Presence Of Alendronate Research Articles

Long-term passage impacts human dental pulp stem cell activities and cell response to drug addition in vitro.

Dental pulp stem cells (DPSCs) possess mesenchymal stem cell characteristics and have potential for cell-based therapy. Cell expansion is essential to achieve sufficient cell numbers. However, continuous cell replication causes cell aging in vitro, which usually accompanies and potentially affect DPSC characteristics and activities. Continuous passaging could alter susceptibility to external factors such as drug treatment. Therefore, this study sought to investigate potential outcome of in vitro passaging on DPSC morphology and activities in the absence or presence of external factor. Human DPSCs were subcultured until reaching early passages (P5), extended passages (P10), and late passages (P15). Cells were evaluated and compared for cell and nuclear morphologies, cell adhesion, proliferative capacity, alkaline phosphatase (ALP) activity, and gene expressions in the absence or presence of external factor. Alendronate (ALN) drug treatment was used as an external factor. Continuous passaging of DPSCs gradually lost their normal spindle shape and increased in cell and nuclear sizes. DPSCs were vulnerable to ALN. The size and shape were altered, leading to morphological abnormality and inhomogeneity. Long-term culture and ALN interfered with cell adhesion. DPSCs were able to proliferate irrespective of cell passages but the rate of cell proliferation in late passages was slower. ALN at moderate dose inhibited cell growth. ALN caused reduction of ALP activity in early passage. In contrast, extended passage responded differently to ALN by increasing ALP activity. Late passage showed higher collagen but lower osteocalcin gene expressions compared with early passage in the presence of ALN. An increase in passage number played critical role in cell morphology and activities as well as responses to the addition of an external factor. The effects of cell passage should be considered when used in basic science research and clinical applications.

Enhancement of isoprene production in engineered Synechococcus elongatus UTEX 2973 by metabolic pathway inhibition and machine learning-based optimization strategy

An engineered Synechococcus elongatus UTEX 2973-IspS.IDI is used to enhance isoprene production through geranyl diphosphate synthase (CrtE) inhibition and process parameters (light intensity, NaHCO3 and growth temperature) optimization approach. A cumulative isoprene production of 1.21 mg/gDCW was achieved with productivity of 12.6 μg/gDCW/h in culture supplemented with 20 μg/mL alendronate. This inhibition strategy improvises the cumulative isoprene production 5.76-fold in presence of alendronate. The maximum cumulative production of isoprene is observed to be 5.22 and 6.20 mg/gDCW (54.4 and 64.6 μg/gDCW/h) at statistical and artificial neural network genetic algorithm (ANN-GA) optimized conditions, respectively. The overall increase of isoprene production is found to be 29.52-fold using an integrated approach of inhibition and ANN-GA optimization in comparison to unoptimized cultures without alendronate. This study reveals that alendronate use as a potential inhibitor and machine learning based optimization is a better approach in comparison to statistical optimization to enhance the isoprene production.

Vascular response to stress: Protective action of the bisphosphonate alendronate.

Since several additional actions of bone bisphosphonates have been proposed, we studied the effect of the bisphosphonate alendronate (ALN) on the vascular response to environmental stress. Primary cultures of endothelial cells (EC) and vascular smooth muscle cells (VSMC) exposed to strained conditions were employed for experimental evaluation. After ALN treatment, cell migration, proliferation, and angiogenesis assays were performed. The participation of signal transduction pathways in the biochemical action of ALN was also assessed. In VSMC cultures, ALN counteracted the stimulation of cellular migration elicited by the proinflammatory agent lipopolysaccharide (LPS) or by high levels of calcium and phosphorus (osteogenic medium). Indeed, ALN reduced the increase of VSMC proliferation evoked by the stressors. When LPS and osteogenic medium were added simultaneously, the enhancement of cell proliferation dropped to control values in the presence of ALN. The mechanism of action of ALN involved the participation of nitric oxide synthase, mitogen-activated protein kinase (MAPK), and protein kinase C (PKC) signaling pathways. The study revealed that ALN exhibits a proangiogenic action. On EC, ALN enhanced vascular endothelial growth factor (VEGF) synthesis, and induced capillary-like tube formation in a VEGF-dependent manner. The presence of vascular stress conditions (LPS or osteogenic medium) did not modify the proangiogenic action elicited by ALN. The findings presented suggest an extra-bone biological action of ALN, which could contribute to the maintenance of vascular homeostasis avoiding cellular damage elicited by environmental stress.

Role of protein tyrosine phosphatase 1B inhibitor in central insulin resistance and associated cognitive deficits

BackgroundProtein tyrosine phosphatase 1B (PTP1B) inhibitors are potential candidates for the treatment of peripheral insulin resistance and diabetes mellitus. Similar to peripheral action within the brain also, PTP1B activation impairs insulin signaling pathways. Activation of PTP1B in brain also accentuates neuroinflammation, oxidative stress and decreases neurotrophic factors in various brain dysfunctions including cognitive decline. ObjectivesThe main objective of our study was to elucidate the role of alendronate, a potent PTP1B inhibitor (blood brain barrier crossing bisphosphonate) in central insulin resistance and associated memory deficits. MethodologyTo induce central insulin resistance, streptozotocin (3 mg/kg) intracerebroventricular (ICV) was administered in two alternate days (1st and 3rd). After 21 days, memory was assessed via using the passive avoidance and Morris water maze paradigm. At the end of behavioral studies, animals were sacrificed to assess a variety of biochemical and molecular parameters in the hippocampus and cerebral cortex region of the brain. Treatment drug alendronate (3 mg/kg/day, p.o) and standard drug donepezil (3 mg/kg/i.p.) were administered from the 3rd day of STZ administration till the end of the study. Inhibition of PTP1B activates phosphoinsotide-3 kinase (PI3 K) (down-stream regulator of insulin signaling pathway).Thus, to illuminate the mechanism of action of alendronate, PI3 K inhibitor, wortmannin was administered in presence of alendronate in one group. ResultsAdministration of alendronate to ICV streprozotocin treated rats resulted in modulation of the insulin signaling pathway and associated behavioral, biochemical and molecular changes in central insulin resistance. However, the protective effect of alendronate was entirely vanished when it was administered in the presence of wortmannin. ConclusionAlendronate can be an important treatment strategy in central insulin signaling pathway dysfunction and associated cognitive deficits. Protective effect of alendronate is via modulation of PI3-K/Akt signaling pathway.

Bone microenvironment-mediated resistance of cancer cells to bisphosphonates and impact on bone osteocytes/stem cells.

Anti-resorptive bisphosphonates (BPs) have been clinically used to prevent cancer-bone metastasis and cancer-induced bone pathologies despite the fact that the phenotypic response of the cancer-bone interactions to BP exposure is "uncharted territory". This study offers unique insights into the interplay between cancer stem cells and osteocytes/osteoblasts and mesenchymal stem cells using a three-dimensional (3D) live cancer-bone interactive model. We provide extraordinary cryptic details of the biological events that occur as a result of alendronate (ALN) treatment using 3D live cancer-bone model systems under specific bone remodeling stages. While cancer cells are susceptible to BP treatment in the absence of bone, they are totally unaffected in the presence of bone. Cancer cells colonize live bone irrespective of whether the bone is committed to bone resorption or formation and hence, cancer-bone metastasis/interactions are though to be "independent of bone remodeling stages". In our 3D live bone model systems, ALN inhibited bone resorption at the osteoclast differentiation level through effects of mineral-bound ALN on osteocytes and osteoblasts. The mineral-bound ALN rendered bone incapable of osteoblast differentiation, while cancer cells colonize the bone with striking morphological adaptations which led to a conclusion that a direct anti-cancer effect of BPs in a "live or in vivo" bone microenvironment is implausible. The above studies were complemented with mass spectrometric analysis of the media from cancer-bone organ cultures in the absence and presence of ALN. The mineral-bound ALN impacts the bone organs by limiting transformation of mesenchymal stem cells to osteoblasts and leads to diminished endosteal cell population and degenerated osteocytes within the mineralized bone matrix.

Alendronate does not prevent long bone fragility in an inactive rat model.

The lack of estrogen and inactivity are both important in the pathogenesis of osteoporosis in elderly women, and there have been no appropriate rodent studies to examine the effects of common bisphosphonates on these two components separately. We compared the efficacy of alendronate (ALN) on the long bones of aged female rats, which were sedentary, estrogen deficient, or both. The rats were either forced to remain in a sitting position or allowed to walk in standard cages with or without ALN administration. The 8-week experimental period began 5weeks after ovariectomy or sham surgery. Parameters of the hindlimb bones were determined by a three-point bending test, peripheral quantitative computed tomography, microfocus computed tomography, confocal laser Raman microspectroscopy, and dynamic histomorphometry. Regardless of ovariectomy, ALN was ineffective against the deterioration of breaking stress caused by sitting even though the trabecular bone mineral density was significantly higher in the sitting-ALN groups. Toughness was significantly deficient in the ovariectomy sitting-ALN group. This was in agreement with the bone geometry with a greater marrow space. Sitting also increased the mineral-to-matrix ratio and the carbonate-to-phosphate ratio, both indicative of aged bone. A greater loss of proteinaceous amide intensity compared with mineral intensity resulted in an increased mineral-to-matrix ratio in the presence of ALN. Sitting resulted in deficits in the quality and the geometry of cortical bone, resulting in fragility. The use of bisphosphonates, such as ALN, may provide a therapy best suited for osteoporotic individuals whose daily activity is not limited.

Bisphosphonates and adipogenesis: Evidence for alendronate inhibition of adipocyte differentiation in 3T3-L1 preadipocytes through a vitamin D receptor mediated effect

Background: Adipocyte and osteoblast derive from the same mesenchimal progenitor. Age-related decrease in bone mass is accompanied by an increase in marrow adipose tissue. Vitamin D3 (VD3) inhibits adipogenesis in 3T3-L1 preadipocytes. Recently it has been demonstrated that alendronate (ALN) inhibits adipogenesis while promoting osteoblast differentiation of mesenchimal stem cells. Aim of the Study: To evaluate the role of ALN on adipocyte differentiation in vitro and the potential synergic role of VD3 co-treatment. Procedures: Murine 3T3-L1 and 3T3-F442A preadipocytes were routinely differentiated in presence of ALN and VD3 10-9 - 10-7 M for 7 days and then stained with Oil Red O. The effect of these treatments on mRNA expression of the main molecular markers of adipocyte differentiation (PPARγ and C/EBPα) and VD Receptor (VDR) were analyzed through RT-PCR. Results: Both ALN and VD3 showed a marked anti-adipogenic effect on 3T3-L1 cells. Co-incubation of ALN 10-8 M and VD3 10-9 M displayed no synergic effect on inhibition of adipogenesis. PPARγ mRNA expression was significantly reduced by ALN and VD3. mRNA expression of C/EBPα was reduced only by VD3 treatment. An increase in VDR mRNA expression of 3T3-L1 cells was observed with both ALN and VD3. On the contrary, 3T3-F442A cells, which are in a more advanced adipogenic differentiation stage compared to 3T3-L1, did not express detectable levels of VDR. Interestingly, adipose differentiation of 3T3-F442A was not affected by ALN nor VD3. These results suggest that VDR may represent the molecular target of the anti-adipogenic effect of ALN. Conclusion: VDR plays a critical role in mediating the anti-adipogenic effect of ALN. Further studies to clarify this mechanism are warranted.

Alendronate promotes plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin degradation sites within the MMP-9 catalytic domain

Irreversible MMP-9 inhibition is considered a significant therapeutic goal in inflammatory, vascular and tumour pathology. We report that divalent cation chelators Alendronate and EDTA not only directly inhibited MMP-9 but also promoted irreversible plasmin-mediated MMP-9 inactivation by exposing cryptic plasmin-degradation sites within the MMP-9 catalytic-domain and producing an inhibitory hemopexin–domain fragment. This effect was also observed using MDA-MB-231 breast cancer cells, which activated exogenous plasminogen to degrade endogenous proMMP-9 in the presence of Alendronate or EDTA. Degradation-mediated inactivation of proMMP-9 occurred in the absence of transient activation, attesting to the incapacity of plasmin to directly activate proMMP-9 and direct MMP-9 inhibition by Alendronate and EDTA. Our study provides a novel rational for therapeutic Alendronate use in MMP-9-dependent pathology characterised by plasminogen activation.

Systemically alendronate was incorporated into dental tissues but did not cause morphological or mechanical changes in rats teeth

This study evaluated the effect of the systemic use of sodium alendronate in rats in vivo. Forty-five Wistar rats aged 36 to 42 days and weighing 200 to 230 g were randomly assigned to a control group (n = 20), which received distilled water, and an experimental group (n = 25), which received 2 weekly doses of 1 mg/kg of chemically pure sodium alendronate. The animals were killed after 60 days of treatment. The tibias were removed for analysis of bone mineral density by dual-energy X-ray absorptiometry (DXA). Then, the maxillary incisors were extracted for analysis of the mineralized dental tissues using fluorescence spectroscopy (FS), scanning electron microscopy (SEM), bright field microscopy (BFM), and cross-sectional microhardness (CSMH) testing. DXA and CSMH data were subjected to statistical analysis by Kruskal-Wallis test (5% significance level). The experimental group presented higher bone mineral density than the control group by DXA. FS analysis revealed presence of alendronate in the mineralized dental tissues of the specimens of the experimental group. Significant morphological differences were not found by SEM and BFM. Enamel and dentin (100 and 300 μm from the dentinoenamel junction) CSMH data did not show significant difference between the control and experimental groups. Based on the obtained results, we conclude that while alendronate increased the bone mineral density and was incorporated into the mineralized dental tissues it did not cause significant alterations in the morphology and microhardness of rat incisor enamel and dentin.

New approaches for the local prevention of osteoporotic fractures

ABSTRACTCombining Bisphophonates (BPs) and Calcium Phosphate Cement (CPC) to form a new medical device for the local treatment of Osteoporosis is a promising challenge. Our formulation was optimized from an apatitic-type CPC and we have shown that the best solution consists in introducing the bisphosphonate (Alendronate) in the calcium deficient apatite (CDA), a solid component of the cement, through a chemical exchange reaction. The cement obtained was characterized by 31P NMR and high frequency impedance for monitoring the CPC setting. The presence of Alendronate in the cement was also demonstrated by 31P NMR which has been also used to characterize the chemical transformation of α-TCP (main component of the apatitic cement) during the setting process. BP absorption/desorption experiments have been realized on cement blocks, under continuous flow condition, to model the release profile of the Alendronate. In vivo experiments showed promising results in terms of resorbability of the Alendronate–loaded cement while promoting new bone formation. The same methodology is considered to introduce gallium, a potential inhibitor of osteoclastic resorption, in a CPC formulation. First experiments have shown that gallium can be incorporated in calcium phosphate ceramics (i.e. β-TCP) where gallium is part of the network.

Reviews of Current Literature

Malden N, Lopes V. An epidemiological study of alendronate-related osteonecrosis of the jaws. A case series from the south-east of Scotland with attention given to case definition and prevalence. J Bone Miner Metab. 2011. [Epub ahead of print[This article presents a close case study series of alendronate-associated osteonecrosis of the jaws (AONJ). From their study the authors draw epidemiologic conclusion regarding AONJ as well as comment on clinical considerations and treatment protocol. Alendronate is the most common bisphosphonate prescribed in the world. Since the first cases of ONJ started to emerge a task force of the American Society of Bone and Mineral Research (ASBMR) proposed recommendations to address key issues with ONJ and bisphosphonates. These include case definition and clinical management as well as epidemiology. In this study, to confirm and stage AONJ, the authors used the definition proposed by American Association of Oral and Maxillofacial Surgeons 2009 position papers on BONJ. The specific details for case definition, as described by ASBMR, fall in to 7 categories of information (A–G): A, patient details—for this article all were white Caucasian except for one, and 82% were female; B, diagnosis/staging of disease—mild to moderate pain, presence of nonvital bone; C, local site and clinical history—64% included a history of dental extraction preceding the condition; D, alendronate dose and duration of presentation—70 mg daily dose; the duration of administration to first ONJ recognition had a mean of 3 years; E, indication for bisphosphonate use—64% were receiving alendronate to counteract the osteoporotic affects of glucocorticosteroids; 36% were receiving the drug for age-related or hormone/menopause-related osteoporosis; an estimate mean of 4.7 years of “disease onset” prior to ONJ onset; F, co-morbidity and duration of ONJ—55% were ASA III; duration of comorbidity to diagnosis of ONJ was difficult to assess, but in 64% it was considered between 5 and 10 years; G, co-medications and duration of ONJ—prednisolone was prescribed in 55% of the cases. Three patients were taking immunosuppressant leflunomide or methotrexate. A known complication of immunosuppressive drugs is oral ulcerations and 27% of patients had concomitant administration of those with alendronate. Four patients with cardiovascular disease were taking statins and two were taking nicorandil. Nicorandil is a drug used in the management of persistent angina pectoris. It has been associated with oral ulcerations, occasionally affecting the alveolar ridges. Thus the authors suggest that drug associated ulcerations, if occurring over bone in the presence of alendronate could initiate the development of ONJ. Thus these drugs should be considered risk factors. 55% of patients who developed ONJ were taking prednisolon and alendronate.Management of ONJ included mild analgesics (64%), antimicrobials in various combinations (73%) that ranged from 1 to 11 weeks, one surgical debridement (64%), chlorhexidine was prescribed to all patients, and 45% of patients discontinued alendronate use at the diagnosis of ONJ. It was decided that the risk associated with discontinuation of alendronate are less then the risks associated with continuation of alendronate. However a case-by-case review with multispecialist discussion is suggested when discontinuation of bisphosphonate therapy is considered as a treatment adjunct to management of AONJ. In this study the duration from diagnosis to resolution ranged from 2 weeks to 20 months (mean of 6.5 months). By tracking alendronate prescriptions the drug patient year (DPY) was calculated. Thus epidemiology could be approximated. In the population studied, the incidence of AONJ as a spontaneous event or in response to a traumatic oral insult is between 0.03% and 0.017% respectively. Where alendronate is prescribed alone or for age-related or hormone-related osteoporosis the prevalence is 0.004%. The presence of comorbidity where comedications are being prescribed with alendronate significantly increases the risk of developing AONJ. It is suggested that rheumatoid arthritis patients receiving alendronate, glucocorticosteroids, and often an immunosuppressant are at an increased risk of ONJ with an incidence of 0.1% of AONJ. This study of 11 patients and an extrapolation to a population of 900 000 with DPY calculations brings our attention to AONJ from another clinical perspective that should benefit our patients and our considerations in their care.

Biofunctional alendronate–Hydroxyapatite thin films deposited by Matrix Assisted Pulsed Laser Evaporation

We applied Matrix Assisted Pulsed Laser Evaporation (MAPLE) in order to synthesize alendronate-hydroxyapatite thin films on titanium substrates. Alendronate-hydroxyapatite composite nanocrystals with increasing bisphosphonate content (0, 3.9, 7.1% wt) were synthesized in aqueous medium. Then, they were suspended in deionised water, frozen at liquid nitrogen temperature and used as targets for MAPLE experiments. The depositions were conducted with a KrF* excimer laser source (l = 248 nm, t FWHM = 25 ns) in mild conditions of temperature and pressure. The obtained thin films had a good crystallinity, which slightly decreases with the increase of alendronate content, and exhibited a porous-like structure. Osteoblast-like MG63 cells and human osteoclasts were cultured on the thin films up to 14 days. In the presence of alendronate, MG63 cells displayed a normal morphology, increased proliferation and higher values of differentiation parameters, namely type I collagen, osteocalcin, and osteoprotegerin/TNF-related activation-induced cytokine receptor ratio. In contrast, osteoclasts showed significantly reduced proliferation, and increased level of Caspase 3. Moreover, the coatings synthesized from hydroxyapatite at relatively high bisphosphonate content (7.1% wt) displayed a reduced production of Tumour Necrosis Factor alpha (TNF- α) and Interleukin 6 (IL-6), suggesting a down-regulatory role of alendronate on the inflammatory reaction. The successful deposition of alendronate modified hydroxyapatite thin films yields coatings with enhanced bioactivity, able to promote osteoblast differentiation and to inhibit osteoclast proliferation.

Alendronate and Pamidronate calcium phosphate bone cements: Setting properties and in vitro response of osteoblast and osteoclast cells

We have investigated the effect of Alendronate and Pamidronate, two bisphosphonates widely employed for the treatment of pathologies related to bone loss, on the setting properties and in vitro bioactivity of a calcium phosphate bone cement. The cement composition includes α-tricalcium phosphate (α-TCP) (90 wt%), nanocrystalline hydroxyapatite (5 wt%) and CaHPO 4 · 2H 2O (5 wt%). Disodium Alendronate and disodium Pamidronate were added to the liquid phase (bidistilled water) at two different concentrations: 0.4 and 1 mM (AL0.4, AL1.0, PAM0.4, PAM1.0). Both the initial and the final setting times of the bisphosphonate-containing cements increase with respect to the control cement. X-ray diffraction analysis, mechanical tests, and SEM investigations were carried out on the cements after different times of soaking in physiological solution. The rate of transformation of α-TCP into calcium deficient hydroxyapatite, as well as the microstructure of the cements, is not affected by the presence of Alendronate and Pamidronate. At variance, the bisphosphonates provoke a modest worsening of the mechanical properties. MG63 osteoblasts grown on the cements show a normal morphology and biological tests demonstrate very good rate of proliferation and viability in every experimental time. In particular, both Alendronate and Pamidronate promote osteoblast proliferation and differentiation, whereas they inhibit osteoclastogenesis and osteoclast function.