Subtype-specific bone mineralization defects and early treatment amelioration in murine models of autosomal recessive osteopetrosis revealed by Raman spectroscopy.

Abstract

Autosomal recessive osteopetrosis is a rare genetic disorder caused by impaired osteoclast function, leading to excessive bone mass, defective remodeling, and fragility fractures. While bone density alterations are well recognized, compositional parameters remain poorly characterized. Here, we applied Raman spectroscopy to characterize bone defects in two murine models, the RANKL-deficient Rankl^-/- (mild form) and the TCIRG1-deficient oc/oc (severe form), compared with healthy controls. Raman analysis revealed a gradient in mineral-to-matrix ratio, progressively showing lower values from healthy to Rankl^-/- and to oc/oc (skull: 2.93 ± 0.02 vs. 2.19 ± 0.31 vs. 1.72 ± 0.15, p < 0.05; long bone: 3.59 ± 0.56 vs. 2.60 ± 0.53 vs. 1.86 ± 0.17, p < 0.05 for all pairs except Rankl^-/- vs oc/oc). Crystallinity showed overall lower values in osteopetrotic bones compared to WT, with partially overlapping distributions between Rankl^-/- and oc/oc mice (skull: 0.050 ± 0.001 vs. 0.046 ± 0.001 vs. 0.048 ± 0.001; long bone: 0.050 ± 0.001 vs. 0.047 ± 0.001 vs. 0.048 ± 0.001 for WT, Rankl^-/- and oc/oc, respectively). Multivariate analysis achieved 95% classification accuracy under exploratory animal-level cross-validation. In oc/oc mice treated with bone marrow transplantation, Raman analysis detected a significantly higher mineral-to-matrix ratio at day 18 compared to untreated oc/oc mice (2.668 ± 0.124 vs. 2.207 ± 0.311; p = 0.040, respectively). These findings identify genotype-specific compositional alterations in ARO models and provide proof-of-concept that Raman spectroscopy can characterize mineralization defects in intact bones ex vivo, suggesting that further development of non-invasive or minimally invasive Raman-based approaches may enable future studies of ARO progression and therapeutic response in clinically relevant settings.