Design of Biomimetic Habitats for Tissue Engineering with P-15, a Synthetic Peptide Analogue of Collagen

Abstract

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.

Covalent attachment of P15 peptide to titanium surfaces enhances cell attachment, spreading, and osteogenic gene expression

“…While a number of earlier studies have highlighted the role of P15 to enhance bone formation, relatively few have focused on how it might be utilized to increase implant integration. 10,11,14,26,28,29 Previous studies by Bhatnagar et al, 11 demonstrated enhanced binding of cells to P15-coupled polyglycolate (PGA) fibers and mesh, compared to the PGA alone. Kubler et al, 30 demonstrated cells plated on P15/ABM formed multilayered sheets that clustered around the hydroxyapatite particles in contrast to cells plated on the other substrates which formed only monolayer sheets on the graft particles.…”

“…11,26 Ti and P15 tethered to a titanium (Ti-P15) foils were statically incubated at 378C for either 1 week or 3, or 6 months in air or phosphate buffered saline (PBS). At each time point, the retrieved foil was washed three times with PBS, incubated 30 min in blocking buffer followed by a 2 h incubation with rabbit anti-P15 (Cerapedics, Co).…”

“…9 Both collagen and peptides DGEA and P15 are thought to bind integrin receptors stimulating osteoblastic differentiation and expression of the osteogenic phenotype. 10,11 P15 is the common name of a synthetic amino acid peptide (GTPGPQGIAGQRGVV) that is charecterstic of the cell-binding domain of human collagen type I protein. 10 Currently, it is used as a coating on the surface of anorganic bone material (ABM), where it promotes osteoblast differentiation and enhances cell adhesion, migration, and survival.…”

Evaluation of ABM/P-15 versus autogenous bone in an ovine lumbar interbody fusion model

“…This product combines P-15, a synthetic 15-amino acid residue with a sequence identical to the cell-binding domain found on the a1(I) chain of Type-I collagen [3], onto an anorganic bovine-derived hydroxyapatite matrix (ABM) through a chemisorptive-coupling procedure [4]. The result is a complex that remains stable under physiologic conditions [5]. A delivery agent is also frequently used.…”

“…The mineral component (ABM) provides the necessary calcium phosphate and three-dimensional matrix needed for cellular invasion, while P-15 modulates cell binding by acting as an attachment factor for the a2-b1 integrin on the surface of boneforming cells. Attachment of these cells initiates a cascade of intra-cellular signaling that leads to extracellular matrix and factor production, cell differentiation, and subsequent osteogenesis [5,[8][9][10][11][12][13]. In an in vitro study by Yang et al [13] they showed that ABM/P-15 significantly stimulated human osteoblast synthesis of bioactive BMP-2 and alkaline phosphatase-specific activity of human bone marrow cells under both basal and osteogenic conditions.…”