Tissue engineering in surgery

Education Section J.P. Garner Biomedical Sciences, Dstl Porton Down, Salisbury, SP4 OJQ Correspondence to: JP Garner; Biomedical Sciences, Dstl Porton Down, Salisbury, SP4 OJQ

Tissue engineering in surgery The demands forrepair and renewal ofworn outor injured human tissue continue to increase and it is now apparent that this demand cannot be met from human donors. Apartial solution may be found in living related and trans-species transplantation butthese approaches invoke the problems ofdisease transfer and ethical dilemmas. Tissue engineering is a new technology that seeks to meet these increasing demands by utilising novel cell culture methods in vitro to provide tissue replacements in vivo. This article reviews the current state of tissue engineering and its potential for use in surgery Keywords: Tissue engineering, breast reconstruction, neointestine, tissue engineered colon, liver assist devices, pancreatic islet transplantation, cryopreservation Surg J R Coli Surg Edinb Irel., 2 Apri/2004, 70-78

INTRODUCTION There is a continuing impetus to improve the quality of life of patients who survive lifethreatening operations or illnesses and are left with considerable morbidity, such as diabetes following pancreatic resection or short bowel syndrome after multiple resections for Crohn's disease. Allied with this are the advancements in surgical science which seek to treat other diseases which have previously had a dismal outcome such as fulminant hepatic failure. Abdominal organ transplantation continues to be a successful treatment for some but it represents only a partial solution to these problems; it is an approach fundamentally limited by the dearth of donor organs. This is addressed to some degree by the increasing use of live-related donors for partial organ donation, but the deficit of organs to potential recipients is still huge. Tissue engineering represents a potential answer to this problem. This article seeks to explain the scientific basis of tissue engineering and describe the current status of research in some areas specifically relevant to the general surgeon. It is a burgeoning field with numerous articles being published, attempting to bring the promise of tissue engineered surgical solutions closer to a clinical reality. Unfortunately, there are still significant hurdles to overcome in many areas before tissue engineering becomes the routine approach to treating functional tissue deficits.

TISSUE ENGINEERING The first accepted example of tissue engineering dates back 70 years to when Bisceglie (1933) wrapped mouse tumour

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cells in a polymer membrane and successfully transplanted them into the chick embryo peritoneum. I One definition of tissue engineering is 'the science of generating tissue, using laboratory molecular biological techniques and the principles of material engineering, to treat a functional or anatomical defect in vivo'. There are three main ways in which this is achieved. The re-introduction into the body of individual or clusters of cells to reproduce a specific - often enzymatic - function. It has been used successfully in pancreatic islet transplantation.' A non-enzymatic example is the transplantation of myoblast cells into the area of a myocardial infarction to limit the area of fibrotic scarring and restore contractile fu nctiorr' Implantation of an acellular biomaterial scaffold onto or into which native cells from adjacent uninjured tissue repopulate. It acts as a template for regeneration - an example is the use of a synthetic polymer tube as a guide to peripheral nerve regeneratiorr' The implantation of pre-formed biomaterial constructs. These are generally cellular and tend to be more representative of the native organ with a greater and more © 2004 Surg J R Coli Surg Edinb Irel 2:2; 70-78

rapid functionality than the other two tissue engineering methods. An example would be the implantation of a binactive glass construct to reconstruct the middle ear ossicular chain ." The cells used in tissue engineering may be autologous, allogeneic or xenogeneic. Autologous cells are derived from the specific individual into which they will be later reimplanted. Such autologous cells require harvest or donation in advance of need, subsequently coupled with in vitro culture. This imparts both technical and time constraints but does avoid the issues of immunogenicity/rejection and transmission of disease. Allogeneic cells are derived from an individual of the same species other than the recipient. Common examples of allogeneic donations are cadaveric organ transplantation and pooled human blood transfusion. Xenogeneic cells arc derived from a species different to that of the recipient. Both allogeneic and xenogeneic cells raise important and potentially insurmountable issues of disease transmission, host rejection and ethical disquiet but retain the potential advantage of being cultured and constructed in advance of need. This would enable the generation of an ' off-the-shelf' replacement tissue. Whatever the source, the cells used may be either adult or embryonic. Embryonic cells arc, by definition, pluripotent and may be channelled into whichever cell line is required but this procedure is not technically straightforward and there is a limited supply of allogeneic embryonic cells available. Adult cells may be pluripotent or 'committed' to following a predetermined cellular heritage; harvest of adult cells may leave anatomical or functional defects at the donor site. Other than small cell clusters, implanted cells require a scaffold to act as a three-dimensional template. Scaffolds may be natural or synthetic, degradable or not. Examples of commonly used scaffolds are: an acellular small intestinal submucosal tube used in the generation of artificial arteries which is a natural non-degradable scaffold, or the polyglactin (Vicrylt'") scaffolds on which ncocartilage is generated - the VicrylTM hydrolyses as the cell population increases. The combination of scaffold and cells is known as a construct. In general, the majority of tissue engineering concepts are still in their infancy. Clinical utility and long-term follow-up is available for only a few products, notably cultured skin substitutes and cartilage renewal systems which arc both outside the scope of this article.??Some organ-specific research is more advanced than others, a state of affairs dictated in part by the relative complexity of the organs concerned and also the strength of the clinical need. Several areas are described below - some in considerable technical detail - in order to illustrate the extent of progress, to date, the volume of work being undertaken and the nature of the problems still to be surmounted before tissue engineering can provide solutions to the problems posed by everyday surgical practice.

© 2004 Surg J R Coli Surg Edinb Irel 2. 2; 70·78

Figure 1: A sheet of small intestinal submucosal scaffold. Animal gut is enzymatically treated torender anacellular wafer thin tissue that can actas a scaffold in a wide range of TE applications including arterial conduits, bladder reconstruction and the regeneration ofsmall intestine itself

Figure 2: A collagen sponge scaffold. Collagen is extracted from pig skins, homogenised and enzymatically treated toproduce a collagen solution free of antigenic material. It can be molded and freeze-dried before reinforcement over the base with polyglycolic acid felt toproduce arobust scaffold. Reproduced from the lnt J ofArtit Organs 2001; 24:5G-4 with pennission from Wichtig Editore.

ARTERIAL CONDUITS Given the large numbers of both coronary and peripheral artery bypass grafting that take place each year, the development of a successful, reliable, artificial small calibre arterial conduit has been termed the' Holy Grail' for cardiovascular surgeons." Whilst Dacron" and PTFE are successful large calibre grafts at diameters below about 6 mm, their patency rates are poor.') Before embarking on a project to engineer the ideal small calibre arterial graft it is useful to consider the properties it should possess. It should be elastic and able to withstand repetitive dynamic loading ie pulsatile flow. It must achieve burst pressures significantly greater than systolic pressure and present a non-thrombogenic surface that retains patency. In addition to these absolute requirements, the ideal conduit would also retain vasoactivity in response to circulating

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Figure 3:Collagen sponge scaffold being tested ina small intestine model. AScm length ofjejunum has been resected and a silicone stent interposed tomaintain a patent tube. A sheet ofcollagen sponge (arrow) is waiting to be wrapped around the tube to mimic the serosal layers.. After 4 weeks the silicone tube is removed endoscopically and the neointestine exposed to gut content. Reproduced from the Int J ofArtif Organs 2001; 24:50-4 with permission from Wichtig Editore.

chemical modulators, and should integrate into the host system rather than represent a persistent foreign body. Finally, and almost uniquely when considering tissue engineering implants, it must be able to function immediately upon implantation. Faced with such a formidable task, what advances has tissue engineering made in constructing the perfect artificial artery? The four general approaches of endothelialisation of synthetic grafts, collagen scaffolds, cell self-assembly and seeded biosynthetic scaffolds are outlined below. Endothelialisation of synthetic grafts: Early attempts to solve the 'Holy Grail' problem were centred on overcoming the inherent thrombogenicity of the luminal surface of small diameter synthetic grafts by seeding them with endothelial cells (ECs). This is the simplest approach, utilising current graft technology and availability, of tackling the thrombogenic nature of its lumina. In order to avoid the issue of immunogenicity, this approach requires an autologous cell source (usually from a previously excised vein or from the microvessels in a liposuction aspirate) which is then cultured in vitro before being seeded onto the graft. This technique has two inherent flaws. Firstly, it is actually very difficult to get ECs to adhere to the luminal surface of the graft, although novel adherence promoters such as a recombinant fibronectin compound have helped." Secondly, it does not confront the issues of vasoactivity and biointegration, but as larger calibre synthetic grafts seem to work well enough without confronting these issues it is open to debate whether this is a major problem. Collagen scaffolds: Two approaches have been used. Weinberg and Bell (1986) have mimicked the advential and medial layers of native artery using culture derived collagen / fibroblast gel sheets and a similar collagen / smooth muscle cell (SMC) sheet wrapped over each other to form a tubular construct. 11 These were then lined by an EC monolayer. This artificial artery represented a major advance in conduit © 2004Surg J R ColiSurg Edinb Ire12: 2; 7MB

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technology and presented theoretical advantages by promoting intercellular signalling via the collagen matrix." Unfortunately, the tensile strength of such constructs was woefully inadequate for physiological use and required external support from a Dacron" framework, which introduced the problems of a lack of remodelling and vasoactivity outlined above. I I The second collagen-based approach employs an acellular scaffold for native in vivo growth. Small intestine may be treated by detergent and enzymatic means to leave an acellular small intestine submucosa (SIS) tube. This may be further treated with cross-linked collagen and heparinised before implantation into animal hosts." The SIS graft represents a robust conduit with good compliance when compared with a standard vein graft. Animal studies have yielded reasonable medium-term patency rates.":" Unfortunately, whereas these animal studies have shown a smooth EC neo-intima when examined at necropsy. it is widely recognised that the human endothelial cells arc significantly more reluctant to populate a thrombogenic surface. This is an area of active current research. 17 Cell selfassembly models: L'Heureaux et at. (1995) have grown flat sheets ofSMCs and fibroblasts and then rolled them up around a central tubular support. This is removed some eight weeks later after maturation of the construct and subsequently followed by EC seeding. This produced a model of a trilaminar artery with an experimental burst pressure of 2000 mmHg. Gratifyingly, this construct method also preserved many cellular functions, often lost in standard culture environments such as von Willebrand factor expression by endothelial cells and good platelet adhesion inhibition in vitro. Unfortunately. this success did not transfer to the in vivo model with occlusion rates of 50% occuring at one week. IX It is also not clear whether this arterial conduit would function vasoactively. However, if autologous cells are used, this completely cell-cultured method would obviate the problem of immunogenicity. Seeded biosynthetic scaffold constructs: By using a © 2004 Surg J R Coif Surg Edinb Irel 2. 2, 70· 78

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degradable scaffold, SMCs can be seeded onto an appropriately sized and shaped template. As the culture progresses the scaffold is gradually replaced by a tubular model of SMCs; these can then be given an EC lining. An important feature of this type of modelling is the use ofbioreactor culture vessels." These impart dynamic mechanical stresses into the culture formulation which has been shown to markedly improve cellular orientation into a manner much more closely resembling a native artery." This results in higher tensile strength and burst pressure. 17 Niklason et af. (1999) used a polyglycolic tubular mesh and imparted a dynamic pulsatile radial distension force during eight weeks of culture; the polyglycolic mesh had by then been replaced by a SMC layer. After EC seeding and further culture, this bilaminar artificial artery had a burst pressure of 2000mmHg and exhibited serotonin and endothelin-I vasoactivity. Patency was maintained for up to four weeks after porcine carotid implantation." Whilst remarkable advances have been made in the pursuit of the artificial small calibre vascular conduit, ther e are significant hurdles still to be overcome before a tissue engineering vessel is available for clinical use . It has been suggested that it will be at least another decade before a marketable artery will be produced."

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BREAST TISSUE An increasing trend towards skin-sparing mastectomy, which maintains the residual skin envelope, has allowed reconstructive options to flourish which potentially lessen the cosmetic impact of autologous tissue tran sfer." Nevertheless, there are often still considerable problems associated with methods of reconstruction such as abdominal wall weakness and herniation after a transabdominal myorecutaneous flap or shoulder weakness and prominent scarring after a latissmus dorsi myorecutaneous reconstruction flap. Given that the problem is one of volume restitution, a cultured adipocyte construct would seem to be an ideal solution. Initial work sought to unravel the cellular and extracellular signalling mechanisms involved in the differentiation of the mesenchymal stem cell via many intermediaries into the adult adipocyte. Amongst a legion of cell-to-cell and intracellular messengers it appears that insulin and insulin-like growth factor-I (IGFI) have pre-eminence." Coupling this discovery with the use of poly(lactic-co-glycolic-acid)-polyethylene glycol (PLAG-PEG) microspheres for insulin and IGF-l delivery, Yuksel et af. (2000) demonstrated significant increases in the weight of rat adipofascial flaps after insulin/1GF-1 delivery sphere implantation, compared with sham spheres." This was due to an increase in the number of adipocytes rather than hypertrophy of existing fat cells and suggests a cellular proliferation and differentiation that was absent from the shamtreated side. Subsequent use of similar spheres in a non -adipose environment demonstrated fat generation de novo ." Th is work was extended to the insulin/lGF-1 microsphere seeding of a PGLA hemispherical scaffold before implantation into rats ." Histologically, after 12 weeks there was no residual scaffold, © 2004 Surg J R Coli Surg Edinb Ire!2: 2: 70-78

only a fibroelastic/ad ipose breast shape, albeit 30% smaller than the original scaffold. Work continues to identify methods of increa sing yield and rate of growth on these breast sca ffolds which ultimately could be used as an aesthetic replacement for the human breast although no evidence regar ding the potential longevity of such implants has accru ed. lUI 110'

SMALL INTESTINE Short bo wel syndrome rem ain s a difficult clin ical problem to manage whether it is a result of congenital atresia or multiple resections for Crohn's disease or infarction. Perm anent total parenteral nutrition has been used effecti vely in som e cases to ameliorate the metabolic and nutritional consequences of short bowel syndrome but is itself beset with problems of hepatic failur e, sepsis and access difficulties." Delaying small bowel transit by segment inversion is only parti ally successful and small bowel transplantation is still in its infancy." There are still problems of donor organ suppl y, immunosuppress ion and rejection - the five-year graft surviva l rate is only 4050%.29 Th is clinical need makes the small intestine one of the most exciting areas of tissue engi neering research - not least bec ause small bowel has been used as a scaffold for eng ineering a variety of other tissues since the 1960s.30 Two differing appro aches have been used . Tavakk et al. (200 1) have grown neo intestinal cyst structures in vivo after implantation of polyglycolic acid scaffolds seed ed with partially denatured small bowel tissue derived fro m an allogenic source.' 1 The cysts are opened after maturation and anastomosed to the in situ inte stine. Examination of this neointestinal tissu e has show n it to regenerate the normal structural features of the small intestine including mucosa, submucos a, a thin muscularis layer and villus form ation ." At a cellular level, ther e is norm al or ientation and distributi on of new enterocytes and cellular transport function is intac t." In addition , after 20 wee ks exposure to the normal intestinal environment in vivo, the neointestine is populated with a normal range of immune cells including B, T and natural killer cell s as well as rnacroph age s." The second approach has two subtypes , both using a collage n scaffold. Chen and Badylak (200 J ) extended the techniques that had util ised an SIS tube for blood vessels, dura mater and urina ry bladder replication and investigated its potential as a scaffold for small intes tinal regenerat ion .Pv' " :" After creation of gut defec ts by excision of 50% circumference of a small intestinal segment in 23 experimental dogs, SIS patches of appro ximately 7x3 cm were sutured in place . A further four dogs underwent a small bowel segmental resection with an interposition graft of approximately 6 em of tubular SIS anastomosed at either end . In the patch group, 20 out of 23 dogs survived, the remainder succumbing to peritonitis from patch leakage . Macroscopic all y, there was excellent integrati on of the patches into nat ive bowel w ith shrinkage of patch size to only 20% of original size at one year . Histological sect ions at six month s demonstrated virtuall y normal small intestinal © 2004 Surg J R Coli Surg EdinbIrel2: 2: 70-78

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wall. The tube grafts were less successful with three out of four dogs suffering anastomotic breakdown and the fourth showing a clinically apparent luminal stenosis of the graft. Hori et al. (200 1) used a collagen sponge scaffold deri ved from homogenized pig skin over a silicone tube to reconstruct a segmen t of previou sly bypa ssed j ej unum in a dog model." The silico ne tub e was removed endo scopically at four weeks. Histological exam ination showed a regenerated submucosal layer lined by intestinal mucosa with a villus pattern . There was a thin mus cularis layer and a firmly adherent serosa. Two out of six dogs had significant stenosis at the graft site. Small intestinal replacement employing tissue engineering is a promi sing appr oach . There are, however, specific area s of conce rn that must be addressed before clinical trials take place: ensuring sufficient musculari s generation for acti ve peristalsis and dem onstration in vivo of acceptable levels of absorpti ve, secretory and immunological function.

LARGE INTESTINE Total colectomy may be associated with significant long-term The Royal Colleges ofSurgeons ofEdinburgh and Ireland

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functional problems including bile salt malabsorption and increased stool frequency." Ileal pouch reconstruction is by no means a panacea with a long-term risk of pouchitis of about 50%.40 Some of these problems may be amenable to tissue engineering solutions. Using a modified version of the small bowel technique described above, implanted colon-seeded scaffolds into the omenta of rats has been carried OUt. 4 1 Four weeks later, the rats were randomised to have formation of an end ileostomy, or ileostomy and an interposition segment of tissue engineered colon just proximal to the stoma. These rats demonstrated significantly less weight loss and longer stool transit times than the standard experimental animals. Furthermore, such animals had a decreased stool water content, evidence of greater bile salt absorption and a more normal short chain fatty acid metabolism than controls. Histologically, the tissue engineered colon pouches were larger than at initial anastomosis with a normal large bowel architecture. Whilst large scale production of tissue engineered colon tubes has many problems to overcome, this experiment has demonstrated by 'proof-of-principle' that functional colon can be tissue engineered.

LIVER The use of tissue engineering in hepatology is twofold. It is recognised that there is a huge shortfall of cadaveric liver donors, compared with the requirements and that living sharedorgan donation is still not sufficient to meet these demands. Additionally, there is no reliable liver support device available to keep a patient alive when acute liver failure supervenes. It is known that in acute liver failure only 20% of those patients managed by medical means will survive and that the only reasonable expectation of cure lies with orthotopic transplantation." Tissue engineering may contribute in two ways - the creation of a liver support device to keep the patient alive whilst definitive treatment (transplantation or regeneration) occurs and secondly, the creation of a functional implantable liver replacement in lieu of orthotopic transplantation. Extracorporeal liver support systems, generically termed bio-artificial livers, generally incorporate a system of isolated hepatocytes in a synthetic suspension through which plasma is driven." Such systems function reasonably well, but there are problems due to the short functional life-span of the hepatocytes; current liver cell cultures remain stable for only up to eight hours before replacement is required. This raises issues, cost being one of them, regarding the suitability of such devices for long-term liver replacement. As there is a shortage of donor livers for orthotopic transplantation, a similar dearth of organs to provide hepatocytes for use in bio-artificiallivers exists; consequently most devices use porcine hepatocytes. Using pig cells raises the possibility of transmission of zoonoses, particularly pig endogenous retrovirus, although to date there have been no cases of in vivo transmission." Initial results of these devices has proved promising. A phase one clinical trial reported the experience of a porcine 4.

bio-artificial liver in three groups of patients requiring liver support." Sixteen out of 18 patients with fulminant hepatic failure survived (15 were bridged to a successful orthotopic transplantation and one recovered native liver function). All three patients who suffered primary failure of an orthotopic transplantation were bridged to re-transplantation and two out of ten patients with decompensation of chronic liver failure not suitable for transplantation were bridged to successful orthotopic transplantation, the remainder succumbing to their underlying disease. The other method ofreplicating hepatic function is by in vivo transplantation of hepatocytes. Several methods and sites for such transplantation have been described: intraportal injection with lodgement in the native liver, subcapsular injection in both kidney and spleen and transplantation into pre-implanted polyvinyl alcohol sponges.v" Notwithstanding the potential shortfall of donor human hepatocytes, this technique appears promising. A clinical case, whereby intraportal injection of human hepatocytes under cyclosporin immunosuppression allowed regeneration of the native liver and recovery from fulminant hepatic failure, has been reported and illustrates the technical feasibility of this technique." It has not yet been defined where the optimal recipient site of engraftment should be, although adjunctive procedures required to provide adequate hepatotrophic stimulation for heterotopically transplanted liver cells have been identified." Allogeneic hepatocytes were transplanted into a polyvinyl alcohol sponge that had previously been implanted into the greater omentum of experimental rats - a technique previously shown to be able to support a whole liver-equivalent cell mass." A portocaval shunt exposed the sponge (in the systemic circulation) to portal blood flow which appears to be vital for hepatotrophic stimulation and significantly increased the volume of graft attachment compared with controls." Concomitant transplantation of Islets of Langerhans cell clusters increased this still further and appears to be the optimal adjunctive regimen for hepatocyte transplantation. 50

PANCREAS Pancreatic replacement focuses on two separate areas; injection of isolated clusters of islets cells and the production of an artificial pancreas. It is notable that all pancreatic tissue engineering research is targeted at the endocrine pancreas, more specifically insulin secretion, and no attempts are made to address the function of the exocrine pancreas. The most successful group, to date, are the Edmonton group who have recently published long-term follow-up of 17 patients who have completed their transplant protocol; II are now insulin independent." Their technique involves intraportal injection of allogeneic cadaveric-derived islet cells. A non-steroidal immunosuppression regimen is used to minimise the side-effect profile but these have still been substantial with evidence of immunosuppression induced renal failure, anaemia and leucopaenia. Nevertheless, they have demonstrated a good clinical outcome in a reasonable number © 2004 Surg J R Coli Surg Edinb /rel2.· 2; 70-78

engineering neobladders, and artificial sphincters are already being used in clinical trials for the treatment of vesico-ureteric reflux.v-" Orthopaedic surgeons use autologous chondrocyte culture systems to repair osteochondral defects.' The treatment of cardiac failure and cardiomyopathy by implantation of tissue engineered muscle cells is an exciting prospect for cardiothoracic surgeons." It is likely that in time some, if not all, of these tissue engineering projects will find a place in the routine practice of most physicians and surgeons.

CURRENT PROBLEMS

Figure 6: To many this was a pinnacle ofachievement in tissue engineering - the first three dimensional realistic construct 'grown'from individual cells; to others it was an abomination and this typifies the problems that surround the perception oftissue engineering amongst the general public.

of patients previously blighted by labile diabetes. In attempts to overcome the problems of immunosuppression, several groups are investigating techniques with which to defeat the human immune system and introduce islet cell clusters safe from immunogenic reflection. One such method is by encapsulating them to prevent host recognition of the implant as foreign - immunoisolation. Several methods have been tried including alginate capsules, hydrogels and semi-permeable membranes but, unfortunately, as well as inhibiting immune recognition, they generate a fibrous foreign body reaction that inhibits oxygen and nutrient diffusion and diminishes islet function and longevity." A novel tissue engineering solution used autologous chondrocytes cultured in vitro to produce a membrane that can encapsulate the islet clusters without inhibiting insulin production." It is hoped that when implanted it will both avoid immune rejection and provide normoglycaemia. By harnessing hydrogel technology, Chae et al. (200 I) have constructeda biohybrid artificial pancreas." The hydrogel compound N-isopropylacrylamide (NiPAAm) mixed with a little with acrylic acid exhibits thermosensitive reversible gelation at temperatures between 30_34 °C. ~4 By suspending islet cell clusters in a NiPAAm matrix in liquid form it can be injected into a subcutaneously implanted biohybrid artificial pancreas. When the insulin secretion from the islets wanes. surface cooling of the pouch renders its contents liquid once more and suitable for aspiration followed by repeat injection of a fresh islet preparation. This list is by no means exhaustive and virtually every human tissue is the subject of tissue engineering research. Urologists may in due course look forward to tissue © 2004 SurgJ R ColiSurg Edmb Irel 2.· 2; 70-78

It is easy to be engulfed by a tide of enthusiasm for these innovative techniques that promise to revolutionise surgical practice, but it is important to recognise the enormity of some of the problems that, as yet, have not been satisfactorily addressed. Time: Many of the tissue culture techniques described herein are, by surgical standards, inordinately slow; for example L'Heureux's artificial artery took 12weeks of culture. Such techniques are not relevant when a surgical solution is required urgently. The ideal would be to manufacture these tissue constructs in advance of need and have them ready to use 'off-the-shelf' . This raises two further interlinked problems: immunogenicity and preservation. lmmunogenicity: The current successful tissue engineering products such as cartilage repair systems utilise autologous cells in elective situations. Unless individuals pre-donate cells to generate a unique bank of 'spare-part organs', the problems of the host immuneresponse to allogeneic or xenogeneictissues must be overcome. Current immunosuppressive regimen are costly and are associated with significant deleterious effects. Approaches such as the autologous chondrocyte wrapping, described above, seem promising. Perhaps an advanced donation of cartilage would be sufficient for an individual to maintain a stock of irnrnunoisolatory chondrocytewrappers to cover allogeneic tissue transplants. Preservation: Assuming that the problems of immunogenicity can be overcome with 'wrappers' or other techniques, a method of preserving these engineered artificial tissues for 'off-the-shelf' use must be identified. Ever since the discovery of cryoprotective agents like glycerol and its successful preservation of humangametesand embryos,science has tried 10 freeze ever more complex tissues." Unfortunately, cryopreservation results in formation of extracellular ice crystals which, on thawing, disrupt the intercellularbonds and signalling mechanisms. rendering them non-functional." To a certain extent, high dose cryoprotective agents overcome this by inducing amorphous solidification (vitrification) but such doses of cryoprotective agents are toxic to human tissue." : 60 Bothtechniquesrequire low-temperature storage and relatively 'high-tech' equipment. Thus, they are unlikely to be useful on a worldwide basis. where perhaps the need is greatest. A potential solution lies in heavy sugar technology. Several lower order biological systems such as seeds. fungi and brine shrimp routinely survivedry dormancyunharmedand it is thought that The Royal Colleges ofSurgeons of Edinburgh and Ireland

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this is related to high concentrations of heavy sugars such as trehalose." Its applicability to the preservation of engineered tissue is being evaluated. Vascularisation and Organogenesis: A fundamental limitation to the size of any implantable living construct is its blood supply. Complex tissues can receive nutrients and oxygen (and eliminate waste) by diffusion over only a few micrometres, Thus, any attempts at fabricating complex organoids must address the problems of angiogenesis. Incorporation of angiogenetic factors such as vascular endothelial growth factor is one potential solution, encouraging accelerated ingrowth of vessels from the host.v' However, even under the influence of vascular endothelial growth factor, this still occurs too slowly to sustain cellular survival. Alternatively, incorporation of preformed branching capillary networks into a tissue construct scaffold and anastomosis to a host vessel would appear feasible. Two techniques have been investigated to create these capillary networks. 'Solid free form fabrication' lays down layer after layer of a PGLA liquid copolymer using a modified ink jet printer," When dry, the powder is leached out leaving a 3-D network of branched channels and spaces throughout the scaffold. This can subsequently be seeded with engineered cells such as hepatocytes and excellent synthetic activity has been demonstrated from hepatocytes organised in this manner.t' A second method employs micromachining of silicone wafers to produce capillary networks down to 10 microns in diameter which are then seeded with endothelial cells. Proliferation and extracellular matrix deposition follows; the cell-sheets can then be lifted from the silicon mold and folded into three-dimensional constructs." Ethics: Many of the tissue engineered projects outlined above utilise cells and tissue from allogeneic and xenogeneic sources, both of which generate intense ethical debate. Similarly, embryonic stem cells, whilst representing an immensely useful tool in the genesis of many tissues, remain an emotive issue for many areas of the public. It would seem a pointless exercise engineering tissues if the intended recipients are unhappy to receive them. The ethical and moral dilemmas surrounding this work need to be clarified and resolved before

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its acceptance by the non-medical public. Isolated cellular transplantation of both pancreatic islets and hepatic cells have both been used on a limited clinical basis but the rest of tissue engineered cells and tissues are still experimental. In the foreseeable future, improvements in hepatocyte culture may deliver a serviceable liver assist device into routine practice, perhaps in the next 5-10 years. The small diameter arterial conduit will take at least another decade to develop and if the eventual aim of a non-immunogenic 'offthe-shelf' vessel is to be realised then 20-years is a more realistic time frame. Somewhere between these two extremes lie the other tissues; intestine, both small and large, is at least a decade away but a reliable breast reconstruction from cultured adipocytes could enter clinical trials within 10 years.

SUMMARY Tissue engineering has made enormous advances in the last decade and the first generation of clinical products are now in the medical marketplace. There are still significant hurdles to overcome before the general surgeon will be able to use a tissue engineering solution for a problem in everyday practice. It is almost inevitable, however, that the surgeon of 2030 will be widely versed in the application of the results of tissue engineering.

ACKNOWLEDGEMENTS I am profoundly grateful to Dr Joseph Vacanti, John Homans Professor of Surgery at Harvard Medical school, Dr Steve Badylak, Director of Pre-Clinical Tissue Engineering at the McGowan Institute for Regenerative Medicine, University of Pittsburgh, and Dr Yoshio Hori, of the Tohuku University Biomedical Engineering Research Organisation, Japan for their generous provision of images to illustrate this review. © British Crown copyright 2004/DSTL - published with the permission ofthe Controller of Her Majesty's Stationery Office

Copyright: 3 March 2004

© 2004Surg J R Coli Surg Edinb Irel 2:2; 70-78