# Titanium Surface Roughness Mediated Macrophages Polarization-Influenced Osteogenic Differentiation of Periodontal Ligament-Derived Mesenchymal Stromal Cells

## TL;DR

This study shows that titanium implant surface roughness affects macrophage behavior, which in turn influences the bone-forming potential of periodontal ligament stem cells.

## Contribution

The novel finding is that medium surface roughness on titanium implants promotes M2 macrophage polarization and enhances osteogenic differentiation of PDL MSCs.

## Key findings

- Medium surface roughness (Ti-MR) reduced pro-inflammatory gene expression in macrophages.
- Ti-MR increased anti-inflammatory and osteogenic gene expression in PDL MSCs.
- Ti-MR led to the highest population of CD163+ macrophages and improved calcium deposition.

## Abstract

Implant surface topography significantly influences cell behavior, including macrophages and bone cell interactions. The polarization of macrophages, key immune cells, is influenced by implant surface characteristics. This research aimed to examine periodontal ligament mesenchymal stromal cells (PDL MSCs) responses to the polarized macrophages induced by titanium surface roughness.

RAW 264.7 macrophages were cultured with various surface roughness of titanium disks. Macrophage adhesion and polarization were evaluated by scanning electron microscope, gene expressions profiling, and flow cytometry. PDL MSCs were treated with conditioned medium of macrophages and analyzed with 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-2H-tetrazolium bromide assay, real-time polymerase chain reaction, and Alizarin red staining.

Data was statistically analyzed using GraphPad Prism 9 for Windows 11. The one-way analysis of variance test was used to compare the groups. Dunn post hoc test was used to compare the difference between the groups when appropriate. Significance was accepted when
p
 < 0.05.

Medium surface roughness (Ti-MR) consistently inhibited tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 gene expressions (
p
 < 0.001) and upregulated transforming growth factor-β, vascular epithelial growth factor, and IL-10 expressions (
p
 < 0.01). Confirmatory flow cytometry analysis showed consistent results, with Ti-HR and Ti-MR exhibiting the highest population of CD163+ cells (99.1 and 90.7%, respectively), while Ti-LR exhibited the lowest M1/M2 ratio (0.93). Furthermore, treatment of RAW 264.7 conditioned medium increased osteopontin, alkaline phosphatase, collagen type-1 A-1 chain, osteocalcin, runt-related transcription factor-2, and bone sialoprotein gene expressions and calcium deposition (
p
 < 0.01).

Titanium implant surface topography influences macrophage polarization and osteogenic differentiation of PDL MSCs, with Ti-MR being the most effective in polarizing macrophages toward M2 and inducing optimal osteogenic responses from PDL MSCs.