Evaluation of hydrophilization on a Ti disk using a handheld-type low-temperature atmospheric pressure plasma device

A small, handheld low-temperature atmospheric pressure plasma device (Piezobrush® PZ2; input power: 30 W, Relyon Plazma GmbH, Regensburg, Germany) with air was used in this study (Fig. 1a). Commercial pure titanium (CpTi) disks (JIS grade 4; Tokyo Titanium, Saitama, Japan; diameter: 13 mm, thickness 2.54 mm) were used to evaluate the effect of plasma application to Ti. The CpTi disks were polished roughly with waterproof polishing paper (#120, #240, #320, #400, #600, #800, and #1200) using a polishing machine (Ecomet 3; Buehler, Lake Bluff, IL, USA) and finished with polishing cloth (3 μm diamond particles and 0.6 μm colloidal silica). All samples were ultrasonically cleaned with acetone for 10 min, 95% ethanol for 10 min, and distilled water for 10 min before use in the experiments. Prior to evaluation, theses samples aseptically stored at room temperature for 1 week to exclude effect of plasma application. The distance between the plasma device and Ti disk was set to 5 mm, and hydrophilizing by plasma treatment was performed for 30 s (Fig. 1b). The evaluation of macro- and micro-level surface observation, wettability, roughness, and temperature change in the Ti disks after plasma treatment was performed using a handheld plasma device (PL) or no treatment (Cont). The micro-level surface of the Ti disks was observed by scanning electron microscopy (SU6600; Hitachi, Tokyo, Japan). The wettability of the Ti disk surface after plasma treatment was evaluated using a contact angle measurement. The pure water droplet volume was 1 μL and the contact angle was measured after 5 s using a contact angle meter (Phoenix α; Meiwa-forces, Tokyo, Japan; n = 5 per group). The two-dimensional arithmetic mean surface roughness (Ra), with a length of 645 μm (cutoff value: 250 μm), and the three-dimensional arithmetic mean roughness (Sa), with a range of 645 × 645 μm (cutoff value: 250 μm) and a cutoff value of 250 × 250 μm, were measured using a three-dimensional laser microscope (LEXT OLS4000; Olympus, Tokyo, Japan; n = 5 per each group). The temperature change of the Ti disk surface was measured using an infrared noncontact thermometer for 5 s after plasma treatment (THK-TOP01; Tahoco, Fujian, China; n = 5 per group).

Fig. 1

Schema of the time schedule for tissue sampling. a Photograph of the plasma device. b The distance between the plasma device and Ti disk was set to 5 mm, and plasma treatment was performed for 30 s. c The sampling time point is indicated as a cross. An implant (diameter 2 mm, length 4.5 mm), plasma treatment to the implant, and an intraoral photograph after implant placement are shown

Dental implant surgery

Five-week-old male Sprague Dawley rats weighing approximately 150 g (Japan SLC Co., Ltd., Tokyo, Japan) were used in this experiment. The bilateral maxillary first molars were extracted under general anesthesia using a combination anesthetic (0.375 mg/kg medetomidine hydrochloride, Nihon Zenyaku Kogyo, Fukushima, Japan; 2.0 mg/kg midazolam, Fuji Pharmaceutical, Tokyo, Japan; and 2.5 mg/kg butorphanol tartrate, Meiji Seika, Tokyo, Japan). For Cont, the Ti implant (JIS grade 4 commercially pure titanium, 2 mm in diameter and 4.5 mm in length; T & I Japan, Saitama, Japan) was immediately placed in the extraction socket on the right side with primary stabilization. For PL, Ti implant picked up with yellow pipets and covered bone contact area and the soft tissue contact area of it hydrophilized a using small, handheld plasma device (Piezobrush® PZ2; Relyon Plazma GmbH) for 30 s with a rolling motion, and then the treated implants were immediately placed in the extraction socket on the left side with primary stabilization (Fig. 1c). The rats were housed until killing while being provided with water and solid food, and the placed implants were checked for bacterial infection, movement, and loosening until tissue sampling. All rats were euthanized under deep anesthesia at each time point: 3, 7, and 14 days after implant surgical treatment. Samples for histological evaluation (n = 5/time point) and total RNA extraction (n = 3/time point) were collected. All experiments were performed according to the Guidelines for the Treatment of Animals at Tokyo Dental College (approval No.: 213303 and 233303).

Histological measurement of the peri-implant soft tissue sealing area

At 3, 7, and 14 days after surgery, the maxillary jaw, including the implant-placed area, was harvested (n = 5/time point). The tissue samples with the implant were fixed in 10% neutral buffered formalin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 1 day and decalcified with ethylenediaminetetraacetic acid (pH 7.0–7.5, 0.5 mol/L; Wako Pure Chemical Industries, Ltd.) at room temperature for 2 weeks. The implant body was carefully removed and the specimens were embedded in paraffin according to the standard protocol. The sections were cut along the coronal plane (thickness: 3 μm) and stained with hematoxylin and eosin (H&E) for histological observation and measurement of the peri-implant soft tissue sealing area. Images of the stained sections were captured for measurement of the peri-implant soft tissue sealing area using a conventional microscope (Axiophot2; Carl Zeiss, Oberkochen, Germany).

For evaluating the peri-implant soft tissue sealing area, three sections at each time points were randomly selected from a sample (n = 5/time point). The axial width of the keratinized epithelium area as the peri-implant sulcus epithelium area (PISE), non-keratinized epithelium area attached to the implant as the peri-implant epithelium area (PIE), and connective tissue area attached to the implant as PICT were measured. In addition, the axial width of the soft tissue sealed area (STSA) was calculated as the total width of PIE and PICT, while that of the peri-implant soft tissue area was calculated as the total width of PISE, PIE, and PICT (Fig. 2).

Fig. 2

Measurement of the axial width at the peri-implant soft tissue sealed area. Representative images of H&E staining to measure the vertical width at the peri-implant soft tissue during wound healing are shown

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Tissue samples for total RNA extraction were collected using a dissecting microscope (SZ61; Olympus). All samples were washed with saline and placed in RNAlater RNA stabilization reagent (Qiagen, Valencia, CA, USA). Tissue homogenization was performed with 5 mm zirconia beads at 30 Hz for 2 min using a tissue lyser (Qiagen).

RT-qPCR was performed to evaluate collagen maturation using type 1 collagen α1 (Col1a1), type 1 collagen α2 (Col1a2), type 3 collagen α1 (Col3a1), and type 12 collagen α1 (Col12a1), and cell adhesion using integrin α2 (Itga2), integrin β1 (Itgb1), integrin α5 (Itga5), fibronectin 1 (Fn1), protein tyrosine kinase 2 (Ptk2), and vinculin (Vcl). The expression levels of target genes were determined by RT-qPCR and normalized against that of glyceraldehyde 3-phosphate dehydrogenase as an endogenous control. Total RNA was reverse-transcribed using the QuantiTect Reverse Transcription kit (Qiagen), and RT-qPCR was performed using the TaqMan Fast Universal PCR Master Mix (Thermo Fisher Scientific Inc. MA, USA) and TaqMan MGB probes (Applied Biosystems, Foster City, CA, USA) (Col1a1: Rn01463848_m1, Col1a2: Rn01526721_m1, Col3a1: Rn01437681_m1, Col12a1: Rn01521220, Itgb1: Rn00566727_m1, Itga2: Rn01489315_m1, Itga5: Rn01761831_m1, Fn1: Rn00569575_m1, Ptk2: Rn00433209_m1, and Vcl: Rn01755886_m1) in the ABI 7500 Fast Prism Sequence Detection System (Thermo Fisher Scientific Inc.). Total RNA was first incubated at 42 °C for 2 min, reverse-transcribed at 42 °C for 15 min, and then inactivated at 95 °C for 3 min. The RT-qPCR conditions were as follows: 95 °C for 20 s, 50 cycles at 95 °C for 30 s, and 60 °C for 30 s. All reactions were performed in triplicate for each sample, and the results were analyzed using the ΔΔCT method. These target gene expression patterns were compared in the Cont and PL groups during wound healing from Day 3 to Day 14.

Picrosirius red staining for collagen fiber detection

The paraffin-embedded sections were stained with Picrosirius Red (Picrosirius Red Stain Kit; ScyTek Laboratories Inc., Logan, UT, USA) for collagen fiber detection at the peri-implant soft tissue area during wound healing. The sections were deparaffinized with xylene and ethanol and stained with Picrosirius Red Solution for 15 min. Stained sections were rinsed with 0.5% acetic acid solution and absolute ethanol. These stained sections were then examined and photographed using a conventional microscope (Axiophot 2; Carl Zeiss) for evaluation.

Immunohistochemical evaluation for integrin α2, α5, and β1 at the biological sealing area

Immunohistochemical expression of integrin α2, integrin α5, and integrin β1 was evaluated by 3,3'-diaminobenzidine (DAB) staining. The paraffin sections deparaffinized with xylene and ethanol and washed with phosphate-buffered saline (PBS) were treated with 0.1% protease (Nichirei, Tokyo, Japan) for antigen retrieval for 3 min at 37 °C. These sections were then treated with 0.3% H2O2 in methanol at room temperature to block endogenous peroxidase and incubated with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) for 30 min to block nonspecific immunoglobulin binding. For primary antibody reaction, sections were incubated with the following primary antibodies for 2 h at room temperature: anti-integrin α2 rabbit polyclonal antibody (1:200; St John’s Laboratory Ltd., London, UK), anti-integrin α5 rabbit polyclonal antibody (1:200; St John’s Laboratory Ltd.), and anti-integrin β1 rabbit polyclonal antibody (1:200; Proteintech Group Inc., Chicago, IL, USA). After washing with PBS, sections were incubated for 30 min at room temperature with secondary antibody: Histofine Simple Stain Rat MAX-PO(R) for rabbit primary antibody (Nichirei). Immunoreactions were visualized using diaminobenzidine reagent (DAB substrate kit; Nichirei), and sections were finally counterstained with hematoxylin. All sections were examined and photographed using a conventional microscope (Axiophot 2; Carl Zeiss).

Statistical analysis

Histological measurement of the peri-implant soft tissue sealing area was performed with three sections for a sample (n = 5/time point). RT-qPCR was repeated three times for the two samples (n = 6 samples/time point). The values are expressed as means ± standard deviations. Data analysis was performed using GraphPad Prism (version 5.04; GraphPad Software Inc., San Diego, CA, USA). The levels of significance were defined as p < 0.01 and p < 0.05. Statistical analysis for the evaluation of Ti disks (wettability, roughness, and temperature change) was performed using Student’s t-test. Statistical analysis of histological measurements at each time point was performed using Mann–Whitney U test, and gene expression patterns of the Cont and PL groups during wound healing from Day 3 to Day 14 was performed using two-way analysis of variance with Bonferroni multiple comparisons.