The analysis of different scaffolds and the benefit of fibrin glue for tendon tissue engineering at different culture times.

This study evaluated a tendon substitute model. Tenocytes were isolated from pig Achilles tendon, seeded onto scaffolds (Opocrin 2%, Typeone 3% and Symatese 2%) and studied by histology, immunofluorescence for collagen type 1 and 3 and biochemical analysis to assess cellularity. The permeability of these compounds was evaluated in the presence or absence of fibrin glue. Opocrin 2% was the best choice for cellular distribution within the scaffolds, which were then cultured for T0, T4, T7 and T10 days. Fibrin glue has been strongly supportive for the survival of cells with a significant increase in DNA content at T10 (P<0.05). Moreover, the synthetic activity of fibrin-free scaffolds was always negative. Lastly, a progressive increase in collagen 1 and 3 with fibrin-glue was observed. However, static culture is not sufficient to support long-term cellular activities and at T10 there is still a lack of organized matrix similar to the native tissue.

Comparison of three novel biphasic scaffolds for one-stage treatment of osteochondral defects in a sheep model.

In the last years, several tissue engineering techniques have been applied to develop different kinds of osteochondral substitutes to overcome the scarce reparative properties of this tissue. The aim of this study was to generate and compare three biphasic scaffolds in an osteochondral lesion in a large-animal model. A critical osteochondral defect was generated in the medial femoral condyle of 18 skeletally mature sheep. Three defects were left untreated, the remaining lesions were divided into three groups: 5 lesions were treated with a biphasic scaffold made of collagen type I and small cylinders of Magnesium Hydroxyapatite; 5 lesions were treated with a biphasic substituted formed by collagen type I and Wollastonite, 5 lesions were treated with a scaffold made of collagen type I and small cylinders of Wollastonite/Hydroxyapatite. Animals were sacrificed after 3 months and samples were analyzed by CT and MRI, macroscopic evaluation and histology. Our study demonstrated that one of these novel biphasic scaffolds possesses the potential for being applied for one-stage procedures for osteochondral defects.

The extracorporeal perfusion of swine uterus as an experimental model: the effect of oxytocic drugs.

The objective of this study was to establish an experimental model for extracorporeal perfusion of swine uterus. In order to validate this model, we examined some biochemical parameters and determined the effect of oxytocic drugs (Oxytoxin, Prostaglandin E (2)) on extracorporeal perfused swine uteri. Thirty swine uteri were perfused with Krebs-Ringer bicarbonate-glucose buffer for a period up to eleven hours with the aim to preserve a viable organ, which should be responsive to hormones. The intrauterine pressure was recorded after administration of various concentrations of oxytocin and prostaglandin E (2). Perfusate pH, perfusate lactate, partial oxygen and carbon dioxide tensions, oxygen saturation, and hydrogencarbonate levels in the perfusate, all indicators of tissue ischemia or cell necrosis, showed good preservation of the organ for up to seven hours. We examined the relation of intrauterine pressure to oxytocin and prostaglandin E (2). Both were able to induce contractions of the uterus, whereas prostaglandin E (2) produced rhythmical contractions of smaller amplitude and a higher frequency. We could demonstrate that our perfusion system was able to preserve the swine uterus in a functional condition appropriate for the study of physiological questions.

Intracellular glutathione and lipid peroxide availability and the secretion of vasoactive substances by human umbilical vein endothelial cells after incubation with TNF-alpha.

BACKGROUND: The major pathophysiologic changes observed in preeclampsia suggest that endothelial cell dysfunction plays an important role in this disorder. The pathway mediating to endothelial cell dysfunction is unknown, however, the pathogenesis of preeclampsia is thought to be related to increased oxidative stress and increased vasoconstriction. The concentration of tumor necrosis factor alpha (TNF-alpha), a cytokine produced by macrophages and many other cell types, has been observed to be significantly increased in preeclampsia. It has been hypothesized that TNF-alpha overproduction by the placenta may then may produce an increase in plasma levels and subsequent endothelial dysfunction in preeclampsia. This study investigated the effect of TNF-alpha on glutathione and lipid peroxide levels and on the secretion of vasoactive substances by human umbilical vein endothelial cells (HUVECs). METHODS: Human umbilical vein endothelial cells were incubated for 24 h in the presence of different concentrations of TNF-alpha (0-1000 pg mL-1) that were shown in an earlier experiment to have no effects on the vitality and proliferation rate of HUVECs. The levels of reduced glutathione (GSH) and lipid peroxides (LPOs), assessed by malondialdehyde and 4-hydroxyalkenal, were measured in endothelial cell lysates. For the measurement of vasoactive substances, levels of prostacyclin (PGI2), determined by 6-keto-prostaglandin F1a, thromboxane A2 (TXA2), measured by thromboxane B2, endothelin-1 (ET-1), and nitric oxide (NO), measured by total nitrite, were assessed in endothelial cell supernatants. RESULTS: At lower concentrations (10-100 pg mL-1), TNF-alpha increases the intracellular content of LPO and GSH, stimulates the secretion of ET-1 and TXA2, but inhibits the secretion of PGI2 in endothelial cells compared with control cells. At concentration of 1000 pg mL-1, TNF-alpha increases the secretion of PGI2 and TXA2, but it decreases the ET-1 concentration. TNF-alpha has no effect on NO secretion. CONCLUSION: These findings demonstrate that at concentrations corresponding to values in plasma from preeclamptic women, TNF-alpha induces oxidative stress and results in altered secretion of vasoactive substances in favour of vasoconstrictors in human endothelial cells. We conclude that TNF-alpha may participate in the pathway leading to endothelial cell dysfunction seen in preeclampsia.

Effect of endothelin-1 on intracellular glutathione and lipid peroxide availability and on the secretion of vasoactive substances by human umbilical vein endothelial cells.

BACKGROUND: The major pathophysiologic changes observed in preeclampsia suggest that endothelial cell dysfunction plays an important role in this disorder. The pathway mediating endothelial cell layer dysfunction is unknown. The concentration of endothelin-1 (ET-1), a potent mammalian vasoconstrictor peptide produced by the vascular endothelium, has been observed to be significantly increased in preeclampsia. In this study, we determined the in vitro effect of endothelin-1 on glutathione and lipid peroxide levels and on the secretion of vasoactive substances by human umbilical vein endothelial cells (HUVECs). METHODS: Human umbilical vein endothelial cells were incubated for 24 h in the presence of different concentrations of ET-1 (0-1000 pmol L(-1)), which were shown in an earlier experiment to have no effects on vitality and proliferation rate of HUVECs. The levels of glutathione (GSH) and lipid peroxides (LPO) were measured in endothelial cell lysates. For the measurement of vasoactive substances, levels of nitric oxide (NO), prostacyclin (PGI2) and thromboxane A2 (TXA2) were measured in endothelial cell supernatants. RESULTS: At lower concentrations (5-50 pmol L(-1)), ET-1 increases the intracellular content of LPO, stimulates the secretion of TXA2, but inhibits the secretion of PGI2 in endothelial cells compared with control cells. At higher concentrations (100-1000 pmol L(-1)), ET-1 increases the intracellular content of GSH, but results in a decrease of LPO, and increase of PGI2, back to control levels. ET-1 has no effect on NO secretion. CONCLUSION: These findings demonstrate that at concentrations corresponding to values in plasma from preeclamptic women, ET-1 induces oxidative stress and results in altered secretion of vasoactive substances in human endothelial cells. We conclude that ET-1 may participate in the pathway leading to endothelial cell dysfunction seen in preeclampsia.

Influence of reduced intracellular glutathione availability on the secretion of vasoactive substances by human umbilical vein endothelial cells.

OBJECTIVE: Oxidative stress might be the reason for endothelial dysfunction in preeclampsia. The glutathione-peroxidase system is one of the primary antioxidants in the endothelium. We tested the effect of oxidative stress by reduction of glutathione availability on the secretion of vasoactive substances in endothelial cells. METHODS: Endothelial cells in culture were incubated with different concentrations of buthionine-[S,R]-sulfoximine (BSO) or 1-chloro-2,4-dinitrobenzene (CDNB), both leading to a reduced intracellular availability of glutathione. The secretion of the vasoactive substances nitric oxide (NO), endothelin-I (ET-1), and prostacyclin (PGI2) was measured with respect to vitality and proliferation rate of the endothelial cells in culture. MAIN OUTCOME MEASURE: Effect of oxidative stress on the secretion of vasoactive substances from endothelial cells. RESULTS: The oxidants CDNB and BSO have (in concentrations before evidence of cytotoxicity) a stimulating effect on the production of PGI2, they inhibit NO availability, and they do not significantly interfere with ET-1 production. Conclusion Oxidative stress in vitro induces an imbalance in the secretion of NO, ET-1, and PGI2 in endothelial cells.

Production of vasoactive substances by human umbilical vein endothelial cells after incubation with serum from preeclamptic patients.

OBJECTIVE: To determine the in vitro effect of serum from preeclamptic patients on the proliferation, viability and secretion of vasoactive substances by human umbilical vein endothelial cells (HUVEC). STUDY DESIGN: HUVEC were incubated for 24h with sera from 16 preeclamptic, 19 healthy pregnant and 8 healthy nonpregnant women. Proliferation rates were determined by cell counting and vitality by trypan blue staining. The vasoactive substances, 6-keto-prostaglandin F(1alpha), nitrite and nitrate, and endothelin-1 (ET-1) were measured in endothelial cell supernatants. RESULTS: The preeclamptic serum had no effect on cell proliferation or vitality compared with control sera. It induced more HUVEC production of ET-1, but not of prostacyclin (PGI2) or nitric oxide compared with control serum. CONCLUSION: Preeclamptic serum appears to contain a factor(s) that specifically stimulates ET-1 secretion from HUVEC without altering cell growth or vitality.

Stimulatory effects of silibinin and silicristin from the milk thistle Silybum marianum on kidney cells.

The biochemical influence of flavonolignans from the milk thistle Silybum marianum has been tested on kidney cells of African green monkeys. Two nonmalignant cell lines were selected, with the focus of the work on the fibroblast-like Vero line. Proliferation rate, biosynthesis of protein and DNA, and the activity of the enzyme lactate dehydrogenase (as a measure of the cellular metabolic activity) were chosen as parameters for the effect of the flavonolignans. Silibinin and silicristin show remarkable stimulatory effects on these parameters, mainly in Vero cells; however, isosilibinin and silidianin proved to be inactive. In vitro experiments with kidney cells damaged by paracetamol, cisplatin, and vincristin demonstrated that administration of silibinin before or after the chemical-induced injury can lessen or avoid the nephrotoxic effects. The results warrant in vivo evaluations of the flavonolignan derivatives.