Elisa Atti

Osteogenic Potential of Mandibular vs. Long-bone Marrow Stromal Cells

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.

Periapical disease and bisphosphonates induce osteonecrosis of the jaws in mice.

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 using 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 using 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 done. Periapical lesions were larger in vehicle‐treated versus 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 tartrate‐resistant acid phosphatase (TRAP)+ cell numbers was observed, but round, detached, and removed from the bone surface cells were present in BP‐treated animals. Although 88% of the BP‐treated 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.

Adverse Effects of Hyperlipidemia on Bone Regeneration and Strength

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 (WT) 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 postsurgery 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 (bone volume [BV]/tissue volume [TV]) was significantly reduced, whereas 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, tumor necrosis factor (TNF)‐α, 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.

Novel oxysterols have pro‐osteogenic and anti‐adipogenic effects in vitro and induce spinal fusion in vivo

Stimulation of bone formation by osteoinductive materials is of great clinical importance in spinal fusion surgery, repair of bone fractures, and in the treatment of osteoporosis. We previously reported that specific naturally occurring oxysterols including 20(S)‐hydroxycholesterol (20S) induce the osteogenic differentiation of pluripotent mesenchymal cells, while inhibiting their adipogenic differentiation. Here we report the characterization of two structural analogues of 20S, Oxy34 and Oxy49, which induce the osteogenic and inhibit the adipogenic differentiation of bone marrow stromal cells (MSC) through activation of Hedgehog (Hh) signaling. Treatment of M2‐10B4 MSC with Oxy34 or Oxy49 induced the expression of osteogenic differentiation markers Runx2, Osterix (Osx), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN), as well as ALP enzymatic activity and robust mineralization. Treatment with oxysterols together with PPARγ activator, troglitazone (Tro), inhibited mRNA expression for adipogenic genes PPARγ, LPL, and aP2, and inhibited the formation of adipocytes. Efficacy of Oxy34 and Oxy49 in stimulating bone formation in vivo was assessed using the posterolateral intertransverse process rat spinal fusion model. Rats receiving collagen implants with Oxy 34 or Oxy49 showed comparable osteogenic efficacy to BMP2/collagen implants as measured by radiography, MicroCT, and manual inspection. Histological analysis showed trabecular and cortical bone formation by oxysterols and rhBMP2 within the fusion mass, with robust adipogenesis in BMP2‐induced bone and significantly less adipocytes in oxysterol‐induced bone. These data suggest that Oxy34 and Oxy49 are effective novel osteoinductive molecules and may be suitable candidates for further development and use in orthopedic indications requiring local bone formation. J. Cell. Biochem. 112: 1673–1684, 2011.

Hyperlipidemia induces resistance to PTH bone anabolism in mice via oxidized lipids

In hyperlipidemia, oxidized lipids accumulate in vascular tissues and trigger atherosclerosis. Such lipids also deposit in bone tissues, where they may promote osteoporosis. We found previously that oxidized lipids attenuate osteogenesis and that parathyroid hormone (PTH) bone anabolism is blunted in hyperlipidemic mice, suggesting that osteoporotic patients with hyperlipidemia may develop resistance to PTH therapy. To determine if oxidized lipids account for this PTH resistance, we blocked lipid oxidation products in hyperlipidemic mice with an ApoA‐I mimetic peptide, D‐4F, and the bone anabolic response to PTH treatment was assessed. Skeletally immature Ldlr−/− mice were placed on a high‐fat diet and treated with D‐4F peptide and/or with intermittent PTH(1–34) injections. As expected, D‐4F attenuated serum lipid oxidation products and tissue lipid deposition induced by the diet. Importantly, D‐4F treatment attenuated the adverse effects of dietary hyperlipidemia on PTH anabolism by restoring micro–computed tomographic parameters of bone quality—cortical mineral content, area, and thickness. D‐4F significantly reduced serum markers of bone resorption but not bone formation. PTH and D‐4F, together but not separately, also promoted bone anabolism in an alternative model of hyperlipidemia, Apoe−/− mice. In normolipemic mice, D‐4F cotreatment did not further enhance the anabolic effects of PTH, indicating that the mechanism is through its effects on lipids. These findings suggest that oxidized lipids mediate hyperlipidemia‐induced PTH resistance in bone through modulation of bone resorption.

Mouse lipin-1 and lipin-2 cooperate to maintain glycerolipid homeostasis in liver and aging cerebellum

The three lipin phosphatidate phosphatase (PAP) enzymes catalyze a step in glycerolipid biosynthesis, the conversion of phosphatidate to diacylglycerol. Lipin-1 is critical for lipid synthesis and homeostasis in adipose tissue, liver, muscle, and peripheral nerves. Little is known about the physiological role of lipin-2, the predominant lipin protein present in liver and the deficient gene product in the rare disorder Majeed syndrome. By using lipin-2–deficient mice, we uncovered a functional relationship between lipin-1 and lipin-2 that operates in a tissue-specific and age-dependent manner. In liver, lipin-2 deficiency led to a compensatory increase in hepatic lipin-1 protein and elevated PAP activity, which maintained lipid homeostasis under basal conditions, but led to diet-induced hepatic triglyceride accumulation. As lipin-2–deficient mice aged, they developed ataxia and impaired balance. This was associated with the combination of lipin-2 deficiency and an age-dependent reduction in cerebellar lipin-1 levels, resulting in altered cerebellar phospholipid composition. Similar to patients with Majeed syndrome, lipin-2–deficient mice developed anemia, but did not show evidence of osteomyelitis, suggesting that additional environmental or genetic components contribute to the bone abnormalities observed in patients. Combined lipin-1 and lipin-2 deficiency caused embryonic lethality. Our results reveal functional interactions between members of the lipin family in vivo, and a unique role for lipin-2 in central nervous system biology that may be particularly important with advancing age. Additionally, as has been observed in mice and humans with lipin-1 deficiency, the pathophysiology in lipin-2 deficiency is associated with dysregulation of lipid intermediates.

RANKL inhibitors induce osteonecrosis of the jaw in mice with periapical disease.

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‐κB 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.

MicroCT Morphometry Analysis of Mouse Cancellous Bone: Intra- and Inter-system Reproducibility

The agreement between measurements and the relative performance reproducibility among different microcomputed tomography (microCT) systems, especially at voxel sizes close to the limit of the instruments, is not known. To compare this reproducibility 3D morphometric analyses of mouse cancellous bone from distal femoral epiphyses were performed using three different ex vivo microCT systems: GE eXplore Locus SP, Scanco μCT35 and Skyscan 1172. Scans were completed in triplicate at 12 μm and 8 μm voxel sizes and morphometry measurements, from which relative values and dependence on voxel size were examined. Global and individual visually assessed thresholds were compared. Variability from repeated scans at 12 μm voxel size was also examined. Bone volume fraction and trabecular separation values were similar, while values for relative bone surface, trabecular thickness and number varied significantly across the three systems. The greatest differences were measured in trabecular thickness (up to 236%) and number (up to 218%). The relative dependence of measurements on voxel size was highly variable for the trabecular number (from 0% to 20% relative difference between measurements from 12 μm and 8 μm voxel size scans, depending on the system). The intra-system reproducibility of all trabecular measurements was also highly variable across the systems and improved for BV/TV in all the systems when a smaller voxel size was used. It improved using a smaller voxel size in all the other parameters examined for the Scanco system, but not consistently so for the GE or the Skyscan system. Our results indicate trabecular morphometry measurements should not be directly compared across microCT systems. In addition, the conditions, including voxel size, for trabecular morphometry studies in mouse bone should be chosen based on the specific microCT system and the measurements of main interest.

A Novel Osteogenic Oxysterol Compound for Therapeutic Development to Promote Bone Growth: Activation of Hedgehog Signaling and Osteogenesis through Smoothened Binding

Osteogenic factors are often used in orthopedics to promote bone growth, improve fracture healing, and induce spine fusion. Osteogenic oxysterols are naturally occurring molecules that were shown to induce osteogenic differentiation in vitro and promote spine fusion in vivo. The purpose of this study was to identify an osteogenic oxysterol more suitable for clinical development than those previously reported, and evaluate its ability to promote osteogenesis in vitro and spine fusion in rats in vivo. Among more than 100 oxysterol analogues synthesized, Oxy133 induced significant expression of osteogenic markers Runx2, osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts and in M2‐10B4 mouse marrow stromal cells. Oxy133‐induced activation of an 8X‐Gli luciferase reporter, its direct binding to Smoothened, and the inhibition of Oxy133‐induced osteogenic effects by the Hedgehog (Hh) pathway inhibitor, cyclopamine, demonstrated the role of Hh pathway in mediating osteogenic responses to Oxy133. Oxy133 did not stimulate osteogenesis via BMP or Wnt signaling. Oxy133 induced the expression of OSX, BSP, and OCN, and stimulated robust mineralization in primary human mesenchymal stem cells. In vivo, bilateral spine fusion occurred through endochondral ossification and was observed in animals treated with Oxy133 at the fusion site on X‐ray after 4 weeks and confirmed with manual assessment, micro‐CT (µCT), and histology after 8 weeks, with equal efficiency to recombinant human bone morphogenetic protein‐2 (rhBMP‐2). Unlike rhBMP‐2, Oxy133 did not induce adipogenesis in the fusion mass and resulted in denser bone evidenced by greater bone volume/tissue volume (BV/TV) ratio and smaller trabecular separation. Findings here suggest that Oxy133 has significant potential as an osteogenic molecule with greater ease of synthesis and improved time to fusion compared to previously studied oxysterols. Small molecule osteogenic oxysterols may serve as the next generation of bone anabolic agents for therapeutic development.

Ostene, a new alkylene oxide copolymer bone hemostatic material, does not inhibit bone healing.

OBJECTIVE In this study, we investigate the effects of a soft bone hemostatic wax comprised of water-soluble alkylene oxide copolymers (Ostene; Ceremed, Inc., Los Angeles, CA) on bone healing in a rat calvaria defect model. We compared the effects with a control (no hemostatic agent) and bone wax, an insoluble and nonresorbable material commonly used for bone hemostasis. METHODS Two bilateral 3-mm circular noncritical-sized defects were made in the calvariae of 30 rats. Alkylene oxide copolymer or bone wax was applied or no hemostatic material was used (control). After 3, 6, and 12 weeks, rats were sacrificed and the calvariae excised. Bone healing, expressed as fractional bone volume (± standard error of the mean), was measured by microcomputed tomography. RESULTS Immediate hemostasis was achieved equally with bone wax and alkylene oxide copolymer. Bone wax-filled defects remained unchanged at all time points with negligible healing observed. At 3 weeks, no evidence of alkylene oxide copolymer was observed at the application site, with fractional bone volume significantly greater than bone wax-treated defects (0.20 ± 0.03 versus 0.02 ± 0.01; P = 0.0003). At 6 and 12-weeks, alkylene oxide copolymer-treated defects continued to show significantly greater healing versus bone wax (0.18 ± 0.04 versus 0.05 ± 0.01 and 0.31 ± 0.04 versus 0.06 ± 0.02, respectively). At all time points, alkylene oxide copolymer-treated and control defects showed good healing with no significant difference. CONCLUSION Alkylene oxide copolymer is an effective hemostatic agent that does not inhibit osteogenesis or bone healing.