Early osteogenic signal expression of rat bone marrow stromal cells is influenced by both hydroxyapatite nanoparticle content and initial cell seeding density in biodegradable nanocomposite scaffolds

Incorporation of hydroxyapatite (HA) within a degradable polymeric scaffold may provide a favorable synthetic microenvironment that more closely mimics natural bone tissue physiology. Both incorporation of HA nanoparticles and alterations of the paracrine cell–cell signaling distance may affect the intercellular signaling mechanism and facilitate enhanced osteogenic signal expression among the implanted cell population. In this study we investigate the effect of the incorporation of HA nanoparticles into poly(propylene fumarate) (PPF) scaffolds on the surface properties of composite scaffolds and early osteogenic growth factor gene expression in relation to initial cell seeding density. The results of surface characterization indicated that HA addition improved the surface properties of PPF/HA composite scaffolds by increasing the roughness, hydrophilicity, protein adsorption, and initial cell attachment. Rat bone marrow stromal cells (BMSCs), which were CD34−, CD45−, CD29+, and CD90+, were cultured on three-dimensional (3-D) macroporous PPF/HA scaffolds at two different initial cell seeding densities (0.33 and 1.00 million cells per scaffold) for 8 days. The results demonstrated that endogenous osteogenic signal expression profiles, including bone morphogenetic protein-2, fibroblast growth factor-2, and transforming growth factor-β1, as well as the transcriptional factor Runx2, were affected by both HA amount and initial cell seeding density. Up-regulated expression of osteogenic growth factor genes was related to subsequent osteoblastic differentiation of rat BMSCs on 3-D scaffolds, as characterized by alkaline phosphatase activity, osteocalcin mRNA expression, and calcium deposition. Thus, the PPF/HA composite scaffold construction parameters, including amount of HA incorporated and initial cell seeding density, may be utilized to induce the osteoblastic differentiation of transplanted rat BMSCs.