A completely biological tissue-engineered human blood vessel
Nicolas L'heureux1,a, Stéphanie Pâqueta, Raymond Labbéa, Lucie Germaina, and François A. Augera,1
a Laboratoire d'Angiogénèse Expérimentale/LOEX, Hôpital du Saint-Sacrement and Department of Surgery, Faculty of Medicine Laval University, Québec City, Québec G1S 4L8, Canada
Mechanically challenged tissue-engineered organs, such as blood vessels, traditionally relied on synthetic or modified biological materials for structural support. In this report, we present a novel approach to tissue-engineered blood vessel (TEBV) production that is based exclusively on the use of cultured human cells, i.e., without any synthetic or exogenous biomaterials. Human vascular smooth muscle cells (SMC) cultured with ascorbic acid produced a cohesive cellular sheet. This sheet was placed around a tubular support to produce the media of the vessel. A similar sheet of human fibroblasts was wrapped around the media to provide the adventitia. After maturation, the tubular support was removed and endothelial cells were seeded in the lumen. This TEBV featured a well-defined, three-layered organization and numerous extracellular matrix proteins, including elastin. In this environment, SMC reexpressed desmin, a differentiation marker known to be lost under standard culture conditions. The endothelium expressed von Willebrand factor, incorporated acetylated LDL, produced PGI2, and strongly inhibited platelet adhesion in vitro. The complete vessel had a burst strength over 2000 mmHg. This is the first completely biological TEBV to display a burst strength comparable to that of human vessels. Short-term grafting experiment in a canine model demonstrated good handling and suturability characteristics. Taken together, these results suggest that this novel technique can produce completely biological vessels fulfilling the fundamental requirements for grafting: high burst strength, positive surgical handling, and a functional endothelium
Key Words: tissue engineering • vascular graft • cultured cells • ascorbate • mechanical strength
Source: The FASEB Journal. 1998;12:47-56.
IN THE LAST 10 TO 15 YEARS, tremendous progress in cell biology and cell culture have led to the birth of the field of tissue engineering. One of the primary objectives in this field is to use cultured human cells to recreate functional tissues and organs in order to provide "replacement parts" that can be grafted into humans. Historically, tissue-engineered organs have relied on synthetic materials to provide a scaffolding effect and mechanical strength. This has been especially true for tissue-engineered vascular grafts where mechanical strength and suturing characteristics are critical. However, despite vast improvements in the field of biomaterials, a completely biocompatible material is still not available for the production of vascular grafts.
The small-diameter synthetic vascular grafts that are presently implanted (inside diameter 5 mm) have shown poor patency rates, particularly in below-the-knee locations where low blood flow and high resistance increase the risks of thrombus formation (1–3). Initial strategies to increase patency focused on graft pacification, using various protein coatings to minimize blood/biomaterial interactions (4–6). More recently, several investigators have seeded the lumen of synthetic vascular grafts with various cell types, mostly endothelial cells, to create a living hemocompatible lining (7–10). Although some of these reports have shown promising results, synthetic grafts may still induce a low-level foreign body reaction/chronic inflammation, which could be a key event in late graft failure (11). Furthermore, vascular grafts made from synthetic materials can lead to bacterial colonization and subsequent graft infection, which is associated with mortality and amputation rates (12, 13).
We hypothesize that a tissue-engineered blood vessel (TEBV)3 composed exclusively of biological ma~terials and autologous vascular cells would have several theoretical advantages. First, a living graft implies a responsive and self-renewing tissue with an inherent healing potential. Second, its biological matrix can be remodeled by the body according to the needs of the environment. Third, the absence of synthetic material will preclude foreign body reaction, allow complete graft integration, limit graft infection, and may result in increased overall patency rates (14–16). In 1986, Weinberg and Bell (17) were the first to produce a completely biological TEBV from animal collagen gels and cultured bovine endothelial cells (ECs), smooth muscle cells (SMC), and fibroblasts. Unfortunately, this model did not display the requisite mechanical strength and, even reinforced with Dacron meshes, failed to show a burst strength that would allow its in vivo grafting. Using similar techniques, our group (18) has developed and reported a completely biological TEBV from cultured human vascular cells and human collagens and encountered the same mechanical limitations. Recently, Hirai and Matsuda (19) constructed a similar canine model that withstood venous grafting when supported by a Dacron mesh, but ruptured when grafted alone.
In this report, we present a radically different approach to TEBV production based exclusively on the use of cultured human cells, without synthetic or exogenous biological material. This is the first biological model to demonstrate sufficient mechanical strength to warrant its in vivo grafting. Our innovative approach takes advantage of the abundant endogenous synthesis of extracellular matrix (ECM) by mesenchymal cells when cultured in the presence of ascorbic acid. This TEBV displayed histological organization, ECM composition, cell differentiation markers, and cellular functions observed in normal human blood vessels. Furthermore, its grafting potential was established by a short-term implantation in a canine model.
Cell isolation and culture
Human umbilical vein endothelial cells were obtained by the method of Jaffe et al. (20). Briefly, umbilical cords were collected from healthy newborns in ice-cold culture media. Veins were cannulated at both ends, washed with calcium-free HEPES buffer (10 mM HEPES, 119 mM NaCl, 6.7 mM KCl, and 11 mM glucose, at pH 7.35), and a warm collagenase solution (0.160 U/ml in HEPES buffer with 5 mM CaCl2) was injected to rinse and fill the vein. After a 15 min incubation at 37°C, the veins were gently massaged and vigorously perfused with medium. The cell solution obtained was centrifuged, and the cell pellet was resuspended in medium for endothelial cells [medium M199 supplemented with 20% fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/ml of heparin, 25 µg/ml of endothelial cell growth factor supplement (Sigma, St. Louis, Mo.), 100 U/ml of penicillin G, and 25 µg/ml of gentamicin]. ECs were then plated on gelatin-coated tissue culture flasks. The endothelial nature of these cells was confirmed by von Willebrand factor expression and acetylated low density lipoprotein (ac-LDL) incorporation (21). Human vascular SMC were isolated from umbilical veins by the explant method of Ross (22) and cultured in standard medium (Dulbecco's modification of Eagle's medium-Ham's F12 modified medium (3:1), 10% FBS and antibiotics). SMC identity was confirmed by SMC specific -actin immunostaining (23). Human skin fibroblasts were obtained from normal adult skin specimens removed during reductive breast surgery of healthy human subjects (15–37 years old). Small skin fragments were then floated on a 500 µg/ml thermolysin solution in HEPES buffer for 2 h at 37 °C. The dermis was separated from the epidermis with forceps, cut into small pieces, and incubated for 20 h at 37 °C in a solution of collagenase (200 U/ml in Dulbecco's modification of Eagle's medium). After centrifugation, fibroblasts were plated into tissue culture flasks in standard medium. Fibroblasts did not express EC or SMC markers. All cells were plated at a density of 104 cells/cm2, maintained at 37°C in a humidified atmosphere (92% air and 8% CO2), and used between passages 3 and 7. Cells were tested at different passages for mycoplasma infection with Hoechst fluorescent staining for cytoplasmic DNA and were always found to be negative (24).
To induce ECM formation, SMC and fibroblasts were cultured in standard culture medium supplemented with 50 µg/ml of sodium ascorbate in 75 cm2 culture flasks. After approximately 30 days, both cell types formed sheets, comprising cells and ECM, that could be manually peeled off from the culture flask. These sheets could be wrapped around an inert tubular support to produce a cylinder composed of concentric sheet layers. After a maturation period, the layers adhered firmly to one another, forming a cohesive tubular tissue. From this basic technique, we then developed a sequential approach to TEBV construction. The first step was to produce an acellular inner membrane (IM) by dehydrating a tubular tissue made with a fibroblast sheet. The second step was to slip the IM around a perforated tubular mandrel (polytetrafluoroethylene, outside diameter 3.0 mm) and roll a sheet of SMC around it to produce a vascular media. At this stage, the construct was placed in a bioreactor designed to provide both luminal flow of culture medium and mechanical support. After a week of maturation, the third step was to roll a sheet of fibroblasts around the vascular media to provide an adventitia. Finally, after a maturation period of at least 8 wk, the inner tubular mandrel was removed and the TEBV was either tested for mechanical strength or cannulated at both ends for luminal endothelial cell seeding as described before (18). During maturation, tissues were cultured in standard medium with 50 µg/ml of sodium ascorbate except after EC seeding, when EC medium was used. Overall, the production of the graft involves a culture period of 3 months: 3 wk for SMC sheet formation, 1 wk for media maturation, 7 wk for adventitial maturation, and 1 wk for EC growth. This does not include IM production or cell expansion.
Five weeks of culture were found to produce fibroblast sheets with an optimal strength to culture time ratio for a given maturation period (see Results). Consequently, all ad~ventitias or IM were produced from 35-day-old sheets. IM were produced in advance from dehydrated adventitias, stored at -20°C, and rehydrated before use. When adventitias were produced separately for mechanical testing, a 4.6 mm styrene mandrel was used.
For EC staining, living tissues were labeled with CMFDA (5-chloromethylfluorescein-diacetate; Molecular Probes, Eugene, Oreg.) for 2 h (10 µM) and with DiI-ac-LDL (DiI=1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine chlorate; BTI, Stoughton, Mass.) for 3 h (7.5 µg/ml) in standard culture conditions. Cells were then fixed in 4% formaldehyde, rinsed in phosphate-buffered saline, permeabilized with 0.1% saponin, and incubated with a mouse anti-human von Willebrand factor monoclonal antibody (Chemicon, El Segundo, Calif.) for 45 min, rinsed, and incubated with a Cascade blue conjugated goat anti-mouse antibody (Molecular Probes) for 30 min. Green, red, and blue stains were taken as separate color micrographs, using appropriate filters on a Nikon epifluoroscopic microscope, and digital images were overlaid as true optical colors. For medial staining, frozen cross sections were fixed in acetone and incubated with mouse anti-desmin monoclonal antibody (Sigma) and Texas red conjugated goat anti-mouse antibody (Molecular Probes). Nuclei were stained blue with Hoechst 33258. For adventitial staining, frozen cross sections were fixed in cold acetone and double stained with rabbit anti-human elastin antibody (A. Grimaud, Institut Pasteur, Lyon, France), mouse anti-vimentin monoclonal antibody (N. Marceau, Hôtel-Dieu, Québec, Canada), FITC conjugated goat anti-rabbit (Cederlane, Hornby, Canada), and Texas red conjugated goat anti-mouse antibody.
Adventitias (inside diameter 4.6 mm) were cannulated on a specially designed system and pressurized with phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4). Hydrostatic pressure was increased by 5 mmHg steps until vessel failure. Fresh human saphenous veins (HSV) were diameter matched with TEBV (inside diameter 3 mm) and free of collaterals. HSV were unused segments of carefully dissected autografts from patients undergoing distal vascular reconstruction.
Gelatinase activity was determined using 10% sodium dodecyl sulfate-polyacrilamide gels copolymerized with 3.5% gelatin as described before (25). Briefly, conditioned culture medium samples were centrifuged at 10000 g and immediately frozen at -20°C until use. After running, gels were rinsed for 15 min in washing buffer (50 mM Tris, pH 7.4) and then for 30 min in buffer containing 2.5% Triton X-100. They were rinsed once more in washing buffer for 15 min and put overnight at 37°C in digestion buffer (50 mM Tris, pH 7.4, containing 10 mM CaCl2 and 100 mM NaCl) under slow agitation. Gels were fixed with a 10% methanol solution containing 10% acetic acid, stained with 0.05% Coomassie blue prepared in the fixative, and scanned. All mediums were conditioned for 48 h and sample volumes were adjusted for total culture medium volumes.
Cell density was obtained by counting endothelial cell nuclei per surface area of Hoechst 33258 stained en face prepara~tions of endothelialized IM using a Nikon epifluoroscopic microscope equipped with appropriate UV filters. Five specimens were used and five randomly chosen fields were counted per specimen. Cell coverage of the luminal surface was calculated from morphometric analysis of hematoxylin stained en face preparation of endothelialized IM. Six specimens were used and three to five randomly chosen fields were counted per specimen. In both cases cells were fixed with 10% formalin for 30 min prior to staining. Prostacyclin (PGI2) synthesis was measured by detection of its stable degradation product 6-keto-prostaglandin F1α with a radioimmonoassay kit (Amersham, U.K.) in culture medium conditioned by confluent ECs on the IM. EC medium was perfused at a rate of 2.6 ml/h for 12 h with or without thrombin (2 U/ml). For platelet adhesion studies, fresh heparinized human blood from healthy male volunteers (14 U/ml) was slowly perfused (0.2 ml/min) in the lumen of an IM or an endothelialized IM with an eight-roller peristaltic pump at 37°C for 30 min. Lumens were gently flushed with culture medium to remove unattached platelets, fixed in 2% glutaraldehyde, and processed for scanning electron microscopy (SEM). Three separate experiments were performed with three endothelialized and unendothelialized vessels. Each vessel was cut into five specimens. Platelets were counted in three random fields from each specimen.
ECs were omitted to avoid acute rejection. Consequently, dogs were anticoagulated with warfarin (days -1 to 7) to raise their prothrombin time to twice the normal value. Dogs also received acetylsalicylic acid (325 mg/day on days -2 to 7) and heparin (100 U/kg on days 0 and 1). Immunosuppression was obtained with cyclosporin-A at a blood concentration of 500 ng/ml (days -2 to 7). Anastomosis was performed using a continuous running suture of 6–0 polypropylene. Graft patency was monitored daily by Doppler signaling techniques. Animal experiments were approved by the Ethics Committee of Laval University in accordance with the guidelines of the Canadian Council on Animal Care.
Macroscopically, the TEBV appeared as a homogeneous tubular tissue strikingly resembling a human artery (Fig. 1A). Histological analysis revealed well-defined tissues: intima, media, and adventitia (Fig. 1B). The adventitia exhibited very dense collagenous layers as well as abundant fibroblasts. In the media, SMC appeared as elongated cells with circumferential or longitudinal orientations. SMC density, although very high for an in vitro model, was still lower than in normal vascular media. SMC did not penetrate the dense IM even though they were in contact with it for more than 8 wk and exposed to a gradient of nutriments leading to the lumen. Metabolic labeling and immunostaining of the endothelium (Fig. 1C) revealed confluent and active ECs as demonstrated by ac-LDL uptake and by von Willebrand factor, two specific EC functions (21, 26–28). SMC stained positively for muscular markers -smooth muscle actin (not shown) and desmin (Fig. 1D) (23, 29). Adventitial cells were negative for desmin or -smooth muscle actin, but fibroblasts expressed vimentin and synthesized high amounts of elastin assembled in small fibers, which were organized in large circular arrays (Fig. 1E). Immunostaining also indicated that the ECM contained type I, III, and IV collagens as well as laminin, fibronectin, and chondroitin sulfates (not shown).
Mechanical strength of the TEBV
Because mechanical strength has been the weak point of previous biological TEBV, we first evaluated this property in our vessel. To maximize its burst strength, we decided to optimize the strength of the engineered adventitia since this layer was clearly stronger than the media. The extent of both culture and maturation periods influenced the mechanical properties of this tissue. The culture period before detachment and rolling of the fibroblast sheet determined the thickness of the resulting adventitia (Fig. 2A). As expected, the burst pressure of the adventitia was similarly dependent on culture time (Fig. 2B). From these results, a culture period of 5 wk was chosen for all fibroblast sheets (IM and adventitias) since it gave the optimal strength to culture time ratio. During the maturation period, the burst strength of the adventitia steadily increased from the 1st to the 7th wk, when it reached a plateau at 2232 ± 251 mmHg (Fig. 2C). This plateau was maintained until at least the 12th wk, and some adventitias were matured for 24 wk without significant change in strength (2382±249 mmHg; n=5). Consequently, TEBV were kept in culture for at least 8 wk after the addition of the adventitia. The burst strength of the complete TEBV (2594±501 mmHg) was comparable to that of the adventitia alone, although the IM itself had a burst strength of over 1000 mmHg (Fig. 2D). Most important, TEBV burst strength was significantly higher than that of human saphenous veins (1680±307 mmHg), which are currently considered the optimal grafts for lower limb vascular reconstruction (3).
We attribute the mechanical strength of this model to the particularly well-organized collagenous matrix of the adventitia (Fig. 3A). Ultrastructural analysis revealed long collagen fibrils, featuring the characteristic 67 nm cross-striation periodicity, organized in closely packed bundles of parallel fibrils as observed in vivo (30). Furthermore, fibril bundles were oriented in perpendicular directions to one another, and a network of 10–12 nm elastin-associated microfibers was observed parallel to the collagen fibrils. Such a level of ultrastructural organization is characteristic of a mature collagen scaffold (30, 31).
Because increased matrix metalloproteinase (MMP) synthesis was proposed as a critical factor in long-term instability of collagen-based engineered organs in vitro (17), we followed collagenase synthesis during our vessel production and maturation. Normal fibroblasts cultured in vitro produce large quantities of MMP-2 (32), also called 72 kDa type IV collagenase/gelatinase or gelatinase A. Gelatin zymography of conditioned culture medium (Fig. 3B) revealed that fibroblast sheets prior to rolling produced mainly 70 kDa and 60 kDa gelatinases (lane 1), likely the pro-enzyme form of MMP-2 and its enzymatically active 62 kDa form (32). SMC sheets produced mostly the 72 kDa form of MMP-2 and also produced a gelatinolytic doublet of about 50 kDa (lane 3). When cell sheets were rolled, a rapid and marked overall decrease in collagenase production was observed for both fibroblasts (lane 1 vs. 2) and SMC (lane 3 vs. 4). However, the rolling step specifically induced expression of the 50 kDa gelatinolytic doublet by fibroblast sheets when rolled either alone (lane 1 vs. 2) or over SMC (lanes 1 + 4 vs. 5). This induction subsided within 2 wk (lane 6). During long-term culture, down-regulation of gelatinase expression was maintained and the remaining activity was observed mainly in the 72 kDa band (lane 7 and 8).
The ECs seeded on the IM covered 99.2 ± 0.7 % (n=6) of the luminal surface at a cell density of 5.4 ± 1.1 x 104 cells/cm2 (n=5), as measured by morphometric analysis. These ECs expressed a functional thrombin receptor since they responded to thrombin stimulation by an 2.9 ± 0.7 (n=5) fold increase in PGI2 synthesis, a potent inhibitor of platelet aggregation. Heparinized human whole blood was used in a functional assay to evaluate the blood compatibility of this endothelium. When ECs were absent, platelets adhered to the IM in small clusters, whereas conflu~ent ECs inhibited platelet adhesion by 93.2 ± 3.6 % and limited the phenomenon to single platelet adhesion in intercellular gaps (Fig. 4).
Grafting of the TEBV
Based on our promising in vitro data, we extended the model to a preliminary in vivo assessment. In addition to the ability to withstand hydrostatic pressure, TEBVs must also resist the various mechanical insults involved in vascular grafting procedures: manipulation with metal instruments and suture-related strains. To assess in vivo graftability, 5 cm-long unendothelialized human TEBV (inner diameter =3 mm) were implanted as interposition femoral grafts in mongrel dogs. TEBV were not endothelialized in order to avoid the immediate thrombosis associated with the acute rejection of endothelialized vascular xenograft (33, 34). Suturability and handling characteristics of the graft were evaluated as `tissue-like' by an experienced vascular surgeon. When blood flow was reestablished, no transmural blood loss was observed and immediate patency was confirmed by a palpable pulse and a visible downstream pulsatile flow. After 7 days, graft patency was assessed by angiography and explantation (Fig. 5). Even with the severe problems associated with xenografts, we were able to demonstrate a 50% patency rate (three out of six grafts). Patent grafts showed a smooth thrombus-free luminal surface and did not show signs of degradation, tearing, or dilatation. In all grafts, intramural blood infiltrations were observed between tissue layers, but these did not correlate with decreased patency; some patent grafts had significant infiltrations (Fig. 5B) whereas some thrombosed grafts had minimal infiltrations. Aside from blood infiltration, histological analysis revealed that the graft's architecture was retained. Graft failures occurred during the first days, as evaluated by Doppler signaling, and were the result of occlusive thrombus formation. This is in keeping with the thrombogenic nature of the collagen matrix observed in vitro in the absence of an endothelium (Fig. 4A).
In this report, we have established the feasibility of producing a completely biological TEBV exclusively from human cells. This construct meets the three fundamental qualities expected of a vascular graft: mechanical strength, blood compatibility, and suturability. The first and the latter depend directly on the complex and naturally organized 3-dimensional ECM of this vessel. Although the proteins from naturally occurring ECM have been isolated and characterized, these molecules cannot be reassembled in vitro into an ECM featuring physiological density and organization. This explains the inability of previously designed biological TEBV to display physiological strength (17–19). Our model displayed a supraphysiological bursting pressure, and we have shown that this property can be modulated by the culture and maturation periods. However, compliance has also been established as a critical factor for long-term patency of vascular graft (35, 36). This TEBV was clearly more compliant than synthetic ePTFE vascular grafts but, as observed during burst pressures experiments, it was much less compliant than HSV. This is not surprising since the TEBV was optimized for strength. Nonetheless, the mechanical properties of the vessel can be modified by changing the thickness of the different layers. However, since mechanical properties of biological vascular grafts have been shown to change dramatically once implanted (37), the optimal compliance of the TEBV will have to be determined in conjunction with an in vivo study.
In their landmark paper describing the first attempt at creating a biological TEBV, Weinberg and Bell (17) hypothesized that collagenase synthesis might be a critical factor in the mechanical stability of tissue-engineered organs in vitro. Indeed, they observed that their collagen gel-based model experienced an abrupt loss of strength ( 50%) between the 4th and 12th wk of maturation in vitro. In our system, we have observed the transient induction of a gelatinolytic doublet near 50 kDa after the rolling step of the fibroblast sheets. This suggests the induction of MMP-1 (interstitial collagenase), which preferentially degrades native collagen but can also exhibit limited gelatinolytic activity (38, 39). The transient nature of the induction and the general long-term down-regulation of gelatinase activity from both SMC and fibroblasts suggest that the ECM was remodeled to provide a stable physiological environment. The long-term mechanical stability of our model is consistent with the decreased level of collagenase activity observed. A possible explanation for the sustained strength of our TEBV might be that a cell-synthesized matrix induces less collagenase expression, or is more resistant to protease activity, than a biochemically extracted and reassembled matrix such as the one used by Weinberg and Bell. Another possibility would be an increased secretion of tissue inhibitors of metalloproteinase (TIMP) by the cells in this model, which would then reduce collagenase activity. However, MMP expression is only one aspect of the question; in order to verify the above hypothesis, an assessment of ECM proteins synthesis and deposition is needed.
We have shown that a confluent endothelium, expressing normal endothelial markers, was established on the luminal surface of the IM. These ECs expressed functional thrombin receptors as judged by their increase in PGI2 synthesis when challenged with thrombin. PGI2 response to thrombin has been shown to be maximal when human ECs are cultured on natural collagen (40). Activation of the thrombin receptor has also been shown to induce nitric oxide production by ECs (41). This suggests that if platelet adhesion/activation occurs after in vivo grafting, the endothelium will respond to the ensuing release of thrombin by an increase in the synthesis of two potent inhibitors of platelet aggregation. Unfortunately, the hemocompatibility of a human endothelium cannot be properly tested in an animal model because ECs can express major histocompatibility complex (MHC) class II proteins and MHC-mismatched vascular grafts have shown very poor results, even with immunosuppression therapy (42, 43; for review, see ref 44). Consequently, we evaluated the hemocompatibility of the endothelium by using an in vitro functional assay. Although the endothelium was exposed to heparinized human blood under conditions that promote platelet adhesion (very low flow rate), ECs drastically inhibited platelet adhesion and activation. However, it is important to remember that the antithrombotic activity of the EC is useful only if the cells remain attached to the luminal surface when exposed to arterial blood flow. Indeed, endothelial cell seeding experiments of vascular grafts have been plagued with this problem for years (45, 46). Recently, two groups (8, 47) have finally reported increased patency rates for seeded small-diameter synthetic vascular grafts in humans by using EC seeding. In both cases, the grafts were heavily coated with a mixture of biological proteins. These results suggest that the biological nature of the ECM of our TEBV may favor EC adaptation to flow by providing the appropriate anchoring molecules.
Since this is the first implantable, totally biological TEBV, it is difficult to hypothesize its behavior after grafting on the basis of results obtained with other models. However, it is important to underscore the critical differences between this graft and others that share some common characteristics. For example, the Sparks-mandril graft is a tubular scar tissue induced by a subcutaneously implanted Dacron mesh (48). This model was used as an autologous small-diameter vascular graft in the middle 1970s, but was associated with high thrombogenecity and aneurysm formation (49). Although autologous in nature, this graft did include a synthetic mesh and was not endothelialized. Furthermore, this graft was essentially a scar tissue. The ECM was therefore synthesized by cells as part of an inflammatory response, which may explain why it was unable to resist the mechanical strains under physiological conditions. Finally, the closing publication on the Sparks-mandril project suggested that its mechanical instability was linked to the absence of elastin fibers and SMC (50), both of which are present in our model. More recently, a completely biological autologous small vascular graft was produced using the small intestine submucosa in dogs (51). Animal experiments showed very good results, but it appears that human intestinal tissue is not suitable for the production of such a graft (52). Nevertheless, these experiments demonstrate that an autologous tissue of nonvascular origin can be transformed to a completely biological vascular graft and successfully remodeled by the body after implantation.
In vivo experiments were undertaken in order to evaluate the suturability and early mechanical stability, which are challenging problems for biological TEBV. Our results showed that this model can be sutured by using conventional surgical techniques and that it does not tear or dilate when exposed to clinical grafting conditions for a week. However, the patency rate observed in these experiments is not indicative of the potential of this TEBV as a vascular graft. Indeed, an autologous endothelialized graft would undoubtedly show superior performances. Consequently, TEBV will have to be produced from animal cells in order to assess the long-term value of this vascular graft in an autologous system. This TEBV offers exciting future possibilities since the nonsynthetic nature of this vascular graft warrants hope of increased long-term patency rates for lower limb vascular reconstructions. Furthermore, a TEBV produced from subcultured cells may be an appealing tool for gene therapy because these cells would be in close proximity to blood and because human ECs, SMC, and fibroblasts can be safely transfected in vitro without using viruses that induce immunogenic response (53–55).
The potential clinical application of such a TEBV raises several technical questions. For example, what cell source will be used? The external jugular vein has already been used successfully to isolate vascular cells for a clinical application (8, 47). In theory, this tissue could provide the three cell types needed for the production of the TEBV. Besides veins, other sources are also available, such as skin (microvascular ECs and fibroblasts) or fat tissue (microvascular endothelial/mesothelial cells and fibroblasts) (56–60). Upscaling this model to the size needed for vascular reconstructions (i.e., 15 cm for coronary bypass and 15 to more than 30 cm for peripheral reconstructions) is also an important question. This process will either involve a strategy based on overlapping sheets or rely on larger culture containers to produce tubes from a single sheet. Upscaling also requires that a relatively large number of cells must be obtained from the initial biopsy. This brings us to the most critical factor: the time needed for TEBV production. It is obvious that this type of engineered autologous tissue is not designed to be used for emergency surgeries. However, in a nonurgent setting, a significant period of time would be available for the production of a TEBV, especially if this vessel provides excellent long-term patency. Nonetheless, the culture period needed for graft production should be as short as possible for maximal clinical applicability. Many factors can be optimized to reduce production time. For example, biopsy size, culture medium components, fetal bovine serum selection, and bioreactor designs are all elements that we are currently testing to decrease culture and maturation periods.
In addition to being a promising graft from a mechanical point of view, our model also demonstrates fundamental morphological, histological, and functional characteristics of blood vessels. For example, we have observed the expression of desmin, a marker for highly differentiated SMC considered to be irreversibly lost in cultured human SMC (61). Furthermore, a similar SMC construct showed contractile responses when challenged with vasoactive agonists (N. L'Heureux, L. Germain, F. A. Auger, and J. C. Stoclet, unpublished results), a property also considered lost in cultured human SMC (61). Another remarkable aspect of this model is the presence of elastin fibers, an important ECM component never reported in other TEBV (17–19). A possible explanation for these observations is that the cell-synthesized matrix of our TEBV may provide appropriate signals to the vascular cells through stimulation of integrin receptors, thus directing them toward a more physiological phenotype (62–64). The coculture of the various vascular cell types in this model may also favorably affect cellular behavior.
This model exhibits unique features that make it a particularly well-suited system for the study of vascular cells in vitro, especially for experiments involving physical forces. For example, it could be used to study the influence of culture conditions on the development of mechanical properties of the resulting tissue (strength, elasticity, permeability, etc.). Another possibility would be to apply mechanical forces to these engineered tissues and observe cell responses to these forces. The 3-dimensional structure and the natural characteristics of the ECM should ensure a more physiological environment for the cells, whereas the unique mechanical strength of these tissues could allow stimulation at physiological levels.
On a microscale, cultured mesenchymal cells such as SMC and fibroblasts recreate a physiological environment by secreting and organizing, in the presence of vitamin C, a 3-dimensional ECM with characteristics similar to those observed in vivo (65–67). We assembled these microstructures into macrostructures to produce engineered tissues featuring significant mechanical strength and complex histological organization. In turn, these tissues can be assembled together and with other cells to produce completely biological tissue-engineered organs. However, the shape and cell content of theses tissues can be modified to create, with the same basic technique, a wide range of completely biological tissue-engineered organs to meet the growing need for human replacement organs.
We gratefully thank the Fondation des Maladies du Coeur and the Fondation de l'Hôpital du Saint-Sacrement for financial support; Dr. C. Matthews (University of Guelph) for helpful discussions concerning the in vivo studies; N. Marceau and A. Grimaud for generously providing antibodies for vimentin and elastin; R. Guidouin and S. Bourassa from the Institut des Biomatériaux de Québec for SEM; K. Baker, L. Martin, A. Pusterla, and L. Vue for technical assistance; C. Marin for photographic assistance; and M. Michel and F. Berthod for careful review of the manuscript. L. Germain and F. A. Auger were recipients of Scholarships from the Fonds de la Recherche en Santé du Québec and N. L'Heureux was recipient of Studentships from the Fonds FCAR du Québec.
1 Correspondence: Laboratoire d'Angiogénèse Expérimentale/LOEX, Hôpital du Saint-Sacrement, 1050, chemin Sainte-Foy, Québec (Québec) G1S 4L8, Canada. E-mail: [email protected] .
3 Abbreviations: TEBV, tissue-engineered blood vessel (or vessels); SMC, smooth muscle cell (or cells); EC, endothelial cell; FBS, fetal bovine serum; ac-LDL, acetylated low density lipoproteins; IM, inner membrane (or membranes); CMFDA, 5-chloromethylfluorescein-diacetate; DiI, 1;1'-dioctadecyl-3;3;3';3'-tetramethylindocarbocyanine chlorate; HSV, human saphenous veins; PGI2, prostacyclin; SEM, scanning electron microscopy; MMP, matrix metalloproteinase; ECM, extracellular matrix; MHC, major histocompatibility complex.
Received for publication June 27, 1997. Accepted for publication October 2, 1997.
Figure 1. Organization of the TEBV. A) Macroscopic view of a mature TEBV (9 wk of adventitial maturation). The vessel is self-supporting when removed from culture medium (open lumen = 3 mm). Note that the various layers now form a continuous vascular wall (inset, graduation = 1 mm). B) Paraffin cross section of the vascular wall stained with Masson's trichrome shows collagen in blue-green and cells in dark purple. Aside from an oversized internal elastic lamina (IM=125 µm), the histology is similar to that of a muscular artery with a large media (M=320 µm) and a surrounding adventitia (A=235 µm). C) En face view of the endothelium seeded on the IM. Cytoplasmic green fluorescence reveals cell viability, metabolic activity, and degree of confluence. Red fluorescence (DiI-ac-LDL uptake) confirms cell viability and the endothelial nature of the cells. Blue fluorescence shows the characteristic von Willebrand factor expression in ECs (orange = red + green; pink = red + green + blue). Scale bar = 25 µm. D) Frozen cross section of the media–adventitia junction (arrows) stained for desmin (nuclei are stained blue). Scale bar = 50 µm. E) Frozen cross section of the adventitia double stained for elastin (green) and vimentin (red). Scale bar = 50 µm.
Figure 2. Mechanical strength of the TEBV. A) Wall thickness of adventitia as a function of culture time before sheet detachment and rolling (maturation period is 5 wk in all cases; *significantly different from 4 wk (P*significantly different from 5 wk (P*significantly different from 5 wk (P*P
Figure 3. Extracellular matrix ultrastructure and collagenase expression. A) Transmission electron micrograph of the adventitial matrix. Uranyl acetate and lead citrate stain. Scale bar = 500 nm. B) Gelatin zymogram showing gelatinase activity in conditioned culture medium. Lanes 1 and 2: fibroblast sheet prior to rolling (FIB) and 48 h after rolling (adventitia = ADV). Lanes 3 and 4: SMC sheet prior to rolling (SMC) and 48 h after rolling on a IM (vascular media = VM). Identical results were obtained without a IM. Lanes 5 to 8: addition of the adventitia over the VM. Note that albumin bands (dark 68 kDa band) are lighter in conditioned mediums from sheets prior to rolling because different sample volumes were used to take into account the total volume of culture medium.
Figure 4. Inhibition of platelet adhesion by the endothelium. Scanning electron micrographs of unendothelialized IM (A) promoted platelet adhesion and activation whereas endothelialized IM (B) almost completely inhibited the process.
Figure 5. In vivo grafting of the TEBV. A) Angiogram of the lower limbs 7 days after implantation. Two patent TEBV are visible (arrows) providing normal blood flow in both legs. B) After angiographic examination, the TEBV was explanted along with adjacent segments of the femoral artery, gently flushed with saline, fixed in formaldehyde, longitudinally slit open, and pinned down to expose the luminal surface. Blood infiltrations are seen inside the vessel wall. The luminal surface is free of thrombus. Anastomosis showed no signs of deterioration. Graduation = 1 mm.