Study of synthetic peptide conjugated hydrogel on bone regeneration

Abstract

Stem cell carrying biomaterials including porous scaffolds, membranes have gained some success in bone regeneration. Among them, injectable hydrogel systems have received much attention due to their versatile and tunable characteristics and ease of application in the defect without the need of surgery. In addition, bioactive agent including protein, peptide and genetic materials also can be loaded into the hydrogel and the controlled release of these agent can favor the bone regenerative potential. Despite the efficacy of these bioactive molecules, however, the effective loading into the gel has been limited to the physical mixing. The simple mixture of bioactive agent and hydrogel have limited release control, due to burst release, therefore the alternative conjugating method of molecules and hydrogel is required. The chemical conjugation of protein with hydrogel might alter the efficacy of the protein, therefore it was anticipated that the synthetic peptide with functional moiety with chemical conjugation would be an alternate for the hydrogel conjugation. Herein, synthetic peptide from osteopontin termed as collagen binding motif (CBM) was attempted as it has defined osteogenic activity from the previous studies. The CBM peptide (CBMP) has osteogenic differentiation capacity to mesenchymal stem cells and thus been chosen in combination with hydrogel. The hydrogel has tyrosine functional group, and was designed to chemically conjugating with matching tyrosine group under the triggering by hydrogen peroxide with horseradish peroxidase (HRP). The CBMP possesses tyrosine functional group, therefore hydrogel crosslinking was attempted with CBMP under the H2O2/HRP condition even containing stem cells. The CBMP-crosslinked hydrogel demonstrated well survival of incorporating stem cells. The CBMP-crosslinked hydrogel increased osteogenic differentiation of cultured stem cells, as reflected by the increased osteogenic marker protein expression, calcein acetoxymethyl ester (calcein AM) staining, alkaline phosphatase activity staining, mineral deposition measured by Energy Dispersive Spectrometer (EDS). The further in vivo study confirmed the stem cells in the CBMP-crosslinked hydrogel significantly improved bone formation capacity in the rat calvarial defect as similar extent to that of BMP-containing hydrogel. Taken together, the CBMP conjugating hydrogel was demonstrated to have increased potential for osteogenic differentiation of stem cells, and moreover can be suggested as a tool as bioactive delivery vehicle of stem cells in tissue regenerative practice.