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Because bisphosphonates (BPs) are potent inhibitors of bone resorption, we hypothesized that they would retard direct remodeling of stress fractures. The aim of this study was to determine the effect of risedronate on direct remodeling and woven bone callus formation following stress fracture formation in the rat ulna. In 135 adult female Wistar rats, cyclic loading of the ulna created stress fractures. Rats were treated daily with oral saline, or risedronate at 0.1 or 1.0 mg/kg. From each bone, histomorphometry was performed on sections stained with toluidine blue at a standard level along the fracture. The high dose of risedronate caused a significant decrease in the percentage of repaired stress fracture and bone resorption along the stress fracture line at 6 and 10 weeks after loading (p < 0.05). At this dose, intracortical resorption was significantly reduced at 10 weeks after loading and intracortical new bone area was significantly reduced at 6 and 10 weeks. Woven bone formation and consolidation phases of stress fracture repair were not affected by low or high doses of risedronate. In conclusion, high dose bisphosphonate treatment impaired healing of a large stress fracture line by reducing the volume of bone resorbed and replaced during remodeling. We also confirmed that periosteal callus formation was not adversely affected by risedronate treatment. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1827–1833, 2011
Bisphosphonates are potent inhibitors of osteoclasts and bone resorption, and are widely used to treat conditions of excessive bone remodeling, including osteoporosis. In osteoporosis, reduced bone mechanical properties can lead to fatigue damage even under normal loading patterns. Many osteoporotic fractures are complete fractures, however some will occur in the form of incomplete fractures similar to stress fractures seen in athletes.1, 2 While bisphosphonates reduce the risk of fractures in osteoporosis, fractures still occur during treatment, so it is necessary to understand the effect of these medications on subsequent fracture healing.2–5
Stress fractures are an important clinical condition commonly affecting athletes, soldiers, and dancers.6 They are a form of fatigue injury associated with repetitive loading. Excessive bone remodeling in response to repetitive loading during training may contribute to the development of stress fractures1, 7 and it is likely that there will be further interest in bisphosphonates for the prevention and treatment of remodeling associated with fatigue injury in bone. The use of bisphosphonates has been proposed as a preventive measure against stress fractures.8, 9 However a trial in army recruits failed to show any preventive efficacy of risedronate therapy.8 Treatment with pamidronate was reported to produce a beneficial effect in clinical stress fractures in young athletes,10 but this study only had small numbers of cases. Of clinical interest, there have been recent case series of displaced transverse diaphyseal femoral fractures with minimal trauma in patients after long-term bisphosphonate treatment.2 These fractures were classified as being typical of stress fractures.4, 11 There is however, very little information available on the effect of bisphosphonate treatment on progression or repair of stress fractures.
There is also a growing focus on the use of bisphosphonates for a variety of orthopedic conditions that can be affected by an imbalance between catabolic and anabolic processes during bone healing. In experimental studies of complete fracture in rats, treatment with bisphosphonates consistently increased the hard callus volume, strength and mineral content.3, 12, 13 In animal models, stress fractures heal by direct remodeling along the fracture line.14, 15 Treatment with bisphosphonates may therefore have a negative effect on stress fracture healing when compared to complete fractures. For example, alendronate reduced the number of resorption spaces observed along stress fracture lines induced by ulnar loading in the rat. The woven bone response was not affected by alendronate treatment initiated before or after loading.9 These effects were, however, only examined at a maximum of 2 weeks following loading. Another study using the rat ulna loading model demonstrated that treatment with aledronate did not affect resorption space area, repaired crack length or woven bone area at 2 and 4 weeks after stress fracture creation but that mineral apposition rate and bone formation rate were decreased at 4 weeks after loading.16 While mechanical properties were not significantly altered by alendronate treatment at 8 weeks after loading, histology was not examined at this later timepoint.16
Repetitive axial loading of the rat ulna has been used to create reliable stress fractures and associated woven bone. The histology and gene expression profile of stress fracture healing in this model has been extensively characterized previously.15 Stress fracture healing in this model consists of two distinct healing components; the rapid woven bone response on the periosteal surface and direct remodeling, in the form of numerous basic multicellular units (BMUs) progressing along the fracture line.14, 15, 17, 18 The aim of this study was to determine the effect of long-term daily risedronate treatment on the healing of stress fractures in the rat ulna. High and low doses were selected to determine if a dose–response existed, and oral treatment was used to represent a clinical protocol. The gastrointestinal tract absorbs bisphosphonates quite poorly. However, most clinical dosing is still by oral administration and effective doses can be administered this way with few adverse effects.19
Previous studies have used thick sections of plastic embedded bone to examine the histomorphometry of stress fracture healing in the rat ulnar loading model.9, 14 This provides a method for measuring flourochrome labels and bone formation indices, but does not allow for detailed examination of resorption and formation indices of healing along the fracture line. We used thin sections of paraffin embedded bone in this study to allow a detailed analysis of the cellular resorption and formation activity within the BMUs, and thus provide valuable information on the effect of bisphosphonates on the progression and activity of cells within a BMU. Stress fracture healing and woven bone response was examined out to 10 weeks after loading to determine the long-term effect of risedronate treatment on stress fracture healing and woven bone consolidation.
We hypothesized that risedronate treatment would impair and delay the resorption and subsequent bone formation within the BMUs during remodeling along the fracture line. We also hypothesized that treatment with risedronate would not prevent the woven bone response but consolidation of the woven bone, as measured by the reduction in woven bone area over time, would be impaired.
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- MATERIALS AND METHODS
Risedronate is one of the nitrogen-containing bisphosphonates, widely used clinically, particularly to treat osteoporosis.21, 22 Bisphosphonates are potent inhibitors of osteoclastic bone resorption and have effects on osteoclast recruitment, differentiation, activity, and apoptosis.23–26 This study examined the effects of bisphosphonate treatment on stress fracture healing over an extended period. In contrast to complete fractures, stress fractures require early resorption during healing to enable new bone formation along the fracture line.15 We observed that daily risedronate treatment, at a dose equivalent to twice that recommended for treatment of osteoporosis, impaired stress fracture healing in the rat ulna up to 10 weeks after loading. Measures of resorption and subsequent new bone formation associated with the fracture line were significantly reduced at 6 and 10 weeks after loading and the length of the repair along the crack was significantly reduced at 2, 4, and 6 weeks after loading.
Most studies elect to use a subcutaneous administration route for risedronate studies in animals. However, we decided to use the oral route as this is the routine method of administration of risedronate in human patients. We selected 2 dose rates to determine if a dose response could be detected and the high dose rate (1.0 mg/kg) was selected to ensure an anti-resorptive effect was likely to occur while remaining within a clinically relevant dose range. Oral dose rates of 0.5 and 2.5 mg/kg risedronate prevented bone loss in an ovariectomy model in rats while 0.1 mg/kg had no effect.27 Reported results of pre-clinical testing for risedronate suggest that the dose rate needed in a rat to be equivalent to the standard 35 mg/kg/week human dose is 0.5 mg/kg.a
Bone resorption along the stress fracture line was clearly depressed by treatment with risedronate. The area of intracortical resorption was significantly reduced in the high-dose group at 10 weeks after loading. The stress fracture in this model heals by remodeling with BMUs progressing along the crack. The length of the crack undergoing healing depends on the progression of both osteoclastic and osteoblastic activity, and that length was significantly reduced in the high-dose treatment groups at all timepoints. New bone formation was also significantly reduced by treatment at the high dose, 6 and 10 weeks after loading. This likely reflects the decreased resorption surface available for new bone formation. The ulnar loading model has provided a method for examining the effects of BP treatment on the progression of focal BMUs over a predictable time-course and direction, at a known anatomical location. These results demonstrate that BPs can delay and reduce the volume of bone resorbed during the initiation and progression of remodeling in response to a large fatigue crack.
The reduction in newly formed bone along the stress fracture line is consistent with previous work demonstrating that alendronate reduced bone formation rate and mineral apposition rate at 4 weeks after loading in this model.16 The reduced bone formation within BMUs along the fracture line in this study was likely to have resulted from the reduced bone resorption during fracture remodeling, rather than a direct effect of risedronate on bone formation. There is some evidence that bisphosphonates can affect bone formation independently of osteoclast activity. Although bisphosphonates promote apoptosis of osteoclasts, they also reduce apoptosis of osteocytes and may inhibit osteoblast activity.28–30 Mineral apposition rate on the periosteal surface of the femur and tibia was reduced with risedronate or alendronate treatment in growing rats. As this periosteal modeling should be independent of osteoclast activity, this may indicate a direct effect of bisphosphonates on osteoblast activity.31 Most bisphosphonate compound is bound to bone mineral and only osteoclasts are able to release and take up bound bisphosphonates.32 The effects of bisphosphonates on osteoclast function are therefore likely to be more prominent than effects of transiently unbound bisphosphonates on other cell types.32, 33
In the current study, treatment with risedronate at high and low doses following stress fracture formation did not change the area or thickness of the woven bone. The woven bone in this model restores the mechanical properties of the ulna by 2 weeks after loading,34, 35 and this woven bone response was unaffected by risedronate treatment even at the high dose. Woven bone formation following rat ulnar loading was also unaffected by alendronate treatment given for 2 weeks prior to loading.9 In complete fractures, proliferative new bone is not affected by treatment with bisphosphonates. In complete fractures in rats, bisphosphonate treatment does not alter the rate of bone union and consistently results in a larger callus.12, 13, 36 Similar results were observed in models using dogs and sheep.37, 38 In these studies, mechanical properties of the callus were improved3, 12, 13, 36 or not significantly changed by treatment with bisphosphonates.38, 39 Woven bone is a prominent feature of clinical stress fractures and provides mechanical protection to a stress fracture line.40, 41
The area of the woven bone was not significantly affected by bisphosphonate treatment in the current study, even at 10 weeks after loading. The woven bone area decreased in the period between 2 and 6 weeks after loading, in all treatment groups. This indicates that the consolidation of this woven bone was not affected by osteoclast inhibition. It is possible that there is such a prolific resorption response that osteoclast recruitment and activity over the large surface area of highly vascular periosteum is able to progress sufficiently to compensate for ongoing inhibition and loss of osteoclasts by bisphosphonates.
Bisphosphonates are not currently used clinically to treat stress fractures in athletes. Nonetheless, there is growing interest in their potential to prevent excessive remodeling that may predispose to stress fracture development. Milgrom et al.8 tested this hypothesis by treating Israeli military recruits with Risedronate during basic training. Recruits were treated with a loading dose of 30 mg of risedronate daily for the first 10 days then the standard dose rate of 30 mg weekly for a further 12 weeks. This did not reduce the incidence of stress fractures compared to placebo treatment. However, the authors suggested that the dose used may have produced an excessive reduction in remodeling.8 The effect of bisphosphonate treatment on the subsequent healing of the stress fractures that did occur was not reported. In the ulnar loading model, 2 weeks of treatment with alendronate, prior to loading, did not protect the bone from fatigue damage.9 However, that effect was tested using a single loading session where remodeling could not have contributed to the development of the resulting stress fractures. The stress fracture produced in the rat ulna model differs in its creation to those observed clinically. In particular, the effects of bisphosphonate treatment on the progressive accumulation of fatigue damage and remodeling over time, prior to fracture creation, cannot easily be examined using this model. Diaphyseal fractures of the femur, in patients on long-term bisphosphonates, have also been described as fatigue fractures.2, 4, 5, 11 Although we have not studied long-term treatment using a clinical dose, our data suggest a possible mechanism whereby inhibition of direct remodeling of the stress fracture line could retard its repair, leading to greater bone fragility.
The results of this study demonstrate that a bisphosphonate can delay and reduce the volume of bone resorbed during the initiation and progression of remodeling in response to a large stress fracture line. However woven bone formation and subsequent consolidation were not affected by high doses of risedronate even over 10 weeks duration. This suggests that the periosteal reaction to stress fracture is not negatively affected by risedronate, even when treatment is continued after the stress fracture was sustained. This woven bone response is able to return the ulna to its original strength within 2 weeks of stress fracture creation in the rat ulna loading model.35, 42 It is therefore possible that woven bone callus is able to protect the site of the fracture and reduce any detrimental effects of a delay in stress fracture remodeling in clinical stress fracture cases.