Non-Canonical Wnt16 and microRNA-145 Mediate the Response of Human Bone Marrow Stromal Cells to Additively Manufactured Porous 3-Dimensional Biomimetic Titanium–Aluminum–Vanadium Constructs
Authors: Cohen David. J. , Berger Michael B. , Deng Jingyao , Jacobs Thomas W. , Boyan Barbara D. , Schwartz Zvi 
Published: February 2025

Metal 3D printing is increasingly being used to manufacture titanium–aluminum–vanadium (Ti6Al4V) implants. In vitro studies using 2D substrates demonstrate that the osteoblastic differentiation of bone marrow stromal cells (MSCs) on Ti6Al4V surfaces, with a microscale/nanoscale surface topography that mimics an osteoclast resorption pit, involves non-canonical Wnt signaling; Wnt3a is downregulated and Wnt5a is upregulated, leading to the local production of BMP2 and semaphorin 3A (sema3A). In this study, it was examined whether the regulation of MSCs in a 3D environment occurs by a similar mechanism. Human MSCs from two different donors were cultured for 7, 14, or 21 days on porous (3D) or solid (2D) constructs fabricated by powder-bed laser fusion. mRNA and secretion of osteoblast markers, as well as factors that enhance peri-implant osteogenesis, were analyzed, with a primary focus on the Wnt family, sema3A, and microRNA-145 (miR-145) signaling pathways. MSCs exhibited greater production of osteocalcin, latent and active TGFβ1, sema3A, and Wnt16 on the 3D constructs compared to 2D, both of which had similar microscale/nanoscale surface modifications. Wnt3a was reduced on 2D constructs as a function of time; Wnt11 and Wnt5a remained elevated in the 3D and 2D cultures. To better understand the role of Wnt16, cultures were treated with rhWnt16; endogenous Wnt16 was blocked using an antibody. Wnt16 promoted proliferation and inhibited osteoblast differentiation, potentially by reducing production of BMP2 and BMP4. Wnt16 expression was reduced by exogenous Wnt16 in 3D cells. Addition of the anti-Wnt16 antibody to the cultures reversed the effects of exogenous Wnt16, indicating an autocrine mechanism. Wnt16 increased miR-145-5p, suggesting a potential feedback mechanism. The miR-145-5p mimic increased Wnt16 production and inhibited sema3A in a 3D porous substrate-specific manner. Wnt16 did not affect sema3A production, but it was reduced by miR-145-5p mimic on the 3D constructs and stimulated by miR-145-5p inhibitor. Media from 7-, 14-, and 21-day cultures of MSCs grown on 3D constructs inhibited osteoclast activity to a greater extent than media from the 2D cultures. The findings present a significant step towards understanding the complex molecular interplay that occurs in 3D Ti6Al4V constructs fabricated by additive manufacturing. In addition to enhancing osteogenesis, the 3D porous biomimetic structure inhibits osteoclast activities, indicating its role in modulating bone remodeling processes. Our data suggest that the pathway mediated by sema3A/Wnt16/miR145-5p was enhanced by the 3D surface and contributes to bone regeneration in the 3D implants. This comprehensive exploration contributes valuable insights to guide future strategies in implant design, customization, and ultimately aims at improving clinical outcomes and successful osseointegration.