Akishige Hokugo

Increased prevalence of bisphosphonate-related osteonecrosis of the jaw with vitamin D deficiency in rats.

Necrotic bone exposure in the oral cavity has recently been reported in patients treated with nitrogen‐containing bisphosphonates as part of their therapeutic regimen for multiple myeloma or metastatic cancers to bone. It has been postulated that systemic conditions associated with cancer patients combined with tooth extraction may increase the risk of osteonecrosis of the jaw (ONJ). The objective of this study was to establish an animal model of bisphosphonate‐related ONJ by testing the combination of these risk factors. The generation of ONJ lesions in rats resembling human disease was achieved under the confluence of intravenous injection of zoledronate (ZOL; 35 µg/kg every 2 weeks), maxillary molar extraction, and vitamin D deficiency [VitD(−)]. The prevalence of ONJ in the VitD(−)/ZOL group was 66.7%, which was significantly higher (p < .05, Fisher exact test) than the control (0%), VitD(−) (0%), and ZOL alone (14.3%) groups. Similar to human patients, rat ONJ lesions prolonged the oral exposure of necrotic bone sequestra and were uniquely associated with pseudoepitheliomatous hyperplasia. The number of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick‐end label–positive (TUNEL+) osteoclasts significantly increased on the surface of post–tooth extraction alveolar bone of the VitD(−)/ZOL group, where sustained inflammation was depicted by [18F]fluorodeoxyglucose micro‐positron emission tomography (µPET). ONJ lesions were found to be associated with dense accumulation of mixed inflammatory/immune cells. These cells, composed of neutrophils and lymphocytes, appeared to juxtapose apoptotic osteoclasts. It is suggested that the pathophysiologic mechanism(s) underpinning ONJ may involve the interaction between bisphosphonates and compromised vitamin D functions in the realm of skeletal homeostasis and innate immunity.

Stimulation of bone regeneration following the controlled release of water-insoluble oxysterol from biodegradable hydrogel

Recently bone graft substitutes using bone morphogenetic proteins (BMPs) have been heralded as potential alternatives to traditional bone reconstruction procedures. BMP-based products, however, are associated with significant and potentially life-threatening side effects when used in the head and neck region and furthermore, are exorbitantly priced. Oxysterols, products of cholesterol oxidation, represent a class of molecules that are favorable alternatives or adjuncts to BMP therapy due to their low side effect profile and cost. In order to establish the optimal clinical utility of oxysterol, an optimal scaffold must be developed, one that allows the release of oxysterol in a sustained and efficient manner. In this study, we prepare a clinically applicable bone graft substitute engineered for the optimal release of oxysterol. We first solubilized oxysterol in water by making use of polymeric micelles using l-lactic acid oligomer (LAo) grafted gelatin. Then, the water-solubilized oxysterol was incorporated into a biodegradable hydrogel that was enzymatically degraded intracorporeally. In this manner, oxysterol could be released from the hydrogel in a degradation-driven manner. The water-solubilized oxysterol incorporated biodegradable hydrogel was implanted into rat calvarial defects and induced successful bone regeneration. The innovative significance of this study lies in the development of a bone graft substitute that couples the osteogenic activity of oxysterol with a scaffold designed for optimized oxysterol release kinetics, all of which lead to better repair of bone defects.

Molecular Effects of Fractional Carbon Dioxide Laser Resurfacing on Photodamaged Human Skin

Objective To elucidate the sequential changes in protein expression that play a role in the clinically beneficial results seen with fractional carbon dioxide (CO(2)) laser resurfacing of the face and neck. Methods Nine healthy volunteers were recruited for participation from the senior authors facial plastic surgery practice. After informed consent was obtained, each volunteer underwent a 2-mm punch biopsy from a discrete area of infra-auricular neck skin prior to laser treatment. Patients then immediately underwent laser resurfacing of photodamaged face and neck skin at a minimal dose (30 W for 0.1 second) with the Pixel Perfect fractional CO(2) laser. On completion of the treatment, another biopsy specimen was taken adjacent to the first site. Additional biopsy specimens were subsequently taken from adjacent skin at 2 of 3 time points (day 7, day 14, or day 21). RNA was extracted from the specimens, and reverse transcriptase-polymerase chain reaction and protein microarray analysis were performed. Comparisons were then made between time points using pairwise comparison testing. Results We found statistically significant changes in the gene expression of several matrix metalloproteinases (MMPs). The data demonstrate a consistent up-regulation of MMPs 1, 3, 9, and 13, all of which have been previously reported for fully ablative CO(2) laser resurfacing. There was also a statistically significant increase in MMP-10 and MMP-11 levels in this data set. Conclusion This study suggests that the molecular mechanisms of action are similar for both fractional and fully ablative CO(2) laser resurfacing.

Animal model of radiogenic bone damage to study mandibular osteoradionecrosis.

OBJECTIVE The objective of the study was to create an animal model to study mandibular osteoradionecrosis (ORN) using high-dose rate (HDR) brachytherapy. METHODS Ten Sprague-Dawley male rats were used in this study. Six rats received a single dose of 30 Gy using an HDR remote afterloading machine via a brachytherapy catheter placed along the left hemimandible. The remaining 4 rats served as controls with catheter placement without radiation (sham). On the day following irradiation or sham, all 3 left mandibular molars were atraumatically extracted. Twenty-eight days after irradiation, mandibles were examined using nondecalcified histology with sequential fluorochrome labeling, decalcified histology, and micro-computed tomography scanning. RESULTS Irradiated rats demonstrated exposed bone at the extraction sockets, whereas the control animals had complete mucosalization. Alopecia was also seen in the irradiated group. Both histologic and radiologic analyses of the mandible specimens demonstrated a reduction in bone formation in the radiated mandibles as compared with controls. CONCLUSIONS Our HDR brachytherapy model incorporating postradiation dental extractions has successfully demonstrated reproducible radiogenic mandibular bone damage analogous to the clinical ORN. Although clinical criteria continue to be used today in describing ORN, this model can serve as a platform for future studies to define ORN and delineate its pathogenesis.

Equilibrium-dependent bisphosphonate interaction with crystalline bone mineral explains anti-resorptive pharmacokinetics and prevalence of osteonecrosis of the jaw in rats

Bisphosphonates (BPs) are chemically stable analogs of pyrophosphate exhibiting strong affinity to bone and have been used for the treatment of diseases characterized by excessive bone resorption. Contrary to the widely accepted BP accumulation model in bone after repeated applications, we report here that an equilibrium-dependent BP-crystalline bone mineral interaction may better explain BP bio-distribution and anti-catabolic bone remodeling and may be relevant to the appearance of osteonecrosis of the jaw (ONJ) in rats. Fluorescent-labeled BP analogs were synthesized and used to evaluate the mode of bone adsorption. After fluorescent-labeled BP adsorbed on crystalline calcium phosphates in vitro, subsequent BP application replaced the previously absorbed BP depending on the dose and the relative binding affinity to hydroxyapatite. The in vivo intravenous zoledronate (ZOL) injection of repeated fractional doses resulted in lower serum CTX and TRAP5b measurements than a single bolus injection in spite of the equivalent cumulative dose. Repeated injections resulted in the distribution of fluorescent-labeled BP on the large area of bone surfaces; whereas the single bolus injection gave rise to the intense BP bio-distribution at selected bone sites such as the alveolar process of jawbones. Necrotic maxillary alveolar bone was predominantly observed in vitamin D deficiency rats treated with bolus ZOL injection. The palatal necrotic bone was characteristically sequestrated by the fistulation of hyperplastic oral epithelium, suggesting the initial development of ONJ-like lesions in rats. Our results suggest that equilibrium-dependent BP-bone interaction may, in part, determine the effectiveness and influence side effects of long-term and repeated applications of BPs.

Trabecular bone deterioration in col9a1+/- mice associated with enlarged osteoclasts adhered to collagen IX-deficient bone.

Introduction: Short collagen IX, the exclusive isoform expressed by osteoblasts, is synthesized through alternative transcription of the col9a1 gene. The function of short collagen IX in bone was characterized in col9a1‐null mutant mice.

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.

Small Cytoskeleton-Associated Molecule, Fibroblast Growth Factor Receptor 1 Oncogene Partner 2/Wound Inducible Transcript-3.0 (FGFR1OP2/wit3.0), Facilitates Fibroblast-Driven Wound Closure

Wounds created in the oral cavity heal rapidly and leave minimal scarring. We have examined a role of a previously isolated cDNA from oral wounds encoding wound inducible transcript-3.0 (wit3.0), also known as fibroblast growth factor receptor 1 oncogene partner 2 (FGFR1OP2). FGFR1OP2/wit3.0 was highly expressed in oral wound fibroblasts without noticeable up-regulation of alpha-smooth muscle actin. In silico analyses, denaturing and nondenaturing gel Western blot, and immunocytology together demonstrated that FGFR1OP2/wit3.0 were able to dimerize and oligomerize through coiled-coil structures and appeared to associate with cytoskeleton networks in oral wound fibroblasts. Overexpression of FGFR1OP2/wit3.0 increased the floating collagen gel contraction of naïve oral fibroblasts to the level of oral wound fibroblasts, which was in turn attenuated by small-interfering RNA knockdown. The FGFR1OP2/wit3.0 synthesis did not affect the expression of collagen I as well as procontractile peptides such as alpha-smooth muscle actin, and transforming growth factor-beta1 had no effect on FGFR1OP2/wit3.0 expression. Fibroblastic cells derived from embryonic stem cells carrying FGFR1OP2/wit3.0 (+/-) mutation showed significant retardation in cell migration. Thus, we postulate that FGFR1OP2/wit3.0 may regulate cell motility and stimulate wound closure. FGFR1OP2/wit3.0 was not up-regulated during skin wound healing; however, when treated with FGFR1OP2/wit3.0 -expression vector, the skin wound closure was significantly accelerated, resulting in the limited granulation tissue formation. Our data suggest that FGFR1OP2/wit3.0 may possess a therapeutic potential for wound management.

A novel oxysterol promotes bone regeneration in rabbit cranial bone defects

Bone morphogenetic proteins (BMPs) have played a central role in the development of regenerative therapies for bone reconstruction. However, the high cost and side‐effect profile of BMPs limits their broad application. Oxysterols, naturally occurring products of cholesterol oxidation, are promising osteogenic agents alternative to BMPs. The osteogenic capacity of these non‐toxic and relatively inexpensive molecules has been documented in rodent models. We studied the impact of Oxy49, a novel oxysterol analogue, on the osteogenic differentiation of rabbit bone marrow stromal cells (BMSCs). Moreover, we evaluated the capacity for in vivo bone regeneration with Oxy49 in rabbit cranial bone defects. We found that rabbit BMSCs treated with Oxy49 demonstrated differentiation along osteogenic pathways, and that complete bone regeneration occurred when cranial defects were treated with Oxy49. Collectively, these results demonstrate that Oxy49 has the ability to induce osteogenic differentiation in rabbit BMSCs with an efficacy comparable to that of BMP‐2 and to promote significant bone regeneration in cranial defects. Oxysterols may be a viable novel agent in bone tissue engineering. Copyright

In vitro study of a novel oxysterol for osteogenic differentiation on rabbit bone marrow stromal cells.

Background: Bone morphogenetic proteins (BMPs) are powerful osteoinductive growth factors but are associated with exorbitant costs and undesirable side effects. Oxysterols are biocompatible cholesterol oxidation products with osteoinductive properties that may represent an alternative to BMP. In this study, the authors examine the osteogenic potential and mechanisms of actions of oxysterol 49, a novel oxysterol analogue, in primary rabbit bone marrow stromal cells. Methods: Bone marrow stromal cells were isolated from the iliac crests of New Zealand White rabbits and then treated with various concentrations of oxysterol 49 or BMP-2, either alone or in combination. Alkaline phosphatase activity and expression of osteocalcin and osteopontin were evaluated. The effect of treatment of cells with cyclopamine, a known hedgehog signaling pathway inhibitor, was also assessed. Results: Alkaline phosphatase activity was increased in cells treated with 1 µM oxysterol 49 relative to cells treated with BMP-2. Expression of osteocalcin and osteopontin in cells treated with oxysterol 49 and BMP-2 was equivalent. Alkaline phosphatase activity was decreased with the addition of cyclopamine. Combined treatment with oxysterol 49 and BMP-2 resulted in additive increases in alkaline phosphatase activity and osteocalcin and osteopontin expression. Conclusions: Oxysterol 49 has osteoinductive properties that are similar to those of BMP-2 in rabbit bone marrow stromal cells. The mechanism of this activity is at least in part related to the hedgehog signaling pathway. The two growth factors demonstrate additive effects when used in combination. Further study is required to examine the potential role of oxysterol 49 as a complement or alternative to BMP-2 in bone tissue engineering.