Immunomodulatory fibrous hyaluronic acid-Fmoc-diphenylalanine-based hydrogel induces bone regeneration

Aim: To investigate the potential of an ultrashort aromatic peptide hydrogelator integrated with hyaluronic acid (HA) to serve as a scaffold for bone regeneration. Materials and Methods: Fluorenylmethyloxycarbonyl-diphenylalanine (FmocFF)/HA hydrogel was prepared and characterized using microscopy and rheology. Osteogenic differentiation of MC3T3-E1 preosteoblasts was investigated using Alizarin red, alkaline phosphatase and calcium deposition assays. In vivo, 5-mm-diameter calvarial critical-sized defects were prepared in 20 Sprague–Dawley rats and filled with either FmocFF/HA hydrogel, deproteinized bovine bone mineral, FmocFF/Alginate hydrogel or left unfilled. Eight weeks after implantation, histology and micro-computed tomography analyses were performed. Immunohistochemistry was performed in six rats to assess the hydrogel's immunomodulatory effect. Results: A nanofibrous FmocFF/HA hydrogel with a high storage modulus of 46 KPa was prepared. It supported osteogenic differentiation of MC3T3-E1 preosteoblasts and facilitated calcium deposition. In vivo, the hydrogel implantation resulted in approximately 93% bone restoration. It induced bone deposition not only around the margins, but also generated bony islets along the defect. Elongated M2 macrophages lining at the periosteum–hydrogel interface were observed 1 week after implantation. After 3 weeks, these macrophages were dispersed through the regenerating tissue surrounding the newly formed bone. Conclusions: FmocFF/HA hydrogel can serve as a cell-free, biomimetic, immunomodulatory scaffold for bone regeneration.