As the global number of patients with osteoporosis and malignant diseases such as breast cancer, multiple myeloma, and prostate cancer increases every year, there is an associated rise in the use of antiangiogenic medications and antiresorptive medications. With increasing frequencies in the use of antiangiogenic and antiresorptive medications, there is an associated increase in the number of medication-related osteonecrosis of the jaw (MRONJ) cases reported from patients. MRONJ has emerged as a significant comorbidity in cancer patients treated with antiangiogenics or high doses of potent antiresorptive agents, such as bisphosphonates (BPs) or denosumab. MRONJ first emerged from BP-treated cancer patients who presented with a spectrum of dental problems, including delayed wound healing following a dental extraction or oral surgery, exposed bone, soft tissue infection and inflammation, anesthesia, paresthesia, odontalgia, sinus pain, and aching bone pain in the mandible, which continues to be a significant source of problems for dentists, physicians, and patients today. A significant number of MRONJ cases secondary to osteoporosis have also been reported in osteoporotic patients receiving antiresorptive medications. The American Association of Oral and Maxillofacial Surgeons (AAOMS) has established diagnostic standards for MRONJ based on pharmacological history, clinical signs, and radiographic findings. However, as the expertise and knowledge base for MRONJ continues to evolve, revisions and refinements for MRONJ pathogenesis and treatment strategies are necessary to reflect the current research status of the disease correctly. This review highlights current scientific information associated with MRONJ to identify and summarize preventative measures, and treatment interventions for reading the impact of this debiliating disorders.

Introduction:In this study, we tested the hypothesis that pre-osteoclast signaling is key in triggering post-traumatic angiogenesis in alveolar bone via the SDF-1/CXCR4 pathway. Interruption of osteoclast differentiation through zoledronate (Zol) disrupts the crosstalk between pre-osteoclasts and endothelial cells, hindering the initial angiogenic reaction following dental trauma. This disruption could therefore play a role in the pathogenesis of medication-related osteonecrosis of the jaw (MRONJ). Methods:The effect of zoledronate on the expression of SDF1 was tested in pre-osteoclasts (POC) in vitro. Then, we tested the effect of pre-osteoclast conditioned medium on HUVEC cell differentiation, migration, tube-formation, and CXCR4 expression and activity in-vitro. Lastly, we quantified the effect of zoledronate treatment on post-traumatic vascular perfusion of alveolar bone, using microCT-angiography and immunohistochemistry. Results:SDF-1 mRNA expression decreased in Zol-treated POCs (p = 0.02). Flow-Cytometry analysis showed a decrease in CXCL-12+ (SDF-1α) expressing POCs with Zol treatment (p = 0.0058). On the other hand, CXCR4 mRNA expression was significantly inhibited in Zol-treated HUVECs (p = 0.0063). CXCR4 protein expression and activity showed a corresponding dose-dependent downregulation HUVEC surface treated with conditioned media from POC treated with Zol (p = 0.008 and 0.03, respectively). Similar inhibition was observed of HUVEC migration (p = 0.0012), and tube formation (p < 0.0001), effects that were reversed with SDF-1. Finally, there was a significant reduction of CD31+ HUVECs in Alveolar bone of Zol-treated rats (p = 0.0071), confirmed by significantly lower percentage of blood vessel volume (p = 0.026), and marginally lower vessel number (p = 0.062) in the alveolar bone. Conclusion:Pre-osteoclasts play a crucial role in the initial angiogenic response in alveolar bone following dental extraction. Disruption of this process may be a predisposing factor to osteonecrosis.

Changes in mandibular CDH5, CXCL1, and PECAM1 expression following exposure to bisphosphonate and molar extraction suggest a loss of vascular endothelial cell barrier integrity plays a role in MRONJ Objectives: Medication related osteonecrosis of the jaw (MRONJ) is a disorder characterized by loss of blood supply to the jaws and death to the bone.In our previous work, we created a rat model of MRONJ by multiple injections of 60ug/Kg zoledronic acid (ZA) via tail vein followed by extraction of a single first molar.We have previously shown in this model a decrease in the vasculature of the jaws and a delay in bone healing after 6 weeks.The current study was designed to look at mRNA expression and immunohistochemical localization of three endothelial cell markers (CDH5, CXCL1 and PECAM1) 6 weeks following ZA injection with and without molar extraction and in saline injected rats as controls.The objective of this study is to determine if the expression of these markers may serve to identify clinically significant changes in vascular endothelium barrier function associated with the onset of MRONJ.Methods: Using RT-PCR, we analyzed the expression of mRNA for angiogenic markers, six weeks after injection with either 60ug/Kg ZA or saline as a control and in ZA injected rats following first molar extraction.Routine immunohistochemical procedures with antibodies specific for rat marker proteins were used to study the immunohistochemical localization of proteins translated from the mRNA angiogenic markers with significant expression.All tissues were processed together under identical conditions.

Selection of Angiogenic Markers that Predict the Transition from Bisphosphonate Exposure to MRONJ in a Rat ModelMedication related osteonecrosis of the jaw (MRONJ) is a disorder characterized by loss of blood supply to the jaws and death to the bone.In our previous work, we created a rat model of MRONJ by two injections of 60ug/Kg zoledronic acid (a powerful bisphosphonate abbreviated as ZA) via tail vein followed by extraction of a single first molar.We have shown in this model (ZA-treated rats plus molar extraction) a decrease in the vasculature of the jaws and a delay in bone healing beyond 4 weeks.Purpose: The current study identified angiogenic factors from the jaws of our MRONJ model where expression was altered independent of exposure to ZA alone.Methods: Using RT-PCR arrays containing 84 different gene sequences related to angiogenesis, we screened RNA isolated from the jaws of Control, ZA-treated rats, and our MRONJ rat model (ZA-treated plus first molar extraction), 3 and 6 weeks after extraction.Heat maps of gene expression were analyzed to identify genes where expression was either maximal or lost in MRONJ rats relative to ZA-treated rats without extraction.Results: Our study demonstrates the loss or gain of expression for 22 genes in the MRONJ rat model relative to rats treated with ZA alone.In MRONJ rats, the loss of expression was seen for 10 genes after 3 weeks and 3 additional genes (13 total) after 6 weeks where maximal expression was seen in rats treated with ZA-alone.This study also identified 5 genes that were maximally expressed in MRONJ rats after 3 weeks and an additional 4 genes (9 total) after 6 weeks that were not expressed in rats treated with ZA alone.Conclusions: Our study identifies genes that predict the transition from asymptomatic bisphosphonate exposure to MRONJ.

Medication-related osteonecrosis of the jaw (MRONJ) is a debilitating side effect of antiresorptive and antiangiogenic medications used to treat osteoporosis and the complications of bone metastasis of several cancers. This manuscript reviews the mechanisms of current drug therapies that exacerbate MRONJ and highlights the most promising treatment options available today. We also discuss new intriguing developments for the treatment of MRONJ that may further improve patient outcomes.

While earlier studies have suggested that cells positive for hematopoietic markers can be found in dental tissues, it has yet to be confirmed. To conclusively demonstrate this, we utilized a unique transgenic model in which all hematopoietic cells are green fluorescent protein+ (GFP+). Pulp, periodontal ligament (PDL) and alveolar bone (AvB) cell culture analysis demonstrated numerous GFP+ cells, which were also CD45+ (indicating hematopoietic origin) and co-expressed markers of cellular populations in pulp (dentin matrix protein-1, dentin sialophosphoprotein, alpha smooth muscle actin [ASMA], osteocalcin), in PDL (periostin, ASMA, vimentin, osteocalcin) and in AvB (Runx-2, bone sialoprotein, alkaline phosphatase, osteocalcin). Transplantation of clonal population derived from a single GFP+ hematopoietic stem cell (HSC), into lethally irradiated recipient mice, demonstrated numerous GFP+ cells within dental tissues of recipient mice, which also stained for markers of cell populations in pulp, PDL and AvB (used above), indicating that transplanted HSCs can differentiate into cells in dental tissues. These hematopoietic-derived cells deposited collagen and can differentiate in osteogenic media, indicating that they are functional. Thus, our studies demonstrate, for the first time, that cells in pulp, PDL and AvB can have a hematopoietic origin, thereby opening new avenues of therapy for dental diseases and injuries.

Bone to mechanical loading elicits a biological response that has clinical significance for several areas in dental medicine, including orthodontic tooth movement, tempromandibular joint disease, and endosseous dental implant osseointegration. Human orthopedic studies of failed hip implant sites have identified increased mRNA expression of several collagen-degrading matrix metalloproteinases (MMPs), while in vitro experiments have shown increases in MMP secretion after exposure to inflammatory mediators. This investigation evaluates the effects of mechanical deformation on in vitro osteoblasts by assessing changes in MMP gene expression and enzyme activity. We seeded mouse neonatal calvarial osteoblasts onto flexible 6-well plates and subjected to continuous cyclic mechanical stretching. The expression and activity of mRNA for several MMPs (2, 3, 9, and 10) was assessed. When subjected to mechanical stress in culture, only mRNA specific for MMP-9 was significantly increased compared to nonstretched controls (P < .005). Measurement of MMP activity by gelatin zymography demonstrated that none of the MMPs showed increased activity with stretching; however, MMP-2 activity decreased. Our results suggest that in response to stretch, MMP-2 responds rapidly by inhibiting conversion of a MMP-2 to the active form, while a slower up-regulation of MMP-9 may play a role in the long-term remodeling of extracellular matrix in response to continuous mechanical loading. This study suggests that the regulation of metalloproteinases at both the mRNA and protein level are important in the response of bone to mechanical stress.

Objectives: Bisphosphonates become adsorbed on hydroxyapatite crystals in the bone matrix. In case of side-effects, stopping the treatment would not affect the bisphosphonates already deposited in bone. This study tests the feasibility of in-vivo targeted removal of bisphosphonates from bone using chelating agents.Design: 32 Sprague Dawley rats were given an injection of fluorescent pamidronate (OsteoSense EX; 0.16 nmol/g). They were treated with either systemic (cadmium) or local [ethylenediaminetetraacetic (EDTA) or citric acid (CA)] chelating agents to induce the removal of the bisphosphonate from bone. We evaluated the decrease in fluorescence in the alveolar bone, femur, tibia, and vertebrae. We also analyzed the systemic effects of treatment.Results: Systemic chelation reduced the pamidronate signal universally. However, the maximum reduction was observed in the alveolar bone and femur (22% and 21%, p values 0.008 and 0.028, respectively). Systemic chelation did not impair calcium homeostasis. The chelation effect was not due to a systemic toxic effect on the liver or kidney. On the other hand local chelation at the extraction site significantly (p = 0.011) decreased the pamidronate signal at bony surfaces of the socket.Conclusions: Systemic and local chelating agents can remove bisphosphonate from bone. This study establishes a new concept for the prevention of side effects of bisphosphonates during high-risk situations. Published by Elsevier Ltd.

Platelet-rich plasma (PRP) is an autogenous source of growth factors shown to facilitate human bone growth. Bio-Oss, an osteoconductive xenograft, is used clinically to regenerate periodontal defects, restore dental alveolar ridges, and facilitate sinus-lift procedures. The purpose of this study was to analyze whether a combination of PRP and Bio-Oss would enhance bone regeneration better than either material alone. PRP and/or Bio-Oss were administered in an 8-mm critical-size defect (CSD) rat calvarial model of bone defect between 2 polytetrafluoroethylene membranes to prevent soft tissue incursion. Eight weeks after the induction of the CSD, histologic sections were stained with hematoxylin and eosin stain and analyzed via light microscopy. Qualitative analyses revealed new bone regeneration in all 4 groups. The Bio-Oss and PRP plus Bio-Oss groups demonstrated greater areas of closure in the defects than the control or PRP-only groups because of the space-maintaining ability of Bio-Oss. The groups grafted with Bio-Oss showed close contact with new bone growth throughout the defects, suggesting a stronger graft. The use of PRP alone or in combination with Bio-Oss, however, did not appear to enhance osseous regeneration at 8 weeks. Areas grafted with Bio-Oss demonstrated greater space-maintaining capacity than controls, and PRP was an effective vehicle for placement of the Bio-Oss. However, at 8 weeks this study was unable to demonstrate a significant advantage of using PRP plus Bio-Oss over using Bio-Oss alone.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) causes bones of the mandible and maxilla to become necrotic and protrude into the oral cavity. Compromised blood supply to bone is also a feature of BRONJ. The design of this study was first to use our established technique of molar extraction and IV bisphosphonate injection to produce features of BRONJ in rats that mimic the human disease; second to confirm vascular changes in the mandible and eye using micro-CT of vascular casts, and image analysis of retina/choroid images; and third to show parallel bisphosphonate-induced changes in the structure and markers of the vasculature of the bone and eye. The results of this study show structural changes in the eye and mandible as well as biochemical changes including the up-regulation of VEGF in response to the bisphosphonate-associated ischemia. These changes are not associated with angiogenesis in either the eye or mandible as determined by reduced vascular complexity. These results suggest that observations of direct changes to the vasculature in the retina/choroid structures of the eye in patients taking bisphosphonates could serve as a window to the progression of debilitating changes occurring as a result of bisphosphonate therapy.

This study aims to develop a reproducible rat model for post-traumatic bisphosphonate-related osteonecrosis of the jaw (BRONJ). In our previous studies using dental extraction as an inducing factor, only 30%-60% of zoledronate-treated animals fulfilled the definition of clinical BRONJ. We modified the zoledronate regimen and introduced repeated surgical extraction to illicit quantifiable BRONJ in all animals. Eighty retired-breeder female Sprague-Dawley rats were divided between the treatment (i.v. zoledronate; 80 μg/kg/week for 13 weeks) and control (saline) groups. On week 13, the left mandibular first molar was surgically extracted, followed by the second molar a week later. Animals were euthanized at 1-week, 2-weeks, and 8-weeks following extraction. The occurrence and severity of BRONJ were scored in each animal based on gross and MicroCT analysis. Parameters of bone formation and osteoclast functions at the extraction site were compared between groups. All zoledronate-treated animals developed a severe case of BRONJ that fulfilled the clinical definition of the condition in humans. Osteoclast attachment continued to be defective eight weeks after stopping the treatment. There were no signs of kidney or liver toxicity. Our data confirmed that repeated surgical extraction (major trauma) by itself consistently precipitated massive bone necrosis in ZA-treated animals, eliminating the need to induce pre-existing infection or comorbidity. These results will be the basis for further studies examining the in-vivo pathogenesis and prevention of BRONJ.

Long-term use of intravenous bisphosphonates, such as zoledronic acid (zoledronate), has been linked to bisphosphonate-related osteonecrosis of the jaw (BRONJ). Invasive dental surgery seems to trigger the bone necrosis in most cases. To determine the effects of zoledronic acid on the vascular structure of the rat mandible. Extracted of the mandibular first molar in rats that received 2 IV injections of zoledronate (20 μg/kg), 4 weeks apart. Zoledronate-treated rats (n = 18) were then compared to a control group of untreated rats (n = 18). At the fourth, eighth, and 12th week after molar extraction, 8 rat mandibles from each group were perfused with 35% radiopaque triphenylbismuth in methyl methacrylate via carotid artery perfusion. Mandibles were harvested and examined by micro-CT to assess the spatial and dimensional changes of the vasculature as a result of zoledronate treatment. The micro-CT analysis showed that zoledronic acid-treated rats had blood vessels that were thicker, less connected, and less ordered than control rats that were not exposed to zoledronic acid. This study demonstrated that treatment with zoledronic acid in rats is associated with vascular changes in alveolar bone. Further studies are underway to explore whether these vascular changes contribute to the pathogenesis of BRONJ.

BACKGROUNDSignificant adverse effects on fibroblast growth and metabolism are observed with nicotine. We investigated the synergistic effects of nicotine and cyclical mechanical strain (CMS) on human gingival fibroblasts (HGFs) in a wound-healing model.METHODSHGFs were isolated and grown in Dulbecco's modified Eagle's medium. Three-millimeter wounds were created on a confluent cell monolayer grown in a media containing 0, 1, 2, or 4 mM nicotine, with or without CMS. The applied deformation regimen remains constant for 6 days. On days 1, 2, 4, and 6, the cells were stained with hematoxylin and eosin Y for the evaluation of wound repopulation.RESULTSThe application of CMS alone demonstrates a biphasic response, with an initial stimulatory effect on wound repopulation (days 1-2) and less repopulation during the later phase (days 4-6). The addition of nicotine clearly demonstrated a time and inverse dose-dependent relationship on wound repopulation, with no effect during the early phase and reduced wound repopulation during the later phase.CONCLUSIONSInitial treatment of HGF wounds with CMS resulted in faster wound repopulation regardless of nicotine presence. By day 6, wound healing of HGF exposed to both nicotine and CMS is delayed. These findings suggest that CMS and nicotine may affect fibroblasts and delay wound healing at other sites in the body as well.

PURPOSE:To investigate the effects of hyperglycemia and metformin (a popular biguanide antidiabetic) on periimplant healing.METHODS:Thirty-six male rats were assigned to 3 groups: (1) nondiabetic Wistar-Kyoto rats (controls), (2) Goto-Kakizaki (GK) spontaneously diabetic rats (GK group), and (3) GK rats were fed metformin (100 mg/kg body weight per day) in their water for 4 weeks (GK + Met group). The right maxillary first molars were extracted and sites were allowed 1 month to heal. Titanium implants (1 × 3 mm) were placed in healed extraction sites. Six rats from each group were analyzed at weeks 1 and 4 by micro computed tomography for bone/implant contact ratio, percent bone volume, trabecular number, and bone mineral density. Blood was also analyzed for glucose, HbA1c, and pyridinoline (PYD).RESULTS:At week 1, glucose levels in the GK-Met rats were high, and all bone parameters were similar to GK rats (lower bone parameters and higher PYD than controls). At week 4, glucose levels in the GK-Met rats and all parameters were similar to controls.CONCLUSIONS:Hyperglycemic GK type 2 diabetic rats showed improved blood glucose and wound healing around oral implants after metformin administration.

The purpose of this study was to develop a rat model predictive of bisphosphonate-related osteonecrosis of the jaw (BRONJ) after exodontias. Thirty female rats were randomized into 2 groups, control and experimental. The experimental group received 2 intravenous injections of zoledronate (20 μg/kg). The mesial root of the right mandibular first molar was extracted. Rats were euthanized at 0, 4, and 8 weeks. Bone mineral density (BMD), collagen breakdown (pyridinium [PYD]), vascular regeneration (VEGF), and histology were examined. A trend toward higher PYD values was suggested in control vs experimental groups after wounding. Serum VEGF increased significantly after wounding for both control and experimental groups. After 8 weeks, VEGF continued to rise for the experimental group only. In the extraction socket area, BMD was significantly lower after wounding in control vs. zoledronate-treated rats. Histology sections from experimental groups showed bacteria and bone necrosis. Consistent findings of BRONJ features similar to those in humans were observed after zoledronate treatment.

Introduction: Endogenous dentin matrix metalloproteinases (MMPs) contribute to extracellular collagen matrix degradation in hybrid layers after adhesive dentin bonding procedures. Endodontic irrigants, including chlorhexidine and ethylenediaminetetraacetic acid (EDTA), might help protect the hybrid layer from this process. The objective of the present study was to determine the exposure time necessary for EDTA to inactivate endogenous MMP activity in human dentin. Methods: Dentin beams (2 x 1 x 3 mm) were prepared from midcoronal dentin of extracted third molars. The beams were demineralized in 10 wt% phosphoric acid, which also activated endogenous MMPs, and were divided into 4 experimental groups on the basis of exposure time to 17% EDTA (0, 1, 2, or 5 minutes). A generic colorimetric MMP assay measured MMP activity via absorbance at 412 nm. Data were evaluated by Kruskal-Wallis analysis of variance, followed by Dunn pair-wise comparisons at alpha = 0.05. Results: All exposure times resulted in significant inhibition (P <.001) compared with unexposed controls. Specifically, percent inhibition for 1-, 2-, and 5-minute exposure times was 55.1% +/- 21.5%, 72.8% +/- 11.7%, and 74.7% +/- 19.7%, respectively. Conclusions: Seventeen percent EDTA significantly inhibits endogenous MMP activity of human dentin within 1-2 minutes. This might minimize hybrid layer degradation after resin bonding procedures in the root canal space. (J Endod 2012;38:62-65)

Aim. Physicochemical mechanical and in vitro biological properties of novel formulations of polymeric calcium phosphate cements (CPCs) were investigated. Methods. Monocalcium phosphate, calcium oxide, and synthetic hydroxyapatite were combined with either modified polyacrylic acid, light activated polyalkenoic acid, or polymethyl vinyl ether maleic acid to obtain Types I, II, and III CPCs. Setting time, compressive and diametral strength of CPCs was compared with zinc polycarboxylate cement (control). Specimens were characterized using X-ray diffraction, scanning electron microscopy, and infrared spectroscopy. In vitro cytotoxicity of CPCs and control was assessed. Results. X-ray diffraction analysis showed hydroxyapatite, monetite, and brushite. Acid-base reaction was confirmed by the appearance of stretching peaks in IR spectra of set cements. SEM revealed rod-like crystals and platy crystals. Setting time of cements was 5-12 min. Type III showed significantly higher strength values compared to control. Type III yielded high biocompatibility. Conclusions. Type III CPCs show promise for dental applications.