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| Background Large volume muscle tissue engineering still remains a major challenge. An axial vascular pedicle and perfusion bioreactor are necessary for development and maintenance of large volume engineered muscle in order to provide circulation within the construct. We aimed to determine whether a novel model to investigate large volume engineered muscle flap from an existing rat groin fat ?ap was developed. Method A fat ?ap based on the super?cial inferior epigastric vascular pedicle in the rat was harvested and placed into a perfusion bioreactor. The ?aps were kept in the bioreactor for up to 7 weeks and meanwhile, transdifferentation of adipose to muscle tissue could have taken place. This system enables a myogenic differentiation medium ?ow through the bioreactor of a constant pH and a constant oxygen concentration. Assessment of viability was performed by immunofluorescence, histological staining, calcein based life/dead test and determination of RNA quantity and quality after 1, 3, 5 or 7 weeks. Result Immunofluorescence staining showed that smooth muscle around vessels were still intact without signs of necrosis, or atrophy. The visual assessment of viability by a calcein based life/dead test revealed a viability of the rat adipose tissue preserved in the bioreactor system with permanent perfusion. In this study, RNA samples from different experimental conditions were quanti?ed by photometry and intact rRNA bands of 18S and 28S were observed by gel electrophoresis, indicating that degradation of RNA was minimal. Conclusion This is the ?rst demonstration that ?ow perfusion maintains rat groin engineered muscle flap long-term viability in vitro and a large volume vascularized muscle could be engineered in a perfusion bioreactor. |
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| Keywords:tissue engineering; ?ow perfusion; ex vivo cultivation; perfusion bioreactor????? |
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