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Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues

Tatsuya Shimizu^*,1, Hidekazu Sekine^*,1, Joseph Yang^*, Yuki Isoi^*, Masayuki Yamato^*, Akihiko Kikuchi^*, Eiji Kobayashi and Teruo Okano^*,2

^* Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan; and
Division of Organ Replacement Research, Center for Molecular Medicine, Jichi University Medical School, Tochigi, Japan

²Correspondence: Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. E-mail: tokano{at}abmes.twmu.ac.jp

SPECIFIC AIMS

Although the field of tissue engineering has expanded rapidly, the lack of effective methods to overcome the limits of mass transport has prevented the creation of thick, viable, cell-dense structures. We sought to develop an approach that could overcome these diffusion limits and allow for the construction of thick, vascularized myocardial tissues.

PRINCIPAL FINDINGS

1. Diffusion limits restrict the viable tissue thickness of layered cardiomyocyte sheets
We previously described a novel method of cell sheet engineering for myocardial tissue reconstruction using cardiomyocyte sheets harvested from temperature-responsive culture dishes. Neonatal rat cardiomyocytes cultured on temperature-responsive dishes can be noninvasively harvested as intact sheets without the need for proteolytic enzymes, such as trypsin. When layered, these cell sheets adhere to each other via their deposited ECM on the basal sheet surface and began to pulsate synchronously and spontaneously after stacking. An increasing number (1–5) of cardiomyocyte sheets were layered and transplanted into the dorsal subcutaneous tissues of athymic rats. However, 1 month after implantation, resected grafts showed that whereas constructs consisting of 1, 2, or 3 cell sheets could survive without necrosis, the 4- and 5-layer constructs showed areas of disordered vasculature, with an increased amount of connective tissues due to primary ischemia. The results demonstrated that the limit for layered cardiomyocyte sheets in vivo appears to be 80 µm and prevents the unlimited stacking of cell sheets for tissue reconstruction.

2. Multi-step transplantation at 1- or 2-day intervals can create synchronously beating six-layer myocardial tissues
In cell sheet construct survival, we noticed that microvessel network formation within the transplanted grafts begins just after implantation and within a few days, a well-defined vascular network had formed within the grafts. We hypothesized that by allowing for an appropriate lag period, repeated transplantations could allow for the creation of tissues thicker than the 80 µm limit (Fig. 1 A). However, another key requirement in the re-creation of functional myocardial grafts is the fabrication of tissues that show synchronous pulsation, similar to the native heart.

View larger version (25K): [in this window] [in a new window]   Figure 1. Schematic illustration of polysurgery of cell sheet grafts to bioengineer vascularized cell-dense tissues. A) Three confluent cell sheets are stacked as the initial graft, then transplanted in vivo. After adequate neovascularization, a second triple-layer graft is overlaid on the first. The resulting multilayer cell-dense tissue construct is now perfused through both grafts by the underlying microvasculature. B) The first graft is transplanted over a surgically accessible artery and vein. In this case, the graft is supplied with new vasculature and blood directly from these existing vessels. After sufficient vascularization has occurred, the second graft is transplanted onto the first. Finally, the microvascularized construct accompanied by graftable vessels harvested from the host is fully perfused by host vessels and surgically resected. Ectopic transplantation of such a graft is then possible.

Using 1-, 2-, or 3-day intervals between the transplantation of triple-layer cardiomyocyte sheets, we discovered that in 1- or 2-day intervals, electrical coupling could occur between the two grafts, creating six-layer synchronously beating constructs (supplemental video 1).

Histological findings demonstrated that when six-layer constructs were implanted with a single procedure that significant amounts of necrotic tissue were present within the grafts. In contrast, with the 1-day interval procedure the two grafts became intimately connected, creating a single uniform cell-dense structure. In the 2-day interval, the grafts were separated by significant amounts of connective tissues but were connected at some points within the grafts, allowing for the observed synchronous beatings. In the 3-day intervals, the two transplanted grafts survived after in vivo implantation but were separated by a thick layer of connective tissues, thereby inhibiting electrical coupling. These results showed that a single synchronously beating myocardial tissue graft composed of six cardiomyocyte sheet layers could be created by multi-step transplantation at 1- or 2-day intervals.

3. Multi-step polysurgery can overcome the thickness limitation to create 1 mm thick myocardial tissues
After discovering that triple-layer cardiomyocyte sheet grafts transplanted at 1- or 2-day intervals could re-create cell-dense myocardial tissues that demonstrated synchronous beatings, we attempted to layer additional 3-layer grafts with the multi-step procedures at 1- or 2-day intervals. Using polysurgery, 10 triple-layer grafts were implanted into the subcutaneous tissues and observed. One wk after the procedures, spontaneous and synchronized graft contraction and relaxation cycles could be observed macroscopically, even without opening of the transplant site (supplemental video 2). Results from echocardiography also revealed pulsatile myocardial tissues (supplemental video 2), with a measured graft thickness of 0.9 ± 0.1 mm, 1 wk after final transplantation. When the implant sites were surgically reopened, the grafts demonstrated vigorous, synchronous pulsations, resembling native cardiac muscle (supplemental video 2); this beating continued for several minutes, even after surgical resection (supplemental video 3). Histological results showed cell-dense thick myocardial tissues with a well-organized microvascular network when 10-times polysurgery with the 1-day interval was used (Fig. 2 ). These results demonstrated that cell sheet integration via polysurgery is able to recreate functional myocardial tissue constructs well beyond the current limits of mass transport. The current findings show a valuable and novel approach for overcoming the vascularization problem that has limited the creation of thick, cell-dense structures with tissue engineering.

View larger version (134K): [in this window] [in a new window]   Figure 2. Thick cell-dense tissues engineered using 10-times polysurgery. Azan staining shows that by using 10-times polysurgery with 1-day intervals, multilayer cell-dense myocardium with well-organized microvessels can be created. Bidirectional arrow indicates viable myocardial cell sheet layers. The grafts were 0.84 ± 0.16 mm thick. Data are presented as mean ± SD (n=3).

4. Polysurgery over host vessels creates surgically accessible thick, myocardial grafts
While cell sheet integration using polysurgery demonstrates the ability to re-create tissues that can overcome the limits of mass transport, in applications to many organs, including the heart, the use of multi-step transplantation directly to the damaged tissues, would likely encounter difficulty due to the relatively high risk of complications from each individual procedure. An appealing alternative is the ectopic fabrication of viable thick tissues at sites that require less invasive procedures, with subsequent transplantation to the site of interest. Our results with polysurgery at subcutaneous compartments can re-create functional tissues, with neovascularization likely induced from the surrounding tissues. In such a case, however, it is impossible to surgically cut and reconnect the host microvessels ectopically to developing vessels at the target site, posing a significant obstruction for successful clinical applications. A method to overcome this limitation would be to create ectopic constructs with surgically accessible arterial and venous conduits that could be connected to the vasculature at the target site. We hypothesized that using multi-step transplantation over an existing host large artery and vein would promote blood perfusion throughout the entire thick tissue construct, thereby inducing neovascularization from the host vessels into the grafts and creating an accessible construct with surgically connectable large blood vessels (Fig. 1B ).

In an athymic rat model, triple-layer cardiomyocyte sheets were transplanted at 1-day intervals over an exposed superficial caudal epigastric artery and vein, branches of the femoral artery and vein. When the implantation sites of the leg were opened 2 wk later, the grafts showed strong and synchronous pulsations, similar to native tissues (supplemental video 4). When India ink was infused from the inlet of the femoral artery, it flowed from the femoral artery into the caudal epigastric artery, then diffused throughout the bioengineered tissue grafts before finally being discharged from the caudal epigastric vein and femoral vein (supplemental video 4). When resected tissues were subjected to histological analyses, cross sections of the ink-perfused myocardial grafts demonstrated that the entire microvascular network within the grafts were stained with the ink, indicating that vascularization was indeed induced from the host and that the entire blood supply was from the underlying large artery (Fig. 3 A, B). Immunohistochemical analysis for connexin 43 showed diffuse staining throughout the thick tissue grafts, indicating the likely presence of gap junctions that allow for electrical communication between the cardiomyocytes of the bioengineered grafts (Fig. 3C) . Anti-troponin T antibody showed diffuse and disorganized staining 3 days after transplantation; and at 7 days, well-defined and organized sarcomeres could be observed (Fig. 3D , E). Transmission electron microscopy showed characteristic structures of normal cardiac tissues, including sarcomeres and intercalated disks (Fig. 3F ).

View larger version (54K): [in this window] [in a new window]   Figure 3. Myocardial tissue grafts created over surgically accessible blood vessels. A) Azan staining shows the multilayer myocardial tissue graft (g) over the caudal epigasiric artery (a) and vein (v), which was fixed 2 wk after the polysurgery procedure. Black ink injected from the host femoral artery stains the vasculature within and surrounding the graft. B) Under high magnification, the area of tissue indicated by the square box in panel A shows complete microvascular networks within the construct, which are stained with black ink, indicating blood supply to the construct via the selected artery. C) Immunohistochemistry for connexin 43 shows diffuse staining throughout the entire myocardial tissues, suggesting gap junction formation within the bioengineered constructs. D) Anti-troponin T antibody staining demonstrated diffuse and disorganized staining 3 days after transplantation. E)Cardiomyocytes were striated and sarcomeres were well organized in the myocardial tissue graft at 7 days. F) TEM results at 7 days showed characteristic structures of myocardium including well-organized myofilaments (Mf), mitochondria (Mt), and intercalated disks (ICD).

Finally, we resected multilayer grafts fabricated by polysurgery over a connectable artery and vein, and transplanted the bioengineered tissues into the neck of another nude rat. To connect the graft vasculature to the host vessels, the protruding femoral artery and vein were anastomosed to the carotid artery and jugular vein of the new host. Seconds after reperfusion had occurred, the transplanted grafts began to pulsate (supplemental video 4). Two weeks after ectopic transplantation, the grafts had survived and could maintain their pulsation, demonstrating the possibility of using polysurgery over a connectable artery and vein for future applications.

CONCLUSIONS AND SIGNIFICANCE

Current methods in tissue engineering are limited by the restrictions of passive diffusion, preventing the creation of thick and viable tissues.

The present results demonstrate a unique method for bioengineering functional tissues of up to 1 mm in thickness and show the possibility of using cell sheet integration methods to overcome the longstanding problems associated with the limits of mass transport. By creating myocardial tissues that resemble native structures morphologically and physiologically, these methods can be applied to various cell-dense tissues. Despite limitations, the demonstration of a novel method of cell sheet integration that allows for the re-creation of thick tissues overcomes a significant hurdle in the progression of tissue engineering.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4715fje;

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