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Investigating In Vivo Roles of Osteocyte Estrogen Receptor beta (Ot-ERβ) in Skeletal Biology and Validation of a Novel Three-dimensional (3D) In Vitro System for Studying Osteocyte Biology
Osteoporosis causes over two million skeletal fractures in the United States every year in people over 50 years of age. Age-related bone loss results from imbalanced bone turnover mainly caused by decreases in sex hormones and skeletal mechanobiology. Estrogen receptor β (ERβ) in osteocytes (Ot) has been proposed to mediate skeletal structural adaptations in response to estrogen and mechanical stimuli. However, direct in vivo studies on Ot-ERβ are lacking, and relevant in vitro studies are mostly made in two-dimensional (2D) culture models, whose cellular environment restricts Ot morphology and biology. To better understand the mechanisms of estrogen-ERs in age-related bone loss, it is important to investigate the role of Ot-ERβ in skeletal turnover in response to sex hormonal and mechanical cues and develop a novel 3D culture model that can reproduce Ot morphology for future in vitro ER studies. The role of Ot-ERβ in bone turnover and skeletal adaptive response to mechanical load were examined in male and female mice at 12wk and 30wk old. Ot-ERβ shows age- and sex-dependent effects on bone morphology. Young male mice with Ot-ERβ deletion (ERβ-dOT) showed increased vertebral cancellous bone, whereas decreased cortical and cancellous vertebral bone mass appeared in adult male ERβ-dOT mice. No difference in bone mass occurred in female mice between genotypes. Ot-ERβ mediates tibial mechanoadaptation in cortical but not cancellous in young and adult male mice but plays an inhibitory role in young female mice during cortical mechanoadaptation. Gonadectomy studies on young adult mice revealed that deletion of Ot-ERβ inhibits the sex hormone withdrawal-induced decreases in bone mass and skeletal strength for male mice but did not play a major role for female mice. Lastly, a novel 3D in vitro culture system was developed using collagen-mineral composites for investigating culture mineralization, osteocyte biology, and osteocyte-osteoblast interaction. Cell viability and cellular differentiation were validated after 3 days and 56 days of culture. Optimal PSC-HA culture conditions were determined based on osteocyte differentiation, gene expression analyses, and tissue mineralization. Overall, this work takes novel steps to demonstrate the in vivo role osteocyte-ERβ plays in skeletal morphology and mechanobiology and develops a novel in vitro 3D culture using PSC-HA composites. These advances will contribute to future mechanistic studies of sex hormone receptors in osteoblasts and osteocytes in age-related bone loss using controlled in vitro environments.