Tissue Engineered Heart Valve Scaffolds Research Articles

Biomechanical and morphological stability of acellular scaffolds for tissue-engineered heart valves depends on different storage conditions

Currently available bioprosthetic heart valves have been successfully used clinically; however, they have several limitations. Alternatively, tissue-engineering techniques can be used. However, there are limited data concerning the impact of storage conditions of scaffolds on their biomechanics and morphology. The aim of this study was to determine the effect of different storage conditions on the biomechanics and morphology of pulmonary valve dedicated for the acellular scaffold preparation to achieve optimal conditions to obtain stable heart valve prostheses. Scaffold can then be used for the construction of tissue-engineered heart valve, for this reason evaluation of these parameters can determine the success of the clinical application this type of bioprosthesis. Pulmonary heart valves were collected from adult porcines. Materials were divided into five groups depending on the storage conditions. Biomechanical tests were performed, both the static tensile test, and examination of viscoelastic properties. Extracellular matrix morphology was evaluated using transmission electron microscopy and immunohistochemistry. Tissue stored at 4 °C exhibited a higher modulus of elasticity than the control (native) and fresh acellular, which indicated the stiffening of the tissue and changes of the viscoelastic properties. Such changes were not observed in the radial direction. Percent strain was not significantly different in the study groups. The storage conditions affected the acellularization efficiency and tissue morphology. To the best of our knowledge, this study is the first that attributes the mechanical properties of pulmonary valve tissue to the biomechanical changes in the collagen network due to different storage conditions. Storage conditions of scaffolds for tissue-engineered heart valves may have a significant impact on the haemodynamic and clinical effects of the used bioprostheses.

Biodegradable and biomimetic elastomeric scaffolds for tissue-engineered heart valves.

Heart valve tissue engineering (HVTE) is of special significance to overcome the drawbacks of current valve replacements. Although biodegradable synthetic elastomers have emerged as promising materials for HVTE, a mature HVTE construct made from synthetic elastomers for clinical use remains to be developed. Hence, this review summarized various types of biodegradable synthetic elastomers and their key properties. The major focus that distinguishes this review from the current literature is the thorough discussion on the key features of native valve microenvironments and various up-and-coming approaches to engineer synthetic elastomers to recreate these features such as anisotropic tri-layered architecture, mechanical anisotropy, biodegradability and biocompatibility. This review is envisioned to inspire and instruct the design of functional HVTE constructs and facilitate their clinical translation.

Degradable Chitosan-Collagen Composites Seeded with Cells as Tissue Engineered Heart Valves

Degradable collagen-chitosan composite materials have been used to fabricate tissue engineered heart valves. The aims of this study were to demonstrate that the collagen-chitosan composite scaffolds are cytocompatible, and endothelial cells can be differentiated from bone marrow mesenchymal stem cells (BMSCs) when seeded onto the scaffolds. The adhesion and biological activities of the seeded cells were also investigated. Collagen-chitosan composite material was used as the cell matrix, and smooth muscle cells, fibroblasts and BMSCs were used as seed cells. After four weeks of in vitro culture, the smooth muscle cells, fibroblasts, and BMSCs were sequentially seeded into the collagen-chitosan composite material. After four weeks in culture, the cellular density and activity were assessed on segments of the tissue engineered heart valve scaffolds to determine the cell viability and proliferation in the collagen-chitosan composite material. The tissue engineered heart valves stained positively for both smooth muscle actin and endothelial cell factor VIII, suggesting that the seeded cells were in fact smooth muscle cells, fibroblasts, and endothelial cells. The 6-ketone prostaglandin content, as measured by radioimmunoassay, of the collagen-chitosan cell culture fluid was higher than that of the serum-free medium (P <0.01). Light and electron microscopy showed that the seeded cells had shapes similar to the morphology of smooth muscle cells, fibroblasts, and endothelial cells. Endothelial cells can be differentiated from BMSCs when seeded onto the collagen-chitosan composite scaffolds. The seeded cells retained their biological activity after being cultured in vitro and seeded into the collagen-chitosan composite material.

Hyaluronan Hydrogels for a Biomimetic Spongiosa Layer of Tissue Engineered Heart Valve Scaffolds.

Advanced tissue engineered heart valves must be constructed from multiple materials to better mimic the heterogeneity found in the native valve. The trilayered structure of aortic valves provides the ability to open and close consistently over a full human lifetime, with each layer performing specific mechanical functions. The middle spongiosa layer consists primarily of proteoglycans and glycosaminoglycans, providing lubrication and dampening functions as the valve leaflet flexes open and closed. In this study, hyaluronan hydrogels were tuned to perform the mechanical functions of the spongiosa layer, provide a biomimetic scaffold in which valve cells were encapsulated in 3D for tissue engineering applications, and gain insight into how valve cells maintain hyaluronan homeostasis within heart valves. Expression of the HAS1 isoform of hyaluronan synthase was significantly higher in hyaluronan hydrogels compared to blank-slate poly(ethylene glycol) diacrylate (PEGDA) hydrogels. Hyaluronidase and matrix metalloproteinase enzyme activity was similar between hyaluronan and PEGDA hydrogels, even though these scaffold materials were each specifically susceptible to degradation by different enzyme types. KIAA1199 was expressed by valve cells and may play a role in the regulation of hyaluronan in heart valves. Cross-linked hyaluronan hydrogels maintained healthy phenotype of valve cells in 3D culture and were tuned to approximate the mechanical properties of the valve spongiosa layer. Therefore, hyaluronan can be used as an appropriate material for the spongiosa layer of a proposed laminate tissue engineered heart valve scaffold.

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.

Fine structure of glycosaminoglycans from fresh and decellularized porcine cardiac valves and pericardium.

Cardiac valves are dynamic structures, exhibiting a highly specialized architecture consisting of cells and extracellular matrix with a relevant proteoglycan and glycosaminoglycan content, collagen and elastic fibers. Biological valve substitutes are obtained from xenogenic cardiac and pericardial tissues. To overcome the limits of such non viable substitutes, tissue engineering approaches emerged to create cell repopulated decellularized scaffolds. This study was performed to determine the glycosaminoglycans content, distribution, and disaccharides composition in porcine aortic and pulmonary valves and in pericardium before and after a detergent-based decellularization procedure. The fine structural characteristics of galactosaminoglycans chondroitin sulfate and dermatan sulfate were examined by FACE. Furthermore, the mechanical properties of decellularized pericardium and its propensity to be repopulated by in vitro seeded fibroblasts were investigated. Results show that galactosaminoglycans and hyaluronan are differently distributed between pericardium and valves and within heart valves themselves before and after decellularization. The distribution of glycosaminoglycans is also dependent from the vascular district and topographic localization. The decellularization protocol adopted resulted in a relevant but not selective depletion of galactosaminoglycans. As a whole, data suggest that both decellularized porcine heart valves and bovine pericardium represent promising materials bearing the potential for future development of tissue engineered heart valve scaffolds.

EMT-Inducing Biomaterials for Heart Valve Engineering: Taking Cues from Developmental Biology

Although artificial prostheses for diseased heart valves have been around for several decades, viable heart valve replacements have yet to be developed due to their complicated nature. The majority of research in heart valve replacement technology seeks to improve decellularization techniques for porcine valves or bovine pericardium as an effort to improve current clinically used valves. The drawback of clinically used valves is that they are nonviable and thus do not grow or remodel once implanted inside patients. This is particularly detrimental for pediatric patients, who will likely need several reoperations over the course of their lifetimes to implant larger valves as the patient grows. Due to this limitation, additional biomaterials, both synthetic and natural in origin, are also being investigated as novel scaffolds for tissue-engineered heart valves, specifically for the pediatric population. Here, we provide a brief overview of valves in clinical use as well as of the materials being investigated as novel tissue-engineered heart valve scaffolds. Additionally, we focus on natural-based biomaterials for promoting cell behavior that is indicative of the developmental biology process that occurs in the formation of heart valves in utero, such as epithelial-to-mesenchymal transition or transformation. By engineering materials that promote native developmental biology cues and signaling, while also providing mechanical integrity once implanted, a viable tissue-engineered heart valve may one day be realized. A viable tissue-engineered heart valve, capable of growing and remodeling actively inside a patient, could reduce risks and complications associated with current valve replacement options and improve overall quality of life in the thousands of patients who received such valves each year, particularly for children.

Gamma Radiation Alters the Ultrastructure in Tissue-Engineered Heart Valve Scaffolds

Xenogenic extracellular heart valve matrices have been suggested as scaffolds for tissue engineering. However, these matrices are immunogenic and stimulate an intense cell-mediated immune response and calcification. Mitigating the immunogenicity was attempted by different doses of gamma irradiation. Mechanical properties of gamma-irradiated porcine matrices and control matrices (nonirradiated) were examined by tensile strength testing. Irradiated matrices (1, 10, 50, and 100 gray [Gy]) and control matrices were implanted subcutaneously in Wistar rats (n = 20). After 24 h, 1, 2, 3, and 4 weeks the explants were examined by light microscopy and transmission electron microscopy. Calcium (Ca) content was determined using inductively coupled plasma-mass spectrometry. Antibody reaction against porcine tissue in the rat serum was determined. Tensile strength increased in irradiated matrices at the expense of elasticity. Ten gray-irradiated leaflets showed minimal lymphocytic inflammatory infiltration with preservation of ultrastructure. Ca levels after 2 weeks were as follows: control (0 Gy), 388 +/- 264 microg/mg; 1 Gy, 240 +/- 95 microg/mg; 10 Gy, 188 +/- 54 microg/mg; 50 Gy, 289 +/- 94 microg/mg; 100 Gy, 651 +/- 57 microg/mg. All implants still elicit an antibody immunoglobulin G reaction. Exposure to 10 Gy gamma irradiation reduces lymphocytic inflammatory infiltrates and Ca levels in acellular porcine matrices with preservation of structural integrity. This could prolong the durability of these matrices.

Construction of Tissue-Engineered Homograft Bioprosthetic Heart Valves In Vitro

Human mesenchymal stem cells (MSCs) differentiate into multiple cell-lineages and may serve as an alternative source of seed cells for tissue engineering. We investigated whether MSCs could be induced to differentiate into endothelial cells (ECs) and function as seed cells for the in vitro construction of tissue-engineered heart valves (TEHVs). Aortic or pulmonary valve homografts were decellularized with 0.1% sodium dodecylsulphate and used as scaffolds for TEHVs. The MSCs were isolated from human bone marrow by Percoll gradient centrifugation (1.073 g/ml), differentiated into ECs with vascular endothelial growth factor (10 ng/ml), and seeded onto a decellularized scaffold (high-density seeding, >10(5) cells/cm2) and grown in static culture for 14 days. Over 90% of the differentiated cells from MSCs stained positively for von Willebrand factor and Tie-2-related antigen. Additionally, Weibel-Palade corpuscle was observed in the cytoplasm of these cells. Levels of reendothelialization in static culture on days 7, 14, and 20, were 73%, 85%, and 95%, respectively. These results show that MSCs from human bone marrow can differentiate, in vitro, into ECs that can then be used to construct TEHVs. Reendothelialization in static culture can be used to provide the basic material for pulsatile-flow cultivation.

SURFACE MODIFICATION FOR DURABLE TISSUE-ENGINEERED HEART VALVES

Purpose: Artificial valves have disadvantages that they cannot grow, repair and remodel. Tissue engineered heart valves are expected to overcome these shortcomings. The durability of tissue-engineered heart valves is now questionable. The purpose of the study is to evaluate of the durability of tissue engineered heart valve with surface modified scaffolds. Method: The scaffold was constructed with poly(D, L lactid-co-glycol-ide) (PGLA). To enhance cell attachment and in-growth, surface properties were modified by immobilization (if fibronectin and adhesion peptide sequence (RGD). For effective surface modification, the scaffolds were CO2 gas plasma treated prior to the immobilization process. The mock circulation system using a pulsatile pump was used for durability test under various shear stresses physiological condition. The cell-polymer construct was evaluated with MTT assays. In addition, scanning electron microscopy (SEMI study of the seeded scaffolds was also performed to document tissue development. Result: MTI assay and SEM evaluation revealed that in surface modified scaffolds, cell attachment was enhanced and cells were well ingrown to he tissue like structure. consequently the result of durability tests showed that the cells on the tissue engineered heart valve scaffolds with modified surface endured the diverse physiological shear stress Acknowledgement: This work was supported by the BK 21 project.