Leigh B. Kleinert

The effects of porosity on endothelialization of ePTFE implanted in subcutaneous and adipose tissue.

Healing of biomaterial implants varies depending on the type and structure of material and the tissue surrounding the implant. In this study we examined structural differences of 30 microm, 60 microm, and 100 microm expanded polytetrafluoroethylene (ePTFE) using scanning electron microscopy, and we also investigated differences in healing for these three different porosity ePTFE grafts implanted within subcutaneous tissue and adipose tissue. Scanning electron microscopic examination of 30 microm, 60 microm, and 100 microm ePTFE revealed structural differences and differences in fiber density within the internodal space. Circular patches (6 mm in diameter) of 30 microm ePTFE were implanted within subcutaneous tissue and epididymal fat pads of male Sprague-Dawley rats. After 5 weeks, the implants were removed and analyzed for fibrous capsule formation, endothelialization, and for activated monocytes and macrophages in association with the material. Histological evaluation revealed dense fibrous capsule formation surrounding only the 30 microm ePTFE subcutaneous implants. From immunohistochemistry data obtained, we generated an Endothelialization Index (measure of neovascularization) and a Monocyte/Macrophage Index (measure of inflammatory response) for each sample. Consistently, 60 microm ePTFE had the greatest Endothelialization Index at both implant sites while 100 microm ePTFE generally had the largest values for the Monocyte/Macrophage Index. These data indicate that both the structure of the material and the site of implant influence the healing characteristics of ePTFE and suggest that activated monocytes and/or macrophages associated with the implant may inhibit endothelialization of ePTFE.

Cellular proliferation and macrophage populations associated with implanted expanded polytetrafluoroethylene and polyethyleneterephthalate

The chronic inflammatory response associated with the abluminal surface of polymeric vascular grafts has been suggested to affect adversely graft neovascularization, the cellular response at the luminal surface of vascular grafts, and overall graft patency. To better understand the source for this chronic inflammation, this study examined two types of macrophages and the amount of cellular proliferation around two widely used graft materials, expanded polytetrafluoroethylene (ePTFE) and polyethyleneterephthalate (PET or Dacron) implanted in the rat for 3 and 5 weeks. Serial sections of explants were analyzed for recruited macrophages (ED1), resident macrophages (ED2), and proliferating cells (PCNA). Results show that Dacron is more inflammatory than ePTFE and that there is a segregated macrophage response; the first 54 micrometer of perigraft tissue were composed predominantly of recruited macrophages (ED1+) while the more distal tissue consisted of resident macrophages (ED2+). Proliferating cells were located predominantly in this same 54 micrometer perigraft region. In subcutaneous tissue they accounted for 23% of all cells present around Dacron after 3 weeks of implantation and 8% after 5 weeks. Conversely, cellular proliferation around ePTFE increased from 4% at 3 weeks to 21% at 5 weeks. In adipose tissue, proliferation levels around the implanted polymers were lower and more similar after 3 and 5 weeks. Serial sections revealed the coordinate expression of PCNA and ED1 antigens by the same individual cells, suggesting that proliferation is a mechanism used to perpetuate the chronic inflammatory response. These results suggest a new target for designing treatments to alter inflammation and improve the healing associated with these biomaterials.

Differential healing and neovascularization of ePTFE implants in subcutaneous versus adipose tissue

The preclinical evaluation of polymer biocompatibility is often performed using animal subcutaneous implant models. The choice of subcutaneous tissue as the implant site is due to a number of factors including simplicity of the surgery involved. Results from subcutaneous implants cannot necessarily be extrapolated to other tissues due to the differences in cellular composition of tissues. We have evaluated and compared the healing characteristics of expanded polytetrafluoroethylene (ePTFE) discs implanted in either subcutaneous tissue or epididymal fat pad tissue in rats. Following 3 and 5 weeks of implantation, the healing characteristics of discs were evaluated histologically with particular emphasis on tissue and polymer neovascularization. Implants placed in subcutaneous tissue exhibited limited formation of new microvascular elements within and directly in contact with the polymer, and the formation of an extensive fibrous capsule. In contrast, ePTFE implanted in the epididymal fat pads of rats exhibited extensive neovascularization of tissue surrounding the polymer, penetration of these microvascular cells into the graft interstices for distances < or = 100 microns and no morphological evidence of a fibrous capsule. The rat epididymal fat pad provides an alternative tissue for polymer healing evaluations. Due to the extensive presence of fat in subcutaneous tissue in humans, we suggest the fat pad model provides a more relevant preclinical evaluation of the healing characteristics of polymers used clinically in anatomic positions which contain significant amounts of fat.

Inflammation and Neovascularization Associated with Clinically Used Vascular Prosthetic Materials

This study was designed to evaluate and compare healing characteristics, specifically neovascularization and inflammation, of polymeric vascular graft materials commonly used in clinical applications. Our hypotheses were (i) polymeric materials used in vascular graft manufacture stimulate chronic inflammation and (ii) inflammation and neovascularization of polymeric materials are related. Impra and Gore-Tex ePTFE, Meadox weavenit and woven Dacron, Hemashield microvel and woven Dacron, and Golaski microknit Dacron were implanted as 6-mm diameter disks within rat subcutaneous and adipose tissue. Following 5 weeks of implantation samples were evaluated by histological and immunocytochemical analysis. Sections were stained using hematoxylin and eosin or reacted with ED1 antibody and GS1 lectin to quantify inflammation and neovascularization. respectively. The extent of inflammation and neovascularization were influenced by both tissue site of implantation and polymer characteristics. For subcutaneous implants, inflammation was graded as follows: Meadox weavenit > Hemashield woven > Meadox woven > Gore-Tex ePTFE > Hemashield microvel > ImpraePTFE > Golaski microknit, while only the Golaski microknit neovascularized. Inflammation was graded as follows for adipose implants: Hemashield woven > Hemashield microvel > Meadox weavenit > Meadox woven > Gore-Tex ePTFE > Golaski microknit > Imnpra ePTFE, while the following order of neovascularization was observed: Impra ePTFE > Gore-Tex ePTFE > Golaski microknit. The degree of inflammation following biomnaterial implantation has a profound effect on implant neovascularization. These data suggest an inverse relationship exists between inflammation and neovascularization.

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.

Endothelial Cell Transplantation onto Polymeric Arteriovenous Grafts Evaluated Using a Canine Model

Prosthetic arteriovenous grafts (AVG) placed for hemodialysis access fail in humans due to the thrombogenicity of the flow surface and development of cellular intimal hyperplasia, particularly at the venous anastomosis. The poor patency rates of prosthetic AVG result in significant morbidity and mortality in dialysis patients. Consequently, investigators have been evaluating methods to improve the patency of prosthetic grafts by examining endothelial cell transplantation as a means of creating an antithrombogenic lining on artificial polymers. A canine model was developed to study the effects of cell transplantation of autologous, fat-derived microvessel endothelial cells (MVEC) onto the luminal surface of expanded polytetrafluoroethylene (ePTFE) grafts. Microvessel endothelial cells were isolated from falciform ligament fat, with each dog receiving its own endothelial cells. Isolated cells were subsequently placed into the lumen of the graft (4 mm by 20 cm ePTFE). The graft lumen was pressurized to 5 pounds per square inch (psi) resulting in the partial denucleation of the graft, due to the flow of buffer into the interstices of the graft, and the forced deposition of cells onto the luminal surface. Animals were maintained on aspirin and persantine during the implant phase. During the implant phase, grafts were evaluated by both duplex ultrasound and magnetic resonance angiography (MRA). At explant, gross observation of the sodded grafts revealed a glistening white flow surface with no evidence of thrombosis. Morphologic and scanning electron microscopic evaluations revealed the presence of a cellular lining on the luminal flow surface that exhibited characteristics of antithrombogenic endothelial cells. Midgraft samples were evaluated by immunocytochemistry and indicated that cells on the luminal surface react positively with antibodies to von Willebrand factor. Results from this study demonstrate that the canine model provides an excellent method of studying the effects of MVEC sodding on the thrombogenicity and hyperplastic response of prosthetic arteriovenous graft.

Anastomotic tissue response associated with expanded polytetrafluoroethylene access grafts constructed by using nonpenetrating clips

PURPOSE The gross, light microscopic, and scanning microscopic appearance of arterial and venous anastomoses in expanded polytetrafluoroethylene (ePTFE) access grafts constructed with nonpenetrating clips were compared with that of those constructed with polypropylene suture. We hypothesized that clip-constructed anastomoses would provide controlled approximation of native vessel intimal and medial components with the ePTFE grafts. We further hypothesized that anastomotic healing with clips would involve primarily an intimal cellular response, as compared with suture-constructed anastomoses in which cells within the media and adventitia walls participate. METHODS Femoral artery to femoral vein arteriovenous (AV) grafts were constructed in five dogs using 4-mm internal diameter ePTFE graft material. Each animal received one AV graft with anastomoses constructed by using polypropylene sutures in one leg and one AV graft with anastomoses constructed with Vascular Closure System clips in the contralateral leg. Animals were given aspirin for the duration of the study, and grafts were explanted at 5 weeks. At the time of explantation, graft segments were grossly evaluated and then underwent light and scanning electron microscopic analysis. RESULTS At the time of explantation, all access grafts were patent. Joining the ePTFE grafts to the native vessels with clips resulted in minimal vessel wall damage. The lumenal contours of the discontinuous approximation were smooth and without gross endothelial disruption. These observations are in contrast to the lumenal compromise and endothelial disturbance associated with the sutured anastomoses. Furthermore, hemostasis was achieved immediately in the clipped grafts, decreasing the incidence of perianastomic hematoma. Finally, cellular reconstitution occurred at the anastomotic cleft in both the sutured and the clipped junctions. The neointima exhibited an endothelial cell lining on the lumenal surface and the presence of alpha-smooth muscle cell actin positive cells within the subendothelial layer. CONCLUSION Vascular Closure System clips are a viable alternative to suture for the approximation of ePTFE AV access grafts to native blood vessels. The use of the clips resulted in a more streamlined anastomosis, with decreased vessel wall damage, immediate hemostasis, and a trend toward shorter procedure times.

Vein patching reduces neointimal thickening associated with prosthetic graft implantation

PURPOSE Modified anastomotic techniques utilizing autogenous vein-cuffs or patches have been devised with the hope of improving prosthetic graft patency. The mechanisms of the presumed improvement in patched anastomoses have never been elucidated and remain speculative. We characterized the healing response of the Taylor vein patch in prosthetic arteriovenous fistulae in a canine model of intimal hyperplasia. METHODS Six adult dogs underwent placement of bilateral (6 patched, 6 control) 4-mm diameter expanded polytetrafluoroethylene loop femoral artery-vein fistulae. Serial duplex ultrasound examinations confirmed graft patency until explant at 6 weeks. Differential light microscopy with computerized image analysis was performed on serial 5-microm sections. Intimal thickness through the venous anastomosis and outflow veins of Taylor patch and control (nonpatched) grafts were compared. Cell type-specific immunocytochemical antibody stains for smooth muscle cells (alpha SMC actin) and endothelial cells (von Willebrand factor) were performed. RESULTS Eleven of 12 grafts remained patent for 6 weeks, 1 control graft thrombosed. Mean duplex-derived peak systolic velocities of patched (96 cm/sec) and control (108 cm/sec) grafts were similar. Microscopy revealed more intimal pannus anastomotic suture line ingrowth in controls (mean thickness = 178 microm) than Taylor patched grafts (mean 147 microm, p = 0.0002). Significantly less intimal thickening was present in the outflow vein of patched (mean thickness = 90 microm) versus control grafts (mean 195 microm, P <0.0001). The intima maintained a single cell layer of vWF + endothelial cells, while the majority of the cells comprising the lesion expressed alpha SMC actin. CONCLUSION Perianastomotic pannus is primarily composed of intimal smooth muscle cells. Neointimal thickening is significantly reduced in prosthetic arteriovenous fistulae created with the Taylor vein patch in a canine model. Reduction in perianastomotic intimal thickening may explain the reported clinical improvement in prosthetic bypass graft patency when modified with vein patch techniques.

Origin of endothelial cells that line expanded polytetrafluoroethylene vascular grafts sodded with cells from microvascularized fat

PURPOSE Cell transplantation onto prosthetic vascular grafts remains an attractive technique to reduce the thrombogenicity of polymeric materials. In this study we evaluated whether autologous cells isolated from falciform ligament fat and transplanted onto the lumenal surface of 4 mm expanded polytetrafluorethylene grafts were the same cells present on the surface of these grafts when they were explanted from canine carotid arteries 3 weeks after their implantation. METHODS The fluorescent dye PKH-26 was used to label transplanted cells to evaluate their fate after implantation of grafts as carotid artery replacements. This fluorescent dye homogeneously labeled all cells in the primary cell isolate. RESULTS In vitro studies indicated that dye labeling was nontoxic, as evidenced by the normal growth characteristics of fluorescently labeled cells compared with nonlabeled cells. Immunocytochemical analysis of microvascularized fat before cell isolation determined that approximately 90% of the cells stained positive for von Willebrand factor2. At the time of explant, seeded grafts exhibited a nonthrombogenic lumenal cell lining as evidenced by the lack of adherent platelets or fibrin. Cells on the lumenal surface of grafts exhibited PKH-26 fluorescence emission. In addition, these cells expressed von Willebrand factor and actively sequestered DiI-acetylated low-density lipoprotein. CONCLUSIONS We conclude that sodding of prosthetic grafts with autologous microvascularized fat-derived cells results in the formation of an endothelial cell lining on the lumenal flow surface. These endothelial cells are the same cells placed on the lumenal surface of the graft at the time of initial cell transplantation. Finally, a confluent monolayer forms after high-density cell sodding by the process of cell adherence and spreading, without the need for cell proliferation.

The neointima formed in endothelial cell sodded ePTFE vascular grafts results from both cellular-hyperplasia and extracellular-hypertrophy.

Endothelial cell transplantation onto polymeric vascular grafts results in the formation of a neointima. The formation of this neointima is often suggested to result from a chronic cellular hyperplasia where the terms intimal hyperplasia and intimal thickening are used interchangeably. While the formation of a midgraft neointima in sodded grafts involves a level of cell proliferation, the synthesis and deposition of extracellular matrix proteins is also a ubiquitous observation in these grafts. To assess the composition of midgraft neointima in sodded grafts, a morphometric method was developed to provide a differential quantitation of the cellular-hyperplastic and extracellular-hypertrophic elements of intimal thickening. The formed neointima on microvessel endothelial cell sodded and control (noncell-treated) ePTFE vascular grafts was quantified after 3, 12, and 52 wk of graft implantation in a canine carotid artery model. Midgraft sections of grafts were evaluated for both intimal thickness (IT) and cell density per unit volume and quantified using a PC-based image analysis program. Sodded grafts explanted at 3 wk exhibited an average neointimal cell density (3 x 10(9) cells/cm3; IT 30 microns) equivalent to cell densities observed in normal arterial media. After 12 wk the mean cell density approached a hyperplastic value (3.7 x 10(9) cells/cm3; IT 76 microns), while grafts explanted after 52 wk exhibited a mean cell density (2.8 x 10(9) cells/cm3; IT 30 microns) similar to 3-wk values. Control grafts that received no cells exhibited no midgraft cellular coverage. These results indicate that neointima formation in the midgraft region of sodded grafts occurred via mechanisms involving both a cellular hyperplasia and an extracellular hypertrophy. Differential responses occur presumably due to localized differences in cellular proliferation and cellular biosynthetic activity.