The literature suggests morphological alterations and molecular biological changes within the cellular milieu of human cells, exposed to microgravity (µ g ), as many cell types assemble to multicellular spheroids (MCS). In this study we investigated juvenile normal human dermal fibroblasts (NHDF) grown in simulated µ g (s-µ g ) on a random positioning machine (RPM), aiming to study changes in cell morphology, cytoskeleton, extracellular matrix (ECM), focal adhesion and growth factors. On the RPM, NHDF formed an adherent monolayer and compact MCS. For the two cell populations we found a differential regulation of fibronectin, laminin, collagen-IV, aggrecan, osteopontin, TIMP-1, integrin-β 1 , caveolin-1, E-cadherin, talin-1, vimentin, α-SM actin, TGF-β 1 , IL-8, MCP-1, MMP-1, and MMP-14 both on the transcriptional and/or translational level. Immunofluorescence staining revealed only slight structural changes in cytoskeletal components. Flow cytometry showed various membrane-bound proteins with considerable variations. In silico analyses of the regulated proteins revealed an interaction network, contributing to MCS growth via signals mediated by integrin-β 1 , E-cadherin, caveolin-1 and talin-1. In conclusion, s-µ g -conditions induced changes in the cytoskeleton, ECM, focal adhesion and growth behavior of NHDF and we identified for the first time factors involved in fibroblast 3D-assembly. This new knowledge might be of importance in tissue engineering, wound healing and cancer metastasis.

SummaryBackground: For tendon tissue engineering, tenocyte-seeded scaffolds are a promising approach. Under conventional 2D culture however, tenocytes show rapid senescene and phenotype loss. We hypothesized that phenotype loss could be counteracted by simulated microgravity conditions. Methods: Human tenocytes were exposed to microgravity for 9 days on a Random Positioning Machine (RPM). Formation of 3D-structures (spheroids) was observed under light microscopy, gene expression was measured by realtime PCR. Cells under conventional 2D-culture served as control group. Results: Simulated microgravity reached a value of as low as 0.003g. Spheroid formation was observed after 4 days, and spheroids showed stable existance to the end of the observation period. After 9 days, spheroids showed a significantly higher gene expression of collagen 1 (Col1A1) compared to adherent cells under microgravity (4.4x, p=0.04) and compared to the control group (5.6x, p=0.02). Gene expression of collagen 3 (COL3A1) was significantly increased in spheroids compared to the control group (2.3x, p=0.03). Gene expressions of the extracellular matrix genes Tenascin C und Fibronectin (TNC and FN) were increased in adherent cells under microgravity compared to the 1g-control group, not reaching statistical significance (p=0.1 and p=0.3). For the gene expression of vimentin, no significant alteration was observed both in the adherent cells and in the spheroids compared to the 1g control group. Gene expression of the tenocyte-specific transcription factor scleraxis (SCX) was significantly increased in spheroids compared to the control group (3.7x, p=0.03). Conclusion: Simulated microgravity could counteract tenocyte senescence in vitro and serve as a promising model for scaffold-free 3D cell culturing and tissue engineering. Level of evidence: V (laboratory study).

VEGF enhances proliferation and expression of tendon-related genes in human tenocytes. It could therefore be a useful addition for tenocyte cultivation.

Background: For tendon tissue engineering, tenocyte-seeded scaffolds are a promising approach. Under conventional 2D culture however, tenocytes show rapid senescene and phenotype loss. We hypothesized that phenotype loss could be counteracted by simulated microgravity conditions. Methods: Human tenocytes were exposed to microgravity for 9 days on a Random Positioning Machine (RPM). Formation of 3D-structures (spheroids) was observed under light microscopy, gene expression was measured by realtime PCR. Cells under conventional 2D-culture served as control group. Results: Simulated microgravity reached a value of as low as 0.003g. Spheroid formation was observed after 4 days, and spheroids showed stable existance to the end of the observation period. After 9 days, spheroids showed a significantly higher gene expression of collagen 1 (Col1A1) compared to adherent cells under microgravity (4.4x, p=0.04) and compared to the control group (5.6x, p=0.02). Gene expression of collagen 3 (COL3A1) was significantly increased in spheroids compared to the control group (2.3x, p=0.03). Gene expressions of the extracellular matrix genes Tenascin C und Fibronectin (TNC and FN) were increased in adherent cells under microgravity compared to the 1g-control group, not reaching statistical significance (p=0.1 and p=0.3). For the gene expression of vimentin, no significant alteration was observed both in the adherent cells and in the spheroids compared to the 1g control group. Gene expression of the tenocyte-specific transcription factor scleraxis (SCX) was significantly increased in spheroids compared to the control group (3.7x, p=0.03). Conclusion: Simulated microgravity could counteract tenocyte senescence in vitro and serve as a promising model for scaffold-free 3D cell culturing and tissue engineering. Level of evidence: V (laboratory study).