Osteogenic activity of vanadyl(IV)–ascorbate complex: evaluation of its mechanism of action

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 M) significantly stimulated osteoblastic proliferation (113–125% basal, p < 0.01) in UMR106 cells, but not in the MC3T3E1 cell line. VOAsc (5–100 M) dose-dependently stimulated type-I collagen production (107–156% basal) in osteoblasts. After 3 weeks of culture, 5–25 M VOAsc increased the formation of nodules of mineralization in MC3T3E1 cells (7.7–20-fold control, p < 0.001). VOAsc (50–100 M) 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 M 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

Characterization of Poly(ε-caprolactone)/Polyfumarate Blends as Scaffolds for Bone Tissue Engineering

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(ε-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 andMC3T3E1 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

La diabetes altera el potencial osteogénico de células progenitoras de médula ósea: efectos del tratamiento con metformina

En este trabajo, estudiamos el efecto de una Diabetes inducida por destrucción parcial de la masa de células beta pancreáticas, sobre el compromiso osteogénico de células progenitoras de médula ósea (CPMO), y su modulación por el tratamiento oral con Metformina. Para ello utilizamos ratas Sprague Dawley, divididas en cuatro grupos: controles [C], controles tratadas con Metformina [M], diabéticas [D], y diabéticas tratadas con Metformina [DM]. La inducción de Diabetes se realizó, por inyección intraperitoneal sucesiva de ácido nicotínico y estreptozotocina. Sobre los cultivos de CPMO se evaluó la actividad específica de Fosfatasa Alcalina (FAL) y la producción de Colágeno tipo 1 (Col-1) en estado basal y en medio de diferenciación osteogénico luego de 15 días. A los 21 días, se evaluaron los depósitos de mineral extracelular. La FAL y el Col-1 de CPMO basales, no mostraron diferencias significativas entre los cuatro grupos experimentales. Al cabo de 15 días, las CPMO de ratas M mostraron un incremento en el Col-1 de 122 % respecto de C; D 30 % respecto de C y DM 68 % respecto de C. La FAL expresó un 171 % para M, 34 % para D; y 125 % para DM todos respecto de C. Luego de 21 días, se observó una disminución en la mineralización de las CPMO de D (65 % respecto del grupo C). El tratamiento con metformina incrementó la mineralización de las CPMO en todos los casos. En conclusión, en nuestro modelo experimental de Diabetes, ésta disminuye el potencial osteogénico de las CPMO, un efecto que es parcialmente revertido por el tratamiento oral con Metformina. Estos hallazgos podrían explicar, al menos en parte, las alteraciones óseas descriptas en el hueso asociadas con la Diabetes.Diabetes mellitus is associated with an increased incidence of skeletal abnormalities, resulting in lower bone formation and/or remodeling. Osteopenia, osteoporosis and an increased incidence of non-traumatic fractures has been particularly observed in patients with type 2 diabetes. Recently, we have demonstrated that metformin has in vitro and in vivo osteogenic effects: (a) it stimulates the proliferation, differentiation and mineralization of osteoblasts in culture, and (b) in non-diabetic rats, it increases the repair of minimal bone lesions and improves femoral trabecular bone microarchitecture. In this study, we evaluated in rats the effect of diabetes induction by a partial destruction of pancreatic beta cells, on the osteogenic commitment of bone marrow progenitor cells (BMPC), and the modulation of this effect by orally administered metformin. We used young male Sprague Dawley rats (200 g), divided into four groups: untreated non-diabetic controls [C], non-diabetic rats treated for 2 weeks with metformin administered in drinking water (100 mg/kg/day) [M], untreated diabetic rats [D], and diabetic rats treated for 2 weeks with metformin (100 mg/kg/day) [DM]. Induction of Diabetes was performed one week prior to treatment with metformin, by successive intraperitoneal injections with 75 mg/kg body weight of nicotinic acid and 60 mg/kg body weight of streptozotocin. At the end of all treatments, blood samples were obtained to confirm the development of Diabetes, after which the animals were sacrificed by cervical dislocation under anesthesia. Femora and/or tibiae were dissected, and bone marrow cells were collected by flushing the bone diaphysal canal with Dulbecco’s modified essential medium (DMEM) under sterile conditions. Adherent cells were grown to confluence in DMEM-10 % fetal bovine serum (FBS), after which we assessed alkaline phosphatase specific activity (ALP) by an enzymatic kinetic method, and type 1 collagen production (Col-1) by a Sirius Red colorimetric method (basal osteoblastic differentiation of BMPC). Subsequently, BMPC were submitted to an osteogenic induction for 15 days with an osteogenic medium (DMEM-10 % FBS containing ascorbic acid and sodium beta-glycerophosphate), after which ALP and Col-1 were evaluated. Basal ALP activity and type 1 collagen production (BMPC without osteogenic differentiation) showed no significant differences between the four experimental groups. After 15 days of culture in osteogenic medium, BMPC from control rats increased their expression of ALP (5 times compared to baseline) and collagen production (11 times compared to baseline). BMPC from diabetic rats after 15 days culture in osteogenic medium, also showed a significant (although smaller) increase in ALP (2-3 fold over basal activity) and collagen production (4-fold compared to baseline). BMPC obtained from rats treated with metformin (groups M and DM) and submitted to osteogenic induction for 15 days, showed an approximately 2-4-fold increase in both ALP and Col-1 (when compared with groups C and D, respectively). After 21 days of osteogenic induction, a decrease was observed in the mineralization of BMPC obtained from group D (65 % of that for group C). Treatment with metformin increased the mineralizing capacity of BMPC in all cases, including a reversal of the inhibitory effect of Diabetes on this parameter. In conclusion, we have found that our model of Diabetes reduces the osteogenic potential of bone marrow progenitor cells, and that this effect is partially reverted by orally administered metformin. These findings could explain, at least in part, the bone alterations that have been associated with Diabetes mellitus

Effect of metformin on bone marrow progenitor cell differentiation: in vivo and in vitro studies

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

Loss of migratory traditions makes the endangered patagonian Huemul deer a year-round refugee in its summer habitat

The huemul (Hippocamelus bisulcus) is endangered, with 1500 deer split into >100 subpopulations along 2000 km of the Andes. Currently occupied areas are claimed-erroneously-to be critical prime habitats. We analyzed historical spatiotemporal behavior since current patterns represent only a fraction of pre-Columbian ones. Given the limited knowledge, the first group (n = 6) in Argentina was radio-marked to examine spatial behavior. Historically, huemul resided year-round in winter ranges, while some migrated seasonally, some using grasslands >200 km east of their current presence, reaching the Atlantic. Moreover, huemul anatomy is adapted to open unforested habitats, also corroborated by spotless fawns. Extreme naivety towards humans resulted in early extirpation on many winter ranges—preferentially occupied by humans, resulting in refugee huemul on surrounding mountain summer ranges. Radio-marked huemul remained in small ranges with minimal altitudinal movements, as known from other subpopulations. However, these resident areas documented here are typical summer ranges as evidenced by past migrations, and current usage for livestock. The huemul is the only cervid known to use mountain summer ranges year-round in reaction to anthropogenic activities. Losing migratory traditions is a major threat, and may explain their presently prevalent skeletal diseases, reduced longevity, and lacking recolonizations for most remaining huemul subpopulations.Fil: Fluck, Werner Thomas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; Argentina. Universidad de Basilea; Suiza. Administración de Parques Nacionales; ArgentinaFil: Smith Flueck, Jo Anne M.. Universidad Nacional del Comahue; Argentina. Parque Protegido Shoonem; Argentina. Deer Lab; ArgentinaFil: Escobar, Miguel E.. Parque Protegido Shoonem; ArgentinaFil: Zuliani, Melina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; Argentina. Fundación Bariloche; ArgentinaFil: Fuchs, Beat. Deer Lab; ArgentinaFil: Geist, Valerius. University of Calgary; CanadáFil: Heffelfinger, James R.. Arizona Game and Fish Department; Estados UnidosFil: Black de Decima, Patricia Ann. Universidad Nacional de Tucumán. Facultad de Ciencias Naturales e Instituto Miguel Lillo; ArgentinaFil: Gizejewski, Zygmunt. Polish Academy of Sciences; ArgentinaFil: Vidal, Fernando. Univerdidad Santo Tomas; Chile. Centro de Conservacion y Manejo de Vida Silvestre; ChileFil: Barrio, Javier. Centro de Ornitología y Biodiversidad; PerúFil: Molinuevo, María Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas; ArgentinaFil: Monjeau, Jorge Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; Argentina. Fundación Bariloche; ArgentinaFil: Hoby, Stefan. Berne Animal Park; SuizaFil: Jiménez, Jaime M.. University of North Texas; Estados Unido

Novel Vanadium-Loaded Ordered Collagen Scaffold Promotes Osteochondral Differentiation of Bone Marrow Progenitor Cells

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 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

Loss of Migratory Traditions Makes the Endangered Patagonian Huemul Deer a Year-Round Refugee in Its Summer Habitat

The huemul (Hippocamelus bisulcus) is endangered, with 1500 deer split into >100 subpopulations along 2000 km of the Andes. Currently occupied areas are claimed-erroneously-to be critical prime habitats. We analyzed historical spatiotemporal behavior since current patterns represent only a fraction of pre-Columbian ones. Given the limited knowledge, the first group (n = 6) in Argentina was radio-marked to examine spatial behavior. Historically, huemul resided year-round in winter ranges, while some migrated seasonally, some using grasslands >200 km east of their current presence, reaching the Atlantic. Moreover, huemul anatomy is adapted to open unforested habitats, also corroborated by spotless fawns. Extreme naivety towards humans resulted in early extirpation on many winter ranges—preferentially occupied by humans, resulting in refugee huemul on surrounding mountain summer ranges. Radio-marked huemul remained in small ranges with minimal altitudinal movements, as known from other subpopulations. However, these resident areas documented here are typical summer ranges as evidenced by past migrations, and current usage for livestock. The huemul is the only cervid known to use mountain summer ranges year-round in reaction to anthropogenic activities. Losing migratory traditions is a major threat, and may explain their presently prevalent skeletal diseases, reduced longevity, and lacking recolonizations for most remaining huemul subpopulations