# Enhanced Osteoblast Adhesion and Proliferation on Vacuum Plasma-Treated Implant Surface
## 4 Discussion  
UV-functionalization is a well-known method that improves bioactivity by changing the physicochemical properties of the implant surface [12; 14; 15; 29]. Based on a large body of evidence indicating UV-functionalization can restore the bioactivity of the aged implant surfaces, UV irradiation devices have been developed as medical devices, capturing the attention of doctors in the dental field. For example, Therabeam(r) SuperOsseo, (Ushio Inc., Tokyo, Japan) and UV Activator (DIO implant Co. Ltd., Busan, Korea) are commercially available UV irradiation devices [30; 31; 32]. Previous studies on UV-functionalization have demonstrated that UV irradiation converts the implant surface to a hydrophilic property known to have a high affinity for protein and cells [15]. It has also been argued that UV irradiation excites the electrons from the valence band to the conduction band and  
Figure 5: (**A**). Fluorescence images of cells labelled with Rhodamine phalloidin, and Hoechst 33,342 adhere on SLA, CaSLA, and PCaSA surfaces (Nucleus images in the top row are shown in grey scale images. Actin images in the middle row are shown in red color. In the bottom row, the merged images show the nucleus in blue color and actin in red color. (**B**). ALP activity of cells on SLA, CaSLA, and PCaSA at the 7 th day of culture. [**_p_ \(<\)0.01, ***_p_ \(<\)0.001]. makes the implant surface, predominantly covered by TiO\({}_{2}\), positively charged [15; 33]. Consequently, these hydrophilic and electrostatic properties have been shown to allow proteins and cells to attach directly to the implant surface, which in turn promotes osseointegration [12; 14; 15; 29; 33]. Plasma treatment, particularly air plasma treatment, is known to result in chemical effects similar to UV functionalization [34; 35]. According to a previous study, N\({}_{2}\) plasma treatment on TiO\({}_{2}\) was shown to reduce Ti\({}^{4+}\) to Ti\({}^{3+}\) as in UV treatment, generating oxygen vacancies and leaving TiO\({}_{2}\) positively charged [34]. It has also been demonstrated that air-based O\({}_{2}\) plasma treatment forms a hydroxyl (OH) group on the TiO\({}_{2}\) surface, which is known to improve the hydrophilicity and binding affinity with proteins [35]. Based on these previous studies, the increase in hydrophilicity, improvement in protein adsorption, and cell adhesion observed in our study can be considered a result of the plasma-induced chemical process on the implant surface. In our in vitro investigation, the plasma-treated implant (PCAsLA) showed significantly higher levels of protein adsorption, osteoblast adhesion, and differentiation than the non-treated implant (CaSLA) (Figures 4 and 5). In addition, when observing the morphology of cells attached to PCaSLA, cells were more evenly and widely attached than that on CaSLA (Figure 5). These results are believed to be relevant to the increased hydrophilicity of the implant by the plasma (Figure 2). [PERSON] et al. have reported that plasma-induced surface modification increased hydrophilicity, cell adhesion, and further upregulated osteogenesis-related genes such as Runx2, ALP, and BMP-2 [36]. In addition, they observed twice as much calcium deposition on the plasma-treated titanium surface compared to the control, indicating a high degree of clinical relevance [36]. As mentioned in the Section 1, hydrocarbon-based impurities are generally known as detrimental to protein adsorption and osteoblast adhesion [12; 13; 14]. [PERSON] et al. (2009) have shown a strong negative correlation between the level of carbon and the attractiveness of protein and cells. Accordingly, they suggested that carbon removal contributed to improving bone-implant integration [14]. Therefore, our experimental results in which plasma treatment reduced the amount of carbon on the implant surface are very encouraging (Figure 3). Plasma contains various high-energy species, including electrons, charged species, reactive oxygen species, metastable atoms, UV photons, etc. [37; 38].