Fracture healing with alendronate treatment in the Brtl/+ mouse model of osteogenesis imperfecta

Abstract

Osteogenesis imperfecta (OI) is a heritable bone dysplasia characterized by increased skeletal fragility. Patients are often treated with bisphosphonates to attempt to reduce fracture risk. However, bisphosphonates reside in the skeleton for many years and long-term administration may impact bone material quality. Acutely, there is concern about risk of non-union of fractures that occur near the time of bisphosphonate administration. This study investigated the effect of alendronate, a potent aminobisphosphonate, on fracture healing. Using the Brtl/+ murine model of type IV OI, tibial fractures were generated in 8-week-old mice that were untreated, treated with alendronate before fracture, or treated before and after fracture. After 2, 3, or 5 weeks of healing, tibiae were assessed using microcomputed tomography (μCT), torsion testing, quantitative histomorphometry, and Raman microspectroscopy. There were no morphologic, biomechanical or histomorphometric differences in callus between untreated mice and mice that received alendronate before fracture. Alendronate treatment before fracture did not cause a significant increase in cartilage retention in fracture callus. Both Brtl/+ and WT mice that received alendronate before and after fracture had increases in the callus volume, bone volume fraction and torque at failure after 5 weeks of healing. Raman microspectroscopy results did not show any effects of alendronate in wild-type mice, but calluses from Brtl/+ mice treated with alendronate during healing had a decreased mineral-to-matrix ratio, decreased crystallinity and an increased carbonate-to-phosphate ratio. Treatment with alendronate altered the dynamics of healing by preventing callus volume decreases later in the healing process. Fracture healing in Brtl/+ untreated animals was not significantly different from animals in which alendronate was halted at the time of fracture.

Keywords: Bisphosphonates; Bone; Fracture repair; Osteogenesis imperfecta; Rodent.

Fig. 1

Study design. Solid arrows indicate when no alendronate injections were given. Dashed lines indicate when weekly subcutaneous injections of alendronate were given. The following groups were tested for both Brtl/+ and wild type (WT) mice.

Fig. 2

Quantitative microcomputed tomography results for callus morphology and densitometry for pooled genotype data. (A) Callus volume and (B) bone volume fraction during healing. Tissue mineral densities (TMD) for the (C) bone in the callus and (D) residual cortical bone. Notations indicate significance (p b 0.05) with respect to no alendronate treatment (n), alendronate treatment before fracture (b) in parts A and B. Notations indicate significance (p < 0.05) with respect to 2 weeks of healing (#) or 3 weeks of healing (+) in parts C and D; i, p = 0.064.

Fig. 3

Representative sagittal planes of microcomputed tomography images from (A) WT mice after 5 weeks of healing and (B) from the intact tibiae of 9-week-old mice. “None” represents no alendronate treatment, “Before” is alendronate before fracture, and “Continued” is treatment before and after fracture.

Fig. 4

Biomechanical changes in fracture calluses based on genotypic and treatment protocol variations at 3 weeks of healing (A–C) and 5 weeks of healing (D–F). Stiffness (A, D), torque at failure (B, E) and energy to failure (C, F) are shown. Notations indicate significance (p < 0.05) with respect to no alendronate treatment (n), alendronate treatment before fracture (b), or between the genotypes (*). In (F), mice that received alendronate before and after fracture compared with those with alendronate before fracture (j, p = 0.053). Genotypes are compared where indicated in A, B, C, and D and are pooled in E and F to compare treatment protocols.

Fig. 5

Mean energy to failure at various timepoints during healing for mice that did not receive alendronate. There was decreased energy to failure in both genotypes after 2 weeks of healing. After 5 weeks of healing, there was a statistically significant difference (p < 0.05) between the fractured and intact tibiae for Brtl/+ mice.

Fig. 6

Parallelism index results for polarized light analysis after 5 weeks of healing. Notations indicate significance with respect to the cortical bone within a genotype (*, p < 0.05). For the cortical bone, there was no difference between the genotypes (w, p = 0.072).

Fig. 7

Raman microspectroscopy results. (A) Crystallinity (inverse full width at half height of 960 cm⁻¹ peak), mineral-to-matrix ratio, and carbonate-to-phosphate ratio for intact tibiae and fracture callus at 3 weeks of healing. (B) Crystallinity vs. mineral-to-matrix ratio in both genotypes. In (C), histograms for spectra taken at the surface (bottom) appear to be a reasonable sampling of spectra taken through the cortical thickness (top). Notations indicate significance with respect to genotype (*, p < 0.05). No significant differences were found by genotype (m, p = 0.076 and n, p = 0.075) or between treatment groups.