Abstract
Recent in vitro studies have revealed that the mechanobiological responses of osteoblasts and osteocytes are fundamentally impaired during estrogen deficiency. However, these two-dimensional (2D) cell culture studies do not account for in vivo biophysical cues. Thus, the objectives of this study are to (1) develop a three-dimensional (3D) osteoblast and osteocyte model integrated into a bioreactor and (2) apply this model to investigate whether estrogen deficiency leads to changes in osteoblast to osteocyte transition, mechanosensation, mineralization, and paracrine signaling associated with bone resorption by osteoclasts. MC3T3-E1s were expanded in media supplemented with estrogen (17β-estradiol). These cells were encapsulated in gelatin-mtgase before culture in (1) continued estrogen (E) or (2) no further estrogen supplementation. Constructs were placed in gas permeable and water impermeable cell culture bags and maintained at 5% CO2 and 37°C. These bags were either mechanically stimulated in a custom hydrostatic pressure (HP) bioreactor or maintained under static conditions (control). We report that osteocyte differentiation, characterized by the presence of dendrites and staining for osteocyte marker dentin matrix acidic phosphoprotein 1 (DMP1), was significantly greater under estrogen withdrawal (EW) compared to under continuous estrogen treatment (day 21). Mineralization [bone sialoprotein (BSP), osteopontin (OPN), alkaline phosphatase (ALP), calcium] and gene expression associated with paracrine signaling for osteoclastogenesis [receptor activator of nuclear factor kappa-β ligand (RANKL)/osteoprotegerin OPG ratio] were significantly increased in estrogen deficient and mechanically stimulated cells. Interestingly, BSP and DMP-1 were also increased at day 1 and day 21, respectively, which play a role in regulation of biomineralization. Furthermore, the increase in pro-osteoclastogenic signaling may be explained by altered mechanoresponsiveness of osteoblasts or osteocytes during EW. These findings highlight the impact of estrogen deficiency on bone cell function and provide a novel in vitro model to investigate the mechanisms underpinning changes in bone cells after estrogen deficiency.
Highlights
Osteoporosis is a debilitating bone disease, in which severe bone loss occurs leading to fractures of the hip, wrist, or vertebrae (Balena et al, 1993)
We demonstrated that the strategy successfully enabled osteoblast to osteocyte differentiation, as evidenced by interconnected dentin matrix acidic phosphoprotein 1 (DMP1) positive dendritic cells that stained positive for αvβ3 and vinculin
Osteocytes cultured under estrogen withdrawal (EW) conditions demonstrated increased DMP1 intensity, cell interconnectivity, mineralization, and paracrine signaling for osteoclastogenesis (RANKL/OPG)
Summary
Osteoporosis is a debilitating bone disease, in which severe bone loss occurs leading to fractures of the hip, wrist, or vertebrae (Balena et al, 1993). Cellular mechanosensors have been identified in osteoblasts and osteocytes, which transduce mechanical signals into the cell through its interactions with the actin cytoskeleton (Ziambaras et al, 1998; Rubin et al, 2006; Malone et al, 2007; Litzenberger et al, 2010; Hoey et al, 2012). WNT/β-catenin signaling promotes bone formation by osteoblasts through inhibition of sclerostin, a protein produced by osteocytes (Papanicolaou et al, 2009; Wijenayaka et al, 2011; Moustafa et al, 2012; Spatz et al, 2015; Thompson et al, 2015; Hemmatian et al, 2018). Osteocytes regulate osteoclastogenesis and bone resorption through the upregulation or downregulation of receptor activator of NFκB ligand (RANKL) and osteoprotegerin (OPG). RANKL is a ligand for receptor activator of NFκB (RANK) present in the cell membrane of osteoclast precursors. OPG is a soluble glycoprotein that acts as a decoy receptor for RANKL, by binding to RANKL and prevents RANK-RANKL association, antagonizing the signaling that promotes osteoclastogenesis (Han et al, 2018)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
