3D printed scaffolds regulated by neural-bone metabolic coupling promote bone unit regeneration

The bone unit (BU) is a multicellular functional unit composed of neuromodulatory networks, bone tissue, and functional blood vessels. As a functional extension of bone regeneration, the BU coordinates neural signal, regeneration of the circulatory network and remodeling of the bone matrix through the neural-bone metabolic coupling mechanism. Currently, effective strategies are lacking to remodel the homeostasis of the neural-bone metabolic coupling, which in turn leads to impaired BU regeneration. Here, we constructed a 3D-printed biomimetic hydrogel scaffold, GGMN-GSE, to synergistically couple nerve-bone metabolism and restore BU regeneration through electrophysiological microenvironment reconstruction and functional cell recruitment. The GGMN layer (Grooved GelMA@MXene@NGF) incorporates conductive MXene to emulate periosteal electrophysiology and features biomimetic microgrooves to guide neural orientation, collectively modulating neural-bone metabolic coupling. The GSE layer (GelMA@SVVYGLR-E7), engineered with a dual-targeting peptide, recruits BMSCs/EPCs to supply functional cells while remodeling the vasculo-osseous regenerative niche, synergistically driving osteogenic regeneration. In vitro, the GGMN-GSE scaffold promoted nerve fiber regeneration and induced secretion of neuropeptides such as CGRP, GHRH, and VIP, and activated Calcrl, Ghr, Vipr , and other receptors on the surface of BMSCs, driving BU regeneration. This effect was mediated by neural-bone metabolic coupling through the PI3K-Akt/STAT5 signaling axis. In vivo, compared with the control group, the density of CGRP + nerves in the GGMN-GSE group increased by 2.9-fold, the density of CD31 + blood vessels in the GGMN-GSE group increased by 1.8-fold, the area of the neogenetic bone matrix expanded by 3.6-fold, and the BV/TV reached 97.1 % in the CT. Confocal analysis of rat calvarial defects established that GGMN-GSE scaffolds exclusively promoted synergistic 3D co-localization of neural networks, functionalized vasculature, and osseous tissue, which drove multi-scale BU regeneration. The 3D-printed scaffolds fabricated in this study effectively remodel neural-bone metabolic coupling homeostasis to treat critical bone defects. This study designed a biomimetic periosteum-bone bilayer hydrogel scaffold system (GGMN-GSE) based on the concept of “neuro-osseous metabolic coupling”, constructed with an electro-mechano-chemical (E-M-C) mode. The biomimetic periosteum layer (GGMN): drives directional neuronal regeneration and functional maturation through MXene and microgroove topological structures; concurrently, NGF enhances the sensitivity of the CGRP/MAPK/ERK pathway, coupling neuro-osseous metabolism with conductive/topographical dual-mode signaling to accelerate bone unit (BU) remodeling. The biomimetic bone layer (GSE): employs the SVVYGLR-E7 peptide for targeted recruitment of BMSCs/EPCs, providing seed cells for BU reconstruction and inducing osteogenic and vasculogenic differentiation. This study promotes bone unit regeneration by constructing a neuro-osseous metabolically coupled biomimetic scaffold, providing a novel direction for the treatment of critical-sized bone defects resulting from various pathologies. • A neuro-osseous coupled bilayer hydrogel (GGMN-GSE) was engineered to deliver integrated multi-stimuli for bone unit regeneration. • The GGMN layer promotes neural regeneration via electro-mechano-chemical cues to regulate bone unit repair. • The GSE layer recruits BMSCs/EPCs via SVVYGLR-E7 peptide to induce osteogenic and vasculogenic differentiation.