Development-Inspired Osteocyte-Derived Acellular Matrix Bioink for 3D-Printed In Situ Bone Regeneration

Cell-laden bioinks are widely used in bioprinting but remain limited by demanding storage, batch variability, immune-safety concerns and high manufacturing costs. To address these translational barriers, we developed MIND, a development-inspired osteocyte-derived acellular matrix bioink designed for additive manufacturing-enabled in situ bone regeneration. Primary osteocytes were pulse-activated with the small molecule S24 to enhance Wnt/β-catenin signaling, allowing deposition of a regenerative extracellular matrix. After mild decellularization, the matrix was incorporated into a GelMA-based photocrosslinkable ink and printed with a PCL framework to fabricate implantable composite scaffolds. Decellularized MIND retained favorable physicochemical and printing properties, with residual DNA <50 ng/mg, collagen/GAG retention >80%, shear-thinning behavior and BMSC viability >95%. Without exogenous cells or growth factors such as BMP-2 or VEGF, MIND promoted multilineage regenerative responses in vitro. BMSC ALP activity increased 3.2-fold, mineralized nodule area increased 4.5-fold, and Runx2, Osx and Ocn were upregulated. HUVEC tube branching increased 3.1-fold, with enhanced VEGFA/ANGPT1 expression and CD31/EMCN-positive type-H vessel signals. SH-SY5Y neuronal-like differentiation increased 2.5-fold, accompanied by TRPV1/TUBB3 upregulation and NeuN/β3-Tubulin-positive neural ingrowth in vivo. MIND also supported remodeling-associated osteoclast signaling, including moderate TRAP-positive multinucleated cell formation and Nfatc1/Ctsk upregulation. Under oxidative stress or spontaneous senescence-like conditions, MIND reduced SA-β-Gal-positive BMSCs by 65%, intracellular ROS by 70%, p16/p21 expression, γH2AX foci and SASP factors including IL-6, IL-1β and IL-8. RNA sequencing and pharmacological intervention with rapamycin and bafilomycin A1 suggested that BNIP3/BNIP3L-associated mitochondrial quality control contributed to the pro-osteogenic and stress-protective effects of MIND. In a rat 5-mm critical-sized calvarial defect model, the acellular MIND scaffold increased BV/TV to 53.5% at 4 weeks versus 3.6% in the blank group, and to 89% at 8 weeks, with improved mineralization, organized collagen birefringence, neurovascular ingrowth and balanced remodeling. These results demonstrate an acellular, matrix-instructive bioink platform that integrates printability, off-the-shelf potential and host-cell recruitment, providing a translational strategy for 3D-printed bone tissue engineering.

CHEN Jiafeng, PhD, Chongqing Medical University, under the supervision of Professor Tu Xiaolin. Her main research interest lies in bone tissue regeneration engineering, with a focus on the fabrication of functionalized decellularized matrix‑based bioinks and their application in critical‑sized bone defect repair.

Her research is dedicated to developing multi‑functional bioinks with immunomodulatory properties. By integrating key strategies such as neurotization and vascularization, the work aims to remodel the microenvironment for bone regeneration.