Hydrocalumite‐Like Material Induces Early Osteogenic Priming and ECM Remodeling Without Effective Mineralization in Preosteoblasts

ABSTRACT Hydrocalumite‐like phases, belonging to the layered double hydroxide family, are potential biomaterials for bone repair; however, their cellular effects remain incompletely understood. Here, we evaluated a hydrocalumite‐like material of composition [Ca 2 Al(OH)₆]Cl· n H 2 O (abbreviated Ca 2 Al–Cl) in preosteoblastic MC3T3‐E1 cells, assessing viability, adhesion/signaling, osteogenic differentiation, extracellular matrix (ECM) remodeling, and cytokine profiles. Across a 0.002–0.2 mg mL −1 range of Ca 2 Al–Cl, preserved cell viability and membrane integrity were observed, with the highest concentration (0.2 mg mL −1 ) eliciting the largest increase in metabolic activity (~270% of control), with a progressive reduction at lower concentrations; quantitative Crystal Violet staining showed increased total attachment at 0.2 and 0.02 mg mL −1 , while early adhesion transcripts (Integrin‐β1, FAK, Src, PP2A, Cofilin) decreased. Cell‐cycle readouts revealed increased p21 and decreased CDK6, with CDK2/4 levels remaining unchanged. During 14‐day differentiation, Osteocalcin increased, and ALP staining revealed more foci, yet Alpl, Runx2, and Osterix/Sp7 decreased, and Alizarin Red showed no visible mineral increase; OPG/RANKL were not modulated, thus the regulatory axis of bone resorption was not altered. The collagen content, as determined by Picrosirius staining, matched that of the Control, whereas the osteogenic medium was lower. Gelatin zymography indicated relatively higher MMP‐2 (pro/intermediate/active) and lower MMP‐9 under Ca 2 Al–Cl. Cytokine profiling revealed increased levels of IL‐6 and IL‐33, accompanied by reduced IL‐1/IL‐1β and IL‐10, and unchanged TNF. Collectively, Ca 2 Al–Cl induces an ECM‐centric, early osteogenic priming phenotype characterized by selective remodeling (MMP‐2‐biased) and cytokine modulation without late mineral accrual under these conditions. These findings position Ca 2 Al–Cl as a potential modulator of the early bone regenerative microenvironment, a property that could be harnessed for applications where controlled matrix organization is prioritized over rapid mineralization. However, further studies quantifying ion release dynamics and utilizing 3D/co‐culture models are essential to elucidate the causal mechanisms and translate this priming effect into a functional regenerative outcome.