Scaffold-based three-dimensional human fibroblast culture provides a structural matrix that supports angiogenesis in infarcted heart tissue.

BACKGROUND: We have developed techniques to implant angiogenic patches onto the epicardium over regions of infarcted cardiac tissue to stimulate revascularization of the damaged tissue. These experiments used a scaffold-based 3D human dermal fibroblast culture (3DFC) as an epicardial patch. The 3DFC contains viable cells that secrete angiogenic growth factors and has previously been shown to stimulate angiogenic activity. The hypothesis tested was that a viable 3DFC cardiac patch would stimulate an angiogenic response within an area of infarcted cardiac tissue. METHODS AND RESULTS: A coronary occlusion of a branch of the left anterior descending coronary artery was performed by thermal ligation in severe combined immunodeficient mice. 3DFCs with or without viable cells were sized to the damaged area, implanted in replicate mice onto the epicardium at the site of tissue injury, and compared with animals that received infarct surgery but no implant. Fourteen and 30 days after surgery, hearts were exposed and photographed, and tissue samples were prepared for histology and cytochemistry. Fourteen and 30 days after surgery, the damaged myocardium receiving viable 3DFC exhibited a significantly greater angiogenic response (including arterioles, venules, and capillaries) than nonviable and untreated control groups. CONCLUSIONS: In this animal model, viable 3DFC stimulates angiogenesis within a region of cardiac infarction and can augment a repair response in damaged tissue. Therefore, a potential use for 3DFC is the repair of myocardial tissue damaged by infarction.

Development and characterization of tissue-engineered aortic valves.

Tissue-engineered aortic valves, known as recellularized heart valves, were developed by seeding human neonatal fibroblasts onto decellularized, porcine aortic valves. Recellularized heart valves were cultured up to 8 weeks in a novel bioreactor that imposed dynamic pulsatile fluid flow to expose the dermal fibroblasts to mechanical forces. Our data showed that, under static or dynamic flow conditions, dermal fibroblasts attached to and migrated into the decellularized, porcine valve scaffolding. The human cells remained viable as indicated by MTT viability staining. Gradual colonization of the decellularized porcine scaffolding by the human dermal fibroblasts was shown histologically by hematoxylin & eosin staining, immunocytochemically using a monoclonal antibody directed against prolyl-4-hydroxylase (an intracellular enzyme expressed by human fibroblasts synthesizing collagen), and quantitative digital image analyses. Thymidine and proline radiolabeled analog studies at 1, 2 and 4 weeks of individual leaflets cultured statically demonstrated that the human fibroblasts were mitotic and synthesized human extracellular matrix proteins, thereby supplementing the existing porcine matrix. The overall approach results in a heart valve populated with viable human cells. In the development of valves that perform in a similar manner as natural biological structures, this approach may present some unique benefits over current medical therapies.

The in vitro growth of a three-dimensional human dermal replacement using a single-pass perfusion system.

A human dermal replacement has been developed by seeding human neonatal dermal fibroblasts onto a biosorbable polyglactin (polyglycolide/polylactide) mesh and culturing in a bioreactor. The mesh provides the proper environment for the cells to attach, grow in a three-dimensional array, and establish a tissue matrix over a 2- to 3-week culture period. The dermal replacement has been characterized and found to contain a variety of naturally occurring dermal matrix proteins, including fibronectin, glycosaminoglycans, and collagen types I and III. To efficiently and reproducibly produce this dermal tissue equivalent, a closed, single-pass perfusion system was developed and compared with a static process. In the single-pas perfusion system, growth medium (containing ascorbic acid) was perfused around the 4 x 6 in. pieces of mesh at specific flow rates determined by nutrient consumption and waste production rates. The flow rates used for this system indicate that a diffusion-limited regime exists with a mean residence time greater than 1 h for essential nutrients and factors. By controlling glucose concentrations in the system to a delta of 0.70 g/L from the inlet to the outlet of the bioreactor, it took 6 fewer days to grow a tissue similar to that produced by the static system.

Characterization, barrier function, and drug metabolism of an in vitro skin model.

We have characterized an in vitro skin model consisting of neonatal keratinocytes and fibroblasts grown on a nylon mesh. To produce a dermal model, fibroblasts were seeded onto nylon mesh and grown for 4 weeks until a physiologic dermal-like matrix was formed. This matrix was found to consist of collagens I and III, fibronectin, and glycosaminoglycans. Keratinocytes were then seeded onto the dermal model and the co-culture was grown at the air/liquid interface. A differentiated epidermis with distinct basal, spinous, granular, and stratum corneum layers was formed. When incubated in the presence of keratinocytes, fibronectin immunofluorescence increased throughout the dermis compared to cultures incubated similarly in the absence of keratinocytes. A basement membrane zone rich in laminin, collagen IV, and heparan sulfate proteoglycan was detected. The epidermis, isolated from the co-culture by thermolysin digestion, was analyzed for differentiation markers. K1 keratin (67-kDa) and involucrin were detected by immunologic techniques. Ceramide lipids (types III and IV), thought to be important in barrier function, were detected by thin-layer chromatography. The permeability of the co-culture to a panel of compounds, including [3H]-water, was determined using Franz and side-by-side diffusion cells. The permeability coefficient for water was of the same order of magnitude as that determined for neonatal foreskin. The co-culture also showed selective permeability to a panel of compounds of differing lipid solubility. This co-culture metabolized [3H]-testosterone to a profile of metabolites similar to that of neonatal foreskin. We believe that this in vitro skin model will be useful for the study of drug permeability and metabolism.

Disinfection of bacteria in water systems by using electrolytically generated copper:silver and reduced levels of free chlorine.

As an alternative disinfectant to chlorination, electrolytically generated copper:silver (400 and 40 micrograms/L copper and silver, respectively) with and without free chlorine (0.3 mg/L) was evaluated over a period of 4 weeks in indoor and outdoor water systems (100 L tap water with natural body flora and urine). Numbers of total coliform, pseudomonas, and staphylococci were all less than drinking water standards in systems treated with copper:silver and free chlorine and systems treated with free chlorine alone (1.0 mg/L). No significant differences (p less than or equal to 0.05) in bacterial numbers were observed between systems with copper:silver and free chlorine and those with free chlorine alone. Overall, free-chlorine treatments (0.3 or 1.0 mg/L) showed significantly lower heterotrophic plate numbers than those without free chlorine. When challenged with a natural Staphylococcus sp. isolate, water with copper:silver and free chlorine had a 2.4 log10 reduction in bacterial numbers within 2 min, while free chlorine alone or copper:silver alone showed 1.5 and 0.03 log10 reductions, respectively. Addition of copper:silver to water systems may allow the concentration of free chlorine to be reduced while still providing comparable sanitary quality of the water.

Evaluation of immunofluorescence techniques for detection of Cryptosporidium oocysts and Giardia cysts from environmental samples.

Cryptosporidium and Giardia species are enteric protozoa which cause waterborne disease. The detection of these organisms in water relies on the detection of the oocyst and cyst forms or stages. Monoclonal and polyclonal antibodies were compared for their abilities to react with Giardia cysts and Cryptosporidium oocysts after storage in water, 3.7% formaldehyde, and 2.5% potassium dichromate, upon exposure to bleach, and in environmental samples. Three monoclonal antibodies to Cryptosporidium parvum were evaluated. Each test resulted in an equivalent detection of the oocysts after storage, after exposure to bleach, and in environmental samples. Oocyst levels declined slightly after 20 to 22 weeks of storage in water, and oocyst fluorescence and morphology were dull and atypical. Oocyst counts decreased after exposure to 2,500 mg of sodium hypochlorite per liter, and fluorescence and phase-contrast counts were similar. Sediment due to algae and clays found in environmental samples interfered with the detection of oocysts on membrane filters. Two monoclonal antibodies and a polyclonal antibody directed against Giardia lamblia cysts were evaluated. From the same seeded preparations, significantly greater counts were obtained with the polyclonal antibody. Of the two monoclonal antibodies, one resulted in significantly lower cyst counts. In preliminary studies, the differences between antibodies were not apparent when used on the environmental wastewater samples. After 20 to 22 weeks in water, cyst levels declined significantly by 67%. Cysts were not detected with monoclonal antibodies after exposure to approximately 5,000 mg of sodium hypochlorite per liter.

Efficacy of copper and silver ions and reduced levels of free chlorine in inactivation of Legionella pneumophila.

Water disinfection systems utilizing electrolytically generated copper and silver ions (200 and 20, 400 and 40, or 800 and 80 micrograms/liter) and low levels of free chlorine (0.1 to 0.4 mg/liter) were evaluated at room (21 to 23 degrees C) and elevated (39 to 40 degrees C) temperatures in filtered well water (pH 7.3) for their efficacy in inactivating Legionella pneumophila (ATCC 33155). At room temperature, a contact time of at least 24 h was necessary for copper and silver (400 and 40 micrograms/liter) to achieve a 3-log10 reduction in bacterial numbers. As the copper and silver concentration increased to 800 and 80 micrograms/liter, the inactivation rate significantly (P less than or equal to 0.05) increased from K = 2.87 x 10(-3) to K = 7.50 x 10(-3) (log10 reduction per minute). In water systems with and without copper and silver (400 and 40 micrograms/liter), the inactivation rates significantly increased as the free chlorine concentration increased from 0.1 mg/liter (K = 0.397 log10 reduction per min) to 0.4 mg/liter (K = 1.047 log10 reduction per min). Compared to room temperature, no significant differences were observed when 0.2 mg of free chlorine per liter with and without 400 and 40 micrograms of copper and silver per liter was tested at 39 to 40 degrees C. All disinfection systems, regardless of temperature or free chlorine concentration, showed increase inactivation rates when 400 and 40 micrograms of copper and silver per liter was added; however, this trend was significant only at 0.4 mg of free chlorine per liter.