Commercially available Ti6Al4V plates (10 × 10 × 2 mm; Kobe Steel Ltd., Kobe, Japan) and Ti6Al4V miniscrews (Φ 1.4 × 4 mm, n = 32; Jeil Medical Corp., Seoul, Korea) were used in this study. To produce a Ca-P coating and a nanotubular surface on the alloy, samples were subjected to APH treatment as previously described.13 Nanotubular arrays were formed by anodization at 20 V for 1 hour in a solution of Glycerol/H₂O/NH₄F (79, 20, and 1 wt% respectively). Ca-P particles were loaded into the arrays by cyclic pre-calcification, which involves soaking the samples repeatedly first in NaH₂PO₄ (0.05 M, 80℃) and then saturated Ca(OH)₂ (100℃) for 1 min at a time. In this case, 30 cycles were completed before a final heat-treatment at 500℃ for 2 hours. Untreated (UT) specimens and TiO₂ nanotubular (AH) specimens (which were subjected to anodization and heat-treatment, but not to cyclic pre-calcification) were prepared as control groups.

The topographical characteristics and chemical composition of the resulting surfaces were observed with a field emission scanning electron microscope (FE-SEM, S-800; Hitachi, Tokyo, Japan) equipped with an energy dispersive X-ray spectrometer (EDS; Bruker, Billerica, MA, USA). X-ray diffraction (XRD, Dmax III-A type; Rigaku, Tokyo, Japan) was used for phase analysis.

As a test of their bioactivity, the samples were immersed in SBF for 3 days while looking for hydroxyapatite (HA) precipitation. The SBF solution was prepared using Hanks' solution (H2387; Sigma-Aldrich Chemical Co., St. Louis, MO, USA), with addition of calcium chloride dihydrate 0.185 g/L, magnesium sulfate 0.09767 g/L, sodium hydrogen carbonate 0.35 g/L (pH 7.4). Changes in the surface microstructure were studied by FE-SEM and EDS.

The mean surface roughness (Ra) was obtained from three repeat measurements using a surface roughness tester (Surftest SV-3000; Mitutoyo Corporation, Kawasaki, Japan). The hydrophilic properties of the surfaces were evaluated by measuring the contact angle of 10 µL SBF solution dropped from 5 mm above the sample surface. Images were obtained by stereoscopic microscope (EZ4D; Leica Microsystems CMS GmbH, Wetzlar, Germany) and the contact angles were measured using Screen Protractor 4.0 (Iconico Inc., New York, NY, USA).

The study was conducted in compliance with the principles of the Helsinki Declaration and ethical clearance was obtained from the Institutional Animal Care and Use Committee of the Chonbuk National University Laboratory Animal Center (approval number: CBU 2012-0027). Experiments were conducted on the tibias of 16 male Wistar rats (250-280 g, 7 weeks old) using 16 UT and 16 APH-treated miniscrews. Miniscrew implantation surgery was performed under general anesthetic using 75 mL/100 g intramuscular zolazepam-tiletamine (Zoletil 50; Virbac, Carros, France) and additional local anesthetic was applied on the surgical site using 2% lidocaine with epinephrine (1:100,000). An incision of 1.0 cm length was made, and the bone surface of the tibia was surgically exposed with a periosteal elevator. The cortical bone was drilled with a 1.2 mm pilot drill bur (Jeil Medical Corp.) at 250 rpm under copious saline irrigation. Miniscrews were inserted into the medial region of the bilateral tibia diaphysis using a self-tapping process until the screw thread was completely implanted in the bone cortex. For each rat, one UT and one APH-treated miniscrew were placed on opposite tibias. The surgical wound was sealed by 3-0 suture silk. Postoperatively, antibiotics (amoxicillin, 6 mg/kg) and anti-inflammatories (nabumetone, 5 mg/kg) were administered orally. Eight rats were sacrificed, both at 3 and 6 weeks after insertion, with an overdose of thiopental (JW Pharmaceutical Corp., Seoul, Korea).

Note that in clinical practice, there are two approaches to loading miniscrews: self-drilling (without pilot drilling) and self-tapping (with pilot drilling).5,9,17,18 The advantages of the self-drilling method are its ease of use, low mechanical involvement, intensive bone-implant contact, and low thermal damage.5,18 However, it tends to cause more microdamage to the cortical bone than the self-tapping method,9,17 and because friction during insertion could have impaired the surfaces, these factors led us to follow the self-tapping approach.

The removal torque value (RTV) was measured at 3 and 6 weeks after insertion. The implant sites in the rat tibia were surgically exposed to the bone and examined after carefully removing any overgrowing bone and soft tissue. Removal torque tests were performed using a digital torque gauge (9810P; Aikoh Engineering Co. Ltd., Tokyo, Japan) with a precision of 0.1 N·cm (n = 6 for each group). After stabilizing the legs, the torque was increased incrementally by slowly rotating the gauge counterclockwise. The peak torque value was recorded by a single examiner when rupture occurred between the miniscrew and the bone. After this test, the extracted miniscrew surfaces were examined with FE-SEM and EDS.

Two miniscrew-containing tibias were removed from both the 3- and 6-week groups, fixed with 10% formalin, stained in Villanueva bone, dehydrated in a series of increasing concentrations of alcohol, and embedded in methyl methacrylate. Subsequently, the blocks were cut parallel to the miniscrew axis and ground to make 40-µm thick sections. The miniscrew-bone interface was examined and bone-implant contact (BIC%) was measured on 5 threads using an optical microscope (DM 2500M, Leica Microsystems CMS GmbH) equipped with the Leica Application Suite (version 1.6.0; Leica Microsystems AG, Heerbrugg, Switzerland) under 100× magnification. BIC% values were determined as the percentage of direct contact between mineralized bone and the miniscrew surface within each thread. Five threads from two miniscrews from both groups were chosen randomly for this evaluation.

After calculating the means and standard deviations of the contact angle, Ra, RTV, and BIC%, independent t-tests were used to compare those results between groups and measuring periods, with the threshold for statistical significance set at a p-value of 0.05. All statistical analyses were carried out using SPSS sortware ver. 12.0 (SPSS Inc., Chicago, IL, USA).

Figure 1A shows the shape of the Ti6Al4V miniscrews used in this study. After anodization, their surface consisted of a highly compact and ordered arrangement of nanotubes, as evidenced in Figure 1B and 1C. As described above, cyclic precalcification was performed in order to induce precipitation of apatite-like Ca-P on these nanotubes. Figure 2A shows that the HA precipitation obtained on nanotubular Ti6Al4V was dense and homogeneous. As shown in the side view (Figure 2B), not only did HA precipitate densely onto the surface layer, but it also filled out empty spaces in the nanotubes.

To evaluate surface bioactivity, the samples were immersed in SBF solution for 3 days. Figure 2C and 2D show that APH samples are densely covered in HA protuberances over the entire surface. Furthermore, EDS analysis revealed Ca and P concentrations of 29.78 ± 2.64 wt.% and 14.70 ± 0.97 wt.%, respectively, such that the Ca/P ratio (1.59) was close to that of HA (1.67). This suggests that APH surfaces accelerate the precipitation of HA, implying high bioactive ability. Likewise, XRD data, presented in Figure 3, reveal the presence of octacalcium phosphate and HA in the APH samples.

Table 1 shows that for all groups (UT, AH and APH), the surface roughness is inversely correlated with the contact angle. Moreover, the surface roughness, and thereby the hydrophilicity, is increased by AH and APH treatments with the latter showing the greater effect.

Figure 4 presents the RTVs of UT and APH groups that were measured 3 and 6 weeks after insertion. The torque values are higher for the APH group at both 3 and 6 weeks than for the UT group (4.8 ± 2.7 Ncm versus 2.3 ± 1.2 Ncm at 3 weeks; 8.4 ± 3.1 Ncm versus 4.1 ± 1.4 Ncm at 6 weeks; p < 0.05).

Subsequently, the surfaces of the extracted miniscrews were examined with FE-SEM and EDS. For the UT group, both after 3 weeks (Figure 5A(a)) and 6 weeks (Figure 5A(c)), the surface predominantly shows interface fracture patterns between new bone and miniscrews. In contrast, the surfaces of the APH group show cohesive fracture patterns within newly formed bone at both stages (Figure 5A(b) and 5A(d)). The Ca and P concentration levels for the UT group are low, and the Ca/P ratio decreased from 3 to 6 weeks after insertion, from 1.21 to 0.50. On the other hand, those for the APH group remain high with a Ca/P ratio of 1.45 at 6 weeks, similar to that of HA.

After 3 weeks, very little bone formation is observed in Figure 6A(a) for the UT miniscrews, with a very low BIC% value (8.25 ± 6.67%), meanwhile the surface of the APH-treated miniscrews, shown in Figure 6B(b), is almost entirely covered with newly formed bone and the BIC% is 84.00 ± 8.47% (p < 0.05). After 6 weeks, bone adhesion is enhanced for both groups, with BIC% values of 61.75 ± 12.81% for the UT group (Figure 6A(c)), and 91.50 ± 3.58% for the APH group (Figure 6A(d)). Significant bone formation therefore occurs for both groups (p < 0.05), but the APH surface shows better and faster ossointegration (p < 0.05). This suggests that APH treatment leads to faster osseointegration and stabilization of the miniscrew.