Improved endothelialization and reduced thrombosis by coating a synthetic vascular graft with fibronectin and stem cell homing factor SDF-1α.

Failure of synthetic small-diameter vascular grafts is determined mainly by the lack of endothelial cells, as these cells inhibit thrombosis and intimal hyperplasia. Coating of graft material with homing factors for circulating stem cells has the potential to improve endogenous endothelialization of these grafts and to reduce graft failure. Synthetic knitted polyester grafts (6mm diameter) were coated with FN and SDF-1α before surgical interposition in the carotid artery of sheep. Similar uncoated vascular grafts were implanted in the contralateral side as internal controls. To study the early attraction of stem cells, grafts were implanted in a first series of nine sheep and explanted after 1 or 3 days. In coated grafts, four times higher fractions of CD34(+) and three to four times higher fractions of CD117(+) cells adhering to the vessel walls were found than in control grafts (P<0.05). When such coated and non-coated grafts were implanted in 12 other sheep and explanted after 3 months, all coated grafts were patent, while one control graft was occluded. EcNOS staining revealed that FN-SDF-1α coating significantly increased coverage with endothelial cells from 27 ± 4% of the graft to 48 ± 4% compared with the controls (P=0.001). This was associated with a significant reduction of intimal hyperplasia (average thickness 1.03 ± 0.09 mm in controls vs. 0.69 ± 0.04 mm in coated grafts; P=0.009) and significantly less adhesion of thrombotic material in the middle part of the graft (P=0.029). FN-SDF-1α coating of synthetic small-caliber vascular grafts stimulated the attraction of stem cells and was associated with improved endothelialization and reduced intimal hyperplasia and thrombosis.

Cyclically stretching developing tissue in vivo enhances mechanical strength and organization of vascular grafts.

Tissue-engineered vascular grafts must have qualities that rival native vasculature, specifically the ability to remodel, the expression of functional endothelial components and a dynamic and functional extracellular matrix (ECM) that resists the forces of the arterial circulation. We have developed a device that when inserted into the peritoneal cavity, attracts cells around a tubular scaffold to generate autologous arterial grafts. The device is capable of cyclically stretching (by means of a pulsatile pump) developing tissue to increase the mechanical strength of the graft. Pulsed (n=8) and unpulsed (n=8) devices were implanted for 10 days in Lovenaar sheep (n=8). Pulsation occurred for a period of 5-8 days before harvest. Thick unadhered autologous tissue with cells residing in a collagen ECM was produced in all devices. Collagen organization was greater in the circumferential direction of pulsed tissue. Immunohistochemical labelling revealed the hematopoietic origin of >90% cells and a significantly higher coexpression with vimentin in pulsed tissue. F-actin expression, mechanical failure strength and strain were also significantly increased by pulsation. Moreover, tissue could be grafted as carotid artery patches. This paper shows that unadhered tissue tubes with increased mechanical strength and differentiation in response to pulsation can be produced with every implant after a period of 10 days. However, these tissue tubes require a more fine-tuned exposure to pulsation to be suitable for use as vascular grafts.

Fluorescent microsphere technique to measure cerebral blood flow in the rat.

The present protocol describes a method for parallel measurement of cerebral blood flow (CBF) using fluorescent microspheres and structural assessment of the same material. The method is based on the standard microsphere technique, embolizing capillaries proportional to the blood flow, but requires dissolution of the tissue to retrieve the microspheres. To link the blood flow to the tissue morphology we modified the technique to fluorescent microspheres, which are quantified in cryo- or vibratome sections, allowing structural analysis by, for example, immunohistochemistry or standard histology. The protocol takes 8 h 50 min, without pauses, to complete, but additional flow measurements or specific protocols can increase the time needed.

Molecular and functional characterization of ovine cardiac valve-derived interstitial cells in primary isolates and cultures.

At present the involvement of cardiac valve interstitial cells (VICs) in growth, repair, and tissue engineering is understudied. Therefore, this study aims at characterizing ovine VICs in order to provide a solid base for tissue engineering of heart valves. Ovine ICs of the four heart valves were isolated by the explant outgrowth method and expanded in vitro up to passage 5. Vimentin and collagen I gene expression from freshly isolated or cultured ICs was measured by reverse transcriptase-polymerase chain reaction. Immunocytochemical stainings of vimentin, alpha-smooth muscle actin (ASMA), smooth muscle myosin, and procollagen I were performed on aortic VICs. In addition, migration and extracellular matrix deposition were studied in vitro in aortic VICs. ICs show stable vimentin and collagen I expression in culture. Expression is approximately doubled in cultured ICs compared with fresh isolates. More than 95% of ICs in each passage stain for vimentin and procollagen I. Freshly isolated ICs are ASMA and myosin negative, but ICs in culture partially stain for these contractile markers. ICs have stable matrix production for up to five passages, associated with stable migration of the cells. We conclude that ovine valve interstitial cells undergo phenotypic modulation to activated myofibroblasts under culture conditions but retain stable matrix production.

Development of a novel fluorescent microsphere technique to combine serial cerebral blood flow measurements with histology in the rat.

The aim of the study was to evaluate the microsphere technique for the quantitative assessment of regional cerebral blood flow (rCBF) at different time points in the same animal. Yellow-green and red fluorescent microspheres with a diameter of 15 microm were injected into the rat at two different time points via a cannula inserted into the left ventricle of the heart. The reproducibility of the rCBF measurements in normocapnic conditions (n=7) and the responsiveness of the flow to hypercapnia induced by 7% CO(2) (n=7) was examined. The fluorescent spheres were counted on 100 microm vibratome sections of perfusion-fixed brains and rCBF was calculated. The median total CBF in normocapnic rats was 224 ml/min/100 g for the first microsphere injection and 216 ml/min/100 g for the second one. In the hypercapnic group CBF amounted to 400 ml/min/100 g and after 30 min of normocapnia decreased to 178 ml/min/100 g. No differences between the left and right hemisphere were found and there was no indication that the first injection might have influenced the second one. The described approach allows combining the assessment of rCBF at different time points in physiological or pathological conditions with histological evaluation of related morphological alterations in the same brain region of the same animal.

Validation and implementation of an internal standard in comet assay analysis.

The comet assay is widely used to detect DNA damage in single cells. However, only moderate attention has been paid to the experimental variability of this assay, especially during electrophoresis. To take into account this variation and to be able to compare measurements from different electrophoretic runs, as would be necessary when large numbers of samples need to be analysed, it is important to integrate an internal standard into the assay. This study presents a first step in the validation and implementation of an internal standard in the alkaline comet assay. Untreated and ethyl methanesulfonate treated cells (K562 human erythroleukemia cell line) were used as negative and positive internal standards, respectively, in each electrophoresis run. Three steps were followed: (1) assessment of the different levels of variability which may influence the damage levels of the internal standards, (2) evaluation of the variability across separate electrophoresis runs on the quantification of DNA damage in the internal standards by three experimenters involved in different studies and (3) proposal of an adequate calculation system to integrate the internal standards into test sample data. The application of the two proposed models to samples from a human biomonitoring study is presented. The model which calibrates the measurements against the negative internal standard is the most useful since this negative standard was the most stable across experiments and among the three experimenters. The percentage of DNA in the tail is the most appropriate parameter to analyse induced DNA damage, because its interelectrophoresis and interexperimenter variation is less pronounced than that of tail length.

Chronic low-dose maternal exposure to methylmercury enhances epileptogenicity in developing rats.

Effects of continuous low-dose maternal methylmercury intoxication on the induction and propagation of ictal epileptiform activity induced by 3-aminopyridine, were investigated on the neocortex of 4-weeks-old offspring rats. Epileptogenicity was significantly increased in offspring of mercury-treated animals compared to those of controls, characterized by more frequent occurrence of periodic ictal activity, a facilitated propagation of epileptiform discharges and a strong tendency to generalization. The latency of first ictal event was slightly shorter and the average duration of individual ictal periods slightly longer in treated animals. However, the amplitude of seizure discharges was significantly smaller in treated animals than in controls. We conclude, that the synaptic and membrane mechanisms responsible for initiation and propagation of paroxysmal activity were probably facilitated, while the efficacy of cortical inhibition, in preventing initiation and spread of epileptiform discharges was reduced by mercury treatment in the developing nervous system. The smaller amplitude of paroxysmal discharges could be a sign of a remarkable loss of cortical neurons.

Appropriateness of self-medication: method development and testing in urban Indonesia.

BACKGROUND: Most illness episodes are treated by self-medication, however, little is known about the appropriateness of this self-medication. Moreover, tools to evaluate the appropriateness of self-medication still need to be developed. In order to monitor the use of drugs by the general public, we developed methodology (for evaluation of the appropriateness of self-medication) that would be reproducible and would therefore allow comparison over time and between regions. METHOD: For each complaint, criteria for appropriate treatment were set, based on evaluation of both the efficacy and the risks of the medications used. To keep cost at a minimum and to ensure reproducibility, no use was made of expert panels. Instead, only internationally recognized printed sources were used. RESULTS: This study used data on self-medication collected in urban Indonesia in 1993. After excluding illness episodes first treated only with traditional drugs, non-drug treatments or treated by a health worker, we found that self-medication used as a first action was appropriate in 16% of the cases. Fifty-six per cent combined appropriate and unnecessary components and 8% included unnecessary components only. Sixteen per cent of treatments were considered potentially harmful. Only 4.5% of the illness episodes were not treated. Analysis of these potentially harmful treatments showed that over use of antihistamine in children under 5 years of age, use of prescription drugs and multiple intake of paracetamol or antihistamines in different medicines were the main problems. The results of this analysis enabled us to set priorities and formulate recommendations to rationalize the use of drugs in self-medication. CONCLUSION: The proposed methodology should allow international comparisons and the evaluation of the impact of future interventions.