Fabrication And Bioactivity Evaluation Of Micro/nano Strontium Loaded Surface Titanium

[Background]Dental implant treatment has become the most ideal treatment for the edentulous patients owing to the comfort and esthetic results. However, there are two important problems affecting the use of dental implant clinically: For one thing, some edentulous patients need implantation always accompanied with bone metabolic disease, for example, osteoporosis(OP) which characterized as reduced formation of bone mass and the deterioration of bone microarchitecture. For another, traditional cycle for implant treatment always extend as long as 6 to 12 months from tooth extraction surgery to prosthesis completion, the overlong duration would result in convenient for the patients. Hence, various studies have focused on above issues how to improve the implant osseointegration under osteoporotic conditions and shorten the treatment cycle for implantation prosthesis.Surface modification is one of the most effective methods to enhance the rate and extent of osseointegration. The surface modification mainly divided into two categories: surface roughness modification and surface chemically modification. Surface roughness modification is the strategy to increase the roughness of implant surface via physical or chemical method. Depending on the scale of the features, the surface roughness of implants can be divided into three: macro-, micro-, and nano-sized topologies. Recently, implant with nano-sized topologies have earned much attention and become a development tendency in the field surface modification. Moreover, a novel viewpoint proposed that a surface with hierarchical micro/nano-textured would possess a biomimetic function. The biomemetic viewpoint considered that hierarchical micro/nano structure which simulated the natural tissues assembled in a highly organized way composed of nano-, micro-, and macro-scale building blocks would be more favorable to promote osteogenesis around implant than micro- or nano- scale alone. Therefore, nano and micro/nano would be the research orientation of surface roughness modification. Surface chemical modification is another strategy to promote osteogenesis around implant via loaded osteogenesis chemical element. Among various chemical elemrnt, strontium have earned much ateetion for its dual effects of promoting bone formation and reducing bone resorption. And strontium ranelate was an effective drug on osteoporosis in clinic. Local application of strontium on implant surface is an alternative method to enhance implant osseointegration and avoid the potential adverse reactions simultaneously. Thus, fabricating strontium on implant surface has been of great interest as a method for modification of chemistry to promote osseointegration.By considering above information, we attempted to combine the advantages of roughness in micro/nano scale and modification of chemistry with strontium loading to promote osseointegration. Therefore, we developed implants with a hierarchical micro/nano topography loaded with strontium, which were treated with HF etching to form the micro surface and later combined with magnetron sputtering to form the nano-scale strontium coating. And in order to compare the bioactivities of surface differ in roughness and chemistry under normal and osteoporotic conditions, we also prepared four surfaces that differ in roughness and chemistry and evaluated each effects on osseointegration via an in vitro cellular study and in vivo animal experiment. Meanwhile, we also attempted to evaluate the activities of the NT-Ti O2 and NT-Sr surface and compared the effects on osseointegration immediate implantation after tooth extraction. [Purpose]We prepared four surfaces that differ in roughness and chemistry and compared the effects of these surfaces on osseointegration via an in vitro cellular study and in vivo animal experiment. And we also attempted to evaluate the bioactivity of the micro/nano Sr-loaded surface developed using HF-acid etching combined with magnetron sputtering. Furthermore, we compared the effects of NT-Ti O2 and NT-Sr on osseointegration immediate implantation after tooth extraction. [Methods]1.Smooth Ti(ST), which is polished Ti without further treatment; micro-textured Ti(MT) formed by treatment in 0.5%(w/v) HF for 30 min; Sr-loaded nano-textured Ti(NT-Sr) formed by magnetron sputtering, and Sr-loaded micro/nano-textured Ti(MNT-Sr) fabricated by HF-etching combined with magnetron sputtering. Magnetron sputtering was performed with an industrial PVD system(SKY Technology Development Co. Ltd, Shengyang, China), with material deposited from a magnetron consisting of Sr Ti O3 powder with a purity of 99.99%. The films were deposited under the following conditions: 80 W power density, 7200 s deposition time, and 10 cm target–substrate distance. After preparation, all the Ti disks were ultrasonically cleaned with acetone, ethanol, and deionized water, sequentially, and then sterilized with the cobalt-60 irradiation. The Ti samples were characterized using field-emission scanning electron microscopy(FE-SEM, HITACHI S-4800), atomic force microscopy(AFM, Innova, Vecco Instrument), X-ray diffraction(XRD, Philips X Pert Pro) and X-ray Photoemission Spectroscopy(XPS, ESCALB MK-II). The interface bonding strength of Sr loaded layers were evaluated by Epoxy resin docking tensile test. The surface hydrophilicity of the Ti samples was measured using a surface-contact-angle measurement machine(DSA30, Kruss, Germany). The PBS solution containing released Sr was analysed using inductively coupled plasma atomic emission spectrometry(ICP-AES, IRIS Advantage ER/S);2.The murine osteoblast cell line MC3T3-E1 were incubated on ST、MT、NT-Sr、MNT-Sr surfaces. Cell adhesion experiment, MTT assay, cell morphology observation via SEM and immunofluorescent staining, ALP staining analysis were carried out to evaluate the in vitro bioactivities of osteoblast, including cell adhesion, proliferation, proliferation;3.The primary bone mesenchymal stem cells(BMSCs) from rat were cultured on ST、MT 、 NT-Sr 、 MNT-Sr surfaces. Cellular function including adhesion, proliferation, proliferation were analysed via cell adhesion experiment, MTT assay, SEM and immunofluorescent staining observation, ALP staining and ECM mineralization analysis;4. Implants with ST、MT、NT-Sr、MNT-Sr surfaces were inserted into the distal femurs of normal rats with one implant for each femur. 3 months after implantation, the animals were sacrificed, and the femurs with implants were retrieved for Micro-CT scanning, histological analysis, fluorescent-labelling observation, and biomechanical test. In order to evaluate new bone deposition by using fluorescent labelling, tetracycline, and calcein were intraperitoneally injected 3 and 14 days before sacrifice, respectively;5. Bilateral ovariectomy(OVX) was performed on six months old female rats, scanning was used to OP animal model approval 3 months after ovariectomy surgery. And subsequently, implants with ST、MT、NT-Sr、MNT-Sr surfaces were inserted into the distal femurs of OP rats with one implant for each femur. 3 months after implantation, all the animals were sacrificed for Micro-CT scanning, histological analysis, fluorescent labeling observation and biomechanical test, and Tetracycline and Calcein were intraperitoneal injected 14 d and 3d respectively before sacrificed to evaluate the periods of new bone deposition;6. Sr-loaded nano-textured Ti(NT-Sr) formed by magnetron sputtering technique with 99.99% Sr Ti O3 as sputtering target. The Ti O2 nanotubes(NT-Ti O2) are fabricated by anodization in 0.5 wt% HF. SEM, contact angle measurement and epoxy resin tensile test were used to evaluate surface character, hydrophily and interface bonding strength; After material analysis, Implants with NT-Sr and NT-Ti O2 surfaces were inserted into the fresh socket immediately after teeth extraction, then Micro-CT scanning, histological analysis and biomechanical test were carried out to investigate the implant ossointegration. [Results]1. There is no obvious micro or nano texture on the ST surface. The MT sample formed with etching treatment in 0.5% HF solution has a micropitted texture with F element. The NT-Sr sample formed with magnetron sputtering treatment has a layer of nanodots with diameters of approximately 20~40 nm. The XPS analysis confirms the Sr loading on the NT-Sr surface. The MNT-Sr sample formed with HF etching combined with magnetron sputtering possesses a micropitted texture on which there is an even distribution of nanodots with diameters of approximately 20~40 nm. Moreover, the cross section of NT-Sr and MNT-Sr samples from SEM scanning indicated that the thickness of Sr layer were about 20-40 nm. The XPS and XRD results confirmed that amorphous Sr loaded on the MNT-Sr surface. AFM results shown that significant differences in the surface roughness were observed among the different Ti samples, as characterized by the surface area difference, root mean square roughness(RMS), and Z range. Generally, MT, NT-Sr, and MNT-Sr show greater surface roughness than ST does, in the order of MNT-Sr > MT > NT-Sr. Furthermore, the interface bonding strength of NT-Sr and MNT-Sr samples were 59.3 ± 7.6MPa and 51.2 ± 4.05 MPa, respectively. The contact angles of the water droplets on the ST as well as the MT, NT-Sr, and MNT-Sr surface are 46.58±4.43°, 15.90±2.54°, 27.69±5.27°, and 17.03±3.58°, respectively. The Sr release kinetics is assessed by immersing the Sr containing samples(NT-Sr and MNT-Sr) in 5 m L of PBS for as long as 14 days. Compared with NT-Sr surface, the released Sr amount from the MNT-Sr surface was found to be similar but slightly higher at all the observation time;2. Compared with other three groups, more MC3T3-E1 osteoblast adhered on MT surface. MTT assay shown MT and MNT-Sr surfaces possessed better cell proliferation at 4d, and the difference become more obvious on MNT-Sr surface at 7d. SEM and fluorescence staining shown following results: ST and NT-Sr surface were more liable for cellular spread, while osteoblasts on NT-Sr surface have more lamellipodia form good intercellular junctions between adjacent cells; Osteoblasts on NT and MNT-Sr surface are polygonous in shape with abundant long lamellipodia and filopodia, while the lamellipodia on MNT-Sr group were stronger than that of MT;3. The cell numbers on MT seemed to be higher than those for the other three groups during the whole process. Furthermore, After 60 min of incubation, the numbers of adherent cells on NT-Sr were significantly higher than those for the smooth group. Although the numbers of adherent cells on MNT-Sr suface appeared higher than NT-Sr surface at 60 min and 120 min, the differences were not statistically significant. MT surface shown better cell proliferation than NT-Sr and MNT-Sr surface on day 4, 7, and the differences become more obvious on day 7, however, there was no statistically significant between MT and NT-Sr on day 4. Meanwhile, the OD value for the NT-Sr surface was higher than MNT-Sr surface statistically significant. BMSCs on ST surface mainly exhibited a spindle fibroblast-like shape and with bigger size than those of other groups, polygonous cells with more pseudopodia were observed on the MT and MNT-Sr surfaces, but the pseudopodia on MNT-Sr surface performed more stereoscopic and intercellular junction generated between the adjacent cells. In addition, although the BMSCs on nano surface shown similar flat polygonous shapes with ST surface, intimate intercellular junction were observed in NT-Sr surface compared to ST surface. Compared with other three groups, MNT-Sr surface lead to higher ALP activity induced the highest degree of mineralization of the four groups;4. Three months after implantation, Tb.Th, Tb.N and BV/TV in MNT-Sr group were higher than other groups; MT group was higher than NT-Sr group in BV/TV. The MAR was 1.6±0.45μm/d、2.32±0.59μm/d、2.92±0.58μm/d、3.66±0.62μm/d in ST、MT、NT-Sr、MNT-Sr, respectively and BIC% was 39.70±6.00% 、57.60±7.79%、46.10±5.51%、70.38±8.61%. For the maximum pull out force, the ST、MT、NT-Sr、MNT-Sr group possessed 108.9±46.50N、176.4±47.85N、136.9±21.65N、231.4±67.66N;5. Micro-CT scanning shown demonstrated OP rat model established successfully 3 months after OVX. 3 month after implantation, MNT-Sr group shown better osseointegration than other groups, although NT-Sr shown better MAR than MT, MT possessed higher BIC% and maximum pull out force;6. Nanotubes with diameter of 15~80nm distributed on NT-Ti O2 surface, while nanoparticles with 20~40nm disposited on NT-Sr surface; Compared to NT-Sr, Ti O2 surface possessed better hydrophily; And interface bonding strength of NT-Sr group was higher than NT-Ti O2 group; However, more bone formation, higher BIC% and maximum pull out force results were observed in NT-Sr group. [Conclusion]1. Microrough surface was prepared by etching in 0.5% HF, the nano-sized roughness and Sr coating were achieved via magnetron sputtering, micro/nano Sr coating can be fabricated by HF etching plus magnetron sputtering. MT, MNT-Sr possess better hydrophily. Sr loaded surface fabricated by magnetron sputtering can achieve continuous Sr release at a low rate;2. Smooth surface may be beneficial to osteoblast spread, better cell adhesion can be attributed to micro roughness, and nano roughness with Sr loaded surface may be favorable to osteoblast osteogenic differentiation. Furthermore, micro/nano Sr loaded can synthesize the effects of microroughness, nanoroughness and Sr;3.Micro roughness possess better BMSCs adhesion and proliferation, however, better adhesion and proliferation would lead to decline of osteogenic differentiation, which enhanced in nanoroughness and Sr loaded surface;4. Sr loaded can result in favorable mineralization effects, micro roughness alone can provided better in vivo osteogenesis than nano roughness alone, which may be attributed to improved mechanical interlock offered by micro roughness. So a combination of microroughness, nanoroughness and Sr would be the best option to promote osseointegration;5. Under osteoporotic condition, MNT-Sr surface can improve the implant osseointegration, we can ascribed the favorable osteogenesis of MNT-Sr to increased roughness and anti-osteoporosis element-Sr;6. Compared to NT-Ti O2, NT-Sr surface would be more suitable to immediate implantation. Nano roughness coating prepared by magnetron sputtering possessed higher interface bonding strength. Stable interface bonding strength would a crucial factor in affecting implant osseointegration, which is a basis of favorable bioactivity.