【摘要】：研究背景：齲病是口腔的一種常見病和多發(fā)病。當(dāng)齲病發(fā)展到牙本質(zhì)層時,致齲菌及其代謝過程中所產(chǎn)生的一些毒性物質(zhì)可沿著牙本質(zhì)小管滲入到牙髓組織中。牙髓組織遭遇到外界刺激時,組織中原有的干細(xì)胞向受損部位遷移、到達(dá)受損部位后,分化為成牙本質(zhì)細(xì)胞并形成修復(fù)性牙本質(zhì)。牙髓組織的這種防御機制可以防止牙髓進(jìn)一步受損,但牙齒本身的這種自我修復(fù)的能力很有限。目前臨床上治療齲病的方法是先去除齲壞的牙體組織,然后使用人工材料對缺損的組織來加以修復(fù)。雖然近年來臨床上使用的充填和修復(fù)材料發(fā)展較迅速,材料無論是在生物學(xué)性能還是強度上都有了明顯的提高,但與天然的牙體組織相比仍然具有較大的差距。而且隨著充填治療時間的延長充填材料會對牙髓組織的活力產(chǎn)生一定的影響。長期的臨床觀察發(fā)現(xiàn)：充填治療后,在充填體和牙體組織之間會有微滲漏或繼發(fā)齲的出現(xiàn),這些最終都會導(dǎo)致充填治療的失敗。干細(xì)胞在組織的缺損修復(fù)和再生的研究中有著重要作用。牙髓干細(xì)胞(dental pulp stem cells,DPSCs)屬于成體性干細(xì)胞,是目前發(fā)現(xiàn)的五種牙源性干細(xì)胞中的一種。1990年,Fitzgerald等發(fā)現(xiàn)當(dāng)牙髓組織受到外界入侵時,牙髓組織中的細(xì)胞可遷移到受損處并迅速形成修復(fù)性牙本質(zhì),他當(dāng)時就提出了牙髓組織中有干細(xì)胞的存在。DPSCs作為一種成體性干細(xì)胞后來的研究發(fā)現(xiàn)其具有成骨、成脂、成牙本質(zhì)等多向分化的潛能,體外增殖速度快,目前的研究較為廣泛。DPSCs在體外較易獲得,培養(yǎng)的方法相對較為成熟。且與胚胎干細(xì)胞不同的是DPSCs的培養(yǎng)不涉及到任何倫理學(xué)方面的問題。目前的研究顯示在體外DPSCs可以冷凍保存較長的時間且復(fù)蘇后細(xì)胞的生物學(xué)性狀無明顯的改變。DPSCs的這些生物學(xué)特性為牙髓組織損傷修復(fù)的研究以及組織工程牙齒的構(gòu)建提供了重大的依據(jù)。在組織工程研究領(lǐng)域,生物性誘導(dǎo)劑必不可少。BMP-7 (bone morphogenic protein 7)作為一種較安全的生物因子,屬于轉(zhuǎn)化生長因子-β (TGF-β)家族的一員。首次在19世紀(jì)70年代被發(fā)現(xiàn),因其具有成骨作用,也被稱為成骨蛋白-1 (osteogenic protein-1,OP-1)。BMP-7作為安全的誘導(dǎo)劑在骨科部分已進(jìn)入臨床研究應(yīng)用的階段,發(fā)現(xiàn)其在半月板損傷的治療中成效較為顯著。也有研究將BMP-7的基因?qū)氲焦撬栝g充質(zhì)干細(xì)胞中,可提高細(xì)胞的成骨效果。在臨床研究中Ashraf Ayoub等將BMP-7負(fù)載到Ⅰ型膠原支架上應(yīng)用到多種類型的牙槽嵴裂缺損的治療中,結(jié)果表明在骨裂的重建修復(fù)中該設(shè)計無論從影像學(xué)的角度還是從臨床的角度治療效果都非常良好。Yang X等將質(zhì)粒編碼的BMP-7基因轉(zhuǎn)染到DPSCs中,然后發(fā)現(xiàn)經(jīng)轉(zhuǎn)染后的細(xì)胞可以向成牙本質(zhì)細(xì)胞表型分化并可以在體外形成硬組織。目前國內(nèi)外有關(guān)BMP-7的研究大多數(shù)都為體內(nèi)動物實驗,雖然也有研究表明高濃度的BMP-7會抑制細(xì)胞的增殖,但BMP-7對DPSCs的增殖和分化是否有影響,以及作用于DPSCs時合適的蛋白作用濃度并不是非常明確。這些問題都有待于進(jìn)一步的實驗研究,為BMP-7應(yīng)用到牙再生提供理論依據(jù)。組織工程的發(fā)展基于生物材料的迅猛發(fā)展,支架材料是組織再生和重建的基礎(chǔ)。首先支架需要在缺損的部位搭建出一個有利于細(xì)胞遷移、黏附、生長和分化的空間結(jié)構(gòu)。同時支架的結(jié)構(gòu)也要有利于新生的組織和血管的長入。這些特性要求應(yīng)用到組織工程的支架材料需要具備良好的三維聯(lián)通立體構(gòu)象、機械強度高、生物安全性能優(yōu)良。在前期我們課題組的研究中采用模板復(fù)制法制備的三維聯(lián)通納米氧化鋯生物陶瓷支架機械強度高,附著骨髓間充質(zhì)干細(xì)胞植入到犬下頜骨缺損部位成骨效果顯著,本研究繼續(xù)使用該支架進(jìn)行進(jìn)一步實驗。研究希望在體外篩選出作用于DPSCs時合適的BMP-7蛋白濃度,將細(xì)胞接種到改善后的納米氧化鋯支架上,評價BMP-7作為誘導(dǎo)劑應(yīng)用到DPSCs的定向誘導(dǎo)中的作用,從而探討其對牙體缺損修復(fù)及應(yīng)用到牙組織工程的可行性。目的：探索BMP-7作為一種安全的生物因子在體外是否會影響DPSCs的增殖和分化,并選擇出BMP-7作用于DPSCs時合適的濃度。將選擇出的合適濃度的BMP-7誘導(dǎo)牙髓細(xì)胞后接種到三維聯(lián)通納米氧化鋯生物陶瓷支架上植入到裸鼠皮下,觀察細(xì)胞的生長、新生的組織情況以及血管再生的能力,評價BMP-7作為誘導(dǎo)劑應(yīng)用于牙組織工程的可行性。方法：(1) hDPSCs的體外分離、培養(yǎng)及成骨成脂鑒定選擇年輕健康成人的第三磨牙,通過組織塊結(jié)合酶消化的方法獲取hDPSCs,觀察細(xì)胞的貼壁生長和克隆形成情況。同時通過成骨和成脂誘導(dǎo)實驗檢測細(xì)胞在體外的多向分化能力,鑒定獲取的原代細(xì)胞中含有干細(xì)胞。(2)不同濃度的BMP-7對hDPSCs形態(tài)的影響配制含不同濃度BMP-7 (Ong/ml,25ng/ml,50ng/ml, 100ng/ml)的培養(yǎng)基,將細(xì)胞在含不同蛋白濃度的培養(yǎng)基中培養(yǎng)。每3-4天換液,連續(xù)培養(yǎng)7天,倒置顯微鏡下觀察各組細(xì)胞的形態(tài)是否有差異。(3)不同濃度的BMP-7對hDPSCs增殖的影響收集處于對數(shù)生長期第三代的hDPSCs細(xì)胞,消化離心計數(shù)后,調(diào)整細(xì)胞懸液濃度。各組細(xì)胞分別加入含不同濃度(Ong/ml,25ng/ml,50ng/ml, 100ng/ml)的BMP-7培養(yǎng)液。連續(xù)培養(yǎng)1,3,7d后通過CCK-8方法檢測細(xì)胞的增殖在各組之間是否有差異。(4)不同濃度的BMP-7對hDPSCs分化的影響通過qPCR、細(xì)胞免疫化學(xué)染色和Western blot的方法檢測牙源性相關(guān)基因和蛋白在不同處理組之間的表達(dá)差異。(5) 降溫速率對納米氧化鋯支架形態(tài)影響的研究在原始的支架高溫?zé)Y(jié)的成形過程中,通過降低支架的降溫速率,觀察溫度對支架的形態(tài)完整性是否有影響。(6)BMP-7誘導(dǎo)處理后的細(xì)胞接種到納米氧化鋯支架上裸鼠皮下研究將選擇出的合適濃度的BMP-7蛋白誘導(dǎo)細(xì)胞后接種到生物陶瓷納米氧化鋯支架上植入裸鼠皮下,觀察細(xì)胞的生長、新生的組織情況以及血管的再生能力。結(jié)果：(1) hDPSCs的體外分離、培養(yǎng)及成骨成脂鑒定通過組織塊結(jié)合酶消化的方法獲得的健康年輕人的牙髓細(xì)胞在體外貼壁生長迅速且克隆形成率高。細(xì)胞表達(dá)間充質(zhì)來源標(biāo)記物,非上皮性來源。在體外經(jīng)成骨和成脂誘導(dǎo)后,可以向成骨和成脂方向分化,具備多向分化的能力,說明獲得的細(xì)胞中有干細(xì)胞的存在。(2)不同濃度的BMP-7對hDPSCs形態(tài)的影響各組細(xì)胞在不同濃度的BMP-7培養(yǎng)基中連續(xù)培養(yǎng)7天后,在倒置顯微鏡下各組細(xì)胞在形態(tài)上都呈類成纖維細(xì)胞樣,多角形態(tài),組間未見到明顯的差異。(3)不同濃度的BMP-7對hDPSCs增殖的影響細(xì)胞增殖在各組隨著體外培養(yǎng)時間的延長都呈現(xiàn)上升的趨勢。對照組細(xì)胞生長情況與不同濃度的BMP-7誘導(dǎo)組之間出現(xiàn)明顯的差異,對照組細(xì)胞增殖在培養(yǎng)期間都高于誘導(dǎo)組。實驗結(jié)果表明一定濃度的BMP-7對DPSCs的增殖隨著處理時間的延長會抑制細(xì)胞的體外增殖能力。(4)不同濃度的BMP-7對hDPSCs分化的影響細(xì)胞免疫化學(xué)染色顯示對照組的DPSCs中DSPP不表達(dá)或個別細(xì)胞表達(dá),DMP-1低表達(dá),ALP有少量細(xì)胞表達(dá)。體外BMP-7誘導(dǎo)DPSCs 7d后,DPSCs中大多數(shù)細(xì)胞DSPP、DMP-1表達(dá)呈陽性,ALP染色出現(xiàn)強陽性。qPCR顯示細(xì)胞經(jīng)不同濃度的BMP-7在體外處理14d后細(xì)胞中成牙本質(zhì)細(xì)胞相關(guān)基因(Col I, DSPP, OCN and Runx2)的表達(dá)情況為DSPP、OCN和Runx2基因在50ng/ml蛋白處理組的表達(dá)量要明顯高于對照組,且差異具有統(tǒng)計學(xué)意義。DSPP的表達(dá)在50ng/ml BMP-7組也高于25ng/ml BMP-7組和100ng/ml BMP-7組。Col I基因在100ng/ml處理組的表達(dá)量最高,高于對照組和其它BMP-7誘導(dǎo)組。蛋白結(jié)果顯示BMP-7處理14天后細(xì)胞中DSPP表達(dá)明顯增加,對照組細(xì)胞中DSPP也出現(xiàn)了低表達(dá)。定量結(jié)果顯示在50ng/ml和100ng/ml BMP-7處理組中DSPP表達(dá)量與對照組相比具有統(tǒng)計學(xué)差異。(5) 降溫速率對納米氧化鋯支架形態(tài)影響的研究支架在首次高溫?zé)Y(jié)時,通過減緩降溫速率后,肉眼觀支架高溫?zé)Y(jié)后首次成形效果明顯改善,支架塌坯的現(xiàn)象沒有出現(xiàn),完整性破壞較少。電鏡下觀察延長降溫時間后,支架呈現(xiàn)出三維網(wǎng)狀聯(lián)通結(jié)構(gòu),內(nèi)部孔隙大小分布較均勻,但支架表面的微裂紋及斷裂現(xiàn)象仍然存在。高倍鏡下氧化鋯顆粒形態(tài)與原燒結(jié)程序下相比未見到明顯的差異。(6)BMP-7誘導(dǎo)后的細(xì)胞接種到納米氧化鋯支架上裸鼠皮下研究細(xì)胞接種到支架上后在支架表面附著伸展。隨著體外培養(yǎng)時間的延長,細(xì)胞增殖迅速,鋪滿支架的內(nèi)部和表面。術(shù)后2個月取材所有標(biāo)本被血管及纖維結(jié)締組織所包繞,支架周圍未見明顯的炎性組織。肉眼觀四組之間未見明顯的差異。髓染色顯示無細(xì)胞組中有纖維結(jié)締樣組織的長入,細(xì)胞組中都在靠近支架邊緣有類似成牙本質(zhì)細(xì)胞樣細(xì)胞及小血管的出現(xiàn),但均未觀察到成熟的牙本質(zhì)樣結(jié)構(gòu)的形成。結(jié)論：(1)在體外BMP-7對DPSCs的增殖有時間和劑量的聯(lián)合效應(yīng)。50ng/ml和100ng/ml的BMP-7在體外具有誘導(dǎo)DPSCs向牙源性方向分化的潛能且不會明顯的抑制細(xì)胞的增殖速率。(2)BMP-7作用于DPSCs時需要掌控好蛋白的使用劑量和作用時間,以便有利于細(xì)胞的生長和分化。(3)三維聯(lián)通納米氧化鋯多孔支架可以為DPSCs提高良好的細(xì)胞外微環(huán)境,顯示出了良好的生物學(xué)性能。(4) DPSCs附著到生物陶瓷納米氧化鋯支架上植入裸鼠皮下后,有望再生牙本質(zhì)樣組織,有待進(jìn)一步的研究。
[Abstract]:Background: caries are a common and frequently occurring disease in the oral cavity. When caries develop to the dentine layer, the cariogenic bacteria and some toxic substances produced in the process of metabolism can infiltrate into the dental pulp tissue along the dentinal tubules. When the dental pulp tissues encounter external stimuli, the original stem cells in the tissue migrate to the damaged part and reach the recipient. When the site is damaged, it divides into odontoblast cells and forms a restorative dentin. This defense mechanism of the dental pulp tissue prevents the dental pulp from further damage, but the tooth itself has limited ability to repair it. At present, the clinical treatment of caries is to remove caries first, and then use artificial materials for the defect group. Although the materials used in the bed have developed rapidly in recent years, materials have been significantly improved in both biological performance and strength, but there is still a big gap compared with natural dental tissues. Stress has a certain effect. Long term clinical observation shows that after filling, there will be microleakage or secondary caries between the filling body and the tooth tissue, which will eventually lead to the failure of the filling treatment. The stem cells have an important role in the study of tissue defect repair and regeneration. Dental pulp stem cells, DPSCs) belongs to adult stem cells. It is a.1990 year of five odontogenic stem cells found. Fitzgerald, etc., found that when the dental pulp tissues were invaded by the outside, the cells in the pulp tissue could migrate to the damaged area and quickly form a restorative dentin. At that time, he proposed the presence of.DPSCs as a stem cell in the dental pulp tissue. A later study of adult stem cells found that it has the potential of multiple differentiation of osteogenesis, fat formation, dentin formation, and so on. The proliferation rate is fast in vitro. The current research on.DPSCs is relatively easy to obtain in vitro, and the methods of culture are relatively mature. And different from embryonic stem cells, DPSCs culture does not involve any ethical aspects. Problems. Current studies show that in vitro DPSCs can be frozen for a long time and after resuscitation, the biological properties of cells have no obvious changes in.DPSCs. These biological characteristics provide a significant basis for the repair of dental pulp tissue damage and the construction of tissue engineering teeth. In the field of tissue engineering, biological inducers Essential.BMP-7 (bone morphogenic protein 7), as a safer biological factor, is a member of the transforming growth factor - beta (TGF- beta) family. It was first discovered in 1870s for its osteogenic effect and also known as osteogenic protein -1 (osteogenic protein-1, OP-1).BMP-7 as a safe inducer in the Department of orthopedics. In the stage of clinical application, it has been found that it is more effective in the treatment of meniscus injury. There is also a study to introduce BMP-7 gene into bone marrow mesenchymal stem cells, which can improve the osteogenic effect of cells. In clinical study, Ashraf Ayoub, etc., applied BMP-7 to type I collagen scaffold to apply to a variety of types of alveolar cleft. In the treatment of damage, the results showed that the design, both from the angle of image and from the clinical point of view, was very good in the reconstruction and repair of the bone fissure. The plasmid encoded BMP-7 gene was transfected into DPSCs, such as.Yang X, and then the transfected cells could differentiate into the odontoid cells and form hard in vitro. Most of the research on BMP-7 at home and abroad is in vivo animal experiments, although there are also studies showing that high concentration of BMP-7 inhibits the proliferation of cells, but the effect of BMP-7 on the proliferation and differentiation of DPSCs, as well as the appropriate concentration of protein acting on DPSCs, is not very clear. These problems need to be further studied. The experimental study provides a theoretical basis for the application of BMP-7 to tooth regeneration. The development of tissue engineering is based on the rapid development of biomaterials, and the scaffolding material is the basis for tissue regeneration and reconstruction. These characteristics require that the scaffolds applied to the tissue engineering need to have good three-dimensional conformation, high mechanical strength and good biological safety performance. In the previous research group, the three-dimensional zirconia bioceramic scaffold prepared by template replication method was used in the research group. High mechanical strength, adherent bone marrow mesenchymal stem cells implanted into the mandible defect site of the dog bone formation effect is significant. This study continues to use the scaffold for further experiments. We hope to screen out the appropriate BMP-7 protein concentration in vitro, and inoculate the cells to the improved nano zirconia scaffold, and evaluate BMP-7 as a lure. The application of the guide to the directional induction of DPSCs to explore the feasibility of dental defect repair and application to dental tissue engineering. Objective: To explore whether BMP-7 as a safe biological factor will affect the proliferation and differentiation of DPSCs in vitro, and select the appropriate concentration of BMP-7 to act on DPSCs. The BMP-7 induced dental pulp cells were implanted into the subcutaneous tissue of nude mice on the three dimensional nano zirconia bioceramic scaffold, observed the growth of the cells, the new tissue and the ability of vascular regeneration, and evaluated the feasibility of BMP-7 as an inducer for dental tissue engineering. (1) the separation, culture and osteogenesis in vitro of (1) in vitro The third molar of young healthy adults was identified by lipid identification. HDPSCs was obtained by tissue block binding enzyme digestion. The cell adhesion and formation of cells were observed. At the same time, the differentiation ability of cells in vitro was detected by osteogenesis and lipid induction. (2) different concentrations of BMP were found. The influence of -7 on the morphology of hDPSCs was prepared with different concentrations of BMP-7 (Ong/ml, 25ng/ml, 50ng/ml, 100ng/ml). The cells were cultured in the medium containing different protein concentrations. The cells were cultured every 3-4 days and cultured for 7 days. Under the inverted microscope, the morphology of the cells in each group was observed. (3) the effect of BMP-7 on the proliferation of hDPSCs in different concentrations was collected. HDPSCs cells in the third generation of logarithmic growth period were collected. After digestion centrifuge counting, the concentration of cell suspension was adjusted. Each cell was added to BMP-7 culture solution containing different concentrations (Ong/ml, 25ng/ml, 50ng/ml, 100ng/ml). After continuous culture 1,3,7d, the proliferation of cells was detected by CCK-8 method. (4) BMP- at different concentrations. 7 Effect on hDPSCs differentiation through qPCR, cellular immunochemistry staining and Western blot method to detect the difference in expression of odontogenic genes and proteins between different treatment groups. (5) the study on the effect of cooling rate on the morphology of nano zirconia scaffold in the process of high temperature sintering of the original scaffold by reducing the cooling of the scaffold Rate, whether the temperature has an effect on the morphological integrity of the scaffold. (6) the cells after BMP-7 induction were inoculated into nude mice subcutaneously on the nano zirconia scaffold, and the suitable concentration of BMP-7 protein was selected to induce the cells to be implanted subcutaneously on the bioceramic nano zirconia scaffold to observe the growth of the cells and the new group. Results: (1) in vitro isolation, culture and identification of hDPSCs in vitro, the dental pulp cells of healthy young people obtained by tissue block binding enzyme digestion are fast growing and cloned in vitro. The cell expression of mesenchymal origin markers, non epithelial sources. After bone and lipid induction, it can differentiate into osteogenesis and fat formation, with the ability to multiform differentiation, indicating the existence of stem cells in the obtained cells. (2) the effects of different concentrations of BMP-7 on the morphology of hDPSCs in each group of cells in different concentrations of BMP-7 medium after 7 days of continuous culture, the cells in each group are in shape under the inverted microscope. (3) the effect of BMP-7 on the proliferation of hDPSCs in each group increased with the prolongation of the culture time in each group. The cell growth of the control group was significantly different from the BMP-7 inducement group with different concentrations, and the cells in the control group increased. The results showed that the proliferation of DPSCs in a certain concentration of BMP-7 inhibited the proliferation of cells in vitro. (4) the effect of BMP-7 on the differentiation of hDPSCs in different concentrations showed DSPP non expression or individual cell expression in DPSCs of the control group and DMP-1 low table. ALP has a small amount of cell expression. After BMP-7 induction of DPSCs 7d in vitro, most of the cells in DPSCs are DSPP, DMP-1 expression is positive, ALP staining appears strong positive.QPCR display cells in different concentrations of BMP-7 in vitro. The expression of the 50ng/ml protein treatment group was significantly higher than the control group, and the difference was statistically significant.DSPP expression in the 50ng/ml BMP-7 group was also higher than the 25ng/ml BMP-7 group and 100ng/ml BMP-7 group.Col I gene expression in 100ng/ml treatment group, higher than the control group and other BMP-7 induction groups. Protein results showed 14 days after treatment. The expression of DSPP in the cells was significantly increased, and the expression of DSPP in the control group also showed low expression. The quantitative results showed that the DSPP expression in the 50ng/ml and 100ng/ml BMP-7 treatment groups was significantly different from that of the control group. (5) the effect of cooling rate on the morphology of nano zirconia scaffold in the first high temperature sintering was reduced by slowing down the cooling rate. After the high temperature sintering of the naked eye view, the first forming effect was obviously improved, the phenomenon of the stents collapsed and the integrity failure was less. Under the electron microscope, the stent presented a three-dimensional network connecting structure and the internal pore size distribution was more uniform, but the micro crack and fracture still existed on the surface of the support frame. The morphology of the zirconia particles was not significantly different from that under the original sintering procedure. (6) the cells after BMP-7 induction were inoculated to the nude mice on the nano zirconia scaffold and were inoculated on the scaffold to extend on the scaffold surface. With the extension of the time of culture in vitro, the cells proliferated rapidly and covered the internal and surface of the scaffold. 2 All specimens were wrapped around the vascular and fibrous connective tissue, and no obvious inflammatory tissue was found around the scaffold. No significant difference was found between the four groups. Pulp staining showed that there were fibrous connective tissue in the cell group, and the cell groups were similar to odontoblast like cells and small vessels near the edge of the scaffold. No mature dentin like structure was observed. Conclusion: (1) the combined effect of time and dose of BMP-7 on the proliferation of DPSCs in vitro,.50ng/ml and 100ng/ml BMP-7 have the potential to induce DPSCs to differentiate into odontogenic direction in vitro and do not significantly inhibit the proliferation rate of the cells. (2) BMP-7 is required to act on DPSCs. The dosage and time of the protein should be controlled in order to help the growth and differentiation of the cells. (3) the three-dimensional porous nano zirconia porous scaffold can improve the good extracellular microenvironment for DPSCs and show good biological properties. (4) it is hopeful that DPSCs attach to the subcutaneous of nude mice on the ceramic nano zirconia scaffold. The dentin like tissue of regenerated teeth needs further study.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R781

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