Abstract
Myocardial infarction (MI) progresses from the acute death of cardiomyocytes and the infiltration of inflammatory cells into granulation, followed by scars. Recently, the identification of stem cells capable of contributing to tissue regeneration has raised the possibility that cell therapy could be employed for repair of damaged myocardium. It was shown that cell transplantation via local intramuscular injection is a promising therapy for patients with myocardial infarction. However, following injection of the dissociated cells, retention of the transplanted cells in the injected area remains a central issue. These facts can be deleterious to cell-transplantation therapy. The purpose of study I was to evaluate using a thermoreversible hydrogel system, treated as a coating on tissue culture polystyrene (TCPS) dishes and developed for harvesting living cell sheets. The hydrogel system was prepared by simply pouring aqueous methylcellulose (MC) solutions blended with distinct salts on TCPS dishes at 20℃. To improve cell attachments, the MC/PBS hydrogel at 37℃ was evenly spread with a neutral aqueous collagen at 4℃. The spread aqueous collagen gradually reconstituted with time and thus formed a thin layer of collagen (the MC/PBS/Collagen hydrogel). After cells reached confluence, a continuous monolayer cell sheet was formed on the surface of the MC/PBS/Collagen hydrogel. Additionally, the developed hydrogel system can be used for culturing a multi-layer cell sheet. Study II examined the hypothesis that the thermo-responsive hydrogel was used as a coating on TCPS dishes and developed for harvesting living cell sheets. The obtained MSC sheets preserved the intercellular junctions and endogenous extracellular matrix and kept their cell phenotype. After injection through a needle, the fragmented MSC sheets maintained intact and retained their activity upon transferring to another growth surface, while the complete cell sheets were torn into pieces. Transplantation of fragmented MSC sheets in the skeletal muscle of a syngeneic rat model via local injection was evaluated. The transplanted MSC sheets were mainly localized at the site of injection, while the dissociated MSCs were scattered around. Additionally, there were more MSCs retained in the local skeletal muscles for the group injected with fragmented MSC sheets than that injected with dissociated MSCs. These results indicated that the fragmented cell sheets may be used as a novel therapeutic cell-carrier for intramuscular administration. In study III, we hypothesized that the use of cell-sheet fragments, with the preservation of extracellular matrix (ECM), may significantly increase cell retention and thus improve cell therapy. Mesenchymal-stem-cell (MSC) sheet fragments with ECM were fabricated. Using a rat model with experimental myocardial infarction, an intramyocardial injection was conducted with a needle directly into the peri-infarct areas. There were four treatment groups (n ≧ 10): sham; PBS; dissociated MSCs; and MSC sheet fragments. The results obtained in the echocardiography and pressure measurements revealed a superior heart function in the MSC-sheet-fragment group compared with the dissociated-MSC group (P < 0.05). The MSC sheet fragments were able to conform and align their inherent ECM along with the interstices of the muscular tissues at the injection sites, while only a few cells were identified in the dissociated-MSC group at 12 weeks postoperatively. Additionally, transplantation of the MSC sheet fragments stimulated a significant increase in vascular density (P < 0.05) and enhanced the graft/host cell connection. Myocardial infarction often leads to left ventricular dilation, thus impairing cardiac functions. To restore the dilated LV, a possible strategy is to replace the infarcted myocardium with bioengineered tissue grafts. The goal of tissue engineering is to repair or replace the damaged organ or tissues by delivering functional cells on supporting scaffolds to areas in need. Therefore, in study IV, a novel bioengineered tissue graft, a porous acellular bovine pericardium sandwiched with multilayered sheets of MSC, was developed for the treatment of MI. It was previously shown by our group that the acellular bovine pericardium fixed with genipin can provide a natural microenvironment for host cell migration and may be used as a tissue-engineering scaffold. We hypothesized that this newly developed tissue graft can provide the required mechanical strength to support the sandwiched multilayered sheets of MSC for tissue regeneration to restore the dilated LV and improve cardiac functions in a syngeneic rat model. The implanted samples were retrieved at 12-week postoperatively (n ≧ 10 per group at each time point) and were used for gross and histological examinations.