Immobilization of collagen on hydroxyapatite discs by covalent bonding and physical adsorption and their interaction with MC3T3-E1 osteoblasts

Hydroxyapatite (HA), a type of calcium phosphate mineral, shows high biological affinity to living bone and is widely used as a substitute for filling bone defects. The purpose of this study was to immobilize collagen on HA discs via covalent bonding and physical adsorption and to evaluate their interaction with MC3T3-E1 osteoblasts. Type I collagen was immobilized on the surface of sintered HA discs in two ways. The first way was to immobilize collagen covalently on the surface of a HA disc (HA-C) through a coupling agent, 3-aminopropyltriethoxysilane. The second one was to immobilize collagen on the surface of a HA disc in the manner of physical adsorption (HAhC). The surface properties of HA immobilized with collagen were characterized and compared with non-immobilized HA (control). The potentiality of collagen-immobilized HA discs for use as bone substitutes or scaffolds were assessed by culturing MC3T3-E1 osteoblasts on the discs. The osteoblasts were cultured for 4 and 68 h on HA discs to examine cell adhesion and proliferation, respectively. Alkaline phosphatase (ALP) activity assay was used to study the in vitro differentiation of MC3T3-E1 osteoblasts when cultured on the modified HA discs. From the results, it was apparent that the osteoblasts had proliferated more on the HA-C disc than on the HA-hC disc. The better proliferation was probably attributed to the higher amount of collagen covalently immobilized than that immobilized by physical adsorption. It was found, from the ALP activity assay, that HA-C showed better osteoblastic differentiation than HA-hC. It was considered that the higher ALP activity of HA-C was mainly expressed by the larger amount of collagen immobilized on the HA surface.