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Zoledronic Acid Improves Femoral Head Sphericity in a Rat Model of Perthes Disease

Posted on: Sunday, 7 August 2005, 03:01 CDT

Abstract

We hypothesized that the bisphosphonate zoledronic acid (ZA) could improve femoral head sphericity in Perthes disease by changing the balance between bone resorption and new bone formation. This study tests the effect of ZA in an established model of Perthes disease, the spontaneously hypertensive rat (SHR).

One hundred and twenty 4-week old SHR rats were divided into three groups of 40: saline monthly, 0.015 mg/kg ZA weekly, or 0.05 mg/kg ZA monthly. At 15 weeks DXA measurements documented that femoral head BMD was increased by 18% in ZA weekly and 21% in ZA monthly compared to controls (p < 0.01). Femoral head sphericity in animals with osteonecrosis was improved in ZA-treatment groups (p < 0.01) as measured by epiphyseal quotient (EQ). The proportion of "flat" heads (EQ ≤ 0.40) was significantly reduced from 32% in saline-treated animals to 12% in weekly ZA and 3% in monthly ZA (p < 0.01). Histologically there was a similar prevalence of osteonecrosis in all groups. The prevalence of ossification delay was significantly reduced by ZA treatment (p < 0.01).

Zoledronic acid favorably altered femoral head shape in this spontaneous model of osteonecrosis in growing rats. Translation of these results to Perthes disease could mean that deformity of the femoral head may be modified in children, perhaps reducing the need for surgical intervention in childhood and adult life.

2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Keywords: Perthes disease; Bisphosphonates; Osteonecrosis; Ossification delay; Histomorphometry

Introduction

Perthes disease is a common idiopathic form of osteonecrosis in childhood, with an incidence of 8.5-21 per 100,000 children per annum [8,13,19]. The etiology of Perthes disease remains unknown, but the consensus of opinion suggests that a vascular insult results in a period of relative ischemia and subsequent osteonecrosis [9]. Rapid resorption of this necrotic bone causes weakening of the growing femoral head, leading to deformity and eventual collapse. Although differing processes may occur in different parts of the same femoral head at any one time, bone formation fails to keep pace with resorption, leading to femoral head deformation [3]. The long- term prognosis in Perthes disease is directly referable to femoral head sphericity. It is well documented that aspherical heads with incongruent joints commonly progress to degenerative osteoarthritis of the hip relatively early in adult life [29]. Therefore, maintenance of femoral head sphericity has become a main focus in the treatment of Perthes disease.

Current treatments aim to restore containment of the femoral head, such that remodeling takes place in close conformation with the acetabulum. Surgical containment procedures meet with limited or mixed success, and the benefit of surgery remains controversial [5,7, 24]. Theoretically, it would be ideal if sphericity and containment were never lost, or were at least minimized. We hypothesized that by preventing or modifying collapse of the necrotic bone with the use of bisphosphonate therapy, improved outcomes for children with Perthes disease could be achieved. In a study on traumatic osteonecrosis in Wistar rats, we showed that zoledronic acid (ZA, Novartis) significantly improved short-term outcome [18]. In the current study we use a well-defined model of spontaneous osteonecrosis, the spontaneously hypertensive rat (SHR). Approximately 50% of SHR rats suffer a spontaneous ischaemic insult and subsequent osteonecrosis of the femoral head epiphysis around 6- 9 weeks of age. As a result, considerable femoral head flattening is seen by the age of 15 weeks [10]. This insult occurs during the early to mid-stages of femoral head development in these growing rats and therefore corresponds to a slightly earlier insult than that seen in most Perthes patients.

Methods

Animal ethical approval was granted for this study (WAEC 103.06- 02). One hundred and twenty 4-week old rats were divided into three groups of 40: 3 doses of saline monthly (saline), 3 doses of 0.05 mg/ kg ZA every four weeks (monthly ZA), or 10 doses of 0.015 mg/kg ZA weekly (weekly ZA). The total dose of ZA at 15 weeks was 0.15 mg/kg in both ZA groups. Rats designated for undecalcified histology (8 per group) were given subcutaneous injections of Calcein 10 mg/kg and Demeclocycline 30 mg/kg at 10 and 3 days prior to cull.

Animals were culled in a CO2 chamber at 15 weeks. This end-point age corresponding to that outlined by Hirano et al. as the time point of considerable flattening and deformity seen in the SHR [10]. Both proximal femora were harvested, radiographs were taken using a Faxitron cabinet X-ray system (Hewlett Packard, McMinnville, OR). X- ray images were digitized and enlarged 12X to be analyzed for sphericity using Bloquant image analysis system (R & M Biometrics Inc., Nashville, TN) connected to a digitizing tablet (GTCO CalComp, Inc., Columbia, MD). Sphericity measurements were derived using a modified epiphyseal quotient (EQ) [20]. The physis was horizontalized and height at centre over width was recorded as EQ.

Femoral lengths were also measured on the digitized X-ray images. Growth after the first ZA dose at 4 weeks was measured in the treated animals from the mineralized lines that remained in the metaphyses and their distance from the growth plate at 15 weeks.

Rat femora designated for decalcified histology (64 specimens per group) were fixed in 4% paraformaldehyde for 24 h at 4 C, and then decalcified in 0.5% paraformaldehyde/14.5% EDTA for approximately 6 weeks. The proximal femur of each sample was processed and embedded in paraffin and 5 m coronal sections through the center of the head produced. Standard H&E stains were used for descriptive histology. Safranin O, light green stain was employed for classification of remaining cartilaginous tissue, Safranin O specifically binds cartilage matrix staining it red, while bone tissue is stained green. Ossification of the epiphysis proceeds from lateral to medial thus the percent of each epiphysis that had ossified from the lateral side was calculated. Epiphyses containing more than 50% cartilage were classified as showing signs of delayed ossification (Fig. 2). The presence of osteonecrosis was determined by observational histology in the central slice, as seen by necrotic osteocytes and empty lacunae. As the exact time at which the ischemic insult occurred in each sample was unknown, many different stages of osteonecrosis were noted within the population at the 15 week time point. Thus, necrotic bone was described simply as the appearance of numerous empty osteocyte lacunae, without any consideration to the state of the surrounding marrow and accompanying reparative processes. Statistical analysis of femoral head sphericity was reported for only those femoral heads that exhibited signs of osteonecrosis.

Rat femora designated for undecalcified histology (16 specimens per group) were stored at 4 C in 70% ethanol. Bone mineral density (BMD) and bone mineral content (BMC) were measured with a pDEXA Sabre scanner (Norland, Ft Atkinson, WI). The specimens were then successively dehydrated in acetone and embedded in methylmethacrylate resin (Medim-Medizinische Diagnostik, Giessen, Germany). Coronal sections (5 m) were cut through the center of the femoral head and fluorescent microscopy was used to determine bone formation rates from the dual fluorochrome labels. Sections were stained by von Kossa for histomorphometric data such as bone volume (BV/TV), trabecular thickness (Tb.Th) and trabecular number (Tb.N). Identification of osteonecrosis in the subset of animals undergoing undecalcified histology could not be reliably performed, and this subset of animals was not included in this outcome analysis. This subset was included in the analysis of this purely mechanistic data, which reflects the effect of ZA on SHR rats with and without osteonecrosis.

Statistics and power

For numeric data such as bone area, BMD and BV/TV, means and standard deviations were calculated and one-way ANOVA applied, with post-hoc t-tests by the least squares difference method (SPSS Inc., Chicago, IL). For analysis of proportions, such as the prevalence of osteonecrosis between groups, a proportional test (chi square) was applied. Significance was set at the 0.05 level.

A power analysis based on EQ showed that power of 0.9 could be achieved with a difference in EQ of 0.03 with 80 femoral heads. The final number of 40 animals per group (80 femoral heads) was chosen as an adequate sample.

Results

Mineralization

There was a range of femoral head deformity from spherical to flat throughout the study population. Many of the samples showed radiographic signs of ossification delay. Observational analyses of the radiographs indicated increases in the amount of epiphyseal ossification in ZA-treated animals. This was confirmed in the 16 femoral heads in each group examined by DXA, with increases in femoral head BMD measuring 18% in the weekly ZA group and 21% in the monthly ZA group over saline (p < 0.01).

Osteonecrosis

Osteonecrosis was observed in 53% of saline femoral heads, 65% of weekly and 52% of monthly ZA femoral heads. This was not significantly different between groups.

Sphericity

Epiphys\eal quotient (EQ) was significantly increased in ZA- treated animals which showed evidence of an osteonecrotic insult, with the mean EQ rising from 0.44 in the saline group to 0.47 in weekly ZA (p < 0.05) and 0.49 in monthly ZA groups (p < 0.01). The proportion of saline femoral heads in this cohort that had an EQ less than or equal to 0.40 was 32%, whereas this was reduced to 12% for weekly ZA (p < 0.05) and 3% for monthly ZA groups (p < 0.01, Fig. 1). These EQ data were also significantly improved for the ZA groups over saline for the entire cohort of animals, ie those with and without osteonecrosis (data not shown).

Ossification delay

Ossification delay is typical in SHR rats (and present to some extent in Perthes disease). Areas of hypertrophie chondrocytes were seen, surrounded by proteoglycan-containing (Safranin O positive) matrix that was partly mineralized on von Kossa sections. Invasion of blood vessels and subsequent endochondral ossification was seen to proceed from lateral to medial in the cpiphyses in these animals (Fig. 2A and B). There was a significant difference in the incidence of ossification delay at 15 weeks between treatment groups in the femoral heads affected by osteonecrosis. While 53% of the saline femoral heads exhibited ossification delay, this was rare in ZA- treated animals, with only 10% of weekly ZA and 21% of monthly ZA- treated animals exhibiting such delay (p < 0.01) (Table 1). There was also a significant decrease in the incidence of ossification delay in the entire cohort of animals with ZA treatment (data not shown).

Fig. 1. Representative radiographs of femoral heads with an EQ of 0.5 (A) and 0.3 (B). Arrow shows region of flat head that is considerably deformed. The mean EQ was higher, and the proportion of severely flattened femoral heads was lower with ZA treatment.

Fig. 2. (A) Ossification delay with large areas of Safranin O staining (red*) cartilage still present in the medial half of the femoral head. The femoral head in (B) is fully ossified (green tissue). Small arrow in A points to the vascular front of the ossification. Large arrow in B shows complete ossification of epiphysis. Safranin O, light green (2.5).

In the subset of femoral heads with both osteonecrosis and ossification delay, there was a marked difference in EQ with treatment. Saline-treated animals in this subset had a mean EQ of only 0.40, while this was increased to 0.49 (p < 0.01) in weekly ZA and 0.49 (p < 0.01) in monthly ZA groups.

Histomorphometry

Static histomorphometric variables for all specimens analyzed by undecalcified histology are listed in Table 2. There was a significant increase in BV/TV in ZA-treated animals based on increased trabecular number, although the trabeculae were significantly thinner in the ZA-treated groups when compared to saline (Fig. 3B, D and F). Bone formation rate (BFR) was significantly reduced compared to saline in both ZA-treated groups (post-hoc p < 0.01).

Growth disturbance

Femoral length was reduced 2.6% in the weekly and 2.3% in the monthly ZA group (p < 0.01) as compared to saline. However, the actual amount of growth at the distal femur from weeks 4-15 was 14.2 mm (SD 0.9) in weekly and 15.0 mm (SD 1.0) in monthly ZA groups. This 5.4% reduction in longitudinal growth in weekly versus monthly was significant (p < 0.01).

Table 1

Proportion of fully ossified femoral heads(a)

Table 2

Histomorphometry(a)

Fig. 3. Representative radiographs (A,C,E) and von Kossa sections magnification 2.5 (B,D,F) from saline (A,B). weekly ZA (C,D) and monthly ZA (E,F) groups. Increased apparent density in ZA-lreated groups on radiographs was due to increased trabecular number. Trabecular thickness was higher in the saline group.

Discussion

Zoledronic acid treatment improves sphericity in SHR rats

We have demonstrated a significant effect of ZA on sphericity of the SHR rat femoral head in this study, with both an increase in mean EQ and a decrease in the number of heads defined as aspherical (EQ ≤ 0.4). As hypothesized, enhanced mineralization was demonstrated in SHR rats treated with ZA. We also found increased bone volume (BV/TV) and an increase in trabecular number (Fig. 3; Table 2). These outcome and mechanistic data are in agreement with previous animal studies on the effect of nitrogen-containing bisphosphonates in models of osteonecrosis.

In a rat bone chamber study, Astrand and Aspenberg showed that high dose alendronatc therapy was able to preserve necrotic bone volume as a scaffold for new bone ingrowth [1]. Total bone volume was doubled in the high dose alendronate group compared to low dose and saline animals. In a study by our group, Wistar rats with traumatic osteonecrosis treated with ZA improved femoral head shape and showed significant increase in trabecular number over saline animals [18]. In a large animal piglet model, indentation studies showed a reduction in stiffness of the infarcted femoral head by 52% at 2 weeks and 75% by 4 weeks, leading to considerable deformity by 8 weeks [23]. In the same model, Kim et al. [15,16] have shown positive effects on femoral head shape and relenlion of trabecular architecture with ibandronatc. Trabecular number was again increased in this study.

Il is of interest that in the current study, trabecular thickness was larger in saline-trealcd controls, this may suggest hypertrophy of those remaining trabeculae which are not resorbed. Grossly thickened trabeculae have also been noted in human pathological specimens of Perthes disease [4]. It is particularly noteworthy that, for the same amount of bone, maintaining trabceular number by thinning results in better strength retention in trabecular bone than does losing trabeculae but retaining trabecular thickness [28]. It is extremely likely that the retention of trabecular number significantly contributed to the improved retention ofoverall femoral head shape.

Effect of zoledronic acid on ossification delay in SHR rats

An unexpected outcome of this study was the increase in the occurrence of complete epiphyseal ossification in the femoral heads of the ZA-treated rats (Fig. 2A and B; Table 1). This was an impressive outcome, with the 53% incidence of ossification delay in femoral heads of saline-treated animals reduced to 10% in weekly and 21% in monthly ZA-treated animals. This indicates that in this model, osteoclast function may not be absolutely required in the process of endochondral ossification of the epiphysis. Further work is needed to fully elucidate the mechanism behind the accelerated epiphyseal ossification, but it has already been established that osteoclasts (and chondroclasts) are only required for remodeling primary bone trabeculae in endochondral ossincation, not for the removal of chondrocytes and unmincralized chondral matrix [6,25,26].

Limitations of the SHR model

We chose the spontaneously hypertensive rat model for this investigation as it more closely resembles Perthes disease than surgically induced models. Extensive studies have been carried out on the occurrence of spontaneous osteonecrosis in the SHR rat. It is of note that weight hearing is a prerequisite for ostconccrosis in this model as ostconccrosis is not observed if the limb is denervated, or if amputation is carried out through the knee [12]. Around 50% of male SHR rats develop avascular changes at around 6-9 weeks of age leading to maximal femoral head deformity by 15 weeks [10]. A limitation of this model is the difficulty in determining which femoral heads had sullercd an ischemic insult. At the 15 week end time point in this study we were able to histologically detect the occurrence of ostconecrosis in the samples. However this excludes those samples that had completely recovered from the insult by this time point.

It has been documented that SIIR nits have a cartilage disorder that results in a delay in ossification of the femoral head epiphysis [14]. Thus, at the age of 6-9 weeks the underdeveloped head cannot withstand normal weight bearing, resulting in occlusion of the lateral epiphyseal vessels (LEVs) and subsequent ischemia and ostconccrosis. Resorption of the necrotic bone in the lateral epiphysis ensues, resulting in reduced mechanical properties and deformation, thus further vascular occlusion. Epiphyscal ossification is therefore further delayed.

We speculate that ZA treatment stabilized the lateral epiphysis by suppressing the resorption of necrotic bone. The un-resorbed trabeculae may have provided the mechanical strength required by the weight bearing lateral epiphysis to resist further deformation. ZA treatment may have thus prevented the weight-bearing induced compression of the LEVs known to be the pathogenesis of ostconecrosis in this rat model [11,21]. Therefore, the vascular cndothelial cells were unhindered in their vascular invasion of the cartilaginous epiphysis, thus subsequent endochondral ossilication of the lemoral heads was able to proceed [17]. Further experiments and analysis will be employed to evaluate this hypothesis. As ZA was administered from week 4, the drug may have had a prophylactic effect. We showed that prophylaxis was more effective than treatment in our previous study on traumatic osteonecrosis [18], assessment of further dosing time-points might also be relevant.

Similar processes may or may not be occurring in Perthes disease, and this could explain why some clinical specimens display multiple infarctions. Whether the ossification delay demonstrated by delayed bone age in Perthes disease [27] is also relevant to the delay in reossification after ischemia is unknown. In some animals in this study with ossification delay, the presence of hypertrophie cartilage in the femoral head was due to primary ossification delay, rather than fibrocartilage repair tissue as seen in Perthes disease after bone resorption. However, in Perthes disease, deformity occurs when there is a mixture of necrotic bone and car\tilage in the femoral head. The SHR rat can still be viewed as a valid model of this particular circumstance. This mixture of cartilage and necrotic bone would be the "softest" group of femoral heads, and it is relevant that in this subset, ZA treatment increased EQ from 0.40 to 0.49.

We adjusted for the various limitations by incorporating a large number of animals in the study. It remains very important to study spontaneously induced osteonecrosis in a growing animal, as this has far more relevance to Perthes disease than traumatic models. The fact that we have similar results and mechanistic data (increased BV/ TV, increased trabecular number) in a traumatic osteonecrosis model in rats gives us encouragement that ZA treatment may be relevant to both circumstances [18].

Potential negative effects of bisphosphonates

Bisphosphonate therapy in children has one potentially worrying negative effect-a decrease in longitudinal growth. In this study a 2.3-2.6% decrease in femoral length with ZA treatment was documented. The animals approximately double the size of the femur during the experimental period, so the growth disturbance can be approximated at around 5% inhibition. We also noted a further 5% decrease in longitudinal growth in weekly over monthly dosing. Close monitoring of longitudinal growth should be performed in all children on bisphosphonates to provide further definitive information on this topic, however clinical studies in children with osteogenesis imperfecta, osteoporosis and fibrous dysplasia show no detectable growth disturbance [2,22,31].

As bisphosphonates avidly bind bone mineral, they have a very long half-life in the skeleton. The effect on bone metabolism is to reduce bone turnover, but when treatment is ceased in adults, slow reversal of the effects are seen [3O]. Further safety data in children will be needed before the application of this potential therapy in Perthes disease.

Summary

This and other studies suggest that a treatment approach utilizing bisphosphonates could prove a valuable adjunct in childhood osteonecrosis. As the safety of potent bisphosphonates becomes more established, it is possible that these compounds could be used to improve outcome and decrease the number of surgical procedures in Perthes disease. Further experimentation in this and other models of Perthes disease and eventual clinical trials will be needed to evaluate the efficacy and safety of potent bisphosphonate therapy in preserving femoral head shape while repair occurs.

Acknowledgement

Funding for this project was received from The National Health and Medical Research Council of Australia. Dr Ian Sharpe and Dr Paul Williams received salary support as Ingham Fellows funded by Ingham Enterprises Pty Ltd. Dr Tony McEvoy received salary support as the Smith & Nephew Fellow funded by Smith and Nephew Orthopaedics. Zoledronic acid was donated by Novartis Pharmaceuticals (Australia). Dr David Little has received patent licensing funding and other research grants from Novartis Pharma AG. We would like to thank Lyndon Mason for his assistance with the femoral length measurements.

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David G. Little (a,b,*), Michelle McDonald (a), Ian T. Sharpe (a,c), Rachel Peat (a), Paul Williams (a,d), Tony McEvoy (a,e)

a Orthopaedic Research and Biotechnology. The Children's Hospital at Westmead 2145, Sydney, NSW, Australia

b Department of Paediatrics and Child Health, University of Sydney, NSW 2006, Australia

c Royal Devon and Exeter Healthcare NHS Trust, Royal Devon and Exeter Hospital (Wonford), Bowmoor House, Barrack Road, Exeter EX2 5DW, UK

d Morriston Hospital, Morriston Hospital NHS Trust, Swansea SA6 6NL, UK

e Royal Preston Hospital, Sharoe Green Lane North, Fulwood, Preston, Lancashire PR2 4QF, UK

Received 14 October 2004; accepted 19 November 2004

* Corresponding author. Address: Orthopaedic Research and Biotechnology, The Children's Hospital at Westmead 2145, Sydney, NSW, Australia. Tel.: +61 2 9845 3352; fax: +61 2 9845 3180.

E-mail address: DavidL3@chw.edu.au (D.G. Little).

Copyright Journal of Bone and Joint Surgery, Inc. Jul 2005

Source: Journal of Orthopaedic Research

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