Preparation of titanium samples
Titanium discs were prepared as described previously 45. Briefly, 216 commercially pure Grade 4 titanium discs (ø = 15 mm, thickness: 2 mm, surface roughness: Ra = 1.3 µm; Ultimate Materials Technology, Hsinchu, Taiwan) were used. After being washed with distilled water and acetone 3 times in a Transsonic ultrasonic bath (Elma Ultrasonic, Singen, Germany), the titanium discs were autoclaved (121°C, 2 atm, 30 min) and dried using laminar flow with UV irradiation overnight.
P. gingivalis inoculation and surface treatments of the titanium discs
The bacterial inoculation was performed as follows. P. gingivalis (ATCC® 33277™) were cultured in a brain heart infusion broth (BD Bacto, REF 237500) supplemented with yeast extract, L-cysteine hydrochloride, and resazurin. Subsequently, P. gingivalis with optical density = 0.1 at 600 nm was inoculated on the titanium discs and then placed in a 24-well plate and cultured under anaerobic conditions for 72 h to allow biofilm formation.
The sterilized titanium discs were divided into 6 groups (Fig. 7). Group 1 did not receive bacterial inoculation or debridement and served as a control. Group 2 received only P. gingivalis inoculation without further treatment. Group 3 received P. gingivalis inoculation followed by 0.12% chlorhexidine (CHX) irrigation (10 mL each time, 30 mL in total). Group 4 received P. gingivalis inoculation followed by periodontal titanium curette (8 mm in diameter, Langer 1/2, item code: 7103, Kohler Medizintechnik, GmbH & Co, Ltd, Stockach, Germany) debridement (30 vertical strokes with hand pressure for each disc) and normal saline irrigation (5 times per 10 strokes, 1 mL each time). Group 5 received P. gingivalis inoculation followed by Er:YAG laser (AdvErL Evo, Morita Corporation, Tokyo, Japan) irradiation using a C600F tip (600 μm in diameter, item code: no. 34-8001703, Morita Corporation) with water cooling at a pulse energy of 80 mJ and frequency of 25 pulses per second after inoculation. The total irradiation time was 10 min and the energy density was 3.87 J/cm2. Group 6 received P. gingivalis inoculation followed by curette debridement and Er:YAG laser irradiation (combining the treatments of Groups 4 and 5).
Contact angle measurement
The surface hydrophilicity of the titanium discs after surface treatment was determined by measuring the contact angle (n = 6) with one drop (0.5 μL) of deionized water using a contact angle goniometer (FTA125; First Ten Ångstroms, Portsmouth, VA, USA). Three independent measurements were performed for each sample.
Surface roughness measurement
To evaluate the surface roughness of the titanium discs, a Surfcorder ET 200 profilometer (Kosaka, Tokyo, Japan) was used (n = 6). The tracing diamond tip was 2 µm with a tracing length of 4 mm, force of 200 µN, tracing speed of 0.2 m/s, and cutoff value of 0.8 mm. Six tracings were performed at different locations on the surface of each specimen. The average surface roughnesses were calculated as Ra values.
Surface morphology observation using scanning electron microscopy
The titanium discs (n = 6) were washed with phosphate-buffered saline (PBS) twice and dehydrated with serial ethanol (50%,70%, 80%, 90%, 95%, 99%, for 15 min each) after surface treatments. After dehydration, the samples were dried using a critical point dryer, mounted on aluminum stubs, sputter-coated with approximately 20-nm-thick gold/palladium, and finally examined using field-emission scanning electron microscopy (FE-SEM; Nova NanoSEM 230, FEI Co, Brno, Czech Republic) with an accelerating voltage of 15 kV.
Detection and quantification of adherent bacteria
The double staining of SYTO 9/PI has been used to quantify viable and dead bacteria. In the present study, adherent bacteria were examined and quantified using the SYTO 9/PI staining technique (LIVE/DEAD BacLight Bacterial Viability Kit; Invitrogen, Carlsbad, CA, USA). After fixation in 4% paraformaldehyde for 10 min, SYTO 9/PI double staining was performed on the titanium discs (n = 6) at 37°C in the dark, followed by incubation for 15 min. The samples were then examined using fluorescence microscopy (Zeiss LSM780 confocal microscope; Zeiss, Oberkochen, Germany). Three independent measurements were performed for each sample.
Quantification of endotoxin residue on the titanium discs
The remaining LPS on the titanium discs (n = 6) after different surface treatments was measured using a toxin sensor chromogenic limulus amebocyte lysate (LAL) endotoxin assay kit (GenScript, Piscataway, NJ, USA). LAL reagents were added to vials containing the treated titanium discs and an endotoxin standard. After incubation at 37°C for 45 min, a chromogenic substrate solution (100 µL) was added and incubated at 37°C for 6 min. A stop solution (500 µL) and color stabilizer (500 µL) were added and measured at 545 nm using a spectrophotometer.
HGF adhesion assay
Gingival tissues were harvested from the maxillary tuberosity of healthy human donors. Ethical approval and informed consent were obtained from all volunteers (IRB NTUH-REC No. 202002055RIND). All methods were performed in accordance with the relevant guidelines and regulations. HGF were seeded on the surface of the treated titanium discs (n = 6) in 24-well plates at a density of 2.5 × 104 cells per disc and cultured for 24, 72, and 120 h for the adhesion assay. To verify and count the cell numbers, ActinGreenTm 488 (Invitrogen) to indicate a cytoskeleton and 4′,6-diamidino-2-phenylindole (DAPI) labeled nucleic acids were used. At each time point, the titanium discs were washed with PBS to remove non-attached cells and fixed in 4% paraformaldehyde for 10 min followed by ActinGreenTm 488 and DAPI. Three fields of view per sample were captured with a Zeiss LSM780 confocal microscope (Zeiss). The cell density (cells/mm2) of HGFs was determined using ZEN Offline (Zeiss). Three independent measurements were performed for each sample.
Statistical analysis
Differences between the control and experimental groups were analyzed using a one-way analysis of variance followed by Tukey’s honest significant difference test for multiple comparisons. All data are presented as means ± standard deviation. Results with p-values < 0.05 were considered to be significant.