In vitro adherence and kinetics studies of adult human endothelial cell seeded polytetrafluoroethylene and gelatin impregnated Dacron grafts.

Lining the luminal surface of small diameter vascular prostheses with living endothelial cells reduces thrombogenicity, decreases infection and improves patency. In vitro adherence and kinetics studies of adult human endothelial cell seeded Polytetrafluoroethylene (ePTFE) and Gelatin impregnated Dacron (Gelseal) were performed. Endothelial cell adherence on ePTFE and Gelseal coated with collagen IV, fibronectin and preclot matrices was compared. Untreated ePTFE and Gelseal were also used. Ten graft segments in each group coated with these matrices were incubated with radio-labelled adult human endothelial cells for 30, 60 and 90 min. Labelled endothelial cells seeded in supra-confluent densities on fibronectin coated ePTFE and Gelseal grafts were used for kinetic studies. Resultant endothelial cell monolayers were then exposed to varying shear stress at flow rates of 200 and 300 ml/min in an artificial flow circuit. Endothelial cell attachment to untreated grafts was poor and a suitable matrix significantly improved adherence with fibronectin and preclot but less so with collagen. A 30 min incubation was sufficient for optimum cell attachment. Cell adherence to ePTFE was significantly better than Gelseal. Scanning electron micrographs (SEM) of ePTFE showed preferential attachment to the nodes whilst on Gelseal, cells conformed to Dacron fibres at different levels and directions. Rapidly formed endothelial cell monolayers on ePTFE and Gelseal grafts resisted shear stress of flow with significant cell retention at 2 h. There was patchy coverage of both grafts with evidence of bridging of gaps between individual fibres in Gelseal.     

Comparison of different vascular prostheses and matrices in relation to endothelial seeding.

Thin-walled expanded polytetrafluoroethylene (ePTFE), woven Dacron and gelatin-impregnated Dacron (Gelseal) vascular grafts were compared, the grafts being coated with three different matrices: collagen IV, fibronectin and preclot matrix. In addition, untreated ePTFE and Gelseal were examined. The graft segments, coated with these matrices, were incubated with radiolabelled adult human endothelial cells for 30, 60 and 90 min. Endothelial cell adherence was calculated from the ratio of radioactive counts in the grafts to counts in grafts plus supernatants. Endothelial cell attachment to untreated grafts was poor, but a suitable matrix significantly improved adherence. All three matrices tested gave good results, although preclot was best; 30-60 min incubation was sufficient for optimum cell attachment. Cell adherence to both Dacron and ePTFE was significantly better than to Gelseal. The type of prosthetic polymer and the substrate protein coating used to promote endothelial cell adherence are two important factors which may determine the ultimate success of endothelial seeding in the operating room.     

Endothelial cell seeding kinetics under chronic flow in prosthetic grafts

Improved patency of endothelial cell seeded grafts relies on good initial adherence and cell retention when the circulation is restored. In this study human adult endothelial cells (HAECs) were used to evaluate the suitability of commercially available prostheses for seeding. Acutely seeded indum-111 oxine labeled HAECs were used to measure cell adherence to plain and fibronectin (FN)-coated expanded polytetrafluoroethylene (ePTFE), gelatin-impregnated Dacron (Gelseal), and collagen-impregnated Dacron (Hemashield) grafts. Cell loss from FN-coated prostheses, when exposed to a simulated human arterial blood flow of 200 ml/min in an artificial pulsatile circulation, was quantified from the loss of gamma activity from the graft over 24 hours, pressure in the circulation being reduced to 15 mm Hg to reduce fluid loss. Initial HAEC adherence (mean [SD]) to plain grafts was 3(1)%, 47(9)%, and 53(9)% for ePTFE, Gelseal, and Hemashield, respectively. This improved significantly with FN coating (78[6]%, 60[8]%, and 76[4]%). Cell retention after 24 hours of flow to FN-coated grafts was 16(10)%, 25(5)%, and 65(4)% and was confirmed qualitatively by scanning electron microscopy and environmental scanning electron microscopy. FN significantly improved initial cell adherence with Dacron grafts showing the better adherence. Cell retention after 24 hours of flow was better with FN-coated Dacron than with ePTFE but was best with Hemashield grafts.     

The response of rapidly formed adult human endothelial-cell monolayers to shear stress of flow: a comparison of fibronectin-coated Teflon and gelatin-impregnated Dacron grafts.

Endothelial cells labeled with indium 111-oxine were seeded in supraconfluent densities onto fibronectin-coated expanded polytetrafluoroethylene (ePTFE) and gelatin-impregnated Dacron graft segments. These grafts with rapidly formed endothelial-cell monolayers were then exposed to varying shear stresses at flow rates of 200 and 300 ml/min, using tissue culture medium in an artificial flow circuit. As the loss of radioactivity represented endothelial-cell loss, cell retention was calculated by the ratio of counts recorded at different time points during 2 hours of flow to initial counts. Although initial cell adherence to gelatin-impregnated Dacron graft segments was poor compared to ePTFE, once cells were attached they resisted shear stress of flow better at 200 ml/min and equally well at 300 ml/min. The cell retention on fibronectin-coated ePTFE was 55.4 +/- 12.9% at 200 ml/min and 56.5 +/- 15.2% at 300 ml/min; cell retention for gelatin-impregnated Dacron graft segments was 69.0 +/- 6.0% and 66.5 +/- 5.5%, respectively. Qualitatively scanning electron microscopy of both ePTFE and gelatin-impregnated Dacron graft segments showed patchy coverage of grafts with cells. There was preferential attachment of endothelial cells to the nodes on ePTFE, although on gelatin-impregnated Dacron graft segments, cells conformed to the Dacron fibers at different levels and directions with evidence of bridging in the gaps between individual fibers. This study shows conclusively that rapidly formed endothelial-cell monolayers on ePTFE and gelatin-impregnated Dacron graft segments resisted a shear stress of flow equal to that seen in a femoropopliteal vein graft with significant cell retention at 2 hours.     

Complete in vitro prosthesis endothelialization induced by artificial extracellular matrix.

This report presents our research on the conditions necessary to substain optimal in vitro prosthetic endothelialization using human endothelium cultures. Human vein endothelial cells were seeded at a concentration of 3 x 10(5)/cm2 in a gelatinized Dacron patch graft coated with a commercial collagen film, using a solution of fibrin glue. Endothelium adhesion, proliferation, and survival were measured by [3H]thymidine incorporation, after 7 days of incubation. Finally, the morphology of prosthetic endothelialization was analyzed by scanning electron microscopy. We observed that the Dacron patch grafts coated with collagen film were able to promote endothelialization better than the prostheses coated with highly concentrated collagen solution or gelatin. We therefore concluded that the collagen film that supports endothelial cell adhesion and proliferation uniformly covers the entire synthetic endoluminal surface of the Dacron graft, thus preventing endothelial cell alterations induced by direct contact with the synthetic prosthetic surface.     

内皮化小口径人工血管的研究

目的 探讨成人大隐静脉内皮细胞(HSVECs)种植到人工血管内表面的可行性.方法 酶消化法获取成人大隐静脉内皮细胞,在体外扩增培养13～15 d,将扩增培养的内皮细胞种植于纤维蛋白胶预衬的聚四氟乙烯(ePTFE)人工血管内表面,继续体外培养9～12 d.在不同的时间点,分别剪取部分内皮化的人工血管,行荧光显微镜和扫描电镜检查.结果 内皮细胞种植于人工血管后,扫描电镜下可见细胞在血管表面黏附、生长、增殖.平均孵育12 d后,人工血管腔面见一层均匀的基质,其表面有内皮细胞单层,内皮细胞排列紧密,呈梭形.结论 成人大隐静脉内皮细胞可以种植到人工血管,在体外增殖形成内皮细胞单层,达到内皮化的效果.     

Adult human endothelial cell coverage of small-caliber Dacron and polytetrafluoroethylene vascular prostheses in vitro

Culture of endothelial cells on synthetic vascular grafts has heretofore met with limited success. We report here a technique which allows attachment and subsequent growth of adult human vascular endothelial cells on the synthetic materials polytetrafluoroethylene (PTFE) and Dacron which are currently used for vascular reconstructive surgery. Studies were conducted on both untreated materials and those pretreated with the extracellular matrix proteins collagen and fibronectin. Collagen was applied to the graft materials with positive pressure and then allowed to gel in the interstices. Fibronectin was added to the collagen-lined lumen followed by a cell suspension. Cell coverage on the grafts was assessed by scanning electron microscopy after various lengths of time. Cells adhered poorly to and did not grow on untreated Dacron and PTFE. Protein-treated materials did allow cell attachment and growth but with distinct differences. On PTFE (n = 30), cells could form a confluent monolayer within 9 days while cell coverage was generally incomplete at this time on the more irregular surface of Dacron (n = 5). Thus, adult human endothelial cells can grow on collagen- and fibronectin-coated prosthetic materials. This approach to lining graft materials in vitro may be useful in improving the performance of small-caliber vascular grafts.     

ePTFE人工血管材料内衬自体骨髓内皮细胞的实验研究

目的:探讨自体骨髓内皮细胞衬里对ePTFE人工血管材料通畅性的影响。方法:24只犬随机分入实验组(12只)和对照组(12只)。实验组取犬自体骨髓,提取单核细胞种植ePTFE人工血管,行左髂动脉人工血管置换术;对照组采用普通ePTFE人工血管。分别于术后1月、3月行彩色多普勒超声观察其通畅率,并行组织染色及扫描电镜观察,比较人工血管表面内皮化情况。结果:术后1月时实验组人工血管通畅率及内膜厚度同对照组均无明显差别;但术后3月时实验组通畅率高于对照组,内膜厚度明显小于对照组(P0.05)。结论:自体骨髓内皮细胞衬里技术可明显提高ePTFE人工血管的短期通畅率,但其对远期通畅率的影响尚有待进一步观察。     

Partially bioresorbable vascular grafts in dogs.

Previous studies have shown the effectiveness of partially resorbable arterial prostheses in the rabbit. This study compares these same compound prostheses with commercial graft materials in the dog. Conduits 4 mm inner diameter X 50 mm in length were woven from composite yarns containing 69% polyglactin 910 (PG910)/31% polypropylene or containing 70% polydioxanone/30% polypropylene. Nonresorbable controls were woven Dacron and expanded polytetrafluoroethylene (ePTFE). Baseline platelet aggregometry to 10(-5) mol/L adenosine diphosphate was performed. Seventy prostheses were implanted into the aorto-ilac positions, and the prosthesis/tissue complexes were harvested serially from 2 weeks to 1 year. Explanted specimens were photographed and fixed for light microscopy and for scanning and transmission electron microscopy. Results showed no aneurysms or perigraft hematomas. Overall patency for the PG910/polypropylene grafts was 18 of 20 (90%) and for polydioxanone/polypropylene was 19 of 22 (86%). For Dacron and ePTFE, 13 of 19 (68%) and 6 of 11 (54%) remained patent at time of explantation. The partially resorbable grafts, as a group, had significantly greater patency than the control grafts (p less than 0.03). Platelet aggregometry was not predictive of graft patency. Histologic analysis of the partially bioresorbable groups showed inner capsules (IC) composed of myofibroblasts and collagen beneath confluent endothelialized surfaces by 1 month. Kinetics of IC formation paralleled the rates of resorption of the resorbable components. IC cellularity and thickness were greater than those within Dacron or ePTFE. This study suggests an enhanced transinterstitial endothelial cell and myofibroblast ingrowth into the ICs of partially resorbable grafts and shows the effectiveness of these prostheses in the dog.     

Comparative analysis of pseudointima biogenesis in Gelseal coated Dacron knitted graft versus crimped and noncrimped graft

Twenty-one grafts (knitted crimped, 7; knitted Gelseal, 7; knitted no crimp, 7) of 7 cm long were inserted into 6 month-old mongrel puppies as infrarenal aortic tube grafts and retrieved at two-month intervals. All grafts were perfusion fixed in situ. Ex vivo arteriograms were obtained prior to processing for light (LM) and transmission electron microscopic (TEM) studies of tissue ingrowth into the grafts. All grafts were patent at the time of harvesting. The thickness of pseudointima plateaued at 10 weeks. There was no demonstrable discrepancy in thickness between grafts (cell 72 +/- 13). The luminal cells were modified myofibroblasts that contained short microvilli, gap junctions, myofilament, and incomplete basal laminae. The pseudointima consisted of an interlamination of myofibroblasts alternating with extracellular matrix of collagen and ground substances. It was more cellular near the lumen and more fibrocollagenous near the graft. Myofibroblasts were found near the lumen and fibroblasts near the graft. Macrophages, vasa vasorum, leukocytes and fibroblasts occupied the interstitial space between the graft fibrils. Similar cellular and extracellular composition existed in the three types of grafts. There was no local inflammatory reaction encountered in the Gelseal treated graft. Gelseal, crimped and noncrimped knitted Dacron grafts had pseudointima of comparable architecture, thickness, cellular and noncellular composition. Gelseal did not hinder pseudointima formation or induce local inflammatory reaction.     

A comparison between fibronectin and matrigel pretreated ePTFE vascular grafts

Two distinct series of experiments were performed to compare the behavior of ePTFE vascular grafts coated with basement membrane gel to that of identical grafts coated with fibronectin. Bilateral carotid interposition grafts (10 cm long) were interposed in 16 conditioned mongrel dogs. In the first series of experiments (n = 10), each graft was seeded with radiolabeled endothelial cells and initial endothelial cell adherence was determined. Following restoration of blood flow in the grafts, endothelial cell retention was measured for 24 hours. Seeding efficiency was 66.48% (+/- 13.2) for fibronectin-coated grafts and 56.58% (+/- 13.51) for gel-coated grafts. There was a slow, constant loss of activity during the first 90 minutes of imaging, and at 24 hours of observation the activity remaining on the fibronectin-coated graft was 13.2 +/- 3.98% of the initial graft activity. Although the basement membrane gel had a higher mean activity at 24 hours (18.9 +/- 7.22%), the difference was not statistically significant at any interval. In the second series of animals (n = 6), radiolabeled platelets were injected within 60 minutes following restoration of flow. Total platelet activity on the explanted grafts was 3.36 (+/- 1.35) x 10(5) counts per gram/0.2 minute for the fibronectin-coated grafts. The gel-coated grafts had 2.74 (+/- 1.33) x 10(5) counts per gram/0.2 minute, a difference that was not statistically significant. Thus, despite its theoretical appeal, basement membrane gel was no better than fibronectin in increasing endothelial cell adherence and retention, and the resulting flow surface of grafts treated with either compound appeared to attract platelets to an equal degree.     

Endothelial seeding of Dacron and polytetrafluoroethylene grafts: the cellular events of healing

To detect cellular differences in the healing of polytetrafluoroethylene (e-PTFE) and Dacron grafts up to 7 months after implantation, we studied 108 aortic graft interpositions in dogs. Each prosthesis was alternately prepared by endothelial seeding or by an unseeded control method. The grafts were perfusion fixed and studied with light, scanning, and transmission electron microscopy at intervals from before to 221 days after implantation. Seeding resulted in the development of an extensive endothelial flow surface in two out of three of the e-PTFE and none out of four of the Dacron grafts by 10 days after implantation (p = 0.053). After 30 days a microfibrillar subendothelial matrix ranging from 5 to 11 mu formed in all but three grafts with endothelial coverage. The inner capsule of mature Dacron grafts was significantly thicker (169 +/- 143 mu) than in e-PTFE grafts (22 +/- 32 mu; p = 0.002). Seeded and unseeded Dacron grafts had predominantly fibroblasts in the outer capsule of the graft by 10 days. Surface endothelium, vasa vasorum, fibroblasts, and myointimal cells appeared in the inner capsule between 10 and 30 days after implantation. In Dacron grafts, fibroblasts and myointimal cells predominated in the inner capsule at 30 days, with smooth muscle cells not being definitely identifiable until after 150 days. Neither fibroblasts nor myointimal cells were common (present but sparse in one of four e-PTFE grafts) at 30 days, and transmural vasa vasorum were never seen. The seeded endothelial cells migrated rapidly from the sites of initial adhesion near the e-PTFE onto the flow surface. Only one of four of the unseeded e-PTFE grafts had surface endothelium after 30 days, and only moderate coverage developed during 180 days. We conclude that endothelial healing is more rapid in seeded e-PTFE grafts than in seeded Dacron grafts and occurs by a different mechanism.     

Proof of fallout endothelialization of impervious Dacron grafts in the aorta and inferior vena cava of the dog

Purpose: The purpose of this study was to prove directly whether cells from the blood stream contribute to endothelialization of isolated, impervious Dacron vascular grafts in the dog. Methods: We designed an 18 cm, three-component graft with two parallel central Dacron limbs; one was made impervious with silicone rubber, and the other was preclotted. This model was implanted in the canine descending thoracic aorta with 30 μm polytetrafluoroethylene grafts anastomosed at each end. An 8 cm, three-component graft completely coated with silicone rubber was implanted in the canine abdominal aorta and inferior vena cava. Implant periods ranged from 4 to 12 weeks. Flow surfaces were studied by use of stereomicroscopy after being stained with silver nitrate, and by use of scanning and transmission electron microscopy, the inner wall and flow surface were studied by light microscopy after hematoxylin-eosin and immunocytochemical staining (the latter for endothelial and smooth muscle cells), and the full wall was studied by light microscopy after hematoxylin-eosin staining. Results: Effective prevention of pannus and transmural ingrowth into the impervious central test grafts was achieved, and scattered islands of endothelial cells were conclusively demonstrated on flow surfaces in each of the three implant sites 4 weeks after implantation. In the descending thoracic aorta, where these grafts were also implanted for 8 and 12 weeks, α-actin – positive cells and microvessels were found beneath some of the endothelial islands. Conclusion: Fallout endothelialization of Dacron vascular grafts occurs in both the arterial and venous systems of the dog. (J VASC SURG 1994;20:546-57.)     

Longevity of expanded polytetrafluoroethylene grafts for superior vena cava

We previously reported that the expanded polytetrafluoroethylene (ePTFE) graft for superior vena cava (SVC) substitution presents the problems of flexion and kinking when the graft is long. We therefore replaced the SVC of dogs with two types of prosthetic substitutes, ePTFE (Gore-Tex) and spiral-supported ePTFE (Im/praflex), and evaluated the long-term patency of the prosthetic substitutes. Total replacement of the SVC was performed in 9 adult mongrel dogs. The substitutes were ePTFE and spiral-supported ePTFE in 5 and 4 dogs, respectively. The animals were killed about 3 years after replacement of the SVC, and the harvested specimens were histologically examined by light microscopy and scanning electron microscopy. Evaluation of ePTFE revealed late occlusion in 1 of 5 dogs. The spiral-supported ePTFE showed patency in all dogs. In the group with ePTFE grafts, light microscopic examination revealed abnormalities of endothelial cells, granulation, and necrosis. There was no hyperplasia of the subendothelial connective tissue near the center. In the animals with spiral-supported ePTFE grafts, the subendothelial connective tissue showed favorable growth even in the center of the reconstructed site. There was no granulation in the spiral-supported ePTFE group. Scanning electron microscopic examination in the ePTFE group showed that endothelial cells were spindle-shaped and had an irregular surface. The spiral-supported ePTFE group showed an almost regular form of endothelial cells and no abnormalities except for the slightly spindled shape in the center. Therefore, we recommend that spiral-supported ePTFE should be used as an SVC substitute in clinical situations.     

Fibronectin binding to gelatin-impregnated Dacron (Gelseal) prostheses

Endothelial cell adherence to uncoated gelatin-impregnated Dacron (Gelseal) is poor but can be significantly improved by precoating with a suitable basement membrane such as fibronectin. To assess the suitability of fibronectin-coated Gelseal for endothelial cell seeding, fibronectin binding to Gelseal and its dissociation kinetics were investigated. Fibronectin binding was quantified by radiolabeling human fibronectin with iodine 125, concentrations of 10, 25, 50, 150, and 250 micrograms/ml being used to coat Gelseal at 30, 60, and 90 min of incubation. The amount of fibronectin bound was directly proportional to the concentration used and increased with time of incubation (p < 0.05). However, the percentage attachment decreased with increasing concentration (p < 0.001). The number of molecules bound per centimeter squared of graft was calculated. In the first 30 min, 75% of bound fibronectin was lost after exposure to a flow rate of 200 ml/min in a pulsatile artificial circulation; thereafter, the fibronectin-Gelseal bond was stable for up to 2 h.     

Sequential studies of healing in endothelial seeded vascular prostheses: Histologic and ultrastructure characteristics of graft incorporation

Chronological events leading to incorporation of endothelial cell seeded prosthetic vascular grafts were documented in this investigation. Forty-one adult dogs underwent thoracoabdominal bypass using double-velour Dacron grafts. Experimental grafts were preclotted with blood containing enzymatically derived endothelium immediately after derivation, or after 14 days of cultivation. Control grafts were preclotted without addition of endothelial cells. Grafts were studied grossly as well as by light, scanning electron, transmission electron, and fluorescence microscopy, 1 to 28 days postimplantation. Control graft healing proceeded from pannus and perigraft ingrowth. Experimental grafts healed from seeded cells as well. Platelets covered all grafts by Days 1 and 2. Thrombus accumulations on control grafts, first evident on Day 4, became maximal by Day 14. Seeded grafts appeared relatively thrombus free with patches of endothelial cells noted by 4 days. These cells were initially separated by gaps, often containing leukocytes. Endothelium became densely packed with cellular migration and proliferation. Subendothelial tissues were composed of fibrin and smooth muscle. Control and experimental grafts were approximately 10 and 80% endothelialized, respectively, by Day 28. Smooth muscle dominated subintimal tissue in experimental grafts. These cells initially appeared fibroblastoid. Endothelial seeding enhances both pseudointimal development and rapid graft incorporation.     

Durability of confluent endothelial cell monolayers on small-caliber vascular prostheses in vitro

Since implantation of small-caliber vascular grafts with preformed confluent endothelial cell monolayers (ECMs) may prevent acute platelet deposition and thrombus formation, we have evaluated the conditions necessary to produce durable ECMs. Cultured human umbilical-vein ECs in buffer were attached to vascular grafts--either expanded polytetrafluoroethylene (ePTFE) or knitted Dacron, both with inside diameters of 4 mm--precoated with collaten type I and perfused in vitro with serum-containing culture medium to achieve cell spreading into confluent monolayers. The number of cells attached was quantified by DNA measurements and indium-111 labeling. Morphology was evaluated by scanning electron microscopy. The maximum number of cells that attached acutely was 3.6 X 10(5) cells/cm2 graft, and the minimum number needed for confluence was 1.4 X 10(5) cells/cm2 graft. Confluence was morphologically complete after 2 hours of in vitro perfusion at 15 ml/min. When ECMs were exposed to varying flow rates, cell retention after 1 hour was 96.1% +/- 5.6% at 50 ml/min, 94.6% +/- 6.1% at 100 ml/min, 77.6% +/- 10.8% at 200 ml/min (p less than 0.001), and 40.1% +/- 10.4% at 400 ml/min (p less than 0.0001). Confluence was maintained for all grafts exposed to flows of 100 ml/min or less. Fewer cells were retained when acutely attached, unspread cells (71.5% +/- 11.5%) were compared with established ECMs (89.9% +/- 6.7%) at a flow rate of 100 ml/min (p less than 0.0001). ECMs on knitted Dacron were more durable than on ePTFE (82.7% +/- 5.3% versus 75.5% +/- 4.8% remained attached at 200 ml/min, P less than 0.05). 111Indium-labeled and unlabeled cells were equivalent with respect to saturation level attachment, spreading time to confluence, and durability under flow at 100 ml/min. We conclude that confluent and durable endothelial cell monolayers can be established on small-caliber vascular grafts within 2 hours.     

Adult human endothelial cell seeding using expanded polytetrafluoroethylene vascular grafts: a comparison of four substrates

Prosthetic small-caliber vascular grafts give poorer patency rates than autogenous vein grafts, possibly because the former never spontaneously form endothelium. Animal studies have shown that endothelialization of prosthetic grafts can be encouraged by seeding endothelial cells into the graft at the time of surgery, resulting in improved patency. Information regarding the attachment characteristics of adult human endothelial cells to prosthetic grafts is, however, sparce. Laboratory experiments were performed by use of cell culture techniques to compare the attachment characteristics of adult human endothelial cells to expanded polytetrafluoroethylene graft material, both untreated and treated, with one of four protein substrates--preclotted blood, fibronectin, laminin, and type 4 collagen. Attachment characteristics were compared quantitatively by use of attachment assays and qualitatively by scanning electron microscopy. Attachment to untreated expanded polytetrafluoroethylene was poor but could be greatly improved by preclotting or precoating with any of the proteins, particularly fibronectin. In preclotted grafts seeded endothelial cells formed a virtually confluent monolayer after 1 hour. Cell attachment to the other grafts coated with protein was patchy and inconsistent. It is concluded that a rapid confluent endothelial lining within a prosthetic vascular graft is possible, and of the substrates examined, preclotted blood best encourages cell attachment to expanded polytetrafluoroethylene.     

Endothelial cell seeding of a new PTFE vascular prosthesis

Previous attempts to line polytetrafluoroethylene (PTFE) prostheses with enzymatically derived endothelial cells have not been as successful as similar work with Dacron grafts because of the failure of such prostheses to develop a satisfactory subendothelium. This article reports our experience with a new, highly porous, unreinforced PTFE prosthesis that appears to circumvent this problem. Segments (4 mm I.D., 10 cm in length) of this new graft were implanted in 41 mongrel dogs as carotid interposition grafts. One graft in each dog was seeded with the dog's own endothelial cells, whereas the contralateral graft was treated in an identical fashion except for the inclusion of endothelium. After a mean period of 7 weeks of implantation, the grafts were harvested, their patencies were noted, the thrombus-free area of their luminal surface was calculated with computerized quantitative planimetry, and graft segments were submitted for scanning and transmission electron microscopy. In seven dogs the luminal surface was scraped from each graft and measured quantitatively. Although seeded grafts failed to show a statistically significant increase in patency during the short course of this experiment, a trend in that direction was quite striking. Furthermore, seeded grafts had a significant increase in thrombus-free area on their luminal surface as well as a significant reduction in the volume of luminal thrombus. Histologically, seeded grafts developed a substantial 75 to 100 microns cellular subendothelium beneath a confluent endothelial lining. No endothelial lining was noted in control grafts. We believe that the superior handling characteristics of this new prosthesis and its ability to develop a substantial subendothelium with a confluent endothelial lining suggest potential future applications and warrant further investigation.     

Role of myofibroblasts in pseudointima formation

Twenty Dacron grafts (10 crimped and 10 noncrimped) were studied with light microscopy, scanning electron microscopy, and transmission electron microscopy (TEM) 1 to 10 months after insertion into the canine thoracoabdominal aorta. Fibroblast-derived pseudointima maintained a smooth luminal lining that approximated the luminal cross-sectional area of the aorta. Turbulent flow at the corrugated surface of crimped grafts was reflected by the nonuniform alignment of the luminal cells. TEM demonstrated that fibroblasts, macrophages, vasa, and nerve fibers had infiltrated the interstices of the grafts. A zone of loose connective tissues and ground substances existed between the graft surface and the pseudointima and occupied the zone between the luminal cells and the body of the pseudointima. The core of the pseudointima consisted of dense fibrous connective tissue elements and myofibroblasts arranged in a laminated pattern. The luminal cells that were seen were myofibroblasts containing microvilli rather than true endothelial cells. A gradual transition of fibroblast to myofibroblast from the graft to the lumen existed. Myofibroblasts with active rough endoplasmic reticula and Golgi complexes were responsible for formation of ground substances and extracellular matrix. Our observation strongly suggests that the cellular lining in healed Dacron prostheses is derived from modified fibroblasts or multipotential cells. These cells have the capability of transforming into endothelioid and myoblastoid cells as dictated by the functional need.