原位保留病变牙骨质联合应用EMD对牙骨质再生的影响
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摘要
背景和目的
牙周病治疗的最终目标是在控制牙周炎症的基础上完成牙周组织的重建。牙周再生一直是牙周领域的研究热点,目前应用于牙周再生的方法主要有：引导组织再生术(guided tissue regeneration, GTR)、骨材料移植、生长因子的应用以及近年来研究较多的组织工程技术。然而这些手段都不能实现牙周组织的完全再生,仅能不同程度地重建部分牙周附着,其中牙骨质的再生显得尤为困难,目前仍不能预测牙骨质再生的数量和质量。
牙骨质是围绕在牙根周围的一薄层矿化结缔组织,是牙周组织的重要组成部分。牙骨质的最主要生理功能是为牙周膜纤维提供附着的锚点。牙骨质中羟基磷灰石占50%,牙骨质基质占50%。由胶原蛋白和非胶原蛋白构成。近些年的研究发现牙骨质基质中含有多种非胶原蛋白和生长因子,如骨涎蛋白(bone sialoprotein, BSP)、骨桥素(osteopontin, OPN)、纤连蛋白(fibronectin, FN)、骨形态蛋白(bone morphogenetic protein, BMP-2,-3,-4)、血小板来源生长因子-α,β (platelet-derived growth factor-a and β, PDGF-a and β)、转化生长因子(transforming growth factor, TGF-β)等,其独特的细胞外基质成分可能为牙骨质的再生提供一个良好的微环境。本课题组的前期实验也发现经EDTA处理的人健康的牙骨质根片能够诱导人牙周膜细胞(human periodontal ligament cells, hPDLCs)向成牙骨质细胞方向转化,从而促进牙骨质新生。然而,这一结论是否同样适用于牙周炎累及的病变牙骨质呢?如果不能,是否有一些生长因子能够增强其促进牙骨质再生的作用呢?
当牙周炎症破坏牙龈胶原纤维到达牙根表面时,牙骨质的结构和成分就遭到了破坏,称为病变牙骨质。虽然有研究发现病变牙骨质在去除牙石菌斑基础上经过简单抛光也能达到牙周健康,但近几年一些学者的研究发现病变牙骨质基质内的一些非胶原蛋白成分发生了改变,如骨涎蛋白(BSP)、骨桥蛋白(OPN)、纤粘连蛋白(FN)等。这些成分的改变势必影响了牙骨质的细胞外基质环境,这种病变牙骨质是否仍有利于牙骨质再生,目前鲜有报道。
在众多应用于牙周再生的生长因子中,釉基质蛋白(enamel matrix proteins, EMPs)被公认具有促进牙骨质再生的作用。EMPs是一组以釉原蛋白为优势的蛋白,它并不是单一的生长因子,它的存在可能一定程度上模仿了牙骨质发育时期的情况。釉基质衍生物(enamel matrix derivative, EMD)是经过纯化的釉基质蛋白,其主要成分为釉原蛋白(amelogenin),商品名为Emdogain,广泛应用于临床和动物实验。动物实验发现EMD能够诱导形成无细胞外源纤维性牙骨质。临床对比试验发现应用EMD比应用屏障膜能够获得更多的临床附着。近几年的研究发现EMD在体外能够对多种细胞的生物学性能产生影响,并能诱导多种细胞,如牙周膜细胞、骨髓基质细胞等向成骨／成牙骨质细胞方向分化。但目前EMD在牙骨质表面和牙本质表面上的作用是否不同尚未见报道。
因此,本研究的目的在于观察原位保留的牙周炎病变牙骨质是否能够促进牙骨质再生,在保留牙骨质的基础上加入EMD是否能强化牙骨质再生的效果。
方法
1.原位保留病变牙骨质联合应用EMD对人牙周膜细胞成牙骨质分化的研究
1.1病变牙骨质原位保留对人牙周膜细胞成牙骨质分化影响的体外研究
收集因牙周炎Ⅲ度松动而拔除的前牙和前磨牙48颗,并沿牙龈边缘和牙周袋底做标记。沿标记截取中间部分,EMS超声器械去除根面牙石菌斑后纵裂为对称的两片,一片用刮治器去除根面牙骨质,暴露根面牙本质(dentin, D组),另一片保留牙骨质(cementum, C组)。所有根片经24%EDTA凝胶酸蚀2min后于20万U／ml青霉素链霉素溶液4℃浸泡48h,无菌PBS冲洗后备用。收集因正畸需要拔除的前磨牙的牙周膜,组织块法原代培养人牙周膜细胞,胰酶消化,收集第二代细胞用于后续实验。C组和D组各取24片置于48孔板内,第二代牙周膜细胞以1×106／ml的浓度接种到根片表面,共培养7天后,每组取四片进行扫描电镜观察,每组剩余20片分别用微量RNA提取试剂盒提取总RNA,荧光实时定量PCR的方法检测两组牙骨质附着蛋白(cementum attachment protein,CAP)和牙骨质蛋白-23(cementum protein-23,CP-23)的mRNA表达量。
1.2原位保留病变牙骨质联合应用EMD对人牙周膜细胞体内形成牙骨质的影响
牙本质组和牙骨质组根片各取24片置于48孔板内,第二代牙周膜细胞以1x106个/ml的浓度接种到根片表面,按照重悬液的不同,将两组根片分为四组：牙骨质片滴加DMEM培养液为C组,牙骨质片滴加EMD培养液为E+C组,牙本质片滴加DMEM培养液为D组,牙本质片滴加EMD培养液为E+D组。每三天换液一次,共培养至第七天时,用聚四氟乙烯膜包裹根片,植入裸鼠背部皮下,每只裸鼠共植入四片,每组一片。三周后处死裸鼠取出根片,10%EDTA液脱钙后进行常规石蜡包埋、5μm切片、HE染色,观察四组标本新生组织的形成情况,对有牙骨质类似物形成的标本进行BSP的免疫组化染色观察。
2.原位保留病变牙骨质联合应用EMD在Ⅲ度根分叉病变牙骨质再生及新附着形成中的作用
选取雄性比格犬八只,双侧下颌第三第四前磨牙(P3P4)作为实验牙位。实验当日前四周,在实验牙位制作高度为5mm的Ⅲ度根分叉骨缺损并置入含有牙周混合菌的棉球2周,以建立Ⅲ度根分叉病变模型。实验当日前2周将棉球取出,并对实验牙位进行龈下刮治以去除菌斑牙石。此后每天用氯己定溶液进行口腔卫生护理。手术当天,未见明显炎症和牙龈退缩,每只比格犬的双侧P3P4按随机原则接受下列处理方法中的一种：C：保留病变牙骨质+EDTA酸蚀2min；D：刮除病变牙骨质+EDTA酸蚀2min；E+C：保留病变牙骨质+EDTA酸蚀2min+EMD凝胶；E+D：刮除病变牙骨质+EDTA酸蚀2min+EMD凝胶。各组均在全麻下颊侧翻瓣,刮除肉芽组织,并按实验要求处理相关根面,并于根分叉入口覆盖海奥胶原膜。基于本实验的目的和实验操作方便的考虑,操作过程中未给以舌侧翻瓣。术后八周4%多聚甲醛灌注法处死动物,取出标本,每组共获8个标本,每组取2个标本进行硬组织包埋处理,切片,甲苯胺蓝染色,进行组织学观察。每组剩余6个标本进行常规脱钙,石蜡包埋,5μm切片,HE染色,进行新生组织的观察和测量。
结果
1.根面保留病变牙骨质联合应用EMD对人牙周膜细胞成牙骨质分化的研究
1.1病变牙骨质原位保留对人牙周膜细胞成牙骨质分化影响的体外研究
牙周膜组织块贴壁7-10天左右,倒置显微镜下可见单个细胞从组织块周边游出,当细胞密度达瓶底80%时可进行传代。传代后的细胞经免疫染色显示波形丝蛋白染色阳性,角蛋白染色阴性,证明培养的细胞是中胚层来源的间充质细胞且无外胚层来源的细胞污染。第二代牙周膜细胞接种于两组根片后的扫描电镜显示：两组根片上细胞生长良好,牙本质片上细胞呈长梭形,牙骨质片上细胞略大,长梭形态不明显。荧光实时定量PCR的结果显示：牙骨质组(C组)CAP mRNA表达量显著高于牙本质组(D组),差异有统计学意义(P=0.001)；C组CP-23mRNA表达量显著高于D组,差异有统计学意义(P=0.003)。
1.2原位保留病变牙骨质联合应用EMD对人牙周膜细胞体内形成牙骨质的影响
裸鼠植入根片后,活动良好,未出现饮食异常。术后一周拆线,除一只裸鼠的左上伤口缝线已开,根片脱离外,其余伤口均愈合良好。聚四氟乙烯膜包绕根片位于裸鼠皮下,无感染,无暴露。三周时取材,D、E+C、E+D三组均获得12个标本,C组获得11个标本。HE染色结果显示：D组与E+D组均无牙骨质类似物形成,新生物均为疏松的纤维结缔组织；C组有7个标本表现为新生牙骨质类似物形成,E+C组有10个标本表现为新生牙骨质类似物形成,两组间差异有统计学意义(P<0.01)。BSP染色结果发现所有的新生牙骨质类似物均对BSP染色阳性。
3.原位保留病变牙骨质联合应用EMD在Ⅲ度根分叉病变牙骨质再生及新附着形成中的作用
我们成功建立了32颗比格犬下颌前磨牙的Ⅲ度根分叉病变模型。术后八周,所有牙位愈合良好,无明显感染或牙龈退缩。组织学测量结果显示,新生牙骨质百分比：C组显著高于D组,而E+C组和E+D组显著高于C组,E+C组和E+D组组间差异不显著。新生骨百分比：C组显著高于D组,而E+C组和E+D组显著高于C组,E+C组和E+D组组间差异不显著。组织学观察发现,D组新生牙周膜纤维稀疏,无主纤维束,与新生牙骨质和牙槽骨平行排列或排列混乱,未插入其中形成良好的新附着；C组新生牙骨质沉积在原有牙骨质表面,牙周膜纤维排列整齐,且有主纤维束分别插入新生牙槽骨和牙骨质中,形成新附着；E+D组与E+C组新生牙骨质数量多,牙周膜纤维粗大,大多建立了良好的新附着,但E+D组多见新生牙骨质与牙本质表面存在裂隙,新生牙周膜纤维排列杂乱。硬组织切片观察结果与石蜡切片结果相似,但未见明显的新生牙骨质与根面分离的情况。
结论
1.经EDTA处理的病变牙骨质能诱导牙周膜细胞分化为成牙骨质细胞,从而促进新生牙骨质的生成,EMD的存在能够增强这一诱导作用。
2.原位保留病变牙骨质有利于牙骨质的再生和新附着的建立,加入EMD能够强化这一效果。
Backgroud and Objective
The ultimate goal of periodontitis treatment is to reconstruct the periodontal tissues on the basis of inflammation control. Nowadays there are several stragies applied in periodontal regeneration, such as guided tissue regeneration (GTR), grafts of bone materials, application of growth factors as well as tissue engineer, which is a hot spot in the field of regeneration. However, these stragies could only result in partial reconstruction of periodontal attachment, the complete regeneration of periodontal tissue is unavailable. The regeneration of cementum seems critically difficult as it is still unable to predict the amount and quality of regenerated cementum.
Cementum, a thin mineralized connective tissue, constitutes the integrity of periodontium with its major function as the site of attachment for principal collagen fibers. Besides the majority of collagens, the extracellular matrix (ECM) of cementum is a rich pool of various growth factors and non-collagen proteins, such as bone sialoprotein (BSP)、osteopontin (OPN)、fibronectin (FN)、bone morphogenetic protein(BMP-2,-3,-4)、 platelet-derived growth factor (PDGF-a and β)、transforming growth factor (TGF-β). These components form the unique microenviroment of cementum. There is accumulating evidence that the preservation of root cementum may favor periodontal regeneration, especially formation of new cementum. The former study in our research group also found that the EDTA conditioned pre-existing healthy cementum may promote hPDLCs differentiate towards cemtoblasts, thus play a role in cementum regeneration. But how about the role of periodontitis affected cementum in cementum regeneration? Is there any growth factor that can be combined to promote its function?
In periodontitis, the structure and composition are affected when the chronic inflammatory process destroys gingival collagen fibers and extends to the root surface, this is so called diseased cementum. Although there are results supporting that the diseased cementum can be periodontal healthy after simple polishing on the basis of removal of calculus and bacterial plaque, the matrix composition of diseased cementum was changed, several non-collagen proteins were affected, such as bone sialoprotein (BSP), osteoprotein (OPN) and fibronectin (FN). The changes in the matrix could result in the alteration of the cementum microenviroment inevitably, the effect of diseased cementum on cementum regeneration is obscure.
Enamel matrix derivative (EMD), which is widely used in regenerative periodontal therapy, is believed to induce the formation of cementum and promote new attachment formation. EMD is an extracellular matrix that contains proteins similar to that derived from Hertwig epithelial root sheath (HERS) and the application of EMD in periodontal defects may mimic the events that take place during the development of root. It was reported that EMD could induce the formation of acellular extrinsic fiber cementum (AEFC) in animal studies, application of EMD could yield more clinical attachment compared with GTR in clinical trials. A number of recent studies explored the effects of EMD on the biological characters of several types of cells in vitro, the results implied that EMD could induce the differentiation of some types of cells, such as periodontal ligament cell and bone marrow stromal cell towards osteoblast/cementoblast lineage. However, there were no reports regarding the comparision of regenerative effects induced by EMD respectively on cementum surface and dentin surface.
Thus, this study aimed to explore the effect of diseased cementum on cementum regeneration, and observe if this effect can be amplified in the presence with EMD.
Methods
1. The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum.
1.1The differential effects of diseased cementum preservation on hPDLCs towards cemetum in vitro.
48extracted teeth due to periodontitis were collected, and notches were made at teeth on the coronal and apical line of the pockets by a pencil immediately after extraction. Root debridement was performed by EMS ultrasonic sealer to completely remove the calculus and plaques. Symmetrical root slices were made using diamond bur and one of the symmetrical slices of each root was further scaled to remove root cementum and distributed into the D (dentin) group, the other symmetrical slice was divided into the C (cementum) group in which the cementum was preserved in situ. All slices were conditioned with24%ethylene diamine tetraacetie acid (EDTA) gel for2min and supplemented with10%penicillin and streptomycin for24hours at4℃for sterilization. hPDLCs were isolated according to a modification of the method of Somerman.24root slices from each group were placed into48-well plates, with one chip in each well. The second passage hPDLCs were harvested and suspended at a concentration of1x10/ml and cell suspension was sparsely seeded onto each root slice. After a7-day co-culture,4slices from each group were selected to undergo SEM observation, and the remained20slices in each group were used for real-time PCR to detect the mRNA expression of cementum attachment protein (CAP) and cementum protein-23(CP-23).
1.2The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum in vivo.
24root slices from C and D group were placed into48-well plate, and hPDLCs with concentration of1x106/ml were seeded onto root slices. Root slices in this part were divided into four groups, cementum slices(group C) and dentin slices (group D) incubated in the DMEM culture medium respectively, as well as cementum slices (group E+C) and dentin slices (group E+D) incubated in DMEM culture medium containing EMD respectively, each group contained12root slices. After a7-day co-culture,12root slices of each group were rinsed with PBS and wrapped with sterilized expanded polytetrafluoroethylene (ePTFE), and then were inserted beneath the dorsal skin of nude mice. Each mouse received four slices, with one from each group. The mice were sacrificed3weeks after surgery, all the specimens were decalcified with10%EDTA and embedded with paraffin,5μm serially slices were made and HE staining was carried out. The newly-formed tissue in four groups were observed and BSP staining was carried out in specimens with newly formed cementum.
2. The effects of diseased cementum preservation in combination with EMD on cementum regeneration and new attachment formation in class III furcation defects.
8male beagle dogs were chosen, and4teeth (the right and left third and fourth premolars in the mandible) from each dog were used for this experiment. Four weeks before the experiment day, class III furcation defects were surgically created (height:5mm) in experiment teeth and cotton balls saturated with anaerobic bacteria were placed in all defects to initiate inflammation. The cotton ball was removed from the defects2weeks before the day of experiment, and then plaque control was achieved by subgingival scaling and oral hygiene with0.2%chlorhexidine gluconate irriation. On the day of experiment, no severe inflammation and gingival recession were observed, four experimental teeth of each dog received one of the following treatments randomly. C:preservation of root cementum in situ+24%EDTA conditioning D:removal of root cementum+24%EDTA conditioning E+C:preservation of root cementum in situ+24%EDTA conditioning+EMD E+D:removal of root cementum+24%EDTA conditioning+EMD All the buccal mucoperiosteal flaps were elevated, and the furactions were treated according to the design, then collagenous membranes were placed on buccal side of all the defects. Flaps on the lingual side were not elevated in consideration of the objective of the study and convenience in operation. All the dogs were sacrificed by4%paraformaldehyde perfusion. All the specimens were harvested and2specimens from each group were embedded with resin and the slices were stained with toluidine blue for histological observation. The remained6Specimens were decalcified with10%EDTA and embedded with paraffin,5μm serially slices were made and HE staining was used for histological observation and histometric study.
Results
1. The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum.
2.1The differential effects of diseased cementum preservation on hPDLCs towards cemetum in vitro..
The cells began to grow out from the periodontal ligament explants after7day to10day. Then they proliferated quickly and the cells were digested when they reached80%confluence. Immunohistochemical staining results showed that the second passage hPDLCs were positive for vimentin and negative for keratin, certificating that they were originated from ectomesenchyme. SEM results showed that the cells seeded on the cementum slices and dentin slices grew well, cells on the dentin surface exhibited a spindle shape while cells on cementum were much larger without typical spindle shape. Both CAP and CP-23expression were significantly higher on the cementum surface than on the dentin surface.(p=0.001for CAP and p=0.003for CP-23respectively).
2.2The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum in vivo.
All the mice survived without inflammation throughout the experiment period,1root slice in group C fell off as the sutures loose, other11specimens in this group were obtained.12specimens of other three groups were obtained. Results of HE staining showed that neither group D nor group E+D had newly formed cementum-like tissue (NFC) formation,7specimens in group C showed NFC formation while10specimens in group E+C showed NFC formation, the results between group C and group E+C were statistically different.
2.The effects of diseased cementum preservation in combination with EMD on cementum regeneration and new attachment formation in class III furcation defects.
We artificially created32inflamed Class III furcation defects in beagle dogs. Within a healing period of8weeks, healing of the wounds in all groups occurred uneventfully, with no infection or suppuration. Histomorphometric results showed that the percentage of regenerated cementum (PRP) and the percentage of regenerated bone (PRB) in C group were statistically higher than those in D group, PRP and PRB in E+D and E+C groups were statistically higher than those in groups without EMD, but there were no differences between E+D group and E+C group. D group:The newly formed periodontal ligament was loosely arranged without principle fiber bundles, and favorable new attachment was not established. C group:Newly formed cementum was laid on the top of old cementum, newly formed periodontal ligament was in orderly arranged, with principle fiber inserting into new bone and new cementum, and favorable new attachment was established. The quality of new attachment in group E+D and E+C was almost good, except for the artifacts between newly formed cementum and dentin surface observed in group E+D. Results from resin slices were similar, but artifacts were not seen.
Conclusion
1. Preservation of diseased cementum promoted the differentiation of inoculated hPDLCs towards cementoblasts, and the effect was amplified by the presence of EMD.
2. Preservation of diseased cementum favored the cementum regeneration as well as formation of new attachment, and the application of EMD could strengthen the effect.
牙周病治疗的最终目标是在控制牙周炎症的基础上完成牙周组织的重建。牙周再生一直是牙周领域的研究热点,目前应用于牙周再生的方法主要有：引导组织再生术(guided tissue regeneration, GTR)、骨材料移植、生长因子的应用以及近年来研究较多的组织工程技术。然而这些手段都不能实现牙周组织的完全再生,仅能不同程度地重建部分牙周附着,其中牙骨质的再生显得尤为困难,目前仍不能预测牙骨质再生的数量和质量。
牙骨质是围绕在牙根周围的一薄层矿化结缔组织,是牙周组织的重要组成部分。牙骨质的最主要生理功能是为牙周膜纤维提供附着的锚点。牙骨质中羟基磷灰石占50%,牙骨质基质占50%。由胶原蛋白和非胶原蛋白构成。近些年的研究发现牙骨质基质中含有多种非胶原蛋白和生长因子,如骨涎蛋白(bone sialoprotein, BSP)、骨桥素(osteopontin, OPN)、纤连蛋白(fibronectin, FN)、骨形态蛋白(bone morphogenetic protein, BMP-2,-3,-4)、血小板来源生长因子-α,β (platelet-derived growth factor-a and β, PDGF-a and β)、转化生长因子(transforming growth factor, TGF-β)等,其独特的细胞外基质成分可能为牙骨质的再生提供一个良好的微环境。本课题组的前期实验也发现经EDTA处理的人健康的牙骨质根片能够诱导人牙周膜细胞(human periodontal ligament cells, hPDLCs)向成牙骨质细胞方向转化,从而促进牙骨质新生。然而,这一结论是否同样适用于牙周炎累及的病变牙骨质呢?如果不能,是否有一些生长因子能够增强其促进牙骨质再生的作用呢?
当牙周炎症破坏牙龈胶原纤维到达牙根表面时,牙骨质的结构和成分就遭到了破坏,称为病变牙骨质。虽然有研究发现病变牙骨质在去除牙石菌斑基础上经过简单抛光也能达到牙周健康,但近几年一些学者的研究发现病变牙骨质基质内的一些非胶原蛋白成分发生了改变,如骨涎蛋白(BSP)、骨桥蛋白(OPN)、纤粘连蛋白(FN)等。这些成分的改变势必影响了牙骨质的细胞外基质环境,这种病变牙骨质是否仍有利于牙骨质再生,目前鲜有报道。
在众多应用于牙周再生的生长因子中,釉基质蛋白(enamel matrix proteins, EMPs)被公认具有促进牙骨质再生的作用。EMPs是一组以釉原蛋白为优势的蛋白,它并不是单一的生长因子,它的存在可能一定程度上模仿了牙骨质发育时期的情况。釉基质衍生物(enamel matrix derivative, EMD)是经过纯化的釉基质蛋白,其主要成分为釉原蛋白(amelogenin),商品名为Emdogain,广泛应用于临床和动物实验。动物实验发现EMD能够诱导形成无细胞外源纤维性牙骨质。临床对比试验发现应用EMD比应用屏障膜能够获得更多的临床附着。近几年的研究发现EMD在体外能够对多种细胞的生物学性能产生影响,并能诱导多种细胞,如牙周膜细胞、骨髓基质细胞等向成骨／成牙骨质细胞方向分化。但目前EMD在牙骨质表面和牙本质表面上的作用是否不同尚未见报道。
因此,本研究的目的在于观察原位保留的牙周炎病变牙骨质是否能够促进牙骨质再生,在保留牙骨质的基础上加入EMD是否能强化牙骨质再生的效果。
方法
1.原位保留病变牙骨质联合应用EMD对人牙周膜细胞成牙骨质分化的研究
1.1病变牙骨质原位保留对人牙周膜细胞成牙骨质分化影响的体外研究
收集因牙周炎Ⅲ度松动而拔除的前牙和前磨牙48颗,并沿牙龈边缘和牙周袋底做标记。沿标记截取中间部分,EMS超声器械去除根面牙石菌斑后纵裂为对称的两片,一片用刮治器去除根面牙骨质,暴露根面牙本质(dentin, D组),另一片保留牙骨质(cementum, C组)。所有根片经24%EDTA凝胶酸蚀2min后于20万U／ml青霉素链霉素溶液4℃浸泡48h,无菌PBS冲洗后备用。收集因正畸需要拔除的前磨牙的牙周膜,组织块法原代培养人牙周膜细胞,胰酶消化,收集第二代细胞用于后续实验。C组和D组各取24片置于48孔板内,第二代牙周膜细胞以1×106／ml的浓度接种到根片表面,共培养7天后,每组取四片进行扫描电镜观察,每组剩余20片分别用微量RNA提取试剂盒提取总RNA,荧光实时定量PCR的方法检测两组牙骨质附着蛋白(cementum attachment protein,CAP)和牙骨质蛋白-23(cementum protein-23,CP-23)的mRNA表达量。
1.2原位保留病变牙骨质联合应用EMD对人牙周膜细胞体内形成牙骨质的影响
牙本质组和牙骨质组根片各取24片置于48孔板内,第二代牙周膜细胞以1x106个/ml的浓度接种到根片表面,按照重悬液的不同,将两组根片分为四组：牙骨质片滴加DMEM培养液为C组,牙骨质片滴加EMD培养液为E+C组,牙本质片滴加DMEM培养液为D组,牙本质片滴加EMD培养液为E+D组。每三天换液一次,共培养至第七天时,用聚四氟乙烯膜包裹根片,植入裸鼠背部皮下,每只裸鼠共植入四片,每组一片。三周后处死裸鼠取出根片,10%EDTA液脱钙后进行常规石蜡包埋、5μm切片、HE染色,观察四组标本新生组织的形成情况,对有牙骨质类似物形成的标本进行BSP的免疫组化染色观察。
2.原位保留病变牙骨质联合应用EMD在Ⅲ度根分叉病变牙骨质再生及新附着形成中的作用
选取雄性比格犬八只,双侧下颌第三第四前磨牙(P3P4)作为实验牙位。实验当日前四周,在实验牙位制作高度为5mm的Ⅲ度根分叉骨缺损并置入含有牙周混合菌的棉球2周,以建立Ⅲ度根分叉病变模型。实验当日前2周将棉球取出,并对实验牙位进行龈下刮治以去除菌斑牙石。此后每天用氯己定溶液进行口腔卫生护理。手术当天,未见明显炎症和牙龈退缩,每只比格犬的双侧P3P4按随机原则接受下列处理方法中的一种：C：保留病变牙骨质+EDTA酸蚀2min；D：刮除病变牙骨质+EDTA酸蚀2min；E+C：保留病变牙骨质+EDTA酸蚀2min+EMD凝胶；E+D：刮除病变牙骨质+EDTA酸蚀2min+EMD凝胶。各组均在全麻下颊侧翻瓣,刮除肉芽组织,并按实验要求处理相关根面,并于根分叉入口覆盖海奥胶原膜。基于本实验的目的和实验操作方便的考虑,操作过程中未给以舌侧翻瓣。术后八周4%多聚甲醛灌注法处死动物,取出标本,每组共获8个标本,每组取2个标本进行硬组织包埋处理,切片,甲苯胺蓝染色,进行组织学观察。每组剩余6个标本进行常规脱钙,石蜡包埋,5μm切片,HE染色,进行新生组织的观察和测量。
结果
1.根面保留病变牙骨质联合应用EMD对人牙周膜细胞成牙骨质分化的研究
1.1病变牙骨质原位保留对人牙周膜细胞成牙骨质分化影响的体外研究
牙周膜组织块贴壁7-10天左右,倒置显微镜下可见单个细胞从组织块周边游出,当细胞密度达瓶底80%时可进行传代。传代后的细胞经免疫染色显示波形丝蛋白染色阳性,角蛋白染色阴性,证明培养的细胞是中胚层来源的间充质细胞且无外胚层来源的细胞污染。第二代牙周膜细胞接种于两组根片后的扫描电镜显示：两组根片上细胞生长良好,牙本质片上细胞呈长梭形,牙骨质片上细胞略大,长梭形态不明显。荧光实时定量PCR的结果显示：牙骨质组(C组)CAP mRNA表达量显著高于牙本质组(D组),差异有统计学意义(P=0.001)；C组CP-23mRNA表达量显著高于D组,差异有统计学意义(P=0.003)。
1.2原位保留病变牙骨质联合应用EMD对人牙周膜细胞体内形成牙骨质的影响
裸鼠植入根片后,活动良好,未出现饮食异常。术后一周拆线,除一只裸鼠的左上伤口缝线已开,根片脱离外,其余伤口均愈合良好。聚四氟乙烯膜包绕根片位于裸鼠皮下,无感染,无暴露。三周时取材,D、E+C、E+D三组均获得12个标本,C组获得11个标本。HE染色结果显示：D组与E+D组均无牙骨质类似物形成,新生物均为疏松的纤维结缔组织；C组有7个标本表现为新生牙骨质类似物形成,E+C组有10个标本表现为新生牙骨质类似物形成,两组间差异有统计学意义(P<0.01)。BSP染色结果发现所有的新生牙骨质类似物均对BSP染色阳性。
3.原位保留病变牙骨质联合应用EMD在Ⅲ度根分叉病变牙骨质再生及新附着形成中的作用
我们成功建立了32颗比格犬下颌前磨牙的Ⅲ度根分叉病变模型。术后八周,所有牙位愈合良好,无明显感染或牙龈退缩。组织学测量结果显示,新生牙骨质百分比：C组显著高于D组,而E+C组和E+D组显著高于C组,E+C组和E+D组组间差异不显著。新生骨百分比：C组显著高于D组,而E+C组和E+D组显著高于C组,E+C组和E+D组组间差异不显著。组织学观察发现,D组新生牙周膜纤维稀疏,无主纤维束,与新生牙骨质和牙槽骨平行排列或排列混乱,未插入其中形成良好的新附着；C组新生牙骨质沉积在原有牙骨质表面,牙周膜纤维排列整齐,且有主纤维束分别插入新生牙槽骨和牙骨质中,形成新附着；E+D组与E+C组新生牙骨质数量多,牙周膜纤维粗大,大多建立了良好的新附着,但E+D组多见新生牙骨质与牙本质表面存在裂隙,新生牙周膜纤维排列杂乱。硬组织切片观察结果与石蜡切片结果相似,但未见明显的新生牙骨质与根面分离的情况。
结论
1.经EDTA处理的病变牙骨质能诱导牙周膜细胞分化为成牙骨质细胞,从而促进新生牙骨质的生成,EMD的存在能够增强这一诱导作用。
2.原位保留病变牙骨质有利于牙骨质的再生和新附着的建立,加入EMD能够强化这一效果。
Backgroud and Objective
The ultimate goal of periodontitis treatment is to reconstruct the periodontal tissues on the basis of inflammation control. Nowadays there are several stragies applied in periodontal regeneration, such as guided tissue regeneration (GTR), grafts of bone materials, application of growth factors as well as tissue engineer, which is a hot spot in the field of regeneration. However, these stragies could only result in partial reconstruction of periodontal attachment, the complete regeneration of periodontal tissue is unavailable. The regeneration of cementum seems critically difficult as it is still unable to predict the amount and quality of regenerated cementum.
Cementum, a thin mineralized connective tissue, constitutes the integrity of periodontium with its major function as the site of attachment for principal collagen fibers. Besides the majority of collagens, the extracellular matrix (ECM) of cementum is a rich pool of various growth factors and non-collagen proteins, such as bone sialoprotein (BSP)、osteopontin (OPN)、fibronectin (FN)、bone morphogenetic protein(BMP-2,-3,-4)、 platelet-derived growth factor (PDGF-a and β)、transforming growth factor (TGF-β). These components form the unique microenviroment of cementum. There is accumulating evidence that the preservation of root cementum may favor periodontal regeneration, especially formation of new cementum. The former study in our research group also found that the EDTA conditioned pre-existing healthy cementum may promote hPDLCs differentiate towards cemtoblasts, thus play a role in cementum regeneration. But how about the role of periodontitis affected cementum in cementum regeneration? Is there any growth factor that can be combined to promote its function?
In periodontitis, the structure and composition are affected when the chronic inflammatory process destroys gingival collagen fibers and extends to the root surface, this is so called diseased cementum. Although there are results supporting that the diseased cementum can be periodontal healthy after simple polishing on the basis of removal of calculus and bacterial plaque, the matrix composition of diseased cementum was changed, several non-collagen proteins were affected, such as bone sialoprotein (BSP), osteoprotein (OPN) and fibronectin (FN). The changes in the matrix could result in the alteration of the cementum microenviroment inevitably, the effect of diseased cementum on cementum regeneration is obscure.
Enamel matrix derivative (EMD), which is widely used in regenerative periodontal therapy, is believed to induce the formation of cementum and promote new attachment formation. EMD is an extracellular matrix that contains proteins similar to that derived from Hertwig epithelial root sheath (HERS) and the application of EMD in periodontal defects may mimic the events that take place during the development of root. It was reported that EMD could induce the formation of acellular extrinsic fiber cementum (AEFC) in animal studies, application of EMD could yield more clinical attachment compared with GTR in clinical trials. A number of recent studies explored the effects of EMD on the biological characters of several types of cells in vitro, the results implied that EMD could induce the differentiation of some types of cells, such as periodontal ligament cell and bone marrow stromal cell towards osteoblast/cementoblast lineage. However, there were no reports regarding the comparision of regenerative effects induced by EMD respectively on cementum surface and dentin surface.
Thus, this study aimed to explore the effect of diseased cementum on cementum regeneration, and observe if this effect can be amplified in the presence with EMD.
Methods
1. The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum.
1.1The differential effects of diseased cementum preservation on hPDLCs towards cemetum in vitro.
48extracted teeth due to periodontitis were collected, and notches were made at teeth on the coronal and apical line of the pockets by a pencil immediately after extraction. Root debridement was performed by EMS ultrasonic sealer to completely remove the calculus and plaques. Symmetrical root slices were made using diamond bur and one of the symmetrical slices of each root was further scaled to remove root cementum and distributed into the D (dentin) group, the other symmetrical slice was divided into the C (cementum) group in which the cementum was preserved in situ. All slices were conditioned with24%ethylene diamine tetraacetie acid (EDTA) gel for2min and supplemented with10%penicillin and streptomycin for24hours at4℃for sterilization. hPDLCs were isolated according to a modification of the method of Somerman.24root slices from each group were placed into48-well plates, with one chip in each well. The second passage hPDLCs were harvested and suspended at a concentration of1x10/ml and cell suspension was sparsely seeded onto each root slice. After a7-day co-culture,4slices from each group were selected to undergo SEM observation, and the remained20slices in each group were used for real-time PCR to detect the mRNA expression of cementum attachment protein (CAP) and cementum protein-23(CP-23).
1.2The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum in vivo.
24root slices from C and D group were placed into48-well plate, and hPDLCs with concentration of1x106/ml were seeded onto root slices. Root slices in this part were divided into four groups, cementum slices(group C) and dentin slices (group D) incubated in the DMEM culture medium respectively, as well as cementum slices (group E+C) and dentin slices (group E+D) incubated in DMEM culture medium containing EMD respectively, each group contained12root slices. After a7-day co-culture,12root slices of each group were rinsed with PBS and wrapped with sterilized expanded polytetrafluoroethylene (ePTFE), and then were inserted beneath the dorsal skin of nude mice. Each mouse received four slices, with one from each group. The mice were sacrificed3weeks after surgery, all the specimens were decalcified with10%EDTA and embedded with paraffin,5μm serially slices were made and HE staining was carried out. The newly-formed tissue in four groups were observed and BSP staining was carried out in specimens with newly formed cementum.
2. The effects of diseased cementum preservation in combination with EMD on cementum regeneration and new attachment formation in class III furcation defects.
8male beagle dogs were chosen, and4teeth (the right and left third and fourth premolars in the mandible) from each dog were used for this experiment. Four weeks before the experiment day, class III furcation defects were surgically created (height:5mm) in experiment teeth and cotton balls saturated with anaerobic bacteria were placed in all defects to initiate inflammation. The cotton ball was removed from the defects2weeks before the day of experiment, and then plaque control was achieved by subgingival scaling and oral hygiene with0.2%chlorhexidine gluconate irriation. On the day of experiment, no severe inflammation and gingival recession were observed, four experimental teeth of each dog received one of the following treatments randomly. C:preservation of root cementum in situ+24%EDTA conditioning D:removal of root cementum+24%EDTA conditioning E+C:preservation of root cementum in situ+24%EDTA conditioning+EMD E+D:removal of root cementum+24%EDTA conditioning+EMD All the buccal mucoperiosteal flaps were elevated, and the furactions were treated according to the design, then collagenous membranes were placed on buccal side of all the defects. Flaps on the lingual side were not elevated in consideration of the objective of the study and convenience in operation. All the dogs were sacrificed by4%paraformaldehyde perfusion. All the specimens were harvested and2specimens from each group were embedded with resin and the slices were stained with toluidine blue for histological observation. The remained6Specimens were decalcified with10%EDTA and embedded with paraffin,5μm serially slices were made and HE staining was used for histological observation and histometric study.
Results
1. The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum.
2.1The differential effects of diseased cementum preservation on hPDLCs towards cemetum in vitro..
The cells began to grow out from the periodontal ligament explants after7day to10day. Then they proliferated quickly and the cells were digested when they reached80%confluence. Immunohistochemical staining results showed that the second passage hPDLCs were positive for vimentin and negative for keratin, certificating that they were originated from ectomesenchyme. SEM results showed that the cells seeded on the cementum slices and dentin slices grew well, cells on the dentin surface exhibited a spindle shape while cells on cementum were much larger without typical spindle shape. Both CAP and CP-23expression were significantly higher on the cementum surface than on the dentin surface.(p=0.001for CAP and p=0.003for CP-23respectively).
2.2The effects of diseased cementum preservation in combination with EMD on the differentiation of hPDLCs towards cementum in vivo.
All the mice survived without inflammation throughout the experiment period,1root slice in group C fell off as the sutures loose, other11specimens in this group were obtained.12specimens of other three groups were obtained. Results of HE staining showed that neither group D nor group E+D had newly formed cementum-like tissue (NFC) formation,7specimens in group C showed NFC formation while10specimens in group E+C showed NFC formation, the results between group C and group E+C were statistically different.
2.The effects of diseased cementum preservation in combination with EMD on cementum regeneration and new attachment formation in class III furcation defects.
We artificially created32inflamed Class III furcation defects in beagle dogs. Within a healing period of8weeks, healing of the wounds in all groups occurred uneventfully, with no infection or suppuration. Histomorphometric results showed that the percentage of regenerated cementum (PRP) and the percentage of regenerated bone (PRB) in C group were statistically higher than those in D group, PRP and PRB in E+D and E+C groups were statistically higher than those in groups without EMD, but there were no differences between E+D group and E+C group. D group:The newly formed periodontal ligament was loosely arranged without principle fiber bundles, and favorable new attachment was not established. C group:Newly formed cementum was laid on the top of old cementum, newly formed periodontal ligament was in orderly arranged, with principle fiber inserting into new bone and new cementum, and favorable new attachment was established. The quality of new attachment in group E+D and E+C was almost good, except for the artifacts between newly formed cementum and dentin surface observed in group E+D. Results from resin slices were similar, but artifacts were not seen.
Conclusion
1. Preservation of diseased cementum promoted the differentiation of inoculated hPDLCs towards cementoblasts, and the effect was amplified by the presence of EMD.
2. Preservation of diseased cementum favored the cementum regeneration as well as formation of new attachment, and the application of EMD could strengthen the effect.
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9. van den Bos T, Handoko G, Niehof A, Ryan LM, Coburn SP, Whyte MP et al. Cementum and dentin in hypophosphatasia. J Dent Res 2005; 84:1021-1025.
lO.Villarreal-Rami'rez E, Moreno A, Mas-Oliva J, Cha' vez-Pacheco JL, Narayanan AS, Gil-Chavarrf a I et al. Characterization of recombinant human cementum protein 1 (hrCEMP1):primary role in biomineralization. Biochem. Biophys. Res. Commun. 2009; 384:49-54.
11. Alvarez Pe'rez MA, Pitaru S, Alvarez Fregoso O, Reyes Gasga J, Arzate H. Anti-cementoblastoma-derived protein antibody partially inhibits mineralization on a cementoblastic cell line. J Struct Biol 2003; 143:1-13.
12. Goncalves PF, Gurge BC, Pimentel SP, Sallum EA, Sallum AW, Casati MZ et al. Effect of two different approaches for root recontamination on new cementum formation following guided tissue regeneration. J Periodont Res 2006; 41:535-540.
13.Goncalves PF, Lima LL, Sallum EA, Casati MZ. Root cementum may modulate gene expression during periodontal regeneration:a preliminary study in humans. J.Periodontol 2008; 79(2):323-331.
14. Aimei Song, Jun Cai, Keqing Pan, Pishan Yang. Pre-existing root cementum may promote cementoblast differentiation of human periodontal ligament cells. Cell Prolif 2012; 45(3):249-258.
15. Poison AM. The root surface and regeneration; present therapeutic limitations and future biologic potentials. J Clin Periodontol 1986; 13:995-999.
16 Adelson LJ, Hanks CT, Ramfjord SJ, Caffesse RG. Cytotoxicity of periodontally diseased root surfaces. J Periodontol 1980; 51:700-704.
17. Aleo JJ, De Renzis FA, Farber PA, Varboncoeur AP. The presence and biologic activity of cementum-bound endotoxin. J Periodontol 1974; 45:672-675.
18. Aleo JJ, De Renzis FA and Farber PA. In vitro attachment of human gingival fibroblasts to root surfaces. J Periodontol 1975; 46:639-645.
19.Nyman S, Sarhed G, Ericsson I, Gottlow J, Karring T. Role of diseased root cementum in healing following treatment of periodontal disease. An experimental study in the dog. J Periodont Res 1986; 21:496-503.
20. Nyman S, Westfelt E, Sarhed G, Karring T. Role of diseased root cementum in healing following treatment of periodontal disease. A clinical study. J Clin Periodontol 1988; 15:464-468.
21. Martin Lao, Victor Marino and P. Mark Bartold. Immunohistochemical Study of Bone Sialoprotein and Osteopontin in Healthy and Diseased Root Surfaces. J Periodontol 2006; 77(10):1665-73
22.Komboli MG, Kodovazenitis GJ, Katsorhis TA.Comparative immunohistochemical study of the distribution of fibronectin in healthy and diseased root surface. J Periodontol 2009; 80(5):824-32.
23.Dantas AA, Fontanari LA, Ishi Ede P, Leite FR. Blood cells attachment after root conditioning and PRP application:an in vitro study. J Contemp Dent Pract 2012; 13(3):332-338.
24.Nevins ML, Camelo M, Schupbach P, Kim SW, Kim DM, Nevins M. Human clinical and histologic evaluation of laser-assisted new attachment procedure. Int J Periodontics Restorative Dent 2012; 32(5):497-507.
25.Koylass JM, Valderrama P, Mellonig JT. Histologic evaluation of a allogenic mineralized bone matrix in the treatment of periodontal osseous defects. Int J Periodontics Restorative Dent 2012; 32(4):405-411.
26.Hammarstrom L. Enamel matrix, cementum development and regeneration. J Clin Periodontol 1997; 24:658-668.
27. Bosshardt DD. Biological mediators and periodontal regeneration:a review of enamel matrix proteins at the cellular and molecular levels. J Clin Periodontol 2008; 35(8 Suppl):87-105.
28. Hammarstrom L, Heijl L, Gestrelius S. Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins. J Clin Periodontol 1997; 24:669-677
29. Sculean A, Chiantella GC, Windisch P, Donos N. Clinical and histologic evaluation of human intrabony defects treated with an enamel matrix protein derivative (Emdogain). Int J Periodontics Restorative Dent 2000; 20:3743-3781
30.Schroeder HE. Biological problems of regenerative cemento-genesis:synthesis and attachment of collagenous matrices on growing and established root surfaces. Int RevCytol 1992; 142:1-59.
31.Bosshardt DD, Sculean A, Windisch P, Pjetursson BE, Lang NP. Effects of enamel matrix proteins on tissue formation along the roots of human teeth. J Periodontal Res 2005; 40:158-167.
32. Al-Hezaimi K, Al-Askar M and Al-Rasheed A. Characteristics of newly-formed cementum following emdogain application. Int J Oral Sci.2011; 3:21-26.
1. Ivanovski S, Gronthos S, Shi S, Bartold PM. Stem cells in the periodontal ligament. Oral Dis 2006; 12:358-363.
2. Aimei Song, Jun Cai, Keqing Pan, Pishan Yang. Pre-existing root cementum may promote cementoblast differentiation of human periodontal ligament cells. Cell Prolif 2012 Jun; 45(3):249-58
3.贾惠梅,欧阳翔英,曹采方。釉基质蛋白对牙周膜细胞在牙骨质根面上生长的影响。中华口腔医学杂志2006年2月第41卷第2期74-76.
4.齐玉萍,黄晶,冯伟,魏美容,杨丕山,宋爱梅。釉基质蛋白对人牙周膜细胞增殖和牙骨质相关蛋白基因表达的影响。中华口腔杂志2012年增刊47卷26-30
5. Freeman E. Periodontium. In:Ten Cate AR, editor. Oral histology:development, structure and function. St. Louis:Mosby; 1994; p:276-312.
6. Carranza FA and Ubios AM. The tooth-supporting structures. In:Carranza FA, Newman MG, editors. Clinical periodontology. Philadelphia:WB Saunders; 1996. p.30-51.
7. Seo BM, Miura M, Gronthos S, Mark Bartold P, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004; 364:149-55.
8. Nagatomo K, Komaki M, Sekiya I, Sakaguchi Y, Noguchi K, Oda S, et al. Stem cell properties of human periodontal ligament cells. J Periodontal Res 2010; 41: 303-10.
9. Singhatanadgit W, Donos N and Olsen I. Isolation and characterization of stem cell clones from adult human ligament. Tissue Eng Part A 2009; 15:2625-36
10. McCulloch CAG, Nemeth E, Lowenberg B, Melcher AH. Paravascular cells in endosteal spaces of alveolar bone contribute to periodontal ligament cell population. Anat Rec 1987; 219:233-242.
11. Polson AM. The root surface and regeneration; present therapeutic limitations and future biologic potentials. J Clin Periodontol 1986; 13:995-999.
12. Bartold PM, McCulloch CAG, Narayanan AS, Pitaru S. Tissue engineering:a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontol 2000 2000; 24:253-269.
13. Blomlof J, Blomlof L, Lind skog S. Smear layer formed by different root planning modalities and its removal by an EDTA gel preparation. Int J Periodontics Restorative Dent 1997; 17(3):242
14. Saito M, Iwase M, Maslan S, Nozaki N, Yamauchi M, Handa K et al. Expression of cementum-derived attachment protein in bovine tooth germ during cementogenesis. Bone 2001; 29:242-248.
15. Arzate H, Jime'nez-Garci'a LF, Alvarez-Pe'rez MA, Landa A, Bar-Kana I, Pitaru S. Immunolocalization of a human cementoblastoma conditioned medium-derived protein. J. Dent. Res.2002; 81:541-546.
16. van den Bos T, Handoko G, Niehof A, Ryan LM, Coburn SP, Whyte MP et al. Cementum and dentin in hypophosphatasia. J Dent Res.2005; 84:1021-1025.
17. Villarreal-Rami'rez E, Moreno A, Mas-Oliva J, Cha'vez-Pacheco JL, Narayanan AS, Gil-Chavarria I et al. Characterization of recombinant human cementum protein 1 (hrCEMP1):primary role in biomineralization. Biochem. Biophys. Res. Commun.2009; 384:49-54.
18. Alvarez Pe' rez MA, Pitaru S, Alvarez Fregoso O, Reyes Gasga J, Arzate H. Anti-cementoblastoma-derived protein antibody partially inhibits mineralization on a cementoblastic cell line. J Struct Biol 2003; 143:1-13.
19. Ke'moun P, Laurencin-Dalicieux S and Rue J. Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivative (EMD) in vitro.Cell Tissue Res 2007; 329(2):283-294
20. Song AM, Shu R, Xie YF, Song ZC, Li HY, Liu XF, et al.A study of enamel matrix proteins on differentiation of porcine bone marrow stromal cells into cementoblasts. Cell Prolif 2007; 40:381-396
21. Saygin NE, Giannobile WV and Somerman MJ. Molecular and cell biology of cementum. Periodontology 2000 2000; 24:73-98.
22. Martin Lao, Victor Marino, and P. Mark Bartold. Immunohistochemical Study of Bone Sialoprotein and Osteopontin in Healthy and Diseased Root Surfaces. J Periodontol 2006; 77(10):1665-73
23. Ruhling A, Wulf J, Schwahn C, Kocher T. Surface wear on cervical restorations and adjacent enamel and root cementum caused by simulated long-term maintenance therapy. J Clin Periodontol 2004; 31:293-298.
24. Marda P, Prakash S, Devaraj CG, Vastardis S. A comparison of root surface instrumentation using manual, ultrasonic and rotary instruments:An in vitro study using scanning electron microscopy. Indian J Dent Res 2012; 23:164-70
1.曹采方.牙周病学[M].人民卫生出版社,2003,75-76
2. Nyman S, Lindhe J and Karring T. Healing following surgical treatment and root demineralization in monkeys with periodontal disease. J Clin Periodontol 1981 Jun; 8(3):249-58.
3. Nyman S, Gottlow J, Karring T, Lindhe J. The regenerative potential of the periodontal ligament. An experimental study in the monkey. J Clin Periodontol 1982 May; 9(3):257-65.
4. Gottlow J, Nyman S, Lindhe J, Karring T. New attachment formation in the human periodontium by guided tissue regeneration: Case reports. J Clin Periodontol 1986; 13(6):604-16.
5. Nyman S, Gottlow J, Karring T, Lindhe J. New attachment formation by guided tissue regeneration. J Periodontal Res 1987; 22(3):252-4.
6. Araujo M, Berglundh T and Lindhe J.The periodontal tissues in healed degree III furcation defects. An experimental study in dogs. J Clin Periodontol 1996; 23: 532-541
7. Araujo MG, Berglundh T and Lindhe J. On the dynamics of periodontal tissue formation in degree III furcation defects. An experimental study in dogs. J Clin Periodontol 1997; 24:738-746.
9. Silvestri M, Sartori S, Rasperini G, Ricci G, Rota C, Cattaneo V. Comparison of infrabony defects treated with enamel matrix derivative versus guided tissue regeneration with a nonresorbable membrane. J Clin Periodontol 2003; 30:386-393.
10.Windisch P, Sculean A, Klein F et al. Comparison of clinical, radiographic, and histometric measurements following treatment with guided tissue regeneration or enamel matrix proteins in human periodontal defects. J Periodontol 2002; 73: 409-417.
11. Bosshardt DD, Sculean A, Windisch P, Pjetursson BE, Lang NP. Effects of enamel matrix proteins on tissue formation along the roots of human teeth. J Periodont Res 2005; 40; 158-167.
12. Al-Hezaimi K, Al-Askar M, Al-Rasheed A. Characteristics of newly-formed cementum following emdogain application. Int J Oral Sci 2011; 3:21-26.
13. Nikolaos D. Gkranias, Filippo Graziani, Anton Sculean, Nikolaos Donos. Wound healing following regenerative procedures in furcation degree III defects: histomorphometric outcomes. Clin Oral Invest 2012; 16:239-249.
14..Nyman S, Sarhed G, Ericsson I, Gottlow J, Karring T. Role of diseased root cementum in healing following treatment of periodontal disease. An experimental study in the dog. J Periodont Res 1986; 21:496-503.
15. Goncalves PF, Gurge BC, Pimentel SP, Sallum EA, Sallum AW, Casati MZ et al. Effect of two different approaches for root recontamination on new cementum formation following guided tissue regeneration. J Periodontal Res 2006; 41:535-540.
16. Inuki T, Katagiri W, Yoshimi R, Osugi M. Noval application of stem cell-derived factors for periodontal regeneration. Biochem Biophys Res Commun.2013; 430(2): 763-768.
17. Effects of EMD in combination with bone swaging and calcium phosphate bone cement on periodontal regeneration in one-wall intrabony defects in dogs. J Periodontol Res 2013; 48:37-43.
18. Fawzy El-Saved KM, Paris S, Becker ST, Neuschl M. Periodontal regeneration employing gingival margin-derived stem/progenitor cells:an animal study. J Clin Periodontol 2012; 39(9):861-870.
19.陈珊,杨军英,冯磊。建立小型猪Ⅱ度根分叉缺损模型的可行性。中国组织工程研究与临床康复。2011；15(33)：6127-30.
20. Yan XZ, Ge SH, Sun QF, Yang PS. A pilot study evaluating the effect of recombinant human BMP-2 and recombinant human β-NGF on the healing of class III furcation defect. J Periodontol 2010; 81(9):1289-98.
21. Takayama S, Murakami S, Shimabukuro Y, Kitamura M, Okada A. Periodontal regeneration by FGF-2 (bFGF) in primate models. J Dent Res 2001; 80:2075-2079.
22. Murano Y, Ota M, Katayama A, Sugito H, Shibukawa Y, Yamada S. Periodontal regeneration following transplantation of proliferating tissue derived from periodontal ligament into class Ⅲ furcation defects in dogs. Biomed Res 2006; 27:139-147.
23. Holt SC, Ebersole J, Felton J, Brunsvold M, Kornman KS. Implantation of Bacteroides gingivalis in nonhuman primates initiates progression of periodontitis. Science 1988; 239:55-57.
24. Adriaens PA, Edwards CA, De Boever JA, Loesche WJ. Ultrastructural observations on bacterial invasion in cementum and radicular dentin of periodontally diseased human teeth. J Periodontol 1988; 59:493-503.
25. Nyman S, Sarhed G, Ericsson I, Gottlow J, Karring T. Role of diseased root cementum in healing following treatment of periodontal disease. An experimental study in the dog. J Periodont Res1986; 21:496-503.
26. Nyman S, Westfelt E, Sarhed G, Karring T. Role of diseased root cementum in healing following treatment of periodontal disease. A clinical study. J Clin Periodontol 1988; 15:464-468.
27. Grzesik WJ, Narayanan AS. Cementum and periodontal wound healing and regeneration. Crit Rev Oral Biol Med 2002; 13:474-484.
28. Lara VS, Figueiredo F, da Silva TA, Cunha FQ. Dentin-induced in vivo inflammatory response and in vitro activation of murine macrophages. J Dent Res 2003; 82:460-465.
29. Nishimura K, Hayashi M, Matsuda K, Shigeyama Y, Yamasaki A, Yamaoka A. The chemoattractive potency of periodontal ligament, cementum and dentin for human gingival fibroblasts. J Periodont Res 1989; 24:146-148.
30. Narayanan AS, Bartold PM. Biochemistry of periodontal connective tissues and their regeneration: a current perspective. Connect Tissue Res 1996; 34:191-201.
31. Bartold PM, McCulloch CAG, Narayanan AS, Pitaru S.Tissue engineering:a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontol 2000 2000; 24:253-269.
32. Kostopoulos L and Karring T. Susceptibility of GTR-regenerated periodontal attachment to ligature-induced periodontitis. J Clin Periodontol 2004; 31:336-340.
2. Bartold PM, Mculloch CA, Narayanan AS, et al. Tissue engineering:a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontal 2000 2000; 24(10):2532-2690.
3. Wang HL and Cooke J. Periodontal regeneration techniques for treatment of periodontal diseases. Dent Clin North Am 2005; 49:637-659.
4. Slavkin HC. What is the role of the host in periodontal disease? Periodontal Abstr 1975; 23:101-103.
5. Lindskog S. Formation of intermediate cementum. Ⅱ:a scanning electron microscopic study of the epithelial root sheath of Hertwig in monkey. J Craniofac Genet Dev Biol 1982; 2:161-169.
6. Wang H, Tannukit S, Zhu D, Snead ML, Paine ML. Enamel matrix protein interactions. J Bone Miner Res 2005; 20:1032-1040.
7. Wojciech J. Grzesik and A.S. Narayanan. Cementum and periodontal wound healing and regeneration. Crit Rev Oral Biol Med 2002; 13; 474-484.
8. Margarita ZD. Regeneration of Periodontal Tissues:cementogenesis revisited. Periodontology 2000 2006; 41:196-217.
9. van den Bos T, Handoko G, Niehof A, Ryan LM, Coburn SP, Whyte MP et al. Cementum and dentin in hypophosphatasia. J Dent Res 2005; 84:1021-1025.
lO.Villarreal-Rami'rez E, Moreno A, Mas-Oliva J, Cha' vez-Pacheco JL, Narayanan AS, Gil-Chavarrf a I et al. Characterization of recombinant human cementum protein 1 (hrCEMP1):primary role in biomineralization. Biochem. Biophys. Res. Commun. 2009; 384:49-54.
11. Alvarez Pe'rez MA, Pitaru S, Alvarez Fregoso O, Reyes Gasga J, Arzate H. Anti-cementoblastoma-derived protein antibody partially inhibits mineralization on a cementoblastic cell line. J Struct Biol 2003; 143:1-13.
12. Goncalves PF, Gurge BC, Pimentel SP, Sallum EA, Sallum AW, Casati MZ et al. Effect of two different approaches for root recontamination on new cementum formation following guided tissue regeneration. J Periodont Res 2006; 41:535-540.
13.Goncalves PF, Lima LL, Sallum EA, Casati MZ. Root cementum may modulate gene expression during periodontal regeneration:a preliminary study in humans. J.Periodontol 2008; 79(2):323-331.
14. Aimei Song, Jun Cai, Keqing Pan, Pishan Yang. Pre-existing root cementum may promote cementoblast differentiation of human periodontal ligament cells. Cell Prolif 2012; 45(3):249-258.
15. Poison AM. The root surface and regeneration; present therapeutic limitations and future biologic potentials. J Clin Periodontol 1986; 13:995-999.
16 Adelson LJ, Hanks CT, Ramfjord SJ, Caffesse RG. Cytotoxicity of periodontally diseased root surfaces. J Periodontol 1980; 51:700-704.
17. Aleo JJ, De Renzis FA, Farber PA, Varboncoeur AP. The presence and biologic activity of cementum-bound endotoxin. J Periodontol 1974; 45:672-675.
18. Aleo JJ, De Renzis FA and Farber PA. In vitro attachment of human gingival fibroblasts to root surfaces. J Periodontol 1975; 46:639-645.
19.Nyman S, Sarhed G, Ericsson I, Gottlow J, Karring T. Role of diseased root cementum in healing following treatment of periodontal disease. An experimental study in the dog. J Periodont Res 1986; 21:496-503.
20. Nyman S, Westfelt E, Sarhed G, Karring T. Role of diseased root cementum in healing following treatment of periodontal disease. A clinical study. J Clin Periodontol 1988; 15:464-468.
21. Martin Lao, Victor Marino and P. Mark Bartold. Immunohistochemical Study of Bone Sialoprotein and Osteopontin in Healthy and Diseased Root Surfaces. J Periodontol 2006; 77(10):1665-73
22.Komboli MG, Kodovazenitis GJ, Katsorhis TA.Comparative immunohistochemical study of the distribution of fibronectin in healthy and diseased root surface. J Periodontol 2009; 80(5):824-32.
23.Dantas AA, Fontanari LA, Ishi Ede P, Leite FR. Blood cells attachment after root conditioning and PRP application:an in vitro study. J Contemp Dent Pract 2012; 13(3):332-338.
24.Nevins ML, Camelo M, Schupbach P, Kim SW, Kim DM, Nevins M. Human clinical and histologic evaluation of laser-assisted new attachment procedure. Int J Periodontics Restorative Dent 2012; 32(5):497-507.
25.Koylass JM, Valderrama P, Mellonig JT. Histologic evaluation of a allogenic mineralized bone matrix in the treatment of periodontal osseous defects. Int J Periodontics Restorative Dent 2012; 32(4):405-411.
26.Hammarstrom L. Enamel matrix, cementum development and regeneration. J Clin Periodontol 1997; 24:658-668.
27. Bosshardt DD. Biological mediators and periodontal regeneration:a review of enamel matrix proteins at the cellular and molecular levels. J Clin Periodontol 2008; 35(8 Suppl):87-105.
28. Hammarstrom L, Heijl L, Gestrelius S. Periodontal regeneration in a buccal dehiscence model in monkeys after application of enamel matrix proteins. J Clin Periodontol 1997; 24:669-677
29. Sculean A, Chiantella GC, Windisch P, Donos N. Clinical and histologic evaluation of human intrabony defects treated with an enamel matrix protein derivative (Emdogain). Int J Periodontics Restorative Dent 2000; 20:3743-3781
30.Schroeder HE. Biological problems of regenerative cemento-genesis:synthesis and attachment of collagenous matrices on growing and established root surfaces. Int RevCytol 1992; 142:1-59.
31.Bosshardt DD, Sculean A, Windisch P, Pjetursson BE, Lang NP. Effects of enamel matrix proteins on tissue formation along the roots of human teeth. J Periodontal Res 2005; 40:158-167.
32. Al-Hezaimi K, Al-Askar M and Al-Rasheed A. Characteristics of newly-formed cementum following emdogain application. Int J Oral Sci.2011; 3:21-26.
1. Ivanovski S, Gronthos S, Shi S, Bartold PM. Stem cells in the periodontal ligament. Oral Dis 2006; 12:358-363.
2. Aimei Song, Jun Cai, Keqing Pan, Pishan Yang. Pre-existing root cementum may promote cementoblast differentiation of human periodontal ligament cells. Cell Prolif 2012 Jun; 45(3):249-58
3.贾惠梅,欧阳翔英,曹采方。釉基质蛋白对牙周膜细胞在牙骨质根面上生长的影响。中华口腔医学杂志2006年2月第41卷第2期74-76.
4.齐玉萍,黄晶,冯伟,魏美容,杨丕山,宋爱梅。釉基质蛋白对人牙周膜细胞增殖和牙骨质相关蛋白基因表达的影响。中华口腔杂志2012年增刊47卷26-30
5. Freeman E. Periodontium. In:Ten Cate AR, editor. Oral histology:development, structure and function. St. Louis:Mosby; 1994; p:276-312.
6. Carranza FA and Ubios AM. The tooth-supporting structures. In:Carranza FA, Newman MG, editors. Clinical periodontology. Philadelphia:WB Saunders; 1996. p.30-51.
7. Seo BM, Miura M, Gronthos S, Mark Bartold P, Batouli S, Brahim J, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004; 364:149-55.
8. Nagatomo K, Komaki M, Sekiya I, Sakaguchi Y, Noguchi K, Oda S, et al. Stem cell properties of human periodontal ligament cells. J Periodontal Res 2010; 41: 303-10.
9. Singhatanadgit W, Donos N and Olsen I. Isolation and characterization of stem cell clones from adult human ligament. Tissue Eng Part A 2009; 15:2625-36
10. McCulloch CAG, Nemeth E, Lowenberg B, Melcher AH. Paravascular cells in endosteal spaces of alveolar bone contribute to periodontal ligament cell population. Anat Rec 1987; 219:233-242.
11. Polson AM. The root surface and regeneration; present therapeutic limitations and future biologic potentials. J Clin Periodontol 1986; 13:995-999.
12. Bartold PM, McCulloch CAG, Narayanan AS, Pitaru S. Tissue engineering:a new paradigm for periodontal regeneration based on molecular and cell biology. Periodontol 2000 2000; 24:253-269.
13. Blomlof J, Blomlof L, Lind skog S. Smear layer formed by different root planning modalities and its removal by an EDTA gel preparation. Int J Periodontics Restorative Dent 1997; 17(3):242
14. Saito M, Iwase M, Maslan S, Nozaki N, Yamauchi M, Handa K et al. Expression of cementum-derived attachment protein in bovine tooth germ during cementogenesis. Bone 2001; 29:242-248.
15. Arzate H, Jime'nez-Garci'a LF, Alvarez-Pe'rez MA, Landa A, Bar-Kana I, Pitaru S. Immunolocalization of a human cementoblastoma conditioned medium-derived protein. J. Dent. Res.2002; 81:541-546.
16. van den Bos T, Handoko G, Niehof A, Ryan LM, Coburn SP, Whyte MP et al. Cementum and dentin in hypophosphatasia. J Dent Res.2005; 84:1021-1025.
17. Villarreal-Rami'rez E, Moreno A, Mas-Oliva J, Cha'vez-Pacheco JL, Narayanan AS, Gil-Chavarria I et al. Characterization of recombinant human cementum protein 1 (hrCEMP1):primary role in biomineralization. Biochem. Biophys. Res. Commun.2009; 384:49-54.
18. Alvarez Pe' rez MA, Pitaru S, Alvarez Fregoso O, Reyes Gasga J, Arzate H. Anti-cementoblastoma-derived protein antibody partially inhibits mineralization on a cementoblastic cell line. J Struct Biol 2003; 143:1-13.
19. Ke'moun P, Laurencin-Dalicieux S and Rue J. Human dental follicle cells acquire cementoblast features under stimulation by BMP-2/-7 and enamel matrix derivative (EMD) in vitro.Cell Tissue Res 2007; 329(2):283-294
20. Song AM, Shu R, Xie YF, Song ZC, Li HY, Liu XF, et al.A study of enamel matrix proteins on differentiation of porcine bone marrow stromal cells into cementoblasts. Cell Prolif 2007; 40:381-396
21. Saygin NE, Giannobile WV and Somerman MJ. Molecular and cell biology of cementum. Periodontology 2000 2000; 24:73-98.
22. Martin Lao, Victor Marino, and P. Mark Bartold. Immunohistochemical Study of Bone Sialoprotein and Osteopontin in Healthy and Diseased Root Surfaces. J Periodontol 2006; 77(10):1665-73
23. Ruhling A, Wulf J, Schwahn C, Kocher T. Surface wear on cervical restorations and adjacent enamel and root cementum caused by simulated long-term maintenance therapy. J Clin Periodontol 2004; 31:293-298.
24. Marda P, Prakash S, Devaraj CG, Vastardis S. A comparison of root surface instrumentation using manual, ultrasonic and rotary instruments:An in vitro study using scanning electron microscopy. Indian J Dent Res 2012; 23:164-70
1.曹采方.牙周病学[M].人民卫生出版社,2003,75-76
2. Nyman S, Lindhe J and Karring T. Healing following surgical treatment and root demineralization in monkeys with periodontal disease. J Clin Periodontol 1981 Jun; 8(3):249-58.
3. Nyman S, Gottlow J, Karring T, Lindhe J. The regenerative potential of the periodontal ligament. An experimental study in the monkey. J Clin Periodontol 1982 May; 9(3):257-65.
4. Gottlow J, Nyman S, Lindhe J, Karring T. New attachment formation in the human periodontium by guided tissue regeneration: Case reports. J Clin Periodontol 1986; 13(6):604-16.
5. Nyman S, Gottlow J, Karring T, Lindhe J. New attachment formation by guided tissue regeneration. J Periodontal Res 1987; 22(3):252-4.
6. Araujo M, Berglundh T and Lindhe J.The periodontal tissues in healed degree III furcation defects. An experimental study in dogs. J Clin Periodontol 1996; 23: 532-541
7. Araujo MG, Berglundh T and Lindhe J. On the dynamics of periodontal tissue formation in degree III furcation defects. An experimental study in dogs. J Clin Periodontol 1997; 24:738-746.
9. Silvestri M, Sartori S, Rasperini G, Ricci G, Rota C, Cattaneo V. Comparison of infrabony defects treated with enamel matrix derivative versus guided tissue regeneration with a nonresorbable membrane. J Clin Periodontol 2003; 30:386-393.
10.Windisch P, Sculean A, Klein F et al. Comparison of clinical, radiographic, and histometric measurements following treatment with guided tissue regeneration or enamel matrix proteins in human periodontal defects. J Periodontol 2002; 73: 409-417.
11. Bosshardt DD, Sculean A, Windisch P, Pjetursson BE, Lang NP. Effects of enamel matrix proteins on tissue formation along the roots of human teeth. J Periodont Res 2005; 40; 158-167.
12. Al-Hezaimi K, Al-Askar M, Al-Rasheed A. Characteristics of newly-formed cementum following emdogain application. Int J Oral Sci 2011; 3:21-26.
13. Nikolaos D. Gkranias, Filippo Graziani, Anton Sculean, Nikolaos Donos. Wound healing following regenerative procedures in furcation degree III defects: histomorphometric outcomes. Clin Oral Invest 2012; 16:239-249.
14..Nyman S, Sarhed G, Ericsson I, Gottlow J, Karring T. Role of diseased root cementum in healing following treatment of periodontal disease. An experimental study in the dog. J Periodont Res 1986; 21:496-503.
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