Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation

Abstract

All procedures that restore missing tissue in patients require some type of replacement structure for the area of defect or injury. This form of therapy accounts for a large part of health-care resources (table). These devices have traditionally been totally artificial substitutes (joints), non-living processed tissue (heart valves), or tissue taken from another site from the patients themselves or from other patients (transplantation).1Langer R Vacanti JP Tissue engineering: the challenges ahead.Sci Am. 1999; 280: 62-65Crossref PubMed Scopus (279) Google Scholar Now a new alternative, tissue engineering, is becoming available to clinicians: the replacement of living tissue with living tissue that is designed and constructed to meet the needs of each individual patient.TableAnnual number of organ and tissue deficiencies and surgical procedures related to these (USA)IndicationProcedures or patients per yearCardiovascular*58 million people in the USA have cardiovascular disease, 14 million have coronary artery disease, and the estimated cost is US$274 billion per year. 65-70% of angioplasty patients need stents, and the cost is US$2 billion per year (data from Guidant Corporation and Reprogenesis Corporation Surveys).Heart—Including coronary artery bypass grafting1 821 000Angioplasty of coronary vessels1 000 000Blood vessels272 000Spinal cord (neural and neuromuscular)469 000Orthopaedic and plastic reconstructiveBone, cartilage, tendon, and ligament1 977 000Breast479 000GastrointestinalLiver, gallbladder, bile duct205 000Pancreas (diabetes)†Estimated cost of care for diabetes per year exceeds US$100 billion (data from American Diabetes Association).728 000Intestinal100 000OtherUrinary system including kidney740 000Skin2 509 000Hernia988 000Dental10 000 000Blood transfusions (units of blood)23 000 000Adapted and updated from Science 1993; 260: 920–26. Updated data from: Vital and Health Statistics, ambulatory and inpatient procedures in the United ‘States, 1996, Series 13: Data from the National Health Care Survey, no 139.Other references: American Association of Blood Banks; American Dental Association; American Diabetes Association; American Liver Foundation; National Center for Health Statistics; National Health Care Survey; National Liver Foundation; and The PTCA 20th Anniversary Project.* 58 million people in the USA have cardiovascular disease, 14 million have coronary artery disease, and the estimated cost is US$274 billion per year. 65-70% of angioplasty patients need stents, and the cost is US$2 billion per year (data from Guidant Corporation and Reprogenesis Corporation Surveys).† Estimated cost of care for diabetes per year exceeds US$100 billion (data from American Diabetes Association). Open table in a new tab Tissue engineering is an interdisciplinary field which applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function,2Langer R Vacanti JP Tissue engineering.Science. 1993; 260: 920-926Crossref PubMed Scopus (9193) Google Scholar Examples of tissue engineering can be found as early as 1933 when Bisceglie3Bisceglie V Uber die antineoplastische immunitat; heterologe Einpflanzung von Tumoren in Huhner-embryonen.Ztschr f Krebsforsch. 1933; 40: 122-140Crossref Scopus (62) Google Scholar encased mouse tumour cells in a polymer membrane and inserted them into the abdominal cavity of a pig.3Bisceglie V Uber die antineoplastische immunitat; heterologe Einpflanzung von Tumoren in Huhner-embryonen.Ztschr f Krebsforsch. 1933; 40: 122-140Crossref Scopus (62) Google Scholar The cells lived long enough to show that they were not killed by the immune system. In 1975, Chick and colleagues4Chick WL Like AA Lauris V Beta cell culture on synthetic capillaries: an artificial endocrine pancreas.Science. 1975; 187: 847-848Crossref PubMed Scopus (134) Google Scholar reported their results of encapsulating pancreatic-islet cells in semipermeable membranes to aid glucose control in patients with diabetes mellitus. Replacement of the skin with cells in collagen gels, or collagen-glycosaminoglycan composites to guide regeneration, was attempted by the early 1980s and these techniques are now in clinical use.5Bell E Ehrlich P Buttle DJ Nakatsuji T Living tissue formed in vitro and accepted as skin-equivalent of full thickness.Science. 1981; 221: 1052-1054Crossref Scopus (843) Google Scholar, 6Burke JF Yannas IV, Quimby WC Bondoc CC Jung WK Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury.Ann Surg. 1981; 194: 413-448Crossref PubMed Scopus (1008) Google ScholarThe next major advance came with the recognition that thicker three-dimensional systems could organise implanted cells to form vascularised tissue in vivo. Synthetic degradable polymers were used as templates for cells to form permanent new tissues.7Vacanti JP Morse MA Saltzman WM Domb AJ Perez-Atayde A Langer R Selective cell transplantation using bioabsorbable artificial polymers as matrices.J Pediatr Surg. 1988; 23: 3-9Summary Full Text PDF PubMed Scopus (508) Google Scholar Systems were designed with highly porous structures to meet the needs for the mass transfer of large numbers of cells. Angiogenesis after implantation produced permanent vascularised new tissue. With the demonstration of experimental success in many different tissues, and the engineering flexibility that synthetic materials provided, tissue engineering has now gained wide attention.2Langer R Vacanti JP Tissue engineering.Science. 1993; 260: 920-926Crossref PubMed Scopus (9193) Google Scholar8Mooney DJ Mikos AG Growing new organs.Sci Am. 1999; 280: 38-43Crossref PubMed Scopus (282) Google Scholar A recent survey of tissue engineering as an emerging industry found that the total capital value of companies in the industry exceeded US$3·5 billion, that the industry growth rate was 22·5% per year, and that it employed over 2500 scientists with an annual expenditure of $450 million.9Lysaght MJ Nguy NAP Sullivan K An economic survey of the emerging tissue engineering industry.Tissue Eng. 1998; 4: 231-238Crossref PubMed Scopus (71) Google Scholar It has been estimated that the total market potential of tissue-engineered products in the USA alone is $80 billion annually (Business Week, July 27, 1998, p 61).Current approaches in tissue engineeringThe primary goal of all approaches in tissue engineering is the restoration of function through the delivery of living elements which become integrated into the patient. Although some techniques of guided tissue regeneration rely on matrices alone, and other approaches rely on cells alone, most investigators in tissue engineering use cells combined with matrices to achieve new tissue formation (figure). Below, we summarise progress with matrices, cells, in-vitro bioreactor systems, and the generation of devices with their own vascular supply. Systems have been designed to be either open and totally integrated into the recipient, or closed (encapsulated) to provide protection from the host's immune system. Tissue-engineered devices can also use controlled drug-delivery methods to release growth factors that may augment angiogenesis or aid in new tissue generation.MatricesMost of the materials used as substrates or encapsulating materials for mammalian cells are either synthetic materials such as lactic-glycolic acid or polyacrylonitrile-polyvinyl chloride, or natural materials such as collagen, hydroxyapatite, or alginate. The former allow control of such material properties as strength, processability, degradation, microstructure, and permeability. Natural materials may be the actual in-vivo extracellular matrix components for cells, and as such would possess natural interactive properties such as cell adhesiveness. One area of research involves synthesis of tissue-regeneration templates that integrate desirable properties of both natural and synthetic materials.10Barrera DA Zylstra E Lansbury PT Langer R Synthesis and RGD peptide modification of a new biodegradable coplymer (polylactic acid-co-lysine).J Am Chem Soc. 1993; 115: 11010-11011Crossref Scopus (460) Google ScholarMatrices used in tissue-engineered devices need to be biocompatible and designed to meet the nutritional and biological needs of the cell populations involved in the formation of new tissue. Many approaches in tissue engineering have relied on synthetic, biodegradable polymer materials. The polymers must have good mechanical characteristics and, in many applications, specific shapes and geometries must be fabricated so as to maintain the polymers’ structure during new tissue formation. Polymers of polyglycolic acid were first shown to be effective as matrices for new tissue formation,7Vacanti JP Morse MA Saltzman WM Domb AJ Perez-Atayde A Langer R Selective cell transplantation using bioabsorbable artificial polymers as matrices.J Pediatr Surg. 1988; 23: 3-9Summary Full Text PDF PubMed Scopus (508) Google Scholar and subsequently found to be adaptable in creating highly specific shapes such as that seen in structural cartilage.11Vacanti CA Langer R Schloo B Vacanti JP Synthetic polymers seeded with chrondocytes provide a template for new cartilage formation.Plastic Reconstr Surg. 1991; 88: 733-759Crossref Scopus (522) Google Scholar Over the past decade, new synthetic materials and material-processing techniques have provided matrices to form tubes for blood vessels and intestine,12Shinoka T Shumtin D Ma PX et al.Creation of viable pulmonary artery autografts through tissue engineering.J Thor Cardiovasc Surg. 1998; 45: 536-545Summary Full Text Full Text PDF Scopus (368) Google Scholar, 13Niklason LE Gao J Abbott WM et al.Functional arteries grown in vivo.Science. 1999; 284: 489-493Crossref PubMed Scopus (1494) Google Scholar, 14Organ GM Mooney DJ Hansen LK Schloo B Vacanti JP Transplantation of enterocytes utilizing polymer-cell constructs to produce neoinstestine.Transplant Proc. 1992; 24: 3009-3011PubMed Google Scholar specific shapes of cartilage and bone for the ear and finger,15Vacanti CA Cima LG Ratkowski D Upton J Vacanti JP Tissue engineered growth of new cartilage in the shape of a human ear using synthetic polymers seeded with chondrocytes.in: 7th edn. Tissue-inducing biomaterials, Materials Research Society Symposium Proceedings. Vol 252. Materials Research Society, Pittsburgh1992: 367-374Google Scholar, 16Isogai N Landis W Kim TH Gerstenfeld LC Upton J Vacanti JP Tissue engineering of a phalangeal joint for application in reconstructive hand surgery.J Bone Joint Surg Am. 1999; 81: 306-316Crossref PubMed Scopus (6) Google Scholar and even heart-valve leafiets.17Shinoka T Ma PX Shum-Tim D et al.Tissue engineered heart valves.Circulation. 1996; 94: 164-168Google ScholarPorous alginate hydrogels have also been used as cell matrices. Their unique ability to change physical states from liquid to gel allows delivery with either catheter-based or endoscope-based systems. Autologous chondrocytes have been delivered endoscopically to cure urinary reflux in children18Diamond DA Caldamone AA Endoscopic treatment of vesicoureteric reflux in children using autologous chondrocytes.in: American Association of Pediatrics Annual Meeting, San Francisco, CA, USA October 1998: 176-177Google Scholar and urinary stress incontinence in women (Frank Gentile, Reprogenesis Corporation, personal communication).Cells used in tissue engineeringVirtually every tissue type in the human body has been investigated in terms of tissue engineering and most studies have looked at specific cell types (figure). For clinical applications, the cells are generally derived from the patients themselves, from close relatives, or other individuals. For example, autologous chondrocyte transplantation for knee repair is in clinical use.19Mayhew TA Williams GR Senica MA Kuniholm G Du Moulin GC Validation of a quality assurance program for autologous cultured chondrocyte implantation.Tissue Eng. 1998; 4: 325-334Crossref PubMed Scopus (56) Google Scholar In the case of tissue-engineered skin, neonatal dermal fibroblasts have been used. However, to provide cells for the different applications a source from which a variety of cells could be derived would be useful.One approach to solve the cell-source difficulty could be the isolation of human stem cells—cells which can be proliferated through multiple generations and made to differentiate into the appropriate cell type. Recent studies showed that stem cells derived from human embryonic blastocysts possessed these characteristics.20Thomson JA Itskovitz-Eldor J Shapiro SS et al.Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12119) Google Scholar Work on stem cells involved in the creation of cartilage, bone, and muscle has shown encouraging results in tissue-engineering applications. Progenitor cells that have been identified can be signalled to turn into cartilage or bone by changing the culturing conditions of the cells before implantation. In addition, small oval cells in the liver have been found, which can turn into either mature liver cells or bile ducts in culture.21Mitaka T Sato F Mizuguchi T Yokono T Mochizuki Y Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells.Hepatology. 1999; 29: 111-125Crossref PubMed Scopus (201) Google ScholarA critical issue for the future, from a tissue-engineering standpoint, is to learn how to control the permanent differentiation of stem-cell populations into the desired cell types, whether we need cartilage, bone, liver, or some other cell type. There are also a number of technical hurdles such as the need for pure stem-cell preparations (ie, those without other cells such as fibroblasts mixed in), methods to reduce cell adhesion during culture, and processes to increase the production of the large numbers of cells needed to create tissue.Another solution may be to create cells that could be used as “universal donors”. One approach uses molecules that mask the histocompatibility proteins on the cell surface that normally identify the donor cells as “non-self”.22Faustman D Coe C Prevention of xenograft rejection by masking donor HLA class I antigens.Science. 1991; 252: 1700-1702Crossref PubMed Scopus (97) Google Scholar This type of approach is being explored to make pig cells acceptable for transplantation to patients with Parkinson's disease.23Pakzaban P Deacon TW Burns LH Dinsmore J Isacson O A novel mode of immunoprotection of neural xenotransplants: masking of donor major histocompatibility complex class I enhances transplant survival in the central nervous system.Neuroscience. 1995; 65: 983-996Crossref PubMed Scopus (95) Google ScholarIn-vitro culture systemsMost of the early work in tissue engineering relied on the use of standard static cell-culture conditions for the in-vitro fabrication of tissue before implantation. However, stirred conditions have been shown to improve the quality of certain tissues, for example, cartilage.24Vunjak-Novakovic G Freed L Biron RJ Langer R Effects of mixing on the composition and morphology of tissue-engineered cartilage.Am Inst Chem Eng J. 1996; 42: 850-860Crossref Scopus (225) Google ScholarThe use of bioreactors enables the in-vitro culture of greater volumes of cells than can be obtained with conventional tissue-culture techniques. Flow and mixing within bioreactors can be controlled to enhance mass transfer of nutrients, gases, metabolites, and regulatory molecules, to regulate the size and structure of the forming tissue. Furthermore, bioreactors can provide mechanical regulatory signals, such as directly applied compression, to stimulate the cells to produce specific biomolecules.For example, one of the difficulties in trying to create thick cartilage constructs has been to control the mass transfer characteristics of nutrients, gases, metabolites, and the provision of physical regulatory signals. However, if cells are cultured on a three-dimensional polymer scaffold in a bioreactor with mixing, the fluid motion inside the reactor increases the mass transfer of nutrients and wastes, and results in cartilage samples that are 30% thicker. Furthermore, this increased mixing appears to stimulate the cells hydrodynamically to produce greater amounts of desired biochemical components, such as glycos-aminoglycans and collagen (increases of 60% and 125%, respectively), which leads to better biomechanical properties.24Vunjak-Novakovic G Freed L Biron RJ Langer R Effects of mixing on the composition and morphology of tissue-engineered cartilage.Am Inst Chem Eng J. 1996; 42: 850-860Crossref Scopus (225) Google ScholarAnother recent study showed that tissue-engineered blood vessels grown in the presence of pulsatile flow—ie, similar to flow in native blood vessels—have far better mechanical properties than those grown under static conditions.13Niklason LE Gao J Abbott WM et al.Functional arteries grown in vivo.Science. 1999; 284: 489-493Crossref PubMed Scopus (1494) Google ScholarThe generation of complex vascularised tissues and organsAlthough the approaches discussed above will meet the needs of many tissue types, the fabrication of large tissue and whole organs de novo remains a major challenge.The use of localised slow release of growth factors is one approach that is being tested. For example, locally released epidermal growth factor over several weeks led to a several-fold increase in the vascularisation and engraftment of liver cells in animal models.25Mooney D Kaufmann PM Sano K et al.Localized delivery of EGF improves the survival of transplanted hepatocytes.Biotechnol Bioeng. 1996; 50: 427-429Google Scholar Another approach involves the creation of devices that have a predesigned branching vascular network from inflow artery through capillary bed to venous outflow. This network would mimic the natural vasculature in its branching generations, size, and flow conditions. It would be generated by vascular endothelial and smooth-muscle cells. Work in this area has begun with technologies developed for solid free-form fabrication.26Griffith LG Wu B Cima MJ Powers MJ Chaignaud B Vacanti JP In vitro organogenesis of liver tissue.Ann NY Acad Sci. 1997; 831: 382-397Crossref PubMed Scopus (139) Google ScholarConcluding remarksTissue engineering is a new technology, but encouraging results are already being reported. The need is enormous and the potential benefits profound. However, much work needs yet to be done, and the research requires close interdisciplinary cooperation among clinical scientists, biologists, materials engineers, and chemists. All procedures that restore missing tissue in patients require some type of replacement structure for the area of defect or injury. This form of therapy accounts for a large part of health-care resources (table). These devices have traditionally been totally artificial substitutes (joints), non-living processed tissue (heart valves), or tissue taken from another site from the patients themselves or from other patients (transplantation).1Langer R Vacanti JP Tissue engineering: the challenges ahead.Sci Am. 1999; 280: 62-65Crossref PubMed Scopus (279) Google Scholar Now a new alternative, tissue engineering, is becoming available to clinicians: the replacement of living tissue with living tissue that is designed and constructed to meet the needs of each individual patient. Adapted and updated from Science 1993; 260: 920–26. Updated data from: Vital and Health Statistics, ambulatory and inpatient procedures in the United ‘States, 1996, Series 13: Data from the National Health Care Survey, no 139. Other references: American Association of Blood Banks; American Dental Association; American Diabetes Association; American Liver Foundation; National Center for Health Statistics; National Health Care Survey; National Liver Foundation; and The PTCA 20th Anniversary Project. Tissue engineering is an interdisciplinary field which applies the principles of engineering and the life sciences toward the development of biological substitutes that restore, maintain, or improve tissue function,2Langer R Vacanti JP Tissue engineering.Science. 1993; 260: 920-926Crossref PubMed Scopus (9193) Google Scholar Examples of tissue engineering can be found as early as 1933 when Bisceglie3Bisceglie V Uber die antineoplastische immunitat; heterologe Einpflanzung von Tumoren in Huhner-embryonen.Ztschr f Krebsforsch. 1933; 40: 122-140Crossref Scopus (62) Google Scholar encased mouse tumour cells in a polymer membrane and inserted them into the abdominal cavity of a pig.3Bisceglie V Uber die antineoplastische immunitat; heterologe Einpflanzung von Tumoren in Huhner-embryonen.Ztschr f Krebsforsch. 1933; 40: 122-140Crossref Scopus (62) Google Scholar The cells lived long enough to show that they were not killed by the immune system. In 1975, Chick and colleagues4Chick WL Like AA Lauris V Beta cell culture on synthetic capillaries: an artificial endocrine pancreas.Science. 1975; 187: 847-848Crossref PubMed Scopus (134) Google Scholar reported their results of encapsulating pancreatic-islet cells in semipermeable membranes to aid glucose control in patients with diabetes mellitus. Replacement of the skin with cells in collagen gels, or collagen-glycosaminoglycan composites to guide regeneration, was attempted by the early 1980s and these techniques are now in clinical use.5Bell E Ehrlich P Buttle DJ Nakatsuji T Living tissue formed in vitro and accepted as skin-equivalent of full thickness.Science. 1981; 221: 1052-1054Crossref Scopus (843) Google Scholar, 6Burke JF Yannas IV, Quimby WC Bondoc CC Jung WK Successful use of a physiologically acceptable artificial skin in the treatment of extensive burn injury.Ann Surg. 1981; 194: 413-448Crossref PubMed Scopus (1008) Google Scholar The next major advance came with the recognition that thicker three-dimensional systems could organise implanted cells to form vascularised tissue in vivo. Synthetic degradable polymers were used as templates for cells to form permanent new tissues.7Vacanti JP Morse MA Saltzman WM Domb AJ Perez-Atayde A Langer R Selective cell transplantation using bioabsorbable artificial polymers as matrices.J Pediatr Surg. 1988; 23: 3-9Summary Full Text PDF PubMed Scopus (508) Google Scholar Systems were designed with highly porous structures to meet the needs for the mass transfer of large numbers of cells. Angiogenesis after implantation produced permanent vascularised new tissue. With the demonstration of experimental success in many different tissues, and the engineering flexibility that synthetic materials provided, tissue engineering has now gained wide attention.2Langer R Vacanti JP Tissue engineering.Science. 1993; 260: 920-926Crossref PubMed Scopus (9193) Google Scholar8Mooney DJ Mikos AG Growing new organs.Sci Am. 1999; 280: 38-43Crossref PubMed Scopus (282) Google Scholar A recent survey of tissue engineering as an emerging industry found that the total capital value of companies in the industry exceeded US$3·5 billion, that the industry growth rate was 22·5% per year, and that it employed over 2500 scientists with an annual expenditure of $450 million.9Lysaght MJ Nguy NAP Sullivan K An economic survey of the emerging tissue engineering industry.Tissue Eng. 1998; 4: 231-238Crossref PubMed Scopus (71) Google Scholar It has been estimated that the total market potential of tissue-engineered products in the USA alone is $80 billion annually (Business Week, July 27, 1998, p 61). Current approaches in tissue engineeringThe primary goal of all approaches in tissue engineering is the restoration of function through the delivery of living elements which become integrated into the patient. Although some techniques of guided tissue regeneration rely on matrices alone, and other approaches rely on cells alone, most investigators in tissue engineering use cells combined with matrices to achieve new tissue formation (figure). Below, we summarise progress with matrices, cells, in-vitro bioreactor systems, and the generation of devices with their own vascular supply. Systems have been designed to be either open and totally integrated into the recipient, or closed (encapsulated) to provide protection from the host's immune system. Tissue-engineered devices can also use controlled drug-delivery methods to release growth factors that may augment angiogenesis or aid in new tissue generation. The primary goal of all approaches in tissue engineering is the restoration of function through the delivery of living elements which become integrated into the patient. Although some techniques of guided tissue regeneration rely on matrices alone, and other approaches rely on cells alone, most investigators in tissue engineering use cells combined with matrices to achieve new tissue formation (figure). Below, we summarise progress with matrices, cells, in-vitro bioreactor systems, and the generation of devices with their own vascular supply. Systems have been designed to be either open and totally integrated into the recipient, or closed (encapsulated) to provide protection from the host's immune system. Tissue-engineered devices can also use controlled drug-delivery methods to release growth factors that may augment angiogenesis or aid in new tissue generation. MatricesMost of the materials used as substrates or encapsulating materials for mammalian cells are either synthetic materials such as lactic-glycolic acid or polyacrylonitrile-polyvinyl chloride, or natural materials such as collagen, hydroxyapatite, or alginate. The former allow control of such material properties as strength, processability, degradation, microstructure, and permeability. Natural materials may be the actual in-vivo extracellular matrix components for cells, and as such would possess natural interactive properties such as cell adhesiveness. One area of research involves synthesis of tissue-regeneration templates that integrate desirable properties of both natural and synthetic materials.10Barrera DA Zylstra E Lansbury PT Langer R Synthesis and RGD peptide modification of a new biodegradable coplymer (polylactic acid-co-lysine).J Am Chem Soc. 1993; 115: 11010-11011Crossref Scopus (460) Google ScholarMatrices used in tissue-engineered devices need to be biocompatible and designed to meet the nutritional and biological needs of the cell populations involved in the formation of new tissue. Many approaches in tissue engineering have relied on synthetic, biodegradable polymer materials. The polymers must have good mechanical characteristics and, in many applications, specific shapes and geometries must be fabricated so as to maintain the polymers’ structure during new tissue formation. Polymers of polyglycolic acid were first shown to be effective as matrices for new tissue formation,7Vacanti JP Morse MA Saltzman WM Domb AJ Perez-Atayde A Langer R Selective cell transplantation using bioabsorbable artificial polymers as matrices.J Pediatr Surg. 1988; 23: 3-9Summary Full Text PDF PubMed Scopus (508) Google Scholar and subsequently found to be adaptable in creating highly specific shapes such as that seen in structural cartilage.11Vacanti CA Langer R Schloo B Vacanti JP Synthetic polymers seeded with chrondocytes provide a template for new cartilage formation.Plastic Reconstr Surg. 1991; 88: 733-759Crossref Scopus (522) Google Scholar Over the past decade, new synthetic materials and material-processing techniques have provided matrices to form tubes for blood vessels and intestine,12Shinoka T Shumtin D Ma PX et al.Creation of viable pulmonary artery autografts through tissue engineering.J Thor Cardiovasc Surg. 1998; 45: 536-545Summary Full Text Full Text PDF Scopus (368) Google Scholar, 13Niklason LE Gao J Abbott WM et al.Functional arteries grown in vivo.Science. 1999; 284: 489-493Crossref PubMed Scopus (1494) Google Scholar, 14Organ GM Mooney DJ Hansen LK Schloo B Vacanti JP Transplantation of enterocytes utilizing polymer-cell constructs to produce neoinstestine.Transplant Proc. 1992; 24: 3009-3011PubMed Google Scholar specific shapes of cartilage and bone for the ear and finger,15Vacanti CA Cima LG Ratkowski D Upton J Vacanti JP Tissue engineered growth of new cartilage in the shape of a human ear using synthetic polymers seeded with chondrocytes.in: 7th edn. Tissue-inducing biomaterials, Materials Research Society Symposium Proceedings. Vol 252. Materials Research Society, Pittsburgh1992: 367-374Google Scholar, 16Isogai N Landis W Kim TH Gerstenfeld LC Upton J Vacanti JP Tissue engineering of a phalangeal joint for application in reconstructive hand surgery.J Bone Joint Surg Am. 1999; 81: 306-316Crossref PubMed Scopus (6) Google Scholar and even heart-valve leafiets.17Shinoka T Ma PX Shum-Tim D et al.Tissue engineered heart valves.Circulation. 1996; 94: 164-168Google ScholarPorous alginate hydrogels have also been used as cell matrices. Their unique ability to change physical states from liquid to gel allows delivery with either catheter-based or endoscope-based systems. Autologous chondrocytes have been delivered endoscopically to cure urinary reflux in children18Diamond DA Caldamone AA Endoscopic treatment of vesicoureteric reflux in children using autologous chondrocytes.in: American Association of Pediatrics Annual Meeting, San Francisco, CA, USA October 1998: 176-177Google Scholar and urinary stress incontinence in women (Frank Gentile, Reprogenesis Corporation, personal communication). Most of the materials used as substrates or encapsulating materials for mammalian cells are either synthetic materials such as lactic-glycolic acid or polyacrylonitrile-polyvinyl chloride, or natural materials such as collagen, hydroxyapatite, or alginate. The former allow control of such material properties as strength, processability, degradation, microstructure, and permeability. Natural materials may be the actual in-vivo extracellular matrix components for cells, and as such would possess natural interactive properties such as cell adhesiveness. One area of research involves synthesis of tissue-regeneration templates that integrate desirable properties of both natural and synthetic materials.10Barrera DA Zylstra E Lansbury PT Langer R Synthesis and RGD peptide modification of a new biodegradable coplymer (polylactic acid-co-lysine).J Am Chem Soc. 1993; 115: 11010-11011Crossref Scopus (460) Google Scholar Matrices used in tissue-engineered devices need to be biocompatible and designed to meet the nutritional and biological needs of the cell populations involved in the formation of new tissue. Many approaches in tissue engineering have relied on synthetic, biodegradable polymer materials. The polymers must have good mechanical characteristics and, in many applications, specific shapes and geometries must be fabricated so as to maintain the polymers’ structure during new tissue formation. Polymers of polyglycolic acid were first shown to be effective as matrices for new tissue formation,7Vacanti JP Morse MA Saltzman WM Domb AJ Perez-Atayde A Langer R Selective cell transplantation using bioabsorbable artificial polymers as matrices.J Pediatr Surg. 1988; 23: 3-9Summary Full Text PDF PubMed Scopus (508) Google Scholar and subsequently found to be adaptable in creating highly specific shapes such as that seen in structural cartilage.11Vacanti CA Langer R Schloo B Vacanti JP Synthetic polymers seeded with chrondocytes provide a template for new cartilage formation.Plastic Reconstr Surg. 1991; 88: 733-759Crossref Scopus (522) Google Scholar Over the past decade, new synthetic materials and material-processing techniques have provided matrices to form tubes for blood vessels and intestine,12Shinoka T Shumtin D Ma PX et al.Creation of viable pulmonary artery autografts through tissue engineering.J Thor Cardiovasc Surg. 1998; 45: 536-545Summary Full Text Full Text PDF Scopus (368) Google Scholar, 13Niklason LE Gao J Abbott WM et al.Functional arteries grown in vivo.Science. 1999; 284: 489-493Crossref PubMed Scopus (1494) Google Scholar, 14Organ GM Mooney DJ Hansen LK Schloo B Vacanti JP Transplantation of enterocytes utilizing polymer-cell constructs to produce neoinstestine.Transplant Proc. 1992; 24: 3009-3011PubMed Google Scholar specific shapes of cartilage and bone for the ear and finger,15Vacanti CA Cima LG Ratkowski D Upton J Vacanti JP Tissue engineered growth of new cartilage in the shape of a human ear using synthetic polymers seeded with chondrocytes.in: 7th edn. Tissue-inducing biomaterials, Materials Research Society Symposium Proceedings. Vol 252. Materials Research Society, Pittsburgh1992: 367-374Google Scholar, 16Isogai N Landis W Kim TH Gerstenfeld LC Upton J Vacanti JP Tissue engineering of a phalangeal joint for application in reconstructive hand surgery.J Bone Joint Surg Am. 1999; 81: 306-316Crossref PubMed Scopus (6) Google Scholar and even heart-valve leafiets.17Shinoka T Ma PX Shum-Tim D et al.Tissue engineered heart valves.Circulation. 1996; 94: 164-168Google Scholar Porous alginate hydrogels have also been used as cell matrices. Their unique ability to change physical states from liquid to gel allows delivery with either catheter-based or endoscope-based systems. Autologous chondrocytes have been delivered endoscopically to cure urinary reflux in children18Diamond DA Caldamone AA Endoscopic treatment of vesicoureteric reflux in children using autologous chondrocytes.in: American Association of Pediatrics Annual Meeting, San Francisco, CA, USA October 1998: 176-177Google Scholar and urinary stress incontinence in women (Frank Gentile, Reprogenesis Corporation, personal communication). Cells used in tissue engineeringVirtually every tissue type in the human body has been investigated in terms of tissue engineering and most studies have looked at specific cell types (figure). For clinical applications, the cells are generally derived from the patients themselves, from close relatives, or other individuals. For example, autologous chondrocyte transplantation for knee repair is in clinical use.19Mayhew TA Williams GR Senica MA Kuniholm G Du Moulin GC Validation of a quality assurance program for autologous cultured chondrocyte implantation.Tissue Eng. 1998; 4: 325-334Crossref PubMed Scopus (56) Google Scholar In the case of tissue-engineered skin, neonatal dermal fibroblasts have been used. However, to provide cells for the different applications a source from which a variety of cells could be derived would be useful.One approach to solve the cell-source difficulty could be the isolation of human stem cells—cells which can be proliferated through multiple generations and made to differentiate into the appropriate cell type. Recent studies showed that stem cells derived from human embryonic blastocysts possessed these characteristics.20Thomson JA Itskovitz-Eldor J Shapiro SS et al.Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12119) Google Scholar Work on stem cells involved in the creation of cartilage, bone, and muscle has shown encouraging results in tissue-engineering applications. Progenitor cells that have been identified can be signalled to turn into cartilage or bone by changing the culturing conditions of the cells before implantation. In addition, small oval cells in the liver have been found, which can turn into either mature liver cells or bile ducts in culture.21Mitaka T Sato F Mizuguchi T Yokono T Mochizuki Y Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells.Hepatology. 1999; 29: 111-125Crossref PubMed Scopus (201) Google ScholarA critical issue for the future, from a tissue-engineering standpoint, is to learn how to control the permanent differentiation of stem-cell populations into the desired cell types, whether we need cartilage, bone, liver, or some other cell type. There are also a number of technical hurdles such as the need for pure stem-cell preparations (ie, those without other cells such as fibroblasts mixed in), methods to reduce cell adhesion during culture, and processes to increase the production of the large numbers of cells needed to create tissue.Another solution may be to create cells that could be used as “universal donors”. One approach uses molecules that mask the histocompatibility proteins on the cell surface that normally identify the donor cells as “non-self”.22Faustman D Coe C Prevention of xenograft rejection by masking donor HLA class I antigens.Science. 1991; 252: 1700-1702Crossref PubMed Scopus (97) Google Scholar This type of approach is being explored to make pig cells acceptable for transplantation to patients with Parkinson's disease.23Pakzaban P Deacon TW Burns LH Dinsmore J Isacson O A novel mode of immunoprotection of neural xenotransplants: masking of donor major histocompatibility complex class I enhances transplant survival in the central nervous system.Neuroscience. 1995; 65: 983-996Crossref PubMed Scopus (95) Google Scholar Virtually every tissue type in the human body has been investigated in terms of tissue engineering and most studies have looked at specific cell types (figure). For clinical applications, the cells are generally derived from the patients themselves, from close relatives, or other individuals. For example, autologous chondrocyte transplantation for knee repair is in clinical use.19Mayhew TA Williams GR Senica MA Kuniholm G Du Moulin GC Validation of a quality assurance program for autologous cultured chondrocyte implantation.Tissue Eng. 1998; 4: 325-334Crossref PubMed Scopus (56) Google Scholar In the case of tissue-engineered skin, neonatal dermal fibroblasts have been used. However, to provide cells for the different applications a source from which a variety of cells could be derived would be useful. One approach to solve the cell-source difficulty could be the isolation of human stem cells—cells which can be proliferated through multiple generations and made to differentiate into the appropriate cell type. Recent studies showed that stem cells derived from human embryonic blastocysts possessed these characteristics.20Thomson JA Itskovitz-Eldor J Shapiro SS et al.Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12119) Google Scholar Work on stem cells involved in the creation of cartilage, bone, and muscle has shown encouraging results in tissue-engineering applications. Progenitor cells that have been identified can be signalled to turn into cartilage or bone by changing the culturing conditions of the cells before implantation. In addition, small oval cells in the liver have been found, which can turn into either mature liver cells or bile ducts in culture.21Mitaka T Sato F Mizuguchi T Yokono T Mochizuki Y Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells.Hepatology. 1999; 29: 111-125Crossref PubMed Scopus (201) Google Scholar A critical issue for the future, from a tissue-engineering standpoint, is to learn how to control the permanent differentiation of stem-cell populations into the desired cell types, whether we need cartilage, bone, liver, or some other cell type. There are also a number of technical hurdles such as the need for pure stem-cell preparations (ie, those without other cells such as fibroblasts mixed in), methods to reduce cell adhesion during culture, and processes to increase the production of the large numbers of cells needed to create tissue. Another solution may be to create cells that could be used as “universal donors”. One approach uses molecules that mask the histocompatibility proteins on the cell surface that normally identify the donor cells as “non-self”.22Faustman D Coe C Prevention of xenograft rejection by masking donor HLA class I antigens.Science. 1991; 252: 1700-1702Crossref PubMed Scopus (97) Google Scholar This type of approach is being explored to make pig cells acceptable for transplantation to patients with Parkinson's disease.23Pakzaban P Deacon TW Burns LH Dinsmore J Isacson O A novel mode of immunoprotection of neural xenotransplants: masking of donor major histocompatibility complex class I enhances transplant survival in the central nervous system.Neuroscience. 1995; 65: 983-996Crossref PubMed Scopus (95) Google Scholar In-vitro culture systemsMost of the early work in tissue engineering relied on the use of standard static cell-culture conditions for the in-vitro fabrication of tissue before implantation. However, stirred conditions have been shown to improve the quality of certain tissues, for example, cartilage.24Vunjak-Novakovic G Freed L Biron RJ Langer R Effects of mixing on the composition and morphology of tissue-engineered cartilage.Am Inst Chem Eng J. 1996; 42: 850-860Crossref Scopus (225) Google ScholarThe use of bioreactors enables the in-vitro culture of greater volumes of cells than can be obtained with conventional tissue-culture techniques. Flow and mixing within bioreactors can be controlled to enhance mass transfer of nutrients, gases, metabolites, and regulatory molecules, to regulate the size and structure of the forming tissue. Furthermore, bioreactors can provide mechanical regulatory signals, such as directly applied compression, to stimulate the cells to produce specific biomolecules.For example, one of the difficulties in trying to create thick cartilage constructs has been to control the mass transfer characteristics of nutrients, gases, metabolites, and the provision of physical regulatory signals. However, if cells are cultured on a three-dimensional polymer scaffold in a bioreactor with mixing, the fluid motion inside the reactor increases the mass transfer of nutrients and wastes, and results in cartilage samples that are 30% thicker. Furthermore, this increased mixing appears to stimulate the cells hydrodynamically to produce greater amounts of desired biochemical components, such as glycos-aminoglycans and collagen (increases of 60% and 125%, respectively), which leads to better biomechanical properties.24Vunjak-Novakovic G Freed L Biron RJ Langer R Effects of mixing on the composition and morphology of tissue-engineered cartilage.Am Inst Chem Eng J. 1996; 42: 850-860Crossref Scopus (225) Google ScholarAnother recent study showed that tissue-engineered blood vessels grown in the presence of pulsatile flow—ie, similar to flow in native blood vessels—have far better mechanical properties than those grown under static conditions.13Niklason LE Gao J Abbott WM et al.Functional arteries grown in vivo.Science. 1999; 284: 489-493Crossref PubMed Scopus (1494) Google Scholar Most of the early work in tissue engineering relied on the use of standard static cell-culture conditions for the in-vitro fabrication of tissue before implantation. However, stirred conditions have been shown to improve the quality of certain tissues, for example, cartilage.24Vunjak-Novakovic G Freed L Biron RJ Langer R Effects of mixing on the composition and morphology of tissue-engineered cartilage.Am Inst Chem Eng J. 1996; 42: 850-860Crossref Scopus (225) Google Scholar The use of bioreactors enables the in-vitro culture of greater volumes of cells than can be obtained with conventional tissue-culture techniques. Flow and mixing within bioreactors can be controlled to enhance mass transfer of nutrients, gases, metabolites, and regulatory molecules, to regulate the size and structure of the forming tissue. Furthermore, bioreactors can provide mechanical regulatory signals, such as directly applied compression, to stimulate the cells to produce specific biomolecules. For example, one of the difficulties in trying to create thick cartilage constructs has been to control the mass transfer characteristics of nutrients, gases, metabolites, and the provision of physical regulatory signals. However, if cells are cultured on a three-dimensional polymer scaffold in a bioreactor with mixing, the fluid motion inside the reactor increases the mass transfer of nutrients and wastes, and results in cartilage samples that are 30% thicker. Furthermore, this increased mixing appears to stimulate the cells hydrodynamically to produce greater amounts of desired biochemical components, such as glycos-aminoglycans and collagen (increases of 60% and 125%, respectively), which leads to better biomechanical properties.24Vunjak-Novakovic G Freed L Biron RJ Langer R Effects of mixing on the composition and morphology of tissue-engineered cartilage.Am Inst Chem Eng J. 1996; 42: 850-860Crossref Scopus (225) Google Scholar Another recent study showed that tissue-engineered blood vessels grown in the presence of pulsatile flow—ie, similar to flow in native blood vessels—have far better mechanical properties than those grown under static conditions.13Niklason LE Gao J Abbott WM et al.Functional arteries grown in vivo.Science. 1999; 284: 489-493Crossref PubMed Scopus (1494) Google Scholar The generation of complex vascularised tissues and organsAlthough the approaches discussed above will meet the needs of many tissue types, the fabrication of large tissue and whole organs de novo remains a major challenge.The use of localised slow release of growth factors is one approach that is being tested. For example, locally released epidermal growth factor over several weeks led to a several-fold increase in the vascularisation and engraftment of liver cells in animal models.25Mooney D Kaufmann PM Sano K et al.Localized delivery of EGF improves the survival of transplanted hepatocytes.Biotechnol Bioeng. 1996; 50: 427-429Google Scholar Another approach involves the creation of devices that have a predesigned branching vascular network from inflow artery through capillary bed to venous outflow. This network would mimic the natural vasculature in its branching generations, size, and flow conditions. It would be generated by vascular endothelial and smooth-muscle cells. Work in this area has begun with technologies developed for solid free-form fabrication.26Griffith LG Wu B Cima MJ Powers MJ Chaignaud B Vacanti JP In vitro organogenesis of liver tissue.Ann NY Acad Sci. 1997; 831: 382-397Crossref PubMed Scopus (139) Google Scholar Although the approaches discussed above will meet the needs of many tissue types, the fabrication of large tissue and whole organs de novo remains a major challenge. The use of localised slow release of growth factors is one approach that is being tested. For example, locally released epidermal growth factor over several weeks led to a several-fold increase in the vascularisation and engraftment of liver cells in animal models.25Mooney D Kaufmann PM Sano K et al.Localized delivery of EGF improves the survival of transplanted hepatocytes.Biotechnol Bioeng. 1996; 50: 427-429Google Scholar Another approach involves the creation of devices that have a predesigned branching vascular network from inflow artery through capillary bed to venous outflow. This network would mimic the natural vasculature in its branching generations, size, and flow conditions. It would be generated by vascular endothelial and smooth-muscle cells. Work in this area has begun with technologies developed for solid free-form fabrication.26Griffith LG Wu B Cima MJ Powers MJ Chaignaud B Vacanti JP In vitro organogenesis of liver tissue.Ann NY Acad Sci. 1997; 831: 382-397Crossref PubMed Scopus (139) Google Scholar Concluding remarksTissue engineering is a new technology, but encouraging results are already being reported. The need is enormous and the potential benefits profound. However, much work needs yet to be done, and the research requires close interdisciplinary cooperation among clinical scientists, biologists, materials engineers, and chemists. Tissue engineering is a new technology, but encouraging results are already being reported. The need is enormous and the potential benefits profound. However, much work needs yet to be done, and the research requires close interdisciplinary cooperation among clinical scientists, biologists, materials engineers, and chemists. Acknowledgments We thank Jamie Lien for her help with background data research.