Mechanotransductive Osteogenesis Through Microarchitectural Stabilization in an Injectable Hydrogel–Mineral System

In the contemporary era of minimally invasive surgery, injectable biomaterial scaffolds have demonstrated significant potential in bone tissue engineering (BTE). Completely injectable hydrogel substratum with microscale bone graft particulates delivered through a needle-shaped orifice shifts a new paradigm for surgical interventions in clinical settings. Despite growing interest in biopolymer-based BTE systems, clinically applicable delivery platforms and a mechanistic understanding of cell–material interactions remain limited. This study developed a dual-syringe auto-mix system capable of generating an in situ cross-linking hydrogel–mineral construct composed of gelatin–hydroxyphenyl propionic acid, hyaluronic acid–tyramine, horseradish peroxidase, hydrogen peroxide, and calcium phosphate particles of varying sizes. Material distribution, rheological and mechanical properties, and swelling were characterized. Goat bone marrow-derived mesenchymal stem cells served as the basis for examining how the composite affected cell viability, morphology, proliferation, contractility, osteogenic differentiation, mineralization, and chemotactic behavior. To determine whether these biological findings were supported mechanically, an ex vivo cone-beam computed tomography model was used to evaluate volumetric stability and resistance to deformation at the graft–host interface. Cross-linking established a stable internal microarchitecture while remaining compatible with cell viability and nutrient-dependent survival. Formation of the gelatin–hyaluronan (GH) network significantly increased the storage modulus relative to gelatin (G) alone, whereas subsequent calcium phosphate incorporation (GH-CP) preserved this mechanical competence while attenuating the volumetric expansion of GH. These physical characteristics were accompanied by more organized cell morphology, enhanced osteogenic differentiation, and mineral deposition throughout a larger portion of the matrix. Heterogeneous interpenetrating gap striation (HIGS) appeared in regions of cellular aggregation and matrix deposition, and a new conceptualization of the osteogenic phenomenon, termed cling osteogenesis, has been proposed. These outcomes support an intricate relationship between early mechanical stabilization, mechanotransduction, and osteogenesis in injectable hydrogel–mineral systems.