| At present, dental implantation has become the most ideal way to restore the missing teeth. However, because of the impaired osseointegration in T2 DM, the failure rate of implantation is significantly higher than healthy patients. It is reported that the osteogenic differentiation suppression of osteoblastic lineage cells around plants in T2 DM patients is the key factor for the impaired osseointegration. Therefore, to improve the osteogenesis ability of osteoblastic lineage cells is the ultimate way to solve the problem. Sema3 A, a newly found distinctive bone protection factor, is able to both promote osteogenesis differentiation and inhibit osteoclast differentiation effectively, which may be an ideal candidate to improve osseointegration in T2 DM. Therefore, in the first part of the study, the Sema3 A is loaded on the surface of titanium implant through chitosan membrane as the carrier.In recent years, because of the wide origin, easy accessing and neglectable trauma, ASCs has become an ideal source of stem cells. The ASCs based cell sheet technology is also considered to be prosperous in the tissue repair. Until now, there is no report yet to use ASCs cell sheet around dental implant. Meanwhile, there are still many disputes about the osteogenesis ability of ASCs. It is generally recognized that the mineralization and calcium deposition of ASCs cannot fully meet the demand of patients with T2 DM and it is essential to improve the osteogenesis ability of ASCs. Therefore, the following part is to test the effects of Sema3 A on ASCs configuration, proliferation and osteogenesis ability. The in vivo confirmation is performed in T2 DM rat model. PART ONE: Enhancement of MG63 osteogenic differentiation on Sema3 A modifiedtitanium implant surface Objectives: To observe the effects on osteoblast behavior on Sema3 A modified titanium implant surface. Methods: The titanium implants were treated by MAO prior to decoration. Chitosan was used to wrap Sema3A(CS/Sema) and connected to the implant surface via silane coupling. Depending on the different complexs loaded on the implant surface, the experiment was divided into four groups: CS/sema-MAO, CS/BSA-MAO, CS-MAO and MAO. Osteoblast MG63 was directly seeded onto sample surface. The cell adhesion number was observed through DAPI staining 2 hours later. Cell viability was determined by MTT test after cultivating for 1, 3, 7 days. Osteogenesis related genes including RUNX2, ALP, OCN and BMP were detected by RT-q PCR after 3 and 7 days induction in osteogenic medium. Mineralized was tested through Alizarin Red staining after inducing for 21 days in osteogenic medium. Results: The cellular adhesion ability in CS/Sema3A-MAO has no significant difference with other control groups. The different coatings had no obvious influence on the cell vitality. The RUNX2 expression ascended more than three times and the other genes expression ascended more than two times after three days induction. After seven days induction, the RUNX2 expression down regulated to normal level whereas other genes increased more than three times. Alizarin Red staining showed that the osteoblast mineralization ability was significantly improved after modification with Sema3 A. Conclusions: Sema3 A modified titanium implant can strongly improve osteogenic differentiation of osteoblasts.PART TWO: Influence of Sema3 A on morphology, proliferation and osteogenicdifferentiation ability of ASCs Objectives: To observe the influence of Sema3 A on ASCs’ morphology, proliferation and differentiation. Methods: The experiment was divided into four groups depending on Sema3 A final concentration in medium: ASCs-0; ASCs-0.25; ASCs-0.5; ASCs-1.0 group. Cell morphology was observed under scanning electron microscopy; Cell proliferation was determined by CCK-8 kit; RT-q PCR was used to test the osteogenesis related genes expression; ALP staining, Sirius Red staining and Alizarin Red staining were used to determine the ALP activity, secretion of collagen and the calcium nodules. Results:(1) Sema3 A had no obvious influence on ASCs’ s morphology.(2) Sema3 A did not affect ASCs’ proliferation activity.(3) The osteogenesis related genes ALP and COL1 expression were significantly upregulated with the increasing of Sema3 A concentration.(4) With the increase of Sema3 A concentration, ALP expression and collagen secretion were significantly increased. Alizarin Red staining results shows that the calcium nodules increased after treated with Sema3 A, but there is no obvious difference in different Sema3 A concentrations. Conclusion: Sema3 A does not affect the morphology and proliferation of ASCs whereas it can tremendously improve osteogenic differentiation ability of ASCs.PART THREE: Evaluation of Sema3 A modified ASCs sheet in improvingosseointegration in T2 DM rat model Objectives: To evaluate the osseointegration in T2 DM rat model by using Sema3 A modified ASCs sheet. Methods: High fat high sugar feed+small doses STZ injection to induce T2 DM rats. ASCs sheet was induced in the presence of Sema3 A. The sheet was wrapped closely around the implant. Then the cell sheet imbedded implant was inserted into the T2 DM rat model. After four weeks and eight weeks of healing, the osseointegration of T2 DM were observed by Micro-CT and Van Gieson staining. Results: T2 DM rat model was induced successfully. The random blood sugar was stable, and all were above 16.7mmol/l. The cell sheet grown as multilayers and can shrink instantly. The sheets wrapped the implant closely. Micro-CT scanning showed that with the increase of Sema3 A concentration, new bone formation around implant augmented significantly. VG staining showed that new bone formation was thicker and more continuous when the Sema3 A concentration increased. Conclusion: Sema3 A modified ASCs sheet is able to promote titanium implant osseointegration in T2 DM condition. |
PDF Full Text Request