Investigation of synergistic effects of inductive and conductive factors in gelatin-based cryogels for bone tissue engineering

Macroporous and biocompatible scaffolds for bone tissue engineering were prepared from 4% gelatin (G) and 4% gelatin/2% nanohydroxyapatite (nHAP), (GN), by cryogelation. The cryogels have interconnected pores with pore size around 100 μm and a high degree of cross-linking. The incorporation of nHAP slightly reduced the porosity, degree of crosslinking, swelling kinetics and equilibrium water uptake, but enhanced the toughness of the cryogel scaffolds. The osteo-regeneration potential of GN cryogels was further enhanced by binding with bone morphogenetic protein (BMP-2) to produce the gelatin/nHAP/BMP-2 (GNB) scaffold. The efficacy of BMP-2 incorporation was tested through in vitro release studies and a sustained release profile could be observed from the cumulative BMP-2 release curve. To elucidate the effect of cryogel composition on cell proliferation and differentiation, rabbit adipose-derived stem cells (ADSCs) were seeded in cryogel scaffolds. In vitro studies demonstrated a reduced proliferation rate and enhanced osteogenic differentiation of ADSCs in GNB cryogel scaffolds from the combined effect of nHAP and BMP-2, judging from the elevated alkaline phosphatase activity and the degree of mineralization. Confocal microscopy confirmed high viability and good cytoskeletal spreading of ADSCs on cryogels while osteocalcin (OCN) protein quantification affirmed the dominance of GNB in the osteogenic differentiation of ADSCs compared to G and GN cryogels. The maximum osteogenesis capability of GNB was also confirmed through the up-regulation of specific bone maker genes of early marker protein collagen I (COL I) and late marker protein osteopontin (OPN). From an in vivo animal model, computed tomography analysis confirmed the superior bone regeneration capability of ADSCs in GNB cryogels by implanting ADSCs/GNB cryogel constructs in rabbit calvarial critical size defects. Histological and immunohistochemical analysis demonstrated new bone formation and continued expression of COL I and OCN bone-specific proteins at the defect site. Taken together, the results demonstrate that G cryogels modified with osteo-conductive nHAP and osteo-inductive BMP-2 could provide cues to synergistically promote the osteogenesis of ADSCs in vitro and in vivo.