Volume 137 Number 1

SURGERY
JANUARY 2005

Surgical research review State-of-the-art tissue engineering: From tissue engineering to organ building
Shyh-Jou Shieh, MD, PhD, and Joseph P. Vacanti, MD, Tainan, Taiwan, and Boston, Mass

From the Division of Plastic and Reconstructive Surgery, Department of Surgery, College of Medicine, National Cheng Kung University, Tainan, Taiwan, and the Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Mass

TISSUE LOSS OR END-STAGE ORGAN FAILURE caused by injury or other types of damage is one of the most devastating and costly problems in human health care. Surgical strategies that have been developed to deal with these problems include organ transplantation from one individual to another, tissue transfer from a healthy site to the diseased site in the same individual, and replacement by using mechanical devices such as joint prosthesis or dialysis machine. Moreover, medical treatment encompassed supplementation of metabolic products of the nonfunctional tissue, such as insulin. Though signicant advances have been achieved in terms of health care by these therapeutic options, many limitations and unsolved issues remain. Organ transplantation is extremely limited by a critical donor shortage1 and the necessity of lifelong immunosuppression and its serious complications. Tissue transfer cannot replace all the functions of the original tissue and bears the risk of
Accepted for publication April 3, 2004. Reprint requests: Joseph P. Vacanti, MD, Department of Pediatric Surgery, MassGeneral Hospital for Children, 1157 Warren, 55 Fruit St, Boston, MA 02114. Surgery 2005;137:1-7. 0039-6060/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.surg.2004.04.002

donor-site complications. Major complications using nonbiological materials include infection, lack of biocompatibility, and limited material durability. When used in children, implanted mechanical devices do not grow as the child does. Chronic supplementation of metabolic products may result in a hormone imbalance due to lack of normal feedback mechanisms, leading to acute or longterm complications. Tissue engineering strives to offer a new solution to tissue loss or organ failure.

RATIONALE FOR TISSUE ENGINEERING Tissue engineering is an interdisciplinary eld that applies the principles of engineering and of life science towards the development of biological substitutes that restore, maintain, or improve tissue or organ function.2 In a classical sense, tissue engineering implies the use of tissue or organspecic cells for seeding a scaffold ex vivo and holds the promise of one day replacing living tissue with living tissue designed and fabricated to meet the individual defects.2,3 This approach is convincing based on several observations for the behavior of tissues and cells: Most tissues undergo remolding, isolated cells tend to form the appropriate tissue structures in vitro under favorable conditions, isolated cells require a template to guide their organization into a proper architecture,
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Fig 1. Tissue engineering process. *Cells: The cells may be tissue or organ specic, differentiated or progenitor cells, adult or embryonic stem cells. They may be autologous, allogeneic, or xenogeneic. In vitro proliferation of chondrocytes is shown. #Matrix: The material may be natural (collagen, brin) or synthetic (polyglycolic acid [PGA]). The matrix can be brous, foam, capsules, gels, or highly complex structures. A PGA brous mesh and poly -caprolactone (PCL) porous sponge are shown. yBioreactor: The bioreactor may be designed with various conditions such as shear stresses, hydrodynamic stimulation, pulsatile or continuous ow to improve seeding efciency and culture conditions in vitro. **Cell/matrix construct (upper, chondrocytes/PGA construct; lower, chondrocytes/PCL construct): Chondrocytes are dynamically seeded and cultured for 6 weeks on a PGA brous mesh and PCL porous sponge. Micrographs of scanning electron microscopy show chondrocytes that adhere to the scaffold and lots of extracellular matrix formation before implantation.

and most tissues cannot be implanted in large volumes due to diffusion limitation.2 Therefore, combining cells of various tissues and types to natural or synthetic degradable matrices to produce living structures has become the most common approach in the eld of tissue engineering (Fig 1). To date, many tissues have been developed in the laboratory, and laboratory-grown organs are beginning to take shape.4 Nevertheless, many obstacles and challenges remain. The fundamental problem involves the mass transfer of oxygen and nutrition, as well as the neovascularization of living tissue in a 3-dimensional structure. It is believed that a successful product of tissue engineering or organ fabrication requires an adequate source of healthy, expandable cells, cell-friendly scaffolds, and the optimal bioreactors. With regard to the application of stem cell biology and material science and microfabrication

technology, and the development of a dynamic in vitro culture system, recent progress and challenges in tissue engineering and organ fabrication are reviewed in this article. CELL SOURCES IN TISSUE ENGINEERING: THE PROMISE OF STEM CELLS Cells play a crucial role to tissue regeneration and repair due to their characteristics of proliferation and differentiation, cell-to-cell interaction, biomolecular production, and extracellular matrix formation. The sources of cells used in tissue engineering can be autologous, allogeneic, or xenogeneic. Ideal donor cells for tissue engineering would be those that are easily accessible, that can easily expand without permanently altering the phenotype and function and without transmitting species-specic pathogens, that are multipotent to differentiate or transdifferentiate into a variety of tissue- or organ-specic cells with

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Fig 2. Therapeutic cloning and its application to tissue or organ engineering. Therapeutic cloning could theoretically provide a limitless source of cells, except mitochondrial DNA, that are genetically identical to the donor.

specialized function, and that have the least immunologic response. Some cells, such as keratinocytes, broblasts, chondrocytes, endothelial cells, smooth muscle cells or skeletal muscle satellite cells, proliferate rapidly. They are good tissue-specic cell sources for tissue engineering. Two Food and Drug Administration (FDA)eapproved living skin products engineered in the laboratory have been applied to a patient with diabetic or venous skin ulcers, and an FDA-approved autologous cell product also has been used to repair an articular cartilage. However, other cells, such as hepatocytes or adult cardiomyocytes, proliferate slowly or not at all. Therefore, alternative sources of cells are needed. Recent advances in stem cell biology have had a marked impact on the progress of tissue engineering.5 Stem cells, which are capable of self-renewal and differentiation into various cell lineages, hold great promise for treating affected tissue in which the source of cells for repair is

limited or not readily accessible. Cells derived from human embryonic blastocysts (after undifferentiated proliferation in vitro for 4-5 months) still maintain the developmental potential to form trophoblast and derivatives of all 3 embryonic germ layers, including gut epithelium (endoderm); cartilage, bone, smooth muscle, and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratied squamous epithelium (ectoderm).6 Although these cell lines should be useful in human regenerative medicine, the ethical and legal issues are still under debate. Adult bone marrow stem cells can replicate as undifferentiated cells that have the potential to differentiate into lineages of mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. They display a stable phenotype, remain as a monolayer in vitro, and could be induced to differentiate exclusively into adipocytic, chondrocytic, or osteocytic lineages.7 To date, the isolation of various autologous adult stem cells,

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including mesenchymal, hematopoietic, neural, muscle, and hepatic stem cells, are being investigated actively because they are immunocompatible and have no ethical concerns. Nevertheless, there are a number of technical obstacles, such as how to isolate stem cell preparations without contamination by other cells, how to control the permanent differentiation to the desired cell types, and how to increase the production of the large number of cells needed to create tissue. The host-immune response to allogenic or xenogeneic cells poses a different but major challenge. Another approach is to create universal donor cells by masking histocompatibility proteins on the cell surface to reduce the cells antigenicity.8 Nuclear transfer, or therapeutic cloning, is a process wherein the nucleus of a somatic cell is injected into an unfertilized, enucleated oocyte (Fig 2). This transformation probably involves deletion of the existing epigenetic state and regression to an embryonic pattern of gene expression. Through this nuclear manipulation, any differentiated somatic cell can potentially be reprogrammed back to totipotency, which results in redifferentiation to the full repertoire of adult cells for any individual tissue repair.9 Although the goal of therapeutic cloning is to generate replacement cells and tissues that are genetically identical to those of the donor, noneself-mitochondria proteins derived from the recipient oocytes could render cloned tissue immunogenic. Some groups have demonstrated that somatic cell nuclear transplantation can give rise to fetal tissues that express recipient oocyte mitochondrial genes but are histocompatible when transplanted back into the nuclear donor.10 These ndings bring closer the promise of therapeutic cloning and tissue engineering. The combination of nuclear transfer, gene therapy, and cell transplantation as a possible applicable paradigm for genetic and phenotypic correction is a challenge to many active scientists worldwide. SCAFFOLDS IN TISSUE ENGINEERING: THIRD-GENERATION BIOMATERIALS (SMART BIOMATERIALS) Scaffolds can be produced from natural materials (collagen, alginate, hydroxyapatite) or synthetic polymers (polyglycolide, polylactide, polylactide coglycolide). Natural materials may closely mimic the native cellular environment, whereas synthetic polymers have the advantages of being able to better control material properties. In general, the ideal scaffold should be 3-dimensional, highly porous with an interconnected pore

network, and biocompatible with a controlled degradation rate; should have an appropriate surface for cell adhesion, proliferation, and differentiation; and should maintain proper mechanical properties. Early work in our laboratory demonstrated that bovine chondrocytes seeded onto a synthetic, biodegradable scaffolding could produce neocartliage after transplantation into athymic mice. We further reported that cartilage can be created in predetermined shapes and dimensions by using cell transplantation on appropriate polymer templates, even in a complex 3-dimensional architecture, like a human ear.11 Tissue engineering of auricular reconstruction with different biodegradable biomaterial trials was subsequently investigated.12 The delicate 3-dimensional polymer scaffolds of high porosity and surface area are crucial to structural tissue engineering, such as bone and cartilage. A new method of 3-dimensional printing involves a technique of solid free-form fabrication that builds complex 3-dimensional polymer structures from a series of extremely thin 2-dimensional layers, starting with a computer model of the desired shape derived from a computerized tomography scan or magnetic resonance imaging of the original tissue. Development of biomaterials poses signicant challenge for tissue engineering. The goal of early or rst-generation biomedical materials, during the 1960s and 1970s, was to attain suitable physical properties to match the replaced tissue with a common feature of biological inertness. Second-generation biomaterials were designed to produce bioactive responses that could elicit a controlled reaction in the physiologic environment. Such bioactive (ceramics, hydroxyapatite) or resorbable (polyglycolide, polylactide) materials have been applied in the medical needs of many elds successfully. Third-generation biomaterials are combining these 2 properties and are being designed to stimulate specic cellular responses at the molecular level.13 Several smart biomaterials for tissue engineering and regeneration are activated by either cells or genes and are designed to improve the complicated biological event of tissue repair. Incorporation of a signal peptide such as RGD (Arg-Gly-Asp) into the biomaterial has attempted to mimic the extracellular matrix, modulate cell adhesion, and induce cell migration. An intermediate density of adhesive ligand is crucial for optimal cell migration. With recent advances in nanotechnology, nanoscale clustering of RGD peptides at surfaces using comb polymer is more effective for inducing cell adhesion and migration.14

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Scaffolds can also be designed to control the release of growth factors that induce cell differentiation in vitro, or in situ tissue regeneration in vivo. Researchers have demonstrated that survival and differentiation of fetal brain cells were improved by the preassembly of neotissues containing cells and nerve growth factorereleasing synthetic particles.15 However, cytokines are unstable and turn over rapidly at the injury site. Gene-activated matrix, the incorporation of plasmid DNAecontaining genes that encode growth factors into polymer matrices, provide a sustained release of growth factors in vivo and result in reproducible tissue regeneration.16 IN VITRO BIOREACTOR SYSTEM: VERSATILE DESIGNS FOR INDIVIDUAL NEEDS Early work in tissue engineering relied on static conditions for in vitro culture to grow tissues in the laboratory. The in vitro dynamic culture design of a bioreactor offers considerable advantages over the static one. Flow and mixing within the bioreactor can be controlled to enhance the mass transfer of nutrients, gases, metabolites, and regulatory molecules, as well as to regulate the size and structure of the forming tissue.3 The modern bioreactor design should simulate the complex physiologic environments in vivo, such as physicochemical parameters (eg, pH, temperature, concentration of nutrients and metabolites), mass transfer rates, and biomechanical conditions, varying only from one tissue to another. For example, physiologic stresses with hydrodynamic stimulation during the engineering of cartilage resulted in a better production of extracellular matrix and an improvement of biomechanical properties. Tissue-engineered blood vessels under shear stresses in pulsatile ow bioreactors had greater burst strength, suture retention, and collagen content than that of nonpulsed culturing.17 In addition, hepatocytes cultured under continuous ow bioreactors showed an increased organization and function compared with those cultured under static conditions.18 SPECIFIC ENGINEERED TISSUES ACHIEVED Over the past 2 decades, our laboratory at Harvard Medical School, in collaboration with the Massachusetts Institute of Technology, has been able to study over 30 tissues of the body, with many showing sophisticated structure and function in animal replacement models.19,20 Five engineered tissues have been approved by FDA; several academic institutes as well as companies are making

efforts to develop new products for regenerative medicine. One skin product, composed of human neonatal dermal broblast grown on a biodegradable scaffold and cryopreserved, has been used to treat foot ulcers. Another product contains multilayered skin, including both dermal and epidermal components. Several types of cartilage replacement therapy, as well as replacement therapies for corneas, blood vessels, and bone, have been successfully used in clinical trials. Injection of autologous chondrocytes to correct vesicoureteral reux in children and patients with urinary incontinence appear to be effective and safe. Our earlier work in tissue engineering of the musculoskeletal system addressing muscle, cartilage, and bone, was focused on using cells in conjunction with synthetic biocompatible scaffolds. Autologous fetal myoblast tissue engineering can be a viable alternative for diaphragmatic replacement in a lamb model.20 The engineered cartilage in the shape of a human ear, a delicate 3-dimensional structure, was rst reported.11 Further in vitro and in vivo studies in auricular tissue engineering bordered on actual clinical application.12 The signicant accumulation of knowledge of optimal conditions for cartilage tissue engineering allows for the ability to engineer other types of cartilage tissues, such as those for nasoseptum, temporomandibular joint disc, composite tracheal tissue, meniscus, and joint resurfacing. For an osteochondral joint defect, in vitro generation of osteochondral tissue composites based on biodegradable polymer scaffolds with chondrogenic and osteogenic cells may provide better osteochondral repair with the development of a well-dened tissue-to-tissue interface. The formation of small phalanges and whole joints from bovine-cell sources transplanted onto biodegradable polymer matrices in athymic mice was further described.20 Moreover, the successful replacement of an avulsed phalanx with tissue-engineered bone suggests that the use of tissue-engineered bone may be an effective approach to the treatment of bone loss due to trauma or disease.21 In cardiovascular tissue engineering, the goal is to develop articial blood vessels and heart valves. For blood vessels, the large diameter (>5 mm in diameter) grafts were commercialized by using Dacron and expanded polytetrauoroethylene. These materials lack growth potential; however, they have a limited use in pediatric cardiovascular surgery. Living vascular grafts engineered from autologous cells and biodegradable polymers functioned well in the pulmonary circulation as demonstrated in lambs.20 This work has evolved

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into the clinical application of transplantation of a tissue-engineered pulmonary artery in a child with a complex congenital heart disease and pulmonary atresia.22 But, the tissue engineering of small-caliber blood vessels has been difcult, and further investigation is ongoing in our laboratory. For tissue engineering of heart valves, we have demonstrated preliminarily that a tissueengineered valve leaet constructed from its cellular components can function in the pulmonary valve position in lambs.23 A whole, tri-leaet, tissueengineered heart valve was then developed and implanted in the pulmonary position with appropriate function for 120 days in a lamb model.24 For nerve tissue engineering, researchers in our group have created a tubular nerve guidance conduit with a biodegradable scaffold and cultured Schwann cells, which possesses the macroarchitecture of a polyfascicular peripheral nerve; work with this model has demonstrated the feasibility of in vivo regeneration through the conduit.20 Furthermore, a biodegradable nerve guidance conduit loaded with growth factor was developed by using materials originally designed for drug delivery applications. Different designs of conduits seeded with Schwann cells are under investigation to promote guided peripheral nerve regeneration. THE CHALLENGE OF ORGAN BUILDING Although the aforementioned approaches in tissue engineering may fulll the needs of several types of tissues, the problem of the fabrication of thick complex tissues (eg, gastrointestinal tract) and whole organs (eg, the liver and kidneys) remains a major challenge. First, the difcult issues regarding isolation, proliferation, and differentiation of multiple cell types to form a complex tissue or whole organ must be overcome. In preliminary studies from our laboratory, intestinal progenitor crypt cells seeded on polymer tubes formed vascularized cysts with a well-differentiated intestinal epithelial lining with mucous secretion.20 Subsequent experiments showed that intestinal crypt cells transplanted heterotopically as epithelial organoid units that consist of a villus structure with overlying epithelium and a core of mesenchymal stromal cells was crucial for development of a composite tissue resembling the small intestine. The epithelial organoid units appeared to maintain the epithelialemesenchymal cellecell interaction, which is important for organotypic morphogenesis and cytodifferentiation. These experiments have evolved into the recent demonstration of good

formation of functional stomach, esophageal, and colon tissues.25 Second, the vascularization of a mass of complex tissue or whole organ in a 3-dimensional structure is a critical and unsolved problem. Tissue implanted with a volume greater than 2 to 3 mm3 cannot obtain sufcient survival of a large mass of transplanted cells to provide function because provision of nutrition, gas exchange, and elimination of waste products are limited by the maximum diffusion distance. Three common approaches have been used for vascularization of bioengineered tissues: incorporation of angiogenic factors, seeding of endothelial cells along with other cell types, and prevascularization of matrices prior to cell seeding.26 However, these methods appear inadequate for thick, complex organs, such as the liver, kidneys, heart, and lungs. Initially we designed a novel, 3-dimensional, synthetic biodegradable polymer scaffold with an intrinsic network of interconnected channels that mimic vascular branching using a 3-dimensional printing technique. Hepatocytes co-cultured with nonparenchymal cells can attach to and survive on these scaffolds in both static and ow conditions.20 Subsequently, using the technologies of microelectromechanical system and standard photolithography, we etched trench patterns reminiscent of the branched architecture of vascular and capillary networks onto silicon and Pyrex surfaces to serve as templates. Microvascular branched architecture, even less than 10 microns in diameter at the capillary level, can be created. Hepatocytes and endothelial cells were then cultured in these tissue-engineered branched vascular channels and showed viable proliferative ability and partial hepatocyte function.27 These templates were then used as molds to replicate the branching patterns onto biodegradable polymer lms, which can then be seeded with endothelial cells to generate a microvasculature. Currently, in collaboration with Draper Laboratories (Cambridge, Mass), we have developed a robust computational model of the vascular circulation, which is based on the fractal nature of network topology, rheology of blood ow, and mass transfer of oxygen and nutrients across the vascular bed.28 These microvascular networks will need to be optimized to provide an even distribution of blood and maximize the mass transfer of oxygen and nutrients to the surrounding tissue, while minimizing resistance to blood ow. We have been able to build a living hepatic system with its own vascular supply by stacking these systems and alternating them with

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hepatocyte compartments. Studies for laboratorygrown kidneys are also underway with a similar approach at our laboratory but remain in their early phase. CONCLUSION Although tremendous progress and encouraging results have been achieved in the eld of tissue engineering, many challenges remain and much work still needs to be done. The ultimate goal of tissue engineering is not only to replace structures and improve the function of diseased tissue, but also to build neo-organs for patients in need. Recent advances in stem cell biology, gene therapy, and engineering technology have allowed an extremely close interdisciplinary collaboration among developmental and cellular molecular biologists, gene therapists, materials engineers, chemists, and physicians, to move the laboratory-grown tissue and organ to clinical application.
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12. Shieh SJ, Terada S, Vacanti JP. Tissue engineering auricular reconstruction: in vitro and in vivo studies. Biomaterials 2004;25:1545-57. 13. Hench LL, Polak JM. Third-generation biomedical materials. Science 2002;295:1014-7. 14. Irvine DJ, Mayes AM, Grifth LG. Nanoscale clustering of RGD peptides at surfaces using Comb polymers. 1. Synthesis and characterization of Comb thin lms. Biomacromolecules 2001;2:85-94. 15. Mahoney MJ, Saltzman WM. Transplantation of brain cells assembled around a programmable synthetic microenvironment. Nat Biotechnol 2001;19:934-9. 16. Bonadio J, Smiley E, Patil P, Goldstein S. Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration. Nat Med 1999;5: 753-9. 17. Niklason LE, Gao J, Abbott WM, Hirschi KK, Houser S, Marini R, et al. Functional arteries grown in vitro. Science 1999;284:489-93. 18. Kim SS, Utsunomiya H, Koski JA, Wu BM, Cima MJ, Sohn J, et al. Survival and function of hepatocytes on a novel 3-dimensional synthetic biodegradable polymer scaffold with an intrinsic network of channels. Ann Surg 1998;228: 8-13. 19. Vacanti JP. Tissue and organ engineering: can we build intestine and vital organs? J Gastrointest Surg 2003;7:831-5. 20. http://www.mgh.harvard.edu/depts/tissue/bibli/main_bib. html 21. Vacanti CA, Bonassar LJ, Vacanti MP, Shufebarger J. Replacement of an avulsed phalanx with tissue-engineered bone. N Engl J Med 2001;344:1511-4. 22. Shinoka T, Imai Y, Ikada Y. Transplantation of a tissue-engineered pulmonary artery. N Engl J Med 2001;344:532-3. 23. Shinoka T, Breuer CK, Tanel RE, Zund G, Miura T, Ma PX, et al. Tissue engineering heart valves: valve leaet replacement study in a lamb model. Ann Thorac Surg 1995; 60:S513-6. 24. Sodian R, Hoerstrup SP, Sperling JS, Daebritz S, Martin DP, Moran AM, et al. Early in vivo experience with tissueengineered trileaet heart valves. Circulation 2000;102: III22-9. 25. Grikscheit TC, Ochoa ER, Ramsanahie A, Alsberg E, Mooney D, Whang EE, et al. Tissue-engineered large intestine resembles native colon with appropriate in vitro physiology and architecture. Ann Surg 2003;238:35-41. 26. Nomi M, Atala A, Coppi PD, Soker S. Principals of neovascularization for tissue engineering. Mol Aspects Med 2002;23:463-83. 27. Kaihara S, Borenstein J, Koka R, Lalan S, Ochoa ER, Ravens M, et al. Silicon micromachining to tissue engineer branched vascular channels for liver fabrication. Tissue Eng 2000;6:105-17. 28. Kaazempur-Mofrad MR, Vacanti JP, Kamm RD. Computational modeling of blood ow and rheology in fractal microvascular networks. Computational Fluid Solid Mechanics 2001;2:864-7.