Acidic phosphate microenvironment within calcium phosphate bone graft substitutes drives ectopic bone formation in mice

Material-induced heterotopic ossification (MIHO) by calcium-phosphate (CaP) bone graft substitutes (BGS) is difficult to anticipate because pore architecture, surface microstructure, and interfacial chemistry are often altered simultaneously. We compared six CaP granule formulations using an in-depth physical and chemical characterization. This included properties like granule size, pore architecture, intragranular concavities, specific surface area and microporosity, phase analysis, and controlled solution exposures that emulate confined fluid in pores to quantify pH, phosphate, calcium, and Ca/P release. These in-vitro readouts were paired with an intramuscular mouse model assessed at 35 and 56 days (early/late ossification centers, spicule density, bone area fraction, and standard host-reaction scores). Rank-based statistics mapped predictors to outcomes. Across materials, MIHO was observed primarily when two domains aligned. Physically, curvature-rich intragranular macropores were positively associated with the initiation of ectopic bone, while micro/nano-textured surface area within those pores was associated with later consolidation. Chemically, the most MIHO-competent materials tended to exhibit a phosphate-rich, mildly acidic interfacial milieu under confined-liquid conditions; this signature-captured by increased phosphate, decreased pH, and a reduced Ca/P release ratio-showed the strongest correlations with late histomorphometric endpoints. Apparent "phase effects" may largely reflect the interfacial chemistry those phases produced rather than intrinsic rankings based on solubility. The data suggest a simple, actionable rule set: concave, well-interconnected pores and micro/nano texture may help localize and sustain an acidic phosphate microenvironment that may bias soft tissues toward ectopic ossification. Practically, measuring ΔpH, ΔPO₄, and Ca/P under confined-liquid conditions may provide candidate in-vitro surrogates for MIHO and a rational basis to design CaP grafts either to promote osteoinduction where desired or to avoid it where it is a risk.