Valvular heart disease is an important source of morbidity. Tissue Engineering of Heart Valves: In Vitro Experiences

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1 Tissue Engineering of Heart Valves: In Vitro Experiences Ralf Sodian, MD, Simon P. Hoerstrup, MD, Jason S. Sperling, MD, Sabine H. Daebritz, MD, David P. Martin, PhD, Frederick J. Schoen, MD, PhD, Joseph P. Vacanti, MD, and John E. Mayer, Jr, MD Departments of Cardiac Research and Cardiac Surgery, Children s Hospital, Harvard Medical School, Boston, Massachusetts Background. Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. Methods. We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent ( mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 m) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. Results. It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. Conclusions. Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure. (Ann Thorac Surg 2000;70:140 4) 2000 by The Society of Thoracic Surgeons Valvular heart disease is an important source of morbidity and mortality world wide [1]. Currently, surgical treatment is the most common therapy of end-stage valvular heart disease. Valve replacement devices include xenografts, mechanical prostheses, or homografts [2]. Each type has limitations, which include their inability to grow, repair, and remodel, which limits their long-term benefit in children and young adults [3 5]. To create a new tissue heart valve from autologous tissue, our laboratory has focused on tissue engineering of cardiovascular structures. The major goal of tissue engineering is to use autologous cells, attach those cells onto a biodegradable scaffold, and ultimately form new functional tissue with the ability to grow and remodel with inherent nonthrombogenicity, which would be a major improvement on the current treatment of heart valve disease [6]. Our group previously reported the implantation of a single tissue-engineered posterior cusp of the pulmonary valve in a lamb model [7, 8]. Following 11 weeks function Accepted for publication Dec 26, Address reprint requests to Dr Mayer, Department of Cardiac Surgery, Children s Hospital, Harvard Medical School, 300 Longwood Ave, Boston, MA of the pulmonary valve in lambs, the cusp resembled native heart valve tissue in several respects: appropriate cellular architecture and developed extracellular matrix similar to the native cusps. However, it was not possible to create a trileaflet heart valve replacement with this construct, because of the stiffness, thickness, and nonpliability of the scaffold. Now we have fabricated a trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (PHA), which was seeded with vascular cells from adult ovine arteries and placed into a pulsatile flow system to induce in vitro neotissue formation for tissue-engineered heart valves. This study was designed to evaluate a new scaffold for tissue engineering of heart valves as well as using a bioreactor to ultimately form viable tissue in vitro. Material and Methods Scaffold Material The scaffold material is a bacterially derived biopolyester, a PHA (Metabolix Inc, Cambridge, MA). PHA is a thermoplastic polymer. It has a low melting point (T m 61 C) and can be molded into almost any shape [9] by The Society of Thoracic Surgeons /00/$20.00 Published by Elsevier Science Inc PII S (00)

2 Ann Thorac Surg SODIAN ET AL 2000;70:140 4 TISSUE ENGINEERING OF HEART VALVES 141 Fig 1. Porous heart valve scaffold and aluminum cast for scaffold fabrication (seen from the top). Furthermore, the material has high elasticity and mechanical strength, which are important in cardiovascular tissue engineering. To give the material a threedimensional seeding surface, we used a salt leaching technique to create a porous scaffold [10]. Sieved sodium chloride crystals (180 to 240 m) were mixed with a polymer solution. This resulted in the formation of PHA with entrapped NaCl crystals, which were leached out with double-distilled water for 3 days at 37 C and 5% CO 2. The resulting porous, three-dimensional polymer was used for the fabrication of a heart valve scaffold for tissue engineering (Fig 1). Cell Harvest Vascular cells were grown from an ovine carotid artery. Sheep were anesthetized with Diprivan (Stuart Pharmaceuticals, Wilmington, DE). Using sterile surgical technique, 5-cm to 6-cm sections of arterial tissue were harvested from the carotid artery. The perivascular connective tissue was removed and only the intima, media, and adventitia of the artery were used for vascular cell harvest. The tissue was washed with phosphate-buffered saline (Gibco BRL, Rockville, MD) and minced into 1-mm pieces, which were distributed in mm Petri dishes (Becton Dickinson Labware, Franklin Lakes, NJ). The cell culture medium consisted of Dulbecco s modified Eagle s medium (Gibco BRL), 10% fetal bovine serum (Sigma, St. Louis, MO), and 1% of an antibiotic solution (gentamycin-penicillin-streptomycin, Sigma). The cell populations were passaged three times to obtain enough cells for cell seeding. The mixed cell population was placed in a humidified incubator at 37 C with 5% CO 2 for 4 weeks. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health publication no , revised 1985). Scaffold Seeding Scaffolds (n 10, pore size: 180 to 240 m) were seeded with 8 million vascular cells. The cells were dripped on to the inside and the outside of the polymeric heart valve scaffold on 4 consecutive days. Thus cells were distributed throughout the whole trileaflet heart valve scaffold. The cell-polymer construct was incubated for an additional day in cell culture medium supplemented with L-proline, L-alanine, glycine, and L-ascorbic acid (Sigma) in an Erlenmeyer flask (Corning). Pulsatile Flow System (Bioreactor) The pulsatile flow system was designed to induce different levels of pulsatile flow under controlled conditions in a humidified incubator (37 C and 5% CO 2 ). The medium we used in our bioreactor system consisted of Dulbecco s modified Eagle s medium (Gibco BRL), 10% fetal bovine serum (Sigma), and 1% of an antibiotic solution (gentamycin-penicillin-streptomycin, Sigma). To adjust the system to certain flow and pressure conditions, pressures were measured by a digital pressure measurement device (Digital Ultrasonic Measurement System, Sono Metrics Inc, London, Ontario, Canada) distal of the tissueengineered heart valve construct. Organization of the Study At the end of day 5 of seeding and incubation the cell-polymer constructs were exposed to pulsatile flow. Two seeded heart valve constructs were placed into the bioreactor for 1 day (group I), 4 days (group II), and 8 days (group III). The bioreactor was started with a low flow of 140 ml/min and a systolic pressure of 10 mm Hg on the first day and increased the flow and the pressure more than 8 days to a flow of 350 ml/min and a systolic pressure of 13 mm Hg on the eighth day. Two control valves were seeded with cells, and incubated under static (no flow) conditions for 1 day (control of experimental group I) and 8 days (control of experimental group III). There was no extra control group for experimental group II. Evaluation of In Vitro Created Tissue A representative portion of the trileaflet heart valve construct was fixed in 10% formalin for histologic examination using hematoxylin and eosin stain for overall morphology and the movat stain to demonstrate the presence of collagen, glycoaminoglycans, and elastin. Another sample was fixed in cacodylic acid buffer (Sigma) and was examined by environmental scanning electron microscopy (ESEM). To assess for the presence of cells and collagenous extracellular matrix formation biochemically, a DNA assay (CyQuant, Molecular Probes, Eugene, OR) [11] and a 4-hydroxyproline assay [12] were performed. Statistical comparison of cell numbers and collagenous extracellular matrix formation after being exposed to different levels of pulsatile flow in a bioreactor were made using the least significant difference test and Bonferroni test. A significant result was assumed if p was less than Results Cell Proliferation (DNA Assay) The DNA assay demonstrated cell proliferation under pulsatile flow conditions as shown in Table 1. Collagen Formation (4-Hydroxyproline Assay) Collagen formation occurred under both static and pulsatile flow conditions as shown in Table 1.

3 142 SODIAN ET AL Ann Thorac Surg TISSUE ENGINEERING OF HEART VALVES 2000;70:140 4 Table 1. Cell Proliferation (DNA Assay) and Collagen Formation (4-Hydroxyproline Assay) Group I (1 day of pulsatile flow) Control Group I (1 day of incubation and no flow) Group II (4 days of pulsatile flow) Group III (8 days of pulsatile flow) Control Group III (8 days of incubation and no flow) Cell number (cells/ mg construct) Collagen formation ( g/mg construct) 11,103 2,365 a 6,540 1,108 17,420 2,318 19,979 1,583 b 9, a p 0.025, significantly different from control group I, least significant difference test (LSD) and Bonferroni. control group III, least significant difference test (LSD) and Bonferroni. b p 0.009, significantly different from Morphology ENVIROMENTAL SCANNING ELECTRON MICROSCOPY. The ESEM examination of the heart valve construct showed that cells attached to the polymer scaffold and grew into the pores created by salt leaching. After 1 day of incubation and 4 days of pulsatile flow exposure, the cells formed a confluent cell layer and the longest of all cell types, which lined the flow exposed side of the scaffold, were oriented in the direction of flow (Fig 2). HISTOLOGY. The histologic examination showed confluent cell layers on both sides of the PHA scaffold in each tested sample (control [no flow], 1 and 4 days of flow). Moreover, the cells grew into pores and formed connective tissue between the inside and outside of the porous heart valve scaffold. Hematoxylin and eosin stain showed single cell necrosis of all cell types (staining pink), but most of all the cells that lined the scaffold appeared viable. Staining for collagen and glycosaminoglycans were positive, but no elastin was found in the control valve or after 1 and 4 days of pulsatile flow exposure (Fig 3). Comment Current heart valve replacement is performed using glutaraldehyde-fixed xenograft valves, mechanical prostheses, or homografts. Each currently used valve type has certain advantages and disadvantages. The most important advantages of xenografts are a low incidence of thrombembolic events and anticoagulantrelated hemorrhage [13]. The major disadvantage of glutaraldehyde-fixed xenograft valves is their progressive degeneration. The leaflets change their mechanical properties, which leads to leaflet failure and finally to central incompetence [14]. Furthermore, the role of valve failure increased in xenograft valve patients who were at a younger age at valve replacement [15, 16]. Mechanical prostheses have the advantage of a good structural durability [17]. Nevertheless, mechanical valves have a high rate of thromboembolic complications, necessitating lifelong anticoagulation and its associated hemorrhagic risk. All valves have a risk of infection (prosthetic valve endocarditis), a potentially fatal complication [18]. Homograft valves have been used for aortic valve replacement since 1962 [19]. The major advantages of homografts over prosthetic valves are freedom from valvular thrombosis and lower risk of infections or reinfections after prosthetic valve endocarditis. The major limitations of homograft valves are that the supply is limited by a worldwide donor shortage and early homograft failure in young children might be associated with specific immune responses [20, 21]. To overcome these disadvantages, the principles of tissue engineering could offer several potential theoretical advantages. The major goal of this approach is to transplant autologous donor cells onto a biodegradable Fig 2. (a) Enviromental scanning electron microscopy (ESEM) of an unseeded, porous polyhydroxyalkanoate scaffold. (b) ESEM shows cell ingrowth into pores (1 day of incubation). (c) ESEM of an almost confluent cell layer after 1 day of incubation and 4 days of pulsatile flow.

4 Ann Thorac Surg SODIAN ET AL 2000;70:140 4 TISSUE ENGINEERING OF HEART VALVES 143 Fig 3. (A) Hematoxylin and eosin staining of a tissue-engineered conduit after 1 day of incubation and 4 days of pulsatile flow: (a) dissolved polymer; (b) cell ingrowth into pores; (c) connective cell layer between two different sides of the polymer. (B) Movat staining shows the presence of collagen, glycosaminoglycan, and elastin (staining yellow, green, and black, respectively). scaffold, condition the seeded scaffold in vitro, and finally to implant a viable tissue construct into the patient. Once in the body, the new tissue can potentially start to remodel and to integrate with surrounding tissue to form a functional structure [22 24]. The new tissue-engineered heart valve would be autologous tissue with normal biological mechanisms for growth and development. The constructs would probably not undergo rejection or foreign body response when the polymer scaffold is completely degraded. Moreover, we assume that the presence of a functional endothelial cell lining theoretically provides a low thrombogenicity with no need for longterm anticoagulation. In previous experiments, our group was able to show the feasibility of the tissue-engineering approach to construct a pulmonary artery conduit and to implant a single tissue-engineered leaflet in the posterior position of the pulmonary valve in a lamb model [7, 25]. The major problems of this leaflet replacement were the stiffness, thickness, and nonpliability of the scaffold. Therefore, it was not possible to create a functional trileaflet heart valve scaffold because of the limitations of the material. We have now fabricated a heart valve scaffold from a porous and elastic material (PHA), seeded vascular cells on to this polymer, and exposed the cell-polymer construct to pulsatile flow. All fabricated heart valve scaffolds showed an appropriate function under subphysiologic and supraphysiologic flow conditions and appeared more pliable than previously described constructs. For this experiment, a porous, three-dimensional polymer was chosen to increase the surface area of the polymer and thereby attach more cells onto the heart valve scaffold. Moreover, the porous nature of the polymer might be important for an in vivo vascularization of the tissue-engineered heart valve after implantation into the circulation. In preliminary studies, we exposed tissue-engineered constructs to pulsatile flow and we were able to show that vascular cells seeded on to combined polymers ([polyglycolic acid] PGA/PHA), have the ability to orient in the direction of flow and to form confluent cell layers under pulsatile flow. Other investigators are currently utilizing special flow systems (bioreactors) to generate vascularlike tissue with significant extracellular matrix formation and supraphysiologic burst pressure before implantation. These observations suggest that flow exposure might be important to guide the development of tissueengineered heart valves or vessels in vitro before implantation into an in vivo model [26 28]. Our results demonstrated that cells seeded onto a porous PHA scaffold attached to this polymer and formed an oriented and confluent cell layer after pulsatile flow exposure. Moreover, our results have demonstrated proliferation of vascular cells (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and glycosaminoglycans as stained by movat under in vitro pulsatile flow conditions. The lack of elastin in our tissue-engineered constructs might be a certain limitation of the approach. Therefore, subsequent experiments are necessary to improve our in vitro techniques to generate an extracellular matrix that would ultimately resemble native vascular tissue. An additional and important observation in this experiment was that the cells migrated into pores, and formed tissue bridges between the inside and the outside of the conduit. The cells appear to be viable on our porous polymer heart valve scaffold. Even though our early in vitro results appear promising and we have created a viable tissue construct on a porous, elastic, and biodegradable polymer in vitro, the results are still preliminary and numerous issues remain to be addressed before the clinical application of tissueengineered heart valves will be possible. A tissueengineered heart valve has to be functional tissue at the time of implantation. Therefore, we have to create a tissue-engineered heart valve with appropriate physiologic characteristics before implantation. We currently do not know which source of cell population or preconditioning technique would have the ideal impact on the formation of a tissue-engineered heart valve. Our group is currently investigating optimal preconditioning techniques for tissue engineering of cardiovascular structures focusing on different cell populations, seeding techniques, pulsatile flow bioreactors, and the use of modified cell culture media (viscosity, concentrations, growth factors), which might have a significant influence on the in vitro tissue formation. We thank Yun Lee from Tufts University for electron microscopy and Metabolix Inc (Cambridge, MA) for providing us with polyhydroxyalkanoates.

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