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.

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.

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.

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.

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.

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.

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.

Severe compromise of preosteoblasts in a surgical mouse model of bisphosphonate-associated osteonecrosis of the jaw.

OBJECTIVES: The effect of amino-bisphosphonates on osteoblastic lineage and its potential contribution to the pathogenesis of bisphosphonate-associated osteonecrosis of the jaw (BONJ) remain controversial. We assessed the effects of zoledronic acid (ZOL) on bone and vascular cells of the alveolar socket using a mouse model of BONJ. MATERIAL AND METHODS: Thirty-two mice were treated twice a week with either 100 µg/kg of ZOL or saline for 12 weeks. The first left maxillary molar was extracted at the third week. Alveolar sockets were assessed at both 3 weeks (intermediate) and 9 weeks (long-term) after molar extraction by semi-quantitative histomorphometry for empty lacunae, preosteoblasts (Osterix), osteoclasts (TRAP), and pericyte-like cells (CD146). Also, the bone microarchitecture was assessed by micro-CT. RESULTS: Osteonecrotic-like lesions were observed in 21% of mice. Moreover, a decreased number of preosteoblasts contrasted with the increased number of osteoclasts at both time points. In addition, osteoclasts display multinucleation and detachment from the endosteal surface. Furthermore, the number of pericyte-like cells increased at the intermediate time point. The alveolar bone mass increased exclusively with long-term ZOL treatment. CONCLUSION: The severe imbalance between bone-forming cells and bone-resorbing cells shown in this study could contribute to the pathogenesis of BONJ.

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.

Medication-Related Osteonecrosis of the Jaw: Basic and Translational Science Updates.

In the late 1990s and the early 2000s, bisphosphonates had become the clinical pillar of excellence for treating metabolic bone disease, and thus their connection with osteonecrosis of the jaw (ONJ) caused significant concern. Over the past decade, progress has been made in understanding what is now referred to as medication-related ONJ (MRONJ), because of its connections to agents other than bisphosphonates, although in many respects the progress has been slow. This review highlights the key basic science and translational (animal) studies in the area of MRONJ and suggests areas of focus as the field moves into the next decade.

Zoledronic acid aggravates kidney damage during ischemia reperfusion injury in rat.

INTRODUCTION: Zoledronic acid (ZA), a bisphosphonate, increases the levels of cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), and reactive oxygen species (ROS) in subjects without cancer. Increased production of ROS, TNF-α, and IL-6 during ischemia and reperfusion (I/R) injury stimulates apoptosis that leads to renal injury. We aimed to investigate whether ZA treatment has a protective effect on renal tissues during I/R. MATERIALS AND METHODS: Twenty-four Sprague-Dawley rats were used in this study, and they were subdivided randomly into three groups, each containing eight rats. Infrarenal abdominal aortic cross ligation was performed on the I/R group. After 2 h of ischemia, 2 h of reperfusion was applied. A single dose of 100 µg/kg ZA was administered intraperitoneally to the ZA group. I/R was performed after 48 h. RESULTS: Whereas TNF-α, IL-6, and nitric oxide (NO) levels of the I/R group were higher than those of the control group, TNF-α, IL-6, and NO levels of the ZA group were higher than those of the I/R group [TNF-α (p=0.038), IL-6 (p=0.012), NO (p=0.002), and caspase-3 (p=0.037)] and the control group [TNF-α (p<0.001), IL-6 (p<0.001), NO (p<0.001), and caspase-3 (p<0.001)]. Whereas the carbonic anhydrase II (CA-II) level of the ZA group was lower than that of the control group (p=0.040), the CA-II level of the I/R group was higher than that of the control group (p=0.020). CONCLUSION: ZA may aggravate renal injury during I/R by increasing cytokine production and apoptosis. It may also increase renal injury and metabolic acidosis during I/R by suppressing CA-II enzyme activities.

Comparative "in vitro" evaluation of the antiresorptive activity residing in four Ayurvedic medicinal plants. Hemidesmus indicus emerges for its potential in the treatment of bone loss diseases.

ETHNOPHARMACOLOGICAL RELEVANCE: Four Indian plants, traditionally used in Ayurvedic medicine: Asparagus racemosus Willd., Emblica officinalis Gaertn., Hemidesmus indicus R. Br., and Rubia cordifolia L. were selected on the basis of their ethnobotanical use and of scientific evidence that suggests a potential efficacy in the treatment of bone-loss diseases. The antiresorptive properties of the four plants have been investigated. The aim was to provide adequate evidence for the exploitation of natural compounds as alternative therapeutics for the treatment of diseases caused by increased osteoclast activity. MATERIALS AND METHODS: Decoctions were prepared from dried plant material according to the traditional procedure and standardization by HPLC was performed using marker compounds for each species. Total polyphenols, flavonoids and radical scavenging activity of the decoctions were also determined. The bioactivity of the plant decoctions was evaluated in subsequent phases. (1) A cytotoxicity screening was performed on the mouse monocytic RAW 264.7 cell line to define the concentrations that could be utilized in the following step. (2) The antiresorptive properties of plant decoctions were compared with that of a "gold standard" drug (alendronate) by measuring osteoclastogenesis inhibition and osteoclast apoptosis. (3) The toxic effect on bone forming cells was excluded by evaluating the impact on the proliferation of osteogenic precursors (mesenchymal stem cells, MSC). RESULTS: All the decoctions inhibited osteoclastogenesis similarly to alendronate at the highest doses, but Hemidesmus indicus and Rubia cordifolia were also effective at lower concentrations. Apoptosis increased significantly when cells were exposed to the highest concentration of Emblica officinalis, Hemidesmus indicus, and Rubia cordifolia. All concentrations of Emblica officinalis tested inhibited the proliferation of osteogenic precursors, while only the highest doses of Asparagus racemosus and Rubia cordifolia were toxic. On the contrary, Hemidesmus indicus did not affect osteogenic precursor growth at any concentration tested. CONCLUSION: Among the medicinal plants included in the study, Hemidesmus indicus showed the greatest antiosteoclastic activity without toxic effect on osteogenic precursors. Therefore, Hemidesmus indicus exhibits the properties of an antiresorptive drug and represents the ideal candidate for further clinical investigations.

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.

Does an alkaline environment prevent the development of bisphosphonate-related osteonecrosis of the jaw? An experimental study in rats.

OBJECTIVE: To investigate the preventive effect of locally applied sodium bicarbonate on bisphosphonate-related osteonecrosis of the jaw (BRONJ). STUDY DESIGN: Thirty-six Sprague-Dawley rats were divided into 4 groups. Animals in group I received 0.1 mg/kg sterile saline 3 times per week for 8 weeks. Groups II, III, and IV received intraperitoneal zoledronate injection in the same manner with the same frequency and duration. The right first molar tooth was extracted in groups III and IV. One mL 8.4% sodium bicarbonate (SB) was applied to the extraction socket at the time of extraction in group IV. The effect of locally applied SB as an alkalizing agent was evaluated by histomorphometric analysis. RESULTS: BRONJ was observed in none of the animals in the control groups, 67% of the animals in the tooth extraction group, and none of the animals in the local SB application group (P < .01). CONCLUSIONS: Administration of locally applied SB had positive effects on the prevention of BRONJ in animals, but further studies are required to verify the effectiveness of this form of treatment before its use in humans.

Cytotoxic Effects of Zoledronic Acid on Human Epithelial Cells and Gingival Fibroblasts

Bisphosphonate-induced osteonecrosis has been related to the cytotoxicity of these drugs on oral mucosa cells. A previous study showed that 5 µM of zoledronic acid (ZA), a nitrogen-containing bisphosphonate, is the highest concentration of this drug found in the oral cavity of patients under treatment. Therefore, in order to simulate an osteonecrosis clinical condition, the aim of this study was to evaluate the highest concentration of ZA applied on human epithelial cells (HaCaT) and gingival fibroblasts. For this purpose, cells (3×104 cells/cm2) were seeded in wells for 48 h using complete culture medium (cDMEM). After 48 h incubation, the cDMEM was replaced by fresh serum-free culture medium (DMEM-FBS) in which the cells were maintained for additional 24 h. Then, 5 µM ZA were added to the DMEM–FBS and the cells incubated in contact with the drug for 48 h. After this period, the number of viable cells (trypan blue), cell viability (MTT assay), total protein (TP) production and cell morphology (SEM analysis) were assessed. Data were analyzed statistically by Mann-Whitney, ANOVA and Tukey's test (α=0.05). ZA caused a significant reduction in the number of viable cells and decreased the metabolic activity of both cell lines. However, decrease of TP production occurred only in the epithelial cell cultures. Morphological alterations were observed in both cell types treated with ZA. In conclusion, ZA (5 µM) was cytotoxic to human epithelial cells and gingival fibroblast cultures, which could be associated, clinically, with the development of bisphosphonate-induced osteonecrosis.

A osteonecrose induzida por bisfosfonatos tem sido associada a um efeito citotóxico destes medicamentos sobre as células da mucosa oral. Um estudo recente demonstrou que 5 µM de ácido zoledrônico (AZ), um potente bisfosfonato nitrogenado, foi a maior concentração encontrada na cavidade oral da pacientes em tratamento com este medicamento. Portanto, para simular esta condição in vivo, o objetivo deste estudo foi avaliar o efeito da aplicação desta concentração de AZ sobre células epiteliais (HaCaT) e fibroblasto de gengiva. As células foram semeadas (3×104 células/cm2) e incubadas por 48 h em placas de 24 compartimentos, utilizando meio de cultura completo (cDMEM). Após permanecer por 24 h em DMEM sem soro fetal bovino (DMEM-SFB), 5 µM do AZ foram adicionados a este meio de cultura, o qual foi incubado em contato com as células por 48 h. Após este período, foram avaliados o número de células viáveis (trypan blue), viabilidade celular (teste de MTT), produção de proteína total e a morfologia celular (MEV). Os dados obtidos foram submetidos aos testes estatísticos de Mann-Whitney e ANOVA complementada por testes de Tukey (p>0,05). Foi demonstrado que o AZ causou diminuição significativa no número de células viáveis, além de redução do metabolismo celular para ambos os tipos celulares avaliados. Porém, redução na produção de proteína total ocorreu apenas para as células epiteliais. Alterações morfológicas foram observadas em ambos os tipos celulares tratados com AZ. Estes dados científicos indicam que a concentração de AZ avaliada neste estudo (5 µM) apresenta ação citotóxica sobre células epiteliais e fibroblastos de gengiva, o que poderia estar associado, clinicamente, ao desenvolvimento da osteonecrose induzida por bisfosfonatos.

Do bisphosphonates inhibit direct fracture healing?: A laboratory investigation using an animal model.

Fracture repair occurs by two broad mechanisms: direct healing, and indirect healing with callus formation. The effects of bisphosphonates on fracture repair have been assessed only in models of indirect fracture healing. A rodent model of rigid compression plate fixation of a standardised tibial osteotomy was used. Ten skeletally mature Sprague-Dawley rats received daily subcutaneous injections of 1 µg/kg ibandronate (IBAN) and ten control rats received saline (control). Three weeks later a tibial osteotomy was rigidly fixed with compression plating. Six weeks later the animals were killed. Fracture repair was assessed with mechanical testing, radiographs and histology. The mean stress at failure in a four-point bending test was significantly lower in the IBAN group compared with controls (8.69 Nmm(-2) (sd 7.63) vs 24.65 Nmm(-2) (sd 6.15); p = 0.017). On contact radiographs of the extricated tibiae the mean bone density assessment at the osteotomy site was lower in the IBAN group than in controls (3.7 mmAl (sd 0.75) vs 4.6 mmAl (sd 0.57); p = 0.01). In addition, histological analysis revealed progression to fracture union in the controls but impaired fracture healing in the IBAN group, with predominantly cartilage-like and undifferentiated mesenchymal tissue (p = 0.007). Bisphosphonate treatment in a therapeutic dose, as used for risk reduction in fragility fractures, had an inhibitory effect on direct fracture healing. We propose that bisphosphonate therapy not be commenced until after the fracture has united if the fracture has been rigidly fixed and is undergoing direct osteonal healing.