Point-of-Care Adipose-Derived Stromal Vascular Fraction Cell Isolation and Expanded Polytetrafluoroethylene Graft Sodding.

Adipose-derived stromal vascular fraction (SVF) cell populations are being evaluated for numerous clinical applications. The current study evaluated a point-of-care technology, the Tissue Genesis "TGI 1000" Cell Isolation System™, to perform an automated isolation of adipose-derived SVF cells to be used in the fabrication of a tissue-engineered vascular graft in the operating room. A total of seven patients were enrolled in this study and received femoral to tibial expanded polytetrafluoroethylene bypass grafts to treat peripheral arterial disease. Lipoaspiration of fat was performed on five patients, and the fat sample was processed immediately in the automated system in the operating room. The mean processing time, from the point of fat delivery into the instrument to removal of the SVF-containing syringe, was 70 min. The SVF cell population was evaluated for cell yield, cell viability, endotoxin levels, and microbial contamination. Samples of the SVF preparation were further subjected to microbiologic evaluation both microscopically before implantation of the graft and through a microbiologic screening using aerobic and anaerobic culture conditions. Mean cell yield was 1E5 cells per cc of fat, and endotoxin levels were below the FDA recognized standards. All SVF preparations were released for graft preparation, and the intimal surface of 90-cm-long grafts was pressure sodded with cells at a concentration of 2E5 cells/cm2. The sodded grafts (n = 5) and control grafts (n = 2) were immediately implanted and graft patency assessed for 1 year. One year patency was 60% for sodded grafts and 50% for control grafts. Automated preparation of autologous adipose-derived SVF cells for immediate use to create cellular linings on vascular grafts is feasible and safe.

Adipose stromal vascular fraction cells isolated using an automated point of care system improve the patency of expanded polytetrafluoroethylene vascular grafts.

We evaluated the use of an automated, point-of-care instrument to derive canine adipose stromal vascular fraction cells, and the subsequent deposition of these cells onto the luminal surface of an expanded polytetrafluoroethylene (ePTFE) vascular graft for use as a bypass graft. The hypothesis evaluated was that an instrument requiring minimal user interface will provide a therapeutic dose of cells to improve the patency of synthetic vascular grafts in an autologous animal model of graft patency. The stromal vascular fraction (SVF) cells were isolated using an automated adipose tissue processing and cell isolation system and cells sodded onto the surface of an ePTFE vascular graft. Control grafts, used off-the-shelf without cell treatment were used as a control to assess patency effects. Each animal received a control, untreated graft implanted in one carotid artery, and the cell-treated graft implanted in the carotid artery on the contralateral side. The grafts were implanted for 6 months utilizing 12 animals. Results indicate a fully automated adipose tissue processing system will consistently produce functional autologous cells for immediate use in the operating room. Cell-sodded polymeric grafts exhibited improved patency compared to control grafts after 6 month implantation in the canine carotid artery model.

Accelerated neovascularization and endothelialization of vascular grafts promoted by covalently bound laminin type 1.

Development of a small diameter (<6 mm) synthetic vascular graft with clinically acceptable patency must overcome the inherent thrombogenicity of polymers and the development of neointimal thickening. Establishment of an endothelial cell lining on the lumenal surface has been hypothesized as a mechanism to improve the function of vascular grafts. The major aim of this study is to evaluate the use of laminin type 1, covalently bound to all surfaces of expanded polytetrafluoroethylene (ePTFE) grafts, on neovascularization of the interstices and lumenal surface endothelialization. One millimeter i.d. vascular grafts were surface modified through covalent attachment of laminin type 1. Grafts were subsequently implanted as interpositional aortic grafts in rats. Following 5-weeks implantation, the grafts were explanted and morphologically evaluated using scanning electron microscopy and light microscopy. Scanning electron microscopy identified an extensive coverage of antithrombogenic cells on the lumenal flow surface of laminin type 1 modified grafts. Histological evaluation confirmed the presence of endothelial cells on the midgraft lumenal surface of laminin 1 modified grafts. Extensive neovascularization of the interstices of the laminin-modified grafts occurred as compared with control grafts. We conclude that surface modification using laminin type 1 accelerates both the neovascularization and endothelialization of porous ePTFE vascular grafts.

Prophylactic gatifloxacin therapy in prevention of bacterial keratitis in a rabbit laser in situ keratomileusis model.

PURPOSE: Use the ID(50) (infectious dose to 50% of experimental animals) to quantify the most effective prophylactic dosing regimen to use with gatifloxacin 0.3% (Zymar) for the prevention of keratitis in a rabbit laser in situ keratomileusis model of Staphylococcus epidermidis infection. SETTING: University Laboratory, University of Arizona, Tucson, Arizona, USA. METHODS: Two groups of rabbits were compared in each of 2 experiments that were separated by 12 months. In the first experiment, rabbits receiving no postoperative antibiotic therapy (Group 1) were compared with rabbits receiving postoperative antibiotic therapy (Group 2). In the second experiment, postoperative antibiotic therapy (Group 3) was compared with preoperative and postoperative antibiotic therapy (Group 4). All antibiotic regimens used gatifloxacin 0.3%. Before antibiotic therapy began, corneal pockets were created in the right eye of each rabbit and all rabbits received balanced salt solution (BSS) only or BSS and S epidermidis inoculations in the corneal pocket. Rabbits were monitored for corneal infiltrates after surgery. RESULTS: The ID(50) of the first, second, third, and fourth groups of rabbits was 10(2), 10(4), 10(5), and 10(7) organisms, respectively. The data showed a statistically significant difference between rabbits receiving BSS only and most rabbits receiving BSS plus inoculate at each postoperative measurement (P<.05). CONCLUSIONS: The findings suggest that the use of both preoperative and postoperative antibiotic therapy may be most effective in preventing infection. Postoperative antibiotic therapy increased the number of S epidermidis necessary to cause infection by at least 100-fold over no antibiotic intervention. Preoperative plus postoperative antibiotic therapy increased the number of bacteria necessary to cause infection by at least 100-fold over postoperative therapy alone and by more than 10000-fold over no antibiotic intervention.

Covalent modification of porous implants using extracellular matrix proteins to accelerate neovascularization.

Healing associated with many polymeric biomedical implants commonly involves the formation of an avascular fibrous capsule. The lack of either formation or persistence of blood vessels in formed fibrous capsules, as well as a lack of new blood vessels within porous polymeric implants, often results in poor performance of the implant. The current study evaluated the use of extracellular matrix protein modification of a commonly used biomedical implant material, expanded polytetrafluoroethylene (ePTFE), as a mechanism to increase the neovascularization both within these porous implants and in tissue that forms in the peri-implant area. Discs of ePTFE were covalently modified with different extracellular matrix proteins including collagen type IV, fibronectin, and laminin type I. Discs were implanted into the adipose tissue of adult rats, and following a 5-week implant phase, histologic analysis of peri-implant tissue angiogenesis and implant neovascularization was performed. Striking differences were observed in angiogenic and neovascularization responses to matrix-modified ePTFE when compared with control, untreated ePTFE. Fibronectin treatment resulted in an extensive inflammatory response but, relative to the degree of inflammation, limited evidence of tissue angiogenesis or polymer neovascularization. Collagen type IV treatment groups exhibited a significant increase in angiogenesis in the peri-implant tissue with minimal evidence of implant neovascularization. In contrast to all other implant modifications, laminin type 1-treated ePTFE samples stimulated an extensive peri-implant tissue angiogenic response and a coordinate neovascularization of the porous interstices of the biomaterial.

Dual porosity expanded polytetrafluoroethylene for soft-tissue augmentation.

BACKGROUND: A variety of nondegradable polymers have been evaluated for use as soft-tissue augmentation devices. This study compared a novel dual porosity expanded polytetrafluoroethylene with current, clinically used devices. METHODS: Studies were performed in a porcine model of soft-tissue healing with both histologic evaluations and determination of biomechanical strength of tissue incorporation. Five different samples of expanded polytetrafluoroethylene were used in this study. Control devices were clinically available soft-tissue augmentation devices manufactured by W. L. Gore and Associates (Newark, Del.). Atrium Medical Corporation (Hudson, NH) manufactured three test devices with modified porosities. A total of 12 animals were used with implant evaluations performed after 1, 3, 6, and 12 months. RESULTS: Significant differences in tissue incorporation were observed morphologically with the dual porosity material, including reduced inflammation and increased cellular and extracellular matrix incorporation of the material. Significant increases in both angiogenesis (new vessel formation in the peri-implant tissue) and neovascularization (blood vessel penetration into the interstices of the implants) were observed with the dual porosity expanded polytetrafluoroethylene material. CONCLUSIONS: This novel dual porosity expanded polytetrafluoroethylene is associated with reduced inflammation and more extensive tissue incorporation as compared with the currently available form. These results suggest a dual porosity expanded polytetrafluoroethylene may provide a superior material for soft-tissue augmentation.

Characterization of angiogenesis and inflammation surrounding ePTFE implanted on the epicardium.

The response of epicardial tissue to the implantation of expanded polytetrafluoroethylene (ePTFE) was evaluated and compared with identical material implanted within subcutaneous and adipose tissues. These two tissue environments were selected for comparison with epicardial implants because they represent tissue often involved in device implantation. Discs of ePTFE (6 mm) were implanted into three different tissue sites in Sprague-Dawley rats. At 5 weeks, polymers and surrounding tissues were harvested and processed for light microscopy. General histology and histochemistry data indicated all polymers to be well incorporated with new tissue. Subcutaneous implants were covered by a dense fibrous capsule (55-70 microm). Epicardial and adipose implants had no fibrous capsule and a significantly greater number of microvessels (arterioles, capillaries, and venules) within the surrounding tissues compared with subcutaneous implants. An increased level of inflammation was also observed around epicardial implants compared with the other implants. Additionally, the new vasculature surrounding epicardially implanted ePTFE revealed an altered microvessel density and vessel type distribution compared with normal (control) epicardium. These results suggest that epicardial tissue responds to implanted ePTFE with a robust inflammatory response that may support the formation of a new microvasculature that is uniquely different from the native epicardial microvasculature.