INTRODUCTION 58 102 103 104 114 178 27 36 74 80 123 14 119 122 176 127 50 64 146 1 35 68 120 67 TABLE 1. Design objectives for and characteristics of replacement heart valves. Feature to optimize Conventional (Mechanical, bioprosthetic) Tissue engineered Closure of leaflets Rapid and complete Rapid and complete Size of orifice area Less than that of natural valves Better Mechanical properties Stable Stable Surgical insertion Easy and permanent Easy and permanent Risk of thrombosis Yes, especially mechanical valves, which require anticoagulation, causing vulnerability to hemorrhage No; endothelial surface to inhibit thrombogenesis Risk of structural dysfunction Degradation of synthetic materials rare with mechanical valves Resistant to degradation and calcification  Tissue degradation and calcification of leaflets with bioprosthetic valves  Risk of Infection Ever present Resistant to infection Viability No Yes, able to repair injury, remodel, and potentially grow with patient FIGURE 1. Specialized ECM enables dynamic aortic valve function. (a) Photograph of the aortic valve in open and closed position (from the aorta). (b) Aortic valve histology emphasizing trilaminar structure and presence of valvular interstitial and endothelial cells. (c) Biomechanical cooperativity between elastin and collagen during valve motion. (d) Schematic depiction of layered aortic valve cuspal structure and configuration of collagen and elastin during systole and diastole. (a) and (b) reproduced by permission from Schoen FJ. “Valvular heart disease: General principles and stenosis,” IN: Cardiovascular Pathology, 3rd Ed, Silver MD, Gotlieb AI, Schoen FJ (eds.), WB Saunders 2001, pp. 402–442; (c) and (d) reproduced by permission from Schoen FJ. Aortic valve structure-function correlations: Role of elastic fibers no longer a stretch of the imagination. J Heart Valve Dis 6: 1–6, 1997.  121 143 1 147 in-vivo in-vivo in-vitro TABLE 2. Key structural elements of heart valves. Element Sub-structure Function Extracellular matrix Collagen Provides strength and stiffness to maintain coaptation during diastole, when cusp has maximal area  Elastin Extends in diastole; contracts in systole to minimize cusp area  Glycosaminoglycans Accomodates shear of cuspal layers, cushions shock during valve cycle Cells Interstitial Synthesize ECM; express MMPs and TIMPs that mediate matrix remodeling  Endothelial Maintain nonthrombogenic blood-tissue interface; regulate immune and inflammatory reactions Blood vessels  Few and focal; valve cusps and leaflets sufficiently thin to be nourished by diffusion from the heart’s blood Nerves  Present, with uncertain function Other principles Corrugations Accordian-like folds in cusps; allows cuspal shape and dimensions to vary with cardiac cycle  Crimp Microscopic collagen folding, allows lengthening at minimal stress  Anisotropy Permits differences in radial and circumferential extensibility  Cords Macroscopic collagen alignment; transfers forces from cusps to aortic wall HEART VALVE FUNCTION AND STRUCTURE 2 185 87 1 ventricularis fibrosa spongiosa 1 145 In vivo 92 93 94 143 1 1 1 1 121 168 169 21 16 17 155 1 169 1 25 121 168 169 1 121 142 148 99 148 in-vitro 169 177 in-vitro 93 HEART VALVE DEVELOPMENT, MATURATION, ADAPTATION, AND REPAIR mesoderm 160 106 174 endocardial cushions mesenchyme 30 epithelial-to-mesenchymal transdifferentiation cardiac jelly 5 124 31 110 5 9 43 78 1 121 117 120 in-vitro in-vivo 118 in-vivo SCAFFOLDS FOR TISSUE ENGINEERING: GENERAL CONCEPTS scaffold cells in-vitro stage bioreactor in-vivo stage 2 77 in-vitro in-vivo in-vivo in-vitro 2 in-vivo in-vivo 3 TABLE 3. Comparative analysis of scaffolds.  Synthetic scaffolds Natural scaffolds Advantages  Control of material structure and properties (e.g. pore size, stability, degradation rate) Maintain architecture of the native tissue (potentially valve)   Easily reproduced Maintain biological information (e.g., reactive sites, growth factors)   Resorbable Potentially resorbable Disadvantages  Difficulty in controlling cell adhesion and tissue reorganization Decellularization may alter physical properties   Inflammation due to incomplete polymer degradation or lack of biocompatibility Difficulty of cell penetration into interior   Space formerly occupied by polymer and its interstices is replaced by fibrosis/scar May induce immunologic reaction   Limited perfusion to deep cells Potential for calcification FIGURE 2. Pathway A in-vitro in-vivo in-vitro in-vivo modified paradigm Pathway B in-vivo  Synthetic Scaffolds 41 53 69 83 100 Polyglycolic acid polylactic acid 48 in-vitro 71 84 85 115 smart 76 79 2 75 N 186 154 92 136 Potential undesirable features of synthetic scaffolds include local tissue inflammation owing to the foreign body reaction and slow and/or incomplete polymer degradation. As the scaffold degrades, the space formerly occupied by a polymer and its interstices is progressively filled by cells and ECM which may eventuate in fibrosis (scar) that poorly resembles specialized native tissue and may contract and distort during maturation. In some cases, cells on the scaffold periphery are healthy and resemble native differentiated parenchymal (i.e., function-specific) tissue whereas cells at the interior become necrotic due to restricted deep delivery of oxygen and nutrients, and removal of wastes. Natural Scaffolds 178 150 151 171 170 66 97 134 187 187 66 166 in-vitro 97 88 129 54 7 133 182 132 130 CELLS FOR TISSUE ENGINEERING: GENERAL CONCEPTS in-vitro in-vivo 2 91 in-vitro 26 2 in-vivo in-vitro 59 37 95 22 46 164 33 81 56 61 125 173 in-vitro 184 70 113 116 86 90 112 in-vitro 107 65 BIOREACTORS FOR TISSUE ENGINEERING in-vitro 52 101 158 181 103 161 96 in-vitro 123 IN-VIVO in-vitro 4 188 in-vitro in-vivo in-vivo TABLE 4. Representative, animal, and clinical implant studies using seeded and non-seeded matrices. Study Scaffold Cells Site In-vitro  (A) Shinoka (1995–96) Polyglycolic acid (PGA) Autologous ovine endothelial cells and fibroblasts Replacement of one pulmonary valve (PV) leaflet in sheep  (B) Hoerstrup (2000) Poly-4-hydroxybutyrate (P4HB) coated PGA Autologous ovine endothelial cells and myofibroblasts Replacement of all three PV leaflets in sheep  (C) Steinhoff (2000) Decellularized pulmonary sheep valves Autologous ovine endothelial cells and myofibroblasts PV conduits implanted into sheep  (D) Dohmen (2002) Decellularized cryopreserved pulmonary allograft Autologous human vascular endothelial cells Reconstruction of the right ventricular outflow tract (RVOT) in a human patient  (E) Perry (2003) P4HB coated PGA Autologous ovine mesenchymal stem cells in vivo  (F) Iwai (2004) Poly(lactic-co-glycolic acid) (PLGA) compounded with collagen microsponge in-vitro Patch implant in canine pulmonary artery  (G) Sutherland (2005) PGA and poly-L-lactic acid (PLLA) Autologous ovine mesenchymal stem cells Replacement of all three PV leaflets in sheep In-vivo in-vitro  (A) Matheny (2000) Porcine small intestinal submucosa N/A Replacement of one PV leaflet in a pig  (B) Elkins (2001) Decellularized (using SynerGraft treatment) human (CryoValve SG) and sheep pulmonary valves N/A SynerGraft-treated and cryopreserved sheep PVs implanted in RVOT in sheep; CryoValve SG human PVs implanted in patients  (C) Simon (2003) Decellularized porcine Synergraft valve N/A Implanted in RVOT in children In-Vitro in-vitro 2 152 153 et al. 52 in-vitro in-vitro in-vitro in-vitro in-vivo 52 in-vivo l in-vitro in-vivo 6 In-Vitro 28 72 141 163 in-vitro 10 158 162 131 in-vivo 95 FIGURE 3. A representative hypothesis for the population of a tissue engineered heart valve by endogenous cells. Key processes include proliferation, differentiation, and mobilization of endothelial progenitor cells within the bone marrow, followed by recruitment in the blood and adhesion to the valve. Subsequently, recruited cells might undergo an epithelial to mesenchymal transdifferentiation within the valve (recapitulating development), followed by differentiation to interstitial cells that ultimately synthesize and remodel the ECM.  HARNESSING THE REPARATIVE POTENTIAL OF CIRCULATING ENDOGENOUS CELLS: UNSEEDED SCAFFOLDS in-vitro in-vivo 2 2 in-vivo 3 in-vivo 73 149 40 126 3 125 180 98 109 18 165 172 108 23 157 82 138 167 135 137 138 139 13 in-vivo 89 60 CLINICAL STUDIES USING ENGINEERED MATRICES AS HEART VALVES 29 156 in-vitro 32 et al. 156 140 CHALLENGES FOR FUTURE TRANSLATION TO THE CLINIC 4 TABLE 5. Critical challenges to clinical translation of heart valve tissue engineering. Challenges Strategy for translation TEHV components and function are complex, heterogeneous and dynamic Develop guidelines for the pre-implantation characterization of TEHV structure, function and quality TEHV function depends upon patient response to implantation and integration with the recipient’s tissues more than conventional valve replacement in-vivo Individuals differ in the speed and effectiveness of their tissue remodeling Assess/control patient variability in tissue remodeling capability Owing to the key role of patient response, animal models may not reliably predict human outcomes Validate suitable animal models that will test key biological processes and correlate with human outcomes Remodeling processes after implantation may release or change seeded cells and recruit host cells Develop tools to monitor the fate of transplanted and endogenous cells (location, function, viability, phenotype) FIGURE 4. in-vitro in-vivo  Numerous steps must be surmounted in the laboratory before heart valve tissue engineered constructs can be made clinically useful. Typical biomaterial-tissue interactions in medical devices, such as thrombosis, infection, and inflammatory interactions, will have to be acceptable. Another important consideration is whether calcification, the major pathologic process in bioprosthetic valve degeneration, will be problematic. Evidence suggests that calcification may not be a major problem as long as polymer or other scaffold is resorbed and/or not intrinsically mineralizable, the interstitial cells are viable, and the ECM is capable of remodeling. 144 in-vivo 179 5 11 24 38 91 FIGURE 5. A hypothesis for inter-individual variability in tissue remodeling. While most individuals will remodel tissue with a usual speed and quality of remodeling, some people will display slow and poor quality of remodeling while others will show fast and better quality of remodeling. Inadequate remodeling could lead to implant failure and its consequences for the patient. The threshold of properties needed for tissue engineered heart valves and the means of conducting post-implantation surveillance of the patient and graft need to consider this variability. Success or failure may be followed and predicted non-invasively.  in-vivo in-vitro in-vivo in-vivo 4 42 39 136 49 175 55 8 44 57 62 63 In-vivo 19 24 in-vivo 12 105 in-vivo 15 47 111 in-vivo in-vitro in-vivo in-vitro in-vitro in-vivo 62 63 in-vivo 51 in-vitro 128 Another key need is the development of science-based approaches to the characterization of fabricated/manufactured engineered tissue products in general and heart valves in particular. These will likely include measurement of mechanical properties of the scaffold and the tissue-scaffold complex, characterization of the dynamic cell phenotypes and ECM components, and the evolution of the final manufactured product, including shelf-life, stability, and shipping considerations. CONCLUSIONS in-vitro in-vivo NOTE ADDED IN PROOF Several studies relevant to and that became available during final production of this manuscript may interest readers. Visconti, R.P., Y. Ebihara, A.C. LaRue, P.A. Fleming, T.C. McQuinn, M. Masuya, H. Minamiguchi, R.R. Markwald, M. Ogawa, and C.J. Drake. An invivo analysis of hematopoietic stem cell potential: hematopoietic origin of cardiac valve interstitial cells. Circ. Res. 98:690–696, 2006. To test the hypothesis that hematopoietic stem cells (HSC) may be a source of adult valve interstitial cells, single lineage-negative (Lin-), c-kit(+), Sca-l(+), CD34- cells from the bone marrow of mice that ubiquitously express enhanced green fluorescent protein (EGFP) were transplanted into a lethally irradiated congenic non-EGFP mouse. Histological analyses of valve tissue from clonally engrafted recipient mice revealed the presence of numerous EGFP+ cells within host valves, some of which exhibited synthetic properties characteristic of fibroblasts (expression of mRNA for procollagen 1 alpha 1). The cells were shown to be the result of HSC-derived cell differentiation and not fusion with host somatic cells. Together, these findings demonstrate a contribution by HSCs to the adult valve interstitial cell population in mice. Cao, F., S. Lin, X. Xie, P. Ray, M. Patel, X. Zhang, M. Drukker, S.J. Dylla, A.J. Connolly, X. Chen, I.L. Weissman, S.S. Gambhir, and J.C. Wu. In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Circulation 113:1005–1014, 2006. As discussed in the body of the present manuscript, monitoring the trafficking and function of stem cells in vivo remains problematic owing to limitations of conventional histological assays and imaging modalities. A recent study demonstrated a method by which embryonic stem (ES) cells could be stably transduced with a lentiviral vector carrying a novel triple-fusion (TF) reporter gene, tracked by positron emission tomography, and monitored for survival, proliferation, and migration. This imaging platform should have broad applications for basic research and clinical studies on stem cell therapy. Kiernan, T.J. Endothelial progenitor cells in 2006 – where are we now? Cardiovasc. Pathol. 15:236–239, 2006. A recent brief review of the current status of endothelial progenitor cells (EPCs), including their role as biomarkers and potential therapeutic applications, may be useful to the reader of the present manuscript. The authors emphasize critical questions relating to the characterization of the biological phenotype of “true” EPCs and the mechanisms of interaction of EPCs with resident cells of the vascular wall.