In-situ synthesis of angiogenic and osteogenic chitosan-whitlockite composite bone filler

The rising demand for efficient and effective substitutes for bone tissue engineering has led to the development of novel in-situ biomaterial composites. Current biomaterials fail to mimic the natural composition of the bone and are limited in their ability to supportadequate revascularization. To address this constraint, we developed a chitosan (CS)-whitlockite (WH) (CS-WH) composite bone filler via an in-situ method. CS mimics the glycosaminoglycans in the extracellular matrix, thereby aiding in the process of regeneration. In addition, WH promotes osteogenesis and angiogenesis, thereby assisting in bone revascularization. The prepared in-situ CS-WH composite bone filler was characterized using FE-SEM, TEM, EDAX, XRD, FTIR, XPS, and TGA/DTG. FE-SEM and TEM analysis of in-situ CS-WH composite bone filler showed an average particle size of 88 nm. EDAX, XRD and XPS analysis confirmed the presence of CS and WH elements in the prepared composite. TGA studies confirmed the thermal stability of CS-WH up to 800 °C. The in vitro ion release pattern of the composite indicated the sustained release of Ca²⁺, Mg²⁺ and PO₄³⁻ ions. The prepared composite showed a reasonably high dielectric constant of about 30 at low frequency (∼1 kHz), and it decreased to 5 at high-frequencies (∼0.8 MHz). The in-situ CS-WH composite bone filler was found to be biocompatible in dental follicle stem cells (DFSCs). In vitro tube formation and cell migration studies in human umbilical vein endothelial cells (HUVECs) demonstrated the angiogenic potential of the prepared composite. The osteogenic potential of the prepared composite was confirmed through expression studies of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), along with alkaline phosphatase activity (ALP) and mineralization studies in DFSCs. Thus, the developed composite bone filler represents a promising candidate for the regeneration and revascularization of alveolar bone defects.