A method for studying MLO-Y4 osteocyte response to simulated microgravity in embedded 3D collagen scaffolds

摘要

The limited number of opportunities for biological research in space has generated the need for ground-based microgravity simulators. The Rotary Cell Culture System (RCCS) has become one of the most widely used devices to simulate microgravity conditions in cultured cells. Nevertheless, the device is known to produce differing effects depending on the cell type, microcarriers used, speed of rotation, vessel dimensions and laboratory conditions. Osteocytes are the mechanosensors of bone, producing biological signals when mechanical forces are applied or removed from their environment. It is thought that these cells play an important role in astronaut osteoporosis, however the cellular mechanisms of the disease have yet to be elucidated. We sought to characterize the response of MLO-Y4 osteocytes in the device using 3D gel scaffolds composed of collagen I and hydroxyapatite nanoparticles to produce a bone-like environment for the cells. The gel was inoculated with a cell suspension and spread onto the walls of a high-aspect ratio vessel (HARV) designed for simulating microgravity in the RCCS. Osteocyte markers of differentiation and mechanical stimulation were studied by quantitative real-time PCR (qRT-PCR) to assess the response of MLO-Y4 osteocytes to the simulated microgravity environment. Two genes were significantly downregulated, Dentin Matrix Protein 1 (DMP1) and Podoplanin (Ell), which are osteocyte and osteoblast markers, respectively. Clinorotation may therefore be influencing MLO-Y4 differentiation. We then characterized the morphology of the cells following simulated microgravity by quantifying the fraction of dendritic cells compared to static controls, however no significant differences were observed. Through these preliminary results, we validated our novel approach combining optimized simulated microgravity methods and 3D culture methods. This work marks an important first step in understanding the role of osteocytes in astronaut bone loss.