**5. Conclusions**

Our data sugges<sup>t</sup> that there is an unknown tension-driven mitochondrial pathway (TDMP), which may control viability of hAMs via triggering mitochondria-mediated apoptosis. This process starts with loss of tissue tension, followed by activation of TDMP, impairment of mitochondria, the release of mitochondrial apoptotic factors, induction of caspase 3-mediated apoptosis and the loss of viability of hAM cells. The presence of tissue distention prolongs the cellular life span of human amniotic membranes. Further studies are required to obtain a detailed time course of activation of apoptosis and to investigate the TDMP to shed more light on the hAM physiology. This knowledge will support optimization of hAM tissue cultures, as the distended hAM comes closer to the in vivo situation than non-distended samples.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/8/12/1641/s1, Supplementary Methods 1: Measuring mitochondrial oxygen consumption using high resolution respirometry. 2: ATP measurement using Bioluminescence Assay Kit CLS II. Table S1: Primer sequences used for mRNA reverse-transcription quantitative PCR analysis.

**Author Contributions:** A.B. and A.W. conceptualized and designed the experiments; L.P., K.S., A.B., A.W. performed experiments; L.P., K.S., A.B., A.W. analyzed data; L.P., A.L., K.S., A.V.K., J.G., S.W., A.B., A.W. interpreted results; L.P., A.B., A.W. drafted the manuscript and figures; L.P., A.L., K.S., A.V.K., J.G., S.W., A.B., A.W. critically read/edited the manuscript; all authors approved the manuscript.

**Funding:** The study was funded by the Austrian Research Promotion Agency (FFG; gran<sup>t</sup> number 867674).

**Acknowledgments:** The authors are grateful for the support of Cornelia Schneider and Simone Hennerbichler-Lugscheider (Blutzentrale Linz). We are also thankful for the histological support from Barbara Schädl and the team of the joint LBI Trauma/MUW histology lab. Special thanks to Anton Dobsak from the Karl Donath Laboratory for Hard Tissue and Biomaterial Research at the University Clinic of Dentistry.

**Conflicts of Interest:** J.G. is the co-founder of Evercyte GmbH and TAmiRNA GmbH. All other authors declare no conflict of interest.
