We performed an in vitro study to demonstrate the effect of UV irradiation on the hydrophilicity of the HA surface, and an in vivo study to demonstrate the effect of UV irradiation on the osteoconductive ability of HA. In the in vivo study, HA implants were inserted into rat femurs, and radiological analyses using microcomputed tomography and histological analyses using undecalcified specimens were performed. The study protocol (ethical code number: M16018) was approved by the Animal Research Committee of Hirosaki University, and all experiments were performed in accordance with the Rules for Animal Experimentation of Hirosaki University.
Hydrophilicity of the hydroxyapatite surface
HA disks (diameter 15 mm, height 3 mm, 0% and 55% porosity: HOYA Technosurgical Inc., Japan) were used to evaluate the hydrophilicity of the HA surface. Four disks of each porosity were treated with UV irradiation for 15 minutes using a photo device (TheraBeam Affinity; Ushio Inc., Japan) (Fig. 1). The light source mounted in the TheraBeam Affinity is a low-pressure mercury (Hg) lamp, which emits 185-nm and 254-nm UV light. Four disks that did not undergo UV irradiation were used as a control.
To examine the change in hydrophilicity on the disk surface after UV irradiation, the wettability was evaluated by measuring the contact angle of a water drop . For this test, 10 μL of water was dropped onto the disk surface before UV irradiation, and at 0 minutes, one hour, three hours, six hours, two weeks, and four weeks after UV irradiation. Moving images were taken at a rate of 240 frames per second (fps) using a high-speed camera (Casio EXILIM EX-ZR 1000, Casio Computer Co., LTD., Japan). Using the still picture immediately after dropping, the angle of the water droplet with respect to the disk surface was measured by the θ/2 method . After the water drop landed on the implant surface, the height (a) and contact diameter (b) of the drop were calculated by image analysis software (ImageJ® v.1.48, National Institutes of Health, USA). Using these measurements, the contact angle (θ) was calculated with the following formula: θ= 2tan– 1 (2a/b). In this analysis, a low contact angle indicates surface hydrophilicity.
Osteoconduction at hydroxyapatite
Twenty HA cylinders (diameter 2.5 mm, length 8 mm, 55% porosity: HOYA Technosurgical Inc., Japan) were used for the in vivo study. Half of the cylinders were treated with UV irradiation for 15 minutes as described above. The remaining 10 cylinders were used without UV treatment as a control.
Ten eight-week-old male Sprague–Dawley rats were used for the animal experiments. The rats were anaesthetised with 1% to 2% isoflurane. Both hind limbs were shaved, and the skin and fascia layers were opened separately. The flat aspect of each distal femur was exposed and used for implantation. The right and left distal femurs were drilled using a 3-mm-diameter drill. UV-irradiated HA implants were inserted into the right femur holes, and HA implants without UV irradiation were inserted into the left ones. After implant placement, the skin and fascia were closed. Two or four weeks after the surgery, the rats were killed by intraperitoneal injection of pentobarbital, and the femurs were harvested. Five specimens were included in each group.
The specimens were fixed in 10% buffered formalin and analysed using microcomputed tomography (Scan Xmate-L090, Comscantecno Co.,Ltd., Japan). The imaging conditions were as follows; voltage: 80kV, current: 100μA, magnification: 4.942 times, resolution: 20.234μm/pixel, and slice thickness: 20.234μm. Three-dimensional bone morphometric analysis was performed using the software (TRI/3D-BON, RATOC system engineering Co., Ltd., Japan). The mineralized bone volume (BV) ratio and tissue volume (TV) within 100 μm from the implant surface were evaluated. The BV/TV ratio (also called the bone volume fraction), which is an important parameter for evaluating the microstructure of bone, was calculated as the bone volume (%) in this area.
After microcomputed tomography, the specimens were embedded in methyl methacrylate without decalcification. The embedded specimens were then cut perpendicular to the long axis of the implant using a microtome. Each section was stained with Villanueva–Goldner to evaluate the bone area, which was stained green and observed by light microscopy (BZ-X700, Keyence Corp., Japan). To evaluate bone formation around the HA, the bone area (green) for each group was measured in ring-shaped regions 100 μm outside and 100 μm inside the HA surface by digital image analysis software (Image J® v.1.48). The bone area ratio was calculated as the bone area divided by the total measured area (the HA area was subtracted), multiplied by 100 (%).
Two-way analysis of variance with Tukey’s post-hoc test was performed to determine differences in the water-drop contact angle. The Wilcoxon signed-rank test was performed to determine differences in the bone volume and bone area ratios between the UV(−) group and UV(+) group at two weeks or four weeks, respectively. The Mann-Whitney U test was also performed to determine differences in the bone volume and bone area ratios between at two weeks and four weeks within each group. Statistical analyses were performed using SPSS (v 22.0; IBM), and p values < 0.05 were considered significant.