Pilot in vitro study of the parameters of artificial niche for osteogenic differentiation of human stromal stem cell pool.

The aim of this research is experimental investigation of the topography and evaluation of some parameters of artificial microterritories promoting osteogenic differentiation of stromal stem cells. A technique of short-term culturing of prenatal human lung stromal cells with fibroblastoid morphology on calcium phosphate substrates with known topography was used. Judging from secretory activity of the cell culture (osteocalcin, alkaline phosphatase), stromal stem cells directly interacting with calcium phosphate discs have advantage in manifestation of osteoblast-like functional activity in comparison with cells cultured on plastic. Rough surfaces of calcium phosphate discs stimulate the formation of spatial human fibroblastoid cell culture. The cells with positive reaction to acid phosphatase are located on spheroliths forming the relief of calcium phosphate coatings. The cells with positive reaction to alkaline phosphatase (marker of osteoblasts) populate hollows (niches) of the artificial surface. The niche for induction of osteogenic differentiation of human multipotent mesenchymal stem cells is apparently a structural and functional formation. It can be characterized by an index calculated as the ratio of the total area occupied by alkaline phosphatase-positive cells to the area of artificial surface occupied by one stained cell.

Osteogenic potential of mesenchymal stem cells from bone marrow in situ: role of physicochemical properties of artificial surfaces.

Correlation analysis demonstrated the role of inorganic parameters of the surfaces of calcium phosphate materials in the regulation of osteogenic differentiation of mesenchymal precursors. The progenitor stromal cells were isolated from syngeneic bone marrow immobilized in vitro on calcium phosphate surfaces with different structure, phasic, and elemental composition. After 45 days of subcutaneous ectopic osteogenesis in BALB/c mice, the tissues grown on these matrixes were characterized histologically. It was found that adhesion of bone marrow cells is the initial stage determining their future proliferation (conduction) over the artificial surface and the area of formed tissue plate. The success of histogenesis depends on surface roughness. The optimal roughness class was 4-5 (Russian State Standards), which enables differentiation of progenitor stromal cells under the specific microenvironmental conditions into the connective and adipose tissue cells. Differentiation of the progenitor cells into the stromal cells producing the hemopoiesis-inducing microenvironment also takes place in the foci of active hemopoiesis. Induction of osteogenic potential of the stromal precursors (osteoinduction) is determined by the ratio between calcium and phosphate atoms in surface coatings. In our experimental system, osteogenic differentiation of stromal mechanocytes was blocked only at Ca/P<0.5.