Biological Designer Self-Assembling Peptide Nanofiber Scaffolds Significantly Enhance Osteoblast Proliferation, Differentiation and 3-D Migration

Akihiro Horii,Xiumei Wang,Fabrizio Gelain,Shuguang Zhang,Mark Isalan

doi:10.1371/journal.pone.0000190

Abstract

A class of self-assembling peptide nanofiber scaffolds has been shown to be an excellent biological material for 3-dimension cell culture and stimulating cell migration into the scaffold, as well as for repairing tissue defects in animals. We report here the development of several peptide nanofiber scaffolds designed specifically for osteoblasts. We designed one of the pure self-assembling peptide scaffolds RADA16-I through direct coupling to short biologically active motifs. The motifs included osteogenic growth peptide ALK (ALKRQGRTLYGF) bone-cell secreted-signal peptide, osteopontin cell adhesion motif DGR (DGRGDSVAYG) and 2-unit RGD binding sequence PGR (PRGDSGYRGDS). We made the new peptide scaffolds by mixing the pure RAD16 and designer-peptide solutions, and we examined the molecular integration of the mixed nanofiber scaffolds using AFM. Compared to pure RAD16 scaffold, we found that these designer peptide scaffolds significantly promoted mouse pre-osteoblast MC3T3-E1 cell proliferation. Moreover, alkaline phosphatase (ALP) activity and osteocalcin secretion, which are early and late markers for osteoblastic differentiation, were also significantly increased. We demonstrated that the designer, self-assembling peptide scaffolds promoted the proliferation and osteogenic differentiation of MC3T3-E1. Under the identical culture medium condition, confocal images unequivocally demonstrated that the designer PRG peptide scaffold stimulated cell migration into the 3-D scaffold. Our results suggest that these designer peptide scaffolds may be very useful for promoting bone tissue regeneration.

Highlights

Regenerative medicine and tissue engineering require two complementary key ingredients: 1) biologically compatible scaffolds that can be readily adopted by the body system without harm, and 2) suitable cells including various stem cells or primary cells that effectively replace the damaged tissues without adverse consequences
We report the functionalization of the RADA16 selfassembling peptide scaffold RADA16 (AcN–RADARADARADARADA–CONH2) through direct solid phase synthesis extension at the C-termini with three short motifs from the osteogenic growth peptide OGP, the Osteopontin cell adhesion motif described above, and a designed 2-unit RGD binding sequence (PRGDSGYRGDS). We showed that these designer peptide scaffolds significantly increased mouse preosteoblast MC3T3-E1 proliferation in comparison to pure RADA16 peptide scaffold
We functionalized RADA16 with three biologically active motifs in order to develop a second generation of self-assembling peptide scaffolds to closer mimic extracellular matrices that enhance preosteoblast cell maintenance and function in vitro

Summary

Introduction

Regenerative medicine and tissue engineering require two complementary key ingredients: 1) biologically compatible scaffolds that can be readily adopted by the body system without harm, and 2) suitable cells including various stem cells or primary cells that effectively replace the damaged tissues without adverse consequences. We previously reported the discovery and development of a class of self-assembling peptides made of only natural amino acids This class of material peptides can undergo spontaneous assembly into well-ordered nanofibers and scaffolds, ,10 nm in fiber diameter with pores between 5–200 nm and over 99% water content[1,2]. These peptide scaffolds have three-dimensional nanofiber structures similar to the natural extracelluar matrices including collagen. When mixed together with other porous polymer scaffolds, the peptide scaffolds enhanced osteoblast growth and differentiation, suggesting possible application for bone tissue engineering[5]

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