title: Mineralization of three-dimensional osteoblast cultures is enhanced by the interaction of 1α,25-dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors.
abstract: 1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] and bone morphogenetic protein-2 (BMP2) are both used to stimulate osteoblastic differentiation. 1α,25(OH)2D3 regulates osteoblasts through classical steroid hormone receptor mechanisms and through rapid responses that are mediated by two receptors, the traditional vitamin D receptor (VDR) and protein disulphide isomerase family A member 3 (Pdia3). The interaction between 1α,25(OH)2D3 and BMP2, especially in three-dimensional (3D) culture, and the roles of the two vitamin D receptors in this interaction are not well understood. We treated wild-type (WT), Pdia3-silenced (Sh-Pdia3) and VDR-silenced (Sh-VDR) pre-osteoblastic MC3T3-E1 cells with either 1α,25(OH)2D3, or BMP2, or with 1α,25(OH)2D3 and BMP2 together, and measured osteoblast marker expression in 2D culture and mineralization in a 3D poly(ε-caprolactone)-collagen scaffold model. Quantitative PCR showed that silencing Pdia3 or VDR had a differential effect on baseline expression of osteoblast markers. 1α,25(OH)2D3 + BMP2 caused a synergistic increase in osteoblast marker expression in WT cells, while silencing either Pdia3 or VDR attenuated this effect. 1α,25(OH)2D3 + BMP2 also caused a synergistic increase in Dlx5 in both silenced cell lines. Micro-computed tomography (μCT) showed that the mineralized volume of untreated Sh-Pdia3 and Sh-VDR 3D cultures was greater than that of WT. 1α,25(OH)2D3 reduced mineral in WT and Sh-VDR cultures; BMP2 increased mineralization; and 1α,25(OH)2D3 + BMP2 caused a synergistic increase, but only in WT cultures. SEM showed that mineralized matrix morphology in 3D cultures differed for silenced cells compared to WT cells. These data indicate a synergistic crosstalk between 1α,25(OH)2D3 and BMP2 toward osteogenesis and mineral deposition, involving both VDR and Pdia3.
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
authors.forename: Jiaxuan
authors.initials: J
authors.lastname: Chen
authors.affiliation: Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Dosier
authors.affiliation: School of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
authors.forename: Jung Hwa
authors.initials: JH
authors.lastname: Park
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
authors.forename: Subhendu
authors.initials: S
authors.lastname: De
authors.affiliation: Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Guldberg
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.|Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: Barbara D
authors.initials: BD
authors.lastname: Boyan
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.|Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: Zvi
authors.initials: Z
authors.lastname: Schwartz
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D002113:Calcification, Physiologic / Q000187:drug effects*; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D051379:Mice; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D018167:Receptors, Calcitriol / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D054457:Tissue Scaffolds / Q000737:chemistry; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D055396:Bone Morphogenetic Protein 2; D012333:RNA, Messenger; D018167:Receptors, Calcitriol; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C118756:dihydroxy-vitamin D3; C499469:recombinant human bone morphogenetic protein-2; D014807:Vitamin D; D000469:Alkaline Phosphatase
keywords: 1α,25(OH)2D3; BMP2; Pdia3; VDR; mineralization; osteoblasts; polycaprolactone scaffolds