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.

Impact of PRP and PRF on Viability of Zoledronic Acid Treated Osteoblasts in 2D and 3D Cell Culture.

BACKGROUND/AIM: Zoledronic acid (ZA) treatment of in vitro cultured osteoblasts (OB) results in reduction in viability, proliferation and differentiation. These effects are slightly attenuated when platelets-rich fibrin and plasma (PRF and PRP) are added. However, it is still unknown whether application of PRP/PRF on ZA-treated OB in a 3D-environment would influence the viability in relation to 2D-cultivation. MATERIALS AND METHODS: Non-treated and ZA-treated OB were cultivated in 2D conditions or seeded in a 3D collagen scaffold with and without PRP/PRF. MTT test was carried out after 5 days of colonization. 4,6-diamidino-2'-phenylindole, dihydrochloride (DAPI)-staining was performed in OB grown in 3D scaffolds to ensure spatial distribution of OB. RESULTS: ZA led to a significant reduction in cell viability compared to the control group. Addition of either PRF or PRP to the 3D colonized and ZA-treated OB significantly enhanced their survival and viability in relation to 2D monolayer cultivation. CONCLUSION: The use of 3D-scaffolds has a positive effect on OB viability, and stimulation by PRF and PRP may provide a therapeutic approach to transfer these results into clinical routine for the treatment of patients with bisphosphonate related osteonecrosis of the jaw (BR-ONJ).

Role of cytochrome P450 2C8 genetic polymorphism and epoxygenase uncoupling in periodontal remodelling affecting orthodontic treatment.

In genome-wide association studies, the CYP2C8 gene locus has been reported to be associated with bisphosphonate-related osteonecrosis of the jaw, a severe devastating side effect of antiresorptive bone treatment. The aim of this study was to elucidate the putative pathomechanism explaining the association between the genetic polymorphism with the alleles CYP2C8*2 and *3 causing low CYP2C8 activity, and disturbed periodontal remodelling in periodontal fibroblasts cultured from patients undergoing orthodontic treatment. CYP2C8 activity, enzyme expression and substrate metabolism were detected in human periodontal fibroblast cultures. Zoledronic acid caused enhanced reactive oxygen species (ROS) production in periodontal fibroblasts, which was enhanced by arachidonic acid as inflammatory signal. Enhanced bisphosphonate-induced uncoupling of the CYP2C8 enzyme was detected in the variant allele (CYP2C8*3) with the result of increased H2 O2 production and lowered substrate oxidation. Conversely, substrate (amodiaquine) addition led to decreased H2 O2 production in isolated CYP2C8 enzymes, but in CYP2C8*3 enzyme, increased H2 O2 was still detected, especially in presence of arachidonic acid. CYP2C8 variants leading to decreased enzyme activity in substrate oxidation may enhance ROS production by reaction uncoupling, and thus, contribute to difficulties in orthodontic treatment and the risk of side effects of antiresorptive drugs.

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.

Effects of Melandrium firmum Rohrbach on RANKL­induced osteoclast differentiation and OVX rats.

Osteoporosis is a systemic skeletal disease characterized by reduced bone mineral density (BMD), which results in an increased risk of fracture. Melandrium firmum (Siebold & Zucc.) Rohrbach (MFR), 'Wangbulryuhaeng' in Korean, is the dried aerial portion of Melandrii Herba Rohrbach, which is a member of the Caryophyllaceae family and has been used to treat several gynecological conditions as a traditional medicine. However, to the best of our knowledge, the effect of MFR on osteoclast differentiation and osteoporosis has not been assessed. To evaluate the effects of MFR on osteoclast differentiation, tartrate­resistant acid phosphatase staining, actin ring formation and bone resorption assays were used. Additionally, receptor activator of nuclear factor­κB ligand­induced expression of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1) and c­Fos were measured using western blotting and reverse transcription­PCR. The expression levels of osteoclast­related genes were also examined. To further investigate the anti­osteoporotic effects of MFR in vivo, an ovariectomized (OVX) rat model of menopausal osteoporosis was established. Subsequently, the femoral head was scanned using micro­computed tomography. The results revealed that MFR suppressed osteoclast differentiation, formation and function. Specifically, MFR reduced the expression levels of osteoclast­related genes by downregulating transcription factors, such as NFATc1 and c­Fos. Consistent with the in vitro results, administration of MFR water extract to OVX rats reduced BMD loss, and reduced the expression levels of NFATc1 and cathepsin K in the femoral head. In conclusion, MFR may contribute to alleviate osteoporosis­like symptoms. These results suggested that MFR may exhibit potential for the prevention and treatment of postmenopausal osteoporosis.

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.

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.

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.

Tablysin-15 inhibits osteoclastogenesis and LPS-induced bone loss via attenuating the integrin αvß3 pathway.

Excessive osteoclast leads to the imbalance in bone reconstruction and results in osteolytic diseases, such as osteoporosis and rheumatic arthritis. Integrin αvß3 abundantly expresses on osteoclast and plays a critical role in the formation and function of osteoclast, therefore, blockage of αvß3 has become an attractive therapeutic option for osteolytic diseases. In this study, we find that Tablysin-15, a RGD motif containing disintegrin, concentration-dependently suppresses RANKL-induced osteoclastogenesis, F-actin ring formation and bone resorption without affecting the cell viabilities. Tablysin-15 binds to integrin αvß3 and inhibits the activation of FAK-associated signaling pathways. Tablysin-15 also suppresses the activation of NF-кB, MAPK, and Akt-NFATc1 signaling pathways, which are crucial transcription factors during osteoclast differentiation. Moreover, Tablysin-15 decreases the osteoclastogenesis marker gene expression, including MMP-9, TRAP, CTSK, and c-Src. Finally, Tablysin-15 significantly inhibits LPS-induced bone loss in a mouse model. Taken together, our results indicate that Tablysin-15 significantly suppresses osteoclastogenesis in vitro and in vivo, thus it might be a excellent candidate for treating osteolytic-related diseases.

Evaluating two nanocarrier systems for the transdermal delivery of sodium alendronate.

Sodium alendronate is a nitrogen-containing bisphosphonate, widely used for osteoporosis treatment. However, due to its several oral administration drawbacks, the transdermal route represents an interesting option. The aim of this study was to formulate sodium alendronate in two submicron delivery systems, microemulsions, and solid-in-oil nanosuspensions, both systems possessing permeation enhancing properties. The composition of microemulsions was determined through the construction of pseudo-ternary phase diagrams. Solid-in-oil nanosuspensions were prepared by an emulsification-freeze-drying method, evaluating the effect of sonication time and the type of surfactant. According to the results of drug loading capacity, droplet/particle size, and polydispersity index, two microemulsions and two nanosuspensions were selected to perform the subsequent evaluations. The results showed that microemulsions allowed a faster release of alendronate than nanosuspensions. The permeation capacity of alendronate formulations was assessed through the synthetic membrane Strat M®, as well as through pigskin, finding higher fluxes with microemulsions than with nanosuspensions. In order to elucidate the effect of the formulations on the permeability barrier of the stratum corneum, techniques such as ATR-FTIR and TEWL were used. Finally, measurements of erythema intensity showed that neither of the two nanosystems caused skin irritation after 2 h of contact. The results suggest that alendronate formulated in a microemulsion can be a viable transdermal nanocarrier for osteoporosis treatment.

Alendronate effect in esophagus, stomach and liver: an animal based pathological study.

Bisphosphonates are commonly used in clinical practice. Their effectiveness is indisputable, however their adverse effects, especially in the GI tract, are still controversial. In our report, we demonstrate pathological findings of the effect of systematic alendronate administration in esophagus, stomach and the liver of an in-vivo animal model of 15 Wistar rats. Light microscopy with immunohistochemistry and electron microscopy were used. Microscopic findings of inflammation of the stomach and mild hepatic dysfunction were observed. Conclusively, alendronate can potentially affect gastric mucosa and liver function on this animal experimental model.

Characterization of urinary biomarkers and their relevant mechanisms of zoledronate-induced nephrotoxicity using rats and HK-2 cells.

The aim of this study was to identify biomarkers of zoledronate-induced nephrotoxicity and to further characterize the mechanisms underlying this process by analyzing urinary metabolites. Twenty-four rats were randomly divided into four groups containing four (two control groups) or eight rats (two zoledronate groups) per group. The rats were injected intravenously with saline or zoledronate (3 mg/kg) singly (single, 3 weeks) or repeatedly eight times (3 weeks/time, 24 weeks). Serum blood urea nitrogen, serum creatinine, creatinine clearance, and kidney injury observed by hematoxylin and eosin and immunohistochemical staining were changed only in the repeated zoledronate group (3 mg/kg, 3 weeks/time, 24 weeks). Urinary levels of S-adenosylmethionine, S-adenosylhomocysteine, l-cystathionine, l-γ-glutamylcysteine, and glutathione related to glutathione metabolism and fumaric acid and succinic acid related to the tricarboxylic acid cycle in the zoledronate-treated group (3 mg/kg, 3 weeks/time, 24 weeks) were significantly lower than those in the control group, suggesting that zoledronate may cause cellular oxidative stress. Besides, urinary levels of uracil and uridine related to pyrimidine metabolism also decreased after zoledronate treatment (3 mg/kg, 3 weeks/time, 24 weeks), while the levels of hypoxanthine related to purine metabolism, histamine related to histamine metabolism, and several amino acids were significantly increased. Moreover, zoledronate-induced enhanced oxidative stress and histamine overproduction were confirmed by reactive oxygen species (ROS) and histamine measurement in a human proximal tubular cell line. Taken together, zoledronate-induced nephrotoxicity may be attributed to it inducing perturbations in glutathione biosynthesis and the tricarboxylic acid cycle, further causing ROS overproduction, oxidative stress, and cellular inflammation, thereby leading to nephrotoxicity.

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.

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.

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.

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 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.

Effects of dexamethasone and nimesulide on bisphosphonate-related osteonecrosis of the jaw: An experimental study.

OBJECTIVE: To evaluate the effects of dexamethasone (DEX) and nimesulide (NIM) on Bisphosphonate-related Osteonecrosis of the Jaw (BRONJ) in rats. DESIGN: BRONJ was induced by zoledronic acid (ZA) infusion (0.2mg/kg) in Wistar rats (n=8), followed by extraction of the left lower first molar (BRONJ groups). Control groups (n=40) received saline (IV). For eight weeks, DEX (0.04, 0.4, 4mg/kg) or saline (SAL) were administered by gavage 24h before each infusion of ZA or saline (IV), or NIM (10.3mg/kg) was administered 24h and 12h before each infusion of ZA or saline (IV). The haematological analyses were conducted weekly. After euthanasia (day 70), the jaws were submitted to radiographic and microscopic analysis. Kidney, liver, spleen and stomach were analysed histopathologically. RESULTS: The BRONJ groups showed a higher radiolucent area compared with the control groups (p<0.05). Histomorphometric analysis revealed healing and new bone formation in the control groups, while the BRONJ groups exhibited devitalized bone with bacterial colonies and inflammatory infiltrate. The BRONJ-DEX 0.4 and 4mg/kg groups had a greater number of bacterial colonies (p<0.05) and an increased polymorphonuclear cell count compared to the saline-BRONJ group, while the BRONJ-NIM group had a lower polymorphonuclear count (p<0.05). The BRONJ groups had leucocytosis, which was reduced by DEX administration. Treatments with DEX with or without ZA caused white pulp atrophy. CONCLUSION: Thus, DEX or NIM therapy was not effective in preventing radiographic and histopathologic events associated with BRONJ. Treatment with DEX attenuated leucocytosis post-infusion with ZA.

Potassium citrate prevents increased osteoclastogenesis resulting from acidic conditions: Implication for the treatment of postmenopausal bone loss.

The extracellular acidic milieu in bones results in activation of osteoclasts (OC) and inhibition of osteoblasts (OB) causing a net loss of calcium from the skeleton and the deterioration of bone microarchitecture. Alkalinization through supplementation with potassium citrate (K citrate) has been proposed to limit the osteopenia progression, even though its pharmacological activity in bone microenvironment is not well defined. We evaluated if K citrate was able to prevent the adverse effects that acidic milieu induces on bone cells. OC and OB were maintained in neutral (pH 7.4) versus acidic (pH 6.9) culture medium, and treated with different K citrate concentrations. We evaluated the OC differentiation at seven days, by counting of multinucleated cells expressing tartrate-resistant acid phosphatase, and the activity of mature OC at 14 days, by quantifying of collagen degradation. To evaluate the effects on OB, we analyzed proliferation, mineralization, and expression of bone-related genes. We found that the low pH increased OC differentiation and activity and decreased OB function. The osteoclastogenesis was also promoted by RANKL concentrations ineffective at pH 7.4. Non-cytotoxic K citrate concentrations were not sufficient to steadily neutralize the acidic medium, but a) inhibited the osteoclastogenesis, the collagen degradation, and the expression of genes involved in RANKL-mediated OC differentiation, b) enhanced OB proliferation and alkaline phosphatase expression, whereas it did not affect the in vitro mineralization, and c) were effective also in OC cultures resistant to alendronate, i.e. the positive control of osteoclastogenesis inhibition. In conclusion, K citrate prevents the increase in OC activity induced by the acidic microenvironment, and the effect does not depend exclusively on its alkalizing capacity. These data provide the biological basis for the use of K citrate in preventing the osteopenia progression resulting from low-grade acidosis.