This study evaluated the effect of ultraviolet functionalization (UV) on bone integration ability in rabbit model, using epifluorescence microscopy. Each of 12 rabbits (n = 6) received randomly four titanium domes prepared with or without ultraviolet for 48 hours (UVC, λ = 250 ± 20 nm; Philips, Tokyo, Japan): (1) turned surface (T), (2) turned surface with UV (T-UV), (3) sandblasted (120 μm aluminum oxide) and etched by 18% hydrochloric acid and 49% sulphuric acid at 60°C for 30 min (SLA) and (4) SLA surface with UV (SLA-UV). Fluorochrome bone labels were marked by oxytetracycline at 25 mg/kg on 13th days and 14th days and calcein at 5 mg/kg on 3th days and 4th days before euthanization. The study samples were sacrified at 2 weeks and 4 weeks. The undecalcified specimens were prepared. The newly formed total bone of cross-sectional area (TB, %), the mineralized trabecular bone of cross-sectional area (MB, %), and the new bone and dome contact (BDC, %) were measured and analyzed by fluorescence microscope and Image Pro Express 6.0. The data of MB and TB showed new bone regeneration was increased in all groups, but no signs of difference were found. However, the means BDC of UV treatment on turned surface at 4 weeks, the UV treated on SLA surface at 2 weeks and 4 weeks were statistically significantly higher than the control group (P < .05). Within the limitations of the study, it can be concluded that ultraviolet functionalization on the titanium surface could enhance the new bone tissues and titanium surface integration.
The loss of single or multiple teeth leads to occlusal collapse, are associated with the vertical and horizontal alveolar bone defect or overerupted molars and premolars.1,2 The application of temporary skeletal anchorage devices (TADs) and dental implants have satisfied a large number of patients. However, there are still limitations, including an invasive surgical procedure, limited insertion sites, and waiting time to osseointegration for TADs.1 Yet, short implants or mini implants were suggested to be inserted in the changing condition instead of standard implants.3
Previous studies4 have pointed out that the improvement of contact osteogenesis between host bone and titanium surface provides better mechanical stability for osseointegration at the early stage. Currently, numerous methods for surface treatment have been developed to improve the percentage of bone–titanium integration. But the results still remain 50%–70%5,6 or 45%–49%,7,8 which are far from the ideal 100% for the implant integration. So it is necessary to focus on the problems of how to improve better bone integration for short implants or mini implants.
Ultraviolet (UV) irradiation, discovered in 1997,9 has been recently applied to titanium surface treatment for its photofuctionalization.10–12 The anatase form of titanium dioxide (TiO2) induced by ultraviolet light demonstrates superhydrophilic surface, changes of surface charge, and removal of various organic compounds for an extremely clean surface.13,14 The changes lead to increase initial cell events, including cell migration, the levels of attachment, and the spread of osteoblasts.15,16 Another possible mechanism might be that UV can create surface oxygen vacancies at the bridging sites, resulting in the changing of Ti4+ sites to Ti3+ sites.16 The titanium surface with or without nanostructural surface can be improved by UV irradiation, for creating surface Ti–OH functional groups.13,17,18 Tsukimura19 stated that UV photofunctionalization might expedite the degree of osseointegration and even elevate the nearly maximum in the levels of bone-titanium integration. However, the mechanisms of UV used on titanium surface is still not clearly known. Numerous researchers have studied on UV-induced photofunctionalization on various titanium surfaces in vitro study instead of in vivo study. Some device of titanium caps, titanium domes, and simple designed implant were applied for animal models.20,21 The possible reasons might be the lack of predictable animal models for better investigation or calculation of the percentage of bone–titanium contact. Therefore, an available animal's model is important for the experiment in vivo study.
Bone integration for new bone tissue and titanium surfaces improves better stability. The incorporation involves multiple steps of induction, revascularization, resorption, osteoid matrix production, and mineralization and remodeling.22 The mineralization of the osteoid matrix may contribute to the most important step for bone regeneration. Different types of fluorochromes injected to the organism were considered to the evolution and observation the available calcium that participated in the mineralized areas.23 Calcein, oxytetracycline, and alizarin were the most used as fluorescent labels in in vivo studies.21,24
Our previous studies have showed that ultraviolet C (UVC; approximately 250 nm) irradiation of an microarc oxidation surface remarkably enhances the amount of Ti–OH groups and better removal of hydrocarbon on titanium surface, compared with ultraviolet A (UVA; approximately 360 nm).18 Other molecular studies of our team have proven that UV treated on sandblasting and acid-etching titanium disk can greatly promote MG63 cell attachment, proliferation, differentiation, and mineralization in in vitro study.15 In the present study, we hypothesize that the UVC treated on titanium surfaces with or without rough surface could improve the contact osteogenesis for integration in the optimal animal model. The objective of this study is to examine the potential effects of UV treatment on turned surfaces and SLA surfaces in in vivo study, using optimal titanium model and epifluorescence electron microscopy.
Materials and Methods
A total of 12 healthy, mature, female New Zealand white rabbits (2.7 and 3.5 kg) were used (n = 6), using a modified animal study design that has been successfully applied in the previous studies.20,25,26 The experimental protocol was approved by the Committee for Animal Research, Province of Guangdong, and China. The surgical procedures were performed according to strict standard operative procedure.
Surface treatment of titanium domes and ultraviolet irradiation
All titanium domes (Grade 2) were characterized of hemispherical shape (internal diameter of 6.0 mm, outer diameter of 7.0 mm, a thickness of 0.5 mm, and a height of 3.0 mm). Some self-tapping screws (vertical height, 2 mm; chamber, 0.45 mm; depth, 0.35 mm) were added on the root of the domes and 2 circular holes (diameter, 0.8 mm) on the top (Figure 1). Each of 48 titanium domes was washed with acetone, absolute alcohol, and deionized water (dH2O) in an ultrasonic cleaner for 15 minutes, respectively. They were randomly allocated to the test group and control group with or without ultraviolet light treatment (Figure 2).
T group: the control group, the inner surface of the pure titanium dome with a turned and clean surface.
SLA group: the control group, domes with inner turned surface were sandblasted by 120 μm aluminum oxide and then etched in a mixture of 18% hydrochloric acid and 49% sulphuric acid at 60°C for 30 minutes.15
SLA-UV group: the test group, the SLA domes were treated with UVC light as described previously for 48 hours.
Experimental animal model and surgical procedure
Study samples were induced by xylazine hydrochloride (Huamu Animal Health Products Co, Ltd, Jilin Province, China) at 0.1 mL/kg. And then they were anesthetized by pentobarbital sodium (Pharmaceutical Group Xinzheng Co, Ltd, Tianjin, China) injection at 40 mg/kg. A sagittal incision was made through the skin and the periosteum of the skull to expose the cranial vertex of the rabbit. Four circular grooves with depth of 1.0 mm were made by a trephine of inside diameter 6.0 mm and outside diameter 7.0 (Stoma, Storz am Mark GmbH Ltd, Emmingen-Liptingen, Germany). The titanium domes were randomly fit stably into grooves (Figure 3). The skin and periosteal flaps were repositioned and closed with a tight suture in layers. All the animals were injected intramuscularly with benzylpenicillin at a dose of 0.3 mg per animal for 3 days after the operation. Fluorochrome bone labels was marked by oxytetracycline at 25 mg/kg on the 13th and 14th days and calcein at 5 mg/kg on the third and fourth days before euthanization.21 The study samples were euthanized using an overdose of anesthetics at 2 weeks and at 4 weeks.
Specimens were removed and fixed in the 10% phosphate buffered formalin solution for 4 days. Then they were thoroughly rinsed in running tap water for 24 hours and processed in the ascending concentrations of ethanol (70%, 80%, 95%, and 100%) for dehydration. The biopsies were infiltrated in methyl methacrylate (MMA, Sinopharm Chemical Reagent Co, Ltd, Shanghai, China) and stored in the refrigerator at 4°C for 3 days. The specimens were embedded in MMA following the ascending degrees (28°C, 32°C, 36°C, and 42°C) for 4 days. The MMA samples were pruned to histological sections with an approximate thickness of 150 μm. Each of the sections was gradually grounded to the thickness of 60–70 μm with silicon carbide sandpaper (No. 600, 800, 1000, 1500, 2000, and 2500 grits). All the sections were analyzed with a fluorescence microscope (Olympus BX51TF, Olympus Co, Tokyo, Japan). Photomultiplicator filters of 460–495 nm wavelength were used for calcein and oxytetracycline.
The following data were measured: (a) the newly formed total bone (including the mineralized tissue and the marrow spaces) of cross-sectional area (TB, %); (b) the mineralized trabecular bone of cross-sectional area (MB, %); and (c) the new bone and domes contact (BDC, %), expressed as percentage of new trabecular in contact with the inner surface of the dome.26,27 The Image Pro Plus digital image analysis system (Image-pro Express 6.0, Media Cybernetics Inc, Bethesda, Md) was connected to a light microscope to analyze the data.
All data obtained from histomorphometric measurements were expressed as the mean ± the standard deviation (n = 6). Independent-sample t tests (SPSS 13.0, Chicago, Ill) were used to analyze the significance of the difference between the ultraviolet treated groups and the controlled groups at different times. It was considered statistically significant if P < .05.
All the titanium domes were anchored securely on the rabbits' calvarium at 2 and 4 weeks. The clinical outcomes disclosed no inflammatory reaction during the healing time.
Histological observations/histomorphometric analysis
The histological pictures of fluorescence microscope displayed that areas labeled in green were found at the areas of inner domes and basic host regions. The calcium labeling green represented the calcium had precipitated in the tissue mineralization (including the new mineralized bone and the host bone). Oxytetracycline (in yellow) was characterized by less density and mostly covered by calcine (in green). Otherwise, it was easily to distinguish the new bone and host bone according the basic line of titanium dome.
The obtained images showed that areas (at the bottom of the titanium dome) labeled in green were presented with newly formed bone trabecular of great density and intense in all groups (Figure 4). The titanium domes were characterized by thin, slender new bone trabeculae, marrow spaces, and some empty spaces. Obviously, the percentage of direct contact between newly bone formation and the inner surface of dome displayed higher in UV treated groups (T-UV, SLA-UV) than UV untreated groups (T, SLA). The majority of the SLA and SLA-UV groups were occupied by a large number of newly formed bone trabeculae, covering nearly one-third of the whole space. The mean values of MB, TB, and BDC were (2.52 ± 1.43% and 3.30 ± 2.15%), (10.06 ± 7.06% and 15.70 ± 11.44%), and (4.34 ± 2.30% and 6.29 ± 1.52%) for T group and T-UV group, respectively. The mean values of MB, TB, and BDC for SLA group and SLA-UV group were (4.24 ± 1.81% and 6.18 ± 3.99%), (17.63 ± 7.93% and 22.63 ± 16.42%), and (8.65 ± 2.81% and 17.00 ± 7.55%), respectively. Only data of BDC were statistically significant between UV light-treated SLA surface and the control group (t = −2.538; P = .029 < .050; 95% CI −15.680˜−1.018) (Figure 7f).
There was a tendency for increasing bone tissue in all domes (Figure 5). The signs of ongoing mineralized bone tissue were always located on the top (Figure 6e through 6g) and sidewalls of the titanium domes (Figure 6a through d). The newly mineralized trabeculae displayed an almost direct contact and continuity with the internal surface of both sidewalls of the domes in T-UV group, SLA group, and SLA-UV group. The mean values of MB, TB, and BDC were (9.59 ± 5.32% and 12.10 ± 5.27%), (32.96 ± 15.62% and 44.71 ± 13.80%), and (13.34 ± 10.32% and 34.02 ± 18.48%) for T group and T-UV group, respectively. The mean values of MB, TB, and BDC for SLA group and SLA-UV group were (9.90 ± 3.73% and 16.86 ± 6.89%), (33.12 ± 12.11% and 59.0 ± 23.99%), and (23.63 ± 15.32% and 43.14 ± 13.35%), respectively. The difference of MB and TB in both compared groups did not reach statistical significance (Figure 7a through d). Compared with the control group, the data of BDC for UV treated on both turned surface (t = −2.394; P = .038 < .050; 95% CI −39.934˜−1.430) and SLA surface (t = −2.352; P = .041 < .050; 95% CI −37.996˜−1.023) at 4 weeks were statistically significant, respectively (Figure 7e and f).
The present animal experiment had demonstrated that the application of UV treatment on the different surface of pure domes improves its osteoconductivity effectively and bone-titanium integration. The prefabricated titanium domes were modified from the guided bone regeneration models, which were originally designed by Lundgren in 1995.20 This kind of dome could make a significant discrimination between the newly formed trabecular bone and the host bone on the rabbit calvarium, named “titanium dome model.” And then, the model had been modified with or without adding some mini-screws or a peripheral collar.25,26 The domes used in the present study were changed. Some self-tapping screws were added at the bottom of the dome instead of the collar or mini-screws, which could reduce the footprint and obtain better stability. Many papers had reported that only 1 or 2 domes could be placed on the skull. However, it was not enough for comparison among more than 3 groups. In the present study, 4 modified domes with an outer diameter of 7 mm were able to place to the same rabbit skull. It was convenient to compare for different groups. The domes also provided the visible line to distinguish the host bone tissue and the vertical new bone tissue. The observation was in agreement with the previous studies.20,25,28 The design model of titanium domes was a practical and optimal method to evaluate the bone-to-titanium integration and the amount of the new bone formation in in vivo study.
The incorporation of implants and new bone tissue are related to the typical 4 steps, including induction, revascularization, resorption, osteoid matrix production, mineralization, and remodeling.22 Numerous studies have pointed out calcium ions are linked to proteoglycules during the process of mineralization. Calcein and oxytetracycline are mostly used as the fluorescent labels. Both of them are considered as the greater affinity to calcium of the mineralized bone tissue. It is possible to visualize the mineralized bone tissue with the special wavelengths of the fluorescence. In the present study, calcein and oxytetracycline were used in samples for labeling the new bone tissue. However, only calcein labeled in green was showed in the all figures (Figures 4, 5 and 6). The probable reason might be that the oxytetracycline (in yellow) was presented with less intense and density, and covered by the calcein (in green). The different regions of calcium precipitation could be another reason for the aforementioned problems. Some previous studies concluded that calcein was always represented with great density in areas of new bone regeneration, whereas oxytetracycline was labeled with more laminar bone formation.23 The figures in our studies showed the areas of inside domes were characterized by thin, slender new bone trabeculae (in green) in all of the groups.
Many histological studies in osseous implants have been widely reported to improve the osseointegration. However, there are still some challenges, such as the need to minimize failure rate and shorten morbidity, and the protracted healing time (4–6 months) and improve the quality and quantity of bone integration.29 The mechanisms underlying bone–titanium integration is poorly understood for the complex process of osseointegration. The UV-treated titanium surface was benefit for better cell activities. The possible mechanisms were the following reasons: (1) increased adsorption of protein, (2) increased osteoblast migration, (3) increased osteoblast attachment, (4) increased proliferation of osteoblast, and (5) promotion of the differentiation.30 However, some previous studies have pointed out that compared with machined surface, titanium's rough surface could promote the differentiation but reduce the proliferation of osteoblast and affect the intracellular tension. These changes might delay or restrict the progression of G1 phase of cell cycle.31 The problems of differentiation and proliferation still need to be overcome in the further research. Our previous studies had focused on these problems. They found that UV-treated microarc oxidation surfaces by a 15 W UVC bacterial lamp could promote the proliferation of MG-63 cells without sacrificing differentiation in the in vitro study.18 The possible mechanisms might be that UV-treated titanium surface enhances intracellular interaction or increases cell signaling pathways.30 The biological effect of the UV–treated titanium surface might imply better bone-to-titanium integration. Some previous studies4 had indicated that the push-in value obtained by UV-treated acid-etched implants at 2 weeks was equivalent to that obtained by untreated acid-etched implants at 8 weeks.4 However, cell activities (protein adsorption, cell attachment, cell spread, cell differentiation, and proliferation) are influenced by many impacts of surfaces characteristics, including surface roughness, surface free energy, and surface charge.15,30,32 To determine whether UV could increase the bone-implant contact percentage for better biochemical anchorage, we have carefully tested new bone formation in the in vivo study on both the smooth and rough titanium surfaces by fluorescent analysis. In other words, the smooth titanium surface without roughness could be a better way to test the efficiency of UV alone. Our present study explored how UV treatment on titanium surface can functionalize and increase bone-titanium integration by fluorescent analysis (Figures 4a and b, 5a and b). At the early stage of 2 weeks, the mineralized bone tissue was in direct contact with the sidewall of the T-UV group. Compared with machined surface, the data of BDC on UV-treated machined surface increased up to 1.5 times at the early stage of healing at 2 weeks. The difference of BDC showed UV treatment on turned surface at 4 weeks were statistically significantly higher than the control group. The data and figures had shown that the application of UV-treated machined surface could enhance the bone-to-titanium interaction. The outcomes partly agreed with our hypothesis and previous findings.13 The possible mechanisms were that UV enhanced more protein absorption and cell activities due to the changes of a positively charged surface for better attraction of proteins with a negative charge. So UV treatment on titanium for better protein adsorption was predominantly regulated by electrostatic property.33 Based on the aforementioned findings, we agreed with the hypothesis that UV-treated machined surfaces could enhance the cell activities and better bone-to-titanium direct connection in the in vivo study without sacrificing proliferation or differentiation.
Interestingly and more importantly, the differences of BDC between the SLA group and the SLA-UV group reached statistical significance at 2 weeks and 4 weeks. At 4 weeks of healing, the total bone tissue (TB) increased in all groups and the mineralized trabeculae (MB) become thicker. The outcomes had implied that UV-treated rough surface also could enhance the bone-to-titanium direct connection. Another possible mechanism might be the reason for explaining the aforementioned problem. The phenomenon of superhydrophilicity induced by ultraviolet was studied by numerous assays.15,34,35 Suketa has reported that the UV photocatalysis on pure titanium not only possesses the ability of surface sterilization, but also promotes reosseointegration.36 More importantly, the aged titanium surface with a hydrophobic state could be transformed into a superhydrophilic surface more easily than the fresh surface. The key point is that UV irradiation can reduce the hydrocarbons accumulated on the aged titanium surface.37,38 However, the mechanism that UV-treated titanium surface could improve hydrophilicity is still not clearly understood. Three possible explanations were proposed in the literature: (1) hydrophilicity is improved by the removal of hydrocarbons or other carbonaceous macerals on the titanium surface, resulting in water adsorption; (2) hydrophilicity is induced by photoproduction, which uses the dissociation of water to obtain a wet surface; and (3) hydrophilicity is ca by the rupture of the Ti-O bonds to generate the high surface-free energy by the photofunctionalization of UV. To a certain degree, the characters of the superhydrophilicity induced by UV improved the total bone formation and mineralization in histological sections. But uncertainty still exists as to which was affected by some of the factors, such as the amount and the quality of the hematoma, the interference of the fibrous tissue, and the individual conditions of the rabbits. These issues need to be further addressed in a future study.
The present study demonstrates that UV photofunctionaliztion of machined and rough surfaces significantly promotes the bone-to-titanium connection. UV-induced improvement of the bioactivity and osteoconductivity of different surface topographies (turned and rough surfaces) demonstrated good contact osteogenesis and bone titanium integration in an in vivo study, probably because of the mechanisms of predominantly electrostatic property and partly because of the removal of hydrocarbons and the oxidation of an abundance of hydroxyl groups. Therefore, the potential ability of UV technology used as one kind of surface treatment might improve osseointegration for commercial implants or mini-implant for orthodontic application.
The study was supported by Stomatological Hospital, Southern Medical University, People's Republic of China (Guangdong Provincial Stomatological Hospital), grants from the Natural Science Foundation of China (No 81170998), and Fund of Guangdong Province Medical Science Research (2016A030310240).
The authors of this study had no conflicts of interest.