Heart Valve Tissue Engineering Research Articles

Biological heart valves

Cardiac valvular pathologies are often caused by rheumatic fever in young adults, atherosclerosis in elderly patients, or by congenital malformation of the heart in children, in effect affecting almost all population ages. Almost 300,000 heart valve operations are performed worldwide annually. Tissue valve prostheses have certain advantages over mechanical valves such as biocompatibility, more physiological hemodynamics, and no need for life-long systemic anticoagulation. However, the major disadvantage of biological valves is related to their durability. Nevertheless, during the last decade, the number of patients undergoing biological, rather than mechanical, valve replacement has increased from half to more than three-quarters for biological implants. Continuous improvement in valve fabrication includes development of new models and shapes, novel methods of tissue treatment, and preservation and implantation techniques. These efforts are focused not only on the improvement of morbidity and mortality of the patients but also on the improvement of their quality of life. Heart valve tissue engineering aims to provide durable, "autologous" valve prostheses. These valves demonstrate adaptive growth, which may avoid the need of repeated operations in growing patients.

Reendothelialization of Human Heart Valve Neoscaffolds Using Umbilical Cord-Derived Endothelial Cells

Heart valve tissue engineering represents a concept for improving the current methods of valvular heart disease therapy. The aim of this study was to develop tissue engineered heart valves combining human umbilical vein endothelial cells (HUVECs) and decellularized human heart valve matrices. Pulmonary (n=9) and aortic (n=6) human allografts were harvested from explanted hearts from heart transplant recipients and were decellularized using a detergent-based cell extraction method. Analysis of decellularization success was performed with light microscopy, transmission electron microscopy and quantitative analysis of collagen and elastin content. The decellularization method resulted in full removal of native cells while the mechanical stability and the quantitative composition of the neoscaffolds was maintained. The luminal surface of the human matrix could be successfully recellularized with in vitro expanded HUVECs under dynamic flow conditions. The surface appeared as a confluent cell monolayer of positively labeled cells for von Willebrand factor and CD 31, indicating their endothelial nature. Human heart valves can be decellularized by the described method. Recellularization of the human matrix resulted in the formation of a confluent HUVEC monolayer. The in vitro construction of tissue-engineered heart valves based on decellularized human matrices followed by endothelialization using HUVECs is a feasible and safe method, leading to the development of future clinical strategies in the treatment of heart valve disease.

The study of a new detergent (octyl-glucopyranoside) for decellularizing porcine pericardium as tissue engineering scaffold

BackgroundThe use of extracellular matrix (ECM) derived from decellularized tissue is increasingly frequent in regenerative medicine and tissue engineering. However, it is recognized that currently used decellularization procedures have negative effects on ECM integrity. The objective of this study was to investigate the impact of a decellularization protocol with a new detergent on the ECM integrity of porcine pericardium (PP) compared with other traditional detergents. Materials and methodsFresh PP were decellularized by sodium deoxycholate in combination with Triton X-100 (SDT), sodium dodecyl sulfate (SDS), and octyl-glucopyranoside (OGP), respectively. Histologic analysis and scanning electron microscopy were performed to confirm the removal of cells and to examine the structure of ECM. DNA content was examined by the method of DNA extraction. Mechanical properties and biochemical compositions of ECM were also studied. ResultsHistologic analysis and DNA determination demonstrated that SDS and OGP completely removed the cells, and the major ECM structure was preserved well for PP treated with 1% (wt/vol) OGP but disrupted for PP treated with SDS; whereas treatment with SDT was insufficient to remove cells from PP. Uniaxial tensile tests showed that PP decellularized by OGP had similar mechanical properties to native PP, whereas the mechanical properties of PP decellularized by SDS and SDT decreased. The biochemical compositions of PP decellularized by OGP were also well conserved, except that glycosaminoglycans markedly decreased. Moreover, the results obtained in the MTT study further indicated that the cytotoxicity of PP decellularized by OGP was significantly lower than that decellularized by SDS and SDT. ConclusionIt is suggested that the environmentally friendly and nontoxic OGP can be used as a decellularizing agent. The OGP method could achieve both complete removal of cells from native PP and preservation of the matrix structure; thus, it might be a suitable approach to preparation of tissue engineering heart valve scaffold.

Percutaneous Tissue-Engineered Pulmonary Valved Stent Implantation: Comparison of Bone Marrow-Derived CD133+-Cells and Cells Obtained from Carotid Artery

The aim of tissue engineering is, for instance, to create a new valve or organ that is similar to the native one. This new construct may have the capability of growth, repair, and remodeling. One instance where tissue engineering offers an enormous advantage is in the treatment of Tetralogy of Fallot. After repair of this defect, pulmonary valve regurgitation with late ventricular dysfunction is not uncommonly observed. In this study, we compared the use of endothelial cells (ECs) and smooth muscle cells (SMCs) derived from the carotid artery (group 1/n=5) with CD133+-cells derived from the bone-marrow (group 2/n=5) of juvenile sheep. Western blot analysis and histology were done before and after percutaneous pulmonary valved stent implantation. After seeding of the pulmonary valved stent with one of the cell groups, the construct was placed in a dynamic bioreactor for 16 days. After incubation, the valved stent was seeded again for 3 days. Finally, the valved stent was implanted percutaneously under angiographic control. In group 1, α-actin and calponin were found in the EC and SMC population by using Western blot analysis. In contrast, CD31 was found in EC, but not in the SMC population. Cells from group 2 demonstrated a pure population. CD133+-cells were detected in the CD133+-cell population and no CD133+-cells were found in the negative fraction. Three months after implantation, the transvalvular gradient was significantly higher in group 1 compared to group 2. CD31-staining demonstrated a confluent monolayer in both groups. Immunohistochemistry revealed strong expression of α-smooth-muscle-actin and an in-growth into the leaflets of both groups (p=ns). CD3-, CD20-, CD45-, and CD68-staining confirmed no signs of inflammation in group 2, whereas in group 1 small amounts of inflammation were detected in all analyzed animals. Von Kossa staining revealed mild to moderate calcifications in the annular region of group 1. In contrast, less calcification was detected in group 2. Autologous CD133+-cells derived from bone marrow had a better outcome with regard to calcification, inflammation, and transvalvular gradient compared to autologous cells derived from the carotid artery. Therefore, CD133+-cells are more advantageous for tissue engineering of heart valves.

Decellularized Tissue-Engineered Heart Valve Leaflets with Recellularization Potential

Tissue-engineered heart valves (TEHV) have been proposed as a promising solution for the clinical needs of pediatric patients. In vivo studies have shown TEHV leaflet contraction and regurgitation after several months of implantation. This has been attributed to contractile cells utilized to produce the extracellular matrix (ECM) during TEHV culture. Here, we utilized such cells to develop a mature ECM in a fibrin-based scaffold that generates commissural alignment in TEHV leaflets and then removed these cells using detergents. Further, we evaluated recellularization with potentially noncontractile cells. A tissue-engineered leaflet model was developed with mechanical anisotropy and tensile properties comparable to an ovine pulmonary valve leaflet. No change in tensile properties occurred after decellularization using 1% sodium dodecyl sulfate and 1% Triton detergent treatment. Cell removal was verified by DNA quantitation and western blot analysis for cellular proteins. Histological and scanning electron microscope imaging showed no significant change in the ECM organization and microstructure. We further tested the recellularization potential of decellularized leaflets by seeding human mesenchymal stem cells (hMSC) on the surface of the leaflets and evaluated them at 1 and 3 weeks in two culture conditions. One medium (M1) was chosen to maintain the MSC phenotype while a second medium (M2) was used to potentially differentiate cells to an interstitial cell phenotype. Cellular quantitation showed that the engineered leaflets were recellularized to the highest concentration with M2 followed by M1, with minimum cell invasion of decellularized native leaflets. Histology showed cellular invasion throughout the thickness of the leaflets in M2 and partial invasion in M1. hMSC stained positive for MSC markers, but also for α-smooth muscle actin in both media at 1 week, with no presence of MSC markers at 3 weeks with the exception of CD90. These results show that engineered leaflets, while having similar tensile properties and collagen content compared to native leaflets, have better recellularization potential.

Use of a special bioreactor for the cultivation of a new flexible polyurethane scaffold for aortic valve tissue engineering

BackgroundTissue engineering represents a promising new method for treating heart valve diseases. The aim of this study was evaluate the importance of conditioning procedures of tissue engineered polyurethane heart valve prostheses by the comparison of static and dynamic cultivation methods.MethodsHuman vascular endothelial cells (ECs) and fibroblasts (FBs) were obtained from saphenous vein segments. Polyurethane scaffolds (n = 10) were primarily seeded with FBs and subsequently with ECs, followed by different cultivation methods of cell layers (A: static, B: dynamic). Group A was statically cultivated for 6 days. Group B was exposed to low flow conditions (t1= 3 days at 750 ml/min, t2= 2 days at 1100 ml/min) in a newly developed conditioning bioreactor. Samples were taken after static and dynamic cultivation and were analyzed by scanning electron microscopy (SEM), immunohistochemistry (IHC), and real time polymerase chain reaction (RT-PCR).ResultsSEM results showed a high density of adherent cells on the surface valves from both groups. However, better cell distribution and cell behavior was detected in Group B. IHC staining against CD31 and TE-7 revealed a positive reaction in both groups. Higher expression of extracellular matrix (ICAM, Collagen IV) was observed in Group B. RT- PCR demonstrated a higher expression of inflammatory Cytokines in Group B.ConclusionWhile conventional cultivation method can be used for the development of tissue engineered heart valves. Better results can be obtained by performing a conditioning step that may improve the tolerance of cells to shear stress. The novel pulsatile bioreactor offers an adequate tool for in vitro improvement of mechanical properties of tissue engineered cardiovascular prostheses.

Effect of biodegradation and de novo matrix synthesis on the mechanical properties of valvular interstitial cell-seeded polyglycerol sebacate–polycaprolactone scaffolds

The development of living heart valves that grow with the patient is a promising strategy for heart valve replacements in pediatric patients. Despite active research in the field of tissue engineered heart valves there have been limited efforts to optimize the balance between biodegradation of the scaffolds and de novo extracellular matrix (ECM) synthesis by cells and study their consequences on the mechanical properties of the cell-seeded construct. This study investigates the effect of in vitro degradation and ECM secretion on the mechanical properties of hybrid polyester scaffolds. The scaffolds were synthesized from blends of fast degrading polyglycerol sebacate (PGS) and slowly degrading polycaprolactone (PCL). PGS–PCL scaffolds were electrospun using a 2:1 ratio of PGS to PCL. Accelerated hydrolytic degradation in 0.1mM sodium hydroxide revealed 2-fold faster degradation of PGS–PCL scaffolds compared with PCL scaffolds. Thermal analysis and scanning electron microscopy demonstrated marginal change in PCL scaffold properties, while PGS–PCL scaffolds showed preferential mass loss of PGS and thinning of the individual fibers during degradation. Consequently, the mechanical properties of PGS–PCL scaffolds decreased gradually with no significant change for PCL scaffolds during accelerated degradation. Valvular interstitial cells (VICs) seeded on PGS–PCL scaffolds showed higher ECM protein secretion compared with PCL. Thus the mechanical properties of the cell-seeded PCL scaffolds did not change significantly compared with acellular scaffolds, probably due to slower degradation and ECM deposition by VICs. In contrast, the PGS–PCL scaffolds exhibited a gradual decrease in the mechanical properties of the acellular scaffolds due to degradation, which was compensated for by new matrix secreted by VICs seeded on the scaffolds. Our study demonstrated that the faster degrading PGS component of PGS–PCL accelerated the degradation rate of the scaffolds. VICs, on the other hand, were able to remodel the synthetic scaffold, depositing new matrix proteins and maintaining the mechanical properties of the scaffolds.

Periodontal Ligament Cells Cultured Under Steady-Flow Environments Demonstrate Potential for Use in Heart Valve Tissue Engineering

A major drawback of mechanical and prosthetic heart valves is their inability to permit somatic growth. By contrast, tissue-engineered pulmonary valves potentially have the capacity to remodel and integrate with the patient. For this purpose, adult stem cells may be suitable. Previously, human periodontal ligament cells (PDLs) have been explored as a reliable and robust progenitor cell source for cardiac muscle regeneration (Pelaez, D. Electronic Thesis and Dissertation Database, Coral Gables, FL, May 2011). Here, we investigate the potential of PDLs to support the valve lineage, specifically the concomitant differentiation to both endothelial cell (EC) and smooth muscle cell (SMC) types. We were able to successfully promote PDL differentiation to both SMC and EC phenotypes through a combination of stimulatory approaches using biochemical and mechanical flow conditioning (steady shear stress of 1 dyne/cm(2)), with flow-based mechanical conditioning having a predominant effect on PDL differentiation, particularly to ECs; in addition, strong expression of the marker FZD2 and an absence of the marker MLC1F point toward a unique manifestation of smooth muscle by PDLs after undergoing steady-flow mechanical conditioning alone, possible by only the heart valve and pericardium phenotypes. It was also determined that steady flow (which was performed using a physiologically relevant [for heart valves] magnitude of ~5-6 dynes/cm(2)) augmented the synthesis of the extracellular matrix collagen proteins. We conclude that under steady-flow dynamic culture environments, human PDLs can differentiate to heterogeneous cell populations that are relevant to heart valve tissue engineering. Further exploration of human PDLs for this purpose is thus warranted.

The Potential of Prolonged Tissue Culture to Reduce Stress Generation and Retraction in Engineered Heart Valve Tissues

In tissue-engineered (TE) heart valves, cell-mediated processes cause tissue compaction during culture and leaflet retraction at time of implantation. We have quantified and correlated stress generation, compaction, retraction, and tissue quality during a prolonged culture period of 8 weeks. Polyglycolic acid/poly-4-hydroxybutyrate strips were seeded with vascular-derived cells and cultured for 4-8 weeks. Compaction in width, generated force, and stress was measured during culture. Retraction in length, generated force, and stress was measured after release of constraints at weeks 4, 6, and 8. Further, the amount of DNA, glycosaminoglycans (GAGs), collagen, and collagen cross-links was assessed. During culture, compaction and force generation increased to, respectively, 63.9% ± 0.8% and 43.7 ± 4.3 mN at week 4, after which they remained stable. Stress generation reached 27.7 ± 3.2 kPa at week 4, after which it decreased to ∼8.5 kPa. At release of constraints, tissue retraction was 44.0% ± 3.7% at week 4 and decreased to 29.2% ± 2.8% and 26.1% ± 2.2% at, respectively, 6 and 8 weeks. Generated force (8-16 mN) was lower at week 6 than at weeks 4 and 8. Generated stress decreased from 11.8 ± 0.9 kPa at week 4 to 1.4 ± 0.3 and 2.4 ± 0.4 kPa at, respectively, weeks 6 and 8. The amount of GAGs increased at weeks 6 and 8 compared to week 4 and correlated to the reduced stress and retraction. In summary, prolonged culture resulted in decreased stress generation and retraction, likely as a result of the increased amount of GAGs. These results demonstrate the potential of prolonged tissue culture in developing functional, nonretracting, TE heart valves.

Editorial introductions

Current Opinion in Pediatrics was launched in 1989. It is one of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of pediatrics is divided into 18 sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. In addition to this, Henry H. Bernstein also invites a prominent authority in the field of Office Pediatrics to write on the subject for each issue. Here we are pleased to introduce the Section Editors for this issue. SECTION EDITORS H. Scott BaldwinH. Scott BaldwinDr Baldwin is the Katrina Overall Chair in Pediatrics and Professor of Pediatrics and Cell and Developmental Biology at Vanderbilt, USA. He serves as Chief of the Division of Pediatric Cardiology and Co-Director of the Pediatric Heart Institute at the Monroe Carell Jr. Children's Hospital, USA. Dr Baldwin received his MD from the University of Virginia, USA, did his pediatric residency and chief residency at the University of Rochester, USA, and completed a pediatric cardiology fellowship at the University of Iowa, USA. He then joined the faculty at the University of Pennsylvania and Children's Hospital of Philadelphia, USA, where he served as Co-Director of Cardiovascular Research before moving to Vanderbilt assuming the role of Vice-Chair of Pediatrics for Laboratory Based Research prior to becoming the Chief of Pediatric Cardiology. Dr Baldwin's interests concentrate on understanding the molecular and genetic etiology of congenital heart diseases. Studies in the laboratory are based on the hypothesis that the developing vasculature provides important patterning information that directs subsequent cardiac and pulmonary morphogenetic events and thus understanding the molecular basis of endothelial heterogeneity is essential in defining the mechanisms and treatment of congenital cardiac disease. His laboratory has developed strategies for endocardial specific gene deletion to allow temporal and spatial gene manipulation throughout the vascular system. He is currently involved in several interdisciplinary collaborations to determine if knowledge about normal valve development can be used to guide development of tissue engineered heart valves for use in the pediatric population. Dr Baldwin is a fellow of the American Heart Association, a fellow of the American College of Cardiology, and a member of the Academic Pediatric Society. He currently serves on the AHA Council of Cardiovascular Diseases in the Young as well as a member of the national AHA Research Oversight Committee. He is also Chair of the Scientific Board for the Sarnoff Foundation, an organization dedicated to providing medical students with an in-depth cardiovascular focused research experience by supporting a one to two year hiatus from medical school to participate in a basic laboratory investigation and he is the principle investigator of an NIH/NHLBI training grant specifically focused on development of both MD and PhD training in disease processes relevant to congenital and acquired heart disease. Robert WyllieRobert WyllieDr Robert Wyllie is the Chief Medical Operations Officer at the Cleveland Clinic, USA, and Vice Chair of the Office of Professional Staff Affairs. Dr Wyllie is a member of the Cleveland Clinic Board of Governors, other offices held include President of the Staff and member of the Board of Trustees. Dr Wyllie was the initial holder of the Calabrese Chair of Pediatrics and served as the Chairman of the Pediatric Institute, and Physician and Phrygian-In-Chief of the Cleveland Clinic Children's Hospital from 2006 to 2011. Dr Wyllie is a Professor in the Lerner College of Medicine, USA, and has participated in more than 250 presentations at scientific meetings, written over 150 manuscripts and chapters, and edits one of the major textbooks in pediatric gastroenterology, now in its fourth edition. Together with his wife, Dr Elaine Wyllie, Director of the Cleveland Clinic's Center for Pediatric Neurology, he raised two sons who are pursuing their own careers in science and technology.

Multi-scale mechanical characterization of scaffolds for heart valve tissue engineering

Electrospinning is a promising technology to produce scaffolds for cardiovascular tissue engineering. Each electrospun scaffold is characterized by a complex micro-scale structure that is responsible for its macroscopic mechanical behavior. In this study, we focus on the development and the validation of a computational micro-scale model that takes into account the structural features of the electrospun material, and is suitable for studying the multi-scale scaffold mechanics. We show that the computational tool developed is able to describe and predict the mechanical behavior of electrospun scaffolds characterized by different microstructures. Moreover, we explore the global mechanical properties of valve-shaped scaffolds with different microstructural features, and compare the deformation of these scaffolds when submitted to diastolic pressures with a tissue engineered and a native valve. It is shown that a pronounced degree of anisotropy is necessary to reproduce the deformation patterns observed in the native heart valve.

In situ heart valve tissue engineering: simple devices, smart materials, complex knowledge

Since the implantation of the first artificial aortic heart valve by Hufnagel et al., the design and construction of heart valve prostheses has undergone continuous and drastic changes to improve t...

Oscillatory shear stress created by fluid pulsatility versus flexed specimen configurations

Oscillatory shear stress (OSS), caused by time-varying flow environments, may play a critical role in the production of engineered tissue by bone marrow-derived stem cells. This is particularly relevant in heart valve tissue engineering (HVTE), owing to the intense haemodynamic environments that surround native valves. In this study, we examined and quantified the role that (i) physiologically relevant scales of pulsatility and (ii) changes in geometry as a function of specimen flexure have in creating OSS conditions. A U-shaped bioreactor capable of producing flow, stretch and flexure was modelled with housed specimens, and computational fluid dynamic simulations were performed. We found that physiologically relevant OSS can be maximised by the application of pulsatile flow to straight, non-moving specimens in a uniform manner. This finding reduces a substantial layer of complexity in dynamic HVTE protocols in which traditionally, time-varying flow has been promoted through specimen movement in custom-made bioreactors.

Microstructural manipulation of electrospun scaffolds for specific bending stiffness for heart valve tissue engineering

Biodegradable thermoplastic elastomers are attractive for application in cardiovascular tissue construct development due to their amenability to a wide range of physical property tuning. For heart valve leaflets, while low flexural stiffness is a key design feature, control of this parameter has been largely neglected in the scaffold literature where electrospinning is being utilized. This study evaluated the effect of processing variables and secondary fiber populations on the microstructure, tensile and bending mechanics of electrospun biodegradable polyurethane scaffolds for heart valve tissue engineering. Scaffolds were fabricated from poly(ester urethane) urea (PEUU) and the deposition mandrel was translated at varying rates in order to modify fiber intersection density. Scaffolds were also fabricated in conjunction with secondary fiber populations designed either for mechanical reinforcement or to be selectively removed following fabrication. It was determined that increasing fiber intersection densities within PEUU scaffolds was associated with lower bending moduli. Further, constructs fabricated with stiff secondary fiber populations had higher bending moduli whereas constructs with secondary fiber populations which were selectively removed had noticeably lower bending moduli. Insights gained from this work will be directly applicable to the fabrication of soft tissue constructs, specifically in the development of cardiac valve tissue constructs.

Application of simple biomechanical and biochemical tests to heart valve leaflets: Implications for heart valve characterization and tissue engineering

A simple biomechanical test with real-time displacement and strain mapping is reported, which provides displacement vectors and principal strain directions during the mechanical characterization of heart valve tissues. The maps reported in the current study allow us to quickly identify the approximate strain imposed on a location in the samples. The biomechanical results show that the aortic valves exhibit stronger anisotropic mechanical behavior than that of the pulmonary valves before 18% strain equibiaxial stretching. In contrast, the pulmonary valves exhibit stronger anisotropic mechanical behavior than aortic valves beyond 28% strain equibiaxial stretching. Simple biochemical tests are also conducted. Collagens are extracted at different time points (24, 48, 72, and 120 h) at different locations in the samples. The results show that extraction time plays an important role in determining collagen concentration, in which a minimum of 72 h of extraction is required to obtain saturated collagen concentration. This work provides an easy approach for quantifying biomechanical and biochemical properties of semilunar heart valve tissues, and potentially facilitates the development of tissue engineered heart valves.

Stem Cell–Based Transcatheter Aortic Valve Implantation: First Experiences in a Pre-Clinical Model

This study sought to investigate the combination of transcatheter aortic valve implantation and a novel concept of stem cell-based, tissue-engineered heart valves (TEHV) comprising minimally invasive techniques for both cell harvest and valve delivery. TAVI represents an emerging technology for the treatment of aortic valve disease. The used bioprostheses are inherently prone to calcific degeneration and recent evidence suggests even accelerated degeneration resulting from structural damage due to the crimping procedures. An autologous, living heart valve prosthesis with regeneration and repair capacities would overcome such limitations. Within a 1-step intervention, trileaflet TEHV, generated from biodegradable synthetic scaffolds, were integrated into self-expanding nitinol stents, seeded with autologous bone marrow mononuclear cells, crimped and transapically delivered into adult sheep (n = 12). Planned follow-up was 4 h (Group A, n = 4), 48 h (Group B, n = 5) or 1 and 2 weeks (Group C, n = 3). TEHV functionality was assessed by fluoroscopy, echocardiography, and computed tomography. Post-mortem analysis was performed using histology, extracellular matrix analysis, and electron microscopy. Transapical implantation of TEHV was successful in all animals (n = 12). Follow-up was complete in all animals of Group A, three-fifths of Group B, and two-thirds of Group C (1 week, n = 1; 2 weeks, n = 1). Fluoroscopy and echocardiography displayed TEHV functionality demonstrating adequate leaflet mobility and coaptation. TEHV showed intact leaflet structures with well-defined cusps without signs of thrombus formation or structural damage. Histology and extracellular matrix displayed a high cellularity indicative for an early cellular remodeling and in-growth after 2 weeks. We demonstrate the principal feasibility of a transcatheter, stem cell-based TEHV implantation into the aortic valve position within a 1-step intervention. Its long-term functionality proven, a stem cell-based TEHV approach may represent a next-generation heart valve concept.

Laser microfabricated poly(glycerol sebacate) scaffolds for heart valve tissue engineering

Microfabricated poly(glycerol sebacate) (PGS) scaffolds may be applicable to tissue engineering heart valve leaflets by virtue of their controllable microstructure, stiffness, and elasticity. In this study, PGS scaffolds were computationally designed and microfabricated by laser ablation to match the anisotropy and peak tangent moduli of native bovine aortic heart valve leaflets. Finite element simulations predicted PGS curing conditions, scaffold pore shape, and strut width capable of matching the scaffold effective stiffnesses to the leaflet peak tangent moduli. On the basis of simulation predicted effective stiffnesses of 1.041 and 0.208 MPa for the scaffold preferred (PD) and orthogonal, cross-preferred (XD) material directions, scaffolds with diamond-shaped pores were microfabricated by laser ablation of PGS cured 12 h at 160°C. Effective stiffnesses measured for the scaffold PD (0.83 ± 0.13 MPa) and XD (0.21 ± 0.03 MPa) were similar to both predicted values and peak tangent moduli measured for bovine aortic valve leaflets in the circumferential (1.00 ± 0.16 MPa) and radial (0.26 ± 0.03 MPa) directions. Scaffolds cultivated with fibroblasts for 3 weeks accumulated collagen (736 ± 193 μg/g wet weight) and DNA (17 ± 4 μg/g wet weight). This study provides a basis for the computational design of biomimetic microfabricated PGS scaffolds for tissue-engineered heart valves.

A Pulsatile Bioreactor for Conditioning of Tissue-Engineered Cardiovascular Constructs under Endoscopic Visualization.

Heart valve disease (HVD) is a globally increasing problem and accounts for thousands of deaths yearly. Currently end-stage HVD can only be treated by total valve replacement, however with major drawbacks. To overcome the limitations of conventional substitutes, a new clinical approach based on cell colonization of artificially manufactured heart valves has been developed. Even though this attempt seems promising, a confluent and stable cell layer has not yet been achieved due to the high stresses present in this area of the human heart. This study describes a bioreactor with a new approach to cell conditioning of tissue engineered heart valves. The bioreactor provides a low pulsatile flow that grants the correct opening and closing of the valve without high shear stresses. The flow rate can be regulated allowing a steady and sensitive conditioning process. Furthermore, the correct functioning of the valve can be monitored by endoscope surveillance in real-time. The tubeless and modular design allows an accurate, simple and faultless assembly of the reactor in a laminar flow chamber. It can be concluded that the bioreactor provides a strong tool for dynamic pre-conditioning and monitoring of colonized heart valve prostheses physiologically exposed to shear stress.

Stem cells for heart valve regeneration

Heart valve tissue engineering holds the potential to overcome limitations of currently used heart valve prostheses. It involves the isolation and expansion of autologous patient cells, the subsequent seeding of these cells onto an appropriate scaffold material, the in vitro incubation and the in vivo implantation of the derived tissue-engineered construct into the patient from whom the cells were taken. While vascular-derived cells require harvest of intact donor tissue and show limited expansion capacities, the use of stem or progenitor cells may overcome these limitations and expand the versatility of the concept of heart valve tissue engineering. Possible sources include cells isolated from blood, bone marrow, adipose tissue, amniotic fluid, chorionic villi, umbilical cord and induced pluripotent stem cells. Here we review different stem cell sources with particular regard to cellular phenotypes and their suitability for application in heart valve tissue engineering.

A novel culture device for the evaluation of three-dimensional extracellular matrix materials

Cell-matrix interactions in a three-dimensional (3D) extracellular matrix (ECM) are of fundamental importance in living tissue, and their in vitro reconstruction in bioartificial structures represents a core target of contemporary tissue engineering concepts. For a detailed analysis of cell-matrix interaction under highly controlled conditions, we developed a novel ECM evaluation culture device (EECD) that allows for a precisely defined surface-seeding of 3D ECM scaffolds, irrespective of their natural geometry. The effectiveness of EECD was evaluated in the context of heart valve tissue engineering. Detergent decellularized pulmonary cusps were mounted in EECD and seeded with endothelial cells (ECs) to study EC adhesion, morphology and function on a 3D ECM after 3, 24, 48 and 96 h. Standard EC monolayers served as controls. Exclusive top-surface-seeding of 3D ECM by viable ECs was demonstrated by laser scanning microscopy (LSM), resulting in a confluent re-endothelialization of the ECM after 96 h. Cell viability and protein expression, as demonstrated by MTS assay and western blot analysis (endothelial nitric oxide synthase, von Willebrand factor), were preserved at maintained levels over time. In conclusion, EECD proves as a highly effective system for a controlled repopulation and in vitro analysis of cell-ECM interactions in 3D ECM.