Effects of Tiludronate on Bone Growth and Remodeling in Young Horses

Margaret A. McNulty, Ashley N. Gillett, Brad A. Goupil, and Colin F. Mitchell Louisiana State University School of Veterinary Medicine, Baton Rouge, LA

Disclosures: The authors have nothing to disclose. INTRODUCTION: Tiludronate, also known as Tildren®, is a bisphosphonate drug utilized in horses of all ages to treat several bone disorders that result in a decrease in bone density, including navicular and dorsal metacarpal diseases. 1, 2 Tiludronate decreases osteoclastic activity by inducing osteoclast apoptosis, thereby inhibiting bone resorption. 3 Since Tildren’s recent approval for use in horses within the U.S., it has been used extensively in the clinical setting. Despite prolific clinical use in young horses, tiludronate’s effects have not been studied on normal bone remodeling processes in this cohort. We hypothesized that tiludronate will inhibit bone resorption in young horses by decreasing osteoclast number, thus adversely affecting normal bone remodeling. METHODS: Young Thoroughbred horses (<5 years old), including both mares and geldings, underwent randomized unilateral tuber coxae biopsies. Following surgery, horses received either tiludronate (TIL, n=5) or saline (SAL, n=5). Sixty days following treatment, the contralateral tuber coxae was biopsied. Prior to obtaining the second biopsies, oxytetracyline was administered for dynamic histomorphometry. Trabecular bone morphology was first evaluated by utilizing micro-computed tomography (µCT) using 30µm voxels. Total volume (TV); bone volume (BV); BV/TV; and trabecular number, spacing, and thickness were evaluated using standard methods and nomenclature. 4 Following µCT evaluation, biopsies were prepared for histology. The first biopsies were prepared for decalcified histology and stained with H&E for subsequent static histomorphometry. The second biopsies were prepared for undecalcified histology; one section was left unstained for dynamic histomorphometry, and the second section was stained with Van Gieson’s picrofushion for static histomorphometry. Histomorphometrical parameters included total area (TA), bone area (BA), BA/TA, osteoclast number & surface, eroded surface, and osteoblast number and surface, and follow standard methods and nomenclature. 5 RESULTS: The biopsy technique outlined in this study provided ample trabecular bone to evaluate via both µCT and histology. However, histopathological evaluations identified substantial numbers of islands of mature hyaline cartilage within the trabeculae of these biopsies. (Fig. 1) These cartilaginous foci were identified in both biopsies of all horses within the study, regardless of sex, age, or treatment. When the horses were split into approximate age groups, there was an age-related trend: younger horses (2 years old) had lower BV/TV values than older (5 years old) horses (p=0.079). When evaluating differences following treatment, there were no significant (p<0.05) differences in any µCT values, however, the data revealed an increase in trabecular number, thickness, connectivity and bone density and a decrease in trabecular spacing in TIL compared to SAL. Static histomorphometry identified a significant decrease in osteoclasts (including osteoclast number, surface, and osteoclast surface/bone surface) following tiludronate administration. (Fig. 2) DISCUSSION: This non-terminal biopsy technique is the first report of obtaining a biopsy that includes both trabecular and cortical bone from a horse that can be used to evaluate bone both via µCT and histology. The origin and nature of the cartilage foci that were identified in the biopsies appears to be related to an apophysis that gives rise to the tuber coxae in horses, however ongoing work is being completed in young (<1 year) and old (skeletally mature) horses to confirm this hypothesis. The presence of this proposed apophysis should be taken into account when utilizing this technique to evaluate bone in the future. While no significant changes were identified in bone morphology as determined by µCT, the data may indicate a potential effect on bone morphology that may not be statistically significant due to low numbers per group or the impact of age on bone morphology that was identified. However, static histomorphometry revealed a significant decrease in osteoclast numbers and related parameters when comparing the first biopsy in tiludronate-treated horses to the second, which indicates that changes in bone morphology may occur over time. Dynamic histomorphometry evaluation is ongoing. SIGNFICANCE: This study is the first to identify potential changes in normal bone morphology and remodeling in young horses following tiludronate treatment.

1. Denoix JM, Thibaud D, Riccio B. 2003. Tiludronate as a new therapeutic agent in the treatment of navicular disease: A double-blind placebo-controlled clinical trial. Equine Vet J 35: 407-413. 2. Carpenter RS. 2012. How to treat dorsal metacarpal disease with regional tiludronate and extracorporeal shock wave therapies in thoroughbred racehorses. 3. Kamm L, McIlwraith W, Kawcak C. 2008. A review of the effect of tiludronate in the horse. J Equine Vet Sci 28: 209-214. 4. Bouxsein ML, Boyd SK, Christiansen BA, et al. 2010. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J Bone Miner Res 25: 1468-1486. 5. Dempster DW, Compston JE, Drezner MK, et al. 2013. Standardized nomenclature, symbols, and units for bone histomorphometry: A 2012 update of the report of the ASBMR histomorphometry nomenclature committee. J Bone Miner Res 28: 2-17.

Figure 1. (A) 3D µCT image of the cartilage foci (arrows) depicted in the H&E stained sample in B. (B) Cartilage foci are seen within the trabecular bone on H&E stained histological sample from Biopsy 1. Bar= 1 mm.

Figure 2. (A) Mean + SEM osteoclast surface/bone surface from biopsies of horses within the Tildren treatment group. * = p=0.016.

ORS 2016 Annual Meeting Poster No. 0694