Mechanical Loading and Phosphate Homeostasis in Rat Bone

Mechanical loading on bones, stimulates bone growth and strengthens the skeleton by activating key genes related to bone and mineral metabolism. Vitamin D and estrogen are crucial for maintaining bone health by regulating calcium, phosphate, and bone turnover. Investigating the interaction between mechanical loading and deficiencies in vitamin D and estrogen is important because these three factors play distinct roles in bone homeostasis, and understanding their combined effects can help improve strategies to prevent bone loss, particularly in conditions like osteoporosis. The objectives of this thesis were to: i) Determine the effects of mechanical loading, using four-point bending on bone expression of phosphate homeostasis-related genes and proteins (FGF23) in rats, ii) Determine the effects of another form of administration of mechanical loading: in vivo axial loading on the expression of phosphate-homeostasis-related genes in the setting of vitamin D deficiency and estrogen deficiency in rats, iii) Determine bone structure, biomechanical competence, and reference point indentation parameters in estrogen deficiency, vitamin D deficiency, and the combination of both in rats. iv) Review the literature on the effects on bone mass and structure of in vivo axial loading models in rats and mice. Mechanical loading influenced phosphate related gene expression by decreasing Fgf23 gene expression and increasing Mepe and Dmp1 gene expression four to eight hours after loading. Mechanical loading decreased serum FGF23 levels, six hours after loading. However, we did not observe loading-related changes in serum FGF23, bone FGF23 or capillary vessels FGF23 at ten days after loading. A single bout of axial loading decreased Fgf23 mRNA and increased Mepe mRNA expression six hours later; however, Dmp1 and Phex transcripts were not affected. We found estrogen deficiency to cause rapid deterioration of the trabecular bone within the four-week study period. We further showed an interaction of vitamin D and estrogen deficiency with regard to bone mineral density (BMD) and trabecular number (Tb.N), which resulted in trabecular bone loss. We also showed that estrogen deficiency affected trabecular bone strength by decreasing failure load in the epiphysis. In terms of the methodological aspects of loading protocols, we showed that peak load, frequency, and loading cycles are the major determinants of the outcomes of bone mass, bone mineral density, and bone formation rate. We identified modifying factors such as age, sex-steroid deficiency, and disuse to be essential determinants of loading experiments and associated bone outcomes. In conclusion, acute mechanical loading affects phosphate homeostasis and bone mineralization by regulating the expression of phosphate-homeostasis-related genes: Fgf23, Mepe, and Dmp1, along with serum FGF23, at six hours after loading. These findings support that mechanical loading provokes both paracrine and endocrine responses in bone. Axial loading of the ulna in these rats affected expression of Fgf23 and Mepe independent of vitamin D and estrogen deficiency. The rat model used in this thesis demonstrated interaction between vitamin D and estrogen deficiencies with regard to BMD and Tb.N, leading to bone loss. A review of the literature on axial loading in rodents revealed that it is important to account for methodological parameters of loading protocols such as peak load, frequency and number of loading cycles, to ensure comparability of mechanoresponse data and bone outcomes. It is also important to adjust these methodological parameters with regard to modifying factors such as age, sex-steroid deficiency, and disuse.