| Part one The processes of biomineralization on titanium surfaceObjective: Implant surface morphology is one of the most important factors in implant osseointegration. The technology of hydroxyapatite coating combines the excellent mechanical properties of metallic materials and biologically active of hydroxyapatite, with good prospects for development.Biomineralization is under the strict control of the organism, selectively absorb various elements from surrounding environment to constructing a highly ordered crystal structure of inorganic minerals in organisms with special features. Biomineralization in vitro is using mineralization like simulated body fluid（SBF）, imitating the crystallization environment in living organisms, and depositing of inorganic minerals. SBF has ion concentrations nearly equal to those of human blood plasma, and it can deposit HA which was similar to bone inorganic constituents in vivo. it shows a better biological activity. Concentrated Growth Factors（ CGF） fiber protein solution contains ion concentrations nearly equal to SBF as well as fibrin and a variety of growth factors, which is closely related to bone regeneration and makes the process similar to that in vivo. In this part, blasting-acid etching titanium surface pretreatment technologies was used, and then immersed the titanium plates into SBF and CGF fiber protein solution respectively for biomineralization. Observe the surface morphology and composition of elements by SEM-EDS.Methods:1 Preparation of SBF: preparation of SBF by the recipe of Kokubo’s simulated body fluid.2 Preparation of CGF plasma: 9ml venous blood was drawn from beagle dogs, centrifuged by Medifuge, and then separated the upper liquid intoasealed disposable plastic tubes.3 Group and treatment of titanium sheets:Pure titanium sheets: circle, with diameter of 8mm, thickness of 1mm,Roughness of 0.172μm, divided into 8 groups, each group of 3.Group A: machining; group B: SLA group; Group C: machining + SBF 7d group, machining titanium sheets merged into SBF for 7d; Group D: SLA +SBF 7d group, SLA titanium sheets merged into SBF for 7d; Group E:Machining + SBF 14 d group, machining titanium sheets merged into SBF for14d; Group F: SLA + SBF 14 d group, SLA titanium sheets merged into SBF for 14d; Group G: machining + CGF group, machining titanium sheets merged into CGF for 7d; Group H: SLA + CGF group, SLA titanium sheets merged into CGF for 7d.4 Morphological Features and Structural AnalysesSEM-EDS was used to observe the topography and analyse the microelements. Nano Measure 1.2 was used to measure the sizes of pores and nanos on the titanium surface. Roughness tester was used to measure the surface roughness.Results: 1 SEM micrograph of Group A: mechanical polishing scratches are in order. Group B: we can see honeycomb structure at low magnification,and multilevel holes with pore size of 0.2-4μm at high magnification. Group C:mechanical polishing scratches are in order, with no significant mineral deposits. Group D: we can see honeycomb structure with a small amount of mineralized nodules at low magnification, irregular mineralized nodules with particle size of 0.3-5μm at high magnification. Group E: mechanical polishing scratches are in order with a small amount of mineralized nodules at low magnification, irregular mineralized nodules with particle size of 0.2-3μm at high magnification. Group F: the titanium surface was covered by large amount of globular sediment at low magnification, and the sediments showed good dispersion with diameter of about 1-3μm at high magnification,large number of small needle-like nanoparticles forming a porous developed pore structure. Group G: mechanical polishing scratches are in order with asmall amount of mineralized nodules at low magnification, irregular mineralized nodules with particle size of 0.2-4μm at high magnification.Group H: we can see honeycomb structure with a small amount of mineralized nodules at low magnification, irregular mineralized nodules with particle size of 0.3-5μm at high magnification; 2 EDS analysis: surface element of group A,B, C is Ti; surface elements of Group D, E, F, G, H are C, O, P, Ca, Ti, Ca atomic percent of which were 3.41 %, 4.62%, 9.73%, 6.28%, 6.81%, P atomic percentage were 2.33%, 2.82%, 6.24%, 4.12%, 4.97%, Ca / P ratios were 1.46,1.64, 1.56, 1.52, 1.37respectively; 3 Results of Nano measure 1.2: surface pore size of group B: the average pore diameter was 1.28μm, proportion of microporous 0.5-3μm was 81%; Group D, E, G, H: the average particle size of surface-mineralized sedimen- tary was respectively 1.55μm, 0.98μm, 1.68μm,2.42μm; Group F: the average particle size was 1.19μm; the average length and width of nanoparticles on surface microsphere were 210 nm and 40 nm,forming a porous developed pore structure. The average pore size was 110nm;4 Surface roughness measurements: Group B> A, the difference was statistically significant（P<0.05）; Group E > G > A, the difference was statistically significant（P<0.05）; Group C> A, the difference was not statistically significant; Group F > D > B, the difference was statistically significant（P<0.05）, Group F > H, the difference was not statistically significant.Part two The effect of the pure titanium surface biomineralization on the proliferation and differentiation of MC3T3-E1 cellsObjective: Proliferation and differentiation of MC3T3-E1 cells are key to osseointegration. Mechanical, chemical, biological and other surface modification technology can effectively improve the compatibility of materials and cells, thus affecting the biological behavior of bone cells. From the perspective of bionics, micro and nano-level HA can significantly increase osteoblast adhesion, proliferation, ALP activity and deposition of calcium and phosphorus, preparing a micro-nano composite topography of titanium surface by means of biological mineralization may be more conducive to theexpression of cell function.In this part of the experiment, we used direct fluorescent staining, MTT method, ALP activity to evaluate the surface adhesion, proliferation and differentiation of MC3T3-E1 cells on titanium sheets, revealed the attachment mechanism of MC3T3-E1 cells in different surface treatment of titanium sheet and functional differentiation mechanism from cellular level.Methods:1 Group and treatment of titanium sheets:Group A: Machining; group; Group B: SLA group; Group C: SLA + SBF7d; Group D: SLA + SBF14 d; Group E: SLA + CGF group, the surface treatments were the same with the firstp art.2 Mineralization induction and identification of MC3T3-E1 cellsMC3T3-E1 cells were induced mineralization in osteogenic medium,identified calcium depositions by Alizarin red staining and nitric acid argentum and HE staining, evaluated ALP activity by ALP staining.3 Evaluated the adhesion, proliferation and differentiation of MC3T3-E1 on titanium surfaces by fluorescent staining, MTT method and ALP activity.Results: 1 Identification of mineralization induction and identification of MC3T3-E1 cells: the result of Alizarin red staining, nitric acid argentum and HE staining and ALP staining of MC3T3-E1 cells induced for 14 days were all positive; 2 Extending situstion of cells on the surfaces of titanium sheet: 48 h after seeding, cells on group A spread like plate-shaped extension which were large and flat polygonal cells, F-actin were throughout the cytoplasm. The F-actin were arranged in an interlocking pattern and intercellular synaptic connections were more wide. The cells on Group D titanium surface had burr-like protrusions extend more and longer pseudopodia; 3 MTT test results:cultured after 1d, OD value of Group D was greater than Group A, B and C,Group E was greater than Group A, B and C, and all these differences were statistically significant（P<0.05）. Group D > Group E, Group C > Group B>Group A, but the differences were not statistically significant. Cultured after3 d, 5d and 7d, Group D > Group E > Group C > Group B> Group A, thedifferences were statistically significant（P<0.05）; 4 ALP activity results:prolonging time of incubation, the 5groups of ALP specific activity increased gradually: culturing after 4d: Group D > Group B> Group A, Group D> Group C and E, the differences were statistically significant（P<0.05）, Group E >Group C, but the differences were not statistically significant. culturing after7 d and 14d: Group D > Group E > Group C > Group B> Group A, the differences were statistically significant（P <0.05）.Part three Analysis of gene expression profile of MC3T3-E1 cells on biomineralization surface of pure titaniumObjective: Gene chip, based on genetic probe hybridization and sequencing techniques, is an efficient nucleic acid sequence analysis technique,of which object analysis is gene sequence. Whole genome expression of m RNA chip can simultaneously detect the transcription level of the almost all known genes of samples, is conducive to filter out changed significantly differentially expressed genes. Mouse embryonic osteoblast MC3T3-E1 is a classical cell to study the mechanism of bone formation. In the second part of the study, we have established a reliable and repeatable induced osteogenesis model of MC3T3-E1 in vitro. In this part of experiment, we detected differentially expressed genes and related signal transduction pathways through the full gene expression difference spectrum analysis technique,which has explored the molecular biological mechanism of the different surface morphology of pure titanium on osteoblasts remodeling.Methods:1 Group and treatment of titanium sheets was the same with the second part.2 Total RNA extraction by using TRIZOL reagent: after cultured for 7d and 14 d of MC3T3-E1, we collected cells on titanium surface, extracted total RNA, and determined its concentration, purity and integrity.3 Experiment on the preparation of whole genome m RNA microarray.4 Microarray scanning and data analysisTranscriptome Analysis Console30 data analysis software was used toscan, filter out up-regulated or down regulated genes above two times. Using Pathway to analyse and screen out differential gene and related signal transduction pathways.Results: 1 Total RNA purity and integrity testing: purity, concentration and integrity of RNA extracted from each group of samples were in line with the requirements of the experiment; 2 Expression profile chip detection analysis results: 2.1 The comparison between Group A and Group B when samples were mineralization induced by 7 days, 4027 differential gene expression were observed with a 2-fold difference（2255 were up-regulated,1772 were down-regulated）. By 14 days, 2934 differential gene expression were observed with a 2-fold difference（1599 were up-regulated, 1335 were down-regulated）. Both in the two time points, up-regulated genes Tgfbr1 were observed; 2.2 The comparison between Group A and Group C when samples were mineralization induced by 7 days, 4087 differential gene expression were observed with a 2-fold difference（2202 were up-regulated, 1885 were down-regulated）. By 14 days, 3135 differential gene expression were observed with a 2-fold difference（1956 were up-regulated, 1179 were down-regulated）.Up-regulated genes were both observed in the two time points as followed:Araf, Sipa1, Map2k7, Rasa3, Thbs1, Apc and Lrp1; 2.3 The comparison between Group A and Group D when samples were mineralization induced by7 days, 4540 differential gene expression were observed with a 2-fold difference（2550 were up-regulated, 1990 were down-regulated）. By 14 days,3043 differential gene expression were observed with a 2-fold difference（1745were up-regulated, 1298 were down-regulated）, and Bmp4 was obviously up-regulated. Up-regulated genes were both observed in the two time points as followed: Prkcd 、Sipa1 and Ski; 2.4 The comparison between Group A and Group E when samples were mineralization induced by 7 days, 4073 differential gene expression were observed with a 2-fold difference（2416 were up-regulated, 1657 were down-regulated）. By 14 days, 3535 differential gene expression were observed with a 2-fold difference（2448 were up-regulated,1087 were down-regulated）, and Runx2 was obviously up-regulated.Up-regulated genes were both observed in the two time points as followed:Smad3 and Sipa1; 2.5 The comparison between Group D and Group C when samples were mineralization induced by 7 days, 912 differential gene expression were observed with a 2-fold difference（461 were up-regulated,451 were down-regulated）. By 14 days, 668 differential gene expression were observed with a 2-fold difference（332 were up-regulated, 336 were down-regulated）. Up-regulated genes were both observed in the two time points as followed: Bmp4 and Foxh1; 2.6 The comparison between Group E and Group C when samples were mineralization induced by 7 days, 2127 differential gene expression were observed with a 2-fold difference（1277 were up-regulated, 850 were down-regulated）. By 14 days, 387 differential gene expression were observed with a 2-fold difference（285 were up-regulated, 102 were down-regulated）. There was no overlap in the genes related to osteogenesis in the two time points. Adjusting the difference multiple of1.5times, up-regulated genes which were related to osteogenesis were both observed in the two time points as followed: Jun and Inhba; 2.7 The comparison between Group D and Group E when samples were mineralization induced by 7 days, 1487 differential gene expression were observed with a2-fold difference（716 were up-regulated, 771 were down-regulated）. By 14 days, 826 differential gene expression were observed with a 2-fold difference（329 were up-regulated, 497 were down-regulated）. Bmp4 were both up-regulated in the two time points. 3 Analysis of differential gene Pathway:Differences genes were involved in 20 ways in which the osteogenic differentiation was closely related with Wnt signaling pathway, MAPK signaling pathway and TGF-β signaling pathway.Part four Effect of biomineralization surface of pure titanium implant on osseointegrationObjective: Treatment of biological mineralization on the surface of dental implants could alter the boneintegration manner of implant and bone tissue, improve the bonding strength, accelerate osseointegration process and shorten implant healing period. And it has good biological activity. In this partof the study, an artificial dental implant model of adult beagle dogs was established. We used metal-bone joint grinding binding technique to observe the integration of implants and bone tissue and, to analyse and evaluate the mechanism of promoting bone union and increasing the bone bonding strength on the surface of bio mineralized implants. All these efforts resulted to provide experimental evidence for the design of dental implant with an excellent surface morphology structure, and improve the success rate of dental implants.Methods:1 Experimental Animals: 15 Adult male Beagle dogs.2 Implant groups and surface treatmentBuried titanium screw implants（diameter 3.5mm, length 8mm）, were divided into five groups of 18, a total of 90, with the same surface treatment method with the second part.3 Establishment of an animal model of delayed implant after tooth extraction in Beagle dogs.Mandibular first, second, third and fourth premolars were extracted from Beagle dogs under general anesthesia. After three months, 90 implants were implanted into the bilateral mandible of beagle dogs.4 Specimens and specimen handlingAnimals were sacrificed at 4, 8 and 12 weeks after surgery, gross observation and X-ray taken to observe bone healing. Half of the specimens were then rotated to test the torsion, and the other half were fixed to prepare hard tissue sections.5 Histological observation and bone metrologyObserving under fluorescent microscope and measuring the bone mineral apposition rate calculation; After the hard tissue sections were stained by the microscope, the images of the interface were observed, and the osseointegration index and the percentage of the calcification area of the implants and the surrounding bone were calculated. Finally, the resulting data were analyzed statistically.Results: 1 X-ray observation after planted: After 4w, 8w, 12 w, X-rayfilms and CBCT observed around the implant were normal transmission image,vertical neck bone resorption are less than 2mm; 2 Double fluorescent labeling results: after 4w, The yellow and green fluorescence were seen scattered around the implant. Yellow fluorescence in group A and group B were mainly concentrated in the planting nest wall, whereas both on the surface of implant and surrounding nests, the yellow fluorescence were visible in group C, D and E; after 8w, fluorescent color range and number increased significantly.When compared with 4w, yellow fluorescence was patchy, and green fluorescence was linear distribution, were in contact with the implant and growed into the interior threaded wall along the osteotomy. Fluoresence distribution could be seen in the bottom part of the thread groove in Group C, D and E; after 12 w,Fluorescent band of Group A and B were mainly in the area around the bone record, less on the implant surface. The implant surface and the corresponding bone surface in Group C, D and E all had yellow fluorescent bands, and were connected together by the green strips; 3 Hard tissue histology observation:after 4w, the planting threads were filled with sparse woven bone, and the bone edge of the self provided hole was inclined to grow on the inclined plane of the thread. When compared with group A and group B,the amount of trabecular bone in group C, D and E is more, and the arrangement is more compact. after 8w, compared with four weeks, the amount of new bone around the implant significantly increased, new bone crawling along the thread flanks into the bottom of the thread, and the new trabecular bone marrow cavity becomes smaller; after 12 w, the new bone was arranged in a dense and orderly manner, forming a lamellar structure. Implant bone contact of group A and B was mainly concentrated on the top of the implant and the inclined wall, while of group C, D and E was in the thread bottom of the groove, meanwhile, the contact osteogenesis phenomenon was more evident; 4 Statistical analysis: 4.1peak torque of unscrewing the implant, after 4w, Group D > Group E > Group C> Group B> Group A, differences between all groups were statistically significant（P <0.05）; after 8w, the peak torque, Group D> Group E > Group C > Group B> Group A, Group B> Group A, Group D > Group E > Group C,and Group C> Group B, all these differences were statistically significant（P<0.05）; postoperative 12 w, peak torque Group D > Group C > Group B >Group A, the difference was statistically significant（P <0.05）, Group D >Group E, Group E > Group C, but the difference was not statistically significant; 4.2 mineral apposition rate（MAR）, postoperative 4w and 12 w,Group D > Group E > Group C> Group B> Group A, differences between groups were statistically significant（P <0.05）; postoperative 8w, Group D >Group E > C Group> Group B> Group A, Groups E and C was no significant difference, the rest of the two groups were statistically significant（P <0.05）;4.3 Osseointegration index（OI%）, postoperative 4w and 8w, Group D > Group E > Group C > Group B> Group A, all the differences were statistically significant（P <0.05）. Postoperative 12 w, Group D > Group E > Group C >Group B> Group A, the comparision between Group E and Group C has no significant difference, the other groups were statistically significant（P <0.05）;4.4 peri-implant bone calcification area percentage（CA%）: after surgery, 4w,Group D > Group E > Group C > Group B> Group A, the comparision between Group E and Group C has no significant difference, the other groups were statistically significant（P <0.05）; when after 8w 12 w, Group D > Group E > Group C > Group B> Group A, differences between groups were statistically significant（P <0.05）.Conclusions:1 Different basic treatment on the surface of pure titanium has influence on biomineralization, and micro nano porous structure can be obtained by the surface treatment of sand blasting and acid etching, which is favorable for the formation of biomineralization.2 Mineralization time has effect on biomineralization, and the mineralization treatment of SBF 2 weeks was better than 1 week.3 The effect of CGF fibrin fluid mineralization treatment is better than SBF.4 In vitro mineralization induction of MC3T3-E1 cells for 14 days can see mineralized bone, MC3T3-E1 cells are reliable cells in vitro study ofbiological materials on cell adhesion, proliferation function.5 Surface treatment of pure titanium forms different morphology and structure, which can affect the extension, proliferation and differentiation of MC3T3-E1 cells.6 The surface S of pure titanium treated by CGF fiber protein solution and SBF were beneficial to the extension, proliferation and differentiation of MC3T3-E1 cells.7 Pure titanium with different surface treatment can lead to gene expression differences in MC3T3-E1 cells. We can infer different surface treatment affect cell adhesion, proliferation, differentiation and osteogenic through different signaling pathways by the gene expression difference analysis.8 The Tgfbr1 gene was up-regulated by sand blasting and acid etching,and it might be related to the TGF-beta signaling pathway of MC3T3-E1 cells.9 The Bmp4 and Map2k7 genes were up-regulated by SBF biomineralization, and it might be related to the Wnt, MAPK and TGF-beta signaling pathway of MC3T3-E1 cells.10 The Smad3 and Runx2 genes were up-regulated by CGF biomineralization, and it might be related to the MAPK and TGF-beta signaling pathway of MC3T3-E1 cells.11 Different surface structure of pure titanium implant can be formed by different treatment methods, which affected the formation of implant bone osseointegration.12 The osseointegration formation of simple mechanical surface was mainly distance osteogenesis; after sandblast and acid etching treatment, the osseointegration formation of pure titanium implant surface has a portion of contact osteogenesis, and the biomineralization surface has bidirectional osteogenesis.13 The amount of new bone formation was the most for SBF biomineralization of 2 weeks, the amount of implant bone formation for CGF biomineralization of 1 week, SBF biomineralization treatment of 1 week, sandblasting and acid etching treatment, mechanical treatment of pure titanium reduced in turn. |
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