Effects of hyperbaric oxygen and inducible nitric oxide synthase inhibitor treatment on femoral head osteonecrosis in a rat model

Introduction: Apoptosis is the final destiny of many cells in the body, though this process has been observed in some pathological processes. One of these pathological processes is femoral head non-traumatic osteonecrosis. Among many pro/anti-apoptotic factors, nitric oxide has recently been an area of further interest. Osteocyte apoptosis and its relation to pro-apoptotic action invite further research, and the inducible form of nitric oxide synthase (iNOS)—which produces a high concentration of nitric oxide—has been flagged. The aim of this study was to investigate the effect of hyperbaric oxygen (HBO) and inducible NOS suppressor (Aminoguanidine) in prevention of femoral head osteonecrosis in an experimental model of osteonecrosis in spontaneous hypertensive rats (SHRs). Methods: After animal ethic approval 34 SHR rats were divided into four groups. Ten rats were allocated to the control group without any treatment, and eight rats were allocated to three treatment groups namely: HBO, Aminoguanidine (AMG), and the combination of HBO and AMG treatments (HBO+AMG). The HBO group received 250 kPa of oxygen via hyperbaric chamber for 30 days started at their 5th week of life; the AMG group received 1mg/ml of AMG in drinking water from the fifth week till the 17th week of life; and the last group received a combination of these treatments. Rats were sacrificed at the end of the 17th week of life and both femurs were analysed for evidence of osteonecrosis using Micro CT scan and H&E staining. Also, osteocyte apoptosis and the presence of two different forms of NOS (inducible (iNOS) and endothelial (eNOS)) were analysed by immunostaining and apoptosis staining (Hoechst and TUNEL). Results: Bone morphology of metaphyseal and epiphyseal area of all rats were investigated and analysed. Micro CT findings revealed significantly higher mean fractional trabecular bone volume (FBV) of metaphyseal area in untreated SHRs compared with all other treatments (HBO, P<0.05, HBO+AMG, P<0.005, and AMG P<0.001). Bone surface to volume ratio also significantly increased with HBO+AMG and AMG treatments when compared with the control group (18.7 Vs 20.8, P<0.05, and 18.7 Vs 21.1, P<0.05). Epiphyseal mean FBV did not change significantly among groups. In the metaphyseal area, trabecular thickness and numbers significantly decreased with AMG treatment, while trabecular separation significantly increased with both AMG and HBO+AMG treatment. Histological ratio of no ossification and osteonecrosis was 37.5%, 43.7%, 18.7% and 6.2% of control, HBO, HBO+AMG and AMG groups respectively with only significant difference observed between HBO and AMG treatment (P<0.01). High concentration of iNOS was observed in the region of osteonecrosis while there was no evidence of eNOS activity around that region. In comparison with the control group, the ratio of osteocyte apoptosis significantly reduced in AMG treatment (P<0.005). We also observed significantly fewer apoptotic osteocytes in AMG group comparing with HBO treatment (P<0.05). Conclusion: None of our treatments prevents osteonecrosis at the histological or micro CT scan level. High concentration of iNOS in the region of osteonecrosis and significant reduction of osteocyte apoptosis with AMG treatment were supportive of iNOS modulating osteocyte apoptosis in SHRs.

iNOS expression and osteocyte apoptosis in idiopathic, non- traumatic osteonecrosis

Background and purpose Non-traumatic osteonecrosis is a progressive disease with multiple etiologies. It affects younger individuals more and more, often leading to total hip arthroplasty. We investigated whether there is a correlation between inducible nitric oxide synthase (iNOS) expression and osteocyte apoptosis in non-traumatic osteonecrosis. Patients and methods We collected and studied 20 human idiopathic, non-traumatic osteonecrosis femoral heads. Subchondral bone samples in the non-sclerotic region (n = 30), collected from osteoarthritis patients, were used as controls. Spontaneously hypertensive rats were used as a model for osteonecrosis in the study. We used scanning electron microscopy, TUNEL assay, and immunohistochemical staining to study osteocyte changes and apoptosis. Results The morphology of osteocytes in the areas close to the necrotic region changed and the number of apoptotic osteocytes increased in comparison with the same region in control groups. The expression of iNOS and cytochrome C in osteocytes increased while Bax expression was not detectable in osteonecrosis samples. Using spontaneously hypertensive rats, we found a positive correlation between iNOS expression and osteocyte apoptosis in the osteonecrotic region. iNOS inhibitor (aminoguanidine) added to the drinking water for 5 weeks reduced the production of iNOS and osteonecrosis compared to a control group without aminoguanidine. Interpretation Our findings show that increased iNOS expression can lead to osteocyte apopotosis in idiopathic, non-traumatic osteonecrosis and that an iNOS inhibitor may prevent the progression of the disease.

Is rheumatoid arthritis a risk factor for oral bisphosphonate-induced osteonecrosis of the jaws?

Bisphosphonate-related osteonecrosis of the jaws is a relevant side-effect of these drugs that has been generating a great concern through increasing reports, worldwide, of this bone necrosis. Among several BRONJ hypothetical co-factors that could play a role in BRONJ pathogenesis, rheumatoid arthritis (RA) has been included as a relevant risk factor for BRONJ; however, until now the relationship between these diseases has not been fully explained. Thus, the purpose of this paper is to establish hypothetical factors that could link these two diseases, considering mainly inflammatory components and the organism effects of medicines used to treat RA, particularly steroids and methotrexate (MTX). (C) 2011 Elsevier Ltd. All rights reserved.

A Role of Oral Bacteria in Bisphosphonate-induced Osteonecrosis of the Jaw

No consensus has yet been reached to associate oral bacteria conclusively with the etio-pathogenesis of bisphosphonate-induced osteonecrosis of the jaw (BONJ). Therefore, the present study examined the effects of oral bacteria on the development of BONJ-like lesions in a mouse model. In the pamidronate (Pam)-treated mice, but not control non-drug-treated mice, tooth extraction followed by oral infection with Fusobacterium nucleatum caused BONJ-like lesions and delayed epithelial healing, both of which were completely suppressed by a broad-spectrum antibiotic cocktail. Furthermore, in both in vitro and in vivo experiments, the combination of Pam and Fusobacterium nucleatum caused the death of gingival fibroblasts (GFs) and down-regulated their production of keratinocyte growth factor (KGF), which induces epithelial cell growth and migration. Therefore, in periodontal tissues pre-exposed to bisphosphonate, bacterial infection at tooth extraction sites caused diminished KGF expression in GFs, leading to a delay in the epithelial wound-healing process that was mitigated by antibiotics.

Oral bisphosphonate-related osteonecrosis of the jaws in rheumatoid arthritis patients: a critical discussion and two case reports

Background: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a clinical condition characterized by the presence of exposed bone in the maxillofacial region. Its pathogenesis is still undetermined, but may be associated with risk factors such as rheumatoid arthritis (RA). The aim of this paper is to report two unpublished cases of BRONJ in patients with RA and to conduct a literature review of similar clinical cases with a view to describe the main issues concerning these patients, including demographic characteristics and therapeutic approaches applied.Methods: Two case reports of BRONJ involving RA patients were discussedResults: Both patients were aging female taking alendronate for more than 3 years. Lesions were detected in stage II in posterior mandible with no clear trigger agent. The treatment applied consisted of antibiotics, oral rinses with chlorhexidine, drug discontinuation and surgical procedures. Complete healing of the lesions was achieved.Conclusions: This paper brings to light the necessity for rheumatologists to be aware of the potential risk to their patients of developing BRONJ and to work together with dentists for the prevention and early detection of the lesions. Although some features seem to link RA with oral BRONJ and act as synergistic effects, more studies should be developed to support the scientific bases for this hypothesis.

Epidemiological aspects of rheumatoid arthritis patients affected by oral bisphosphonate-related osteonecrosis of the jaws

This literature review aims to evaluate the epidemiologic profile of patients with rheumatoid arthritis (RA) that developed a bisphosphonate-related osteonecrosis that affect the jaws (BRONJ), including demographic aspects, as well as clinical and therapeutic issues. A search of PUBMED/MEDLINE, Scopus, and Cochrane databases from January 2003 to September 2011 was conducted with the objective of identifying publications that contained case reports regarding oral BRONJ in RA patients. Patients with RA who develop oral BRONJ are usually women above 60 years taking steroids and long-term alendronate. Most of them have osteoporosis, and lesions, triggered by dental procedures, are usually detected at stage II in the mandible. Although there is no accepted treatment protocol, these patients seem to have better outcomes with conservative approaches that include antibiotic therapy, chlorhexidine, and drug discontinuation.

Accurate Approach in the Treatment of Oral Bisphosphonate-Related Jaw Osteonecrosis

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is not completely understood and difficult to treat. Even though the occurrence of BRONJ is mainly related to the therapy with intravenous bisphosphonates (BPs), this article reports a case of long-term oral BP use and BRONJ occurrence. In addition, a literature review provides some additional information about BPs, BRONJ, and also a guideline for the prevention and treatment of BRONJ stages. A 79-year-old patient presented intense and persistent pain, purulent secretion, and exposed bone at the right side of the lower jaw. Thus, she was submitted to anamnesis, radiologic and tomographic examinations, and bacterial culture and sensitivity tests. These procedures were followed by surgical debridement of the bone and surrounding tissues/cyst and antibiotic prescription and histopathologic analysis of the fragments. Together, the examinations performed showed the occurrence of stage 2 BRONJ. Moreover, the antibiotic prescription, discontinuation of oral BP, and surgical procedures ensure that the patient had no more symptoms. Therefore, considering the presented case, we believe that an accurate approach is promising to diagnose and treat stage 2 BRONJ and other associated pathologic findings.

Management of Osteonecrosis of the Jaws in Patients With History of Bisphosphonates Therapy

Bisphosphonates are compounds used in the treatment of various metabolic and malignant bone diseases. The relation between the use of bisphosphonates and ostenonecrosis of the jaws as an adverse effect of the drug has been intensely discussed during the last few years, and up to this moment, there is no consensus concerning an ideal treatment modality for this condition. Nevertheless, there is an agreement among researchers that the standard goal for controlling jaw osteonecrosis is to prevent it. Otherwise, the rationale for a randomized controlled trial is that current treatment has proven to be suboptimal, and no consensus has been reached yet on the best strategies to repair the exposed bone once bone necrosis is developed. This article is focused on reporting a case of moderate osteonecrosis of the upper jaw induced by bisphosphonates and discusses a possible role for surgical debridement associated to platelet-rich plasma, hyperbaric oxygen therapy, and the cessation of the bisphosphonate use in managing this type of lesion. Moreover, the dentist, the oral surgeon, and the oncologist need to work together to reach better outcomes.

Mandibular cortical bone evaluation on cone beam computed tomography images of patients with bisphosphonate-related osteonecrosis of the jaw

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Experimental development of bisphosphonate-related osteonecrosis of the jaws in rodents

Osteonecrosis of the jaw (ONJ) following the use of bisphosphonates has become of increased interest in the scientific community, due in particular to its as-yet-unsolved pathogenesis. An experimental model of ONJ was induced in normal male rats [alendronate (ALN); 1 mg/Kg/day; n = 10] and matched controls (saline solution; n = 10). After 60 days of drug treatment, all animals were subjected to extractions of the left first lower molars and were euthanized at 3 and 28 days postsurgery. The following analyses were performed: (i) descriptive and quantitative (scores) histological evaluation, (ii) stereometry of distal sockets and (iii) biochemical measurement of C-telopeptide cross-linked collagen type I (CTX) and bone-specific alkaline phosphatase (BALP). The results showed that 28 days postsurgery the animals treated with ALN had areas of exposed and necrotic bone, associated with significant infection, especially in the interalveolar septum area and crestal regions, compared with controls. The levels of CTX, BALP and bone volume, as well as the degrees of inflammation and vascularization, were significantly reduced in these animals. Therefore, analysis of the data presented suggests that ALN therapy is associated with the development of osteonecrosis in the jaws of rodents after tooth extraction. © 2012 International Journal of Experimental Pathology.

Spontaneous osteonecrosis of the jaws in the maxilla of mice on antiresorptive treatment: A novel ONJ mouse model

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mandibular cortical bone evaluation on cone beam computed tomography images of patients with bisphosphonate related osteonecrosis of the jaws

Objectives The objective of this study was to develop a technique for detecting cortical bone dimensional changes in patients with bisphosphonate-related osteonecrosis of the jaw (BRONJ). Study Design Subjects with BRONJ who had cone-beam computed tomography imaging were selected, with age- and gender-matched controls. Mandibular cortical bone measurements to detect bisphosphonate-related cortical bone changes were made inferior to mental foramen, in 3 different ways: within a fixed sized rectangle, in a rectangle varying with the cortical height, and a ratio between area and height. Results Twelve BRONJ cases and 66 controls were evaluated. The cortical bone measurements were significantly higher in cases than controls for all 3 techniques. The bone measurements were strongly associated with BRONJ case status (odds ratio 3.36-7.84). The inter-rater reliability coefficients were high for all techniques (0.71-0.90). Conclusions Mandibular cortical bone measurement is a potentially useful tool in the detection of bone dimensional changes caused by bisphosphonates. Long-term administration of bisphosphonates (BPs) affects bone quality and metabolism following accumulation in bone.1 Since the first cases of bisphosphonate-related osteonecrosis of the jaw (BRONJ) were published in 2003,2 there has been a search for factors that can predict the onset of the condition. Oral and intravenous BPs reduce bone resorption, increase mineral content of bone, and alter bony architecture.3, 4, 5 and 6 Previous studies have demonstrated these changes both radiographically and following histologic analysis.1, 3, 7, 8, 9 and 10 The BP-related jaw changes may present radiological features, such as thickening of lamina dura and cortical borders, diffuse sclerosis, and narrowing of the mandibular canal3 and 11; however, oral radiographs of patients taking BPs do not consistently show radiographic changes to the jaws.11 and 12 The challenge is to find imaging tools that could improve the detection of changes in the bone associated with BP use. Various skeletal radiographic features associated with BRONJ in conventional periapical and panoramic radiographs, computed tomography, magnetic resonance imaging, and nuclear bone scanning have been described.3, 8, 9, 10 and 11 There has also been a search for BP-related quantitative methods for the evaluation of radiographic images, to avoid observer subjectivity in interpretation. Factors thought to be important include trabecular and cortical structure, and bone mineralization.4 Consequently, measurable bone data have been reported in subjects taking BPs through many techniques, including bone density, architecture, and cortical bone thickness.1, 4, 7 and 13 Trabecular microarchitecture of postmenopausal women has been evaluated with noninvasive techniques, such as high-resolution magnetic resonance images showing less deterioration of the bone 1 year after initiation of oral BP therapy.4 A decrease in bone turnover and a trend for an increase in the bone wall thickness has been detected by histomorphometry in subjects taking BPs.1 Alterations in the cortical structure of the second metacarpal have been detected in digital x-ray radiogrammetry of postmenopausal women treated with BPs.7 Mandibular cortical width may be measured on dental panoramic radiographs, and it has been suggested as a screening tool for referring patients for bone densitometry for osteoporosis investigation.14 and 15 Inhibition of the intracortical bone remodeling in the mandible of mice taking BPs has been reported.16 Thus, imaging evaluation of the mandibular cortical bone could be a biologically plausible way to detect BP bone alterations. Computed tomography can assess both cortical and trabecular bone characteristics. Cone-beam computed tomography (CBCT) can provide 3-dimensional information, while using lower doses and costing less than conventional CT. The CBCT images have been studied as a tool for the measurement of trabecular bone in patients with BRONJ.13 Therefore, cortical bone measurements on CBCT of the jaws might also help to understand bone changes in patients with BRONJ. There is no standard in quantifying dimensional changes of mandibular cortical bone. We explored several different approaches to take into consideration possible changes in length, area, and volume. These led to the 3 techniques developed in this study. This article reports a matched case-control study in which mandibular cortical bone was measured on CBCT images of subjects with BRONJ and controls. The aim of the study was to explore the usefulness of 3 techniques for detecting mandibular cortical bone dimensional changes caused by BP.

Mandibular Bisphosphonate-Related Osteonecrosis After Dental Implant Rehabilitation: A Case Report

It has been a matter of debate as to whether dental implant therapies are suitable for patients subjected to long-term use of bisphosphonates (BPs). This report presents a case of a 76-year-old woman who developed BPs-related osteonecrosis of the jaw (BRONJ) in the left hemimandible after dental implant exposure. The implants and the necrotic crestal bone were removed, and postoperatively, a delay in tissue healing with bone exposure was noticed. The histologic analysis of the block biopsies revealed a lamellar bone tissue exhibiting necrotic areas and bacterial colonies associated with the bone outer surface. The bone-implant interface showed viable lamellar bone with enlarged vascular spaces in the areas between the implant threads. The possible mechanisms for the loss of implants in BRONJ patients are discussed, and the potential protocols for dental implant rehabilitation for patients under BP therapies are presented. (Implant Dent 2012;21:449-453)

Association of laser phototherapy with PRP improves healing of bisphosphonate-related osteonecrosis of the jaws in cancer patients: A preliminary study

The aim of this study was to compare retrospectively the effect of three different treatments on the healing outcome of bisphosphonate-related osteonecrosis of the jaws (BRONJ) in cancer patients. Twenty-two cancer patients were treated for BRONJ with one of the following protocols: clinical (pharmacological therapy), surgical (pharmacological plus surgical therapy), or PRP plus LPT (pharmacological plus surgical plus platelet rich plasma (PRP) plus laser phototherapy (LPT). The laser treatment was applied with a continuous diode laser (InGaAlP, 660 nm) using punctual and contact mode, 40 mW, spot size 0.042 cm(2), 6 J/cm(2) (6 s) and total energy of 0.24 J per point. The irradiations were performed on the exposed bone and surrounding soft tissue. The analysis of demographic data and risk factors was performed by gathering the following information: age, gender, primary tumor, bisphosphonate (BP) used, duration of BP intake, history of chemotherapy, use of steroids, and medical history of diabetes. The association between the current state of BRONJ (with or without bone exposure) and other qualitative variables was determined using the chi-square or Fisher's exact test. In all tests, the significance level adopted was 5%. Most BRONJ lesions occurred in the mandible (77%) after tooth extraction (55%) and in women (72%). A significantly higher percentage of patients reached the current state of BRONJ without bone exposure (86%) in the PPR plus LPT group than in the pharmacological (0%) and surgical (40%) groups after 1-month follow-up assessment. These results suggest that the association of pharmacological therapy and surgical therapy with PRP plus LPT significantly improves BRONJ healing in oncologic patients. Although prospective studies with larger sample sizes are still needed, this preliminary study may be used to inform a better-designed future study. (C) 2011 Elsevier Ltd. All rights reserved.

Clinical and image findings in bisphosphonate-related osteonecrosis of the jaws

Abstract: Bisphosphonate-related osteonecrosis of the jaws (BRONJ) is characterized as exposed bone in the jaws for more than 8 weeks in patients with current or previous history of therapy with bisphosphonates (BPs) and no history of radiotherapy in the head and neck. We report a case series of 7 patients with BRONJ and analyze the variations of clinical and imaging signs, correlating them with the presence or absence of bone exposure. Among the patients, 6 were women and 1 was a man, aged 42–79 years. Five of the patients were using zoledronic acid and the other 2 alendronate. The use of BPs varied from 3 to 13 years. In 5 patients, tooth extraction was the triggering event of injuries. Panoramic radiographs and computed tomography (CT) were evaluated by a radiologist blinded to the cases. There were persistent unremodeled extraction socket even several months after tooth extraction in 3 of the cases that were consistent wit CT findings that also showed areas of osteosclerosis and osteolysis. Patients were treated according to the recommendations of the AAOMS, with surgical debridement and antibiotic coverage with amoxicillin in the symptomatic patients. The follow-up of these patients ranged from 8 to 34 months, with a good response to treatment. The image findings in this case series were not specific and showed no difference between each stages of BRONJ (AAOMS, 2009). The image features were similar in presence or absence of exposed bone.