Effects of hydroxyapatite in 3-D chitosan–gelatin polymer network on human mesenchymal stem cell construct development

Human mesenchymal stem cells (hMSCs) have great potential in bone tissue engineering, and hydroxyapatite (HA), a natural component of human hard tissues, is believed to support hMSC growth and osteogenic differentiation. In this study, two types of biomimetic composite materials, chitosan–gelatin (CG) and hydroxyapatite/chitosan–gelatin (HCG), were fabricated and compared to examine the effects of HA on hMSC adhesion and 3-D construct development. The 2-D membranes were prepared to examine the influence of HA on adhesion efficiency of hMSCs, while 3-D porous scaffolds were produced to investigate the effects of HA on material adsorption properties and 3-D hMSC construct development. HA was found to promote protein and calcium ion adsorption of the 3-D porous scaffolds in the complete tissue culture media. HMSCs exhibited higher initial cell adhesion efficiency to 2-D HCG membranes, and maintained higher proliferation rates in the 3-D porous HCG than CG scaffolds with 3.3 times higher final DNA amount in HCG scaffolds over a 35-day period. Colony forming unit-fibroblast (CFU-F) assays showed that higher percentages of cells maintained their progenicity in the 3-D porous HCG scaffolds over the 35-day culture period. Differentiation assays indicated that the multi-lineage differentiation potential of the hMSCs was preserved in both 3-D porous scaffolds. However, higher alkaline phosphate activity was detected in the 3-D porous HCG scaffolds upon osteogenic induction indicating improved osteogenic differentiation potential. The results demonstrate that enhanced protein and calcium ion adsorption properties of HA in the CG polymer network improve initial cell adhesion and long-term growth, favor osteogenic differentiation upon induction, as well as maintain the progenicity of the 3-D hMSC constructs.