Polyethylenimine-Bisphosphonate-Cyclodextrin Ternary Conjugates: Supramolecular Systems for the Delivery of Antineoplastic Drugs.

Bisphosphonates (BPs) are bone-binding molecules that provide targeting capabilities to bone cancer cells when conjugated with drug-carrying polymers. This work reports the design, synthesis, and biological evaluation of polyethyleneimine-BP-cyclodextrin (PEI-BP-CD) ternary conjugates with supramolecular capabilities for the loading of antineoplastic drugs. A straightforward, modular, and versatile strategy based on the click aza-Michael addition reaction of vinyl sulfones (VSs) allows the grafting of BPs targeting ligands and ßCD carrier appendages to the PEI polymeric scaffold. The in vitro evaluation (cytotoxicity, cellular uptake, internalization routes, and subcellular distribution) for the ternary conjugates and their doxorubicin inclusion complexes in different bone-related cancer cell lines (MC3T3-E1 osteoblasts, MG-63 sarcoma cells, and MDA-MB-231 breast cancer cells) confirmed specificity, mitochondrial targeting, and overall capability to mediate a targeted drug transport to those cells. The in vivo evaluation using xenografts of MG-63 and MDA-MB-231 cells on mice also confirmed the targeting of the conjugates.

A Potential Bone-Targeting Hypotoxic Platinum(II) Complex with an Unusual Cytostatic Mechanism toward Osteosarcoma Cells.

Osteosarcoma (OS) is the most common primary pediatric bone tumor lethal to children and adolescents. Chemotherapeutic agents such as cisplatin are not effective for OS because of their poor accessibility to this cancer and severe systemic toxicity. In this study, a lipophilic platinum(II) complex bearing a bisphosphonate bone-targeting moiety, cis-[PtL(NH3)2Cl]NO3 {BPP; L = tetraethyl [2-(pyridin-2-yl)ethane-1,1-diyl]bisphosphonate}, was prepared and characterized by NMR, electrospray ionization mass spectrometry, and single-crystal X-ray crystallography. The cytotoxicity of BPP toward OS cell lines U2OS and MG-63 was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. BPP exhibits moderate inhibition against U2OS cells through a mechanism involving both DNA binding and a mevalonate pathway. The acute toxicity of BPP to mice is 7-fold lower than that of cisplatin. The relative low systemic toxicity may result from the steric hindrance of the ligand, which blocks BPP approaching the bases of DNA. The results suggest that incorporating bisphosphonates into a platinum complex not only enhances its bone-targeting property but also minimizes its reactivity toward DNA and thereby lowers the systematic toxicity of the complex. The diminished cytotoxicity of BPP could be compensated for by increasing the therapeutic dose with marginal harm. This strategy provides a new possibility for overcoming the ineffectiveness and systemic toxicity of platinum drugs in the treatment of OS.

The sarin-like organophosphorus agent bis(isopropyl methyl)phosphonate induces ER stress in human astrocytoma cells.

Organophosphorus (OP) compounds such as sarin are toxic agents that irreversibly inhibit the enzyme acetylcholinesterase. A recent study showed that OP compounds also have multiple toxicity mechanisms, and another suggested that endoplasmic reticulum (ER) dysfunction contributes to OP toxicity. However, the signaling pathway and mechanisms involved are poorly understood. We examined whether the sarin-like OP agent bis(isopropyl methyl)phosphonate (BIMP), which exhibits toxicity similar to that of sarin, induced ER stress in human astrocytoma CCF-STTG1 cells. Our results demonstrate that BIMP exposure reduced cell viability. Moreover, it induced changes in mitochondrial membrane potential and increased cleavage of caspase 3. Treatment with BIMP increased the mRNA levels of the ER stress marker genes binding immunoglobulin protein (BiP) and the transcription factor C/EBP homologous protein (CHOP). Furthermore, BIMP increased the protein expressions and phosphorylation of BiP, CHOP, and protein kinase RNA-like ER kinase and the phosphorylation of eukaryotic translation initiation factor 2. Compared to BIMP treatment alone, pretreatment with the CHOP siRNA, siCHOP, decreased BIMP-dependent CHOP expression and improved CCF-STTG1 cell viability. Our findings suggest that BIMP induced mitochondrial dysfunction and apoptotic cell death event mediated by ER stress in CCF-STTG1 cells and that treatment targeted at managing ER stress has the potential to attenuate the toxicity of OP nerve agents.

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.

[Decrease in toxicity and therapeutic effect of zoledronic acid in combination therapy with different antioxidant extracts]. / Disminución de la toxicidad y del efecto terapéutico del ácido zoledrónico en el tratamiento combinado con diferentes extractos antioxidantes.

BACKGROUND: Zoledronic acid is used in the treatment of cancer-related diseases, although its use has been associated with avascular osteonecrosis. AIMS: To determine the possible protective effect of a range of antioxidant substances against the inhibition of human prostate epithelial cell growth (PNT2) and transgenic adenocarcinoma mouse prostate tumour cells (TRAMP-C1), in treatments combining zoledronic acid and ionising radiation (IR). MATERIAL AND METHOD: Cell survival is studied via cell viability assays (MTT) for 2 cell lines in isolated and combined treatments with zoledronic acid and/or IR, as well as the effect of adding 3 antioxidant substances. RESULTS: Zoledronic acid displays a significant cytotoxic effect over PNT2 and TRAMP-C1 cells (P<.001). The administration of antioxidants together with the zoledronic acid shows a protective effect for normal prostate cells, yet not so for prostate tumour cells. However, the administration of rosmarinic acid and apigenin in treatments combined with zoledronic acid provides a protective effect from the harmful effects of applying ionizing radiation, not only for normal PNT2 cells, but also for tumour cells. CONCLUSION: The use of antioxidant substances decreases the cytotoxic effect of zoledronic acid over non-tumour cells, and as such could be used in benign diseases. Furthermore, in the combined treatment using ionising radiation, these antioxidants also produced a protective effect in tumour cells, thus reducing the therapeutic effect sought by combining the treatment with radiation.

Zoledronate induces bisphosphonate-related osteonecrosis of the jaw in osteopenic sheep.

OBJECTIVES: Bisphosphonate-related osteonecrosis of the jaw (BP-ONJ) occurs in 1 % of patients with medication-induced osteoporosis treated with bisphosphonates. Sheep are an established large animal model for investigating osteoporotic skeletal changes. Zoledronate significantly reduces tissue mineral variability in ovariectomized sheep. The aim of this study was to analyze bone healing after tooth extraction in sheep with induced osteopenia and zoledronate administration. MATERIALS AND METHODS: Eight adult ewes were randomly divided into two groups of four animals. All sheep underwent ovariectomy and a low-calcium diet. Dexamethasone was administered weekly for 16 weeks. Zoledronate was then given every third week for a further 16 weeks in four sheep; these infusions were repeated after extraction of two lower premolars. Four sheep without zoledronate administrations served as controls. RESULTS: Due to general health conditions, two sheep of the zoledronate group had to be excluded before surgery. The remaining two sheep of this group developed BP-ONJ lesions at the extraction site and various other sites in both jaws. Control group animals showed uneventful wound healing. Histology of the alveolar processes as well as lumbar spine revealed larger portions of old bone and smaller portions of new bone in the zoledronate group. CONCLUSIONS: This animal study showed uneventful wound healing after tooth extraction in osteopenic sheep whereas zoledronate treatment leads to development of BP-ONJ-like lesions. CLINICAL RELEVANCE: As bisphosphonate administration is a standard treatment for glucocorticoid-induced osteoporosis, this model can be used for further research in pathogenesis and management of bisphosphonate-related adverse events.

Experimental study of influence of direct bisphosphonate administration on bone substitute.

Articular cartilage has a low self-repair ability and natural healing cannot be expected. Treatment using various bone substitutes has been performed, but these have various disadvantages. Compared to autologous bone substitutes, the osteochondral repair ability of allogeneic bone substitutes is low, but the effective and safe utilization of these as a bone substitute may be possible by improving their osteochondral repair ability. We prepared a full-thickness osteochondral defect in the patellar fossa of rabbits, added a bisphosphonate preparation to bovine tooth-derived Demineralized Dentin Matrix (DDM), which has its own osteochondral repair ability, and investigated osteochondral repair ability in the defects. As a result, we suggest that the addition of high-dose BPs inhibits the osteochondral repair ability of DDM.

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.

Toxicity of a dental adhesive compared with ionizing radiation and zoledronic acid.

BACKGROUND: To determine the toxicity of aqueous dilutions of a universal self-priming dental adhesive (DA) and comparing these with those elicited by exposure to ionizing radiation (IR), Zoledronic acid (Z) treatment and the synergic effects of the combined treatment with IR+Z. MATERIAL AND METHODS: The genotoxic effect of DA was determined by the increase in the frequency of micronuclei in cytokinesis-blocked in cultured human lymphocytes before and after exposure to 2Gy of X-rays. The cytotoxic effect was studied by using the MTT cell viability test in normal prostate cell lines (PNT2) after exposure to different X-ray doses (0Gy-20Gy). The cell lines divided into different groups and treated with different test substances: DA in presence of O2, DA in absence of O2, Z-treated and control. RESULTS: An in vitro dose-dependent and time-dependent cytotoxic effect of DA, Z and IR on PNT2 cells (p>0.001) was demonstrated. DA without-O2, following the recommendations of manufacturers, had a more pronounced effect of increasing cell death than DA with-O2 (p<0.001). In the genotoxicity assay, DA at 25% of its original concentration significantly increased chromosome damage (p<0.001). The samples studied were found to be toxic, and the samples photo-polymerized in absence of O2 showed a bigger cytotoxic effect comparable to the additive toxic effect showed by the combined treatment of IR+Z. CONCLUSIONS: Additional effort should be carried out to develop adhesives, which would reduce the release of hazardous substances; since toxic effects are similar to that reported by other agents whose clinical use is controlled by the health authorities.

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.

Zoledronate inhibits receptor activator of nuclear factor kappa-B ligand-induced osteoclast differentiation via suppression of expression of nuclear factor of activated T-cell c1 and carbonic anhydrase 2.

Bisphosphonates (BPs) are widely used in the prevention of skeletal-related events (SRE), including osteoporosis, skeletal metastases of malignant tumours, and multiple myeloma. Osteonecrosis of the jaw (ONJ) is frequently reported as a major adverse effect induced by BP treatment. The receptor activator of the nuclear factor kappa-B ligand (RANKL) inhibitor, denosumab, has recently been used to prevent SRE, but the frequency of ONJ induced by denosumab is similar to that by BPs. This finding suggests that the inhibition of RANKL-mediated osteoclastogenesis may have a close relationship with the occurrence of ONJ. We therefore investigated the expression status of RANKL-inducible genes in zoledronate-treated mouse osteoclast precursor cells. The molecular targets of zoledronate in the RANKL signal pathway and additional factors associated with osteoclastogenesis were analysed by genome-wide screening. Microarray analysis identified that among 31 genes on 44 entities of RANKL-inducible genes, the mRNA expression level of two genes, i.e., nuclear factor of activated T-cells c1 (NFATc1) and carbonic anhydrase 2 (CAII), was decreased in zoledronate-treated cells. Subsequent analyses verified that these two genes were significantly silenced by zoledronate treatment and that their expression was restored following inhibition of zoledronate action by geranylgeraniol. Zoledronate inhibited RANKL-induced osteoclast differentiation by suppression of NFATc1 and CAII gene expression. Our results suggest that these genes might be common targets for zoledronate and denosumab in the mechanism underlying RANKL-induced osteoclast differentiation. A clear understanding of the common molecular mechanisms of bone-remodelling agents is thus essential for prevention of ONJ.

Lipopolysaccharide aggravates bisphosphonate-induced osteonecrosis in rats.

The pathogenesis of bisphosphonate-related osteonecrosis of the jaw (BRONJ) is highly controversial. We have previously reported the development of osteonecrosis by periodontal pathogenic stimulation in the jaw and femur of rats treated with bisphosphonate. Since the major toxicity factor of Gram-negative bacteria is lipopolysaccharide (LPS), the present study aimed to evaluate the relationship between osteonecrosis and LPS in a rat model of BRON-like lesions. Seventeen male rats were injected subcutaneously with zoledronic acid weekly for 4 weeks and divided into three groups: LPS (LPS administered into the bone marrow of the mandible and femur) and LPS plus polymyxin B (PMB) and saline groups (given neutralized LPS with PMB or saline, respectively, using the same protocol). At 4 weeks after the procedure, harvested specimens were analyzed using histomorphology (n=5 from each group) and histochemistry (n=1 each from LPS and LPS plus PMB groups). There was a significantly wider area of osteonecrosis in the LPS group as compared to the saline and LPS plus PMB groups in both the mandible (P=0.030 and P=0.009, respectively) and femur (P=0.002 and P=0.020, respectively). Our results indicate that LPS stimulation is deeply involved in the development and promotion of BRON.

Laser-enhanced cytotoxicity of zoledronic acid and cisplatin on primary human fibroblasts and head and neck squamous cell carcinoma cell line UM-SCC-3.

INTRODUCTION: Low-level laser therapy (LLLT) is used in parodontitis treatment in combination with an antimicrobial photosensitizer. The purpose of this study was to investigate the combination of LLLT with cisplatin and zoledronic acid as potential photosensitizer in-vitro. MATERIALS AND METHODS: Primary human fibroblasts (PHF) and head and neck squamous cell carcinoma cells (HNSCC, exactly UM-SCC-3) were treated with different concentrations of zoledronatic acid and cisplatin and irradiated twice with a diode laser (wavelength 670 nm, 2 min). Cell viability was tested by XTT assay and histomorphological analysis with HE staining. RESULTS: LLLT increased bioviability for both cell lines (p < 0.001). LLLT lowered PHF viability at the highest concentrations of cisplatin (p = 0.027 and p = 0.005) and zoledronic acid (p < 0.001). For HNSCCs, LLLT reduced cell viability at every concentration of cisplatin (all p < 0.05). In cases of incubation with zoledronic acid, similar to fibroblasts, laser therapy lowered cell viability at the highest concentration only (p < 0.001). CONCLUSIONS: Within the limits of this study, it can be concluded that LLLT enhances the effect of cisplatin and zoledronic acid in the discussed cells in order to develop new therapeutic options for cysts in the cranio-maxillofacial region and other appropriate indications.

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.

Arylamino methylene bisphosphonate derivatives as bone seeking matrix metalloproteinase inhibitors.

The complexity of matrix metalloproteinase inhibitors (MMPIs) design derives from the difficulty in carefully addressing their inhibitory activity towards the MMP isoforms involved in many pathological conditions. In particular, specific metalloproteinases, such as MMP-2 and MMP-9, are key regulators of the 'vicious cycle' occurring between tumor metastases growth and bone remodeling. In an attempt to devise new approaches to selective inhibitor derivatives, we describe novel bisphosphonate bone seeking MMP inhibitors (BP-MMPIs), capable to be selectively targeted and to overcome undesired side effects of broad spectrum MMPIs. In vitro activity (IC50 values) for each inhibitor was determined against MMP-2, -8, -9 and -14, because of their relevant role in skeletal development and renewal. The results show that BP-MMPIs reached IC50 values of enzymatic inhibition in the low micromolar range. Computational studies, used to rationalize some trends in the observed inhibitory profiles, suggest a possible differential binding mode in MMP-2 that explains the selective inhibition of this isoform. In addition, survival assay was conducted on J774 cell line, a well known model system used to evaluate the structure-activity relationship of BPs for inhibiting bone resorption. The resulting data, confirming the specific activity of BP-MMPIs, and their additional proved propensity to bind hydroxyapatite powder in vitro, suggest a potential use of BP-MMPIs in skeletal malignancies.

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.

In vitro cytotoxicity of zoledronate (nitrogen-containing bisphosphonate: NBP) and/or etidronate (non-NBP) in tumour cells and periodontal cells.

OBJECTIVE: Nitrogen-containing bisphosphonates (NBPs), the first-choice drugs for diseases that cause enhanced bone resorption, may injure jawbones and gastrointestinal tissues. In rodents, NBPs cause necrosis at injection sites. Bisphosphonates accumulate within bones, especially where there is inflammation. We hypothesized that if jawbone-accumulated NBPs are released, they may directly injure cells around the jawbones. To examine this hypothesis, we compared the direct effects of zoledronate (NBP) and/or etidronate (non-NBP) on various cells, including periodontal cells. DESIGN: Various human tumour cells (such as squamous carcinoma cells and prostate adenocarcinoma cells) and periodontal cells (such as gingival fibroblasts and periodontal ligament cells) were incubated with or without zoledronate and/or etidronate. Cell viability and cytotoxicity were determined by tetrazolium dye assay and by FITC-Annexin V/propidium iodide assay, respectively. RESULTS: Zoledronate, at 100µM, was toxic to all types of cells tested, while its toxicity varied among cells at both 1 and 10µM. There was no clear difference between tumour cells and non-tumour cells in sensitivity to the cytotoxicity of zoledronate. In contrast, etidronate was not toxic at 1-100µM in any of the cells tested. Interestingly, etidronate reduced the cytotoxicity of zoledronate in many cell-types, including gingival fibroblasts. CONCLUSIONS: These results, together with those reported by others and those from our previous in vivo experiments, suggest that NBPs, upon release from jawbones (e.g., during dental surgery or bone infection), may directly injure various cells located around the jawbones, and that etidronate may be protective against the cytotoxicity of NBPs in periodontal tissues.

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 × 10(4) 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.

Osteomalacia: the missing link in the pathogenesis of bisphosphonate-related osteonecrosis of the jaws?

BACKGROUND: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a well-documented adverse event from treatment with nitrogen-containing bisphosphonates (NBPs). During a preliminary histomorphometric study aimed at assessing the rate of bone remodeling in the jaws of patients with surgically resected BRONJ, we found a defect of bone mineralization (unpublished data). We hypothesized that osteomalacia could be a risk factor for BRONJ in patients taking NBPs. Therefore, we looked for static and dynamic histomorphometric evidence of osteomalacia in biopsies from subjects with and without BRONJ. METHODS: This case-control study used histomorphometric analysis of bone specimens of patients using NBPs (22 patients with BRONJ and 21 patients without BRONJ) who required oral surgical interventions for the treatment/prevention of osteonecrosis. Patients were given tetracycline hydrochloride according to a standardized protocol before taking bone biopsies from their jaws. Biopsies with evidence of osteomyelitis or necrosis at histology were excluded from the study. Osteomalacia was defined as a mineralization lag time >100 days, a corrected mean osteoid thickness >12.5 mm, and an osteoid volume >10%. RESULTS: In all, 77% of patients with BRONJ were osteomalacic compared with 5% of patients without BRONJ, according to histomorphometry. Because osteomalacia was found almost exclusively in NBP users with BRONJ, this is likely to be a generalized process in which the use of NBPs further deteriorates mechanisms of bone repair. CONCLUSIONS: Osteomalacia represents a new and previously unreported risk factor for disease development. This finding may contribute to a better understanding of the pathogenesis of this disease and help with the development of strategies to increase the safety of NBP administration.