Abstract: BACKGROUND: High cumulative remnant cholesterol levels are associated with the risk of various metabolic diseases, but their impact on bone quality remains to be explored.
OBJECTIVE: To investigate the effects of high cumulative remnant cholesterol levels on bone mass, microstructure, and biomechanical properties in high-fat diet treated mice.
METHODS: Ten healthy male SPF-grade C57BL6 mice were randomly allocated into normal control group and high-fat diet group. The normal control group was fed a normal diet for 20 weeks, while the high-fat diet group was fed a high-fat, high-cholesterol diet for 20 weeks. Mouse body mass was detected every week. After 20 weeks of feeding, samples were collected to measure serum total cholesterol, residual cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, cross-linked carboxy-terminal telopeptide of type I collagen, and N-terminal propeptides of procollagen type I levels. Micro-CT was used to assess the microstructure of trabecular and cortical bone in the femur. The three-point bending test was employed to determine the elastic modulus and maximum stress of the femur. RT-qPCR was used to detect mRNA expression of RUNT-related transcription factor 2, type I collagen, osteocalcin, alkaline phosphatase, osteoprotegerin, nuclear factor κB receptor activator ligand, activated T cell nuclear factor 1, and cathepsin K in the tibia. The Pearson correlation method was used to analyze the correlation between residual cholesterol, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol levels and bone mineral density, as well as the correlation between residual cholesterol levels and bone volume fraction, trabecular number, structural model index, and trabecular separation. 
RESULTS AND CONCLUSION: (1) Compared with the normal control group, the high-fat diet group showed significant increases in body mass, levels of serum remnant cholesterol and cross-linked carboxy-terminal telopeptide of type I collagen (P < 0.05). (2) Micro-CT analysis revealed that compared with the normal control group, the high-fat diet group showed more obvious degeneration of bone microstructure, specifically manifested as a significant decrease in trabecular bone mineral density, bone volume fraction, trabecular connectivity density, and trabecular number, and a significant increase in trabecular separation, structural model index, and trabecular pattern factor (P < 0.05), but no significant changes in cortical bone thickness, volume, and area (P > 0.05). Biomechanical analysis results indicated no significant differences in the elastic modulus and maximum stress of the femur between the two groups of mice (P > 0.05). (3) RT-qPCR analysis showed that compared with the normal control group, the mRNA expression levels of RUNT-related transcription factor 2, type I collagen, osteocalcin, alkaline phosphatase, and osteoprotegerin were significantly downregulated in the high-fat diet group (P < 0.05), while the mRNA expression levels of activated T cell nuclear factor 1 and cathepsin K were significantly upregulated in the high-fat diet group (P < 0.05). (4) Pearson correlation analysis revealed a negative correlation between remnant cholesterol and total cholesterol with bone mineral density (P < 0.05), with remnant cholesterol showing a significantly negative correlation with bone volume fraction and trabecular number (P < 0.05) and a significantly positive correlation with structural model index and trabecular separation (P < 0.05). These findings indicate that remnant cholesterol may impact bone quality by disrupting the balance of bone turnover.

Key words: remnant cholesterol, bone mineral density, bone volume fraction, trabecular numbe, structural model index, trabecular separation, connectivity density, trabecular pattern factor, biomechanics