Improving bone marrow stromal cell attachment on chitosan/hydroxyapatite scaffolds by an immobilized RGD peptide

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Abstract
Ample cell adhesion to scaffolds is essential for effective bone tissue engineering. Chitosan/hydroxyapatite (CS/HA) scaffolds with channel-shaped and spherically shaped pore morphologies were prepared via in situ compositing hybridization in combination with lyophilization. The sizes of channel-shaped and spherically shaped pores of the CS/HA scaffolds were 150-650 µm and 3-15 µm, respectively. The RGD peptide (Arg-Gly-Asp) was bound to the surface of CS/HA scaffolds via physical adsorption. More than 63% of RGD present in a PBS solution spontaneously adsorbed onto CS/HA scaffolds. High numbers of viable bone marrow stromal cells (BMSCs) were observed by confocal and fluorescence microscopy for cells cultured on CS/HA scaffolds with and without RGD for 3 days. BMSCs on CS/HA scaffolds with RGD (RGD-CS/HA) were incubated for 4 h under standard culture conditions, and the degree of cell adhesion was calculated. Cell adhesion to RGD-CS/HA scaffolds with different RGD concentrations was 71.6% and 80.7%, respectively. This was 30.9% and 47.5% higher than adhesion to the CS/HA scaffold without RGD, respectively. BMSCs cultured on the scaffolds for 14 days with osteogenic supplements expressed 103% higher alkaline phosphatase on the RGD-CS/HA scaffold (0.001 97 ± 0.000 31 U/L/ng), than on the unmodified scaffold (0.000 97 ± 0.000 25 U/L/ng) (p < 0.01), indicating that a RGD peptide significantly promotes osteogenic differentiation of BMSCs on CS/HA scaffolds. The results of this study indicate that RGD-CS/HA scaffolds promote initial cell adhesion, spread and differentiation toward an osteogenic phenotype.

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