Cell and Material Specific Dual-Functional Peptides for Enhancing Mesenchymal Stem Cell Migration for Bone Tissue Engineering

Abstract

Millions of bone grafting procedures are performed annually to repair bone defects. While autografts and allografts are used clinically for such purposes, their limitations have motivated research into synthetic graft materials. Although numerous biocompatible materials have been developed, lack of bioactivity often results in suboptimal results. The use of bioactive peptides on the surface of a material can help create an interface between materials and specific cells in living tissue. Using phage-display technology, a dual-functional peptide DPI-VTK (GGDPIYALSWSGMAGGGSVTKHLNQISQSY) that contains both a mesenchymal stem cell (MSC) binding domain DPIYALSWSGMA (DPI) and a hydroxyapatite binding domain VTKHLNQISQSY (VTK) was developed. Because of the importance of adhesion molecules for cellular migration, such a peptide could not only enhance the adhesion of MSCs to hydroxyapatite but could also promote the specific migration of host MSCs, bypassing the need for cell transplantation. Additionally, this peptide could be combined with BMP peptides to create dual-functional osteoconductive and osteoinductive interfaces that encourage the homing of MSCs along with their subsequent differentiation. The overall hypothesis of this dissertation is that dual-functional MSC and apatite binding peptide DPI-VTK along with a dual-functional BMP and apatite binding peptide KIP-VTK will increase bone regeneration with mineralized scaffolds by increasing the migration of MSCs and their subsequent osteogenic differentiation. This dissertation proposed the following aims to test this hypothesis: 1) determine if peptide DPI-VTK can increase MSC specific migration in vitro and in vivo to support bone formation; 2) determine if peptide DPI-VTK has a synergistic effect with BMP derived peptide KIP-VTK to increase osteogenic differentiation. In transwell assays, human iPS-MSCs showed significantly greater migration (6-fold increase) over no peptide controls when DPI-VTK was used as a chemoattractant. Non-MSC controls such as MC3T3 cells and mouse dermal fibroblasts (MDFs) showed no statistically significant effect on migration from DPI-VTK. When MSCs were incubated in transwells with peptides DPI-VTK, DPI, and VTK, migration was equivalent for DPI-VTK and DPI, while VTK had no difference from the control, demonstrating that the DPI peptide, but not VTK, can induce migration. Primary calvarial cells had a significant increase in migration (>2-fold increase) when exposed to DPI-VTK. After 1 week in-vivo, immunohistochemical staining for MSC markers demonstrated increased migration of CD90 and CD200 positive cells. After 8 weeks, micro-CT demonstrated a significant increase in regenerated bone volume in the DPI-VTK group versus no peptide control. Coupling peptide VTK to BMP derived peptide KIP increased absorption to mineral. When co-absorbed with DPI-VTK, KIP-VTK failed to generate a synergistic effect on osteogenic differentiation. In conclusion, this research has demonstrated that targeting specific cell populations with cell and material binding peptides such as DPI-VTK is possible. Due to the widespread use of hydroxyapatite-based biomaterials in craniofacial bone grafting, DPI-VTK could be useful clinically for enhancing their ability to promote migration of host MSCs. Developing dual-functional material that both recruits host cells and induces differentiation could be a key strategy for regenerating large-volume defects without the need for exogenous cells or autografts.