(485bc) Osteoinductive Effects of Hydroxyapatite-Chitosan-Gelatin Hydrogel On Human Mesenchymal Stem Cells

Human mesenchymal stem cells (hMSCs) are a potent osteogenic cell source, but hMSC based bone constructs require a biomimetic culture substrate that provides both mechanical stability and osteoinduction of proliferating hMSCs. Hydroxyapatite-chitosan-gelatin (HCG) hydrogel provides a 3D environment where hMSCs can adhere, proliferate, migrate, excrete ECM and undergo osteogenic differentiation. HCG composite material has many advantages, including chemical similarity to natural ECM, adsorption of serum protiens, excellent biocompatibility and biodegradability, and intrinsic osteoinductive properties. Preliminary cell and material investigations evaluated hMSC response to the HCG hydrogel culture substrate. hMSC growth analysis by metabolic assay, SEM and confocal microscopy revealed rapid proliferation and cell migration into the HCG hydrogel matrix. Cell growth kinetics demonstrate that HCG material supports hMSC attachment and proliferation, over 30 days without exhibitting a growth plateau. Osteoinduction of hMSCs cultured in HCG hydrogel is confirmed by alkaline phospatase (ALP) activity and calcium mineralization quantification. hMSCs cultured in HCG hydrogel express the characteristic ALP peak of osteoinduction, without any additional chemical osteoinduction agents.

On-going studies focus on further characterizing the cell-material interactions of the HCG hydrogel, by immunohistochemistry staining and confocal microscopy. Analysis of integrin protein, stem cell protein and, osteo-specific protein expression will reveal how hMSCs interact with the HCG hydrogel and the differentiation state of the cells. hMSC migration into the HCG hydrogel will be systematically examined, by confocal microscopy and modified transwell assay. Quantitative rt-PCR will be used to analyze variations in protein expression. Additionally, osteoinduction enhancing BMP-2-DNA is incorporated, as naked plasmid or as chitosan-DNA nanoparticles, and hMSC growth and osteogenic differentiation will be evaluated. This composite biomaterial can theoretically facilitate bone healing over several months, by providing the progenitor cell population with an osteoinductive physiological and mechanical microenvironment to rebuild the native bone tissue as the implanted HCG material is degraded.