Preparation of A Spaceflight: Apoptosis Search in Sutured Wound Healing Models

To prepare the ESA (European Space Agency) spaceflight project "Wound healing and Sutures in Unloading Conditions", we studied mechanisms of apoptosis in wound healing models based on ex vivo skin tissue cultures, kept for 10 days alive in serum-free DMEM/F12 medium supplemented with bovine serum albumin, hydrocortisone, insulin, ascorbic acid and antibiotics at 32 degrees C. The overall goal is to test: (i) the viability of tissue specimens; (ii) the gene expression of activators and inhibitors of apoptosis and extracellular matrix components in wound and suture models; and (iii) to design analytical protocols for future tissue specimens after post-spaceflight download. Hematoxylin-Eosin and Elastica-van-Gieson staining showed a normal skin histology with no signs of necrosis in controls and showed a normal wound suture. TdT-mediated dUTP-biotin nick end labeling for detecting DNA fragmentation revealed no significant apoptosis. No activation of caspase-3 protein was detectable. FASL, FADD, CASP3, CASP8, CASP10, BAX, BCL2, CYC1, APAF1, LAMA3 and SPP1 mRNAs were not altered in epidermis and dermis samples with and without a wound compared to 0 day samples (specimens investigated directly post-surgery). BIRC5, CASP9, and FN1 mRNAs were downregulated in epidermis/dermis samples with and/or without a wound compared to 0 day samples. BIRC2, BIRC3 were upregulated in 10 day wound samples compared to 0 day samples in epidermis/dermis. RELA/FAS mRNAs were elevated in 10 day wound and no wound samples compared to 0 day samples in dermis. In conclusion, we demonstrate that it is possible to maintain live skin tissue cultures for 10 days. The viability analysis showed no significant signs of cell death in wound and suture models. The gene expression analysis demonstrated the interplay of activators and inhibitors of apoptosis and extracellular matrix components, thereby describing important features in ex vivo sutured wound healing models. Collectively, the performed methods defining analytical protocols proved to be applicable for post-flight analyzes of tissue specimens after sample return

Co-regulation of proteins identified in human thyroid cells cultivated under simulated microgravity

Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains

Suture in Space: Preparation of an Experiment on the Healing of Sutured Wounds on Board the ISS

Wound healing (WH) is a process strictly regulated and highly conserved throughout evolution because it is indispensable for surviving injuries. On Earth WH has been studied in depth, nevertheless the role of mechanical factors in regulating the process and the mechanisms that, in adult mammals, lead to scarring instead of tissue regeneration are not well understood. In weightlessness WH has been poorly studied, and the effect of loading/unloading on the healing mechanisms is quite completely unknown. Preliminary studies showed microgravity-induced alterations in mechanisms underlying tissue repair. The implementation of procedures and tools to manage emergency surgery, trauma, serious burns, wounds and sutures is mandatory for future human deep space exploration missions at distances which are incompatible with medical evacuation to Earth. Therefore, studies on WH in weightlessness are needed and they are also an unique opportunity for understanding healing mechanisms still not completely known. The Suture in Space experiment, which will be performed on board the International Space Station (ISS), was selected by ESA (ESA-AO-ILSRA-2014) and supported by ASI in its development phase. It aims to study in weightlessness the behavior and healing of ex vivo sutured wound models prepared from skin and blood vessels biopsies derived from plastic and vascular surgery in healthy subjects. The experiment preparation required intense research activity on ground in order to: i) standardize procedures for collection of biopsies, model preparation, tissue culturing and monitoring, postflight analysis of samples; ii) define the requirements for hardware development. To ensure tissue viability throughout the in-flight experiment (4 weeks), we studied and developed a new tissue culture technique based on enriched culture media and a device able to model the physiological mechanical tension in the tissues and monitor its changes during WH, thus enabling the study of suture mechanical properties. The culture technique and WH models developed for the Suture in Space experiment can be applied to study: i) mechanical properties of tissues, tissue constructs, wounds and sutures in different loading conditions; ii) the role of gravity in tissue repair; iii) the relationship between biochemical and mechanical factors in repair mechanisms; iv) the influence of mechanical factors on scar quality; v) the effectiveness of treatments promoting WH, when applied in different loading conditions. The results of the experiment are expected to help in defining: i) strategies to manage wounds and promote healing in Space and on Earth; ii) suture techniques and materials to be used in space environment

Suture in Space: Preparation of an Experiment on the Healing of Sutured Wounds on Board the ISS

Wound healing (WH) is a process strictly regulated and highly conserved throughout evolution because it is indispensable for surviving injuries. On Earth WH has been studied in depth, nevertheless the role of mechanical factors in regulating the process and the mechanisms that, in adult mammals, lead to scarring instead of tissue regeneration are not well understood. In weightlessness WH has been poorly studied, and the effect of loading/unloading on the healing mechanisms is quite completely unknown. Preliminary studies showed microgravity-induced alterations in mechanisms underlying tissue repair. The implementation of procedures and tools to manage emergency surgery, trauma, serious burns, wounds and sutures is mandatory for future human deep space exploration missions at distances which are incompatible with medical evacuation to Earth. Therefore, studies on WH in weightlessness are needed and they are also an unique opportunity for understanding healing mechanisms still not completely known. The Suture in Space experiment, which will be performed on board the International Space Station (ISS), was selected by ESA (ESA-AO-ILSRA-2014) and supported by ASI in its development phase. It aims to study in weightlessness the behavior and healing of ex vivo sutured wound models prepared from skin and blood vessels biopsies derived from plastic and vascular surgery in healthy subjects. The experiment preparation required intense research activity on ground in order to: i) standardize procedures for collection of biopsies, model preparation, tissue culturing and monitoring, postflight analysis of samples; ii) define the requirements for hardware development. To ensure tissue viability throughout the in-flight experiment (4 weeks), we studied and developed a new tissue culture technique based on enriched culture media and a device able to model the physiological mechanical tension in the tissues and monitor its changes during WH, thus enabling the study of suture mechanical properties. The culture technique and WH models developed for the Suture in Space experiment can be applied to study: i) mechanical properties of tissues, tissue constructs, wounds and sutures in different loading conditions; ii) the role of gravity in tissue repair; iii) the relationship between biochemical and mechanical factors in repair mechanisms; iv) the influence of mechanical factors on scar quality; v) the effectiveness of treatments promoting WH, when applied in different loading conditions. The results of the experiment are expected to help in defining: i) strategies to manage wounds and promote healing in Space and on Earth; ii) suture techniques and materials to be used in space environment