilage. Whether by remodelling of defect filling tissue collagen type X positive areas will further diminish or even disappear from repair cartilage at later stages has to be evaluated in a longer follow-up study.
Monolayer expansion of human articular chondrocytes (HAC) is known to result in progressive dedifferentiation and loss of stable cartilage formation capacity in vivo. For optimal outcome of chondrocyte based repair strategies, HAC capable of ectopic cartilage formation may be required. Thus, the aim of this study was to establish appropriate quality control measures capable to predict the ectopic cartilage formation capacity of HAC from culture supernatants. This strategy would avoid the waste of cells for quality control purposes, in order to improve cell therapy and tissue-engineering approaches for the repair of joint surface lesions. Standardized medium supernatants (n=5) of freshly isolated HAC and chondrocytes expanded for 2 (PD2) or 6 population doublings (PD6) were screened for 15 distinct interleukins, 8 MMPs and 11 miscellaneous soluble factors by a multiplexed immunoassay. Cartilage differentiation markers like COMP and YKL-40 were determined by ELISA. Corresponding HAC were subcutaneously transplanted into SCID-mice and their capacity to form stable ectopic cartilage was examined histologically 4 weeks later. While freshly isolated chondrocytes generated stable ectopic cartilage positive for collagen type II, none of the PD6 transplants formed cartilaginous matrix. Loss of ectopic stable cartilage formation capacity between PD0 and PD6 correlated with a drop of MMP3 secretion to <
10% of initial levels, while changes for other investigated molecules were not predictive. Chondrocytes from donors with low MMP3 levels (<
10%) at PD2 failed to regenerate ectopic cartilage at PD2, indicating that MMP3 levels of cultured chondrocytes, independent of the number of cell doublings and the time in culture, predicted ectopic cartilage formation. In conclusion, loss of stable ectopic cartilage formation capacity in the course of HAC dedifferentiation can be predicted by determination of relative MMP3 levels demonstrating that standardized culture supernatants can be used for quality control of chondrocytes dedicated for cell therapeutic approaches.
