Hyaluronic acid oligosaccharides promote micro-vessel growth and regenerative ossification in biomimetic calvarial bone scaffolds

Bone regeneration requires well-orchestrated cellular and molecular interactions, particularly vascularization and osteoprogenitor migration and differentiation. Calvarial bone healing is featured by a temporal uncoupling of angiogenesis and osteogenesis: (ⅰ) osteoprogenitors derived from periosteum differentiate into osteoblasts at the injury site, followed by (ⅱ) osteogenic cells invade into the vascularized lesion. Promoting intra-defect micro-vessel growth may enhance osteogenic migration and accelerate ossification during calvarial bone repair. In this study, hyaluronic acid oligosaccharides (oHAs) were incorporated into tissue scaffolds, serving as the stimulators of micro-vessel formation and rising blood perfusion for calvarial bone repair. First, oHAs were tethered in chitosan (oHAs-CTS) and mineralized collagen (oHAs-mHAP) and then fabricated into porous bone scaffolds. The scaffolds' morphology, physicochemical/mechanical properties and degradation behaviors were systematically characterized. After 10 weeks in vivo implantation, oHAs-functionalized scaffolds achieved a 5.1-fold defect closure over blank control and a 2.6-fold over non-oHAs-functionalized groups. This enhancement of regenerative ossification was mechanistically linked to robust micro-vessel invasion within the scaffolds, as evidenced by quantitative analysis of vascular markers using molecular biology methods. While the mechanism underlying angiogenesis-osteogenesis coupling remains incompletely understood, our findings reveal that oHAs-functionalized scaffolds promote micro-vessel invasion and regenerative ossification during calvarial bone repair. STATEMENT OF SIGNIFICANCE: Low-molecular-weight hyaluronic acid oligosaccharides (oHAs), functioning as damage-associated molecular patterns (DAMPs), demonstrate paradoxical pro-angiogenic bioactivity through modulating endothelial cell behaviors, including proliferation, migration, and microvascular morphogenesis. Capitalizing on this mechanism, we engineered oHA-functionalized biomimetic bone scaffolds that significantly enhanced neovascularization in calvarial defect models. This strategy concurrently addresses two critical challenges: the inherent avascularity of engineered bone grafts and the impaired self-repair capacity of calvarial defects by promoting intra-construct microvascularization.