Eur J Cardiothorac Surg 2002;22:238-243
© 2002 Elsevier Science NL
A novel bioartificial myocardial tissue and its prospective use in cardiac surgery
Theo Kofidis1*,
Payam Akhyari1,
Björn Wachsmann,
Jan Boublik,
Knut Mueller-Stahl,
Rainer Leyh,
Stefan Fischer,
A. Haverich
Division of Thoracic and Cardiovascular Surgery and the Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Carl Neuberg Strasse 1, 30625 Hannover, Germany
Received 26 November 2001;
received in revised form 13 February 2002;
accepted 22 March 2002.
* Corresponding author. Tel.: +49-511-532-6581; fax: +49-511-532-5404
e-mail: kofidis{at}thg.mh-hannover.de
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Abstract
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Background: Congenital heart defects such as atrial septal defect, ventricular septal defect, double outlet ventricles and the hypoplastic left heart syndrome as well as ischemic heart disease are associated with aplastic, defective or necrotic myocardial structures. In many of these instances patch closure, reconstruction of the defect or revascularization is required. We have developed a contractile bioartificial myocardial tissue, which offers new perspectives for various reconstructive surgical interventions, including congenital heart surgery. Methods: Neonatal rat cardiomyocytes were seeded in vitro in a commercially available collagen scaffold. Histological examination and ultrastructural evaluation were performed. Protein and mRNA analysis were carried out by two-dimensional electrophoresis and reverse transcription–polymerase chain reaction (RT–PCR). Force measurements of contractions from the spontaneously beating or the pharmacologically stimulated bioartificial myocardial patch were obtained. Results: A solid matrix of 20x15x2 mm with spontaneous contractions resulted 36 h after cardiomyocyte seeding. Histology showed a tight mesh of collagen fibrils. Two-dimensional electrophoresis and RT–PCR revealed cardiotypical proteins (actin, tropomyosin, creatine kinase, ventricular light chain) and mRNA (myosin heavy chain, Connexin 43). The elasticity curve during passive stretch was similar to that of myocardium. Contractile force increased after topical administration of Ca2+ and adrenaline. However, stretch led to the highest levels of contractile force. Conclusions: Our novel contractile bioartificial tissue can be engineered in vitro and may open novel avenues for myocardial tissue replacement in congenital and reconstructive heart surgery. From the current standpoint autologous or allogeneic cells would be preferred over xenogeneic sources.
Key Words: Tissue engineering • Congenital heart surgery • Artificial myocardial tissue
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1. Introduction
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In the last 20 years cardiac surgery has achieved significant improvements in diagnosing diseases and defects, surgical approaches to the treatment of ischemic or valvular disease, reconstruction of cardiac anomalies and long term outcome. Many of cardiac defects require patch closure or reconstruction of a chamber wall (Table 1). Usually surgeons utilize either Dacron or other synthetic materials, which are not vital and do not have the potential to grow with the growing host organism. In addition, synthetic patch materials often cause a significant inflammatory response [1,2]. Elastic properties of such substitutes are generally restricted and do not imitate the function of the host tissue. Also, active contraction of the reconstructive tissue would be desirable in a number of instances, especially in the intra- and extracardiac Fontan circulation. With the advent of such substitutes by concepts of tissue engineering, reconstructive surgery for congenital defects may lead to improved results, when biological, contractile and conductive material is used.
The main obstacle in cardiovascular tissue engineering is the lack of proliferative activity of adult cardiomyocytes. Some investigators focus on the initiation of cardiomyocyte proliferation using pluripotent sources, such as mesenchymal stem cells or embryonic stem cells. Others have achieved important progress in constructing spontaneously beating myocardial tissue equivalents [3–5]. Compared with monolayer cardiomyocyte cultures, it has been suggested that three-dimensional (3D) multilayered cultures of cardiac myocytes resemble cardiac tissue with respect to cellular differentiation and electrical properties [4,6,7,9,10]. Li et al. have shown that cardiomyocytes can attach to scaffolds to form contractile cell-polymer constructs [5,11]. This group and other investigators have constructed a viable cardiac graft that contracted spontaneously in tissue culture. In most cases, cells were derived from fetal rat ventricular myocardium and seeded into a biodegradable material [4,5,9,11]. Bursac et al. have examined the effects of specific variations in a cell-polymer bioreactor model system performing electrophysiological studies to compare engineered constructs with native cardiac tissue, in order to assess their suitability as potential equivalents for tissue repair [4]. Eschenhagen et al. developed 3D-cultured cardiomyocyte constructs to study various features of cardiac disease such as hypertrophy [8]. They also carried out pharmacological studies on engineered cardiac tissue [3].
These matrices have several drawbacks in common such as weak cellular integrity, inhomogeneous seeding, restricted viability, and short duration of contractility. Here, we report on the development of a novel tissue engineered myocardial construct with improved function and discuss its potential clinical application as an alternative matrix for cell and tissue transfer in cardiac surgery.
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2. Materials and methods
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2.1. Isolation of cardiomyocytes
Neonatal Wistar rats (day 1–3) were killed by decapitation according to NIH and USDA Guidelines for the Care and Use of Laboratory Animals. Cardiomyocytes were isolated as described by Eschenhagen et al. [3]. Cardiomyocyte yield and cellular vitality was assessed microscopically.
2.2. Preparation of bioartificial myocardial tissue patch (BMTP)
Our 3D culture system is based on a clinically approved collagen-based matrix, which is a ‘tissue fleece’ initially designed as a hemostypticum for local surgical use (Tissue Fleece®, bovine collagen type I, Baxter, Bioscience GmbH, Heidelberg, Germany). Segments of this preformed collagen scaffold (20x15x2 mm) were placed into quadratic wells of the same size surrounded by silicon rubber (Dow Corning, Wiesbaden, Germany) in cell culture dishes. A 1-ml aliquot of the cell suspension containing 2x106 cells was added to one segment of tissue fleece. The mixture was allowed to gel at 37 °C for 4 h before 4 ml of culture medium per well were added. BMTPs were cultured in modified Eagle's medium (MEM; Life Technologies, Karlsruhe, Germany) plus 10% fetal calf serum (FCS; PAA, Linz, Austria) and 110 µM 5-bromo-2'-deoxyuridine (Sigma Chemicals, Deisenhofen, Germany). Microscopic assessment of cellular distribution and viability, monitoring of contractility, and exchange of culture medium were performed daily.
2.3. Histology
BMTPs were removed from the silicone wells and fixed in 3% formaldehyde (Sigma Chemicals) in phosphate-buffered saline (PBS), pH 7.4. After dehydration according to standard protocols, BMTPs were embedded in paraffin blocks. Furthermore, perpendicular and cross sections (10 µm) of the BMTPs were cut and stained with hematoxylin–eosin (H&E).
2.4. Ultrastructural studies
Transmission electron microscopy was applied for ultrastructural examination using the EM 301, Philips, Micrion-Philips FEI Deutschland, Kassel, Germany. Samples were fixed in Karnovsky's fixative (0.1 M sodium cacodylate with 2% paraformaldehyde and 2.5% glutaraldehyde, pH 7.4), postfixed in 2% osmium tetroxide, dehydrated in ethanol in propylene oxide, and embedded in Poly/Bed812 (Polysciences). Finally, sections were cut into 60-nm slices, stained with lead citrate and uranyl acetate, and examined ultramicroscopically.
2.5. Force measurements
BMTPs were cut into 10x1.5x2-mm slices and were mounted in an experimental device as described by Kraft et al. [12,16]. The BMTP stripes were glued with Histoacryl (Braun, Melsungen, Germany) on metal holding arms on both sides of the chamber, which was filled with culture medium. The temperature of the chamber (and, thus, of the culture medium) was controlled by two water-cooled Peltier elements. Moreover, O2 and CO2 levels in the medium were kept constant at 37 °C by external aeration. One of the stripe holders was connected to a force transducer (SensoNor, Horten, Norway). The opposite holder was flexible to enable stretch of the BMTP stripes. BMTP-based force generation was recorded digitally and stored for off-line evaluation. After baseline measurement, different stimuli such as Ca2+ and epinephrine were added to the culture medium and the generated force was measured. Stretching at various lengths was performed for 10 s each. In order to study the elasticity of BMTPs we have matched ten BMTP stripes to ten stripes of native rat myocardium and performed the stretching maneuvers until burst of the tissue.
Two separate electrodes were placed into the patch and connected to a standard ECG device. A constant electrode distance of 5 mm was used in all experiments. Bipolar signals from spontaneous and stimulated contractions were recorded.
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3. Results
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3.1. BMTPs’ shape, texture and stability
Gelation of the collagen-cell mixture in culture lasted until day 2. The resulting size of BMTPs was about 20x15x2 mm. This shape remained stable throughout the total culture period. We did not observe any cellular detachment or disarrangement. After gelation, the BMTP-texture was flexible but markedly solid (Fig. 1a) . BMTPs were removable from the petri dish without laceration or damage. BMTPs displayed significant elasticity: stretching the BMTP up to 150% of the original length was achievable.
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Fig. 1. (a) A contracting bioartificial myocardial tissue unit in the petri dish, here with electrodes for the detection of electrical activity (b).
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3.2. Cell distribution within the BMTPs
Trypan blue staining revealed 80–90% cellular viability at the end of the cell preparation, prior to 3D seeding. Microscopical analysis (H&E) and confocal microscopy of BMTP-sections revealed a tight homogenous arrangement of cells as well as intercellular contacts in the 3D structure of the scaffold at an early stage of culture (Fig. 2a)
. Both single cells and cell groups of different sizes were seen within the BPs. Electron microscopical studies demonstrated a tight junction-like intercellular contact cells and collagen fibrils, which explains the high degree of integrity and stability of BMTP (Fig. 2b). The collagen ultrastructure itself is composed by fibrillar units embedded in a loose mass. Fibrils seem to penetrate deep into the collagenous ground mass and, thereby, achieve anchorage and stability (Fig. 2c). A series of specimens were stained with MF 20 which revealed a tight population of cardiac cells, arranged along collagen fibers of all layers (Fig. 2d).
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Fig. 2. Cellular seeding of an BMTP. (a) Arrangement of cells in 3D collagen structure. (b) Junction between cellular body and collagen fibril, demonstrated by electron microscopy (x8000). (c) Collagen fibrils are embedded in the amorphous collagen matrix (x63 000). (d) cells attach along collagen fibrils and are distributed homogenously even in deeper layers of BMTP (MF-20 stain).
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3.3. Contractility of BMTPs
Contractions became first apparent 36 h after casting and reached maximal frequency and strength on day 4 in 87% BMTPs (n=87 out of 100 BMTPs). Contractions were ubiquitous and synchronous along the entire length of BMTP. As observed by microscopy and EM, the visible vectorial contractions were transmitted wavelike. Forty percent of the BMTPs displayed continuous contractility for 12 weeks in culture. The frequency of contraction was measured daily and ranged between 40 and 220 beats/min, with an average frequency of 125±35 beats/min.
3.4. Force measurements
BMTP stripes contracted spontaneously and continuously. Force recordings were performed on ten different BMTPs before and after stretching (Fig. 3a)
. Force generation in unstimulated BMTPs reached 8.6±3.6 µN. Stretching of the BMTPs resulted in a significant increase of force (P=0.0001). Maximal force was achieved at 2,5 mm elongation of the BMTP, leading to 119% increase in force (18.8±3.7 µN) compared with unstretched BMTPs (Fig. 3b). Stretch, Ca2+ or epinephrine administration did not affect the contraction rate. Beside stretching, Ca2+ and epinephrine were administered to stimulate BMTPs. Increase of Ca2+ concentration in the culture medium from 1.8 to 2.8 mM resulted in a 103% increase in maximal force to 14.10±3.9 µN (Fig. 3a). Epinephrine stimulated force generation in a separate group of BMTP stripes, and resulted in a maximal force increase from 6.1±0.9 to 10.1±3.4 µN (65.6%). Elasticity testing revealed comparable results for both BMTP and rat myocardium. In eight out of ten tests, myocardium tore first (Fig. 4)
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Fig. 3. (a) Impact of stretch and pharmacological agents on force development (left column: baseline; right column: after stimulus administration). Depicted is force development in unstimulated BMTPs, after 10 s of stretch at 2.5 mm, after addition of further 1 mM Ca2+ in culture medium or after administration of 0.1 mg epinephrine (n=10 each). (b) Stretching of BMTPs results in increase of contractile force, depending on the length of stretch. We measured force immediately after stretching BMTP stripes for 10 s (n=10).
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3.5. Electrographic recordings
Electrographic assessment showed amplitudes of 0.5–4.6 mV, with an average of 2.1±1.3. Morphologically, ECG curves resembled bundle branch blocks and ectopic ventricular activity (Fig. 1b).
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4. Discussion
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Here we report on a novel and simplified method to engineer a highly effective cardiomyocyte scaffold. So far, it is unknown whether cell transplantation strategies will be able to restore myocardial defects and its clinical application is questioned. However, in vitro engineered myocardium-like tissue requires further improvements to enable its clinical use. Potential drawbacks of previously engineered 3D cultures are insufficient size, inadequate geometry, low viability, weak physical properties such as elasticity, integrity and plasticity, low biocompatibility and high production costs.
We have engineered a myocardium-like 3D tissue which can be manufactured in various shapes. By transplanting cells in an one-component scaffold, we have simplified and shortened the seeding procedure enormously. ‘tissue fleece’, in contrast to polylactide/polyglucotide and various other components, frequently employed into the field of bioengineering research, has widely been used clinically for many years. In our system, myocardial cells migrated into a ‘tissue fleece’ to improve mechanical stability compared to various other materials. The mechanical stability of the bioartificial patch maintained for the entire culture period. The mechanical stability was comparable to native cardiac muscle.
Intercellular contacts in BMTPs are strong enough to allow intact conduction of electrical signals. The resulting histological pattern demonstrates a compact multilayer structure. Furthermore, a well-balanced distribution of contracting cardiomyocytes has been demonstrated not only in peripheral areas of the bioartificial patch but also in the center. Low cell density in the central portion of other engineered myocardial systems has been explained by oxygen and nutrient deficiency in the thicker central areas of the unvascularized 3D cultures [5,7,8]. Although central areas of the bioartificial patch are 2 mm of thickness, we did not observe lower cell densities in these areas compared to the edges. Whether this is due to a better nutrient patency of tissue fleece compared to other scaffold mixtures remains to be studied.
One major characteristic of myocardium is the ability to express continuous rhythmic contractions. Our bioartificial patches displayed synchronous and macroscopically visible contractions for up to 12 weeks in vitro. This, to our knowledge, is the longest duration of in vitro contractile activity of engineered bioartificial heart tissue reported in the literature [3,11]. Force recordings of native and stretched bioartificial patches revealed stretch-dependent regulation of contractile force in accordance to the law of Frank–Starling. Furthermore, regulation of contractile force by extrinsic factors was measurable. Increase of [Ca2+] and additional administration of epinephrine to the culture medium in a physiological range lead to enhanced force development. The successful electrographic recording of a common vectorial signal propagation is in concordance with reports of Eschenhagen and Bursac [4,5] and provides further evidence for the cardiac muscle-like function of our 3D myocard tissue culture [4,11]. In contrast to reports by other investigators, beating activity in the bioartificial patch was not transient but continuous, even without stimulation or pacing. The obtained amplitudes were higher than these previously reported by others and may indicate a more physiological structure and, consequently, function of our engineered heart tissue [4].
Bursac et al. have used various scaffolds and culture media to engineer heart tissue [4]. However, the material they have utilized (polyglycolic acid) degrades very slowly, is not approved for the clinical use in cardiovascular surgery and is too rigid to allow contraction of cardiomyocytes. Transplantation of fetal cardiomyocytes is reported to result in formation of a beating cardiac graft in the surrounding scar tissue [11,14]. Even though enlargement of the graft was documented, cellular viability greatly depended on the age of the study animal and the intensity of angiogenesis in the area of injection. Cells did not survive for longer than 3 weeks. Another limitation of cell transfer in to infracted myocardium is that improvement of cardiac contractility cannot be directly attributed to the injected cells. Using large scale myocardial replacement by BMTP, augmentation of myocardial contractility could be visualized. BMTP is stable enough to be entirely implanted in various locations of atrial or ventricular myocardium, as demonstrated by our stretching tests. Moreover, BMTPs remained viable for more than 12 weeks.
The engineered heart tissue reported by Eschenhagen et al. [3,8] consists of Matrigel, which is an expensive material and does not possess the mechanical resistance of BMTP. By our seeding method, we were able to reduce production costs down to 10%, compared to preparations with Matrigel, thereby achieving constructions of larger size and higher numbers. By the use of clinically approved collagen tissues, we simplify the seeding process and offer the possibility of implanting BMTP as well as other 3D tissues in humans. And other important observation was that when liquid substances which were injected into our scaffold did not cause disintegration of the construct, their use was possible in various combinations and concentrations. Such experiments are subjected to ongoing studies at our institution.
Until now, no bioartificial myocardial equivalents have been found suitable for clinical applications. A major limitation is that adult cardiomyocyte populations can not be expanded in vitro. Recent progress in the differentiation of mesenchymal stem cells (MSCs) towards beating cardiomyocytes offers a promising approach to overcome this limitation. Several groups demonstrated the evolvement of cardiomyocyte-like cells from rodent MSC-containing bone marrow stromal cells [13,14]. Like rodent mesenchymal stem cells, autologous human MSCs can easily be retrieved from bone marrow. The purification and in vitro expansion can be performed on a regular basis [15]. Whereas methods to differentiate MSCs to osteoblasts and chondrocytes are well established [15], we and others have currently developed suitable differentiation protocols to convert human mesenchymal stem cells into spontaneously beating cardiomyocytes [14,17].
In conclusion, we have developed an improved type of engineered myocard-like tissue which resembles native cardiac muscle in many aspects. Bioartificial myocardial tissue may offer a powerful tool for the replacement of myocardial structure and function. Future progress in stem cell technology or identification of factors responsible for the proliferation inhibition in adult cardiomyocytes combined with suitable techniques of gene transfer may allow the production of autologous bioartificial myocard-like tissue, capable to restore congenital or other cardiac defects or impaired heart function. Additional studies are expected to evaluate the thrombogenicity of our biomatrix before clinical implantation, which at the present time could potentially lead to thrombogenic scaffold–blood interactions. Finally, vascularization of in vitro engineered tissues may result in the generation of bioartificial, contractile chambers and, ultimately, to the bioartificial heart.
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Footnotes
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1 Both authors contributed equally to this study. 
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Abstract
1. Introduction
