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

Highlights

• Brtl/+ osteogenesis imperfecta mice were treated with bisphosphonates during fracture healing.

• Biomechanical testing and histomorphometric analysis showed altered healing dynamics.

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.

Introduction

Osteogenesis imperfecta (OI) is a genetic bone dysplasia that results in increased susceptibility to fractures during childhood and adolescence. The majority of mutations that cause OI are dominant defects in the genes encoding type I collagen [1], although recessive inheritance has been described [2], [3]. In most forms, these mutations cause a decrease in the amount of collagen or produce structurally abnormal type I collagen molecules and result in skeletal fragility. Most OI patients heal fractures normally [4], although there are reports of hypertrophic callus formation and delayed healing in some OI patients [5], [6], [7].

Bisphosphonates are antiresorptive medications used to treat OI patients in an effort to reduce fracture rates. These medications have been shown to be effective in reducing compression fractures in the spine [8]. A histomorphometric study of biopsies from OI patients showed increased bone formation and resorption parameters, suggesting the utility of bisphosphonate therapy in OI to control excessive bone turnover [9]. Osteoblasts and osteoclasts are both affected by bisphosphonates [10]. Bisphosphonates have a high affinity for bone mineral, and in an early assessment of alendronate, were calculated to have a half-life of over 10 years [11]. Bisphosphonate retention depends on the mechanism of delivery [12] and the rate of bone turnover. In pediatric patients, pamidronate has been detected in urine up to 8 years after cessation of treatment [13]. Furthermore, prolonged bisphosphonate treatment may play a role in abnormal bone modeling [14], radiographic metaphyseal sclerosis, osteonecrosis of the jaw, and possible accumulation of microdamage. These concerns have led to caution in bisphosphonate treatment in growing OI patients [15].

A number of clinical fracture studies with bisphosphonates have investigated the risk that disruption of underlying cellular activity with these antiresorptive agents might interfere with the normal fracture healing process. One randomized, double-blind, placebo-controlled trial with zoledronic acid following hip fracture did not show an increased risk for delayed union [16]. Similarly, in pediatric OI patients, bisphosphonate treatment is not usually associated with altered fracture healing, although intravenous pamidronate may lead to delayed osteotomy healing [17], [18]. Bisphosphonate treatment is often withheld during healing following osteotomy procedures [8], [19], [20], [21], [22].

Animal studies using bisphosphonates such as alendronate, zoledronic acid, incandronate and pamidronate generally indicate an increase in callus size and structural biomechanical changes [23], [24], [25], [26], [27], [28], [29], [30]. The biomechanical properties of the callus compared to intact bone have been shown to be similar or even improved [23], [31]. With these differences in callus mechanical properties and with concerns about impaired remodeling in the fracture healing process, fracture healing in growing OI bone in the presence of bisphosphonates was investigated.

We report here the results of a controlled fracture healing experiment with alendronate treatment using the Brtl/+ mouse model of OI. This knock-in model contains a glycine substitution mutation, models the small size and skeletal fragility that are clinical hallmarks of OI, and responds to alendronate therapy in a similar fashion to clinical patients [32], [33], [34], [35]. Treatment groups were halted at the time of fracture or continued during healing to investigate the most common clinical options. The dynamic callus process was examined at four timepoints during healing. The primary goals were to investigate fracture healing in the Brtl/+ mouse and the impact of alendronate on regenerating tissue.