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Introduction: We previously showed that a 3-week oral metformin (MET) treatment enhances the osteogenic potential of bone marrow stromal cells (BMSCs) and improves several bone histomorphometric parameters in Wistar rats with metabolic syndrome (MetS). However, the skeletal effects of extended periods of MET need to be completely elucidated. Hence, in this study, the impact of a prolonged (3-month) MET treatment was investigated on bone architecture, histomorphometric and biomechanics variables, and osteogenic potential of BMSCs in Wistar rats with or without MetS., Materials and Methods: Young male Wistar rats (n=36) were randomized into four groups (n=9) that received either 20% fructose ( F ), MET ( MET ), F plus MET treatments ( FMET ), or drinking water alone ( Veh ). Rats were euthanized, blood was collected, and bones were dissected and processed for peripheral quantitative computed tomography (pQCT) analysis, static and dynamic histomorphometry, and bone biomechanics. In addition, BMSCs were isolated to determine their osteogenic potential., Results: MET affected trabecular and cortical bone, altering bone architecture and biomechanics. Furthermore, MET increased the pro-resorptive profile of BMSCs. In addition, fructose-induced MetS practically did not affect the the structural or mechanical variables of the skeleton., Conclusion: A 3-month treatment with MET (with or without MetS) affects bone architecture and biomechanical variables in Wistar rats., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Aims: Our objective was to study the vascular smooth muscle cells (VSMC) osteoblastic transdifferentiation in AGE exposed cells or those from diabetic animals, and its response to metformin treatment., Methods: VSMC were obtained from non-diabetic rats, grown with or without AGE; while VSMC of in vivo-ex vivo studies were obtained from non-diabetic control animals (C), diabetic (D), C treated with metformin (M) and D treated with metformin (D-M). We studied the osteoblastic differentiation by evaluating alkaline phosphatase (ALP), type I collagen (Col) and mineral deposit., Results: In vitro, AGE increased proliferation, migration, and osteoblastic differentiation of VSMC. Metformin cotreatment prevented the AGE induced proliferation and migration. Both AGE and metformin stimulated the expression of ALP and Col. AGE induced mineralization was prevented by metformin. VSMC from D expressed a higher production of Col and ALP. Those from D-M showed an ALP increase vs C and M, and a partial decrease vs D. Cultured in osteogenic medium, ALP, Col and mineralization increased in D vs C, remained unchanged in M, and were prevented in D-M animals., Conclusion: Both AGE and DM favor VSMC differentiation towards the osteogenic phenotype and this effect can be prevented by metformin., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The present study shows a novel copolymer synthesis, its application in the membrane design and the physicochemical and biological characterization of the biomaterial obtained. Terpolymer starting diisopropyl fumarate (F), vinyl benzoate (V) and 2-hydroxyethyl methacrylate (H) was prepared by thermal radical polymerization. This polymer (FVH) was obtained in several monomer ratios and characterized by spectroscopic and chromatographic methods (FTIR, 1 H-NMR and SEC). The best relationship of F:V:H was 5:4:1, which allows efficient interaction with chitosan through cross-linking with borax to achieve scaffolds for potential biomedical applications. The membranes were obtained by solvent casting and analyzed by scanning electron microscopy (SEM), swelling behavior and mechanical properties. In addition, we studied the possible cytotoxicity and biocompatibility of these materials using a murine macrophage-like cell line (RAW 264.7) and bone marrow mesenchymal progenitor cells (BMPC), respectively, taking into account their intended applications. The results of this study show that the terpolymer obtained and its combination with a natural polymer is a very interesting strategy to obtain a biomaterial with possible applications in regenerative medicine and this could be extended to other structurally related systems., (© 2021 Wiley Periodicals LLC.)

Recent studies have shown the relevance of growing mesenchymal stem cells (MSCs) in three-dimensional environments with respect to the monolayer cell culture on an adherent substrate. In this sense, macroporous scaffolds and hydrogels have been used as three-dimensional (3D) supports. In this work, we explored the culture of MSCs in a 3D environment created by microspheres, prepared with a fumarate-vinyl acetate copolymer and chitosan. In this system, the environment that the cells feel has similarities to that found by the cells encapsulated in a hydrogel, but the cells have the ability to reorganize their environment since the microspheres are mobile. We evaluated their biocompatibility in vitro using RAW 264.7 macrophages and bone marrow mesenchymal stem cells (BMSCs). The results with RAW 264.7 cells showed good cell viability, without evident signs of cytotoxicity. BMSCs not only proliferate, but also rearrange to grow in clusters, thus highlighting the advantages of microspheres as 3D environments., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The objective of this study was to test a regenerative medicine strategy for the regeneration of articular cartilage. This approach combines microfracture of the subchondral bone with the implant at the site of the cartilage defect of a supporting biomaterial in the form of microspheres aimed at creating an adequate biomechanical environment for the differentiation of the mesenchymal stem cells that migrate from the bone marrow. The possible inflammatory response to these biomaterials was previously studied by means of the culture of RAW264.7 macrophages. The microspheres were implanted in a 3 mm-diameter defect in the trochlea of the femoral condyle of New Zealand rabbits, covering them with a poly(l-lactic acid) (PLLA) membrane manufactured by electrospinning. Experimental groups included a group where exclusively PLLA microspheres were implanted, another group where a mixture of 50/50 microspheres of PLLA (hydrophobic and rigid) and others of chitosan (a hydrogel) were used, and a third group used as a control where no material was used and only the membrane was covering the defect. The histological characteristics of the regenerated tissue have been evaluated 3 months after the operation. We found that during the regeneration process the microspheres, and the membrane covering them, are displaced by the neoformed tissue in the regeneration space toward the subchondral bone region, leaving room for the formation of a tissue with the characteristics of hyaline cartilage., (© 2019 Wiley Periodicals, Inc.)

We developed a composite hydrogel based on chitosan and carboxymethyl cellulose with nanometric hydroxyapatite (nHA) as filler (ranging from 0.5 to 5%), by ultrasonic methodology to be used for bone regeneration. The 3D porous-structure of the biocomposite scaffolds were confirmed by Scanning Electron Microscopy and Microtomography analysis. Infrared analysis did not show specific interactions between the organic components of the composite and nHA in the scaffold. The hydrogel properties of the matrices were studied by swelling and mechanical tests, indicating that the scaffold presented a good mechanical behavior. The degradation test demonstrated that the material is slowly degraded, while the addition of nHA slightly influences the degradation of the scaffolds. Biocompatibility studies carried out with bone marrow mesenchymal progenitor cells (BMPC) showed that cell proliferation and alkaline phosphatase activity were increased depending on the matrix nHA content. On the other hand, no cytotoxic effect was observed when RAW264.7 cells were seeded on the scaffolds. Altogether, our results allow us to conclude that these nanobiocomposites are promising candidates to induce bone tissue regeneration., (© 2020 Wiley Periodicals, Inc.)

This article presents the preparation of matrices from two new families of fumaric copolymers and the effect of structural differences on their physicochemical and biological behavior. Diisopropyl fumarate (DIPF) and poly(ethylene glycol) methyl ether methacrylate (OEGMA) or N-isopropylacrylamide (NIPAM) were copolymerized by conventional radical and RAFT polymerization to obtain lineal or start architectures, respectively. These copolymers were characterized by spectroscopic (FTIR and 1 H-NMR) and chromatographic methods. The thermal stability was studied by thermal gravimetric analysis, showing some differences in relation to the architecture and chemical nature of copolymers. SEM morphological analysis demonstrated that the surface of the matrices derived from OEGMA exhibited an irregular and rough surface, while DIPF-NIPAM copolymers presented smooth surface with nearly no significant porosity. The matrix obtained of both kinds of copolymers presented no cytotoxicity as it was evaluated using a model of macrophages on culture. Moreover, cell growth was better on the surfaces of the DIPF-NIPAM matrices, suggesting a good interaction with this matrix and its potential application as matrices for tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 195-203, 2019., (© 2018 Wiley Periodicals, Inc.)

Long-term diabetes mellitus can induce osteopenia and osteoporosis, an increase in the incidence of low-stress fractures, and/or delayed fracture healing. Strontium ranelate (SrR) is a dual-action anti-osteoporotic agent whose use in individuals with diabetic osteopathy has not been adequately evaluated. In this study, we studied the effects of an oral treatment with SrR and/or experimental diabetes on bone composition and biomechanics. Young male Wistar rats (half non-diabetic, half with streptozotocin/nicotinamide-induced diabetes) were either untreated or orally administered 625 mg/kg/day of SrR for 6 weeks. After sacrifice, femora from all animals were evaluated by a multi-scale approach (X-ray diffraction, Fourier transform infrared spectroscopy, inductively coupled plasma optical-emission spectrometry, static histomorphometry, pQCT, and mechanical testing) to determine chemical, crystalline, and biomechanical properties. Untreated diabetic animals (versus untreated non-diabetic) showed a decrease in femoral mineral carbonate content, in cortical thickness and BMC, in trabecular osteocyte density, in maximum load supported at rupture and at yield point, and in overall toughness at mid-shaft. Treatment of diabetic animals with SrR further affected several parameters of bone (some already impaired by diabetes): crystallinity index (indicating less mature apatite crystals); trabecular area, BMC, and vBMD; maximum load at yield point; and structural elastic rigidity. However, SrR was also able to prevent the diabetes-induced decreases in trabecular osteocyte density (completely) and in bone ultimate strength at rupture (partially). Our results indicate that SrR treatment can partially but significantly prevent some bone structural mechanical properties as previously affected by diabetes, but not others (which may even be worsened).

Advanced glycation end products (AGE) have been demonstrated to induce the osteogenic trans-differentiation of vascular smooth muscle cells (VSMC). Strontium ranelate (SR) is an anti-osteoporotic agent that has both anti-catabolic and anabolic actions on bone tissue. However, in the last years SR has been associated with an increase of cardiovascular risk. We hypothesize that SR can increase the osteoblastic trans-differentiation of VSMC and the induction of extracellular calcifications, an effect that could be potentiated in the presence of AGE and inhibited by simultaneous administration of vitamin D. The present results of our in vitro experiments demonstrate that AGE and SR alone or in combination, stimulate L-type calcium channels, causing an increase in reactive oxygen species and activation of both ERK and NFkB, with the final effect of promoting the osteogenic shift of VSMC. Importantly, these in vitro effects of AGE and/or SR can be prevented by co-incubation with vitamin D., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Natural and synthetic cross-linked polymers allow the improvement of cytocompatibility and mechanical properties of the individual polymers. In osteochondral lesions of big size it will be required the use of scaffolds to repair the lesion. In this work a borax cross-linked scaffold based on fumarate-vinyl acetate copolymer and chitosan directed to osteochondrondral tissue engineering is developed. The cross-linked scaffolds and physical blends of the polymers are analyzed in based on their morphology, glass transition temperature, and mechanical properties. In addition, the stability, degradation behavior, and the swelling kinetics are studied. The results demonstrate that the borax cross-linked scaffold exhibits hydrogel behavior with appropriated mechanical properties for bone and cartilage tissue regeneration. Bone marrow progenitor cells and primary chondrocytes are used to demonstrate its osteo- and chondrogenic properties, respectively, assessing the osteo- and chondroblastic growth and maturation, without evident signs of cytotoxicity as it is evaluated in an in vitro system., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: Deleterious effects of metabolic syndrome (MS) on bone are still controversial. In this study we evaluated the effects of a fructose-induced MS, and/or an oral treatment with metformin on the osteogenic potential of bone marrow mesenchymal stromal cells (MSC), as well as on bone formation and architecture., Methods: 32 male 8week-old Wistar rats were assigned to four groups: control (C), control plus oral metformin (CM), rats receiving 10% fructose in drinking water (FRD), and FRD plus metformin (FRDM). Samples were collected to measure blood parameters, and to perform pQCT analysis and static and dynamic histomorphometry. MSC were isolated to determine their osteogenic potential., Results: Metformin improved blood parameters in FRDM rats. pQCT and static and dynamic histomorphometry showed no significant differences in trabecular and cortical bone parameters among groups. FRD reduced TRAP expression and osteocyte density in trabecular bone and metformin only normalized osteocyte density. FRD decreased the osteogenic potential of MSC and metformin administration could revert some of these parameters., Conclusions: FRD-induced MS shows reduction in MSC osteogenic potential, in osteocyte density and in TRAP activity. Oral metformin treatment was able to prevent trabecular osteocyte loss and the reduction in extracellular mineralization induced by FRD-induced MS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In recent years, there has been an increasing interest in the design of biomaterials for cartilage tissue engineering. This type of materials must meet several requirements. In this study, we apply ultrasound to prepare a compatibilized blend of polyelectrolyte complexes (PEC) based on carboxymethyl cellulose (CMC) and chitosan (CHI), in order to improve stability and mechanical properties through the inter-polymer macroradicals coupling produced by sonochemical reaction. We study the kinetic of the sonochemical degradation of each component in order to optimize the experimental conditions for PEC compatibilization. Scaffolds obtained applying this methodology and scaffolds without ultrasound processing were prepared and their morphology (by scanning electron microscopy), polyelectrolyte interactions (by FTIR), stability and mechanical properties were analyzed. The swelling kinetics was studied and interpreted based on the structural differences between the two kinds of scaffolds. In addition we evaluate the possible in vitro cytotoxicity of the scaffolds using macrophage cells in culture. Our results demonstrate that the ultrasound is a very efficient methodology to compatibilize PEC, exhibiting improved properties compared with the simple mixture of the two polysaccharides. The test with murine macrophage RAW 264.7 cells showed no evince of cytotoxicity, suggesting that PEC biomaterials obtained under ultrasound conditions could be useful in the cartilage tissue engineering field., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Patients with long-term type 1 and type 2 diabetes mellitus (DM) can develop skeletal complications or "diabetic osteopathy". These include osteopenia, osteoporosis and an increased incidence of low-stress fractures. In this context, it is important to evaluate whether current anti-diabetic treatments can secondarily affect bone metabolism. Adenosine monophosphate-activated protein kinase (AMPK) modulates multiple metabolic pathways and acts as a sensor of the cellular energy status; recent evidence suggests a critical role for AMPK in bone homeostasis. In addition, AMPK activation is believed to mediate most clinical effects of the insulin-sensitizer metformin. Over the past decade, several research groups have investigated the effects of metformin on bone, providing a considerable body of pre-clinical (in vitro, ex vivo and in vivo) as well as clinical evidence for an anabolic action of metformin on bone. However, two caveats should be kept in mind when considering metformin treatment for a patient with type 2 DM at risk for diabetic osteopathy. In the first place, metformin should probably not be considered an anti-osteoporotic drug; it is an insulin sensitizer with proven macrovascular benefits that can secondarily improve bone metabolism in the context of DM. Secondly, we are still awaiting the results of randomized placebo-controlled studies in humans that evaluate the effects of metformin on bone metabolism as a primary endpoint.

Bone and cartilage regeneration can be improved by designing a functionalized biomaterial that includes bioactive drugs in a biocompatible and biodegradable scaffold. Based on our previous studies, we designed a vanadium-loaded collagen scaffold for osteochondral tissue engineering. Collagen-vanadium loaded scaffolds were characterized by SEM, FTIR, and permeability studies. Rat bone marrow progenitor cells were plated on collagen or vanadium-loaded membranes to evaluate differences in cell attachment, growth and osteogenic or chondrocytic differentiation. The potential cytotoxicity of the scaffolds was assessed by the MTT assay and by evaluation of morphological changes in cultured RAW 264.7 macrophages. Our results show that loading of VOAsc did not alter the grooved ordered structure of the collagen membrane although it increased membrane permeability, suggesting a more open structure. The VOAsc was released to the media, suggesting diffusion-controlled drug release. Vanadium-loaded membranes proved to be a better substratum than C0 for all evaluated aspects of BMPC biocompatibility (adhesion, growth, and osteoblastic and chondrocytic differentiation). In addition, there was no detectable effect of collagen or vanadium-loaded scaffolds on macrophage viability or cytotoxicity. Based on these findings, we have developed a new ordered collagen scaffold loaded with VOAsc that shows potential for osteochondral tissue engineering.

Bisphosphonates such as alendronate are antiosteoporotic drugs that inhibit the activity of bone-resorbing osteoclasts and secondarily promote osteoblastic function. Diabetes increases bone-matrix-associated advanced glycation end products (AGEs) that impair bone marrow progenitor cell (BMPC) osteogenic potential and decrease bone quality. Here we investigated the in vitro effect of alendronate and/or AGEs on the osteoblastogenic, adipogenic, and chondrogenic potential of BMPC isolated from nondiabetic untreated rats. We also evaluated the in vivo effect of alendronate (administered orally to rats with insulin-deficient Diabetes) on long-bone microarchitecture and BMPC multilineage potential. In vitro , the osteogenesis (Runx2, alkaline phosphatase, type 1 collagen, and mineralization) and chondrogenesis (glycosaminoglycan production) of BMPC were both decreased by AGEs, while coincubation with alendronate prevented these effects. The adipogenesis of BMPC (PPAR γ , intracellular triglycerides, and lipase) was increased by AGEs, and this was prevented by coincubation with alendronate. In vivo , experimental Diabetes (a) decreased femoral trabecular bone area, osteocyte density, and osteoclastic TRAP activity; (b) increased bone marrow adiposity; and (c) deregulated BMPC phenotypic potential (increasing adipogenesis and decreasing osteogenesis and chondrogenesis). Orally administered alendronate prevented all these Diabetes-induced effects on bone. Thus, alendronate could improve bone alterations in diabetic rats by preventing the antiosteogenic, antichondrogenic, and proadipocytic effects of AGEs on BMPC.

Strontium ranelate (SR) is an orally administered and bone-targeting anti-osteoporotic agent that increases osteoblast-mediated bone formation while decreasing osteoclastic bone resorption, and thus reduces the risk of vertebral and femoral bone fractures in postmenopausal women with osteoporosis. Osteoblastic alkaline phosphatase (ALP) is a key enzyme involved in the process of bone formation and osteoid mineralization. In this study we investigated the direct effect of strontium (SR and SrCl2) on the activity of ALP obtained from UMR106 osteosarcoma cells, as well as its possible interactions with the divalent cations Zn(2+) and Mg(2+). In the presence of Mg(2+), both SR and SrCl2 (0.05-0.5 mM) significantly increased ALP activity (15-66 % above basal), and this was dose-dependent in the case of SR. The stimulatory effect of strontium disappeared in the absence of Mg(2+). The cofactor Zn(2+) also increased ALP activity (an effect that reached a plateau at 2 mM), and co-incubation of 2 mM Zn(2+) with 0.05-0.5 mM SR showed an additive effect on ALP activity stimulation. SR induced a dose-dependent decrease in the Km of ALP (and thus an increase in affinity for its substrate) with a maximal effect at 0.1 mM. Co-incubation with 2 mM Zn(2+) further decreased Km in all cases. These direct effects of SR on osteoblastic ALP activity could be indicating an alternative mechanism by which this compound may regulate bone matrix mineralization.

Objective: The aims of this study were: first, to evaluate the possible effects of a fructose rich diet (FRD)-induced metabolic syndrome (MS) on different aspects of long bone histomorphometry in young male rats; second, to investigate the effects of this diet on bone tissue regeneration; and third, to correlate these morphometric alterations with changes in the osteogenic/adipogenic potential and expression of specific transcription factors, of marrow stromal cells (MSC) isolated from rats with fructose-induced MS., Materials/methods: MS was induced in rats by treatment with a FRD for 28 days. Halfway through treatment, a parietal wound was made and bone healing was evaluated 14 days later. After treatments, histomorphometric analysis was performed in dissected femoral and parietal bones. MSC were isolated from the femora of control or fructose-treated rats and differentiated either to osteoblasts (evaluated by type 1 collagen, Alkaline phosphatase and extracellular nodule mineralization) or to adipocytes (evaluated by intracellular triglyceride accumulation). Expression of Runx2 and PPARγ was assessed by Western blot., Results: Fructose-induced MS induced deleterious effects on femoral metaphysis microarchitecture and impaired bone regeneration. Fructose treatment decreased the osteogenic potential of MSC and Runx2 expression. In addition, it increased the adipogenic commitment of MSC and PPARγ expression., Conclusions: Fructose-induced MS is associated with deleterious effects on bone microarchitecture and with a decrease in bone repair. These alterations could be due to a deviation in the adipogenic/osteogenic commitment of MSC, probably by modulation of the Runx2/PPARγ ratio., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Advanced glycation endproducts (AGEs) accumulate with age in various tissues, and are further increased in patients with Diabetes mellitus, in which they are believed to contribute to the development and progression of chronic complications that include a decrease in bone quality. Bisphosphonates are anti-osteoporotic drugs that have been used for the treatment of patients with diabetic bone alterations, although with contradictory results. In the present study, we have evaluated the in vitro alterations on osteoblastic morphology by environmental scanning electron microscopy, in actin cytoskeleton and apoptosis induced by AGEs, as well as the modulation of these effects by alendronate (an N-containing bisphosphonate). Our present results provide evidence for disruption induced by AGEs of the osteoblastic actin cytoskeleton (geodesic domes) and significant alterations in cell morphology with a decrease in cell-substratum interactions leading to an increase in apoptosis of osteoblasts and a decrease in osteoblastic proliferation. High concentrations of alendronate (10(-5)M, such as could be expected in an osteoclastic lacuna) further increase osteoblastic morphological and cytoskeletal alterations. However, low doses of alendronate (10(-8)M, compatible with extracellular fluid levels to which an osteoblast could be exposed for most of its life cycle) do not affect cell morphology, and in addition are able to prevent AGEs-induced alterations and consequently apoptosis of osteoblasts., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Aims: Diabetes mellitus is associated with metabolic bone disease and increased low-impact fractures. The insulin-sensitizer metformin possesses in vitro, in vivo and ex vivo osteogenic effects, although this has not been adequately studied in the context of diabetes. We evaluated the effect of insulin-deficient diabetes and/or metformin on bone microarchitecture, on osteogenic potential of bone marrow progenitor cells (BMPC) and possible mechanisms involved., Methods: Partially insulin-deficient diabetes was induced in rats by nicotinamide/streptozotocin-injection, with or without oral metformin treatment. Femoral metaphysis micro-architecture, ex vivo osteogenic potential of BMPC, and BMPC expression of Runx-2, PPARγ and receptor for advanced glycation endproducts (RAGE) were investigated., Results: Histomorphometric analysis of diabetic femoral metaphysis demonstrated a slight decrease in trabecular area and a significant reduction in osteocyte density, growth plate height and TRAP (tartrate-resistant acid phosphatase) activity in the primary spongiosa. BMPC obtained from diabetic animals showed a reduction in Runx-2/PPARγ ratio and in their osteogenic potential, and an increase in RAGE expression. Metformin treatment prevented the diabetes-induced alterations in bone micro-architecture and BMPC osteogenic potential., Conclusion: Partially insulin-deficient diabetes induces deleterious effects on long-bone micro-architecture that are associated with a decrease in BMPC osteogenic potential, which could be mediated by a decrease in their Runx-2/PPARγ ratio and up-regulation of RAGE. These diabetes-induced alterations can be totally or partially prevented by oral administration of metformin., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Accumulation of advanced glycation endproducts (AGEs) in bone tissue occurs in ageing and in Diabetes mellitus, and is partly responsible for the increased risk of low-stress bone fractures observed in these conditions. In this study we evaluated whether the anti-osteoporotic agent strontium ranelate can prevent the deleterious effects of AGEs on bone cells, and possible mechanisms of action involved. Using mouse MC3T3E1 osteoblastic cells in culture we evaluated the effects of 0.1mM strontium ranelate and/or 100 μg/ml AGEs-modified bovine serum albumin (AGEs-BSA) on cell proliferation, osteogenic differentiation and pro-inflammatory cytokine production. We found that AGEs-BSA alone decreased osteoblastic proliferation and differentiation (P<0.01) while increasing IL-1β and TNFα production (P<0.01). On its own, strontium ranelate induced opposite effects: an increase in osteoblast proliferation and differentiation (P<0.01) and a decrease in cytokine secretion (P<0.01). Additionally, strontium ranelate prevented the inhibitory and pro-inflammatory actions of AGEs-BSA on osteoblastic cells (P<0.01). These effects of strontium ranelate were blocked by co-incubation with either the MAPK inhibitor PD98059, or the calcium channel blocker nifedipine. We also evaluated by Western blotting the activation status of ERK (a MAPK) and b-catenin. Activation of both signaling pathways was decreased by AGEs treatment, and this inhibitory effect was prevented if AGEs were co-incubated with strontium ranelate (P<0.01). On its own, strontium ranelate increased both pERK and activated b-catenin levels. In conclusion, this study demonstrates that strontium ranelate can prevent the deleterious in vitro actions of AGEs on osteoblastic cells in culture by mechanisms that involve calcium channel, MAPK and b-catenin activation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objectives: The formation of biofilms on titanium dental implants is one of the main causes of failure of these devices. Streptococci are considered early colonizers that alter local environment favouring growing conditions for other colonizers. Chlorhexidine (CHX) is so far the most effective antimicrobial treatment against a wide variety of Gram-positive and Gram-negative organisms as well as fungi. This study was designed to develop a CHX delivery system appropriate for healing caps and abutments, with suitable drug release rate, effective as antimicrobial agent, and free of cytotoxic effects., Methods: Polybenzyl acrylate (PBA) coatings with and without CHX (Ti/PBA and Ti/PBA-CHX, respectively) and different drug loads (0.35, 0.70, and 1.40%, w/w) were assayed. The cytotoxic effect of CHX released from the different substrates on UMR106 cells was tested by alkaline phosphatase specific activity (ALP), and microscopic evaluation of the cells. Non-cytotoxic drug load (0.35%, w/w) was selected to evaluate the antimicrobial effectiveness of the system using a microbial consortium of Streptococcus species., Results: The kinetic profile of CHX delivered by Ti/PBA-CHX showed an initial fast release rate followed by a monotonic increase of delivered mass over 48 h. The number of attached bacteria decreased in the following order: Ti>Ti/PBA>Ti/PBA-0.35., Conclusions: PBA-0.35 coating is effective to inhibit the adhesion of early colonizers on Ti without any cytotoxic effect on UMR-106 cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Long-term treatment with the insulin-sensitizer rosiglitazone reduces bone mass and increases fracture risk. We have recently shown that orally administered metformin stimulates bone reossification and increases the osteogenic potential of bone marrow progenitor cells (BMPC). In the present study we investigated the effect of a 2-week metformin and/or rosiglitazone treatment on bone repair, trabecular bone microarchitecture and BMPC osteogenic potential, in young male Sprague-Dawley rats. Compared to untreated controls, rosiglitazone monotherapy decreased bone regeneration, femoral metaphysis trabecular area, osteoblastic and osteocytic density, and TRAP activity associated with epiphyseal growth plates. It also decreased the ex vivo osteogenic commitment of BMPC, inducing an increase in PPARγ expression, and a decrease in Runx2/Cbfa1 expression, in AMP-kinase phosphorylation, and in osteoblastic differentiation and mineralization. After monotherapy with metformin, with the exception of PPARγ expression which was blunted, all of the above parameters were significantly increased (compared to untreated controls). Metformin/rosiglitazone co-treatment prevented all the in vivo and ex vivo anti-osteogenic effects of rosiglitazone monotherapy, with a reversion back to control levels of PPARγ, Runx2/Cbfa1 and AMP-kinase phosphorylation of BMPC. In vitro co-incubation of BMPC with metformin and compound C-an inhibitor of AMPK phosphorylation-abrogated the metformin-induced increase in type-1 collagen production, a marker of osteoblastic differentiation. In conclusion, in rodent models metformin not only induces direct osteogenic in vivo and ex vivo actions, but when it is administered orally in combination with rosiglitazone it can prevent several of the adverse effects that this thiazolidenedione shows on bone tissue., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Hydroxyapatite (HAP)-containing poly-ε-caprolactone (PCL)-polydiisopropyl fumarate (PDIPF) composite (Blend) was developed as an alternative for bone tissue engineering. The physicochemical, mechanical and biocompatibility properties of these composites were evaluated using two osteoblast-like cell lines (UMR106 and MC3T3E1) and compared with the blend without HAP and PCL/HAP films. The increment in the elastic modulus and the decrease in the elongation-at-break of Blend-HAP suggest that the mechanical properties of the HAP scaffolds have improved significantly. The addition of HAP to both PCL and Blend significantly improves the cell biocompatibility and osteogenicity of the scaffolds. Evidence for this notion is based in several observations: (a) HAP-polymer increases proliferation of osteoblastic cells; (b) HAP included in the blend increases the ALP expression in UMR106 cells; (c) HAP-Blend increases the type-I collagen production in both cell lines, and d) higher levels of the osteogenic transcription factor Runx-2 were detected when MC3T3E1 osteoblasts were induced to differentiate and mineralize on HAP-polymer scaffolds. In conclusion, a novel biocompatible HAP-Blend composite with uniform dispersion of semi-nano HAP particles and good interphase compatibility has been prepared successfully. The development of HAP-Blend composite, with improved physical, mechanical and osteoinductive properties, may potentially be used in bone tissue-engineering applications., (Copyright © 2011 John Wiley & Sons, Ltd.)

Diabetes mellitus is associated with bone loss. Patients with type 2 diabetes are frequently treated with oral antidiabetic drugs such as sulfonylureas, biguanides, and thiazolidinediones. Rosiglitazone treatment has been shown to increase adipogenesis in bone marrow and to induce bone loss. In this study we evaluated the effect of in vivo and in vitro treatment with metformin on bone marrow progenitor cells (BMPCs), as well as the involvement of AMPK pathway in its effects. The in vitro effect of coincubation with metformin and rosiglitazone on the adipogenic differentiation of BMPCs also was studied. In addition, we evaluated the effect of in vivo metformin treatment on bone regeneration in a model of parietal lesions in nondiabetic and streptozotocin-induced diabetic rats. We found that metformin administration both in vivo and in vitro caused an increase in alkaline phosphatase activity, type I collagen synthesis, osteocalcin expression, and extracellular calcium deposition of BMPCs. Moreover, metformin significantly activated AMPK in undifferentiated BMPCs. In vivo, metformin administration enhanced the expression of osteoblast-specific transcription factor Runx2/Cbfa1 and activation of AMPK in a time-dependent manner. Metformin treatment also stimulated bone lesion regeneration in control and diabetic rats. In vitro, metformin partially inhibited the adipogenic actions of rosiglitazone on BMPCs. In conclusion, our results indicate that metformin causes an osteogenic effect both in vivo and in vitro, possibly mediated by Runx2/Cbfa1 and AMPK activation, suggesting a possible action of metformin in a shift toward the osteoblastic differentiation of BMPCs., (Copyright 2010 American Society for Bone and Mineral Research.)

There is considerable interest in the design of polymeric biomaterials that can be used for the repair of bone defects. In this study, we used ultrasound to prepare a compatibilized blend of poly(epsilon-caprolactone) (PCL) and poly(diisopropyl fumarate) (PDIPF). The formation of post-sonication inter-polymer coupling products was verified by SEC analysis of a blend with azo-labeled PDIPF. We also analyzed the physicochemical and mechanical properties of the compatibilized blend. When compared to PCL alone, the PCL/PDIPF blend showed no difference in its resistance as evaluated by the elastic modulus, although it did show a 50% decrease in ultimate tensile stress (P < 0.05) and an 84% decrease in elongation-at-break (P < 0.05). However, the mechanical properties of this blend were comparable to those of trabecular bone. We next evaluated biocompatibility of the PCL/PDIPF blend, and of homo-polymeric PCL and PDIPF films for comparison, with UMR106 and MC3T3E1 osteoblastic cells. Osteoblasts plated on the compatibilized blend adhered and proliferated more than on either homo-polymer, showed a greater number of cellular processes with a better organized actin cytoskeleton and expressed more type-I collagen and mineral, both markers of osteoblast phenotype. These results support the hypothesis that this new compatibilized blend could be useful in future applications for bone regeneration.

Patients with long-standing Diabetes mellitus can develop osteopenia and osteoporosis. We have previously shown that advanced glycation endproducts reduce the bone-forming activity of osteoblasts. Bisphosphonates are used for the treatment of various bone disorders, since they reduce osteoclastic function and survival, and stimulate osteoblastic bone-forming capacity. In this work we have investigated whether bisphosphonates are able to revert advanced glycation endproducts-induced deleterious effects in osteoblasts. MC3T3E1 and UMR106 osteoblastic cells were incubated with control or advanced glycation endproducts-modified bovine serum albumin, in the presence or absence of different doses of the bisphosphonates Alendronate, Pamidronate or Zoledronate. After 24-72 h of culture, we evaluated their effects on cell proliferation and apoptosis, type-1 collagen production, alkaline and neutral phosphatase activity, and intracellular reactive oxygen species production. Advanced glycation endproducts significantly decreased osteoblast proliferation, alkaline phosphatase activity and type 1 collagen production, while increasing osteoblastic apoptosis and reactive oxygen species production. These effects were completely reverted by low doses (10(-8) M) of bisphosphonates. High doses of bisphosphonates (10(-4)-10(-5) M) were toxic for osteoblasts. Nifedipine (L-type calcium channel blocker) did not affect the advanced glycation endproducts-induced decrease in osteoblastic proliferation, although it blocked the reversion of this effect by 10(-8) M Alendronate. Both advanced glycation endproducts and Alendronate inhibited the activity of intracellular neutral phosphatases. In conclusion, we show that bisphosphonates revert the deleterious actions of advanced glycation endproducts on osteoblastic cells, and that these effects of bisphosphonates depend on: (a) Ca(2+) influx through L-type voltage-sensitive channels, and (b) blockage of advanced glycation endproducts-induced reactive oxygen species generation.

Advanced glycation endproducts (AGEs) are implicated in the complications of diabetes and ageing, affecting several tissues, including bone. Metformin, an insulin-sensitizer drug, reduces the risk of life-threatening macrovascular complications. We have evaluated the hypothesis that metformin can abrogate AGE-induced deleterious effects in osteoblastic cells in culture. In two osteoblast-like cell lines (UMR106 and MC3T3E1), AGE-modified albumin induced cell death, caspase-3 activity, altered intracellular oxidative stress and inhibited alkaline phosphatase activity. Metformin-treatment of osteoblastic cells prevented these AGE-induced alterations. We also assessed the expression of AGE receptors as a possible mechanism by which metformin could modulate the action of AGEs. AGEs-treatment of osteoblast-like cells enhanced RAGE protein expression, and this up-regulation was prevented in the presence of metformin. Although the precise mechanisms involved in metformin signaling are still elusive, our data implicate the AGE-RAGE interaction in the modulation of growth and differentiation of osteoblastic cells.

Vanadium is a trace element widely distributed in the environment. In vertebrates it is mainly stored in bone tissue. The unique cellular environment in the bone and the variety of interactions that mediate cancer metastasis determine that certain types of cancer, such as breast and prostate cancer, preferentially metastize in the skeleton. Since this effect usually signifies serious morbidity and grave prognosis there is an increasing interest in the development of new treatments for this pathology. The present work shows that vanadium complexes can inhibit some parameters related to cancer metastasis such as cell adhesion, migration and clonogenicity. We have also investigated the role of protein kinase A in these processes.

Biodegradable and biocompatible polymeric scaffolds have been recently introduced for tissue regeneration purpose. In the present study we aimed to develop polymeric-based scaffolds for bone regeneration. Two polyesters, poly-beta-propiolactone (PBPL), poly-epsilon-caprolactone (PCPL) and two polyfumarates, polydiisopropyl fumarate (PDIPF), polydicyclohexyl fumarate (PDCF) were chosen to prepare films which can support osteoblastic growth. Scanning electron microscopy and water contact angle were used to characterize the matrices. Biodegradation studies were performed both in PBS buffer and using an in vitro macrophage degradation assay. Mouse calvaria-derived MC3T3E1 cells and UMR106 rat osteosarcoma cell lines were used to perform biocompatibility and cytotoxicity studies. The polyesters, the most hydrophilic polymers studied, showed a rougher and more porous surfaces than the polyfumarates. Under acellular conditions, only PBPL was degraded by hydrolytic mechanisms. However, macrophages performed an active degradation of all polymeric films. Osteoblasts developed well-defined actin fibres without evidence of cytotoxicity when growing on the films. The number of UMR106 osteoblasts that adhered to the PBPL-based film was higher than that of the cells attached to the PECL and polyfumarates (PDIPF and PDCF) matrices. Both UMR106 and MC3T3E1 osteoblastic lines showed protein levels comparable to control conditions, demonstrating that they grew well on all surfaces. However, UMR106 cells showed a significant increase in proliferation on polyester-derived scaffolds (PBPL and PECL). The alkaline phosphatase activity of UMR106, an osteoblastic marker, was significantly higher than that of control plastic dishes. MC3T3E1 cells expressed similar levels of this differentiation marker in all polymeric matrices. We found similar collagen protein content after 48 h culture of UMR106 cells on all surfaces. However, important differences were evident in the MC3T3E1 line. In conclusion, the synthetic polymeric-based scaffold we have developed and studied supports adhesion, growth and differentiation of two osteoblastic cell lines, suggesting that they could be useful in bone tissue regeneration., (Copyright (c) 2008 John Wiley & Sons, Ltd.)

Advanced glycation end products (AGEs) have been proposed as the pathological mechanisms underlying diabetic chronic complications. They may also play a role in the pathogenesis of diabetic osteopenia, although their mechanisms of action remain unclear. We investigated the protein (immunofluorescence) and gene expression (realtime RT-PCR) of two receptors for AGEs, RAGE and galectin-3, as well as their regulation by AGEs, and the apoptotic effect on osteoblast-like cells (UMR106 and MC3T3E1) in culture. AGEs up-regulated the expression of RAGE and galectin-3 in both cells lines. These effects were accompanied by an increase in the corresponding mRNA in the non-tumoral MC3T3E1 but not in the osteosarcoma UMR106 cells. Finally, we demonstrated that a 24 h exposure to AGEs induced apoptosis in both cell lines. Thus, AGEs-receptors may play important roles in the bone alterations described in aging and diabetic patients.

Bisphosphonates are nonhydrolysable pyrophosphate analogues that prevent bone loss in several types of cancer. However, the mechanisms of anticancer action of bisphosphonates are not completely known. We have previously shown that nitrogen-containing bisphosphonates directly inhibit alkaline phosphatase of UMR106 rat osteosarcoma cells. In this study, we evaluated the effects of alendronate on the migration of UMR106 osteosarcoma using a model of multicellular cell spheroids, as well as the alendronate effect on neutral phosphatases. Alendronate significantly inhibited the migration of osteoblasts in a dose-dependent manner (10(-6)-10(-4) M). This effect was also dependent on calcium availability. The spheroid morphology and distribution of actin fibers were also affected by alendronate treatment. Alendronate dose-dependently inhibited neutral phosphatase activity in cell-free osteoblastic extracts as well as in osteoblasts in culture. Our results show that alendronate inhibits cell migration through mechanisms dependent on calcium, and that seem to involve inhibition of phosphotyrosine-neutral-phosphatases and disassembly of actin stress fibers.

An association has been previously established between uncompensated diabetes mellitus and the loss of bone mineral density and/or quality. In this study, we evaluated the effects of metformin on the growth and differentiation of osteoblasts in culture. Treatment of two osteoblast-like cells (UMR106 and MC3T3E1) with metformin (25-500 microM) for 24 h led to a dose-dependent increase of cell proliferation. Metformin also promoted osteoblastic differentiation: it increased type-I collagen production in both cell lines and stimulated alkaline phosphatase activity in MC3T3E1 osteoblasts. In addition, metformin markedly increased the formation of nodules of mineralization in 3-week MC3T3E1 cultures. Metformin induced activation and redistribution of phosphorylated extracellular signal-regulated kinase (P-ERK) in a transient manner, and dose-dependently stimulated the expression of endothelial and inducible nitric oxide synthases (e/iNOS). These results show for the first time a direct osteogenic effect of metformin on osteoblasts in culture, which could be mediated by activation/redistribution of ERK-1/2 and induction of e/iNOS.

We have previously shown that different vanadium(IV) complexes regulate osteoblastic growth. Since vanadium compounds are accumulated in vivo in bone, they may affect bone turnover. The development of vanadium complexes with different ligands could be an alternative strategy of use in skeletal tissue engineering. In this study, we have investigated the osteogenic properties of a vanadyl(IV)-ascorbate (VOAsc) complex, as well as its possible mechanisms of action, on two osteoblastic cell lines in culture. VOAsc (2.5-25 microM) significantly stimulated osteoblastic proliferation (113-125% basal, p<0.01) in UMR106 cells, but not in the MC3T3E1 cell line. VOAsc (5-100 micrioM) dose-dependently stimulated type-I collagen production (107-156% basal) in osteoblasts. After 3 weeks of culture, 5-25 microM VOAsc increased the formation of nodules of mineralization in MC3T3E1 cells (7.7-20-fold control, p<0.001). VOAsc (50-100 microM) significantly stimulated apoptosis in both cell lines (170-230% basal, p<0.02-0.002), but did not affect reactive oxygen species production. The complex inhibited alkaline and neutral phosphatases from osteoblastic extracts with semi-maximal effect at 10 microM doses. VOAsc induced the activation and redistribution of P-ERK in a time- and dose-dependent manner. Inhibitors of the mitogen activated protein kinases (MAPK) pathway (PD98059 and UO126) partially blocked the VOAsc-enhanced osteoblastic proliferation and collagen production. In addition, wortmanin, a PI-3-K inhibitor and type-L channel blocker nifedipine also partially abrogated these effects of VOAsc on osteoblasts. Our in vitro results suggest that this vanadyl(IV)-ascorbate complex could be a useful pharmacological tool for bone tissue regeneration.

Bone homeostasis is the result of a tight balance between bone resorption and bone formation where macrophage activation is believed to contribute to bone resorption. We have previously shown that a vanadyl(IV)-aspirin complex (VOAspi) regulates cell proliferation and differentiation of osteoblasts in culture. In this study, we assessed VOAspi and VO effects and their possible mechanism of action on a mouse macrophage cell line RAW 264.7. Both vanadium compounds inhibited cell proliferation in a dose-dependent manner. Nifedipine completely reversed the VOAspi-induced macrophage cytotoxicity, while it could not block the effect of VO. VOAspi also stimulated nitric oxide (NO) production, the oxidation of dihydrorhodamine 123 (DHR-123) and enhanced the expression of both constitutive and inducible isoforms of nitric oxide syntases (NOS). All these effects were abolished by nifedipine. Altogether our finding give evidence that VOAspi-induced macrophage cytotoxicity is dependent on L-type calcium channel and the generation of NO though the induction of eNOS and iNOS. Contrary, the parent compound VO exerted a cytotoxic effect by mechanisms independent of a calcium entry and the NO/NOS activation.

Bisphosphonates (BPs) are drugs widely used in the treatment of various bone diseases. BPs localize to bone mineral, and their concentration in resorption lacunae could reach almost millimolar levels. Bone alkaline phosphatase (ALP) is a membrane-bound exoenzyme that has been implicated in bone formation and mineralization. In this study, we investigated the possible direct effect of three N-containing BPs (alendronate, pamidronate, and zoledronate) on the specific activity of bone ALP obtained from an extract of UMR106 rat osteosarcoma cells. Enzymatic activity was measured by spectrophotometric detection of p-nitrophenol product and by in situ visualization of ALP bands after an electrophoresis on cellulose acetate gels. Because ALP is a metalloprotein that contains Zn2+ and Mg2+, both of which are necessary for catalytic function, we also evaluated the participation of these divalent cations in the possible effect of BPs on enzymatic activity. All BPs tested were found to dose-dependently inhibit spectrophotometrically measured ALP activity (93-42% of basal) at concentrations of BPs between 10-5 M and 10-4 M, the order of potency being zoledronate approximately equals alendronate > pamidronate. However, coincubation with excess Zn2+ or Mg2+ completely abolished this inhibitory effect. Electrophoretic analysis rendered very similar results: namely a decrease in the enzymatic activity of the bone-ALP band by BPs and a reversion of this inhibition by divalent cations. This study shows that N-containing BPs directly inhibit bone-ALP activity, in a concentration range to which this exoenzyme is probably exposed in vivo. In addition, this inhibitory effect is most possibly the result of the chelation of Zn2+ and Mg2+ ions by BPs.

The accumulation of irreversible advanced glycation endproducts (AGEs) on long-lived proteins, and the interaction of AGEs with cellular receptors such as AGE-R3/galectin-3 and RAGE, are considered to be key events in the development of long-term complications of diabetes mellitus, Alzheimer's disease, uremia and ageing. The aim of this study was to investigate the expression and sub-cellular distribution of galectin-3, as well as its possible modulation by AGEs, in MC3T3E1 mouse calvaria-derived osteoblasts and in UMR 106 rat osteosarcoma cells. Both osteoblastic lines were cultured either with control bovine serum albumin (BSA) or with AGEs-BSA for 48 h. Cells were evaluated for galectin-3 expression by fixing and immunofluorescent microscopic analysis; or Western blot analysis of whole cell extracts, sub-cellular fractions and culture media. Both cell lines express 30 kDa (monomeric) galectin-3, although expression was about 15-fold lower in the UMR106 osteosarcoma cells. Dimeric (70 kDa) galectin-3 was additionally observed in the UMR106 cells. Immunofluorescent analysis of galectin-3 distribution showed a diffuse cytoplasmic and strong nuclear pattern in MC3T3E1 osteoblasts, and a patchy cytoplasmic pattern in UMR106 cells. Western blot analysis for both cell lines showed that galectin-3 was mainly found in the cytoplasm and in minor amounts in the microsomal fraction, while considerable amounts were secreted into the culture media. Exposure to 100-200 microg/mL AGEs-BSA increased the cellular content of 30 kDa galectin-3 (20-25% for MC3T3E1 and 35-70% for UMR106 versus control BSA, p < 0.05), and decreased the culture media levels of galectin-3 (10-20% for MC3T3E1 and for UMR106 versus control BSA, p < 0.05). These results confirm the expression of galectin-3 in osteoblastic cells, and suggest different levels and sub-cellular distribution of this protein in transformed versus non-transformed osteoblasts. Osteoblastic exposure to AGEs alters their expression and secretion of galectin-3, which could have significant consequences on osteoblast metabolism and thus on bone turnover.

Ions released from metallic dental materials used in orthodontic appliances could induce undesirable effects on cells and tissues. This study evaluates the biocompatibility of two of the most labile components of metallic dental alloys on osteoblastlike cells. The influence of protein and ions on metal dissolution properties is also investigated using different electrolyte solutions. Morphological alterations, cell growth, and differentiation of osteoblasts were assessed after exposure to pure metals (Ag, Cu, Pd, Au) and Ni-Ti alloy and correlated with the kinetics of elements released into the culture media. Results showed that Cu and Ag were the most cytotoxic elements and the other metals were biocompatible with the osteoblasts. The parameters of biocompatibility were correlated with the levels of ions detected into the culture media. Metal ions induced cell death through early mitosis arrest, apoptotic phenomena, and necrotic processes. Voltammograms showed that anions and proteins interfered in the corrosion process. Fetal bovine serum (FBS) strongly affected the electrochemical process, decreasing the oxidation rate of the metals. In conclusion, copper and silver ions showed a time-dependent low biocompatibility, which correlated with the concentration of released ions. The dissolution of the metallic materials was dependent on the composition of the simulated biological media.

The adhesion of osteoblasts to bone extracellular matrix, of which type-I collagen constitutes >85%, can modulate diverse aspects of their physiology such as growth, differentiation and mineralisation. In this study we examined the adhesion of UMR106 rat osteoblast-like cells either to a control (Col) or advanced-glycation-endproduct-modified (AGEs-Col) type I collagen matrix. We investigated the possible role of different integrin receptors in osteoblastic adhesion, by co-incubating these cells either with beta-peptide (conserved sequence 113-125 of the beta subunit of integrins) or with two other peptides, RGD (Arg-Gly-Asp) and DGEA (Asp-Gly-Glu-Ala), which are recognition sequences for the alpha-subunits of alpha(1,5)beta(1) and alpha(2)beta(1) integrins. Collagen glycation inhibited the adhesion of UMR106 osteoblasts to the matrix (40% reduction versus Col, P > 0.001). beta-Peptide showed a dose- and glycation-dependent inhibitory effect on adhesion, and at a concentration of 100 microM decreased the attachment of UMR106 cells to both matrices (42% to Col, P<0.001and 25% to AGEs-Col, P<0.01). The synthetic peptides RGD (1mM) and DGEA (5mM) inhibited the attachment of UMR106 cells to Col (30 and 20%, P > 0.01 and P< 0.001, respectively), but not to AGEs-Col. beta-Peptide induced an increase in UMR106 cell clumping and a decrease in cellular spreading, while DGEA increased spreading with cellular extensions in multiple directions. These results indicate that both alpha and beta integrin subunits participate in osteoblastic attachment to type-I collagen, probably through the alpha(1,5)beta(1) and alpha(2)beta(1) integrins. AGEs-modification of type-I collagen impairs the integrin-mediated adhesion of osteoblastic cells to the matrix, and could thus contribute to the pathogenesis of diabetic osteopenia.

Background: Vanadium derivatives have been reported to display different biological effects, and in particular antineoplastic activity has been demonstrated in both in vivo and in vitro studies. PURPOSE. To study the effect of two new organic vanadyl(IV) complexes (one with glucose, GluVO, and the other with naproxen, NapVO) in osteosarcoma cells., Methods: UMR106 osteosarcoma cells and, for comparison, nontransformed MC3T3E1 osteoblasts were used. Proliferation and differentiation were assessed using the crystal violet assay and ALP specific activity, respectively. Morphological alterations were assessed by light microscopy. Lipid peroxidation was evaluated in terms of production of thiobarbituric acid-reactive substances (TBARS) and apoptosis was measured using annexin V. Extracellular regulated kinase (Erk) activation was investigated by Western blotting., Results: Vanadium complexes caused morphological alterations and they strongly inhibited UMR106 cell proliferation and differentiation. In contrast, in MC3T3E1 cells, these vanadium derivatives had a relatively weak action. In UMR106 tumoral cells there was a significant increase in TBARS production. Both vanadium complexes induced apoptosis and activation of Erk. PD98059, an inhibitor of Erk phosphorylation, did not block the vanadium-induced antitumoral action. However, the antioxidants vitamins C and E abrogated the apoptosis and TBARS production induced by the vanadium complexes., Conclusions: GluVO and NapVO exerted an antitumoral effect in UM106 osteosarcoma cells. They inhibited cell proliferation and differentiation. While the Erk cascade seems not to be directly related to the bioactivity of these vanadium derivatives, the action of both vanadium complexes with organic ligands may be mediated by apoptosis and oxidative stress.

An increase in the interaction between advanced glycation end-products (AGEs) and their receptor RAGE is believed to contribute to the pathogenesis of chronic complications of Diabetes mellitus, which can include bone alterations such as osteopenia. We have recently found that extracellular AGEs can directly regulate the growth and development of rat osteosarcoma UMR106 cells, and of mouse calvaria-derived MC3T3E1 osteoblasts throughout their successive developmental stages (proliferation, differentiation and mineralisation), possibly by the recognition of AGEs moieties by specific osteoblastic receptors which are present in both cell lines. In the present study we examined the possible expression of RAGE by UMR106 and MC3T3E1 osteoblastic cells, by immunoblot analysis. We also investigated whether short-, medium- or long-term exposure of osteoblasts to extracellular AGEs, could modify their affinity constant and maximal binding for AGEs (by 125I-AGE-BSA binding experiments), their expression of RAGE (by immunoblot analysis) and the activation status of the osteoblastic ERK 1/2 signal transduction mechanism (by immunoblot analysis for ERK and P-ERK). Our results show that both osteoblastic cell lines express readily detectable levels of RAGE. Short-term exposure of phenotypically mature osteoblastic UMR106 cells to AGEs decrease the cellular density of AGE-binding sites while increasing the affinity of these sites for AGEs. This culture condition also dose-dependently increased the expression of RAGE and the activation of ERK. In proliferating MC3T3E1 pre-osteoblasts, 24-72 h exposure to AGEs did not modify expression of RAGE, ERK activation or the cellular density of AGE-binding sites. However, it did change the affinity of these binding sites forAGEs, with both higher- and lower-affinity sites now being apparent. Medium-term ( 1 week) incubation of differentiated MC3T3E1 osteoblasts with AGEs, induced a simultaneous increase in RAGE expression and in the relative amount of P-ERK. Mineralising MC3T3E1 cultures grown for 3 weeks in the presence of extracellular AGEs showed a decrease both in RAGE and P-ERK expression. These results indicate that, in phenotypically mature osteoblastic cells, changes in ERK activation closely follow the AGEs-induced regulation of RAGE expression. Thus, the AGEs-induced biological effects that we have observed previously in osteoblasts, could be mediated by RAGE in the later stages of development, and mediated by other AGE receptors in the earlier pre-osteoblastic stage.

Vanadium compounds show interesting biological and pharmacological properties. Some of them display insulin-mimetic effects and others produce anti-tumor actions. The bioactivity of vanadium is present in inorganic species like the vanadyl(IV) cation or vanadate(V) anion. Nevertheless, the development of new vanadium derivatives with organic ligands which improve the beneficial actions and decrease the toxic effects is of great interest. On the other hand, the mechanisms involved in vanadium bioactivity are still poorly understood. A new vanadium complex of the vanadyl(IV) cation with the disaccharide trehalose (TreVO), Na(6)[VO(Tre)(2)].4H(2)O, here reported, shows interesting insulin-mimetic properties in two osteoblast cell lines, a normal one (MC3T3E1) and a tumoral one (UMR106). The complex affected the proliferation of both cell lines in a different manner. On tumoral cells, TreVO caused a weak stimulation of growth at 5 microM but it inhibited cell proliferation in a dose-response manner between 50 and 100 microM. TreVO significantly inhibited UMR106 differentiation (15-25% of basal) in the range 5-100 microM. On normal osteoblasts, TreVO behaved as a mitogen at 5-25 microM. Different inhibitors of the MAPK pathway blocked this effect. At higher concentrations (75-100 microM), the complex was a weak inhibitor of the MC3T3E1 proliferation. Besides, TreVO enhanced glucose consumption by a mechanism independent of the PI3-kinase activation. In both cell lines, TreVO stimulated the ERK phosphorylation in a dose- and time-dependent manner. Different inhibitors (PD98059, wortmannin, vitamins C and E) partially decreased this effect, which was totally inhibited by their combination. These results suggest that TreVO could be a potential candidate for therapeutic treatments.

A recently synthesized vanadyl(IV) complex with aspirin [VO(aspirin)ClH2O]2, has been thoroughly investigated by physicochemical techniques. In order to support the proposed structure, stoichiometry and the coordination sphere of the vanadium center, some studies such as elemental analysis, electronic (diffuse reflectance) and vibrational (infrared) spectroscopies, magnetic susceptibility, as well as the thermal behavior, were carried out. The bioactivity of the vanadium complex (VOAspi) was evaluated on two osteoblast-like cell lines in culture, being its cytotoxic effects stronger than the vanadyl cation as assessed by morphological changes and lipid peroxidation. These effects may be partially explained through the induction of the expression of Erks (Extracellular signal-regulated kinases) and the inhibition of the PTPases (Phosphotyrosine phosphatases) present in the cellular extracts.

The synthesis and spectral and magnetic characterization of VO(2+) complexes with Ibuprofen (2-(4-isobutylphenyl)propionic acid), Naproxen (6-methoxy-alpha-methyl-2-naphthalene acetic acid) and Tolmetin (1-methyl-5-(4-methylbenzoyl)-1H-pyrrole-2-acetic acid) were studied. The complexes [VO(Ibu)(2)] x 5CH(3)OH, [VO(Nap)(2)] x 5CH(3)OH and [VO(Tol)(2)] were obtained from methanolic solutions under nitrogen atmosphere. The biological activities of these complexes on the proliferation of two osteoblast-like cells in culture (MC3T3E1 and UMR106) were compared with that of the vanadyl(IV) cation. The complexes exhibited different effects depending on the concentration and the cellular type, while no effect was observed for their parent drugs.

In chronically uncompensated diabetes mellitus, an increase has been observed in the content of advanced glycation endproduct (AGE)-modified proteins in various tissues, including bone. This increase can lead to a local imbalance in the secretion of cytokines and growth factors, and has been implicated in the pathophysiology of the longterm complications of diabetes. We have previously shown that the proliferation and differentiation of UMR106 rat osteosarcoma and MC3T3E1 mouse calvaria-derived cell lines are regulated by AGE-modified proteins, possibly through the recognition of these AGEs by specific membrane-associated receptors. In the present study, we investigated the effects of AGE-proteins on the secretion of insulin-like growth factor-I (IGF-I) and its binding proteins (IGFBPs) by both osteoblast-like cell lines. In the case of MC3T3E1 cells, this was studied throughout their successive stages of development: proliferation, differentiation and mineralisation. For every condition, cells were incubated 24 hours with increasing concentrations of either bovine serum albumin (BSA) or AGE-BSA. IGF-I in conditioned media was separated from IGFBPs by acid gel filtration-centrifugation, and measured by radioimmunoassay. IGFBPs in conditioned media were analysed by a semi-quantitative western ligand blot. In UMR106 cells, low doses of AGE-BSA significantly decreased the secretion of both IGF-I (56% of control) and a 24 kDa IGFBP (80% of control). Results for MC3T3E1 cells, which predominantly secrete 29 kDa IGFBPs, were dependent on the stage of development. In proliferating preosteoblastic cells, AGE-BSA decreased the secretion of IGF-I (34%-37% of control) while increasing the secretion of IGFBP (124%-127% of control). On the other hand, secretion of these components of the IGF system by mature (differentiated) cells was unaffected by the presence of AGE-BSA. When these cells finally attained mineralisation, incubation with AGE-modified BSA provoked an increase both in IGFBP (131%-169% of control) and in IGF-I secretion (119%-123% of control). The presented evidence suggests that the modulation of growth and development by AGE-modified proteins, previously described for both cell lines, could be the result of an autocrine-paracrine mechanism involving the IGF-IGFBP system.

BACKGROUND: Oxidative modification of low density lipoproteins (LDL) is recognized as one of the major processes involved in atherogenesis. The in vitro standardized measurement of LDL oxidative susceptibility could thus be of clinical significance. The aim of the present study was to establish a method which would allow the evaluation of oxidative susceptibility of LDL in the general clinical laboratory. RESULTS: LDL was isolated from human plasma by selective precipitation with amphipathic polymers. The ability of LDL to form peroxides was assessed by measuring thiobarbituric acid reactive substances (TBARS) after incubation with Cu2+ and H2O2. Reaction kinetics showed a three-phase pattern (latency, propagation and decomposition phases) which allowed us to select 150 min as the time point to stop the incubation by cooling and EDTA addition. The mixture Cu2+/H2O2 yielded more lipoperoxides than each one on its own at the same time end-point. Induced peroxidation was measured in normal subjects and in type 2 diabetic patients. In the control group, results were 21.7 +/- 1.5 nmol MDA/mg LDL protein, while in the diabetic group results were significantly increased (39.0 +/- 3.0 nmol MDA/mg LDL protein; p < 0.001). CONCLUSION: a simple and useful method is presented for the routine determination of LDL susceptibility to peroxidation in a clinical laboratory.

Background: The tissue accumulation of protein-bound advanced glycation endproducts (AGE) may be involved in the etiology of diabetic chronic complications, including osteopenia. The aim of this study was to investigate the effect of an AGE-modified type I collagen substratum on the adhesion, spreading, proliferation and differentiation of rat osteosarcoma UMR106 and mouse non-transformed MC3T3E1 osteoblastic cells. We also studied the role of reactive oxygen species (ROS) and nitric oxide synthase (NOS) expression on these AGE-collagen mediated effects., Results: AGE-collagen decreased the adhesion of UMR106 cells, but had no effect on the attachment of MC3T3E1 cells. In the UMR106 cell line, AGE-collagen also inhibited cellular proliferation, spreading and alkaline phosphatase (ALP) activity. In preosteoblastic MC3T3E1 cells (24-hour culture), proliferation and spreading were significantly increased by AGE-collagen. After one week of culture (differentiated MC3T3E1 osteoblasts) AGE-collagen inhibited ALP activity, but had no effect on cell number. In mineralizing MC3T3E1 cells (3-week culture) AGE-collagen induced a decrease in the number of surviving cells and of extracellular nodules of mineralization, without modifying their ALP activity. Intracellular ROS production, measured after a 48-hour culture, was decreased by AGE-collagen in MC3T3E1 cells, but was increased by AGE-collagen in UMR106 cells. After a 24-hour culture, AGE-collagen increased the expression of endothelial and inducible NOS, in both osteoblastic cell lines., Conclusions: These results suggest that the accumulation of AGE on bone extracellular matrix could regulate the proliferation and differentiation of osteoblastic cells. These effects appear to depend on the stage of osteoblastic development, and possibly involve the modulation of NOS expression and intracellular ROS pathways.

A delivery system for vanadium was developed using poly(beta-propiolactone) (PbetaPL) films. The release kinetics of a complex of vanadium (IV) with aspirin (VOAspi) was evaluated with films prepared from polymers of different molecular weights, as well as with variable drug load. A sustained release of vanadium over 7 days was achieved. The drug release kinetics depends on contributions from two factors: (a) diffusion of the drug; and (b) erosion of the PbetaPL film. The experimental data at an early stage of release were fitted with a diffusion model, which allowed determination of the diffusion coefficient of the drug. VOAspi does not show strong interaction with the polymer, as demonstrated by the low apparent partition coefficient (approximately 10(-2)). UMR106 osteosarcoma cells were used as a model to evaluate the anticarcinogenic effects of the VOAspi released from the PbetaPPL film. VOAspi-PbetaPL film inhibited cell proliferation in a dose-response manner and induced formation of approximately half of the thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation. compared to that with free VOAspi in solution. The unloaded PbetaPL film did not generate cytotoxicity, as evaluated by cell growth and TBARS. Thus, the polymer-embedded VOAspi retained the antiproliferative effects showing lower cytotoxicity than the free drug. Results with VOAspi-PbetaPL films suggest that this delivery system may have promising biomedical and therapeutic applications.

Nitric oxide (NO) has been shown to act as a mediator of cytokines in bone tissue. We have previously demonstrated that vanadium compounds are insulin- and growth factor-mimetic compounds in osteoblasts in culture, although high doses are toxic to these cells. In this study, we measured NO production in two osteoblast-like cells (UMR106 and MC3T3E1) incubated with different concentrations (2.5-100 microM) of vanadate. Vanadate induced NO release in a biphasic manner, with levels being significantly increased at concentrations over 50 microM. The NO donor, sodium nitroprusside, mimicked the vanadate effect: it inhibited cell growth and alkaline phosphatase activity in a dose-dependent manner. Vanadate enhanced the NO synthases, the endothelial and inducible (eNOS and iNOS) isoforms, in a dose-dependent manner. Experiments performed with the ionophore A23187 and EGTA suggested that vanadate-induced NO production involves Ca(2+)-dependent and -independent mechanisms. Altogether, our results suggest that NO may play a critical role in the bioactivity of vanadium in osteoblast-like cells.