Designing and synthesis of In-Situ hydrogel based on pullulan/carboxymethyl chitosan containing parathyroid hormone for bone tissue engineering

Abstract

There has been some confusion in daily life regarding craniofacial bone defects caused by aging, malignant lesions, or trauma. Injectable hydrogels have emerged as a promising method for healing bone defects and accelerating regeneration. In this study, we initiated the conversion of Chitosan (CS) to Carboxymethyl Chitosan (CMCS), followed by Pullulan (PL) to Pullulan Oxide (OPL). A rapidly in situ forming injectable oxidized carboxymethyl chitosan/pullulan (CMCS/OPL) hydrogel was developed through crosslinking. Additionally, to enhance osteogenesis, Parathyroid Hormone (PTH) was loaded into CMCS/OPL to obtain the (CMCS/OPL/PTH) hydrogel.In vitro evaluations involved the examination of sample structure and biological features through FTIR, SEM, swelling test, degradation test, PTH release, rheological behavior, MTT, live & dead assay, and alizarin Red S test. The results confirmed the successful creation of the Schiff base reaction. Notably, the hydrogel exhibited appropriate viscosity, porosity exceeding 90 %, and suitable degradation over 28 days accompanied by a continuous peptide release in both groups. Cell morphology and biocompatibility were favorable, with cell proliferation on day 7 in the CMCS/OPL/PTH hydrogel matching that of the control group. Furthermore, calcium deposition in the CMCS/OPL/PTH hydrogel after 21 days exceeded that in the group without PTH. Inspired by these results, the hydrogel was implanted into the mandibular bone defect in Sprague-Dawley rats. The in vivo study revealed accelerated bone regeneration and angiogenesis attributed to the rapid gelation rate and sustained release of PTH. In conclusion, this in-situ forming hydrogel exhibits considerable potential as a universal bone filler in clinical applications, facilitating osteoregeneration.