Dual-functioning peptides discovered by phage display increase the magnitude and specificity of BMSC attachment to mineralized biomaterials

Abstract

Design of biomaterials for cell-based therapies requires presentation of specific physical and chemical cues to cells, analogous to cues provided by native extracellular matrices (ECM). We previously identified a peptide sequence with high affinity towards apatite (VTKHLNQISQSY, VTK) using phage display. The aims of this study were to identify a human MSC-specific peptide sequence through phage display, combine it with the apatite-specific sequence, and verify the specificity of the combined dual-functioning peptide to both apatite and human bone marrow stromal cells. In this study, a combinatorial phage display identified the cell binding sequence (DPIYALSWSGMA, DPI) which was combined with the mineral binding sequence to generate the dual peptide DPI-VTK. DPI-VTK demonstrated significantly greater binding affinity (1/KD) to apatite surfaces compared to VTK, phosphorylated VTK (VTKphos), DPI-VTKphos, RGD-VTK, and peptide-free apatite surfaces (p < 0.01), while significantly increasing hBMSC adhesion strength (τ50, p < 0.01). MSCs demonstrated significantly greater adhesion strength to DPI-VTK compared to other cell types, while attachment of MC3T3 pre-osteoblasts and murine fibroblasts was limited (p < 0.01). MSCs on DPI-VTK coated surfaces also demonstrated increased spreading compared to pre-osteoblasts and fibroblasts. MSCs cultured on DPI-VTK coated apatite films exhibited significantly greater proliferation compared to controls (p < 0.001). Moreover, early and late stage osteogenic differentiation markers were elevated on DPI-VTK coated apatite films compared to controls. Taken together, phage display can identify non-obvious cell and material specific peptides to increase human MSC adhesion strength to specific biomaterial surfaces and subsequently increase cell proliferation and differentiation. These new peptides expand biomaterial design methodology for cell-based regeneration of bone defects. This strategy of combining cell and material binding phage display derived peptides is broadly applicable to a variety of systems requiring targeted adhesion of specific cell populations, and may be generalized to the engineering of any adhesion surface.

Cell and Material‐Specific Phage Display Peptides Increase iPS‐MSC Mediated Bone and Vasculature Formation In Vivo

“…Moreover, bone formed by cells transplanted on DPI‐VTK coated constructs exhibited increased cellularity and vascularization compared to uncoated, VTK and acellular controls (Figure ). Although seeding efficiency was greater on DPI‐VTK and serum‐coated BLM compared to VTK (Figure a, p < 0.05), the DPI peptide promotes cell specific attachment of MSC populations compared to RGD‐VTK . There was also a greater percentage of iPS‐MSCs within the innermost region of DPI‐VTK coated constructs compared to RGD‐VTK, VTK, and BLM (Figure ), indicating greater migration into the interior of the scaffold.…”

“…To meet the objective of selectively binding MSCs to mineralized biomaterials, we used phage display coupled with bioinformatic approaches and in vitro screening techniques to identify MSC‐specific [DPIYALSWSGMA, DPI] and apatite‐specific [VTKHLNQISQSY, VTK] sequences which were combined into a dual peptide [GGDPIYALSWSGMAGGGSVTKHLNQISQSY, DPI‐VTK] for cell specific adhesion to mineralized biomaterials . DPI‐VTK efficiently targets apatite and increases adhesion strength and specificity to human and murine MSCs and induced pluripotent stem cell derived mesenchymal stem cells (iPS‐MSCs) .…”

A specific affinity cyclic peptide enhances the adhesion, expansion and proliferation of rat bone mesenchymal stem cells on β‑tricalcium phosphate scaffolds

“…Based on the different binding affinity abilities of the cyclic peptide in a loop-constrained heptapeptide (Ph.D.™-C7C) phage display library and the target cell, the peptide that specifically bound to the BMSCs was selected through biopanning of the phage display technology. The biopanning procedure was performed following a previous described method with modifications (23,40).…”

“…Unfortunately, the low usage efficiency of exogenous BMSCs due to their poor adhesion on biomaterial scaffolds is a complication in BMSC-based tissue engineering (18,20,21). Bioactive molecules, particularly polypeptides, have been used to modify biomaterial scaffolds (19,(22)(23)(24). Compared with linear peptides which these studies have focused on (19,(22)(23)(24), cyclic peptides have A specific affinity cyclic peptide enhances the adhesion, expansion and proliferation of rat bone mesenchymal stem cells on β-tricalcium phosphate scaffolds several favorable properties including high affinity, selectivity and stability to protein targets (25).…”

“…Bioactive molecules, particularly polypeptides, have been used to modify biomaterial scaffolds (19,(22)(23)(24). Compared with linear peptides which these studies have focused on (19,(22)(23)(24), cyclic peptides have A specific affinity cyclic peptide enhances the adhesion, expansion and proliferation of rat bone mesenchymal stem cells on β-tricalcium phosphate scaffolds several favorable properties including high affinity, selectivity and stability to protein targets (25). Phage display technology has been widely used to screen small peptides with high affinity and specificity for targets of interest, including small molecules, cells, tissues and materials, through biopanning procedures (26)(27)(28).…”