Medication-related osteonecrosis of the jaw (MRONJ): a review of pathogenesis hypothesis and therapy strategies.

Medication-related osteonecrosis of the jaw (MRONJ), a severe side effect caused by antiresorptive antiangiogenic medication, particularly bisphosphonates (BPs), has become a challenging disease with serious and profound effects on the physical and mental health of patients. Although it occurs with high frequency and is harmful, the exact mechanism of MRONJ remains unknown, and systematic and targeted approaches are still lacking. Maxillofacial surgeons focus on the etiology of osteonecrosis in the mandible and maxilla as well as the appropriate oral interventions for high-risk patients. Adequate nursing care and pharmacotherapy management are also crucial. This review provides a current overview of the clinicopathologic feature and research of MRONJ caused by BPs, with an emphasis on the potential mechanisms and current therapy and prevention strategies of the disease. We are of the opinion that an in-depth comprehension of the mechanisms underlying MRONJ will facilitate the development of more precise and efficacious therapeutic approaches, resulting in enhanced clinical outcomes for patients.

Zoledronate promotes inflammatory cytokine expression in human CD14-positive monocytes among peripheral mononuclear cells in the presence of γδ T cells.

Bisphosphonates distributed to bone exert toxic effects specifically towards osteoclasts. On the other hand, intravenous administration of a nitrogen-containing bisphosphonate (N-BP) such as zoledronate induces acute-phase reactions (APRs), including influenza-like fever 1 day later, indicating an interaction with immunocompetent cells circulating blood. Although it has been reported that activation of γδ T cells is pivotal to induce an APR following treatment with zoledronate, downstream events, including the production of inflammatory cytokines after activation of γδ T cells, remain obscure. We investigated the effects of zoledronate on inflammatory cytokine expression in human peripheral blood mononuclear cells (PBMCs) in vitro. While zoledronate induced mRNA expressions of tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 and interferon-γ (IFN-γ) in PBMC, depletion of γδ T cells abolished that zoledronate-induced expression of those cytokines, indicating the necessity of γδ T cells for expression induction by zoledronate. However, which types of cells were responsible for the production of those cytokines in blood remained unclear. As it is generally accepted that monocytes and macrophages are primary sources of inflammatory cytokines, CD14+ cells from PBMC were exposed to zoledronate in the presence of PBMC, which resulted in induced expression of mRNAs for IL-1ß, IL-6 and IFN-γ, but not for TNF-α. These results indicate that CD14+ cells are responsible, at least in part, for the production of IL-1ß, IL-6 and IFN-γ in blood exposed to zoledronate. This suggests that CD14+ cells play an essential role in the occurrence of APRs following N-BP administration.

Sonic hedgehog signaling is associated with resistance to zoledronic acid in CD133high/CD44high prostate cancer stem cells.

Cancer stem cells (CSCs) are a unique population that has been linked to drug resistance and metastasis and recurrence of prostate cancer. The sonic hedgehog (SHH) signal regulates stem cells in normal prostate epithelium by affecting cell behavior, survival, proliferation, and maintenance. Aberrant SHH pathway activation leads to an unsuitable expansion of stem cell lineages in the prostate epithelium and the transformation of prostate CSCs (PCSCs). Zoledronic acid (ZOL), one of the third-generation bisphosphonates, effectively prevented bone metastasis and treated advanced prostate cancer despite androgen deprivation therapy. Despite strong evidence for the involvement of the SHH in human PCSCs survival and drug resistance, the roles of SHH in the PCSCs-related resistance to ZOL remain to be fully elucidated. The present study aimed to investigate the role of the SHH pathway in ZOL resistance of PCSCs in 2D and three 3D cell culture conditions. For this purpose, we isolated CD133high/ CD44high PCSCs using a flow cytometer. Following ZOL treatment, mRNA and protein expressions of the components of the SHH signaling pathway in PCSCs and non-CSCs were analyzed using qRT-PCR and Immunofluorescence staining, respectively. Our finding suggested that SHH signaling may be activated by different mechanisms that lead to avoidance of the inhibition effect of ZOL. Thereby, SHH pathways may be associated with the resistance to ZOL developed by prostate CSCs. Inhibition of CSCs-related SHH signaling along with ZOL treatment should be considered to achieve improvement in survival or delayed treatment failure and prevention of the CSCs-related drug resistance.

Bisphosphonate-related osteonecrosis induced change in alveolar bone architecture in rats with participation of Wnt signaling.

OBJECTIVE: This work aimed to study the role of inflammation in medication-related osteonecrosis of the jaw (MRONJ) in rats with focus on Wnt signaling. METHODS: A total of 36 female Wistar rats (12 weeks ± 200 g) were divided into 2 groups (n = 6) in 3 experiments: saline (SAL) and zoledronic acid (ZOL). For MRONJ induction, rats received 0.1 mg/kg of ZOL (ip) 3×/week for 9 weeks. Animals from the SAL group received 0.1 mg/kg of 0.9% SAL, ip 3×/week for 9 weeks. On the 8th week, 3 left upper molars were extracted, and on the 11th week, they were euthanized. Maxillae were evaluated by macroscopic and histopathological analyses; scanning electron microscopy (SEM); immunohistochemistry for DKK-1, Wnt 10b, and caspase-3; and Raman spectrometry. Gingiva was also collected for TNF-α e IL-1ß quantification. RESULTS: Bone necrosis was confirmed by healing impairment, reduced number of viable osteocytes, increased caspase-3 immunoexpression, and increased number of empty lacunae (p < 0.05). ZOL enhanced inflammation and increased gingival levels of IL-1ß and TNF-α (p < 0.05). Irregular indentations were seen on bone after ZOL administration. Bone necrosis was marked by reduced amount of total and type I collagen. ZOL reduced the mineral/matrix ratio and increased carbonate/phosphate ratio. It was observed a significant reduction on Wnt10b and beta-catenin immunolabeling in the bone tissue of ZOL group. CONCLUSION: In summary, MRONJ model caused bone necrosis due to intense inflammation. Wnt signaling seems to play an important role in this process. CLINICAL RELEVANCE: New therapeutic strategies focusing on Wnt pathway can provide an interesting approach for future treatments.

Suppression of Bone Necrosis around Tooth Extraction Socket in a MRONJ-like Mouse Model by E-rhBMP-2 Containing Artificial Bone Graft Administration.

Medication-related osteonecrosis of the jaw (MRONJ) is related to impaired bone healing conditions in the maxillomandibular bone region as a complication of bisphosphonate intake. Although there are several hypotheses for the onset of MRONJ symptoms, one of the possible causes is the inhibition of bone turnover and blood supply leading to bone necrosis. The optimal treatment strategy for MRONJ has not been established either. BMP-2, a member of the TGF-ß superfamily, is well known for regulating bone remodeling and homeostasis prenatally and postnatally. Therefore, the objectives of this study were to evaluate whether cyclophosphamide/zoledronate (CY/ZA) induces necrosis of the bone surrounding the tooth extraction socket, and to examine the therapeutic potential of BMP-2 in combination with the hard osteoinductive biomaterial, ß-tricalcium phosphate (ß-TCP), in the prevention and treatment of alveolar bone loss around the tooth extraction socket in MRONJ-like mice models. First, CY/ZA was intraperitoneally administered for three weeks, and alveolar bone necrosis was evaluated before and after tooth extraction. Next, the effect of BMP-2/ß-TCP was investigated in both MRONJ-like prevention and treatment models. In the prevention model, CY/ZA was continuously administered for four weeks after BMP-2/ß-TCP transplantation. In the treatment model, CY/ZA administration was suspended after transplantation of BMP-2/ß-TCP. The results showed that CY/ZA induced a significant decrease in the number of empty lacunae, a sign of bone necrosis, in the alveolar bone around the tooth extraction socket after tooth extraction. Histological analysis showed a significant decrease in the necrotic alveolar bone around tooth extraction sockets in the BMP-2/ß-TCP transplantation group compared to the non-transplanted control group in both MRONJ-like prevention and treatment models. However, bone mineral density, determined by micro-CT analysis, was significantly higher in the BMP-2/ß-TCP transplanted group than in the control group in the prevention model only. These results clarified that alveolar bone necrosis around tooth extraction sockets can be induced after surgical intervention under CY/ZA administration. In addition, transplantation of BMP-2/ß-TCP reduced the necrotic alveolar bone around the tooth extraction socket. Therefore, a combination of BMP-2/ß-TCP could be an alternative approach for both prevention and treatment of MRONJ-like symptoms.

PHNQ from Evechinus chloroticus Sea Urchin Supplemented with Calcium Promotes Mineralization in Saos-2 Human Bone Cell Line.

Polyhydroxylated naphthoquinones (PHNQs), known as spinochromes that can be extracted from sea urchins, are bioactive compounds reported to have medicinal properties and antioxidant activity. The MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell viability assay showed that pure echinochrome A exhibited a cytotoxic effect on Saos-2 cells in a dose-dependent manner within the test concentration range (15.625-65.5 µg/mL). The PHNQ extract from New Zealand sea urchin Evechinus chloroticus did not induce any cytotoxicity within the same concentration range after 21 days of incubation. Adding calcium chloride (CaCl2) with echinochrome A increased the number of viable cells, but when CaCl2 was added with the PHNQs, cell viability decreased. The effect of PHNQs extracted on mineralized nodule formation in Saos-2 cells was investigated using xylenol orange and von Kossa staining methods. Echinochrome A decreased the mineralized nodule formation significantly (p < 0.05), while nodule formation was not affected in the PHNQ treatment group. A significant (p < 0.05) increase in mineralization was observed in the presence of PHNQs (62.5 µg/mL) supplemented with 1.5 mM CaCl2. In conclusion, the results indicate that PHNQs have the potential to improve the formation of bone mineral phase in vitro, and future research in an animal model is warranted.

Removal of Pre-Existing Periodontal Inflammatory Condition before Tooth Extraction Ameliorates Medication-Related Osteonecrosis of the Jaw-Like Lesion in Mice.

Medication-related osteonecrosis of the jaw (MRONJ) is a rare but detrimental intraoral lesion that predominantly occurs in patients with long-term use of antiresorptive agents, such as bisphosphonate and denosumab, a human anti-receptor activator of NF-κB ligand (RANKL) monoclonal antibody (Ab). Surgical intervention, such as tooth extraction, is a known risk factor for MRONJ, which is often performed to eliminate preexiting pathologic inflammatory conditions, such as periodontal diseases. Nonetheless, it remains unknown whether pre-existing periodontal disease condition exacerbates, or removal of such condition ameliorates, MRONJ development after tooth extraction. In this study, we combined the ligature-induced periodontitis and the tooth extraction mouse models under the administration of zoledronic acid (ZOL) or anti-RANKL Ab, and provide experimental evidence that a pre-existing pathologic inflammatory condition exacerbates MRONJ development after tooth extraction in mice. Under ZOL administration, tooth extraction alone induced ONJ lesions; however, extraction of a ligature-placed tooth further exacerbated ONJ development. When the ligature was removed and the inflammatory condition was deescalated, ONJ development was ameliorated. Anti-RANKL Ab administration resulted in similar outcomes. Interestingly, unlike ZOL-administered mice, anti-RANKL Ab-administered mice exhibited complete absence of osteoclasts, suggesting that physical presence of osteoclasts is not directly involved in ONJ development. Collectively, our study demonstrated that periodontal disease is a functionally linked risk factor that predisposes ONJ development after tooth extraction in the presence of bisphosphonate and denosumab.

Zoledronic acid induces cytogenetic toxicity in male germline cells of Swiss albino mice.

This study mainly focuses on the cytogenetic toxicity induction by zoledronic acid (ZA), a nitrogen containing bisphosphonate (N-BPs) in the male germline cells of Swiss albino mice. A single intraperitoneal exposure with three different doses of ZA (2, 4, and 8 mg/kg body weight), toxicity was assessed by analyzing spermatogonial metaphase chromosome aberrations at 24 h, aberrant primary spermatocytes at week 4, and abnormal spermatozoa at week 8 posttreatment. Cyclophosphamide (40 mg/kg) and 0.9% NaCl were used as positive and vehicle controls respectively in the study. The results showed that there was a significant induction in the number of chromosomal aberrations especially at two doses of ZA (4 and 8 mg/kg) after 24 h in the spermatogonial cells (p < 0.001) compared to vehicle control. The transmission genetic damages were noticed as aberrant spermatocytes with atypical bivalents (X-Y/autosomal asynapsis) at 4 mg/kg of ZA (p < 0.01) and at 8 mg/kg of ZA (p < 0.001) at week 4 posttreatment. A statistically significant higher number of abnormal spermatozoa (sperm) were also noticed at week 8 posttreatment of both at 4 and 8 mg/kg of ZA (p < 0.001). Hence, from these genotoxicity studies, it can be concluded that ZA is genotoxic in male germline cells and has the potential of transmitting the genotoxic effects from spermatogonial cells to sperm in male Swiss mice.

Effects of teriparatide on morphology of aortic calcification in aged hyperlipidemic mice.

Calcific aortic vasculopathy correlates with bone loss in osteoporosis in an age-independent manner. Prior work suggests that teriparatide, the bone anabolic treatment for postmenopausal osteoporosis, may inhibit the onset of aortic calcification. Whether teriparatide affects the progression of preexisting aortic calcification, widespread among this patient population, is unknown. Female apolipoprotein E-deficient mice were aged for over 1 yr to induce aortic calcification, treated for 4.5 wk with daily injections of control vehicle (PBS), 40 µg/kg teriparatide (PTH40), or 400 µg/kg teriparatide (PTH400), and assayed for aortic calcification by microcomputed tomography (microCT) before and after treatment. In a followup cohort, aged female apolipoprotein E-deficient mice were treated with PBS or PTH400 and assayed for aortic calcification by serial microCT and micropositron emission tomography. In both cohorts, aortic calcification detected by microCT progressed similarly in all groups. Mean aortic 18F-NaF incorporation, detected by serial micropositron emission tomography, increased in the PBS-treated group (+14 ± 5%). In contrast, 18F-NaF incorporation decreased in the PTH400-treated group (-33 ± 20%, P = 0.03). Quantitative histochemical analysis by Alizarin red staining revealed a lower mineral surface area index in the PTH400-treated group compared with the PBS-treated group ( P = 0.04). Furthermore, Masson trichrome staining showed a significant increase in collagen deposition in the left ventricular myocardium of mice that received PTH400 [2.1 ± 0.6% vs. control mice (0.5 ± 0.1%), P = 0.02]. In summary, although teriparatide may not affect the calcium mineral content of aortic calcification, it reduces 18F-NaF uptake in calcified lesions, suggesting the possibility that it may reduce mineral surface area with potential impact on plaque stability. NEW & NOTEWORTHY Parathyroid hormone regulates bone mineralization and may also affect vascular calcification, which is an important issue, given that its active fragment, teriparatide, is widely used for the treatment of osteoporosis. To determine whether teriparatide alters vascular calcification, we imaged aortic calcification in mice treated with teriparatide and control mice. Although teriparatide did not affect the calcium content of cardiovascular deposits, it reduced their fluoride tracer uptake.

Bone mineralization and vascularization in bisphosphonate-related osteonecrosis of the jaw: an experimental study in the rat.

OBJECTIVES: Pathogenesis of bisphosphonate-related osteonecrosis of the jaws (BRONJ) is not fully explained. An antiangiogenic effect of bisphosphonates (BPs) or an altered bone quality have been advocated. The aims of the present study were to analyze alveolar mandibular vascularization and bone quality in rats with BRONJ. MATERIALS AND METHODS: Thirty-eight Sprague-Dawley rats were randomized into two groups: zoledronic acid (ZA), n = 27, and control (CTRL) n = 11. The ZA group received a weekly IV injection of ZA (100 µg/kg) during 10 weeks. The CTRL group received saline. After 6 weeks, extraction of the right mandibular molars was performed. Rats were sacrificed after 14 weeks. Microtomography characterized bone lesions and vascularization after injection of a radio-opaque material. Raman microspectroscopy evaluated bone mineralization. RESULTS: Fifty-five percent of ZA rats presented bone exposure and signs of BRONJ. None sign was found at the left hemimandible in the ZA group and in the CTRL group. Vascular density appeared significantly increased in the right hemimandibles of the CTRL group compared to the left hemimandibles. Vascularization was reduced in the ZA group. A significantly increased of the mineral-to-amide ratio was found in the alveolar bone of ZA rats by Raman microspectroscopy. CONCLUSIONS: In a rat model of BRONJ, microtomography evidenced osteonecrosis in BRONJ. Raman spectroscopy showed an increased mineralization. Vascularization after tooth extraction was impaired by ZA. CLINICAL RELEVANCE: Prolonged BP administration caused an increase in the mineralization and a quantitative reduction of the vascularization in the alveolar bone; both factors might be involved concomitantly in the BRONJ pathophysiology.

Bone Therapeutics: Safety Considerations, Session Summary.

This session was a series of presentations focused on safety considerations for late stage or currently marketed bone therapeutic agents. The first presentation was an overview of a major regulatory requirement in the nonclinical filing package for bone therapeutics, studies designed to assess the impact of an agent on bone quality. Two presentations focused on safety issues associated with drugs whose primary mechanism of action is inhibition of bone resorption. Typical findings associated with this class of agents in general and reproductive toxicology studies were reviewed, highlighting INHAND (International Harmonization of Nomenclature and Diagnostic Criteria) nomenclature. This was followed by an overview of safety issues that have been identified largely through clinical experience. Similar presentations followed emphasizing safety and regulatory issues associated with classes of drugs whose primary mechanism of action is stimulation of bone formation known broadly as bone anabolic agents. The major focus of these discussions was carcinogenicity risk assessment. The final presentation was an introduction to a rapidly evolving area in bone therapeutics, treatment of rare genetic bone diseases, and the developmental challenges associated with these indications and novel therapeutic modalities.

Comparing the incidence of bone tumors in rats chronically exposed to the selective PTH type 1 receptor agonist abaloparatide or PTH(1-34).

Prolonged treatment with human parathyroid hormone (hPTH) in rats results in development of bone tumors, though this finding has not been supported by clinical experience. The PTH type 1 receptor agonist abaloparatide, selected for its bone anabolic activity, is under clinical development to treat postmenopausal women with osteoporosis. To determine the carcinogenic potential of abaloparatide, Fischer (F344) rats were administered SC daily abaloparatide at doses of 0, 10, 25, and 50 µg/kg or 30 µg/kg hPTH(1-34) as a positive control for up to 2 years. Robust increases in bone density were achieved at all abaloparatide doses and with hPTH(1-34). Comprehensive histopathological analysis reflected a comparable continuum of proliferative changes in bone, mostly osteosarcoma, in both abaloparatide and hPTH(1-34) treated rats. Comparing the effects of abaloparatide and hPTH(1-34) at the 25 and 30 µg/kg respective doses, representing similar exposure multiples to the human therapeutic doses, revealed similar osteosarcoma-associated mortality, tumor incidence, age at first occurrence, and metastatic potential. There were no increases in the incidence of non-bone tumors with abaloparatide compared to vehicle. Thus, near life-long treatment with abaloparatide in rats resulted in dose and time dependent formation of osteosarcomas, with a comparable response to hPTH(1-34) at similar exposure.

Strontium ranelate causes osteophytes overgrowth in a model of early phase osteoarthritis.

BACKGROUND: Osteoarthritis (OA) involves cartilage changes as well as modifications of subchondral bone and synovial tissues. Strontium ranelate (SR), an anti-osteoporosis compound, which is currently in phase III clinical trial for treatment of OA. Evidences suggest that SR preferably deposited in osteophyte, other than in subchondral bone in early phase of OA. This phenomenon raises concern about its utility for OA treatment as a disease-modifying drug. To evaluate the effect of SR on cartilage, subchondral bone mass and subchondral trabecular bone structure in medial meniscectomized (MNX) guinea pigs. METHOD: Thirty-six 3-month-old male Dunkin Hartley albino guinea pigs received either sham or medial meniscectomy operations. One week after the procedure, meniscectomized animals began 12 weeks of SR (625 mg/kg, daily) treatment by oral gavage for MNX + SR group, or normal saline for MNX + V group. All animals were euthanized 12 weeks later, cartilage degeneration and subchondral bone micro-architecture was analyzed. RESULTS: Both OARSI scores (P = 0.523 for marcoscopic scores, P = 0.297 for histological scores) and Cartilage thickness (P = 0.335) in MNX + SR group were comparable to MNX + V group. However, osteophyte sizes were larger in MNX + SR group (P = 0.014), and collapsed osteophytes in MNX + SR group (7 by 12) were significantly more than in MNX + V group (1 by 12) (P = 0.027), while immunohistochemistry indicates catabolic changes in osteophyte/plateau junction. Micro-CT analysis showed bone mineral density (BMD) (P = 0.001), bone volume fraction (BV/TV) (P = 0.008), trabecular spacing (Tb.Sp) (P = 0.020), trabecular thickness (Tb.Th) (P = 0.012) and structure model index (SMI) (P = 0.005) levels to be significantly higher in the MNX + SR group than in the MNX + V group. CONCLUSIONS: SR (625 mg/kg/day) did not protect cartilage from degeneration in MNX guinea pigs but subchondral bone was significantly enhanced. In early phase OA, SR administration causes osteophyte overgrowth, which may be related to incorporation into mineralizing osteophytes. This adverse effect is important for future studies of SR in OA.

Protective Effects of Simvastatin Against Alendronate-Induced Gastric Mucosal Injury in Rats.

BACKGROUND: It has been reported that simvastatin, a statin commonly prescribed for its anti-inflammatory and antioxidant effects, has gastroprotective effects in indomethacin and ethanol-induced gastric ulcers. However, the effects of simvastatin on alendronate-induced gastric mucosal injury remain unexplored. AIM: This study investigated the use of simvastatin for the treatment of alendronate-induced gastric ulcers in rats. METHODS: Female rats were pretreated with vehicle or simvastatin (20 and 60 mg/kg p.o.). After 1 h, the rats received alendronate (50 mg/kg p.o.). Simvastatin was administered once daily for 7 days, and from the fourth day of simvastatin treatment, alendronate was administered once daily for 4 days. On the final day of treatment, 4 h after alendronate administration, animals were euthanized, their stomachs were removed, and gastric damage was measured. Samples of the stomach were fixed in 10 % formalin immediately after their removal for subsequent histopathological assessment. Unfixed samples were weighed, frozen at -80 °C until assayed for glutathione (GSH), malondialdehyde (MDA), and cytokine levels and myeloperoxidase (MPO) activity. A third group was used to measure mucus and gastric secretion. RESULTS: Pretreatment with simvastatin prevented alendronate-induced macroscopic gastric damage and reduced the levels of MDA and GSH, TNF-α and IL-1ß, MPO activity, and mucus levels, in the stomach. CONCLUSIONS: This study demonstrates the protective effects of simvastatin against alendronate-induced gastric ulceration. Maintenance of mucosal integrity, inhibition of neutrophil activity, and reduced oxidative stress associated with decreased gastric acidity may explain the gastroprotective effects of simvastatin.

Role of microcracks in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw.

OBJECTIVES: The aim of this study was to investigate the potential role of microcrack accumulation in the pathogenesis of bisphosphonate-related osteonecrosis of the jaw (ONJ) through an animal model. MATERIALS AND METHODS: Twenty-four ovariectomized rats were randomly divided into a bisphosphonate group (n = 19) and control group (n = 5) and weekly injected with zoledronic acid and normal saline, respectively. After 6 weeks, surgical intervention was performed, and the injections were continued for eight additional weeks. Then, the animals were sacrificed, and ONJ lesions were inspected for the presence of microcracks using scanning electron microscopy. Measurements included bone dimension, number of cracks, crack length, and normalized indices; crack density (Cr.Dn) and crack surface density (Cr.S.Dn) were used for group comparison. RESULTS: Both number of cracks and crack length in the bisphosphonate group were greater than those in the control group (P < 0.05). Of the 19 rats injected with bisphosphonates, 13 rats (68.4 %) were classified into the ONJ group. Cr.Dn and Cr.S.Dn were significantly greater in the ONJ group than in the non-ONJ group, indicating accumulation of unrepaired microcracks (P < 0.05). Seventy-two percent of microcracks in the ONJ group conformed to the defined length that was considered significant according to a previous literature (30-80 µm); whereas 12 % of microcracks in the non-ONJ group were considered significant (P < 0.05). CONCLUSION: Accumulation of unrepaired microcracks was significantly associated with the development of bisphosphonate-related ONJ. Further research is required to determine the role of microcracks in the pathogenesis of bisphosphonate-related ONJ. CLINICAL RELEVANCE: Long-term bisphosphonates use may deteriorate the biomechanical and physiological bone integrity, contributing to the pathogenesis of bisphosphonate-related ONJ.

Bisphosphonate treatment of type I diabetic mice prevents early bone loss but accentuates suppression of bone formation.

Type I (T1) diabetes is an autoimmune and metabolic disease associated with bone loss. Previous studies demonstrate that T1-diabetes decreases osteoblast activity and viability. Bisphosphonate therapy, commonly used to treat osteoporosis, is demonstrated to inhibit osteoclast activity as well as osteoblast apoptosis. Therefore, we examined the effect of weekly alendronate treatments on T1-diabetes induced osteoblast apoptosis and bone loss. Bone TUNEL assays identified that alendronate therapy prevents the diabetes-induced osteoblast death observed during early stages of diabetes development. Consistent with this, alendronate treatment for 40 days was able to prevent diabetes-induced trabecular bone loss. Alendronate was also able to reduce marrow adiposity in both control diabetic mice compared to untreated mice. Mechanical testing indicated that 40 days of alendronate treatment increased bone stiffness but decreased the work required for fracture in T1-diabetic and alendronate treated mice. Of concern at this later time point, bone formation rate and osteoblast markers, which were already decreased in diabetic mice, were further suppressed in alendronate-treated diabetic mice. Taken together, our results suggest that short-term alendronate treatment can prevent T1-diabetes-induced bone loss in mice, possibly in part by inhibiting diabetes onset associated osteoblast death, while longer treatment enhanced bone density but at the cost of further suppressing bone formation in diabetic mice.

Nephrotoxicity of ibandronate and zoledronate in Wistar rats with normal renal function and after unilateral nephrectomy.

A previous animal study compared the nephrotoxic effect of ibandronate (IBN) and zoledronate (ZOL), but interpretation of these study results was limited because of the model of minimal nephrotoxic dosage with a dosage ratio of 1:3. The present study investigated the nephrotoxicity of ibandronate and zoledronate in a 1.5:1 dose ratio, as used in clinical practice and compared the nephrotoxicity in rats with normal and with mildly to moderately impaired renal function. We compared rats with normal renal function (SHAM) and with impaired renal function after unilateral nephrectomy (UNX), treated either with ibandronate 1.5mg/kg, zoledronate 1mg/kg or placebo once (1×) or nine (9×) times. Renal function and markers of tubular toxicity were measured over a 27 week period. After last bisphosphonate treatment the rats were sacrificed and kidneys examined histologically. All bisphosphonate treated animals showed a significant tubular toxicity, which was temporary except in the ZOL-UNX-9×-group. Also the renal function was only transiently reduced except in the ZOL-UNX-9×-group. Histologically, bisphosphonate treatment led to cortical tubuloepithelial degeneration/necrosis and medullary tubuloepithelial swelling which were slightly more pronounced in ibandronate treated animals, when compared to zoledronate treated animals, especially with impaired renal function. In contrast to the previous study we found a similar nephrotoxicity of ibandronate and zoledronate in rats with normal renal function. In rats with impaired renal function the peak of toxicity had not even been fully reached until end of experiment in the zoledronate treated animals. The peak of toxicity seems to be more severe and delayed in rats with impaired renal function compared with rats with normal renal function.

Bisphosphonates inhibit cell functions of HUVECs, fibroblasts and osteogenic cells via inhibition of protein geranylgeranylation.

OBJECTIVES: Bisphosphonate-associated osteonecrosis of the jaw is a severe side effect in patients receiving nitrogen-containing bisphosphonates (N-BPs). One characteristic is its high recurrence rate; therefore, basic research for new therapeutic options is necessary. N-BPs inhibit the farnesylpyrophosphate synthase in the mevalonate pathway causing a depletion of the cellular geranylgeranyl pool, resulting in a constriction of essential functions of different cell lines. Geranylgeraniol (GGOH) has been proven to antagonise the negative biological in vitro effects of bisphosphonates. MATERIAL AND METHODS: This study analyses the influence of the isoprenoids eugenol, farnesol, R-limonene, menthol and squalene on different functions of zoledronate-treated human umbilicord vein endothelial cells (HUVEC), fibroblasts and osteogenic cells. In addition to the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl 2H-tetrazolium bromide (MTT) vitality test, the migration capacity was analysed by scratch wound assay and the morphological architecture of the treated cells by phallacidin staining. RESULTS: In contrast to GGOH, none of the other tested isoprenoids were able to prevent cells from having negative zoledronate effects. CONCLUSIONS: Despite structural analogy to GGOH, the investigated isoprenoids are not able to prevent the N-BP effect. The negative impact of zoledronate on fibroblasts, HUVEC and osteogenic cells is due to inhibition of protein geranylgeranylation since the substitution of squalene and farnesyl did not have any effect on viability and wound healing capacity whereas GGOH did reduce the negative impact. CLINICAL RELEVANCE: These data suggest the importance and exclusiveness of the mevalonate pathway intermediate GGOH as a potential therapeutic approach to bisphosphonate-associated osteonecrosis of the jaws.

Early effects of parathyroid hormone on bisphosphonate/steroid-associated compromised osseous wound healing.

SUMMARY: Administration of intermittent parathyroid hormone (PTH) promoted healing of tibial osseous defects and tooth extraction wounds and prevented the development of necrotic lesions in rats on a combined bisphosphonate and steroid regimen. INTRODUCTION: Osteonecrosis of the jaw (ONJ) has emerged in association with antiresorptive therapies. The pathophysiology of ONJ is unknown and no established cure currently exists. Our objective was to determine the effect of intermittent PTH administration on early osseous healing in the jaw and long bones of rats receiving bisphosphonate and steroid treatment. METHODS: Ovariectomized rats received the combination therapy of alendronate and dexamethasone (ALN/DEX) for 12 weeks. Osseous wounds were created in the jaw and tibia. PTH was administered intermittently and healing at 2 weeks post-op was compared between the jaw and tibia by microcomputed tomography and histomorphometric analyses. RESULTS: ALN/DEX treatment was associated with necrotic open wounds in the jaw but had no negative effects on healing and promoted bone fill in tibial defects. PTH therapy prevented the development of necrotic lesions in the jaw and promoted healing of the tibial defects. PTH therapy was associated with the promotion of osteocyte survival in osseous wounds both in the jaw and tibia. CONCLUSIONS: Wound healing was impaired in the jaw in rats on a combined bisphosphonate and steroid regimen, and PTH therapy rescued necrotic lesions. These findings suggest that PTH therapy could be utilized to prevent ONJ from occurring in patients on combination antiresorptive and steroid therapy.

Alendronate-induced atypical bone fracture: evidence that the drug inhibits osteogenesis.

WHAT IS KNOWN AND OBJECTIVE: Alendronate (ALN) is used for the treatment of post-menopausal osteoporosis. By reducing bone turnover, it increases bone mineral density. However, recent reports suggest an increased risk of atypical bone fractures after long-term ALN administration. Despite its well-known anti-osteoclastic activity, it is unclear whether ALN also suppresses human mesenchymal stem cell (hMSC)-mediated osteogenesis, thus possibly resulting in atypical bone fragility. We hypothesized that ALN does this and we look at its in vitro effects on osteogenesis. METHODS: Morphological analysis, reverse transcriptase polymerase chain reaction, cell viability, alkaline phosphatase (ALP) activity and mineralization assays were investigated in hMSCs treated with a wide range of ALN. RESULTS AND DISCUSSION: After treatment with high concentrations of ALN for 3 and 7 days, cell viability was significantly reduced and cell morphology was altered. Osteogenic differentiation of hMSCs was also substantially suppressed as demonstrated by decreased ALP activity although ALN did not affect osteogenic-related genes tested. Furthermore, ALN at all concentrations tested drastically inhibited alizarin red S-positive mineralized matrix. WHAT IS NEW AND CONCLUSION: ALN has a strong inhibitory effect on hMSC-mediated osteogenesis by suppressing cell proliferation, osteoblast differentiation and function. The insight gained may help in the development of safer alternatives.