Engineering True Bone Regeneration with Functionalized Decellularized Extracellular Matrix: From Microenvironment Programming to 3D Bioprinting

Critical-sized bone defects remain a major clinical challenge due to the limited bioactivity and poor morphological adaptability of current graft materials. Here, we present a “microenvironment functional programming” strategy to engineer a smart decellularized extracellular matrix (dECM)‑based construct that recapitulates the native osteogenic niche and drives true bone regeneration.

Osteocytes were transiently activated with a small‑molecule Wnt agonist (S24) to upregulate Wnt/β‑catenin signaling, followed by gentle decellularization to obtain functionalized osteocyte‑derived dECM (WAO‑DM/MIND). This dECM was combined with GelMA to form a printable bioink and integrated with 3D‑printed polycaprolactone (PCL) scaffolds. The resulting construct retained >80% of collagen and glycosaminoglycans, with residual DNA <50 ng/mg, while preserving endogenous Wnt ligands and matrix‑bound bioactive cues.

In vitro, the functionalized dECM alone (without exogenous growth factors) simultaneously promoted osteogenic differentiation of BMSCs (3.2‑fold increase in ALP activity), induced moderate osteoclastogenesis, enhanced HUVEC tube formation and migration, and drove SH‑SY5Y neuronal differentiation. In vivo, implantation into rat critical‑sized calvarial defects achieved 89% bone volume/tissue volume at 8 weeks, with newly formed bone closely resembling native tissue in morphology and maturity. In a more challenging rat femoral segmental defect model, the construct achieved repair outcomes comparable to autograft. Mechanistically, the dECM activated BNIP3/BNIP3L‑mediated mitophagy in aged/stressed BMSCs, clearing damaged mitochondria, reducing ROS and DNA damage, reversing senescence phenotypes, and restoring osteogenic capacity—revealing a previously unrecognized anti‑senescence function of biomaterial‑derived ECM.

This work demonstrates, for the first time, the synergistic coordination of osteogenesis, osteoclastogenesis, angiogenesis, and neurogenesis on a single material platform, overcoming the limitation of single‑function bone substitutes. The cell‑free, off‑the‑shelf, and 3D‑printable system offers a transformative strategy for personalized regeneration of complex bone defects, particularly in aging populations.