Eur J Cardiothorac Surg 2003;24:906-911
© 2003 Elsevier Science NL
Bioartificial grafts for transmural myocardial restoration: a new cardiovascular tissue culture concept
Theo Kofidisa,b,c*,
Andre Lenza,c,
Jan Boublika,c,
Payam Akhyaria,c,
Bjoern Wachsmanna,c,
Knut Mueller Stahla,c,
Axel Havericha,b,c,
Rainer G. Leyha
a Department of Thoracic and Cardiovascular Surgery, Hannover Medical School, Hannover, Germany
b ARTISS GmbH, Hannover, Germany
c Leibniz Laboratories for Biotechnology and Artificial Organs, Hannover, Germany
Received 8 January 2003;
received in revised form 10 June 2003;
accepted 21 July 2003.
* Corresponding author. Cardiothoracic Surgery/Falk Research Center, 2 fl, Stanford University Medical School, 300 Pasteur Dr., Stanford CA 94305, USA. Tel.: +1-650-725-3828; fax: +1-650-725-3846
e-mail: tkofidis{at}stanford.edu
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Abstract
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Objective: Survival of bioartificial grafts that are destined to restore cardiac function stands and falls with their nutrient supply. Engineering of myocardial tissue is limited because of lack of vascularization. We introduce a new concept to obtain bioartificial myocardial grafts in which perfusion by a macroscopic core vessel is simulated. Methods: We have designed an experimental reactor with multiple chambers for the production of bioartificial tissue or tissue precursors. By introduction of in- and output lines of distinct diameter and insertion of a core vessel into each chamber, we established pulsatile, continuous flow through the embodied three-dimensional tissue culture. In the present study, collagen components served as the ground matrix wherein neonatal rat cardiomyocytes were inoculated. For the assessment of cellular viability and distribution in comparison to static, non-perfused culture, fluor-desoxy-glucose-positron-emission-tomography and life/dead assays were employed. Results: We obtained 3D constructs of 8-mm thickness, which display high viability and metabolism (6.0±1.3e-03 in the perfused vs. 4.0±0.3e-03 in the unperfused chambers). The core vessel has the size of a human coronary and remained patent during the entire culture process. We observed centripetal migration of the embedded cardiomyocytes to the site of the core vessel. Cardiomyocytes partially resumed a spindle like form without additional stretch. Conclusions: The present dynamic tissue culture concept is highly effective in manufacturing thick, viable grafts for cardiac muscle restoration, which could be surgically anastomosable. The bioreactor may accommodate multiple types of cells and tissues for innumerable in vitro and in vivo applications.
Key Words: Myocardial grafts • Tissue engineering • Cardiomyocytes
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1. Introduction
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Morbidity and mortality in the Western population are largely related to loss of functional tissue due to ischemia or end stage organ failure. This is a problem with great socioeconomic relevance. Surgery or transplantation are increasingly necessary as therapeutic measures. Unfortunately, transplantation is limited by lack of suitable donor organs and typically requires lifelong immunosuppression to suppress rejection episodes. The limitations related to transplantation have fueled major attempts in the areas of tissue retention and tissue and organ replacement [1,2]. One major research endeavor in tissue and organ replacement involves the use and better adaptation of xenogeneic organs. This option is limited by infectiological and ethical entanglements. In parallel, experience is growing in the multidisciplinary field of bioartificial tissue manufacture for organ restoration or replacement [2–4,19].
The applicability of bioartificial tissue developed in vitro is often limited by insufficient thickness to allow for transmural tissue replacement in vivo [5,6]. Earlier reports describe engineered tissue grafts of a maximal thickness of 2 mm, which is not sufficient to restore the ventricular wall transmurally [6,15,16,18]. Lack of perfusion, which would guarantee nutrient support for the inoculated cells, greatly limits the thickness of engineered tissue [12–17]. Moreover, poor perfusion of bioartificial tissue inevitably leads to loss of function and cellular viability. Promoting only angiogenesis is considered an insufficient approach for manufacturing of heart muscle equivalents, for the reason that cardiac muscle is a hypermetabolic tissue with high demand on blood supply [7,8]. Thus microcapillaries might not be capable of transporting sufficient amounts of nutrients to the cells. A novel concept of 3D tissue culture should evolve, which would involve a large size supporting vessel, which ideally would branch off in vessels of smaller size into the bioartificial graft. Moreover, the natural dynamic processes that support the specific tissue's functions should be mimicked. In the case of myocardium, pulsatile perfusion, paracrine effects and perfusion gradients should be applied so that the nature of myocardial perfusion and metabolism is imitated with the highest possible fidelity.
Thus, the objective of the culture concept was to provide a methodology for producing improved three-dimensional bioartificial tissue that contains a pulsatile, anastomosable vessel and proves higher viability of inoculated cells compared to grafts in static culture conditions. The potential of the concept to address a broad spectrum of issues in the field of tissue engineering, angiogenesis research, and the design of future preclinical big animal studies is discussed.
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2. Materials and methods
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2.1. Bioreactor design and principle of function
The bioreactor consisted of even, circular chambers with transparent walls. Multiple chambers could be assembled parallel to each other, without communication (Fig. 1
a). Each chamber had a closeable inlet and an outlet. A vessel of natural origin, in this case a rat aorta, was mounted between the inlet and outlet lines at the beginning of the study. Side branches of the aorta were cut off to allow for egress of flow medium later during the experiment. Next, syngeneic rat cardiomyocytes were inoculated into a liquid collagen compound (Tisseel, Baxter Bioscience, Heidelberg, Germany) within the chamber. The general concept of the process is that at least one vessel is inserted into the bioartificial cardiac tissue at the beginning of its production. The vessel was supplied with perfusate by a pulsatile micropump (Ismatec, Wertheim, Germany). Because the engineered tissue was permeated by a vessel during its culture, a pulsatile distribution of oxygen and nutrient substances was expected. Perfusion under pressure can also be accomplished in other ways with suitable pumps and throttling elements. Finally, the bioreactor had two different covers. One was transparent glass, allowing for visual inspection during the culture procedure. This was the ‘maintenance or culture cover’. The other cover had diaphragms arranged at a distance from each so that they fit exactly above every chamber. This cover is the ‘drug administration cover’. The diaphragms can be penetrated for the purpose of adding supplements or aspirating culture medium.
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Fig. 1. (a) A bioreactor overview: consolidation of the matrix–cell mixture starts within minutes after seeding the components into the chamber and is accomplished in the demonstrated form after ca 36 h. (b) A 8-mm-thick graft penetrated by a macroscopic vessel results.
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The herein seeded collagen and cell mixture was perfused continuously for 2 weeks. After 2 weeks, the contents of the bioreactor were analyzed by FDG-PET. Following these studies, the perfusion was terminated and the contents of each chamber were fixed in 3% formaldehyde and stored for histological studies. In the present study, five bioreactors with and five bioreactors without perfusion were evaluated for cellular viability and distribution.
2.2. Cell seeding and preparation of the core vessel
All animal procedures were performed in accordance with the national guidelines for the care and use of laboratory animals. Neonatal Wistar rat hearts were harvested from 1- to 3-day-old animals after decapitation. The hearts were placed in phosphate buffered saline (PBS) and minced. After several steps of centrifugation and enzymatic digestion as described previously [9], cardiomyocytes were stored on ice until assembly of the bioreactor chambers. The core vessel was a syngeneic rat aorta in the present study. Adult Wistar rats were euthanized and a midline laparotomy was performed. The abdominal aorta was identified and resected by simply cutting the abdominal branches. The aortic lumen was washed thoroughly with PBS and stored in 4 °C cold PBS.
In a sterile hood, two blunt needles were inserted into the input and output foramens of each chamber. The input needle was larger in diameter than the output needle (16 G vs. 18 G). This facilitated pulsatile flow under diffusion pressure. An approximately 2-cm segment of rat aorta was mounted inside the chamber on the blunt ends of the needles using histoacryl glue (Braun, Melsungen, Germany) and was tied in place with a 5.0 Prolene suture (Ethicon, Norderstedt, Germany). A fibrin glue applicator (Baxter Bioscience, Heidelberg, Germany) was used to deliver the cell/matrix mixture. One of the two syringes provided was filled with 2 ml of culture medium containing 107 cells/ml and the other syringe was filled with fibrin glue (Tisseel, Baxter Bioscience, Heidelberg, Germany). The mixture of cells, culture medium, and fibrin glue was administered slowly into the chamber in sufficient amounts to cover the core vessel of the bioreactor. Subsequently, it was left to consolidate. A flask was filled with 100 ml minimal essential medium (MEM, Life Technologies, Karlsruhe, Germany) and fetal calf serum (10%), penicillin/streptomycin (1%), and BrDU (1%) (Sigma, Munich, Germany) were added. This bottle served as the supply reservoir and was connected to the input line of the bioreactor chamber by soft silicon tubing. A collection bottle was connected to the outlet on the opposite side of the chamber. The supply and collection bottles were changed every 48 h for a total period of 2 weeks. After 30 min of consolidation, perfusion was initiated. Perfused and unperfused bioreactors were placed, along with their corresponding perfusion lines and supply flasks, in an incubator at 37 °C with 5% CO2. No supplemental substances, such as growth factors were added to the perfusate or tissue block at any time point of the seeding or perfusion process.
2.3. Fluorodeoxyglucose-positron emission tomography (FDG-PET)
The bioreactor was transferred while under perfusion to the Department of Nuclear Medicine and placed into a Siemens Positron Emission Tomograph (Siemens, Munich, Germany) at 30 °C. First, a transmission scan was obtained to assess the density of the block inside every single chamber. During this step, perfusion was stopped. The next step was to administer FDG (9-11 MBq per chamber) and perfusion was started again. Scanning was performed for the wash-in and wash-out modus and FDG was then removed from the system by perfusing the chambers with fresh MEM medium (Life Technologies, Karlsruhe, Germany). The radioactivity was transformed automatically from kBq/ml into ‘ECAT’-counts from the picture reconstruction scanner, which was an ECAT/HR-Siemens system (Siemens, Munich, Germany). After the measurement, the solid engineered tissue block was removed from the chamber for histologic analysis.
2.4. Histology
The solid cell–matrix construct was sectioned immediately after removal from the chamber into two major units. The first was further divided in two subunits for longitudinal and transverse sectioning. The resulting sections were incubated in a solution of PBS which contained calcein (1:1000, Molecular Probes, Leiden, Netherlands) and ethidium homodimer (1:8000, Molecular Probes, Leiden, Netherlands). The second unit was placed in a solution of PBS and MitoTracker (1:2500, Molecular Probes, Leiden, Netherlands). After 45 min of incubation at 37 °C the solutions were removed and the units were rinsed three times in PBS. The sections were then embedded in Tissue Tek Sakura Finetek, Zoeterwoude, Netherlands), frozen and further sectioned to slices of 10 µm. Immunofluorescence was performed as described previously [20,21]. Cell counts were obtained from six section zones. Three longitudinal sections starting from the center of the block and vessel, along the vector of the core vessel (zones 1–3) and three sections in increasing distance from the core vessel to the side margins of the block (250, 500 and 750 µm away from the core vessel, zones 4–6).
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3. Results
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The consolidation process of the seeded mixture began within minutes after seeding of the mixture components into the chambers (Fig. 1a). Thirty-six hours after the start of perfusion, we obtained solid engineered bioartificial grafts of 8.5±1.2 mm thickness (Fig. 1b). Mechanical stability was significant enough to allow for vigorous manipulation without decomposition of the graft. The solid graft was further perfused in the same chamber for up to 2 weeks and did not loose its integrity. The mean cellular population of each block was 10 million cells/mm3. The mean diameter of the core vessel was 1.8±0.4 mm and the continuous and pulsatile flow of culture medium was set at 100 ml/h at a rate of 120 pulsations/min.
The inoculated cells found anchorage on the collagen fibers of the block (Fig. 2
a). Mean population of living cells as evaluated by life/dead assay in the various zones of the block are demonstrated in Table 1. A significantly higher population of living cells were found in the perfused chambers (P: 0.003, Table 1). A centripetal migration of the cardiomyocytes was evident. The cells aligned along the core vessel at a higher density compared to the cell density observed at more distant regions from the core vessel (Table 1). Some of the inoculated cardiomyocytes assumed a spindle-like shape without any additional mechanical stimulation. The population of living cells was significantly higher in zone 4, i.e. in the vicinity of the core vessel in perpendicular sections (P: 0.02). There was a significant decrease in cell density with increasing distance from the center to the rear of the tissue block. In the transversally defined zones 1, 2 and 3, the population of living cells was similar (Table 1). Similarly, the FDG-PET procedure revealed higher metabolic rates in the perfused chambers (Fig. 2b). At 7 days after start of perfusion and 4000 s of activity monitoring, radioactivity counts averaged 6.0±1.3e-03 in the perfused chambers as opposed to the non-perfused chambers, which displayed an ECAT count averaged at 4.0±0.3e-03 (Fig. 2c, P=0.015). In control chambers seeded only with fibrin glue, the signals averaged at 3.7±0.2e-03. The activity curves revealed a peak uptake phase, which resolved during the subsequent washout phase.
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Table 1. Significantly higher populations of living cells (life/dead assay) were found in the perfused chambers, with most significant differences in zone 3 (P=0.03)
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4. Discussion
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The introduced novel concept of perfusion proves effectively enhances viability and metabolism of cells, which are embedded in a solid matrix. It also facilitates a significant increase of the maximal possible graft thickness produced bioartificially. The obtained tissue displays high mechanical stability. Unlike conventional means of tissue culture, this dynamic culture process is not limited by a need for human supervision and control of media flow. Large amounts of tissue can be treated in a parallel simultaneous fashion. Furthermore, by assembling multiple chambers, one can perform comparative studies concurrently.
In a model of acute or chronic ischemia, the diseased or necrotic part of the cardiac muscle could be resected and the graft could be implanted and left to engraft. The anastomoses could be carried out by either bioartificial or venous grafts to the closest coronaries to allow for blood supply. The contained core vessel of the graft could be of biological or synthetic origin, the latter carrying pores to allow for diffusion processes. This way, necrotic regions of the myocardium, where a bypass graft would not appear beneficial for the patient, could now be replaced. Ongoing studies in our laboratory address the issue of engraftment, mechanical stability, resorption kinetics and contractility of the construct in a porcine infarction model. Our previous studies have shown that mechanical pre-conditioning of bioartificial constructs is the most promising way to enhance metabolic and contractile performance over periods of time as long as 12 weeks in vitro. The final goal will be to obtain contractile constructs of the same thickness prior to implantation, using a device that has been co-developed in our laboratory.
Another issue that still has to be addressed is the endothelialization of the inner surface of the graft. There have been excellent reports on the establishment of continuous endothelial layers on bioartificial surfaces, which withstand high hemodynamic strain [4,18]. The endothelialization of the obtained constructs in our bioreactor would be inevitable to prevent thrombus formation, for example, in the ventricular cavity of the heart. Moreover, the heart constitutes a syncitium of cell types. Therefore, additional work is being carried out to establish the optimal conditions for co-culture of cardiomyocytes and endothelial cells.
By supplying the core vessel with a suitable nutrient solution or blood, we would expect additional vessels to grow during in the course of cultivation, assuming that all necessary components, such as endothelial cells and growth factors are present. The formation of microtubular, capillary-like structures in gels has been shown in still cultures previously [10]. For the engineering of heart tissue, an advantage of our system is that for the first time tissue segments exceeding the thickness of a few millimeters can be developed, since the survivability of the perfused, three-dimensional tissue is distinctly superior to that of non-perfused tissue. The inherently porous vessel can be prepared at points with a material or means providing a stimulus for angiogenesis, such as the protein vascular endothelial growth factor (VEGF) [11].
Electrical stimulation of cells can be accomplished by submerging platin electrodes into the individual culture chamber; the flat construction and parallel connection of the chambers makes it possible for multiple experimental reactors to be stacked, so that a complex culture and perfusion system can be assembled of multiple modules. This would allow for large-scale production of autologous tissue for the recipient. The size and construction principle are such that the construction cost remains small but modifiable, and in that way the dynamic culture can be put into operation quickly. The limitation of the present study is that implantation of the obtained tissue in big animals is carried out currently and data are awaited. Therefore, it constitutes a preliminary introduction of a novel culture concept for the ultimate goal to restore myocardial tissue in a large scale using full-thickness grafts, and might accelerate our common efforts to achieve the first vascularized, transmural replacement of the free right or left ventricular wall.
In conclusion, the dynamic culture process introduced here, distinguishes itself primarily by combining established tissue culture features with new ones to make the manufacture of implantable bioartificial myocardial tissue of sufficient thickness and preconditionable properties easier.
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Abstract
1. Introduction
