## 2 Materials and Methods
### Biological Testing  
_Staphylococcus epidermidis_ (14990(r)ATCC(tm)) cells were cultured in heart infusion (HI) broth (Nissui, Tokyo, Japan) at 37 \({}^{\circ}\)C for 18 h and titrated by colony-forming assay using brain heart infusion (BHI) agar (Nissui). Aliquots of \(1\times 10^{7}\) bacteria were diluted in 10 \(\upmu\)L of phosphate-buffered saline (PBS) at physiological pH and ionic strength. The samples underwent preliminary UV sterilization and were distributed into wells. To each well, 1 mL of bacteria culture was added, and samples were incubated at 37 \({}^{\circ}\)C under aerobic conditions for 12, 24, and 48 h [23].  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Substrate** & \begin{tabular}{c} **Voltage** \\ **(V)** \\ \end{tabular} & \
\begin{tabular}{c} **Pulse Time** \\ **(ms)** \\ \end{tabular} &
\begin{tabular}{c} **Laser Spot Size** \\ **(mm)** \\ \end{tabular} & **Layers** & **Gas** \\ \hline LDPE & 225 & 10 & 2 & 1 & N\({}_{2}\) \\ Ti6 Al4V & 425 & 10 & 2 & 3 & N\({}_{2}\) \\ ZTA & 325 & 10 & 2 & 3 & N\({}_{2}\) \\ Y-TZP & 250 & 10 & 2 & 3 & N\({}_{2}\) \\ \hline \hline \end{tabular}
\end{table}
Table 1: Deposition parameters for various substrates. LDPE, low-density polyethylene; Ti6 Al4V, titanium; ZTA, zirconia-toughened alumina; Y-ZTP, yttria-stabilized zirconia.  
Cell viability was evaluated using a tetrazolium-based assay from a microbial viability assay kit (WST-8; Dojindo). Substrates with _Staphylococcus epidermidis_ were collected at 24 and 48 h after cultivation and soaked in 1000 \(\upmu\)L of PBS in 12-well plates. WST-8 solution was added to each well and Optical Density (OD) values were measured (the absorbance at 490 nm) using a plate reader EMax (molecular devices, San Jose, CA, USA) after incubation for 30-60 min.  
SaOS-2 human osteosarcoma cells were first cultured and incubated in 4.5 g/L glucose DMEM (D-glucose, L-glutamine, phenol red, and sodium pyruvate) supplemented with 10% fetal bovine serum. They were allowed to proliferate within Petri dishes for about 24 h at 37 \({}^{\circ}\)C. The final SaOS-2 concentration was \(5\times 10^{5}\) cell/mL. The cultured cells were then deposited on the top surface of samples previously sterilized by exposure to UV-C light for 30 min. In osteeconductivity tests, cell seeding took place in an osteogenic medium which consisted of DMEM supplemented with about 50 \(\upmu\)g/mL ascorbic acid, 10 mM \(\beta\)-glycerol phosphate, 100 mM hydrocortisone, and \(\sim\)10% fetal bovine calf serum. The samples were incubated up to 14 days at 37 \({}^{\circ}\)C. The medium was changed twice a week during the incubation period. Subsequently, the cells were stained for fluorescence microscopy with green dye to identify osteocalcin (Monoclonal, Clone 5-12H, dilution 1:500, TakaraBio, Kusatsu-shi, Japan) and red dye to show osteopontin (Osteepontin, O-17, Rabbit IgG, 1:500, Immuno-Biological Laboratories (IBL) Inc., Maebashi-Shi, Gunma, Japan) [23].  
After exposure to osteoblasts, each batch of samples was observed using fluorescence microscopy (BZ-X700; Keyence, Osaka, Japan). Prior to examination, the sample surfaces were treated with different immunostaining reagents, including Hoechst 33,342 (Dojindo, Kumamoto, Japan), anti-Human Osteocalcin Clone2H9F11F8, and Isotype IgG, Rabbit polyclonal antibody (IBL, Gunma, Japan). Hoechst 3342, a cell nucleus stain (DAPI 4\({}^{\prime}\),6-Diamidino-2-phenylindole, dihydrochloride, solution), served to visualize cell proliferation, while the other two antibodies were used to stain mineralization and bone matrix formation concentration quantifying the matrix proteins osteoclastcin and osteopontin, respectively. Subsequently, a secondary antibody, Goat anti-Mouse IgG1 Antibody FITC Conjugated (Bethyl Laboratories) was added to enhance signal detection and visualization.