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.     

Tissue Engineering of Human Heart Valve Leaflets: A Novel Bioreactor for a Strain-Based Conditioning Approach

Current mechanical conditioning approaches for heart valve tissue engineering concentrate on mimicking the opening and closing behavior of the leaflets, either or not in combination with tissue straining. This study describes a novel approach by mimicking only the diastolic phase of the cardiac cycle, resulting in tissue straining. A novel, yet simplified, bioreactor system was developed for this purpose by applying a dynamic pressure difference over a closed tissue engineered valve, thereby inducing dynamic strains within the leaflets. Besides the use of dynamic strains, the developing leaflet tissues were exposed to prestrain induced by the use of a stented geometry. To demonstrate the feasibility of this strain-based conditioning approach, human heart valve leaflets were engineered and their mechanial behavior evaluated. The actual dynamic strain magnitude in the leaflets over time was estimated using numerical analyses. Preliminary results showed superior tissue formation and non-linear tissue-like mechanical properties in the strained valves when compared to non-loaded tissue strips. In conclusion, the strain-based conditioning approach, using both prestrain and dynamic strains, offers new possibilities for bioreactor design and optimization of tissue properties towards a tissue-engineered aortic human heart valve replacement.     

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.     

Heart Valve Tissue Engineering: Concepts, Approaches, Progress, and Challenges

Potential applications of tissue engineering in regenerative medicine range from structural tissues to organs with complex function. This review focuses on the engineering of heart valve tissue, a goal which involves a unique combination of biological, engineering, and technological hurdles. We emphasize basic concepts, approaches and methods, progress made, and remaining challenges. To provide a framework for understanding the enabling scientific principles, we first examine the elements and features of normal heart valve functional structure, biomechanics, development, maturation, remodeling, and response to injury. Following a discussion of the fundamental principles of tissue engineering applicable to heart valves, we examine three approaches to achieving the goal of an engineered tissue heart valve: (1) cell seeding of biodegradable synthetic scaffolds, (2) cell seeding of processed tissue scaffolds, and (3) in-vivo repopulation by circulating endogenous cells of implanted substrates without prior in-vitro cell seeding. Lastly, we analyze challenges to the field and suggest future directions for both preclinical and translational (clinical) studies that will be needed to address key regulatory issues for safety and efficacy of the application of tissue engineering and regenerative approaches to heart valves. Although modest progress has been made toward the goal of a clinically useful tissue engineered heart valve, further success and ultimate human benefit will be dependent upon advances in biodegradable polymers and other scaffolds, cellular manipulation, strategies for rebuilding the extracellular matrix, and techniques to characterize and potentially non-invasively assess the speed and quality of tissue healing and remodeling.     

Optical mapping of impulse propagation in engineered cardiac tissue

Cardiac tissue engineering has a potential to provide functional, synchronously contractile tissue constructs for heart repair, and for studies of development and disease using in vivo-like yet controllable in vitro settings. In both cases, the utilization of bioreactors capable of providing biomimetic culture environments is instrumental for supporting cell differentiation and functional assembly. In the present study, neonatal rat heart cells were cultured on highly porous collagen scaffolds in bioreactors with electrical field stimulation. A hallmark of excitable tissues such as myocardium is the ability to propagate electrical impulses. We utilized the method of optical mapping to measure the electrical impulse propagation. The average conduction velocity recorded for the stimulated constructs (14.4 +/- 4.1 cm/s) was significantly higher than that of the nonstimulated constructs (8.6 +/- 2.3 cm/s, p = 0.003). The measured electrical propagation properties correlated to the contractile behavior and the compositions of tissue constructs. Electrical stimulation during culture significantly improved amplitude of contractions, tissue morphology, and connexin-43 expression compared to the nonsimulated controls. These data provide evidence that electrical stimulation during bioreactor cultivation can improve electrical signal propagation in engineered cardiac constructs.     

Engineered cardiac organoid chambers: toward a functional biological model ventricle

A growing area in the field of tissue engineering is the development of tissue equivalents as model systems for in vitro experimentation and high-throughput screening applications. Although a variety of strategies have been developed to enhance the structure and function of engineered cardiac tissues, an inherent limitation with traditional myocardial patches is that they do not permit evaluation of the fundamental relationships between pressure and volume that characterize global contractile function of the heart. Therefore, in the following study we introduce fully biological, living engineered cardiac organoids, or simplified heart chambers, that beat spontaneously, develop pressure, eject fluid, contain residual stress, exhibit a functional Frank-Starling mechanism, and generate positive stroke work. We also demonstrate regional variations in pump function following local cryoinjury, yielding a novel engineered tissue model of myocardial infarction. With the unique ability to directly evaluate relevant pressure-volume characteristics and regulate wall stress, this organoid chamber culture system provides a flexible platform for developing a controllable biomimetic cardiac niche environment that can be adapted for a variety of high-throughput and long-term investigations of cardiac pump function.     

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.     

A Tri-Leaflet Nitinol Mesh Scaffold for Engineering Heart Valves

The epidemiology of valvular heart disease has significantly changed in the past few decades with aging as one of the main contributing factors. The available options for replacement of diseased valves are currently limited to mechanical and bioprosthetic valves, while the tissue engineered ones that are under study are currently far from clinical approval. The main problem with the tissue engineered heart valves is their progressive deterioration that leads to regurgitation and/or leaflet thickening a few months after implantation. The use of bioresorbable scaffolds is speculated to be one factor affecting these valves’ failure. We have previously developed a non-degradable superelastic nitinol mesh scaffold concept that can be used for heart valve tissue engineering applications. It is hypothesized that the use of a non-degradable superelastic nitinol mesh may increase the durability of tissue engineered heart valves, avoid their shrinkage, and accordingly prevent regurgitation. The current work aims to study the effects of the design features on mechanical characteristics of this valve scaffold to attain proper function prior to in vivo implantation.     

Design and Hydrodynamic Evaluation of a Novel Pulsatile Bioreactor for Biologically Active Heart Valves

Biologically active heart valves (tissue engineered and recellularized tissue-derived heart valves) have the potential to offer enhanced function when compared to current replacement value therapies since they can possibly remodel, and grow to meet the needs of the patient, and not require chronic medication. However, this technology is still in its infancy and many fundamental questions remain as to how these valves will function in vivo. It has been shown that exposing biologically active tissue constructs to pulsatile pressures and flows during in vitro culture produces enhanced extracellular matrix protein expression and cellularity, although the ideal hydrodynamic conditioning regime is as yet unknown. Moreover, in vitro organ-level studies of living heart valves aimed at studying the remodeling processes require environments that can accurately reproduce in vivo hemodynamics under sterile conditions. To this end, we have developed a system to study the effects of subjecting biologically active heart valves to highly controlled pulsatile pressure and flow waveforms under sterile conditions. The device fits inside a standard incubator and utilizes a computer-controlled closed loop feedback system to provide a high degree of control. The mean pressure, mean flow rate, driving frequency, and shape of the pulsatile pressure waveform can be changed automatically in order to simulate both physiologic and nonphysiologic hemodynamic conditions. Extensive testing and evaluation demonstrated the device's ability to subject a biologically active heart valve to highly controlled pulsatile waveforms that can be modulated during the course of sterile incubation.     

Physiologic Pulsatile Flow Bioreactor Conditioning of Poly(ethylene glycol)-based Tissue Engineered Vascular Grafts

Mechanical conditioning represents a potential means to enhance the biochemical and biomechanical properties of tissue engineered vascular grafts (TEVGs). A pulsatile flow bioreactor was developed to allow shear and pulsatile stimulation of TEVGs. Physiological 120 mmHg/80 mmHg peak-to-trough pressure waveforms can be produced at both fetal and adult heart rates. Flow rates of 2 mL/sec, representative of flow through small diameter blood vessels, can be generated, resulting in a mean wall shear stress of ∼6 dynes/cm² within the 3 mm ID constructs. When combined with non-thrombogenic poly(ethylene glycol) (PEG)-based hydrogels, which have tunable mechanical properties and tailorable biofunctionality, the bioreactor represents a flexible platform for exploring the impact of controlled biochemical and biomechanical stimuli on vascular graft cells. In the present study, the utility of this combined approach for improving TEVG outcome was investigated by encapsulating 10T-1/2 mouse smooth muscle progenitor cells within PEG-based hydrogels containing an adhesive ligand (RGDS) and a collagenase degradable sequence (LGPA). Constructs subjected to 7 weeks of biomechanical conditioning had significantly higher collagen levels and improved moduli relative to those grown under static conditions. These authors contributed equally to this work     

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.     

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.     

Estimation of the Shear Stress on the Surface of an Aortic Valve Leaflet

The limited durability of xenograft heart valves and the limited supply of allografts have sparked interest in tissue engineered replacement valves. A bioreactor for tissue engineered valves must operate at conditions that optimize the biosynthetic abilities of seeded cells while promoting their adherence to the leaflet matrix. An important parameter is shear stress, which is known to influence cellular behavior and may thus be crucial in bioreactor optimization. Therefore, an accurate estimate of the shear stress on the leaflet surface would not only improve our understanding of the mechanical environment of aortic valve leaflets, but it would also aid in bioreactor design. To estimate the shear stress on the leaflet surface, two-component laser-Doppler velocimetry measurements have been conducted inside a transparent polyurethane valve with a trileaflet structure similar to the native aortic valve. Steady flow rates of 7.5, 15.0, and 22.5 L/min were examined to cover the complete range possible during the cardiac cycle. The laminar shear stresses were calculated by linear regression of four axial velocity measurements near the surface of the leaflet. The maximum shear stress recorded was 79 dyne/cm², in agreement with boundary layer theory and previous experimental and computational studies. This study has provided a range of shear stresses to be explored in bioreactor design and has defined a maximum shear stress at which cells must remain adherent upon a tissue engineered construct. © 1999 Biomedical Engineering Society. PAC99: 8719Rr, 8768+z, 8719Hh, 4262Be, 4727Nz, 0630Gv     

Tissue engineering for myocardial regeneration

 Recent progress in stem cell biology has shown the possibility of implantable human myocardial cell sources. It has encouraged myocardial tissue engineering for rescuing damaged hearts. The present strategy is to repair not all of the myocardial tissue, but part of it. There are two approaches. The first is direct injection of dissociated cell suspensions via the pericardium, coronary arteries, or endocardium. Studies using animal models have found improved heart function after transplantation of various types of cells. Myoblasts or bone marrow cells have already been transplanted into patients suffering from severe ischemic heart disease. In direct transplantation of dissociated cells, it is difficult to control the shape, size, and location of the grafts. To solve these problems, further therapies to transplant tissue-engineered three-dimensional (3-D) heart grafts have been investigated. The most popular technique in tissue engineering is to use 3-D biodegradable scaffolds as alternatives to the extracellular matrix. On the basis of this concept, poly(glycolic acid)(PGA), gelatin, alginate, and collagen have been used as scaffolds to fabricate 3-D heart tissues. A new method consisting of layering cell sheets to construct 3-D tissues without any artificial scaffolds has also been applied to myocardial tissue engineering. Electrically communicative pulsatile heart tissues have been achieved both in vitro and in vivo by layering cardiomyocyte sheets. Although myocardial tissue engineering has rapidly progressed, there are several problems to be solved with regard to the source of myocardial cells, tissue reconstruction, neovascularization, and transplantation technology. Further interdisciplinary research will solve these problems, and transplantation of cells or engineered heart tissues will become one of the major treatments for severe heart failure in the near future. Received: October 1, 2002 Correspondence to:T. Okano     

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。     

Progress in developing a living human tissue-engineered tri-leaflet heart valve assembled from tissue produced by the self-assembly approach

The aortic heart valve is constantly subjected to pulsatile flow and pressure gradients which, associated with cardiovascular risk factors and abnormal hemodynamics (i.e. altered wall shear stress), can cause stenosis and calcification of the leaflets and result in valve malfunction and impaired circulation. Available options for valve replacement include homograft, allogenic or xenogenic graft as well as the implantation of a mechanical valve. A tissue-engineered heart valve containing living autologous cells would represent an alternative option, particularly for pediatric patients, but still needs to be developed. The present study was designed to demonstrate the feasibility of using a living tissue sheet produced by the self-assembly method, to replace the bovine pericardium currently used for the reconstruction of a stented human heart valve. In this study, human fibroblasts were cultured in the presence of sodium ascorbate to produce tissue sheets. These sheets were superimposed to create a thick construct. Tissue pieces were cut from these constructs and assembled together on a stent, based on techniques used for commercially available replacement valves. Histology and transmission electron microscopy analysis showed that the fibroblasts were embedded in a dense extracellular matrix produced in vitro. The mechanical properties measured were consistent with the fact that the engineered tissue was resistant and could be cut, sutured and assembled on a wire frame typically used in bioprosthetic valve assembly. After a culture period in vitro, the construct was cohesive and did not disrupt or disassemble. The tissue engineered heart valve was stimulated in a pulsatile flow bioreactor and was able to sustain multiple duty cycles. This prototype of a tissue-engineered heart valve containing cells embedded in their own extracellular matrix and sewn on a wire frame has the potential to be strong enough to support physiological stress. The next step will be to test this valve extensively in a bioreactor and at a later date, in a large animal model in order to assess in vivo patency of the graft.     

Development of a novel pulsatile bioreactor for tissue culture

The construction of tissue-engineered parts such as heart valves and arteries requires more than just the seeding of cells onto a biocompatible/biodegradable polymeric scaffold. It is essential that the functionality and mechanical integrity of the cell-seeded scaffold be investigated in vitro prior to in vivo implantation. The correct hemodynamic conditioning would lead to the development of tissues with enhanced mechanical strength and cell viability. Therefore, a bioreactor that can simulate physiological conditions would play an important role in the preparation of tissue-engineered constructs. In this article, we present and discuss the design concepts and criteria, as well as the development, of a multifunctional bioreactor for tissue culture in vitro. The system developed is compact and easily housed in an incubator to maintain sterility of the construct. Moreover, the proposed bioreactor, in addition to mimicking in vivo conditions, is highly flexible, allowing different types of constructs to be exposed to various physiological flow conditions. Initial verification of the hemodynamic parameters using Laser doppler anemometry indicated that the bioreactor performed well and produced the correct physiological conditions.     

Bioreactors for Cardiovascular Cell and Tissue Growth: A Review

Heart disease is a major cause of death in the Western world. In the past three decades there has been a number of improvements in artificial devices and surgical techniques for cardiovascular disease; however, there is still a need for novel devices, especially for those individuals who cannot receive conventional therapy. The major disadvantage of current artificial devices lies in the fact that they cannot grow, remodel, or repair in vivo. Tissue engineering offers the possibility of developing a biological substitute material in vitro with the inherent mechanical, chemical, biological, and morphological properties required in vivo, on an individual patient basis. In order to develop a true biological cardiovascular device a dynamic physiological environment needs to be created. One approach that employs the use of a simulated biological environment is a bioreactor in which the in vivo biomechanical and biochemical conditions are created in vitro for functional tissue development. A review of the current state of the art bioreactors for the generation of tissue engineered cardiovascular devices is presented in this study. The effect of the simulated physiological environment of the bioreactor on tissue development is examined with respect to the materials properties of vascular grafts, heart valves, and cardiac muscles developed in these bioreactors. © 2003 Biomedical Engineering Society. PAC2003: 8768+z, 8719Hh, 8717Ee, 8719Ff, 8780Rb     

采用微载体技术大规模培养组织工程种子细胞

如何获得大规模、具有再生活力的种子细胞已经成为当前组织工程研究面临的最关键的限制性因素;针对这一限制性因素,研究人员对多个细胞培养系统进行了研究,其中包括微载体细胞培养系统,该系统最初主要用于细菌的大规模培养研究,但近年来的研究发现,微载体细胞培养系统也有助于种子细胞的体外大规模扩增;本对应用微载体技术大规模培养组织工程种子细胞进行了简要综述。     

软骨组织工程生物反应器的研究进展

体外软骨构建是软骨组织工程产业化发展及临床应用的重要手段。然而,采用现有的体外构建技术无法构建功能接近正常的软骨。生物反应器能够在一定程度上模拟体内环境,有望弥补现有体外构建技术的弊端。研究发现,流体剪切力、静态液压力和直接压缩力是体内软骨发育和成熟的重要力学因素,常用软骨生物反应器均据此设计而产生。由于不同类型生物反应器各具特点,研究和开发新型复合式生物反应器将成为未来的发展方向。对目前软骨组织工程生物反应器的研究现状做一综述。