Design of biomimetic habitats for tissue engineering with P-15, a synthetic peptide analogue of collagen

In tissues, collagen forms the scaffold for cell attachment and migration, and it modulates cell differentiation and morphogenesis by mediating the flux of chemical and mechanical stimuli. We are constructing biomimetic environments by immobilizing a collagen-derived high-affinity cell-binding peptide P-15 in three-dimensional (3-D) templates. The cell-binding peptide can be expected to transduce mechanical forces. In their physiological environment, periodontal ligament fibroblasts (PDLF) are subject to significant mechanical forces. We have examined the behavior of human PDLF in culture on particulate bovine anorganic bone mineral (ABM) coated with P-15 (ABM-P-15). Greater numbers of cells associated with ABM-P-15 compared to ABM alone. Higher levels of incorporation of radiolabeled precursors in DNA and protein were consistent with the presence of larger numbers of cells on ABM-P-15 compared to ABM cultures. Scanning electron microscopic examination showed that cultures on ABM-P-15 generated highly oriented 3-D colonies of elongated cells and formed copious amounts of fibrous as well as membranous matrix reminiscent of ligamentous structures. PDLF cultured on ABM formed sparse monolayers with little order and a meager matrix. Alizarin Red stained the matrix of particle associated cells and inter-particle cellular bridges in P-15-associated cultures, indicating mineralization. 3-D colony formation and ordering of cells along with increased mineralization suggests that the coupling of cells to the ABM matrix through P-15 may provide a biomimetic environment permissive for cell differentiation and morphogenesis. Our studies suggest that ABM-P-15 templates may be effective as endosseous grafts, and, when seeded with PDLF, these matrices may serve as tissue engineered substitutes for autologous bone grafts.