| BackgroundThe implant denture has became a new popular treatment for dentition defect and edentulous patients with high success rate, although with individual failure. The way towards a successful treatment is to create a good osseointegration, in which the dynamic balance between osteogenic osteobalst and osteoclastic osteoclast plays a key role. The biomechanical factors also affects the osseointegration. The occlusal force transmits to the implant-bone interface through the implant loaded. Over loading could result in bone resorption around implant and then the implant will be loose and lost. Therefore, many researches focused on what had affected the transmission of occlusal force, such as the bone mass in jaws, the implant shape, design of the crown, the type of occlusal force and materials etc. The two or three dimensional finite element analysis and photoelastic experiment were the main research methods. However, there are researches seldom focusing on the transmission and effect of biomechanics on the implant-bone interface, except previous studies of our group.The osteocyte in the lacuno-canalicular network of bone tissue was responsive to the mechanotransduction. When bone or implants were loaded, the resulting deformation of the non-calcified generates fluid shear stress (FSS) went across the surface of osteocyte. Osteocyte had been demonstrated to be sensitive to the FSS. Osteoblast had shown sensitivity to the FSS, as well.It is not clear that which molecules play a key role in the signal transduction process of the biological response, which is induced in the convertion from morphology to function under FSS loading on the implant-bone interface. Since the Wnt/β-catenin signaling pathway has a close relation with the proliferation of tumor cells, it was the target spot of studies aiming to tumor treatments. Recently it is found that some bone diseases are caused by the mutation of a co-receptor of the Wnts called low density lipoprotein receptor-related protein5(Lrp5). Loss of Lrp5could lead to a decrease number of osteoblast in bone, insensetive response to mechanical loading and lower bone mass; while activation of Lrp5could lead to a increased number of osteoblast, sensitive response to mechanical loading and high bone mass. In addition, mutation of other molecules in this pathway, such as Wnts, Lrp6, DKK1and P-catenin etc, could also produce all kinds of abnormal bone metabolism. Based on these findings, Wnt/β-catenin signaling pathway gradually becomes a new cosset in the cell biomechanics researches about bone tissue. However, it still has no unified understanding of the role of Wnt/β-catenin signaling pathway in bone formation due to the different experimental condition adopted by different researchers.Chapter one:Effects of fluid shear stress on mRNA expression of key molecules of Wnt/p-catenin signaling pathway in primary osteoblasts on different modified titanium surfacesObjective: We established a stress model in vitro and investigated the effect of fluid shear stress and surface microtopography on the Wnt/β-catenin signaling pathway in primary osteoblasts on the implant-cell interface.Methods1. Primary osteoblasts were isolated from neonatal SD rats skull and cultured with the twice enzyme digestion continuous tissue culture method. The osteoblasts were identified by ALP staining and mineralized tuberosis staining.2. Polished Titanium surfaces and sandblasted and acid-alkali treated Titanium surfaces were prepared via different modification, labeled as P group and S group respectively. The surface topography was observed by scanning electron microscopy (SEM).3. The primary osteoblasts were cultured and seeded on glass slides (group G), polished titanium surface (group P) and sandblasted and acid-alkali treated titanium surface (group S) respectively for72h, then the experimental groups were loaded under12dynes/cm2fluid shear stress for Oh,0.25h,0.5h,1h,2h,4h respectively; the control groups were cultured in medium with2%fetal bovine serum for0h,0.25h,0.5h,1h,2h,4h respectively.4.The cell total RNA was extracted after treatments above. The mRNA expression of Lrp5and β-catenin were detected by real-time fluorescence quantitative PCR.Results1. A relatively huge number of primary osteoblasts could be acquired in a short time by the culture method of applied. The cells grew well, ALP staining presented bluish violet and the mineralized tuberosis staining presented bright red. All of these conformed to the biological characteristics of osteoblast.2. Titanium plates demonstrated different surface microtopography. P group was characterized by mechanical grooves with the same direction. S group had a hierarchical micro/nano-textured multiporous topgraphy.3. Factor analysis had shown that modified surface, the stimulus and duration of FSS had significant effects on the expression of Lrp5and β-catenin mRNA (all of the p-valure were less than0.001). It was implied that the expression of Lrp5and β-catenin mRNA were surface-dependent, mechanics-dependent and time-dependent. Level of gene expression was higher in experimental group than that in control group, and higher on S group than the other two groups. Both the factors had a peak value of gene expression during0.5-1h under FSS loading. Lrp5mRNA had a peak value at0.5h both on the two titanium surfaces and at1h on G group under loading, while β-catenin mRNA reached the peak at0.5h on all three surfaces. Both the level of Lrp5and P-catenin mRNA presented an early dramatic rising followed by a drop and then a slow rising again on the S surface. It was suggested that both FSS and sandblasted and acid-alkali treated titanium surface could promote the up-regulation of expression of Lrp5and β-catenin mRNA in primary osteoblasts, of which the peak value might appeared at early loading stage.Chapter two:Effects of fluid shear stress on mRNA expression of key molecules of Wnt/p-catenin signaling pathway in MG63cells on different modified titanium surfacesObjectiveAccording to the comparison of the response of human osteoblast-like cell and primary osteoblast to the same treatment, the role of Wnt/β-catenin signaling pathway in osteoblasts would be further dicussed.Method1.Polished Titanium surfaces and sandblasted and acid-alkali treated Titanium surfaces were prepared via different modification, labeled as P group and S group respectively.2. The MG63cells were cultured and seeded on glass slides (group G), polished titanium surface (group P) and sandblasted and acid-alkali treated titanium surface (group S) respectively for72h, then the experimental groups were loaded under12dynes/cm2fluid shear stress for Oh,0.25h,0.5h,1h,2h,4h respectively; the control groups were cultured in medium with2%fetal bovine serum for Oh,0.25h,0.5h,1h,2h,4h respectively.3. The cell total RNA was extracted after treatments above. The mRNA expression of Lrp5and β-catenin were detected by real-time fluorescence quantitative PCR.Results1. In present study, the Lrp5mRNA was not affected by the mechanical loading, but well affected by different surfaces. Herein the P value is at the critical value, statistical difference may be acquired by increasing sample size. Lrp5mRNA was down regulated on S group in control group during0-4h. Under loading, Lrp5mRNA level presented two peaks on G group from Oh to4h, while it was a gradual up-regulation trend on the two Ti surfaces with a linear correlation between time and gene level. It was inferred that the Lrp5mRNA would be continuously up-regulated after a4h duration of loading.2. Compared to Lrp5, β-catenin mRNA could be up-regulated by12dynes/cm2FSS. But different surfaces had no significant effect on it. Under loading, β-catenin mRNA level also presented two peaks on G group from Oh to4h, while it was a gradual up-regulation trend on the two Ti surfaces with a linear correlation between time and gene level. It was inferred that the β-catenin mRNA also would be continuously up-regulated after a4h duration of loading. 3. Combinative analysis, FSS could up-regulate β-catenin mRNA level but had no effect on Lrp5mRNA level. The Lrp5mRNA level could be affected by different surfaces and down-regulated on sandblasted and acid-alkali treated titanium surface. β-catenin mRNA level was not affected by the surface. Under loading, both the two factors mRNA level presented time-dependent. Two peak value of mRNA appeared at0.5h and2h respectively on G surface, while the gene level increased with the loading time prolonged on the two Ti surfaces.Chapter two:Effects of fluid shear stress on expression of key proteins of Wnt/β-catenin signaling pathway in MG63cells on different modified titanium surfacesObjectiveAccording to detect the change of related protein of Wnt/β-catenin signaling pathway in MG63cells, the effect of FSS and titanium surfaces on this pathway would be further dicussed.Method1. Polished Titanium surfaces and sandblasted and acid-alkali treated Titanium surfaces were prepared via different modification, labeled as P group and S group respectively.2. The MG63cells were cultured and seeded on glass slides (group G), polished titanium surface (group P) and sandblasted and acid-alkali treated titanium surface (group S) respectively for72h, then the experimental groups were loaded under12dynes/cm2fluid shear stress for Oh,0.25h,0.5h,1h,2h,4h respectively; the control groups were cultured in medium with2%fetal bovine serum for Oh,0.25h,0.5h,1h,2h,4h respectively.3. After treatments above, cells were digested and collected to extract the cytoplasmic protein and nuclear protein prepared for the Western blot of Lrp5and P-catenin respectively.Results1. Lrp5protein level was higher on unloading group suggesting that12dynes/cm2FSS could inhibit the expression of Lrp5in MG63cell. Protein expression on the two Ti surfaces was lower than that on glass surface, while there was no difference between the rough Ti surface and smooth Ti surface. It could be inferred that expression of Lrp5protein might be inhibited by Ti surface but not be changed by the rough property. Lrp5protein level was different among different time points both in the condition of loading and unloading. Moreover, there was linear correlation between time factor and protein level under loading. Protein level was down-regulated on G group and P group with the extension of time while it was up-regulated on S group.2. Expression of P-catenin protein was dependent on mechanical loading and different surfaces, and there was statistical difference among different time points in the condition of loading (P<0.05). Protein level was higher on loading group, suggesting that12dynes/cm2FSS could up-regulate the level of β-catenin in nuclear. In the condition of unloading, protein level was lowest on G group and had no difference between S group and P group. However, under loading, it was highest on S group and lowest on P group. It might be concluded that P-catenin level in nuclear could be influenced by different surfaces and the rough Ti surface could promote the effect of FSS on the translocation of β-catenin. There was no linear correlation between time factor and β-catenin protein level. The peak of expression was present at early stage under loading on all the three surfaces.3. In summary,12dynes/cm2FSS could promote the translocation of P-catenin and also inhibited the expression of cytoplasmic Lrp5protein. The expression of Lrp5protein might be inhibited by Ti surface but not be changed by the rough property. However, the rough Ti surface could promote the effect of FSS on the translocation of β-catenin. Both the expression of the two proteins would change with the extension of time under loading.ConclusionThere was a different response to mechanical loading and different modified surfaces between the rat primary osteoblast and the human osteoblast-like cell MG63cell, and that resulted in difference on the signal transduction of molecule level. The Lrp5and β-catenin mRNA level in primary osteoblast might could be up-regulated by appropriate FSS and sandblasted and acid-alkali treated titanium surface, thus the Wnt/β-catenin signaling pathway might be activated and the sandblasted and acid-alkali treated titanium surface might promote the sensitivity of this signaling pathway to the FSS. In MG63cell, β-catenin signaling pathway might be activated by FSS not through the Wnt/Lrp5signal. However, the signaling pathway might would be changed with the loading time on different surfaces. The fact that Lrp5mRNA and protein level were down-regulated on sandblasted and acid-alkali treated titanium surface, suggested an inhibition of canonical Wnt/Lrp5signal in MG63cell. |
