Nondestructive and noninvasive assessment of mechanical properties in heart valve tissue engineering.

Despite recent progress, mechanical behavior of tissue-engineered heart valves still needs improvement when native aortic valves are considered as a benchmark. Although it is known that cyclic straining enhances tissue formation, optimal loading protocols have not been defined yet. To obtain a better understanding of the effects of mechanical conditioning on tissue development, mechanical behavior of tissue constructs should be monitored and controlled during culture. However, currently used methods for mechanical characterization (e.g., tensile and indentation tests) are destructive and are only performed at the end-stage of tissue culture. In this study, an inverse experimental-numerical approach was developed that enables a noninvasive and nondestructive assessment of mechanical properties of engineered heart valves. The applied pressure and volumetric deformation of an engineered heart valve were measured during culture, and served as input for the estimation of mechanical properties using a computational model. To validate the method, six heart valves were cultured, and the mechanical properties obtained from the inverse experimental-numerical approach were in good agreement with uniaxial tensile test data. The method provides a real-time, noninvasive and nondestructive functionality and quality check of tissue-engineered heart valves and can be used to monitor and control the evolution of mechanical properties during tissue culture.

Quantitative assessment of collagen assembly by live cells.

Changes in supramolecular assembly of matrix, specifically collagen, have important functional consequences, especially for tissues requiring high mechanical strength. Thus modulation of collagen assembly could be used as a therapeutic intervention or to control the development of tissue-engineered constructs containing natural matrix. Quantitative methods that monitor such effects currently are lacking. Using live cultured cells, we developed a convenient way either to visualize by fluorescence microscopy or to measure directly, using a high throughput fluorescence assay, the supramolecular assembly of FITC-labeled collagen monomers. The wide applicability of this assay was confirmed by testing the assay using two major collagen sources, rat tail and bovine skin, and vascular smooth muscle cells from two different origins, mouse aorta and human saphenous vein. We further determined that treatments that interfere with the function of the cytoskeleton modulate collagen assembly. Use of positive and negative regulators of lysyl-oxidase indicated that while the assay does not require active production of endogenous collagen, it can be used to monitor the incorporation of such de novo synthesized collagen into labeled fibrils. Thus we have designed a novel quantitative assay that can monitor assembly of exogenous and endogenous collagen by live cells and reveal the effects of various interventions upon this process.

Direct assessment and diminished production of morphine stimulated NO by diabetic endothelium from saphenous vein.

AIM: To directly measure in real time basal and stimulated levels of NO released from human saphenous vein endothelium and to quantify the expression of the mu opiate receptor, which has been linked with NO release. METHODS: Saphenous vein segments from patients with type 2 diabetes (n=12) and patients without diabetes (n=8) were obtained. The release of NO was measured directly from the endothelium using a NO-specific amperometric probe. N(Omega)-nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L), a NO synthase (NOS) inhibitor, or morphine (1 mumol/L), a stimulant, was administered and the measurements were repeated. Values were reported relative to the mean initial measurement of NO release from diabetic endothelium, which was defined as the relative zero level of NO release. A RT-PCR was then performed on the endothelium to measure mu opiate receptor expression. RESULTS: Diabetic patients (n=12) showed a relative and significantly diminished basal level of released NO, (0.049+/-0.012) nmol/L, compared with non diabetic patients (n=8), (0.42+/-0.12) nmol/L (P<0.05). Application of L-NAME to nonstimulated tissues resulted in no change in NO release from the diabetic group and a decrease in NO release of (0.21+/-0.09) nmol/L from the non diabetic group (P<0.05). Morphine stimulation of the diabetic endothelium resulted in a lower peak and shorter duration of NO release compared to the non-diabetic tissue, (21+/-6) nmol/L vs (38+/-4) nmol/L and (7.3+/-1.4) min vs (12.2+/-2.2) min, respectively (P<0.01). Lastly, evaluation of the mu opiate receptor expression was found to be diminished in the diabetics by 59.1 %. CONCLUSION: Maturity-onset diabetes attenuates both the constitutive basal and morphine stimulated NO release from human saphenous vein endothelium. In this study, after NOS inhibition, the actual basal NO release in diabetes was negligible. One explanation for the impaired capacity of diabetic endothelium to release NO was the diminished mu opiate receptor that was seen in diabetic endothelium.

Adult human saphenous vein endothelial cells: assessment of their reproductive capacity for use in endothelial seeding of vascular prostheses.

Autogenous endothelial seeding (AES) of vascular prostheses (VP) using venous endothelial cells (EC) reduces platelet-VP interactions and improves patency rates in small caliber VP in dogs. To conserve patients' veins for use in coronary or limb bypass surgery, human trials of AES should require proof that adequate numbers of EC with the growth capacity to cover VP can be harvested from acceptably small pieces of peripheral vein. EC were isolated from excess saphenous vein segments remaining after coronary bypass surgery by filling veins with 0.1% CLS II collagenase at 37 degrees C for 15 min and removing EC by flushing the veins with culture medium. EC were cultured on fibronectin-coated dishes in medium 199 with 30% human serum and 300 micrograms/ml of endothelial cell growth factor. These cells grew to form confluent monolayers, and were identified as EC by tests for factor VIII antigen. Veins from 53 patients with a mean age of 55.8 +/- 9.8 (SD) years yielded vein segments with an average area of 1.9 +/- 0.6 cm2, from which an average of 5.3 +/- 2.8 X 10(4) cells were removed per cm2 of vein area. EC in culture underwent 14.3 +/- 1.4 population doublings with an average population doubling time of 1.8 +/- 0.3 days (N = 14 cultures), which allowed an 100-fold increase in cell number to occur in 11 to 12 days. These data suggest that the EC available from small vein segments in adult humans have the growth capacity to cover areas comparable in size to the luminal areas of VP commonly used in arterial surgery.