Type I collagen provides a structural framework for connective tissues and plays a central role in the temporal cascade of events leading to the formation of new bone from progenitors. The aim of this study was to examine the ability of the cell-binding domain of type I collagen (P-15 peptide) to promote human bone marrow stromal cell adhesion, proliferation, and differentiation on three-dimensional scaffolds. Human bone marrow stromal cells were selected, expanded, and cultured on particulate microporous ABM ("pure" hydroxyapatite) phase adsorbed with or without P-15 under basal or osteogenic conditions. Immobilized P-15 increased alkaline phosphatase activity and bone morphogenetic protein 2 (BMP-2) gene expression after 1 and 5 days as determined by real-time polymerase chain reaction. P-15 promoted human bone marrow stromal cell attachment, spreading, and alignment on ABM as well as alkaline phosphatase-specific activity in basal and osteogenic cultures. The presence of mineralized bone matrix, extensive cell ingrowth, and cellular bridging between three-dimensional matrices adsorbed with P-15 was confirmed by confocal microscopy, scanning electron microscopy, and alizarin red staining. Negligible cell growth was observed on ABM alone. In vivo diffusion chamber studies using MF1-nu/nu mice showed bone matrix formation and organized collagen formation after 6 weeks. These studies indicate the potential of P-15 to generate appropriate biomimetic microenvironments for osteoblasts and demonstrate the potential for the exploitation of extracellular matrix cues for osteogenesis and, ultimately, bone regeneration.

The process of bone growth, regeneration, and remodeling is mediated, in part, by the immediate cell-matrix environment. Osteoblast stimulating factor-1 (OSF-1), more commonly known as pleiotrophin (PTN), is an extracellular matrix-associated protein, present in matrices, which act as targets for the deposition of new bone. However, the actions of PTN on human bone progenitor cells remain unknown. We examined the effects of PTN on primary human bone marrow stromal cells chemotaxis, differentiation, and colony formation (colony forming unit-fibroblastic) in vitro, and in particular, growth and differentiation on three-dimensional biodegradable porous scaffolds adsorbed with PTN in vivo. Primary human bone marrow cells were cultured on tissue culture plastic or poly(DL-lactic acid-co-glycolic acid) (PLGA; 75:25) porous scaffolds with or without addition of recombinant human PTN (1 pg-50 ng/ml) in basal and osteogenic conditions. Negligible cellular growth was observed on PLGA scaffold alone, generated using a super-critical fluid mixing method. PTN (50 μg/ml) was chemotactic to human osteoprogenitors and stimulated total colony formation, alkaline phosphatase-positive colony formation, and alkaline phosphatase-specific activity at concentrations as low as 10 pg/ml compared with control cultures. The effects were time-dependent. On three-dimensional scaffolds adsorbed with PTN, alkaline phosphatase activity, type I collagen formation, and synthesis of cbfa-1, osteocalcin, and PTN were observed by immunocytochemistry and PTN expression by in situ hybridization. PTN-adsorbed constructs showed morphologic evidence of new bone matrix and cartilage formation after subcutaneous implantation as well as within diffusion chambers implanted into athymic mice. In summary, PTN has the ability to promote adhesion, migration, expansion, and differentiation of human osteoprogenitor cells, and these results indicate the potential to develop protocols for de novo bone formation for skeletal repair that exploit cell-matrix interactions.