Electrospun hydroxyapatite-containing chitosan nanofibers crosslinked with genipin for bone tissue engineering

Bone tissue regeneration remains a clinical challenge, particularly for large defects in orthopedic and craniofacial regions. Current solutions, such as autografts, face limitations due to donor site complications and limited availability. Alternative materials often fail to integrate effectively with native bone tissue. This integration issue is partly attributed to the absence of the periosteum, a critical layer of bone tissue that supports osteoblast activity and tissue remodeling. Prior research has shown that successful bone regeneration requires a scaffold that mimics the natural extracellular matrix in both structure and composition. However, no prior work had resolved how to emulate the periosteum's role in promoting osteoblast maturation. This gap motivated the development of composite scaffolds that combine structural and biochemical cues. The need for a scaffold that supports both mechanical integrity and osteogenic differentiation remains unmet. By integrating mineral components and crosslinking agents, researchers aim to address these limitations.

The goal of this study was to develop a composite scaffold that mimics the structural and biochemical properties of the periosteum to support bone regeneration. The researchers aimed to create a scaffold that provides a physical and mineralized environment similar to non-weight-bearing bone tissue. This scaffold was designed to promote osteoblast differentiation and maturation. The study focused on combining chitosan with hydroxyapatite nanoparticles and crosslinking with genipin. The specific problem addressed was the lack of integration between current scaffolds and host bone tissue. The motivation was to create a material that supports both mechanical stability and osteogenic activity. By using electrospinning, the researchers aimed to produce a nanofibrous structure that closely resembles natural extracellular matrices. The study also sought to evaluate how this composite material influences osteoblast behavior in vitro.

The researchers used electrospinning to fabricate nanofibrous scaffolds from chitosan. These scaffolds were modified to include hydroxyapatite nanoparticles and crosslinked with genipin. Scanning electron microscopy was employed to assess the ultrastructure of the fibers. Spectroscopic techniques confirmed the presence of hydroxyapatite within the composite fibers. The mechanical properties of the scaffolds were measured using Young's modulus analysis. The average fiber diameters were recorded before and after crosslinking with genipin. The study compared the performance of pure chitosan scaffolds with composite scaffolds containing hydroxyapatite. Mouse 7F2 osteoblast-like cells were cultured on these scaffolds to evaluate adhesion, proliferation, and osteogenic differentiation.

The composite scaffolds had an average fiber diameter of 227 ± 154 nm before crosslinking and increased to 335 ± 119 nm after genipin treatment. X-ray diffraction and Fourier transformed infrared spectroscopy confirmed the presence of hydroxyapatite in the composite fibers. The Young's modulus of the composite scaffolds was 142 ± 13 MPa, matching the mechanical properties of the natural periosteum. Cells cultured on composite scaffolds showed higher alkaline phosphatase activity compared to pure chitosan scaffolds. By day 14, alkaline phosphatase activity was 2.4 times higher in cells on composite scaffolds (p < 0.05). Osteonectin mRNA expression was also higher in cells on composite scaffolds over two weeks. Both pure and composite scaffolds supported cell adhesion and proliferation. The composite scaffolds demonstrated enhanced osteoinductivity compared to pure chitosan scaffolds.

The study found that crosslinking electrospun hydroxyapatite-containing chitosan with genipin produced composite scaffolds with mechanical properties similar to the natural periosteum. These scaffolds supported the adhesion, proliferation, and osteogenic differentiation of mouse 7F2 osteoblast-like cells. Alkaline phosphatase activity and osteonectin mRNA expression were significantly higher in cells cultured on composite scaffolds. The results suggest that the composite scaffolds create a microenvironment that emulates the physical and mineralized structure of non-weight-bearing bone extracellular matrix. The presence of hydroxyapatite in the scaffolds appears to enhance osteoinductivity. The genipin crosslinking improved the mechanical stability of the scaffolds. The researchers propose that these scaffolds might be useful for the repair and regeneration of maxillofacial defects and injuries. The findings indicate that the composite scaffolds could facilitate the proliferation, differentiation, and maturation of osteoblast-like cells.