A chondromimetic microsphere for in situ spatially controlled chondrogenic differentiation of human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have been identified as a viable cell source for cartilage tissue engineering. However, to undergo chondrogenic differentiation hMSCs require growth factors, in particular members of the transforming growth factor beta (TGF-β) family. While in vitro differentiation is feasible through continuous supplementation of TGF-β3, mechanisms to control and drive hMSCs down the chondrogenic lineage in their native microenvironment remain a significant challenge. The release of TGF-β3 from an injectable microsphere composed of the cartilage-associated extracellular matrix molecule hyaluronan represents a readily translatable approach for in situ differentiation of hMSCs for cartilage repair. In this study, chondromimetic hyaluronan microspheres were used as a growth factor delivery source for hMSC chondrogenesis. Cellular compatibility of the microspheres (1.2 and 14.1 μm) with hMSCs was shown and release of TGF-β3 from the most promising 14.1 μm microspheres to control differentiation of hMSCs was evaluated. Enhanced accumulation of cartilage-associated glycosaminoglycans by hMSCs incubated with TGF-β3-loaded microspheres was seen and positive staining for collagen type II and proteoglycan confirmed successful in vitro chondrogenesis. Gene expression analysis showed significantly increased expression of the chondrocyte-associated genes, collagen type II and aggrecan. This delivery platform resulted in significantly less collagen type X expression, suggesting the generation of a more stable cartilage phenotype. When evaluated in an ex vivo osteoarthritic cartilage model, implanted hMSCs with TGF-β3-loaded HA microspheres were detected within cartilage fibrillations and increased proteoglycan staining was seen in the tissue. In summary, data presented here demonstrate that TGF-β3-bound hyaluronan microspheres provide a suitable delivery system for induction of hMSC chondrogenesis and their use may represent a clinically feasible tissue engineering approach for the treatment of articular cartilage defects.

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