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INTRODUCTION: Hypertrophic chondrocytes mediate the enduring pathway for endochondral bone formation. However, challenges in tissue-engineered bone constructs include functionality under the mechanical environment inside in vivo conditions. We aim to fabricate a mechanically stable Silk fibroin (SF) carrier incorporating decellularized cartilage derived extracellular matrix (CD-ECM) and hypertrophic chondrocytes as a model of endochondral ossification and characterize superior bone-like tissue formation non-destructively in the case of in vitro studies to find the optimal time point when the constructs should be implanted.
METHODS: Human bone marrow stem cells (hBMSC’s) were seeded onto CD-ECM/SF or SF constructs and primed for 2-week chondrogenesis following a further 8-week hypertrophy or 4-week hypertrophy + 2-week osteogenesis in differentiation medium. Biochemical assays, SEM/EDX, RT-qPCR, Biomechanical tests followed by non-destructive mCT scanning were used as methods at 4 and 8 weeks to determine hypertrophy mediated ossification.
RESULTS: Calcium deposition biochemically determined increased significantly from 4-8 weeks in both SF and CD-ECM/SF constructs (p < 0.05) and retention of sGAG’s were observed in CD-ECM/SF constructs. SEM/EDX revealed calcium and phosphate crystal localization by hBMSC’s under all conditions. Compressive modulus reached a maximum of 70 KPa in 8-week hypertrophic induction group than 4-week hypertrophy+2-week osteogenesis group (p<0.05). mCT scanning at 8 weeks indicated a cloud of denser
minerals in groups only induced for 8-week hypertrophic induction. Gene expression by RT-qPCR revealed that hBMSC’s expressed hypertrophic markers VEGF, COL10, RUNX2 but absence of early hypertrophic marker ChM1 and presence of later hypertrophic marker TSBS1. Further induction of osteogenesis indicated presence of osteogenic markers ALPL, IBSP, OSX under all conditions.
DISCUSSION & CONCLUSIONS: Our data indicates a new method to prime hBMSC’S into late hypertrophic stage in vitro in mechanically stable constructs for endochondral ossification mediated bone tissue regeneration.
ACKNOWLEDGEMENTS: Funding by Life science calls LSC16-024 from NÖ Forschungs- und Bildungsges.m.b.H (NFB) and the provinical government of Lower Austria.
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