Characterization of the microstructural properties that are predictive of regain in strength in phosphate-deficient mice
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Fracture healing occurs in a discrete set of steps, which recapitulates embryonic endochondral bone formation. Pathophysiologies of the fracture healing that prolong the fracture repair or result in non-union may be associated with either environmental or congenital deficiencies. Phosphate deficiency resulting from either dietary or genetic perturbations can impede proper fracture healing and if prolonged can result in delayed union. The clinical assessment of regain of mechanical function is determined by measuring weight-bearing ability, palpation and various radiological approaches. These methods in general only provide qualitative evidence but are lacking in quantitative evidence of the regain in mechanical strength. The aim of this study was to characterize the microstructural properties obtained by micro-computed tomography of fracture calluses at various stages of healing and develop correlations between these structural parameters and mechanical properties that define regain of function. Transverse, mid-diaphyseal fractures were produced on the right leg in three different murine genetic strains—A/J (AJ), C57BL/6J (B6), and C3H/HeJ (C3). Each mouse was either fed a control or phosphate deficient diet that produces a hypophosphatemic state and generates an environmental state that impairs fracture healing. Those on a phosphate deficient diet were kept on this diet for 14 days post-fracture. Fractured limbs were studied at four different post-operative time points—14, 21, 35, and 42. These four time points were based on callus stability and various phases in callus development. Contralateral limbs served as a control, representing full regain of strength. Day 0 contralateral limbs were used for the control group. Contralateral limbs were imaged and torsion tested for each strain on both control and phosphate-deficient diets. The data reveals that in regards to bone volume fraction, bone mineral density, and tissue mineral density all three strains show a progressive return to non-fractured, control values but even by post-operative day 42 do not show a 100% regain in microstructural properties. While there are interactions between specific post-operative time points and the dietary restriction, by post-operative day 42 microstructural properties showed no significant differences between the two groups, suggesting that the effects of phosphate deficiency are reversible upon a return to normal dietary conditions. The AJ and B6 strains show significant interaction between post-operative time point and dietary restrictions earlier in the fracture repair process (post-operative days 14 and 21), whereas the C3 mice show these interactions at later time points, at post-operative 35 and 42 days. Phosphate deficiency induces an overshoot in mechanical properties at post-operative day 21 for AJ and B6 strains and at post-operative day 35 for the C3 strain that appears to be part of a process in which maximum torque and work to failure undergo a compensatory phase in which these two mechanical properties are significantly higher than in non-fractured, control limbs. The overshoot in maximum torque and work to failure is part of an adaptive process in which the callus first overshoots and subsequently returns to non-fractured control values. These results suggest that while microstructural properties and mechanical properties are often affected by diet, this is a reversible phenomenon, which holds implications for those with phosphate deficiency due to either a metabolic or dietary disorder. If normal phosphate intake and absorption are achieved by the period at which couple remodeling in initiated (14 post-fracture) the effects on microstructural and weight bearing levels are reversible. The slower healing seen in C3 mice evidenced by the later regain in microstructural and mechanical integrity may provide a model for patients whose fractures show delayed healing in the clinic. The microstructural properties discussed have the potential to play a role in the clinic to assess fracture healing. With the advent of greater resolution CT imaging assessing these microstructural properties can be useful in determining the progression of healing.