Abstract
Devitalized hypertrophic cartilage matrix (DCM) is an attractive concept for an off-the-shelf bone graft substitute. Upon implantation, DCM can trigger the natural endochondral ossification process, but only when the hypertrophic cartilage matrix has been reconstituted correctly. In vivo hypertrophic differentiation has been reported for multiple cell types but up-scaling and in vivo devitalization remain a big challenge. To this end, we developed a micro tissue-engineered cartilage (MiTEC) model using the chondrogenic cell line ATDC5. Micro-aggregates of ATDC5 cells (approximately 1000 cells per aggregate) were cultured on a 3% agarose mold consisting of 1585 microwells, each measuring 400 µm in diameter. Chondrogenic differentiation was strongly enhanced using media supplemented with combinations of growth factors e.g., insulin, transforming growth factor beta and dexamethasone. Next, mineralization was induced by supplying the culture medium with beta-glycerophosphate, and finally we boosted the secretion of proangiogenic growth factors using the hypoxia mimetic phenanthroline in the final stage of in vivo culture. Then, ATDC5 aggregates were devitalized by freeze/thawing or sodium dodecyl sulfate treatment before co-culturing with human mesenchymal stromal cells (hMSCs). We observed a strong effect on chondrogenic differentiation of hMSCs. Using this MiTEC model, we were able to not only upscale the production of cartilage to a clinically relevant amount but were also able to vary the cartilage matrix composition in different ways, making MiTEC an ideal model to develop DCM as a bone graft substitute.
Highlights
Endochondral ossification is a fascinating phenomenon in which a cartilage template is remodeled into bone tissue by a highly regulated mechanism
4 different formulations were used: (1) basic medium (BM); (2) BM supplemented with 1× insulin-transferrin-selenium (ITS) solution (IM); (3) BM supplemented with 0.1 μM dexamethasone (Dex) and 10 ng/mL transforming growth factor beta 3 (TGFβ3, R&D systems, 243-B3-010) (BC); and BM supplemented with 1× ITS, 0.1 μM Dex and 10 ng/mL TGFβ3 (IC)
ATDC5 micro-aggregates were cultured in basic medium (BM) or basic medium supplemented with 1× ITS (IM medium) for 4 weeks on agarose chips
Summary
Endochondral ossification is a fascinating phenomenon in which a cartilage template is remodeled into bone tissue by a highly regulated mechanism. The scaffold induced vascularization and de novo mineral accumulation in a mouse ectopic model and formed full bridging (4 out of 8 animals) in a rat critically-sized femoral defect model [19] Taken all together, these experiments demonstrated the feasibility of an osteoinductive material made of devitalized hypertrophic cartilage matrix (DCM), either harvested from biological sources or cultured in the lab. Our long-term goal is to create an osteoinductive material made of devitalized ATDC5-derived hypertrophic cartilage that offers the ideal micro-environment for en route endochondral ossification To reach this goal, in this study we optimized the in vivo production of MiTEC qualitatively and quantitatively. We demonstrated the usability of the MiTEC model in optimizing and upscaling the in vivo culture of hypertrophic cartilage
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