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

Proliferative and morphological changes induced by vanadium compounds on Swiss 3T3 fibroblasts

Vanadium compounds are shown to have a mitogenic effect on fibroblast cells. The effects of vanadate, vanadyl and pervanadate on the proliferation and morphological changes of Swiss 3T3 cells in culture are compared. Vanadium derivatives induced cell proliferation in a biphasic manner, with a toxic-like effect at doses over 50 mM, after 24 h of incubation. Vanadyl and vanadate were equally potent at 2.5–10 mM. At 50 mM vanadate inhibited cell proliferation, whereas slight inhibition was observed at 100 mM of vanadyl. At 10 mM pervanadate was as potent as vanadate and vanadyl in stimulating fibroblast proliferation, but no effect was observed at lower concentrations. A pronounced cytotoxic-like effect was induced by pervanadate at 50 mM. All of these effects were accompanied by morphological changes: transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation; and loss of lamellar processes. The magnitude of these transformations correlates with the potency of vanadium derivatives to induce a cytotoxic-like effect: pervanadate > vanadate > vanadyl. These data suggest that the oxidation state and coordination geometry of vanadium determine the degree of the cytotoxicit

Proliferative and morphological changes induced by vanadium compounds on Swiss 3T3 fibroblasts

Vanadium compounds are shown to have a mitogenic effect on fibroblast cells. The effects of vanadate, vanadyl and pervanadate on the proliferation and morphological changes of Swiss 3T3 cells in culture are compared. Vanadium derivatives induced cell proliferation in a biphasic manner, with a toxic-like effect at doses over 50 mM, after 24 h of incubation. Vanadyl and vanadate were equally potent at 2.5–10 mM. At 50 mM vanadate inhibited cell proliferation, whereas slight inhibition was observed at 100 mM of vanadyl. At 10 mM pervanadate was as potent as vanadate and vanadyl in stimulating fibroblast proliferation, but no effect was observed at lower concentrations. A pronounced cytotoxic-like effect was induced by pervanadate at 50 mM. All of these effects were accompanied by morphological changes: transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation; and loss of lamellar processes. The magnitude of these transformations correlates with the potency of vanadium derivatives to induce a cytotoxic-like effect: pervanadate > vanadate > vanadyl. These data suggest that the oxidation state and coordination geometry of vanadium determine the degree of the cytotoxicity.Facultad de Ciencias Exacta

Effect of hypothyroidism on the composition and turnover rate of islet phospholipids

It has already been demonstrated that the pancreatic B cells of hypothyroid rats have a reduced capacity to release insulin in response to glucose (l). This impaired B cell function may be partly due to a diminished rate of glucose oxidation and net calcium uptake associated with ultrastructural alteration of the pancreatic islets (2). To identify further other factors responsible for this diminished B cell secretory function, we studied the composition and the turnover rate of phospholipids in islets obtained from hypothyroid rats.Facultad de Ciencias Médica

Effect of hypothyroidism on the composition and turnover rate of islet phospholipids

It has already been demonstrated that the pancreatic B cells of hypothyroid rats have a reduced capacity to release insulin in response to glucose (l). This impaired B cell function may be partly due to a diminished rate of glucose oxidation and net calcium uptake associated with ultrastructural alteration of the pancreatic islets (2). To identify further other factors responsible for this diminished B cell secretory function, we studied the composition and the turnover rate of phospholipids in islets obtained from hypothyroid rats.Facultad de Ciencias Médica

Synthesis of a new vanadyl(IV) complex with trehalose (TreVO): insulin-mimetic activities in osteoblast-like cells in culture

Vanadium compounds show interesting biological and pharmacological properties. Some of them display insulin-mimetic effects and others produce antitumor 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), Na6 [VO(Tre)2].4H2O, 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 lM but it inhibited cell proliferation in a dose-response manner between 50 and 100 lM. TreVO significantly inhibited UMR106 differentiation (15–25% of basal) in the range 5–100 lM. On normal osteoblasts, TreVO behaved as a mitogen at 5–25 lM. Different inhibitors of the MAPK pathway blocked this effect. At higher concentrations (75–100 lM), 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

Effect of vanadium compounds on acid phosphatase activity

The direct effect of different vanadium compounds on acid phosphatase (ACP) activity was investigated. Vanadate and vanadyl but not pervanadate inhibited the wheat germ ACP activity. These vanadium derivatives did not alter the fibroblast Swiss 3T3 soluble fraction ACP activity. Using inhibitors of tyrosine phosphatases (PTPases), the wheat germ ACP was partially characterized as a PTPase. This study suggests that the inhibitory ability of different vanadium derivatives to modulate ACP activity seems to depend on the geometry around the vanadium atom more than on the oxidation state. Our results indicate a correlation between the PTPase activity and the sensitivity to vanadate and vanadyl cation.Facultad de Ciencias Exacta

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

Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic development and oxidative stress

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

Effect of hypothyroidism on the composition and turnover rate of islet phospholipids

It has already been demonstrated that the pancreatic B cells of hypothyroid rats have a reduced capacity to release insulin in response to glucose (l). This impaired B cell function may be partly due to a diminished rate of glucose oxidation and net calcium uptake associated with ultrastructural alteration of the pancreatic islets (2). To identify further other factors responsible for this diminished B cell secretory function, we studied the composition and the turnover rate of phospholipids in islets obtained from hypothyroid rats.Facultad de Ciencias Médica