Changes in Human Foetal Osteoblasts Exposed to the Random Positioning Machine and Bone Construct Tissue Engineering

Abstract

Human cells, when exposed to both real and simulated microgravity (s-µg), form 3D tissue constructs mirroring in vivo architectures (e.g., cartilage, intima constructs, cancer spheroids and others). In this study, we exposed human foetal osteoblast (hFOB 1.19) cells to a Random Positioning Machine (RPM) for 7 days and 14 days, with the purpose of investigating the effects of s-µg on biological processes and to engineer 3D bone constructs. RPM exposure of the hFOB 1.19 cells induces alterations in the cytoskeleton, cell adhesion, extra cellular matrix (ECM) and the 3D multicellular spheroid (MCS) formation. In addition, after 7 days, it influences the morphological appearance of these cells, as it forces adherent cells to detach from the surface and assemble into 3D structures. The RPM-exposed hFOB 1.19 cells exhibited a differential gene expression of the following genes: transforming growth factor beta 1 (TGFB1, bone morphogenic protein 2 (BMP2), SRY-Box 9 (SOX9), actin beta (ACTB), beta tubulin (TUBB), vimentin (VIM), laminin subunit alpha 1 (LAMA1), collagen type 1 alpha 1 (COL1A1), phosphoprotein 1 (SPP1) and fibronectin 1 (FN1). RPM exposure also induced a significantly altered release of the cytokines and bone biomarkers sclerostin (SOST), osteocalcin (OC), osteoprotegerin (OPG), osteopontin (OPN), interleukin 1 beta (IL-1β) and tumour necrosis factor 1 alpha (TNF-1α). After the two-week RPM exposure, the spheroids presented a bone-specific morphology. In conclusion, culturing cells in s-µg under gravitational unloading represents a novel technology for tissue-engineering of bone constructs and it can be used for investigating the mechanisms behind spaceflight-related bone loss as well as bone diseases such as osteonecrosis or bone injuries.

Simulated Microgravity Influences VEGF, MAPK, and PAM Signaling in Prostate Cancer Cells

“…A possible explanation for this finding could be that the cells try to increase the ECM to resist the s-µg conditions, because the ECM provides structural support for the cells [74], which they lack after detaching because of the RPM-exposure. Our results concerning the ECM fit the findings of a study of human fetal osteoblasts exposed to s-µg, where the cells showed collagen deposition in spheroids and increased FN1 and LAMA1 gene expression [70]. However, the gene expression of COL4A5, FN1, and LAMB2 was reduced in s-µg-exposed adult retinal pigment epithelial cells [31].…”

“…An increase in the density of F-actin was also found in A431 cells, although the actin filaments became less organized [69]. Just like PC-3 cells, human fetal osteoblasts showed formation of filopodia and lamellipodia [70]. In contrast, osteoblasts had reduced cortical actin and fewer stress fibers [71].…”

Real Microgravity Influences the Cytoskeleton and Focal Adhesions in Human Breast Cancer Cells

“…The cytoskeleton is discussed as the initial gravity sensor [26]. For example, bone cells exposed to r- and s-µ g show changes in their cytoskeleton and focal adhesions, two major mechanosensitive structures [27,28]. Similar results were obtained when investigating thyroid cancer and melanoma cells cultured under µ g -conditions [14,29].…”

“…r-µ g -induced changes in the cytoskeleton and/or in focal adhesion components of human cells as for example cancer cells and normal cells like osteoblasts, chondrocytes and cancer cells are the two major mechanosensitive responses [8,14,28]. The cytoskeleton responds to changes in the mechanical environment because of its connection to the ECM through focal adhesions.…”

Modeling the Impact of Microgravity at the Cellular Level: Implications for Human Disease

“…Bone cell morphology is significantly modified following exposure to microgravity when compared to control cells (Guignandon et al, 1995;Hughes-Fulford, 2003). To adapt to the new mechanical environment the bone cells have reduced transcription and translation of cytoskeletal and cytoskeletalassociated proteins (Xu et al, 2017;Mann et al, 2019), decreased focal adhesion formation, together resulting in the increased formation of osteoclast resorption pits (Nabavi et al, 2011). Furthermore, the actin cytoskeleton of osteoblasts subjected to 4 days of microgravity exposure completely collapsed (Hughes-Fulford, 2003), significantly impacting multiple downstream signaling pathways, most notably, the inhibition of bone morphogenic protein (BMP) signaling axis (Patel et al, 2007;Xu et al, 2017).…”