Injectable NGF-loaded double crosslinked collagen/hyaluronic acid hydrogels for irregular bone defect repair via neuro-guided osteogenic process

The structural and functional repair of critical-sized irregular bone defect is still considered a serious challenge. Most bone implant materials used in current clinic are not able to induce reconstruction of functional neural network, which is the key in bone regeneration via neural-osteogenic pathway. In this study, we aimed to manufacture a novel injectable double-crosslinked collagen/hyaluronic acid hydrogel loaded with nerve growth factor (NGF), which was capable of well filling irregularly shaped bone defects and inducing reconstruction of neural network. Hyaluronic acid (HA) was sequentially oxidized by sodium periodate and modified by methacrylic anhydride to prepare methacrylated oxidized HA (MOH) as a crosslinker. Subsequently, the Collagen-MOH@NGF (CMHN) hydrogel was established by Schiff base reaction and double-bond photocrosslinking. Our results demonstrated the excellent microporous structure, injectable, self-healing, in-situ rapid crosslinking properties of CMHN hydrogel along with suitable mechanics, rheology, swelling, degradation and biocompatibility. Significantly, the introduction of NGF endowed CMHN hydrogel with better biological properties than that without NGF. In vitro, CMHN hydrogel promoted DRG neuron growth and osteogenic differentiation of BMSCs mediated by NGF-calcitonin gene-related peptide (CGRP) axis. In vivo, CMHN hydrogel possessed a better bone repair effect than free NGF and blank hydrogel, with a long-lasting NGF-promoting neuro-osteogenic action within 8 w in a rat cranial critical defect model. Overall, the prepared injectable NGF-loaded collagen/HA dual network hydrogel is a promising material to facilitate functional and structural repair of complex bone defects.