Foreign body reaction (FBR), an immune-mediated complex healing process, plays a crucial role in integrating implants into the body. Macrophages, as the first line of immune system interaction with implant surfaces, play a bidirectional role in modulating the inflammation-regeneration balance. For a deep understanding and the evaluation of the reactions between implant materials and immune responses, reliable in vitro methods and protocols are pivotal. Among different in vitro models, primary monocyte-derived macrophages (MDMs) present an excellent model for investigating macrophage-implant interactions. We have implemented an experimental protocol to evaluate the polarization of MDMs into M1 (classically activated) and M2 (alternatively activated) macrophages on implant surfaces. We isolated blood monocytes from healthy donors and differentiated them into macrophages using macrophage colony-stimulating factor (M-CSF). Differentiated macrophages were cultured on implant surfaces and polarized into M1 and M2 subtypes. M1 polarization was achieved in the presence of interferon (IFN)-γ and lipopolysaccharide (LPS), while M2 polarization was performed in a medium containing interleukin (IL)-4 and IL-13. We evaluated macrophage phenotypes by Enzyme-linked Immunosorbent Assay (ELISA), confocal laser scanning microscopy (CLSM), and quantitative real-time PCR (qRT-PCR) based on panels of secreted cytokines, cell surface markers, and expressed genes. The extracted RNA was transformed into complementary DNA (cDNA), and qRT-PCR was used to quantify mRNA related to M1 and M2 macrophages. Accordingly, M1 macrophages have been characterized by higher expression of proinflammatory Tumor necrosis factor (TNF-α) cytokine and CCR7 surface marker compared to M2 macrophages, which exhibited higher levels of CD209 and CCL13. Consequently, CCR7 and CD209 were identified as specific and reliable markers of M1 and M2 macrophage subtypes by immunostaining and visualizing by CLSM. Further confirmation was achieved by ELISA detecting elevated TNF-ɑ level in M1 and increased CCL13 in M2 cells. The proposed markers and experimental setup can be used effectively to assess the immunomodulatory potential of implants.

Physicochemical modification of titanium implants aims to enhance early osseointegration by improving bioactivity. This study deposited and evaluated an anatase TiO₂ film on clinically relevant sandblasted, acid-etched titanium (Ti-SLA) to enhance in vitro bioactivity and osteogenic responses. An ~8 µm TiO₂-anatase coating was deposited on Ti-SLA by reactive pulsed DC magnetron sputtering. Surface characterization included FE-SEM, helium ion microscopy, and XRD. Wettability and surface free energy (SFE) were evaluated by contact angle analysis. In vitro bioactivity was assessed by hydroxyapatite (HA) formation in twofold-concentrated simulated body fluid (2× SBF). Osteoblast responses were evaluated through cell adhesion, viability, alkaline phosphatase activity, gene expression, and mineralization. The coating produced hierarchical multi-globular microstructures decorated with faceted anatase nanocrystals. Ti-SLA’s initial hydrophobicity converted to a superhydrophilic, high-energy surface with increased polar SFE. Homogeneous HA crystallites deposited exclusively on SLA-anatase in 2× SBF. SAOS-2 cells showed enhanced metabolic activity, ALP activity, osteogenic gene upregulation, and improved mineralized matrix, while primary human osteoblasts exhibited increased metabolic activity and calcium deposition. The anatase coating produced a superhydrophilic, high-energy micro-nano surface that accelerates HA formation and enhances osteoblast function in vitro, warranting in vivo validation for early osseointegration.

The success of titanium dental implants rely on osseointegration, influenced by surface properties and early immune responses. While sandblasted and acid-etched (SLA) titanium surfaces have shown clinical success, macrophage-mediated immune responses at these interfaces remain poorly understood. Anatase nanostructures have been shown to influence macrophage polarization on smooth titanium, but their effects on micro-rough SLA surfaces are not fully explored. This study investigates the immunomodulatory effects of micro-nanostructured anatase coatings on SLA titanium using human monocyte-derived macrophages (MDMs). M0-MDMs, were cultured and polarized to M1 and M2- macrophages on Ti-machined, Ti-SLA, Ti-SLA-anatase, and coverslip control surfaces for 48 h. Macrophage behavior was assessed using CCK-8 assay, confocal microscopy, SEM, ELISA, and qRT-PCR. All surfaces demonstrated excellent cytocompatibility, with similar macrophage viability across all investigated groups. M1 macrophages showed upregulation of CCR7 and TNF-α, while M2 macrophages expressed CD209 and CCL13 across all surfaces. Importantly, Ti-SLA-anatase did not significantly alter M1 or M2 markers, cytokine secretion, or gene expression, and did not exacerbate inflammatory responses. Micro-nanostructured anatase coatings on SLA titanium are immunologically well-tolerated and do not increase inflammation. These findings, combined with previously reported enhanced osteogenic properties, suggest the clinical potential of anatase-coated SLA surfaces.