Session:

Titanium nanotubular surfaces have been extensively studied for use in biomedical implants due to their ability to promote osseointegration and bone formation. However, despite the large body of literature on the subject, the impact of the biochemical environment, in particular the glucose levels, and potential synergistic/antagonistic effects with surface physicochemical cueing remains poorly understood. In this study, we investigated the effects of cellular preconditioning in elevated glucose environments on the interaction of human MG63 osteoblast-like cells with nanotubes of 20 nm and 80 nm, as well as with a two-tier honeycomb (HC) structure composed of smaller 20 nm domains nested within larger tubes of approximately 90 nm in diameter. The design and production of these nanotubular surfaces saw significant improvement in this study over previous work, with surfaces demonstrating enhanced order, homogeneity, and reproducibility. Experiments utilizing standard TC-treated well plates were used to effectively separate environmental and surface effects. In determining preconditioning parameters, MG63 cells were cultured in low glucose (5mM) and high glucose (25mM) environments over a three-week period, and a portion of every passage was seeded on TC treated 48-well plates, monitoring differentiation through ALP assays and cell viability using the PrestoBlue viability assay. Normalization of these two markers was well established after two weeks of preconditioning. Preconditioned cells were successively seeded on 20nm, 80nm, and HC surfaces. Cell proliferation, morphology, and viability were assessed through Ki67 and actin staining (48 hrs), along with the PrestoBlue assay (Days 1, 3 and 7). Our results indicated that MG63 cells cultured in high glucose conditions on TC culture plates had significantly lower cell viability compared to those cultured in normal glucose conditions, as previously shown by relevant literature. This effect, however, was mitigated or reversed completely by interaction with the nanotubular surfaces. A parallel outcome was observed in wound healing assays, where migration rates, slowed by the high glucose, were restored or elevated upon exposure to the nanotubular surfaces. The 80 nm and HC surfaces demonstrated significantly higher cell viability (Day 7) and elevated ALP activity (Days 7 and 14) relative to the other surfaces. This trend in differentiation was further explored through the expression of osteogenic markers: osteocalcin (OCN) and osteopontin (OPN), analyzed using fluorescence microscopy and Western blot staining, as well as the degree of mineralization, quantified via the Alizarin Red absorbance assay. Our results demonstrate that high glucose preconditioning significantly influences cell surface dynamics, altering rates of proliferation, migration, mineralization and expression of differentiation markers. Prolonged exposure to high glucose media increases cell metabolic activity and total osteogenic marker expression relative to immediate transition from low to high glucose environments, effectively addressing a gap in the literature where information regarding prolonged exposure to high glucose prior to experimentation is missing or arbitrarily chosen. This normalization aids in distinguishing environmental effects of increased glucose concentration from the physicochemical cueing induced via cell-surface interactions. Although nanotubular surfaces largely outperformed, the multi-hierarchical HC surface’s elevated levels of differentiation and proliferative markers in both high and low glucose conditions, as well as ease of production, shows promising signs of being an optimal surface, thereby highlighting the potential of the 2-tier structure for tissue engineering and regenerative medicine. Taken together, our study demonstrates the potential to counteract certain effects of negative environmental stimuli through careful control of nanotopographical surface features.

Co-Author(s): Fabio Variola (University of Ottawa)

Ryan Berthelot

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