Bone Apatite Composition Of Osteonecrotic Trabecular Bone From The Femoral Head Of Immature Pigs

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1 Bone Apatite Composition Of Osteonecrotic Trabecular Bone From The Femoral Head Of Immature Pigs Olumide O. Aruwajoye 1,2, Harry KW Kim, MD 1, Pranesh B. Aswath, PhD 2. 1 Texas Scottish Rite Hospital, Dallas, TX, USA, 2 University of Texas at Arlington, Arlington, TX, USA. Disclosures: O.O. Aruwajoye: None. H.K. Kim: None. P.B. Aswath: None. Introduction: Ischemic osteonecrosis of the femoral head can lead to excessive resorption of the trabecular bone and collapse of the femoral head as a structure during the healing process. Prior to the resorption and collapse of the femoral head, the calcified cartilage and trabecular bone in the necrotic femoral head become hypermineralized [1]. The chemical composition of the hypermineralized necrotic trabecular bone is unknown. We hypothesized that the apatite composition changes in necrotic bone and contribute to increased osteoclast resorption. The purpose of this study was to identify differences in the mineral apatite composition of actively resorbed necrotic bone versus areas of normal bone and newly formed bone. Methods: The local Institutional Animal Care and Use Committee approved the study. Ischemic osteonecrosis of the right femoral head was surgically induced in 5 piglets (6 to 8 weeks of age) by placing a ligature tightly around the femoral neck to disrupt the blood flow to the femoral head. Unoperated left femoral heads were used as normal controls [2]. At eight weeks following the induction of osteonecrosis, when osteoclastic resorption is expected, the animals were euthanized and the left and right femoral heads were retrieved and prepared for histology. The femoral heads were embedded in methyl methacrylate (MMA) for sectioning and McNeal tetrachrome staining using standard procedures. The remaining portions of the embedded block were deplasticized and analyzed for apatite composition with X-ray absorbance near edge structure (XANES), and Raman spectroscopy. Regions of normal, necrotic, and newly formed bone within the femoral heads were identified for analysis based on corresponding histology staining. Peak height ratios from Raman spectra were averaged from five pairs of femoral heads. A Wilcoxon signed rank test was used to compare the normal vs necrotic bone, and the newly formed bone vs the normal bone. A p<0.05 was considered significant. Results: The histology of the unoperated side showed a spherical femoral head with normal bone formation throughout the femoral head. The operated side showed a flattening deformity and resorptive changes in the necrotic bone. Higher magnification of the normal bone showed osteoblasts lining the osteoid, which is indicative of bone formation. Furthermore, the necrotic region of the operated side showed trabeculae with large resorption pits and a lack of osteoblasts. However, new ossification within the operated head was noted in the periphery of the femoral head with osteoblasts lining the surface of the bone. (Fig.1) XANES spectra of the normal, necrotic, and newly formed bone showed contributions of phosphorous and oxygen containing species in the chemical composition of bone. The contributions were assessed by comparing the chemistry of known model compounds with necrotic and normal bone. Phosphorous spectra indicated that there were similarities in the phosphorous coordination in both normal and necrotic regions. However, oxygen-containing species such as carbonate and phosphate showed increased contribution of carbonate in the necrotic trabecular bone (Table 1).

2 In order to determine the extent of substitution of carbonate in the apatite structure the Raman spectroscopy was also used. The peak intensity ratio between the carbonate 1070 cm 1 and phosphate 960 cm 1 were used to estimate the relative amounts of carbonates to phosphates. The ratio was lowest for newly formed bone and highest for the necrotic bone. In fact, the carbonate to phosphate ratio was significantly higher in the necrotic versus normal bone and necrotic versus newly formed bone(p=0.043) (Fig. 2). Discussion: The purpose of this study was to assess the apatite composition of the necrotic and the normal bone and to investigate differences between areas of increased osteoclast resorption and normal bone formation. The differences in normal, necrotic, and newly formed bone can be seen by relative carbonate to phosphate content. Increase in carbonate has been linked to aging bone [3]. In addition, carbonate substitution causes local distortions in the crystal structure and may contribute to micro-fracture of necrotic bone. The reason for increased resorption of necrotic bone can only be speculated. However, carbonated apatite as opposed to noncarbonated apatite is indeed more soluble and thus easier to break down. Furthermore, resorption of carbonated substituted hydroxyapatite has been shown to be significantly increased when the carbonate content is increased in in-vitro studies [4]. We believe the compositional changes due to carbonate substitution in the necrotic bone also contribute to increased resorption by osteoclasts. Significance: Local changes in the apatite composition due to carbonate substitution may play a role in the increased resorption of the necrotic bone due to its increase in solubility. A better understanding of the apatite composition of necrotic bone could shed light on osteoclast activity and potentially improve therapeutic treatments that target excessive resorption of bone.

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5 ORS 2015 Annual Meeting Poster No: 0831