Scaffolds for tissue engineering of cardiac valves

Tissue engineered heart valves offer a promising alternative for the replacement of diseased heart valves avoiding the limitations faced with currently available bioprosthetic and mechanical heart valves. In the paradigm of tissue engineering, a three-dimensional platform – the so-called scaffold – is essential for cell proliferation, growth and differentiation, as well as the ultimate generation of a functional tissue. A foundation for success in heart valve tissue engineering is a recapitulation of the complex design and diverse mechanical properties of a native valve. This article reviews technological details of the scaffolds that have been applied to date in heart valve tissue engineering research.     

6-Month aortic valve implantation of an off-the-shelf tissue-engineered valve in sheep

Diseased aortic valves often require replacement, with over 30% of the current aortic valve surgeries performed in patients who will outlive a bioprosthetic valve. While many promising tissue-engineered valves have been created in the lab using the cell-seeded polymeric scaffold paradigm, none have been successfully tested long-term in the aortic position of a pre-clinical model. The high pressure gradients and dynamic flow across the aortic valve leaflets require engineering a tissue that has the strength and compliance to withstand high mechanical demand without compromising normal hemodynamics. A long-term preclinical evaluation of an off-the-shelf tissue-engineered aortic valve in the sheep model is presented here. The valves were made from a tube of decellularized cell-produced matrix mounted on a frame. The engineered matrix is primarily composed of collagen, with strength and organization comparable to native valve leaflets. In vitro testing showed excellent hemodynamic performance with low regurgitation, low systolic pressure gradient, and large orifice area. The implanted valves showed large-scale leaflet motion and maintained effective orifice area throughout the duration of the 6-month implant, with no calcification. After 24 weeks implantation (over 17 million cycles), the valves showed no change in tensile mechanical properties. In addition, histology and DNA quantitation showed repopulation of the engineered matrix with interstitial-like cells and endothelialization. New extracellular matrix deposition, including elastin, further demonstrates positive tissue remodeling in addition to recellularization and valve function. Long-term implantation in the sheep model resulted in functionality, matrix remodeling, and recellularization, unprecedented results for a tissue-engineered aortic valve.     

组织工程心脏瓣膜支架材料的研究和展望

背景：当前应用于临床的生物瓣和机械瓣都存在着一定缺陷,而组织工程心脏瓣膜将避免这些问题成为理想的生物瓣膜替代物。 目的：综述近年来组织工程心脏瓣膜支架材料的研究进展及面临的问题。 方法：应用计算机检索1990至2011年万方数据库相关文章,检索词为“组织工程,心脏瓣膜,支架材料”,并限定文章语言种类为中文。同时计算机检索1990至2011年 PubMed数据库相关文章,检索词为“tissue engineering,heart valve,scaffold materials”,并限定文章语言种类为English。共检索到文献147篇,最终纳入符合标准的文献61篇。 结果与结论：人工心脏瓣膜置换是目前治疗心脏瓣膜性病变的主要外科治疗手段,但现有机械瓣和生物瓣都不是理想的心脏瓣膜置换物,在耐久性,增长潜力,相容性,抗感染方面有着显著的缺陷。组织工程心脏瓣膜是一个活体器官,并具有和自体心脏瓣膜同样的生长,修复和重建能力,这一新概念的提出为构建理想的心脏瓣替换物带来了希望。     

New pulsatile bioreactor for in vitro formation of tissue engineered heart valves

Two potential obstacles to the creation of implantable tissue engineered heart valves are inadequate mechanical properties (ability to withstand hemodynamic stresses) and adverse host-tissue reactions due to the presence of residual nondegraded polymer scaffold. In an attempt to address these problems, we developed an in vitro cell culture system that provides physiological pressure and flow of nutrient medium to the developing valve constructs. It is anticipated that in vitro physical stress will stimulate the tissue engineered heart valve construct to develop adequate strength prior to a possible implantation. Long-term in vitro development will be realized by an isolated and thereby contamination-resistant system. Longer in vitro development will potentially enable more complete biodegradation of the polymeric scaffold during in vitro cultivation. This new dynamic bioreactor allows for adjustable pulsatile flow and varying levels of pressure. The system is compact and easily fits into a standard cell incubator, representing a highly isolated dynamic cell culture setting with maximum sterility, optimal gas supply and stable temperature conditions especially suited for long-term experiments.     

组织工程心脏瓣膜支架材料的选择与应用

背景：支架材料的选择在组织工程心脏瓣膜中起着至关重要的作用,支架材料的选择也就影响着组织工程心脏瓣膜的构建效果。 目的：评价组织工程心脏瓣膜支架材料的的优缺点,并对其选择进行总结。 方法：以 “组织工程,心脏瓣膜,支架材料,生物相容性”,为中文关键词;以:“tissue engineering,heart valves, scaffold material, biocompatibility” 为英文关键词,采用计算机检索1993-01/2009-10相关文章。纳入与有关生物材料与组织工程心脏瓣膜的相关的文章;排除重复研究及Meta分析类文章。 结果与结论：人工合成高分子材料有更大的可控性,可预先塑性,大量制备,孔径和孔隙率较容易控制,成本低廉;天然生物材料和合成高分子材料都存在一定不足,将人工可降解材料与天然材料相结合构建瓣膜支架,发挥两者各自的优势构建出性能良好的组织工程心脏瓣膜。组织工程心脏瓣膜的研究前景广阔。但距离临床应用还有很长的路要走,相信随着研究的不断深入以及支架材料的不断优化对组织工程心脏瓣膜构建方法的改进,在不远的将来造福于广大心脏瓣膜病患者。     

Artificial aortic valves: an overview

This review discusses strategies that may address some of the limitations associated with replacing diseased or dysfunctional aortic valves with mechanical or tissue valves. These limitations range from structural failure and thromboembolic complications associated with mechanical valves to a limited durability and calcification with tissue valves. In pediatric patients there is an issue with the inability of substitutes to grow with the recipient. The emerging science of tissue engineering potentially provides an attractive alternative by creating viable tissue structures based on a resorbable scaffold. Morphometrically precise, biodegradable polymer scaffolds may be fabricated from data obtained from scans of natural valves by rapid prototyping technologies such as fused deposition modelling. The scaffold provides a mechanical profile until seeded cells produce their own extra cellular matrix. The microstructure of the forming tissue may be aligned into predetermined spatial orientations via fluid transduction in a bioreactor. Although there are many technical obstacles that must be overcome before tissue engineered heart valves are introduced into routine surgical practice these valves have the potential to overcome many of the shortcomings of current heart valve substitutes.     

Bioreactors for Development of Tissue Engineered Heart Valves

Millions of people worldwide are diagnosed each year with valvular heart disease, resulting in hundreds of thousands of valve replacement operations. Prosthetic valve replacements are designed to correct narrowing or backflow through the valvular orifice. Although commonly used, these therapies have serious disadvantages including morbidity associated with long-term anticoagulation and limited durability necessitating repeat operations. The ideal substitute would be widely available and technically implantable for most cardiac surgeons, have normal hemodynamic performance, low risk for structural degeneration, thrombo-embolism and endocarditis, and growth potential for pediatric patients. Tissue engineered heart valves hold promise as a viable substitute to outperform existing valve replacements. An essential component to the development of tissue engineered heart valves is a bioreactor. It is inside the bioreactor that the scaffold and cells are gradually conditioned to the biochemical and mechanical environment of the valve to be replaced.     

Approaches to heart valve tissue engineering scaffold design

Heart valve disease is a significant cause of mortality worldwide. However, to date, a nonthrombogenic, noncalcific prosthetic, which maintains normal valve mechanical properties and hemodynamic flow, and exhibits sufficient fatigue properties has not been designed. Current prosthetic designs have not been optimized and are unsuitable treatment for congenital heart defects. Research is therefore moving towards the development of a tissue engineered heart valve equivalent. Two approaches may be used in the creation of a tissue engineered heart valve, the traditional approach, which involves seeding a scaffold in vitro, in the presence of specific signals prior to implantation, and the guided tissue regeneration approach, which relies on autologous reseeding in vivo. Regardless of the approach taken, the design of a scaffold capable of supporting the growth of cells and extracellular matrix generation and capable of withstanding the unrelenting cardiovascular environment while forming a tight seal during closure, is critical to the success of the tissue engineered construct. This paper focuses on the quest to design, such a scaffold.     

利用人骨髓基质干细胞构建组织工程心脏瓣膜的体外实验

BACKGROUND:Nowadays, mechanical or biological valve recipients used in the clinic are still at the risk of infection, hemorrhage, thrombosis and reoperation owing to valve stenosis. Tissue-engineered heart valve with biological activity can overcome the disadvantages above. While, the optimal choice of scaffolds and seeding cells remains disputable. OBJECTIVE:To explore the feasibility to construct tissue-engineered heart valve with acellularized porcine aortic valve scaffold and human bone marrow stromal stem cells in vitro. METHODS:The porcine aortic valves were decellularized with the detergent and enzymatic extraction process to remove the cellular components. Human bone marrow stromal stem cells were aspirated from sternum of the patients with simple congenital heart malformation, and then the cells were seeded on the acellularized porcine aortic valve scaffold and cultured for 5 days. RESULTS AND CONCLUSION:Flow cytometry identified that the characteristics of surface antigen of the inoculated seed cells were in line with those of human bone marrow stromal stem cells. Light microscopy and electron microscopy confirmed that the cellular components in the porcine aortic valves could be removed to obtain the complete acellular fiber mesh stent. There was no significant difference in biomechanical property between before and after acellularization. The human bone marrow stromal stem cells implanted on the acellularized porcine aortic valve scaffold could form a continuous cell layer on the surfaces of the scaffold. The inoculated bone marrow stromal stem cells could be differentiated into fibroblasts. The implantation of human bone marrow stromal stem cells on the acellularized porcine aortic valve scaffold can construct the tissue-engineered heart valve.     

组织工程化心脏瓣膜的研究进展

心脏瓣膜置换术是外科治疗瓣膜性心脏病的主要方法，但目前临床应用的人工瓣膜的远期效果尚不满意。近年来，随着组织工程学技术的进展，利用培养的自身组织细胞种植于支架材料表面，体外重新构建理想的心脏瓣膜植物日益成为研究热点。本文简述了心脏瓣膜工程的定义，细胞支架材料的选择，种子细胞的培养、种植方法以及组织工程化心脏瓣膜的评估，并指出下一步研究中尚需解决的问题。     

Electrospun bioresorbable heart valve scaffold for tissue engineering

Currently marketed mechanical or biological prosthetic heart valves are regarded as valid substitutes for native heart valves suffering from degenerative pathologies. These devices require strict follow-up due to dysfunctions or post-surgical complications. Potential drawbacks of these medical devices are calcification, tearing of the cusps, thromboembolism and hemolysis. In this context, a tissue engineering approach offers a promising alternative scenario. In this paper, a trileaflet poly(epsilon-caprolactone) (PCL) heart valve scaffold prototype has been manufactured by electrospinning technique using a custom-made rotating target. Process parameters were selected in order to achieve suitable microstructure and mechanical performance. The electrospun heart valve prototype was functionally characterized by means of a pulse duplicator in order to evaluate the mechanical/hydraulic response to the imposed testing conditions. Leaflets synchronously opened in the ejection phase and the proper apposition of the leaflets prevented high leakage volumes in the diastolic phase. This preliminary study suggests a successful perspective for the proposed approach in designing a novel tissue engineered bioresorbable heart valve.     

Heart Valves from Polyester Fibers vs. Biological Tissue: Comparative Study <Emphasis Type="Italic">In Vitro</Emphasis>

Transcatheter aortic valve implantation (TAVI) has become a popular alternative technique to surgical valve replacement for critical patients. Biological valve tissue has been used in TAVI procedures for over a decade, with over 100,000 implantations to date. However, with only 6 years follow up, little is known about the long-term durability of biological tissue. Moreover, the high cost of tissue harvesting and chemical treatment procedures favor the development of alternative synthetic valve leaflet materials. Textile polyester is one such material which provides outstanding folding and strength properties combined with proven biocompatibility, and could therefore be considered as a candidate to replace the biological valve leaflets in TAVI procedures. For that purpose, in addition to the mechanical properties, the hemodynamic properties of the synthetic material should be comparable to the properties of biological tissue. An ideal replacement heart valve would provide low static and dynamic regurgitation, ensure laminar flow across the valve, and limit the turbidity of flow downstream of the valve. The purpose of the present work is to compare in vitro the mechanical and hemodynamic performances of textile woven polyester valves with biological ones. Testing results indicate that textile valves trade elasticity for superior mechanical strength, relative to biological tissue. Despite this, the dynamic flexibility of textile valve leaflets strongly resembled what was seen with biological leaflets. Regurgitation, as well as slightly modified turbulent patterns, in textile valves was higher than biological valves due to the increased porosity, but, rapid tissue ingrowth post-implantation would likely mitigate this effect. Together these findings provide additional evidence favoring the use of textile polyester as a synthetic heart valve leaflet material.     

Antigen clearing from porcine heart valves with preservation of structural integrity

Bioprostheses currently used for replacement of diseased cardiovascular tissue are preserved and partially protected from immune rejection through chemical fixation. However, after implantation, chemically preserved (fixed) material has limited durability and lacks the ability to revitalize through cellular ingrowth and remodeling. As an alternative to fixation, we aimed at thoroughly removing antigens from tissue, leaving an intact scaffold, suitable for integration and revitalization in the host. Extensive washing of porcine heart valves with a mixture of two detergents (SDS and Triton X-100) yielded an intact matrix devoid of cells and depleted of soluble proteins that was minimally immunogenic in rabbits. A detailed characterization of the biomechanics and durability of the tissue is under way. If the lack of immunogenicity is confirmed in primates, our results would suggest that a detergent-washed, unfixed porcine heart valve can be an attractive non-inflammatory scaffold for heart valve regeneration in humans.     

Fabrication of a trileaflet heart valve scaffold from a polyhydroxyalkanoate biopolyester for use in tissue engineering

Previously, we reported the implantation of a single tissue engineered leaflet in the posterior position of the pulmonary valve in a lamb model. The major problems with this leaflet replacement were the scaffold's inherent stiffness, thickness, and nonpliability. We have now created a scaffold for a trileaflet heart valve using a thermoplastic polyester. In this experiment, we show the suitability of this material in the production of a biodegradable, biocompatible scaffold for tissue engineered heart valves. A heart valve scaffold was constructed from a thermoplastic elastomer. The elastomer belongs to a class of biodegradable, biocompatible polyesters known as polyhydroxyalkanoates (PHAs) and is produced by fermentation (Metabolix Inc., Cambridge, MA). It was modified by a salt leaching technique to create a porous, three-dimensional structure, suitable for tissue engineering. The trileaflet heart valve scaffold consisted of a cylindrical stent (1 mm X 15 mm X 20 mm I.D.) containing three valve leaflets. The leaflets were formed from a single piece of PHA (0.3 mm thick), and were attached to the outside of the stent by thermal processing techniques, which required no suturing. After fabrication, the heart valve construct was allowed to crystallize (4 degrees C for 24 h), and salt particles were leached into doubly distilled water over a period of 5 days to yield pore sizes ranging from 80 to 200 microns. Ten heart valve scaffolds were fabricated and seeded with vascular cells from an ovine carotid artery. After 4 days of incubation, the constructs were examined by scanning electron microscopy. The heart valve scaffold was tested in a pulsatile flow bioreactor and it was noted that the leaflets opened and closed. Cells attached to the polymer and formed a confluent layer after incubation. One advantage of this material is the ability to mold a complete trileaflet heart valve scaffold without the need for suturing leaflets to the conduit. Second advantage is the use of only one polymer material (PHA) as opposed to hybridized polymer scaffolds. Furthermore, the mechanical properties of PHA, such as elasticity and mechanical strength, exceed those of the previously utilized material. This experiment shows that PHAs can be used to fabricate a three-dimensional, biodegradable heart valve scaffold.     

Tissue engineering in the cardiovascular system: progress toward a tissue engineered heart

Achieving the lofty goal of developing a tissue engineered heart will likely rely on progress in engineering the various components: blood vessels, heart valves, and cardiac muscle. Advances in tissue engineered vascular grafts have shown the most progress to date. Research in tissue-engineered vascular grafts has focused on improving scaffold design, including mechanical properties and bioactivity; genetically engineering cells to improve graft performance; and optimizing tissue formation through in vitro mechanical conditioning. Some of these same approaches have been used in developing tissue engineering heart valves and cardiac muscle as well. Continued advances in scaffold technology and a greater understanding of vascular cell biology along with collaboration among engineers, scientists, and physicians will lead to further progress in the field of cardiovascular tissue engineering and ultimately the development of a tissue-engineered heart.     

Intermittent straining accelerates the development of tissue properties in engineered heart valve tissue

Tissue-engineered heart valves lack sufficient amounts of functionally organized structures and consequently do not meet in vivo mechanical demands. To optimize tissue architecture and hence improve mechanical properties, various in vitro mechanical conditioning protocols have been proposed, of which intermittent straining is most promising in terms of tissue properties. We hypothesize that this is due to an improved collagen matrix synthesis, maturation, and organization, triggered by periodic straining of cells. To test this hypothesis, we studied the effect of intermittent versus constrained conditioning with time (2-4 weeks), using a novel model system of human heart valve tissue. Temporal variations in collagen production, cross-link density, and mechanical properties were quantified in engineered heart valve tissue, cyclically strained for 3-h periods, alternated with 3-h periods rest. In addition, an innovative method for vital collagen imaging was used to monitor collagen organization. Intermittent straining resulted in increased collagen production, cross-link densities, collagen organization, and mechanical properties at faster rates, as compared to constrained controls, leading to stronger tissues in shorter culture periods. This is of utmost importance for heart valve tissue engineering, where insufficient mechanical properties are currently the main limiting factor.     

组织工程心脏瓣膜的构建：现状与前景

背景：目前临床上应用的心脏生物瓣和机械瓣都存在一些缺陷和不足,而组织工程心脏瓣膜有可能避免这些问题的出现,成为瓣膜替代物的理想选择。 目的：探讨构建组织工程心脏瓣膜的实验研究进展。 方法：应用数据库检索的方法分析关于组织工程心脏瓣膜的实验研究文献,组织工程心脏瓣膜的三大要素为种子细胞、支架材料和细胞种植。 结果与结论：心脏瓣膜修复和置换是目前治疗心脏瓣膜性疾病的主要外科手段。目前,主要用于构建组织工程心脏瓣膜的种子细胞有血管内皮细胞、内皮祖细胞以及骨髓间充质干细胞等。经脱细胞处理的支架具有良好的生物力学性能和组织相容性,细胞种植后支架表面会形成一层连续的细胞层,其构建的组织工程心脏瓣膜是可行的。组织工程心脏瓣膜有着良好的应用前景,但目前还有很多问题需要解决,还处于研究的初级阶段。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Founder's Award, 25th Annual Meeting of the Society for Biomaterials, perspectives. Providence, RI, April 28-May 2, 1999. Tissue heart valves: current challenges and future research perspectives.

Substitute heart valves composed of human or animal tissues have been used since the early 1960s, when aortic valves obtained fresh from human cadavers were transplanted to other individuals as allografts. Today, tissue valves are used in 40% or more of valve replacements worldwide, predominantly as stented porcine aortic valves (PAV) and bovine pericardial valves (BPV) preserved by glutaraldehyde (GLUT) (collectively termed bioprostheses). The principal disadvantage of tissue valves is progressive calcific and noncalcific deterioration, limiting durability. Native heart valves (typified by the aortic valve) are cellular and layered, with regional specializations of the extracellular matrix (ECM). These elements facilitate marked repetitive changes in shape and dimension throughout the cardiac cycle, effective stress transfer to the adjacent aortic wall, and ongoing repair of injury incurred during normal function. Although GLUT bioprostheses mimic natural aortic valve structure (a) their cells are nonviable and thereby incapable of normal turnover or remodeling ECM proteins; (b) their cuspal microstructure is locked into a configuration which is at best characteristic of one phase of the cardiac cycle (usually diastole); and (c) their mechanical properties are markedly different from those of natural aortic valve cusps. Consequently, tissue valves suffer a high rate of progressive and age-dependent structural valve deterioration resulting in stenosis or regurgitation (>50% of PAV overall fail within 10-15 years; the failure rate is nearly 100% in 5 years in those <35 years old but only 10% in 10 years in those >65). Two distinct processes-intrinsic calcification and noncalcific degradation of the ECM-account for structural valve deterioration. Calcification is a direct consequence of the inability of the nonviable cells of the GLUT-preserved tissue to maintain normally low intracellular calcium. Consequently, nucleation of calcium-phosphate crystals occurs at the phospholipid-rich membranes and their remnants. Collagen and elastin also calcify. Tissue valve mineralization has complex host, implant, and mechanical determinants. Noncalcific degradation in the absence of physiological repair mechanisms of the valvular structural matrix is increasingly being appreciated as a critical yet independent mechanism of valve deterioration. These degradation mechanisms are largely rationalized on the basis of the changes to natural valves when they are fabricated into a tissue valve (mentioned above), and the subsequent interactions with the physiologic environment that are induced following implantation. The "Holy Grail" is a nonobstructive, nonthrombogenic tissue valve which will last the lifetime of the patient (and potentially grow in maturing recipients). There is considerable activity in basic research, industrial development, and clinical investigation to improve tissue valves. Particularly exciting in concept, yet early in practice is tissue engineering, a technique in which an anatomically appropriate construct containing cells seeded on a resorbable scaffold is fabricated in vitro, then implanted. Remodeling in vivo, stimulated and guided by appropriate biological signals incorporated into the construct, is intended to recapitulate normal functional architecture.     

Pediatric tubular pulmonary heart valve from decellularized engineered tissue tubes

Pediatric patients account for a small portion of the heart valve replacements performed, but a pediatric pulmonary valve replacement with growth potential remains an unmet clinical need. Herein we report the first tubular heart valve made from two decellularized, engineered tissue tubes attached with absorbable sutures, which can meet this need, in principle. Engineered tissue tubes were fabricated by allowing ovine dermal fibroblasts to replace a sacrificial fibrin gel with an aligned, cell-produced collagenous matrix, which was subsequently decellularized. Previously, these engineered tubes became extensively recellularized following implantation into the sheep femoral artery. Thus, a tubular valve made from these tubes may be amenable to recellularization and, ideally, somatic growth.The suture line pattern generated three equi-spaced leaflets in the inner tube, which collapsed inward when exposed to back pressure, per tubular valve design. Valve testing was performed in a pulse duplicator system equipped with a secondary flow loop to allow for root distention. All tissue-engineered valves exhibited full leaflet opening and closing, minimal regurgitation (<5%), and low systolic pressure gradients (<2.5 mmHg) under pulmonary conditions. Valve performance was maintained under various trans-root pressure gradients and no tissue damage was evident after 2 million cycles of fatigue testing.     

Tri-layered elastomeric scaffolds for engineering heart valve leaflets

Tissue engineered heart valves (TEHVs) that can grow and remodel have the potential to serve as permanent replacements of the current non-viable prosthetic valves particularly for pediatric patients. A major challenge in designing functional TEHVs is to mimic both structural and anisotropic mechanical characteristics of the native valve leaflets. To establish a more biomimetic model of TEHV, we fabricated tri-layered scaffolds by combining electrospinning and microfabrication techniques. These constructs were fabricated by assembling microfabricated poly(glycerol sebacate) (PGS) and fibrous PGS/poly(caprolactone) (PCL) electrospun sheets to develop elastic scaffolds with tunable anisotropic mechanical properties similar to the mechanical characteristics of the native heart valves. The engineered scaffolds supported the growth of valvular interstitial cells (VICs) and mesenchymal stem cells (MSCs) within the 3D structure and promoted the deposition of heart valve extracellular matrix (ECM). MSCs were also organized and aligned along the anisotropic axes of the engineered tri-layered scaffolds. In addition, the fabricated constructs opened and closed properly in an ex vivo model of porcine heart valve leaflet tissue replacement. The engineered tri-layered scaffolds have the potential for successful translation towards TEHV replacements.