The effect of pre-coating human bone marrow stromal cells with hydroxyapatite/amino acid nanoconjugates on osteogenesis

Aqueous colloidal suspensions of positively charged, amino acid-functionalized hydroxyapatite (HAp) nanoparticles (HAp/alanine and HAp/arginine) were added to a HBMSC suspension to effect non-specific cell surface deposition due to favourable attractive electrostatic interactions. Subsequent maintenance of these hybrid precursors under in vitro basal (non-osteogenic) culture conditions for up to 21 days, either as a monolayer or as a 3D pellet culture system, resulted in significantly increased levels of markers of osteoblast differentiation in comparison with uncoated cells. In monolayer culture, osteogenic activity could be further enhanced in a dose-dependent manner by surface derivatization of the amino acid-stabilized nanoparticles with the cell surface-specific binding peptide arginine-glycine-aspartic acid (RGD). Significantly, in 3D pellet culture conditions all HAp nanoconjugates promoted osteoblast differentiation, whereas for uncoated cells even soluble osteogenic culture additives were ineffectual. We therefore tested these constructs for in vivo activity by subcutaneous implantation in immunocompromised mice. New osteoid formation was observed in samples recovered after 21 days, comparable to the extensive areas of mineralized extracellular matrix produced in vitro. Overall, these studies outline the potential of biomolecular/hydroxyapatite nanoconjugates to promote osteogenic cell differentiation in vitro and hence provide new models to examine skeletal cell differentiation and function. Moreover, the pre-coating of HBMSCs enables the formation of viable hybrid multicellular 3D constructs with demonstrable activity both in vitro and in vivo.