Review Reports

Figure 8 depicts the 3D micro-CT images of TiO₂ NT and Pt-TiO₂ NT with and without 850 nm NIR light irradiation. Following two and six weeks of implantation, widespread bone formation (yellow color in Figure 8) was observed surrounding the TiO₂ NT or Pt-TiO₂ NT surface-treated implant (white color in Figure 8), and the periosteum was elevated. Figure 9 presents the findings of the 3D micro-CT analysis, indicating a tendency for an increase in the bone volume with 850 nm NIR irradiation. However, no statistically significant difference was observed depending on light irradiation (P > 0.05).

Figure 8. Three-dimensional micro-CT images and analysis of TiO₂ NT- and Pt-TiO₂ NT-treated implants after (A) two and (B) six weeks of implantation (White color: TiO₂ NT or Pt-TiO₂ NT surface-treated implant, yellow color: bone)

Two weeks after implantation (Figure 10), we could not find a significant histological difference between TiO₂ NT and Pt-TiO₂ NT regardless of 850 nm NIR light irradiation. Instead, the immature bone matrix was observed at the implant-surrounding bone interface in both groups (Black arrows in Figure 10). After six weeks of implantation (Figure 11), histological differences between the groups were not found regardless of 850 nm NIR light irradiation. However, mature new bone matrix, including osteocytes, appeared at the interface between the implant and existing bone (Blue arrows in Figure 11).

Abstract: Photobiomodulation (PBM) therapy is known to have the potential to improve bone regeneration after implant surgery. However, the combinatory effect of the nanotextured implant and PBM therapy on osseointegration has not yet been proved. This study evaluated the photobiomodulation-based synergistic effects of Pt-coated titania nanotubes (Pt-TiO₂ NT) and 850 nm near-infrared (NIR) light on osteogenic performance in vitro and in vivo. The FE-SEM and the diffuse UV-Vis-NIR spectrophotometer were used to perform the surface characterization. The live-dead, MTT, ALP, and AR assays were tested to perform in vitro tests. The removal torque testing, the 3D-micro CT, and the histological analysis were used to conduct in vivo tests. The live-dead and MTT assay resulted that Pt-TiO₂ NTs were biocompatible. The ALP activity and AR assays demonstrated that the combination of Pt-TiO₂ NT and NIR irradiation significantly enhanced osteogenic functionality (P < 0.05). However, the results of in vivo test, employing the removal torque testing, the 3D-micro CT, and histological analysis, showed overall improved outcomes; however, no significant difference was observed between the control and experimental groups (P>0.05). Therefore, we confirmed the possibility of the combination of Pt-TiO₂ NT and NIR light as a promising technology for implant surgery in dentistry. 

Keywords: photobiomodulation; Pt, titania nanotubes; near-infrared; osseointegration

Especially when considering the effect of PBM therapy in the clinical setting, several factors, such as the dose of light, the wavelength of light, and the safety from the light irradiation, play an essential role in the success of PBM therapy

. The null hypotheses of this study are that there are no significant differences in the results of (1) in vitro tests and (2) in vivo tests according to the presence or absence of Pt coating and 850 nm NIR light irradiation.

Based on all results of the in vitro and in vivo tests, the first null hypothesis established before the experiment was rejected, but the second null hypothesis was accepted. In vitro tests showed that the combination of Pt-TiO₂ NT and 850 nm NIR light irradiation improved osteogenic performance. However, the results of in vivo tests indicated that this combination did not show any noticeable effect of enhancing osseointegration.

2.2.2. MTT assay

Cell toxicity was assessed using an MTT assay kit (Sigma-Aldrich, St Louis, MO, USA). The same cell culture and NIR light irradiation conditions as those utilized for the live/dead assay were employed for the MTT assay. The testing and evaluation of the MTT assay were performed according to the protocol specified in ISO 10993-5, Annex C [29]. After additional 24 and 48 h of incubation, 100 mg/mL of MTT solution was added to each well, and the samples were cultured at 37°C in a 5% CO₂ incubator. After 4 h, the formazan produced by the MTT solution was dissolved in DMSO (Sigma-Aldrich, St Louis, MO, USA), and the absorbance was measured at 570 nm using an ELISA reader (Spectra MAX 250; Molecular Devices Co., Sunnyvale, CA, USA). If the cell viability value of the specimen is higher than 70% of the control (hMSCs cultured on the cell culture dish), the specimen is determined to be biocompatible according to the decision of ISO 10993-5. The MTT assay was performed on four samples from each group.

Figure 4 presents images of calcein-AM- and EthD-1-stained hMSCs cultured on TiO₂ and Pt-TiO₂ NT, along with MTT assay results, with and without 850 nm NIR light irradiation. The images showed no evidence of damaged or dead cells (indicated by red fluorescence) in any of the experimental groups after 24 and 48 h of cultivation. Moreover, the elongation ratio of the filopodia of hMSCs cultured on Pt-TiO₂ NT under 850 nm NIR light irradiation was higher than those of the other groups. The MTT assay results indicated no significant differences among all experimental groups (P > 0.05), and the cell viability values of all experimental groups were above 70% compared to control group (hMSCs cultured on cell culture dish), indicating good biocompatibility in all experimental groups.

The results of the removal torque test, micro-CT, and histological analysis indicated that the combination of 850 nm light irradiation and Pt-TiO₂ NT did not improve significantly bone formation in vivo compared to in vitro. Significantly, several NIR light irradiation variables, such as the light intensity, irradiation time, and irradiation distance, are limited to produce the results of in vivo tests like those of in vitro tests. Additionally, because the SD rats used in the animal experiments were constantly moving, irradiating the femur (implanted place) with the same light intensity for a specific time was impossible. To minimize the variations in the aforementioned variables, a small chamber accommodating one SD rat was fabricated as shown Figure 1, and NIR light irradiation was performed for 15 min in this chamber. Although the in vivo test was performed in an improved experimental environment, we could not obtain in vivo results similar to those obtained in vitro. Therefore, improvements in the research design limitations (equipment and light irradiation devices) for ensuring consistent light irradiation are necessary, and related experiments are currently in progress.

Due to the diversity of the material, the limited cytotoxicity evaluation cannot evaluate the comprehensive cytotoxicity of the material. Therefore, cytotoxicity evaluation in various ways should be performed, and we will evaluate the cytotoxicity of the experimental specimen through various cytotoxicity evaluation methods [37].

Also, we expect to try this combination for patients with exceptional cases like immune deficiencies and evaluate the reparative mechanisms contributing to defining an osseointegration procedure [40]