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The surface topography and chemistry of titanium–aluminum–vanadium (Ti6Al4V) implants play critical roles in the osteoblast differentiation of human bone marrow stromal cells (MSCs) and the creation of an osteogenic microenvironment. To assess the effects of a microscale/nanoscale (MN) topography, this study compared the effects of MN-modified, anodized, and smooth Ti6Al4V surfaces on MSC response, and for the first time, directly contrasted MN-induced osteoblast differentiation with culture on tissue culture polystyrene (TCPS) in osteogenic medium (OM). Surface characterization revealed distinct differences in microroughness, composition, and topography among the Ti6Al4V substrates. MSCs on MN surfaces exhibited enhanced osteoblastic differentiation, evidenced by increased expression of RUNX2, SP7, BGLAP, BMP2, and BMPR1A (fold increases: 3.2, 1.8, 1.4, 1.3, and 1.2). The MN surface also induced a pro-healing inflammasome with upregulation of anti-inflammatory mediators (170–200% increase) and downregulation of pro-inflammatory factors (40–82% reduction). Integrin expression shifted towards osteoblast-associated integrins on MN surfaces. RNA-seq analysis revealed distinct gene expression profiles between MSCs on MN surfaces and those in OM, with only 199 shared genes out of over 1000 differentially expressed genes. Pathway analysis showed that MN surfaces promoted bone formation, maturation, and remodeling through non-canonical Wnt signaling, while OM stimulated endochondral bone development and mineralization via canonical Wnt3a signaling. These findings highlight the importance of Ti6Al4V surface properties in directing MSC differentiation and indicate that MN-modified surfaces act via signaling pathways that differ from OM culture methods, more accurately mimicking peri-implant osteogenesis in vivo. 

Abstract This study examined the effects of 24R,25‐dihydroxyvitamin D₃(24R,25(OH)₂D₃) in estrogen‐responsive laryngeal cancer tumorigenesis in vivo, the mechanisms involved, and whether the ability of the tumor cells to produce 24R,25(OH)₂D₃locally is estrogen‐dependent. Estrogen receptor alpha‐66 positive (ER+) UM‐SCC‐12 cells and ER− UM‐SCC‐11A cells responded differently to 24R,25(OH)₂D₃in vivo; 24R,25(OH)₂D₃enhanced tumorigenesis in ER+ tumors but inhibited tumorigenesis in ER− tumors. Treatment with 17β‐estradiol (E₂) for 24 h reduced levels of CYP24A1 protein but increased 24R,25(OH)₂D₃production in ER+ cells; treatment with E₂for 9 min reduced CYP24A1 at 24 h and reduced 24R,25(OH)₂D₃production in ER− cells. These findings suggest the involvement of E₂receptor(s) in addition to ERα66. To investigate if 24R,25(OH)₂D₃can act locally, ER+ and ER− cells were treated with 24R,25(OH)₂D₃after inhibiting putative 24R,25(OH)₂D₃receptors, and the cells were assessed for effects on DNA synthesis (proliferation) and p53 production (apoptosis). Specific inhibitors were used to assess downstream secondary messenger signaling pathways and requirements for palmitoylation and caveolae in both cell lines. The results show that 24R,25(OH)₂D₃binds to a complex of receptors, including TLCD3B2, VDR, and protein disulfide‐isomerase A3 (PDIA3) in ER+ UM‐SCC‐12 cells. The mechanism requires palmitoylation, and PLD, PI3K, and LPAR are involved. The anti‐tumorigenic effects of 24R,25(OH)₂D₃in ER− UM‐SCC‐11A cells involve a membrane‐receptor complex consisting of VDR, PDIA3, and ROR2 within caveolae to activate a yet‐to‐be‐elucidated downstream signaling cascade. This work demonstrates a driving mechanism for the therapeutic agent 24R,25(OH)₂D₃that may be used for laryngeal cancer patients.