The presence of skeletal hypomineralization was confirmed in mice lacking the gene for bone alkaline phosphatase, ie, the tissue-non-specific isozyme of alkaline phosphatase (TNAP). In this study, a detailed characterization of the ultrastructural localization, the relative amount and ultrastructural morphology of bone mineral was carried out in tibial growth plates and in subjacent metaphyseal bone of 10-day-old TNAP knockout mice. Alizarin red staining, microcomputerized tomography (micro CT), and FTIR imaging spectroscopy (FT-IRIS) confirmed a significant overall decrease of mineral density in the cartilage and bone matrix of TNAP-deficient mice. Transmission electron microscopy (TEM) showed diminished mineral in growth plate cartilage and in newly formed bone matrix. High resolution TEM indicated that mineral crystals were initiated, as is normal, within matrix vesicles (MVs) of the growth plate and bone of TNAP-deficient mice. However, mineral crystal proliferation and growth was inhibited in the matrix surrounding MVs, as is the case in the hereditary human disease hypophosphatasia. These data suggest that hypomineralization in TNAP-deficient mice results primarily from an inability of initial mineral crystals within MVs to self-nucleate and to proliferate beyond the protective confines of the MV membrane. This failure of the second stage of mineral formation may be caused by an excess of the mineral inhibitor pyrophosphate (PPi) in the extracellular fluid around MVs. In normal circumstances, PPi is hydrolyzed by the TNAP of MVs' outer membrane yielding monophosphate ions (Pi) for incorporation into bone mineral. Thus, with TNAP deficiency a buildup of mineral-inhibiting PPi would be expected at the perimeter of MVs.

Osteonecrosis of the jaw (ONJ) is a well-recognized complication of antiresorptive medications, such as bisphosphonates (BPs). Although ONJ is most common after tooth extractions in patients receiving high dose BPs, many patients do not experience oral trauma. Animal models utilizing tooth extractions and high BP doses recapitulate several clinical, radiographic and histologic findings of ONJ. We and others have reported on rat models of ONJ utilizing experimental dental disease in the absence of tooth extraction. These models emphasize the importance of dental infection/inflammation for ONJ development. Here, we extend our original report in the rat, and present a mouse model of ONJ in the presence of dental disease. Mice were injected with high dose zoledronic acid and pulpal exposure of mandibular molars was performed to induce periapical disease. After 8 weeks, quantitative and qualitative radiographic and histologic analyses of mouse mandibles were executed. Periapical lesions were larger in vehicle- vs. BP treated mice. Importantly, radiographic features resembling clinical ONJ, including thickening of the lamina dura, periosteal bone deposition and increased trabecular density, were seen in the drilled site of BP treated animals. Histologically, osteonecrosis, periosteal thickening, periosteal bone apposition, epithelial migration and bone exposure were present in the BP treated animals in the presence of periapical disease. No difference in TRAP+ cell numbers was observed, but round, detached, and removed from the bone surface cells were present in BP animals. Although 88% of the BP animals showed areas of osteonecrosis in the dental disease site, only 33% developed bone exposure, suggesting that osteonecrosis precedes bone exposure. Our data further emphasize the importance of dental disease in ONJ development, provide qualitative and quantitative measures of ONJ, and present a novel mouse ONJ model in the absence of tooth extraction that should be useful in further exploring ONJ pathophysiological mechanisms.

Antiresorptive medications are essential in treating diseases of pathologic osteoclastic bone resorption, including bone cancer and osteoporosis. Bisphosphonates (BPs) are the most commonly used antiresorptives in clinical practice. Although inhibition of bone resorption is important in regulating unwanted malignant and metabolic osteolysis, BP treatment is associated with potential side effects, including osteonecrosis of the jaws (ONJ). Recently, non-BP antiresorptive medications targeting osteoclastic function and differentiation, such as denosumab, have entered the clinical arena. Denosumab treatment results in a similar rate of ONJ as BPs. Animal models of ONJ, using high-dose BP treatment in combination with tooth extraction or dental disease, provide valuable tools and insight in exploring ONJ pathophysiology. However, the ability of other antiresorptives to induce ONJ-like lesions in animal models has not been explored. Such studies would be beneficial in providing support for the role of osteoclast inhibition in ONJ pathogenesis versus a direct BP effect on oral tissues. Here, we tested the ability of the receptor activator of NF-kB ligand (RANKL) inhibitors RANK-Fc (composed of the extracellular domain of RANK fused to the fragment crystallizable [Fc] portion of immunoglobulin G [IgG]) and OPG-Fc (composed of the RANKL-binding domains of osteoprotegerin [OPG] linked to the Fc portion of IgG) to induce ONJ in mice in the presence of periapical disease, but in the absence of dental extractions. We demonstrate radiographic evidence of ONJ in RANK-Fc–treated and OPG-Fc–treated mice, including inhibition of bone loss, increased bone density, lamina dura thickening, and periosteal bone deposition. These findings closely resembled the radiographic appearance of an ONJ patient on denosumab treatment. Histologic examination revealed that RANK-Fc treatment and OPG-Fc treatment resulted in absence of osteoclasts, periosteal bone formation, empty osteocytic lacunae, osteonecrosis, and bone exposure. In conclusion, we have successfully induced ONJ in mice with periapical disease, using potent osteoclast inhibitors other than BPs. Our findings, coupled with ONJ animal models using high-dose BPs, suggest that osteoclast inhibition is pivotal to the pathogenesis of ONJ.

Although fundamentally similar to other bones, the jaws demonstrate discrete responses to developmental, mechanical, and homeostatic regulatory signals. Here, we hypothesized that rat mandible vs. long-bone marrow-derived cells possess different osteogenic potential. We established a protocol for rat mandible and long-bone marrow stromal cell (BMSC) isolation and culture. Mandible BMSC cultures formed more colonies, suggesting an increased CFU-F population. Both mandible and long-bone BMSCs differentiated into osteoblasts. However, mandible BMSCs demonstrated augmented alkaline phosphatase activity, mineralization, and osteoblast gene expression. Importantly, upon implantation into nude mice, mandible BMSCs formed 70% larger bone nodules containing three-fold more mineralized bone compared with long-bone BMSCs. Analysis of these data demonstrates an increased osteogenic potential and augmented capacity of mandible BMSCs to induce bone formation in vitro and in vivo. Our findings support differences in the mechanisms underlying mandible homeostasis and the pathophysiology of diseases unique to the jaws.

Hyperlipidemia increases the risk for generation of lipid oxidation products, which accumulate in the subendothelial spaces of vasculature and bone. Atherogenic high-fat diets increase serum levels of oxidized lipids, which are known to attenuate osteogenesis in culture and to promote bone loss in mice. In this study, we investigated whether oxidized lipids affect bone regeneration and mechanical strength. Wild type and hyperlipidemic (Ldlr−/−) mice were placed on a high-fat (HF) diet for 13 weeks. Bilateral cranial defects were introduced on each side of the sagittal suture, and 5 weeks post-surgery on the respective diets, the repair/regeneration of cranial bones and mechanical properties of femoral bones were assessed. MicroCT and histological analyses demonstrated that bone regeneration was significantly impaired by the HF diet in WT and Ldlr−/− mice. In femoral bone, cortical bone volume fraction (BV/TV) was significantly reduced while cortical porosity was increased by the HF diet in Ldlr−/− but not in WT mice. Femoral bone strength and stiffness, measured by three-point bending analysis, were significantly reduced by the HF diet in Ldlr−/−, but not in WT mice. Serum analysis showed that the HF diet significantly increased levels of parathyroid hormone, TNF-alpha, calcium and phosphorus, whereas it reduced procollagen type I N-terminal propeptide, a serum marker of bone formation, in Ldlr−/−, but not in WT mice. The serum level of carboxyl-terminal collagen crosslinks, a marker for bone resorption, was also 1.7-fold greater in Ldlr−/− mice. These findings suggest that hyperlipidemia induces secondary hyperparathyroidism and impairs bone regeneration and mechanical strength.