Tissue Engineering von vaskularisiertem Skelettmuskelgewebe — ein in vivo Ansatz

Abstract

Introduction: For a successful tissue engineering of skeletal muscle vascularization plays a crucial role besides the specific features of native skeletal muscle tissue, such as innervation and parallel alignment of myofibers.

If dimensions of a tissue engineered construct exceed the limits of diffusion cell survival is dependant of vascularisation. Therefore approaches to axial vascularisation are necessary in order to promote cell survival as well as providing the possibility to construct transplantation. One promising approach to axial vascularisation is the microsurgical model of an arteriovenous loop (AV-loop) that has been applied for engineering vascularised skeletal muscle tissue. Furthermore biocompatibility of the matrix to be used should be considered for later clinical application. Materials and methods: In vitro studies were conducted to first establish a suitable matrix composition of a bioresorbable fibrin matrix. Cell viability and differentiation were estimated by DAPI/desmin staining and RT-PCR analysis. For in vivo studies 12 AV-loops were generated microsurgically in the groin of syngenic Lewis-rats followed by incorporation into a fibrin matrix in side a Teflon isolation chamber. In the contralateral groin identical isolation chambers were implanted without an AV-loop. After a period of 14d allowing for vascularisation of the fibrin matrix, CFDA-labelled mybolasts, which had been expanded through 3 passages previously, were injected into the matrices. Explantation of the matrices was performed after 2, 4 and 8 weeks post-cell injection. Only animals with patent AV-loops at the time of secondary cell injection were included in the study. Results: In dependance of the fibrin matrix composition different proliferaetion rates were observed, while differentiation markers were positive. In vivo a significant resorbtion rate of the fibrin matrices was observed. However transplanted CFDA-positive myoblasts could be detected still after 8 weeks in the AV-loop group. In contrast there were no CFDA-positive cells detectable in the control matrices. The identified grafted cells were mostly positive for the myogenic transcription factor MyoD, suggesting retention of myogenic differentiation. RT-PCR analysis demonstrated mRNA expression of desmin and MEF-2 after 4 weeks as well as MyoD after 8 weeks, while expression of other more differentiated muscle genes was negative. There was no evidence of myoblast fusion to contractile myofibers. Conclusion: Further adjustment to the matrix should reduce the resorbtion rate of the fibrin matrix, while incorporation of neurogenic stimuli, such as a perioheral nerve branch could promote myogenic differentiation. However the model of the AV loop seems to be a promising approach to overcoming the barrier of vascularisation in the field of skeletal muscle tissue engineering.