The Effect Of Ultraviolet Photocatalysis On Pure Titanium Domes With Different Surfaces:in Vivo Study On Rabbits Calvarial Bone At The Early Stage

BACKGROUNDThe rapid development of Oral Implantology is attributed to the application and practice of the osseointegration theory. In order to satisfy the different needs of different patients, clinical scholars have put forward the four basic goals of modern implantology, including the normal chewing, speech, healthy and beauty. Base on the previous studies, the researchers has worked on the optimization of the titanium surface modification techniques to achieve more rapid and effective osseointegration to fulfill the growing clinical requirement and needs.There are two different healing modes for bone formation around the implant, called contact osteogenesis and distance osteogenesis. The process of contact osteogenesis refers that new bone matrix is released by osteoblasts at the implant surface. Bone trabeculae is quickly formed and in direct contact with the implant surface. The direction of newly bone formation is from the implant surface to host bone tissues. For the second mode, new bone is formed on the surfaces of old bone in the peri-implant site. According to Davies, both of the healing process reveals that osteoconduction and de novo bone formation are the crucial stages for osseointegration; since exactly at these phases, the implant is anchored within the wound site by the newly formed bone matrix. The crucial stage of contact osteogenesis is to establish a biological matrix (hematoma) as early as possible, through which mesenchymal stem cells (MSCs) can migrate and attach to the implant surface, then proliferate and differentiate into osteoblasts that can ultimately lay down a new bone matrix on the implant surface.The optimization of the required condition for contact osteogenesis as early as possible would contribute to improve new bone tissues deposition. The rate and extent of healing around an implant depend on the degree of contact osteogenesis that occurs at the implant surface. Pokharau pointed out that the de novo bone tissue by contact osteogenesis provided better mechanical stability of the implant at the early stages.The first application of UV treatment as one of the surface treatment on titanium dioxide film was recorded by Wang, implying that the transformation of the properties of the titanium dioxide film experienced from hydrophobic to super-hydrophilic. However, Carlsson et al. revealed the hydrophilic surface of the implant was difficult to exhibit higher osseointegration in vivo study. Still Buser et al. pointed out that the implants with hydrophilic surface have higher bone-implant integration compared with the traditional one. Then the application of ultraviolet photocatalysis as a non-chemical, non-biological processing method has gradually become one of the implant surface modification, in order to improve the osteoconduction for better bone titanium integration, accelerate the process of bone deposition by contact osteogenesis and reduce clinical healing periods. Previous groundwork demonstrated an increase in the occupancy of carbon result in a reduction in osteoblast attachment and protein absorption on bulk titanium surfaces over storage time in a sterile atmosphere after surface preparation including machined and acid-etched surface. Currently used commercial titanium implants, including clinical and experimental implants, are found to contain hydrocarbons contaminated and the degradation of biological activity on the titanium surface was statistically correlated with an increase in the occupancy of carbon.With taking some consideration about the different kinds of surface treatment used in recent years, combined with a variety of surface treatment methods has become a popular trend for the optimization of the advantage of technique, which has the ultimate goal for better biocompatibility of implant and improving bone tissues deposition directly on the implant surface. Compared with the conventional surface treatment, UV photofunctionalization is expected to be a new approach of surface enhancement to circumvent conventional surface modification technologies, due to its technical simplicity, high efficacy and low-cost, since it does not require additional chemical or mechanical processing of the original titanium surfaces.Our previous studies had been done some research on the chemical experiments and in vitro study of sandblasted and acid-etched titanium surface by UV irradiation, which indicated that UV treatment do not change the topography and roughness of the surface and obtain a large number of hydrophilic groups. The biological activity of MG63osteoblasts in vitro study was detected on hydrophobic SLA surface and hydrophilic SLA by UV treatment at time points1d,7d, and14d. The improvement of cell adhesion, proliferation, differentiation and mineralization had been reported after UV treatment.Base on the previous study, we hypothesized that UV light treatment of pure titanium would enhance its osteoconductive capacity and improve bone deposition. The objective of the present study was to examine the effect of UV treatment on different pure titanium surfaces (turned surface and sandblasted and acid-etched surface) in vivo study.OBJECTIVE1. The aim of the study was to evaluate the effect of the optimization of the tranditional pure titanium dome on rabbit calvarial bone surface and establish a reliable, efficient, simple and good repeatability animal model for implant surface treatment.2. The objective of the present study was also to examine the effect of UV treatment on different pure titanium surfaces (turned surface and sandblasted and acid-etched surface) in vivo study at the early stage at2w,4w and6w and provide evidence for the clinical application from animal studies.METHORD1. Titanium domes and animalsEighteen healthy, mature, female New Zealand rabbits were supplied by the Animal Experiment Center, Guangdong, Academy of Medical Sciences. The pure titanium plate Grade2was purchased from Titanium Metal Corporation, USA. The prefabricated pure titanium domes were processed by Geefull Metal&Plastic products Co.Ltd. The prefabricated pure titanium domes, in hemispherical shape with inner empty, using the Grade2, were performed by special precision machine The domes were featured an internal diameter of6.0mm, outer diameter of7.0mm, tickness of0.5mm, a height of3.0mm, adding some self-tapping screws on the root of the domes (the vertical height of2mm, the chamber of0.45mm, the depth of0.35mm) and two circular holes on the top (diameter0.8mm).2. Surface treatment All the inner surfaces of the pure titanium domes had the same basic form and topography. The inner surfaces of titanium domes were treated by the method with reference to the previous study. Two groups of equal number of were randomly allocated to four different surface ctreatment, turned (T), and turned and ultraviolet irradiation (T-UV). Half of the experimental samples were treated by a mixed solution of49%H2SO4and18%HCl;60℃30min after sandblasted by120μm Al2O3. Part of the SLA domes and T domes were irradiated by UV for48h.3. Animal modelEighteen New Zealand rabbits were anesthetized and then treated following the standard operative procedure. The rabbit skull bone surfaces were carefully exposed then prepared four circular grooves by the trephine drill. Four domes with different surface treatment were placed in calvarial bone surface of each rabbit. Surgical sites were closed in layers and then all the animals were injected antibiotics to prevent infection after surgery in the following five days. The animals were sacrified after a healing period of2w,4w and6w following the pure titanium dome placement. A t the13th,14th day and the3th and4th day before scarifice, the rabbits were administrated with tetracycline at30mg/kg and with calcein at6mg/kg. The four titanium domes on each rabbit were primaryly prepared and then fixed at10%neutral formalin solution.4. Prepared the undecalcified histological sectionsAll the specimens were thoroughly rinsed in running water after fixiation and then dehydrated in the ascending concentrations of ethanol. Following that the biopsies were embedded in methacrylate and then processed the exact cutting by LEICA SP1600with the thickness of150μm in each section. Each section was grounded to30μm. All the sections were stained with methylene blue-acid fuchsin. The new bone tissue morphology was observed by an optical microscope and obtained the data of related indicators of morphological measurements through image measurement software detection indicators.5. Histomorphometric measurements①the newly formed mineralized bone percentage (MB%)=the newly mineralized formed bone tissues/the total spaces of the inner dome*100%②the newly formed total bone percentage (TB%)=the newly formed trabecular bone and marrow spaces/the total spaces of the inner dome*100%③Bone-implant contact percentage (BIC)=the length of new trabecular bone in contact with inner surface of dome/the total length of the inner periphery of the dome*100%6.Statistical analysisThe obtained data were classified, integrated, and analysis using statistical software SPSS13.0. The time periods (2w,4w and6w) and the different surfaces (turned and sandblast and acid-etching surfaces) and the treatment (with UV or without UV) were assessed by a univariate analysis. If the interaction effect was significant then the difference between time periods, surfaces and treatment were analysis by two independent samples t-test. A result was considered statistically significant if P<0.05.RESULTS1. Genneral observationAll the animals were alive and tired after surgery, less activity and less food within5days. The animals almost returned to normal after one week. All the titanium domes had a good stability. Specimens observed were wrapped around the titanium domes by a large number of soft tissues. Some fibrous tissues occupied some of inner spaces of the titanium through the small hoes at the top of the dome.2. Fluorescence microscope observationThe new bone tissues with bright green light were observed by fluorescence microscopy. The trabecular bone tissues were deposited along the inner sidewalls and the bottom of the titanium domes. The visible boundary was seen between new bone formation and the calvarial bone. Mineralization of bone tissues with highlighted green light were an apparent contrast with the marrow spaces and soft tissues. At2weeks, only a small amount of thin trabeculae was deposited along the calvarial bone surface. The SLA and SLA-UV group showed more new bone tissues than T group and SLA group, respectively, and the mineralized trabeculae were closely contacted with the titanium walls. At4weeks, the amount of bone trabeculae was increasing, the same as the soft tissue. New bone trebeculae of SLA-UV dome and T-UV dome, in direct contact with the sidewall of the dome, even climbed up to the top1/3of the dome.The total bone formation in SLA-UV dome was the better than the other three groups. At6weeks, the rate of the newly bone formation was slowed down. The fluorescent displayed no difference in the SLA-UV group and the SLA group.The total bone tissues in T-UV group were more than in the T group.3. Histology observationThe mineralization new mature trabecular bone tissues were observed in red, the osteoid and fibrous tissues in blue from the sections with methylene blue-acid fuchsin staining under the light microscopy. New bone tissues increased during the time period2-4weeks were faster those during4-6weeks. At2weeks, bone formation characterized with thin trabeculae laid on the calvarial bone surface with different shape. Compared with the T group, a direct contact between the thin mineralized trabeculae and the internal surface of the dome was observed at a varying degree in all the other groups. At4weeks, the newly bone foramtion was characterized by thicker trabeculae with signs of ongoing remodeling and large marrow spaces. At the same time, some soft tissues had spreaded into the inner spaces of the dome. The newly bone trabeculae was fully integrated to the preexisting calvarial bone surface. The mineralized trabecular bone was in contact with sidewall at different height in the dome, except the T group.The newly bone in SLA-UV and T-UV domes displayed the highest and even reached the top1/3of the dome. At6weeks, the spaces for the newly trabecular bone were limited by the growing soft tissues. Thicker trabeculae with signs of ongoing remodeling and large marrow spaces still could be observed in all groups. The total newly trabecular bone tissues and newly bone in direct contact with the inner sidewall in T-UV group and SLA-UV group were better than those in the T group and SLA group, respectively.4. Outcomes of statistical analysisThe results of univariate analysis displayed that the difference was obvious at time, various surface and treatment. Then the differences betweengroups at each time were caculated by the application of two independent samples t-test, resulting in the following outcomes.At2weeks, the means of MB%were as follows, T-UV (3.800±1.640)%> T (2.350±1.411)%, SLA-UV (16.178±3.989)%> SLA (4.239±1.812)%. Based on the t-test, both of them did not have a statistically significant effect, respectively. The means of TB%were as follows, T-UV (16.363±8.7590)%> T (10.062±7.057)%, SLA-UV (22.625±16.417)%> SLA (17.627±7.933)%. Both of them did not have a statistically significant effect, respectively, either. The means of BIC were as follows, T-UV (6.622±1.297)%>T (4.208±2.228), SLA-UV (17.003±7.552)%> SLA (8.653±2.813)%. Both of them,(P=0.048<0.05) and (P=0.029 <0.05)had a statistically significant effect.At4weeks,the means of MB%were as follows,T-UV(12.596±5.028)%>T (9.590±5.317)%,SLA-UV(16.825±6.892)%>SLA(9.899±3.729)%.Based on the t-test,both of them did not have a statistically significant effect,respectively. The means of TB％were as follows,T-UV(44.714±13.803)%>T(32.975±15.619)%,SLA-UV(59.006±23.990)%>SLA(33.115±12.111)%.The previous one did not have a statistically significant effect while the latter one reached the statistical significant(P=0.048<0.05).The means of BIC were as follows,T-UV (30.850±13.741)%>T(15.168±6.648)%,SLA-UV(43.144±13.352)%>SLA (23.634±15.321)%.Both of them,(P=0.039<0.05)and(P:0.041<0.05),had a statistically significant effect,respectivel y.At6weeks,the means of MB%were as follows,T-UV(13.956±3.740)%>T (10.094±2.470)%,SLA-UV(17.087±3.097)%>SLA(13.021±2.639)%.Based on the t-test,the previous one did not have a statistically significant effect while the latter one reached the statistical significant(P=0.034<0.05).The means of TB%were as follows,T-UV(45.353±16.423)%>T(33.057±4.300)%SLA-UV (63.556±19.870)%>SLA(55.296±15.819)%.The previous one had a statistically significant effect(P=0.031<0.05)while the latter did not.The means of BIC were as follows,T-UV(25.805±8.332)%>T(16.167±2.879)%,SLA-UV (53.961±14.257)%>SLA(35.778±9.819)%.Both of them,(P=0.036<0.05) and(P=0.030<0.05),had a statistically significant effect.CONCLUSION1.The modified pure domes had simply processing,good repeatability and the identical basic form and topography.The addition of self-trapping screws instead of the adding fixture device did really some help to increase the stability of the pure titanium domes when placed.The application of the pure domes with self-trapping screws on rabbit calvarial bone surface had demonstrated to be advantage of reducing the surgical trauma and simplifing the operation steps, improving the efficiency, The optimized pure titanium dome was more objective and reliable to reflect the new bone formation among groups with different surface treatment in terms of implant biocompatibility, osteoconductivity and bone titanium integration. The optimized pure titanium dome with self-trapping screw was a kind of application prospect in animal experimental model for implant surface treatment.2. UV treatment on the pure titanium dome with different surfaces demonstrated to be capacity of osteoconductivity and good biocompatibility. The osteoconduction of the T-UV group, SLA-UV group was better than T group, SLA group respectively. The combination of UV treatment with turned surface or sandblast acid-etching surface had a capacity to significantly ehance osteoconductivity of titanium and promote bone tissues directly deposition on the titanium suface and bone mineralization on the titanium-bone interface.3. UV irradiation, as one of the surface treatment methods with simple, clean technology, with no need to add or increase chemical change on topography of the titanium, would be a prospect and profound technology in the future.