[Endothelialization of 6 mm vascular prostheses with microvascular endothelial cells from fatty tissue of the omentum]. / Endothelialisierung von 6-mm-Gefässprothesen mit mikrovaskulären Endothelzellen aus dem Fettgewebe des Omentums.

The seeding of 6 mm Dacron prostheses with microvascular endothelial cells from omental adipose tissue leads to endothelialized prostheses, 5 weeks later with a complete coverage of functional and thrombus free endothelium. The method is ready for clinical trials.

[Development of a small-lumen vascular prosthesis coated with autologous endothelial cells]. / Entwicklung einer kleinlumigen mit autologen Endothelzellen beschichteten Gefässprothese.

A new compliant prosthesis with a monolayer of autologous endothelial cells (ENC) has been developed. It consists of a porous polyurethane-siloxane-copolymer reinforced by a polyester network to prevent excessive dilatation. On the inner surface an ENC monolayer is established before implantation by a cell culture procedure. The prosthesis displays compliance (13.2 +/- 3.0 x 10(-4) mmHg-1) comparable to native arteries. It is non-kinkable (minimal radius of curvature less than 5 mm). Burst resistance, remaining deformation, cut out force and tensile strength are superior to standard values. ENC-coverage in excess of 95% of the inner surface was produced in vitro using a lining procedure. The monolayer of confluent cells was demonstrated to consist of endothelial cells by their characteristic cobblestone morphology, the expression of factor VIII related antigen and the specific uptake of Dil-Ac-LDL. The unstimulated prostacyclin production was similar both in native veins as well as in lined prostheses. Antithrombogenicity of the endothelial cell lining was demonstrated in 24 h animal implants.

An improved procedure for enzymatic harvesting of highly purified canine venous endothelial cells for experimental small diameter vascular prostheses.

We developed a new device, the vein holder, to improve yield and purity of enzymatic harvests of venous endothelial cells. External jugular veins of mongrel dogs were dissected by a no-touch technique. In vitro length and circumference of the vein segments were decreased to about half of the in situ dimensions. The vein holder enabled mounting of the veins at 80% of their in situ length during endothelial cell harvesting. Trypan blue staining and scanning electron microscopic observations revealed that vein eversion as well as the new vein holder technique successfully removed the endothelium. Endothelial cell harvests by the eversion technique were, however, low and varied in size, viability, and purity. In contrast, the defined handling by the new vein holder technique regularly provided markedly increased amounts of endothelial cells. Most of the cells attached and developed cultures consisting of endothelial cells only, as shown by the uptake of DilAcLDL. Prostacyclin production of confluent cultures was similar to that of native veins. It is concluded that minimal handling, defined mounting, and prevention of overfilling the vein markedly improves endothelial cell harvests, providing greater amounts of viable and purified endothelial cells.

A compliant small-diameter vascular prosthesis lined with functional venous endothelial cells.

The long-term patency of small-diameter vascular grafts is still unsatisfactory. In contrast to native arteries, they are inelastic and lack active antithrombogenicity. To improve long-term patency, a new 4 mm internal diameter prosthesis was developed which is compliant and lined with functional endothelial cells (ENC). The wall of this prosthesis consists of a microporous polyurethane-siloxane copolymer reinforced with a polyester network. It displays compliance (13.2 x 10(-4) mmHg-1) comparable to native arteries, is nonkinkable (minimum radius of curvature = 5 mm), burst resistant, and easily suturable. Using a lining procedure, coverage of prostheses by ENC was in excess of 95%. The ENC populations were found to be highly pure (by factor VIII-related antigen, DilAcLDL uptake) and to produce about 0.3 ng prostacyclin per cm2. In vitro tests of shear stress resistance demonstrated that ENC monolayers on the new elastic prosthesis remain intact for 3 hr in physiologically pulsating culture medium (Vmax = 50 cm/sec). Lined prostheses implanted for 24 hours in mongrel dogs as an arteriovenous shunt demonstrated the antithrombogenicity of the cultured ENC. The results suggest that small-diameter vascular prostheses which are compliant, porous, and actively antithrombogenic are feasible.

[Development of small-diameter vascular prostheses covered with human endothelial cells: Possibilities and limits of in vitro-tests]

There is an increasing demand for small-diameter vascular prostheses for the replacement of arteriosclerotic coronary arteries. They may be replaced by autologous blood vessels, usually parts of the saphenous vein. Prostheses of synthetic materials and an inner diameter of less than 4 to 6 mm are unsatisfactory and, therefore, not implanted for coronary arteries. A substantial improvement is, however, expected for prostheses covered with human autologous endothelial cells. It has to be proved that this new type of vascular prostheses is an adequate replacement for small arteries. Tests of the new prosthesis should comprise cell and tissue compatibility of the synthetic materials as well as normal function of the endothelial cells. The aim of the present paper was to reduce the number of animal experiments in this development by establishing new in vitro tests for endothelial cell compatibility of synthetic materials and for the adherence of endothelial cell on the prosthesis. Physiologically haemodynamic streaming conditions are in vitro produced by self-constructed circulatory systems. First results demonstrate that physiologic shear stress is achieved. Limits and relevance of the in vitro tests are discussed in relation to animal experiments and clinical studies.

Glucose uptake and phosphorylation in fat cells of fasted and hypophysectomized rats.

Fat cells of hypophysectomized and fasted rats metabolize 10 times less glucose than adipocytes of normal rats in the presence of insulin. Glucose transport (3-O-methylglucose influx), transport plus phosphorylation (2-deoxyglucose uptake), hexokinase, pyruvate dehydrogenase and glucose-6-phosphate dehydrogenase activities were determined in an attempt to localize the metabolic defects. Insulin stimulates 3-O-methylglucose influx 5-fold in normal cells and 3-fold in cells of fasted rats. The basal influx in cells of fasted rats is increased and even more so in cells of hypophysectomized rats where the rate of basal influx is the same as that in cells of normal rats under maximal insulin stimulation. It cannot be further stimulated by insulin. In contrast to 3-O-methylglucose influx, basal uptake and phosphorylation of 2-deoxyglucose in cells of fasted and hypophysectomized rats is drastically decreased and stimulation by insulin is abolished. Total hexokinase and pyruvate dehydrogenase activities are drastically reduced in the homogenate of fat cells of hypophysectomized and fasted rats. Phosphorylation by hexokinase appears to become one of the rate-limiting steps of glucose metabolism in cells of hypophysectomized rats.

Microalbuminuria and nerve conduction velocity in type-I diabetics during conventional therapy and during continuous i.v. insulin infusion.

The objective of this study was to follow the development of microalbuminuria and nerve conduction velocity under continuous i.v. insulin therapy over a limited period of 4 months. For this purpose, 8 labile type I diabetics were selected (age 33 +/- 8 years, duration of diabetes 16 +/- 9 years) and treated conventionally with two insulin injections daily over 4 months. Afterwards, the same patients were treated with continuous i.v. insulin infusion and finally again with two injections daily over 4 months each. This procedure allowed each diabetic to serve as his own control. HbA1, microalbuminuria, nerve conduction velocity and relative refractory period of the ulnar nerve were checked at monthly intervals. During the continuous i.v. infusion over 4 months, blood sugar values were significantly lower, glucosuria had disappeared almost completely and the glycosylated hemoglobin had fallen to near normal values. The mean rate of albumin excretion was 16 +/- 5 micrograms/min at rest and 76 +/- 26 micrograms/min during exercise (normal: 3.9 +/- 0.4 and 4.8 +/- 1.2 microgram/min, respectively) and did not change significantly. Nerve conduction velocity in the ulnar nerve rose significantly under i.v. insulin therapy from 47.9 +/- 0.6 m/sec to 52 +/- 0.6 m/sec. Similarly, the relative refractory period of the same nerve fell significantly from 3.7 +/- 0.2 to 1.9 +/- 0.1 msec (i.e. to within normal range). It is concluded that functional disturbances of peripheral nerve can regress by improved blood sugar control with continuous i.v. insulin infusion over 4 months. On the other hand, incipient microangiopathy measured as microalbuminuria remains unchanged over the same period of time. If an improvement is at all possible, considerably longer periods of euglycemia are likely to be necessary.