The effect of plasma treatment on the osseointegration of rough titanium implant: A histo-morphometric study in rabbits.

ABSTRACT BACKGROUND/
PURPOSE: The surface properties, such as hydrophilicity and functional OH groups, play an important role in bone fixation in vivo. In our previous study, the plasma treatments of large grit and acid etching (SLA) method produce functional OH groups on the rough surface. There is no report in discussing the integration between basic Ti-OH groups and bone-to-implant contact (BIC). The aim of this study was to evaluate the effect of the functional OH groups on the rough surface both in vitro and in vivo.
MATERIALS AND METHODS: Functional hydroxyl groups were produced on a SLA-treated surface. The surface topography, roughness, wettability, and chemical composition were examined using various techniques. Twenty-four implants were inserted into the proximal tibia of four New Zealand white rabbits. The biological responses were measured in terms of histomorphometric analysis 4 and 8 weeks post-implantation.
RESULTS: The surface morphology and roughness were similar among all groups. However, the concentration of OH groups and hydrophilicity were found increased in the plasma treatment. The cell morphology in RF-plasma treated groups had more polygonal type and higher expression of actin and vinculin. The bone-to-implant contact (BIC) ratios of RF-200W were significantly higher than other groups (P < 0.05). The relationship between basic OH groups and BIC showed linear correspondence.
CONCLUSION: The Ti-OH groups introduced on the rough surface by plasma treatments can trigger cell adhesion which further initiate new bone apposition. We propose that RF-plasma treatment can help to enhance bone healing at 4 and 8 weeks.

Introduction

Artificial implants with good osseointegration is an important factor in the success of implantation. Osseointegration refers to bone-implant interface bonding. Direct contact between an implant and surrounding bone often depends on the implant surface properties.1, 2, 3 Sand-blasting with large grit and acid etching (SLA) is commonly used as a surface treatment because it produce a high contact ratio with micron and submicron roughness surface. SLA is often used in commercial implant surface treatment; however, since bone healing time takes about 8 weeks, the surface properties need to be further optimized. Compared with SLA treated implants, those with chemically modified SLA (modSLA) can reduces healing time to 3–4 weeks because the surface becomes more hydrophilic. Therefore, to speed up osseointegration, plasma treatment can be considered SLA-implant surfaces to introduce functional groups and enhance hydrophilicity.

Study have shown that hydrophilic SLA surface influence osteoblast behavior by altering protein absorption and focal adhesion, which can directly induce cell differentiation through a subsequent intracellular signaling cascade. Additional surface plasma treatment showed many advantages like introduce functional groups, change wettability, and eliminate contamination, making it an ideal and effective modification method for improving physicochemical properties. The amount of surface hydroxyl (OH)s groups that contains acidic OH groups and basic OH groups increased by plasma treatment; such groups are a key factor in osteoblast–titanium interaction. After plasma treatment, the surface becomes hydrophilic and contains abundant hydroxyl OH groups. Cells live on the OH-rich surface exhibit excellent growth and differentiation.9, 10, 11 Canullo et al. reported that plasma treatment can improve protein adsorption and cell adhesion on a rough titanium substrate. MacDonald et al. used radio-frequency plasma treatment to increase shear strength (determined using a pull-out test) and increase implant-femur bonding.

The success of a clinical implant is mainly determined by the bonding strength and osseointegration between the implant and new surrounding bone tissues. In our previous study, the plasma treatments of large grit and acid etching (SLA) method produced functional OH groups on the rough surface. Since there is no report in discussing the integration between basic Ti—OH groups and bone-to-implant contact (BIC). The present study uses simple and quick plasma treatment to introduce functional OH groups onto an implant surface and examines the cell cytoskeleton in vitro and bone-to-implant contact in vivo. The surface properties of plasma- and SLA-treated samples were also examined. Cell adhesion was investigated in vitro using immunofluorescence staining. Finally, bone-to-implant contact was measured to evaluate bone healing in a rabbit model.

Materials and methods

Sample preparation

Commercially pure Ti discs (cpTi, ASTM F67, Grade II) measuring 12.7 mm in diameter and 2 mm in thickness were used in vitro. A pure titanium implant measuring 11.52 mm in length and 2.48 mm in diameter and with a small head was used in vivo. The discs and implants were sand-blasted with large grit Al2O3 (particle size: 355–425 μm) at a pressure of 4 kg/cm2. Then, a mixed acid (HCl/H2SO4 in ratio of 1:3) was used to treat the samples for 30 min at 80 °C. The samples were then washed with acetone, ethanol, and distilled water in sequence.

Plasma treatments

The SLA-treated samples (flat specimens and implants) were separately modified using plasma treatments under an atmosphere of oxygen. The DC and RF plasma treatment procedures followed those reported in a previous study. The plasma-treated specimens were placed in the oxygen atmosphere for more than 10 min and then used absolute alcohol to keep its original properties before being analyzed.

The samples are denoted as follows: control (only SLA), DC-50W (SLA-treated titanium treated using DC plasma at 50 W), RF-50W (SLA-treated titanium treated using RF plasma at 50 W), and RF-200W (SLA-treated titanium treated using RF plasma at 200 W). The wettability, functional OH groups, and protein adsorption values are listed in Table 1. The surface roughness was evaluated using a surfcorder (SE 1200; Kosaka Laboratory Ltd., Japan). The surface morphology was examined using scanning electron microscopy (SEM, JSM-6390LV, JEOL, Japan).

Table 1

Surface characteristics of control, DC-50W, RF-50W, and RF-200W (n = 5).

	Roughness (μm)	Contact angle (°)	XPS analysisa			Protein adsorptiona (μg)
			Basic Ti—OH (%)	Acidic OH (%)	O1s (%)
Control	2.63 ± 0.25	112 ± 6	10.00	8.21	81.79	0.23
DC-50W	2.57 ± 0.28	<10	19.08	29.80	51.12	0.26
RF-50W	2.81 ± 0.17	<10	21.91	25.70	52.39	0.34
RF-200W	2.71 ± 0.34	<10	33.05	23.86	43.09	0.47

From our previous study.

Cell cytoskeleton development

A fluorescence microscope (LSM780, Zeiss, Germany) was used to observe the cell morphology and cytoskeletal distribution. Cells were cultured at a seeding density of 5 × 103 cells/cm2 on the specimens (four groups). Vinculin was stained using a mouse monoclonal antibody (Sigma, USA) and anti-mouse IgG conjugated with Alexa fluor 594 (Molecular Probes, USA). F-actin was labeled with Alexa fluor 488-labled phalloidin (Molecular Probes, USA). The samples were stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear dye and finally fixed using ProLong® Gold antifade reagents (Molecular Probes, USA) before fluorescence microscopy analysis.

Animals and surgical procedure

The animal experiment followed the animal approval (IACUC #103305) of National Cheng Kung University (Tainan, Taiwan). Four adult male New Zealand white rabbits weighing 3.0–3.5 kg were used in this study. The surgical process was based on that published in a previous study. The animals were anesthetized intramuscularly with a mixture of tiletamine/zolazepam (Zoletil, Virbac, France) and xylazine (Rompun, Bayer, USA). Three implants were implanted into each tibia. All implants were inserted through the first cortical layer. After surgery, all the rabbits were injected with topical analgesics and antibiotics. All the animals were kept in individual cages in the animal experiment center of National Cheng Kung University. They were given the standard laboratory diet. 4 or 8 weeks post-implantation, the rabbits were given an overdose of tiletamine/zolazepam (Zoletil) (10 ml, i.m.); the rabbits were then euthanized using potassium chloride (10 ml, i.m.). Two rabbits were euthanized for each time period (4 and 8 weeks).

Histological and histomorphometric evaluations

After the animals were euthanized, the tibia of two rabbits were removed at the specified time periods for histomorphometric analysis. The sample preparation protocol followed Lillie's method. Histomorphometric analysis was conducted and bone-to-implant contact (BIC) ratio was calculated based on backscattered electron image of scanning electron microscopy BEI-SEM (JSM-6390LV, JEOL, Japan). The BIC ratio (%) was calculated by dividing the length from implant head edge to the second thread by the distance of the bone in direct contact with the implant surface. The BIC ratio of the implant was calculated using the software Image J (National Institutes of Health, USA) after image processing.

Statistical analysis

The BIC data are shown as means ± standard deviation (SD). Significant differences between control, DC plasma treatment, and RF plasma treatment groups were determined using one-way analysis of variance (ANOVA). The data were evaluated using Statistical Analysis System software (SAS; SAS Institute Inc., Cary, NC, USA). Results were considered significant at P < 0.05. The Duncan test was used to determine the significance level to specify treatment effects.

Results

Surface characterization

Fig. 1 SEM images showed all samples have the similar surface structure, namely a mountain and valley structure with micron- and submicron-scale notches. The morphology was the same for all groups and the surface roughness values, determined by the surfcorder, showed no statistically difference (P > 0.05). Wettability was estimated using the contact angle test. The mean contact angle of the control was 112°; a contact angle of over 90° indicates hydrophobicity. The plasma-treated samples had a contact angle of 0° (see Fig. 1), indicating that they were hydrophilic (Table 1). The OH group ratios, determined using X-ray photoelectron spectroscopy (XPS), were cited from our previous study (see Table 1). Result showed that the basic Ti—OH ratio increased as plasma power increase. RF-200W had the highest basic OH ratio among the four groups. The acidic OH content increased after DC or RF plasma treatment. RF-200W had the highest OH group content. In this study, DC and RF plasma samples with various OH group content levels were used to examine the effect of functional OH groups in vitro and in vivo.

Figure 1

SEM morphology images of A. control (only SLA), B. DC-50W, C. RF-50W, and D. RF-200W. All specimens show mountain and valley structure with micron- and submicron-scale notches. Morphologies are the same but surface becomes hydrophilic after DC or RF plasma treatment.

Cell adhesion and spreading examined using immunofluorescence

A fluorescence microscope was used to examine cell morphology and cytoskeletal organization. MG63 cells fluorescence images cultured on the four kinds of specimen for 1 and 24 h are shown in Figure 2, Figure 3, respectively. The cells show the complex focal adhesion characteristic pattern with dense phalloidin-stained f-actin (green) and vinculin (red).

Figure 2

Fluorescence images of MG63 cells cultured on four types of surface after 1 h of incubation: A. control, B. DC-50W, C. RF-50W, and D. RF-200W. F-actin (green) was stained with Alexa Flour 488 phalloidin; vinculin (red) was stained with Alexa Fluor 594; nucleus (blue) was stained with DAPI. Actin filaments and vinculin expression are abundant after RF plasma treatment.

Figure 3

Fluorescence images of MG63 cells cultured on four types of surface after 24 h of incubation: A. control, B. DC-50W, C. RF-50W, and D. RF-200W. F-actin (green) was stained with Alexa Flour 488 phalloidin; vinculin (red) was stained with Alexa Fluor 594; nucleus (blue) was stained with DAPI. MG63 cells on RF-200W showed elongated morphology; cells in control group showed round morphology.

After 1 h, the MG63 cells were attached to all surfaces. Compared with the control surface, the cells were rounder and had a smaller adhesion area than the plasma treatment groups (Fig. 2). For RF-50W and RF-200W, actin filaments appeared at the edges of MG63 cells. On RF-200W, the MG63 cells are well spread and formed noticeable focal adhesions. Cells cultured on the control and DC-50W specimens showed few stress fibers, indicating limited interaction with the substrate (Fig. 2A,B). The actin filaments exhibited a flattened polygonal morphology spread over a wider area compared to control (Fig. 2C,D).

After 24 h, cells on the RF-50W and RF-200W specimens exhibited long extensions of cytoplasmic membranes, making the cells fully spreading out shape (Fig. 3C,D). Vinculin-positive focal adhesions were observed in the cell plasma and elongated projections (Fig. 3C,D). The cell shape at 1 h (round) was different from that at 24 h (elongated) in all groups. The plasma treatment also induced the extension of the lamellipodia on RF-200W specimens (1 h vs. 24 h; Fig. 3C, white arrow).

Bone-to-implant contact evaluation

At 8 weeks, the backscattered electron image of scanning electron microscopy (BEI-SEM) image of an RF-200W implant are shown in Fig. 4. Fig. 4 shows that bone tissues were ingrowth well on the thread of the implant. RF-200W implants displayed good direct bone-to-implant contact in most regions of the implant surfaces.

Figure 4

BEI-SEM image of RF-200W. New bone formation is dense and bone contact ratio is high at 8 weeks.

At 4 weeks (see Fig. 5B), there were statistically no significant differences between paired specimens (RF-200W, RF-50W) and (RF-50W, DC-50W); whereas the differences among all other specimen pairs were statistically significant. At 8 weeks, the BIC ratio of RF-200W showed highest value (P < 0.05). There was no significant different in BIC ratio among the control, DC50W, and RF-50W specimens.

Figure 5

A. Bone-to-implant contact ratios after 4 and 8 weeks of healing. Bars labeled with underlines indicate significant difference between groups according to Duncan test (n = 3, P < 0.05). Duncan grouping of BIC at B. 4 weeks and C. 8 weeks.

Discussion

The loading condition is positively correlate with good osseointegration. The osseointegration rate of titanium dental implants depends on implant composition and surface roughness. Previous studies reported that SLA treatment of implants leads to better bone healing at 4 or 6 weeks post-implantation than other treatments (polishing or only acid etching). The modSLA can improve bone healing thus shortening the healing time.17, 18, 19 In this study, a rough surface with micron and submicron structures was created using the SLA method (Fig. 1). This plasma treatment did not affect surface roughness. The roughness values for the four groups showed no statistically difference (Table 1). In addition, the functional OH groups on the SLA-roughened surface did not change the contact angle. These results are consistent with the previous research.,,, This implied that the functional OH groups had a great effect on wettability and thus enhanced the biological response. Most studies have end discussion in in vitro cell behavior, few continue to discuss the effect of OH groups in vivo.

In this study, the MG63 cells cultured on RF-50W and RF-200W exhibited more spreading (Fig. 2) compared to the control group. Plasma treatment can improve cell adhesion, particularly on the hydrophilic surface. The cytoskeletal organization, governs osteoblastic cell adhesion, may be influenced by a rough titanium surface. Feng et al. reported that the basic hydroxyl (OH)b groups and the polar component are more important than the acidic hydroxyl (OH)a groups and the dispersion component of the total surface energy. Thus, (OH)b groups play a crucial role in osteoblast–titanium interaction. Titanium surfaces with dissociated (OH)b groups have more positive charges. Previous studies reported that a high content of OH groups, a rough structure, and a charged surface can induce bone-related cells to express adhesion proteins, especially actin and vinculin.22, 23, 24 The initial attachment of cells on the substrate is a key factor in biomaterial design and development.

The protein adsorption of DC-50W and RF-50W are statistically different in our previous study shown in Table 1. Cell behavior including cell attachment and spreading is strongly influenced by the protein adsorption. Fig. 2B,C showed attachment morphology containing actin filaments and there is more actin filaments of RF-50W than DC-50W at 1 h although the Ti—OH ratio showed no significant difference. The cell cytoskeleton was both flatter for RF-50W and RF-200W at 1 h but RF-200W expressed higher actin filaments on at 24 h. The in vitro cell response may be correlated with in vivo bone healing and regeneration. This means that good cell adhesion triggers subsequent responses, including new bone formation. Dense bone formation appeared on DC-50W, RF-50W, and RF-200W samples, indicating better osseointegration than the control. However, at 8 weeks, only RF-200W showed statistically difference (Fig. 5), which may be due to the different type of plasma treatment and plasma power. At both 4 and 8 weeks, there was a linear correlation between basic Ti—OH and the BIC ratio (Fig. 6).

Figure 6

Effects of basic Ti—OH on BIC ratio. Increasing basic Ti—OH increased BIC ratios.

In conclusion, plasma treatment was used to introduce functional OH groups on SLA-treated titanium surfaces without changing the surface topography and roughness. The abundant functional OH groups made the surface hydrophilic, enhancing cell adhesion and promoting the cytoskeleton to express focal adhesions. BIC analysis showed RF plasma treatment created surface with good cell affinity and in vivo examines more new bone formation than other groups. Furthermore, the abundant OH groups introduced by plasma treatments and basic Ti—OH groups had positive effects on BIC. Moreover, the basic Ti—OH showed a greater effect on bone regeneration. We propose that RF-plasma treatment can help to enhance bone healing at the early stage.

Conflicts of interest

The authors have no conflicts of interest relevant to this article.