We compared DMF and Titanium plasma spray (TPS) coatings of CoCr alloy surface both in-vitro and in-vivo, to find if there is difference in terms of cell morphology, biocompatibility, cell proliferation rate, shear strength and histomorphometry. For in vitro study, with AM technology-based DMF method, Pure Ti (CPTi powder grade 2, ASTM F1580) powders between size of 45-150um were melted and laminated using selective laser on CoCr alloy surface. Computer assisted design (CAD) program was used prior to executing actual coating process to design porous structure to simulate the porous properties of cancellous bone (NX-based coating CAM for Insstek, Siemens). Laser irradiated surface of CoCr alloy formed a melted pool, by following the path of a pre-programmed grid-shaped tool with 80W laser power, 1.5 m/min scan speed, and 2.2 g/min power delivery rate. Next, metal powders were sprayed and laminated onto the melted surface, which is different from selective laser melting (SLM) and electron beam melting (EBM) (15). To give porous surface an irregularity of thickness and shape, coating layer was twice coated; once with thickness of 300 um and then with thickness of 500 um. In plasma spraying method which we utilized to compare to DMF, Ti powder for coating was injected into plasma gas stream which is heated up to 20,000℃. With high kinetic force, powder was shot onto the substrate and then melted, forming porous structure. Scanning electron microscopy was used to assess the structure and morphology of the produced surfaces (11).
Osteoblasts derived from human mesenchymal cells were prepared (18). 5×104 osteoblasts were seeded onto DMF and TPS CoCr coating specimens. After 6 h of seeding of cells in each implant, the media was removed and then the cells were washed with PBS 3 times. After adding 2% glutaraldehyde-PBS solution, these cells were stabilized for 2 hours. The cells were then washed with dextrose water solution 3 times. At 30-min intervals, the cells were dehydrated with 50%–100% ethanol solutions. The ethanol was removed, and the cells were left at room temperature to allow for complete ethanol evaporation. Two surfaces were then characterized by scanning electron microscope (JEOL JSM-6700F; JEOL, Ltd, Tokyo, Japan) after the test specimens had been coated with platinum.
The seeded cells on the coated surfaces were incubated for 24, 48, 72, and 96 hours. The medium was replaced with fresh medium before measuring cell proliferation using the Cell Titer 96 Nonradioactive Cell Proliferation Assay (Promega Corp, Madison, WI), according to the manufacturer's instructions. Cell proliferation assay is a colorimetric method for determining the number of viable cells. In this study, the number of viable cells was measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek Instruments, Inc, Winooski, VT) (19).
The differentiation of osteoblast cells was evaluated by immunofluorescence staining for the Runx-2 and fibronectin genes(5, 20). After 21 days of incubation, irrigation with PBS three times, and stabilization with 4% paraformaldehyde for 10 min, the cells were incubated to use primary antibodies to RUNX-2 and fibronectin (1:100, Abcam, Cambridge, England) overnight at 4°C. After incubation with primary antibodies, cells were incubated with secondary Alexa Fluor 594 goat anti-rabbit and mouse (Invitrogen, CA, USA) for 1 hour at room temperature. The cells were mounted with DAPI mount for 10 min, and the cells were washed with PBS. We confirmed the differentiation of osteoblast cells with colocalization by expression of DAPI, RUNX-2 and fibronectin under high-powered magnification via a confocal microscope (Olympus, Tokyo, Japan).
For in vivo study, 20 full-grown rabbits (>3.2 kg) were assigned as experimental subjects. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the institution. A DMF and a TPS coated rods were inserted surgically into intramedullary canals of each distal femur separately. Specimens were harvested 3 months after the surgery and push-out test and histomorphometric analysis was conducted. Each harvested distal femur was sliced at the two ends of the rods, and foreign substances were removed. To test shear strength of bone-implant interface of the products, jig of universal testing machine (Daekyung tech DTU-900MH30kN, Korea) was positioned vertically along the long axis of the rod and then push-out test was performed at a push rate of 1 mm/min (Fig. 1). The push strength was recorded until the rod became dissociated with the femur or breakage of the femur occurred (3).
The harvested bone tissue was dehydrated with alcohol in stages and soaked in Technovit 7200 resin (Heraeus Kulzer, Germany). The soaked tissue was embedded in paraffin for curing via a light system (Exakt, Germany). The block was sliced into 200-μm-thick sections with a hard tissue slicer (Struers, Germany). These sections were then stained with hematoxylin and eosin (H&E; Sigma-Aldrich). Microscopy images were obtained by x12.5, x40, x100 (BX51, Olympus, Japan). The specimens from each implant were analyzed by determining the percentage of direct contact between mineralized bone and the CoCr alloy surface from intersection counting, using an integrative eyepiece with parallel sampling lines at a magnification of x100 (21).
For statistical analysis, we compared the cell proliferation assays on the two surfaces, mean interfacial shear strength & bone-to-implant contact percentage of the two different surfaces using a Wilcoxon signed-rank test. Statistical analysis was performed using SPSS® 18.0 software (SPSS, Inc., Chicago, IL.).