牙龈间充质干细胞影响即刻种植骨缺损修复的实验研究

发布时间：2018-06-19 10:55

本文选题：牙龈干细胞 + 种植体　；参考：《山东大学》2014年博士论文

【摘要】：背景和目的：随着口腔种植的飞速发展,为了不断满足广大缺牙患者的实际需求,将拔牙和种植体植入同步进行-即刻种植技术已成为修复缺失牙的一种治疗趋势。即刻种植的疗程短、手术次数少、手术创伤小、硬组织保存较好和软组织美学效果好并且其成功率与延期种植相近,受到越来越多的患者和临床医生的青睐。然而由于种植体的大小形态往往与拔牙窝不相符,使得拔牙后即刻种植的种植体与牙槽窝骨壁之间存在不同程度的间隙,或者患牙受炎症、外伤的影响,致使周围牙槽骨缺损甚至吸收,这些情况都严重影响着即刻种植种植体初期稳定性的获得。临床工作即刻种植手术过程中,当种植体与拔牙窝之间的间隙大于2mm时,通常建议进行骨引导再生(Guided bone regeneration,GBR)或联合使用骨替代材料。如何加快骨整合的速度,缩短种植体-骨界面的愈合时间,从而缩短种植疗程,并且尽早实现种植体的早期抗力成为现阶段口腔种植领域的研究热点。骨组织工程的发展为口腔种植体周围骨缺损的修复带来了新的契机。所谓的组织工程就是联合使用种子细胞、各种生长因子和支架材料促进组织的修复与再生。种子细胞的选择则是最基本与最关键的环节。间充质干细胞(Mesenchymal stem cells, MSCs)具有高度自我更新、复制能力以及多向分化的潜能,来源于发育早期的中胚层,广泛存在于全身多种组织,在组织工程中常被用作种子细胞。骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells, BMSCs)来源于骨髓,在骨组织工程中最早被用作种子细胞,但是骨髓间充质干细胞的取材时需要抽取供者的骨髓,取材过程非常痛苦,且其随着供者年龄的增长,其细胞数量和扩增、分化能力会出现明显下降趋势。寻找一种能够替代骨髓间充质干细胞并且可以弥补其缺陷的间充质干细胞,成为干细胞研究专家的头等大事。牙龈干细胞(Gingival derived mesenchymal stem cells,GMSCs)的成功提取,让更多的学者开始关注研究GMSCs。因为GMSCs取材方便简单、扩增容易、核型稳定,在口腔再生研究中受到研究者的追捧,甚至有学者推测GMSCs在再生医学中的修复能力甚至超过了BMSCs。富血小板纤维蛋白(Platelet rich fibrin,PRF)是法国学者Choukroun等在PRP的基础上发展了新一代血小板浓缩物,是将血液置于不含抗凝剂的试管中离心后形成的富含血小板和白细胞的纤维蛋白凝胶。PRF的凝固反应类似于生理的过程,其纤维蛋白的三维空间结构呈柔韧多孔状,可以有效地网聚血小板因子和白细胞,并能缓慢释放其内的多种生长因子。这些因子能够促进细胞增殖、基质改建及血管再生等,是组织愈合的重要调节因子。同时纤维蛋白可作为骨髓间叶细胞迁移和分化的基质。众多的实验研究已经证明,PRF能促进间充质干细胞以及众多前体细胞的增殖和分化,有潜在的新骨形成能力。目前尚未见到关于牙龈干细胞在即刻种植种植体骨缺损修复过程中作用的相关报道。本实验利用比格犬进行即刻种植,在种植体周围制造4mm×4mm×3.5mm大小的骨缺损,将GMSCs注射到缺损区的底部的骨髓腔,然后将PRF凝胶填充到骨缺损的区域,通过硬组织切片以及脱钙后的组织切片观察即刻种植后种植体周围骨缺损愈合的过程,初步探讨GMSCs在即刻种植种植体周围骨缺损修复中的作用。材料与方法： 1.牙龈干细胞的分离、培养和鉴定本实验从临床上阻生齿拔除牙上获得健康的牙龈组织,采用组织块法获得原代细胞。通过传代培养后,取第4代细胞用于实验。采用有限稀释法,挑选单克隆获得干细胞特性的牙龈干细胞进行传代培养。通过流式细胞仪检测第4代GMSCs表面抗原CD34,CD45,CD29,CD90, CD105以及STRO-1的表达。通过成纤维细胞克隆形成单位(CFU-F)实验检测牙龈干细胞的克隆形成能力。三向分化能力鉴定：在体外以1μM地塞米松、200μM吲哚美辛、10μM胰岛素和0.5mM3-异丁基-1-甲基黄嘌呤诱导骨髓基质干细胞向脂肪细胞分化,2周后用油红O(Oil Red O)染色检测脂肪滴的形成；以10ng/ml转化生长因子β1(transforming growth factor-β1,TGF-β1)、50nM抗坏血酸磷酸盐、0.1μM地塞米松诱导牙龈干细胞向软骨细胞分化,3周后通过甲苯胺蓝染色检测软骨特异性蛋白聚糖的形成；以0.1μM地塞米松、10mMβ-甘油磷酸钠、50mg/L抗坏血酸磷酸盐诱导牙龈干细胞向成骨细胞分化,4周后进行茜素红染色鉴定钙化结节的形成；体外分化诱导实验以不含诱导因子的完全培养基培养的同代牙龈干细胞为对照。 2.植入种植体建立即刻种植种植体周围骨缺损后注射牙龈干细胞健康雄性成年Beagle犬8只,行全身麻醉后小心拔除犬双侧下颌第二、第三、第四前磨牙。于远中根拔牙窝即刻植入直径3.6mm,长度8mm Super Line种植体。每个牙位远中根拔牙窝处植入的种植体均紧贴牙槽窝远中骨壁及颊舌骨壁,获得初期稳定性。在种植体近中制作深约4mm,颊舌向4mm,近远中向3.5mm的类似箱型的缺损区域,将第四代GMSCs以密度为1×108/ml重悬于新鲜的培养液中,实验组在缺损区的底部注射1ml细胞悬液,然后用PRF充填缺损区,对照组直接在缺损区充填PRF后缝合。术后2周、4周、8周处死动物,取得含种植体的下颌骨组织块,固定后,经过硬组织切片以及组织块脱钙后组织切片通过亚甲基蓝-碱性品红染色以及HE染色,观察缺损区种植体骨结合率以及种植体周围新骨生成率,评价牙龈干细胞对即刻种植种植体周围骨缺损修复的作用。结果： 1.牙龈干细胞的分离、培养和鉴定利用组织块法可成功分离人牙龈干细胞,经过传代培养获得的第四代干细胞。流式细胞仪检测其表面标记牙龈干细胞表达CD105(85.14%), CD29(95.11%),CD90(91.78%)以及Stro-1(16.21%)；而不表达CD.34(0.09%)和CD45(0.03%)。成纤维细胞克隆形成单位实验显示牙龈干细胞具有较强的克隆形成能力。人牙龈干细胞骨向诱导4周后进行茜素红染色,发现有致密红染的细胞外基质沉积和矿化结节的形成；脂向诱导2周后进行油红0染色,可见大量红色脂滴形成；成软骨诱导3周后进行甲苯胺蓝染色,观察到细胞胞质内有异染蓝色,以上结果提示本实验分离培养的牙龈干细胞具有多向分化的潜能。 2.建立即刻种植种植体周围骨缺损动物模型以及牙龈干细胞的注射种植体植入后的2周,种植体周围骨缺损区域可见大量的纤维结缔组织,未见明显的骨结合,但是缺损区远离种植体的区域可见少量的新骨生成。随着种植体植入时间的延长,缺损区种植体周围可以发现明显的骨结合,在种植体周围以及远离种植体的区域均可见到新骨的生成。种植体植入4周后,缺损区实验组与对照组的种植体骨结合率分别为51.08±0.98%,40.79±0.65%；8周后的骨结合率分别为72.83±1.09%,61.17±2.79%,其差异具有统计学意义(p0.05)。种植体植入2周后,缺损区新骨生成率实验组与对照组分别为17.08±0.49%,14.30±1.25%；4周后的新骨生成率实验组与对照组分别为47.33±3.21%,37.04±2.29%；8周后缺损区内的新骨生成率实验组与对照组分别为65.96±3.60%,58.83±3.36%,其差异具有统计学意义(p0.05)。结论： 1、人牙龈干细胞可以在体外成功分离培养、扩增与纯化,方法简单,易于操作。分离培养所获得牙龈干细胞具有较强的克隆能力以及三向(成脂、成骨、成软骨)分化潜能。 2、在即刻种植骨缺损修复的动物模型上牙龈干细胞表现出卓越的成骨能力,能够加速种植体周围骨缺损的修复,可以作为种子细胞应用于种植体周围骨缺损的修复。
[Abstract]:Background and Purpose :

In order to satisfy the actual needs of patients with missing teeth , immediate implant technology has become a therapeutic trend in the restoration of missing teeth .

How to accelerate the speed of bone integration and shorten the healing time of implant - bone interface can shorten the treatment course , and realize the early resistance of implant as a hot spot in the field of oral planting .

The development of bone tissue engineering brings new opportunities for the repair of bone defects around oral implants . The so - called tissue engineering is the combination of seed cells , growth factors and scaffold materials to promote the repair and regeneration of tissue . The selection of seed cells is the most basic and crucial link .

Bone marrow - derived mesenchymal stem cells were originally used as seed cells in bone tissue engineering , but the bone marrow of bone marrow mesenchymal stem cells was used as seed cells .

Platelet rich fibrin ( PRF ) , a French scholar , developed a new generation of platelet concentrates on the basis of PRP .

In order to investigate the effect of GMSCs on bone defect healing immediately after implant , the effects of GMSCs on the repair of bone defects around the implant were investigated .

Materials and Methods :

1 . Isolation , culture and identification of gingival stem cells

In this experiment , the healthy gingival tissues were obtained from the tooth extraction teeth . The primary cells were obtained by tissue block method . After subculture , the 4th generation cells were used for the experiment . The expression of CD34 , CD29 , CD90 , CD105 and STRO - 1 was detected by flow cytometry .
transforming growth factor - 尾1 , transforming growth factor - 尾1 , TGF - 尾1 , 50nM ascorbic acid phosphate and 0.1渭M dexamethasone on differentiation of gingival stem cells into chondrocytes , and detecting the formation of cartilage - specific protein by means of methylene blue staining after 3 weeks ;
After 4 weeks , alizarin red staining was used to identify calcified nodules .
In vitro differentiation - induced experiments were compared with the same - generation gingival stem cells cultured in a complete medium without an inducing factor .

2 . Implantation of implants for immediate implant of peri - implant bone defects and injection of gingival stem cells

In order to evaluate the effect of gingival stem cells on bone defects around the implant .

Results :

1 . Isolation , culture and identification of gingival stem cells

CD105 ( 85.14 % ) , CD29 ( 95.11 % ) , CD90 ( 91.78 % ) and Stro - 1 ( 16.21 % ) were detected by flow cytometry .
The results showed that gingival stem cells had strong clonal formation ability . After 4 weeks of induction , alizarin red staining was performed in human gingival stem cells , and the formation of dense red - stained extracellular matrix deposition and mineralized nodules was found .
After 2 weeks of lipopolysaccharide , the oil was stained with oil red 0 , and a large amount of red lipid droplets were observed .
The results suggested that the cultured gingival stem cells had multi - directional differentiation potential after 3 weeks of chondrogenic induction . The results suggested that the cultured gingival stem cells had the potential of multi - directional differentiation .

2 . Establishment of an animal model for immediate implant of peri - implant bone defects and injection of gingival stem cells

There was a large number of fibrous connective tissue around the implant in 2 weeks after implant implantation , no obvious bone union was seen in the area of the defect , but a small amount of new bone formation could be seen around the implant . As the implant implantation time was prolonged , the bone union rate of the implant was 51.08 卤 0.98 % and 40.79 卤 0.65 % , respectively .
After 8 weeks , the bone union rate was 72.83 卤 1.09 % , 61.17 卤 2.79 % , the difference was statistically significant ( p < 0.05 ) . After 2 weeks of implant implantation , the new bone formation rate in the defect area was 17.08 卤 0.49 % and 14.30 卤 1.25 % , respectively .
The new bone formation rate after 4 weeks was 47.33 卤 3.21 % , 37.04 卤 2.29 % in the experimental group and the control group , respectively .
The new bone formation rate in the defect area after 8 weeks was 65.96 卤 3.60 % , 58.83 卤 3.36 % in the experimental group and the control group , respectively ( p < 0.05 ) .

Conclusion :

1 , human gingival stem cells can be successfully isolated , amplified and purified in vitro , and the method is simple and easy to operate .

and 2 , the gingival stem cells show excellent osteogenic capacity on an animal model immediately planted with bone defect , can accelerate the repair of the bone defect around the implant , and can be used as a seed cell to be applied to the repair of the bone defect around the implant .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R783.6

【相似文献】