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

Abstract

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 °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.

Morphological and Molecular Changes in Juvenile Normal Human Fibroblasts Exposed to Simulated Microgravity

“…2J). Fibronectin is the main mediator of cell-ECM interaction 35 and an important factor involved in early wound repair 36,37 . In addition, the corresponding protein was elevated in RPM-AD and RPM-MCS (Fig.…”

“…Furthermore, an enhanced fibronectin mRNA is detectable in abnormal wound healing when keloids are formed 45 . Astronauts suffer of an abnormal wound healing and thus, it is important to increase the current knowledge about the changes in fibroblasts cultured in µ g in regard to their ECM synthesis 35 . ECM proteins, adhesion molecules, and cytoskeletal proteins form a dynamic network interacting with signalling molecules as an adaptive response to µ g .…”

Role of Apoptosis in Wound Healing and Apoptosis Alterations in Microgravity

“…Programmed cell death, also known as apoptosis, is a biological process occurring in multicellular organisms (Riwaldt et al, 2017). Apoptosis is necessary to maintain the body balance and to remove cells during the organ development phase.…”

Pathway Analysis Hints Towards Beneficial Effects of Long-Term Vibration on Human Chondrocytes

“…The first strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, US) was used for reverse transcription. qPCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Darmstadt, Germany) according to routine protocols [16,17]. cDNA-selective-primers were synthesized by TIB Molbiol (Berlin, Germany) and are listed in Table 1.…”