Toward a new blood vessel

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1 Toward a new blood vessel Briain D MacNeill a, Irina Pomerantseva a,b, Harry C Lowe a, Stephen N Oesterle a,b and Joseph P Vacanti b,c Abstract: Strategies to treat atherosclerotic coronary artery disease include coronary artery bypass grafting (CABG), in which grafts are used to bypass atherosclerotic vessels and restore blood ow to the ischemic myocardium. The grafts used include healthy arteries or veins harvested from a separate site. Results with arterial grafts have been superior to venous grafts; promoting the practice of total arterial revascularization using only arterial grafts. Suitable arterial grafts, however, are scarce and harvest procedures add to morbidity and cost. Tissue engineering combines the principles of engineering with life sciences for the development of biological substitutes and restore, maintain or improve tissue function. Advances in this eld have included the development of tissue-engineered blood vessels, with the potential to serve as arterial grafts, conduits or stulae. This review describes the history of tissue engineering arteries, the techniques used, and progress to date. The source of cells and the future direction of this eld are explored. Key words: atherosclerosis; bypass; coronary artery disease; revascularization; tissue engineering Introduction Atherosclerosis is the commonest cause of premature mortality in the Western world, with more than two of every ve Americans dying of cardiovascular disease. 1 Coronary artery atherosclerosis is estimated to cause over a million myocardial infarctions annually in the USA alone. 2 The number of patients with stable angina has been estimated as Current surgical treatment strategies include the use of coronary artery bypass grafting (CABG) using autologous arteries and veins to bypass areas of coronary atherosclerosis. Over CABG procedures are carried out annually in the USA. Surgical revascularization with autologous arteries is complicated, however, by the need for a harvest procedure and by a paucity of suitable harvest sites. The long-term patency of venous grafts is markedly reduced by graft atherosclerosis, lack of vasomotor tone and damage through manual handling at the time of bypass. 3 Graft sites are prone to infection and poor healing, particularly in diabetic patients. 4 Large caliber arterial bypass with synthetic grafts has reached clinical application. The same cannot be said for vessels under 6 mm, where the use of synthetic materials as conduits has yielded little success. 5 Advances in tissue engineering have therefore led investigators to search for a biologically engineered functional substitute for vascular conduits. 6 a Division of Cardiology, b Tissue Engineering Laboratories and c Department of Surgery, Massachusetts General Hospital, Boston, MA, USA Address for correspondence: JP Vacanti, Department of Surgery, Warren 1157, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA. Tel: ; jvacanti@partners.org Ó Arnold 2002 Historical perspective Synthetic grafts have been in use since the 1950s for the treatment of peripheral arterial disease. 7 While successful for large caliber vessels, the rate of acute and subacute thrombosis in smaller vessels yielded inferior results. Graft modi cation with protein lining and polymer resurfacing reduced thrombosis and neo-intimal hyperplasia but did little to improve long-term arterial graft patency. 8 In 1986, Weinberg and Bell reported the rst tissue-engineered blood vessel created from collagen gels combined with bovine endothelial cells, broblasts and smooth muscle cells. 9 The technique was expanded and applied to human tissue-engineered vessels with results that displayed increased graft strength, to such an extent that a mechanical support or scaffold was redundant. 1 0 An in vitro model demonstrated that mechanical strength and contraction were further increased in constructs exposed to continuous cyclical strain. 1 1 Exposure of cultured constructs to constant pulsatile pressure, as during fetal vasculogenesis, using a biomimetic system yielded improved mechanical characteristics and superior patency rates after implantation. 1 2 Normal blood vessels Arteries have a trilamellar structure in which each layer plays an important role in the normal function of the vessel. The three layers are the tunica intima, tunica media, and tunica adventitia. The tunica intima, or endothelial layer, consists of endothelial cells that line the lumen. Among its many functions, the endothelial layer operates as a transport barrier, a lter, a regulator of vascular tone and prevents spontaneous thrombosis. The tunica media, or medial layer, is formed from smooth muscle cells, collagen, elastin and proteoglycans. It confers mechanical strength to the vessel and controls vessel caliber by contracting or relaxing. The tunica adventitia, or adventitial layer, comprises broblasts / x02vm433ra

2 242 BD MacNeill et al and extra-cellular matrix and is important in supporting the vasavasorum and vascular innervation. Techniques in tissue-engineered vessels As with other tissue-engineered substitutes the ideal design of manufactured organs is one that incorporates all essential functions of the native tissue. An engineered blood vessel requires a biological pro le that will not harm its host as well as durability to survive implantation and prolonged hemodynamic pressures. Vasoactivity of the graft would be useful in regulating blood ow through the vessel. 1 3 To accurately mimic a blood vessel, all of these elements must be considered. The techniques used in tissue-engineering small vessels can be broadly divided into the formation of the three layers of a normal vessel, the supportive scaffold used, in vitro evaluation, implantation and nally in vivo study. Intimal layer Evidence from both in vitro and in vivo experiments con- rms that endothelial cells play a pivotal role in regulating vascular repair after injury. 8, Endothelial cells were rst harvested from human umbilical veins in 1973 by Jaffe et al. 1 9 Before the 1980s successful culture of endothelial cells was con ned to animal models. The discovery of an endothelial cell growth factor (ECGF) permitted improved human endothelial culture techniques. 2 0 Subsequent studies highlighted the importance of numerous supplementary growth factors that are currently commercially available from human or bovine serum. Endogenous and exogenous factors such as heparin, camp, and isobutyl methylxanthine have all been shown to in uence the growth and proliferation of endothelial cells in culture Initial cell attachment is promoted by precoating culture asks with denatured bovine collagen or other components of extracellular matrix; however, after 6 hours of culture cell attachment occurs through endogenous matrix components. Endothelial cell characteristics can broadly be categorized as dependent or independent of harvest site. Differing replication rate, growth rate, protein synthesis and cellular morphology have been described in endothelium cultured from various donor sites. 2 4 Diverse responses of endothelial cells from different sources to varied growth factors has also been found. 2 5 Similarly, the response of the cultured endothelial cells to vasoactive mediators, secretion of prostaglandin I 2 and secretion of plasminogen-plasmin inhibitors differs among tissue sites. 2 6 In contrast, some endothelial cell characteristics are universal regardless of the donor site but are not unique to endothelial cells. These include secretion of angiotensin converting enzyme (ACE), platelet endothelial cell adhesion molecule (PECAM-CD 3 1 ) and the uptake of oxidized low-density lipoproteins. 2 7 The presence of Weibel Palade bodies, which secrete P-selectin and von Willebrand factor, is both unique to endothelial cells and displayed by all endothelial cells regardless of their source. 2 8 Routine protocols for endothelial cell characterization involve demonstration of von Willebrand factor, Weibel Palade bodies and uptake of oxidized low-density lipoprotein. One of the fundamental tasks following successful tissue implant is distinction of cultured cells from the native cells. To facilitate identi cation, methods of cell labeling have been developed using retroviral vectors expressing marker proteins and transfecting primary cell lines prior to culture. 2 9 These techniques allow accurate identi cation of transplanted cell lines and also demonstrate the potential of incorporating gene therapy into biograft design Medial layer Vascular smooth muscle is the dominant cell type normally found in the media of human arteries. In the normal state, vascular smooth muscle cells exhibit a contractile phenotype, existing in a non-proliferating differentiated state. In this state, cytoskeletal marker proteins (smooth muscle alpha-actin, myosin heavy chains and calponin) are expressed, and the vascular smooth muscle contracts in response to chemical and mechanical stimuli. 3 4 In atherosclerosis, however, the phenotype can change in response to various growth factors and assume a synthetic or secretory phenotype. This change has been termed modulation. 3 5 Following modulation smooth muscle cells are capable of proliferation, as during vasculogenesis in the fetus. In the adult, such proliferation gives rise to neointimal hyperplasia and restenosis. 3 6 In almost all medium to large arteries the elastic laminae forms a mechanical barrier between the smooth muscle cells of the media and the intimal and adventitial layers, a feature that is exploited in isolating smooth muscle cells. Techniques for isolating smooth muscle cells include enzymatic disaggregation using elastase to digest the elastic laminae and collagenases to digest the surrounding extracellular matrix. 3 4 Using a novel bioreactor system to condition cultured cells with pulsatile ow, similar to that encountered in physiological conditions, improved structural integrity and functional development could be achieved, conferring greater mechanical strength to the engineered vessel. 1 2 Identi cation of smooth muscle cells relies on the morphological hill and valley appearance in culture and the demonstration of alpha actin. Other characteristics include the presence of myosin heavy chains and calponin, the latter being con ned to functional cells that display a contractile phenotype. As described with endothelial cell labeling, a retrovirus expressing green uorescent protein (GFP) can be used to tag cultured smooth muscle cells. Genetic modi cation of smooth muscle cells is useful to identify bioengineered cells in vitro, and it demonstrates the feasibility of therapeutic gene transfer. Accordingly, using smooth muscle cells as hosts to express therapeutic recombinant genes could allow modi cation of their behavior and their role in intimal hyperplasia and restenosis Adventitial layer The adventitia consists predominantly of broblasts and extracellular matrix. Many investigators have developed engineered grafts without including an adventitial layer; however, combining sheets of broblasts and smooth muscle cells before seeding with endothelial cells can confer increased mechanical strength. 1 0 Fibroblast harvest is easily achieved from simple skin scrapings. Fibroblast growth is superior if culture media is supplemented with sodium ascorbate, eventually growing in sheets that can be manipulated. Adventitial staining and identi cation can be carried

3 Toward a new blood vessel 243 out in frozen section using anti-elastin antibody and antivimentin antibody. 1 0 Scaffold Tissue engineering techniques generally require the use of a scaffold to serve as a three-dimensional template for cell attachment, proliferation, and maintenance of differentiated function. A scaffold is an exogenous extra-cellular matrix designed to mimic the architecture of the normal tissue and to support structured growth of cultured cells. Materials such as collagen and polyglycolide were initially used; however, as the tissue developed it rapidly outgrew its nutrient supply. The discovery of branched microporous polymers allowed maximization of surface area and optimal nutrient supply to the growing cells. 3 9 Historically, the use of biomaterials in medicine has been con ned to functional or non-biological roles. Essentially, their role has been to facilitate sliding, direct blood ow, transmit loads to ll spaces; roles that require the materials used to be durable and thus bioinert. Ideally, however, a vascular scaffold should degrade on completion of its role as a structural support, yet should not stimulate an immune response in the process of degradation. Consequently, small vessel scaffold requires controlled reactivity, structural integrity to withstand implantation and subsequent physiological pressure, and a porous interconnectivity to allow adequate nutrient supply to the developing tissue. 4 0 Compliance matching in synthetic vascular grafts has been shown to promote long-term graft patency. 4 1 Compliance mismatch gives rise to altered ow, increased shear rates and downstream turbulence that, over time, gives rise to endothelial damage and intimal hyperplasia. 4 2 The stress/strain relationship has been shown to be dependent not only on the scaffold type but also on the extent of the cells seeded, demonstrating that the mechanics and the biological features of the graft are interdependent. 4 3 Currently the most frequently used polymers for tissueengineering scaffolds include poly(l-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(dl-lactic-co-glycolic acid) (PLGA), and poly-4-hydroxybutyrate (P4HB). More recently, combinations of these have demonstrated superior cell attachment and mechanical properties. 4 4 Many groups have had success without traditional scaffolds. 1 0,4 5,4 6 Branched grafts have been constructed pouring a mixture of smooth muscle cells and collagen onto a tubular mold with subsequent thermal gelation. 4 5 The addition of an adventitial layer enabled the development of a construct composed exclusively of cultured human cells without the use of a scaffold, with microscopic and pharmacological properties similar to a native vessel. 1 0 Intraperitoneal culture on silastic tubing created a layer of mesothelial cells which, after removal of the tubing, could be everted and fashioned into an arterial conduit. 4 6 Evaluation In vitro evaluation can be carried out on microscopic, biochemical, mechanical and pharmacological levels. Microscopic assessment evaluates the general appearance of the construct and its similarity to a native trilamellar structure. It allows assessment of the attachment of cells to the scaffold and gauges the degree of degradation of biodegradable scaffolds. Construct staining allows assessment of the constituents of the extracellular matrix such as elastin, collagen and glycosaminoglycans (GAGs). Speci c staining allows characterization of each cell type from different layers, as discussed above. Immunohistochemical staining permits detection of proliferating cells. Electron microscopy provides further information on the endothelial cell alignment, and evidence of cellular phenotype throughout the construct. Biochemical evaluation quantitatively measures protein content, hydroxyproline for collagen, elastin and GAGs. Matrix metalloproteinases (MMPs), which mediate the breakdown of type I and III collagens, can be measured using an ELISA-based assay. More recently, tissue-derived inhibitors of metalloproteinases (TIMPs) have been studied. The dynamic relationship between MMPs, TIMPs, and the proliferating smooth muscle cell appear to have a pivotal role in the formation of neo-intimal hyperplasia following graft insertion. With the addition of radiometric metalloproteinase assay it is possible not only to measure the levels but also the activity of MMPs and TIMPs. 4 7,4 8 Tensile strength, graft compliance and burst pressure are mechanical characteristics that are important in construct evaluation. Tensile strength has been measured with a dynamic mechanical analyzer in circumferential and longitudinal sections while graft compliance has been assessed in static and pulsatile states using a video motion analyzer. 1 2 Burst pressure of the tissue-engineered graft, which was measured by increasing hydrostatic pressure until graft failure, compared favorably with saphenous vein as a control. 1 0 Pharmacological assessment is best detailed in L Heureux s study in which the relaxant effect of cyclical AMP, the role of CA 2 + contraction, and the effect of numerous drugs on the conduit were assessed. 4 9 Dose response curves for tissue-engineered constructs and normal vessels were compared and found to be similar, although the tension generated was of a much lower amplitude in tissueengineered vessels, possibly due to a lower number of vascular smooth muscle cells in the tissue-engineered grafts. 4 9,5 0 Implantation Translating in vitro projects to in vivo models raises important issues in tissue engineering: such as the similarity of the animal model to human physiology and the homogeneity between donor and recipient. Tissue-engineered conduits were cultured from human vascular cells and inserted as femoral grafts in mongrel dogs. Acute rejection, and subsequent thrombosis, was deemed likely if an endothelial monolayer of human cells was a component of the vascular graft. Accordingly, the endothelial layer was omitted. To prevent thrombosis in the absence of a functional endothelial layer, the animals were anticoagulated with warfarin. Patency rates were 50% after 7 days, but importantly the construct displayed manual handling and suturability compatible with normal tissue. 1 0 Autologous and xenograft constructs were inserted into the saphenous artery of Yucatan miniswine, treated with daily aspirin. All implants remained patent for 2 weeks; autologous grafts that had been cultured under pulsatile conditions remained patent for 4 weeks. 1 2 In a novel approach, vascular constructs were cultured in the recipient s own peritoneal cavity and implanted as interpositional carotid artery grafts into adult rats and rab-

4 244 BD MacNeill et al Table 1 Evaluation used in in vivo studies: 10,12,46 comparison of methods used for in vivo evaluation in three trials. Description Technique Evaluation Comment Human cells cultured without Unendothelialized constructs 3 Doppler signaling, Results displayed mechanical scaffold and implanted into mm in diameter and 5 cm in angiography and explantation properties similar to a normal canine model 10 length, inserted displayed a patency rate of vessel and good handling and 50% at 7 days suturability properties. High rate of thrombosis assumed to relate to lack of endothelium Bovine and swine cells Autologous and xenograft Doppler analysis, digital Patency con rmed up to the cultured in a PGA scaffold and vessels cultured under subtraction angiography and time of explanation in grafts implanted into the saphenous pulsatile and static conditions explanation after 4 weeks. Ex cultured under pulsatile artery of four miniswine 12 vivo contraction studies and conditions. Non-pulsed grafts histological analysis developed thrombosis at 3 weeks. Signi cantly less in ammatory response noted in autologous grafts Intraperitoneal culture of Immune reaction used to Clinical evaluation, histological A novel technique of autologous grafts implanted stimulate cell attachment on a analysis and contractile autologous culture with into the carotid artery of rat silastic tubing inserted studies were carried out. resultant grafts resembling and rabbit models 48 intraperitoneally Overall patency rates were normal blood vessels. Patency 67% up to 4 months noted Figure 1 The potential future of tissue-engineered blood vessels. (1) Progenitor cells are harvested from peripheral blood, bone marrow or adipose tissue and (2) cultured under sterile conditions until con uent sheets are formed. (3) The cells are then seeded onto a synthetic polymer which acts as a three-dimensional template for cellular proliferation during (4) dynamic culture in a biomimetic system that exposes the construct to physiological pressure to improve construct strength and functional development. (5) Finally, the construct is evaluated histologically and mechanically prior to (6) clinical application as a bypass graft conduit.

5 Toward a new blood vessel 245 bits. Grafts remained patent for at least 4 months, developing a structure similar to a native vessel. 4 6 In vivo evaluation (Table 1) Perioperative patency is con rmed by the demonstration of distal pulsatile ow. Postoperative patency is easily ascertained with Doppler ultrasound and angiography, including digital subtraction imagery. Ex vivo evaluation provides a histological comparison to native vessel, allows identi - cation of transplanted cells, and facilitates pharmacological and mechanical testing, though the latter has been hindered by the development of brous tissue at the outer surface of the construct. Cell sources Recent debate on the use of stem cells in medical research has examined the roles of embryonic and adult stem cells for therapeutic application. The fundamental difference between these cell sources is that embryonic cells are pluripotent meaning that they can give rise to any kind of cell in the body, while adult stem cells are multipotent meaning that they can give rise to many but not all cell types in the body. To date the sources of adult stem cells have included bone marrow cells, peripheral blood cells, adipose tissue, placental tissue and brain tissue. Initially, it was thought that adult stem cells, once differentiated, could not de-differentiate, until the ability of adult neural stem cells to transform into hematopoetic precursor cells was demonstrated. Adult stem cells displayed greater plasticity than previously thought. 5 1 Stem cells have not been widely used in the tissue engineering of blood vessels. Endothelial precursor cells derived from peripheral blood have recently been cultured and shown to display characteristics typical of endothelial cells. Cellular components have been removed from blood vessels through a process of trypsinization and extraction, resulting in an acellular blood vessel. The acellular vessel was then seeded with endothelial precursor cells and preconditioned in vitro by exposure to shear stress. The seeded grafts were then implanted and displayed a maintained patency with a non-thrombogenic luminal surface of autologous endothelial cells derived from precursor cells. 5 2 Alternative sites for adult stem cell retrieval have included bone marrow, epithelial tissues and more recently adipose tissue These studies demonstrate the ability to obtain endothelial cells from peripheral progenitor cells, thereby eliminating the need for an initial surgical harvest from a vessel (Figure 1). Conclusion The burden of cardiovascular disease combined with limitations in our ability to treat small vessel atherosclerosis adequately has spurred progress in tissue engineering of blood vesssels. 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