自体骨髓干细胞移植狗牙周缺损处引导组织再生的实验观察

目的本文对应用自体骨髓干细胞移植引导组织再生的动物实验的观察进行评价。方法6只成年狗,实验组,对照组各18颗牙。分别在每条狗抽取骨髓1ml,在实验室内进行原代骨髓干细胞培养,培养液为内含15%小牛血清(FCS)和0.5%青-链霉素抗生素的a-MEM培养液。第1代细胞转移到18块大小为6×2mm2胶原膜上,约每张胶原膜上1×107个细胞,培养24小时后相差显微镜下观察细胞在膜上附着情况。在人工制造的牙周缺损中进行体外培养的自体骨髓干细胞移植结合GTR方法(实验组)和单纯GTR方法(对照组)。在6周后切片行牙周组织学观察。结果实验组新生牙槽骨新生牙周膜组织及新生牙骨质的修复再生的效果明显好于对照组(P<0.05),形成了的牙周结构,只是引导再生的牙周组织基本恢复到正常的牙周组织高度。实验组牙槽骨再生高度平均为4.50±0.13mm;对照组为3.09±0.28mm。结论应用自体骨髓干细胞移植结合e-pTFE膜引导牙周组织再生可促进牙周组织的再生、加快正常骨结构组织的建立并缩短修复再生时间。     

组织工程用于修复慢性牙周组织缺损的动物实验研究

目的:应用细胞型和/或非细胞型组织工程化牙周组织移植修复慢性牙周缺损的动物实验,探讨其用于牙周再生治疗的可行性。方法:人工构建5只成年杂种狗慢性牙周缺损病变模型,分别随机采用:引导组织再生治疗术+富血小板血浆+B ioOss(GTR+PRP+B ioOss)、引导组织再生治疗术+富血小板血浆+B ioOss+自体牙周膜细胞(GTR+PRP+B ioOss+PDLCs)、引导组织再生治疗术+自体牙周膜细胞(GTR+PDLCs)和GTR治疗,其中GTR组6颗牙,其余3组各为8颗牙。12周后作病理切片,HE染色观察牙周组织再生情况。结果:动物实验发现GTR组的新生牙槽骨、牙骨质和牙周组织高度分别为(0.52±0.21)mm、(0.8±0.13)mm、(1.9±0.10)mm。而另外3组的新生牙槽骨、牙骨质和牙周组织高度分别为GTR+PRP+B ioOss组:(1.36±0.17)mm、(1.92±0.18)mm、(2.62±0.16)mm;GTR+PRP+B ioOss+PDLCs组:(1.42±0.22)mm、(2.07±0.19)mm、(2.68±0.20)mm;GTR+PDLCs组:(1.39±0.19)mm、(1.82±0.16)mm、(2.55±0.12)mm,这3组牙槽骨、牙骨质和牙周组织的修复再生效果均明显优于GTR组(P<0.05),而它们之间的差别无显著性。结论:应用GTR技术结合组织工程可显著促进狗牙周组织缺损的再生。     

狗牙周缺损处移植自体骨髓干细胞的骨化实验观察

目的 对应用自体骨髓干细胞移植的动物骨化实验的观察进行评价。方法 将6只成年狗的36颗牙，分为实验组和对照组（各18颗牙）。有人工制造的牙周缺损中进行体外培养的自体骨髓干细胞移植，结合引导组织再生（guided tissue regeneration，GTR）方法（实验组）和单纯GTR方法（对照组）。6周后对切片行牙周组织学观察。结果 实验组新牙槽骨骨化效果明显好于对照组。实验组建立了正常钙化骨结构，Masson染色为红色；对照组虽然建立了正常骨结构，但钙化程度极低，基本为胶原性骨结构，Masson染色为蓝绿色。结论 应用自体骨髓干细胞移植结合聚四氟乙烯膜引导牙周组织再生可促进牙周组织的再生、加快正常骨组织结构的建立，并缩短修复再生时间。     

PRP和自体骨髓基质细胞移植修复狗牙Ⅱ度根分叉病变的研究

目的：应用富血小板血浆和／或骨髓基质细胞移植修复根分又病变的动物实验,探讨其用于牙周再生治疗的可行性。方法：人工构建5只成年杂种狗慢性Ⅱ度根分叉病变模型,分别随机采用：引导组织再生治疗术 富血小板血浆 Bio-Oss(GTR／PRP／Bio-Oss)、引导组织再生治疗术 富血小板血浆 Bio-Oss 自体骨髓基质细胞(GTR／PRfl／Bio-Oss/MSCs)、引导组织再生治疗术 骨髓基质细胞(GTR／MSCs)和GTR治疗,每组各7颗牙。12周后作病理切片,HE染色观察牙周组织再生情况。结果：动物实验发现GTR组的新生牙槽骨、牙骨质和牙周膜高度与GTR／PRfl／Bio-Oss组、GTR／MSCs组无显著性差异;而GTR／PRPl／Bio-0ss／MSCs组与上3组有显著性差异。结论：在本实验条件下,MSCs和胶原膜复合,PRP与Bio-Oss复合对GTR的引导牙周组织再生作用没有显著的促进作用。     

牙周膜细胞松质骨基质支架复合移植促进牙周组织再生的研究

目的观察牙周膜细胞（PDLCs）接种松质骨基质（CBM）支架复合移植对牙周组织再生修复的影响和意义。方法将体外培养的狗自体PDLCs接种到CBM三维支架上,体外进行细胞计数和扫描电镜观察,并植人狗人工牙周组织缺损处,表面覆盖聚四氟乙烯膜（e-PTFE）,以单纯翻瓣组和只覆盖e-PTFE组作为对照。术后8周对动物组织标本进行组织学观察和测量,分析比较各组牙周组织的再生情况。结果PDLCs在CBM支架材料上形成良好的贴附并增殖,扫描电镜可见CBM具有良好的多孔网状结构,细胞在CBM上生长旺盛,伸展充分。自体PDLCs／CBM／e-PTFE膜复合植入组较单纯翻瓣组和e—PTFE组有更多的新生牙槽骨、新生牙周膜和新生牙骨质生成,且未见上皮长入。结论PDLCs接种松质骨基质支架复合移植能更有效地促进牙周组织再生和重建,CBM有望用作牙周组织工程的支架材料。     

引导牙周组织再生膜根间隙中细胞钙化与膜放置时间的关系

目的 :研究在引导牙周组织再生技术中膜根间隙中再生组织钙化与时间的关系 ,为不可吸收膜放置时间和可吸收膜的吸收时间提供依据。方法 :4只狗的 2 4颗牙用于实验 ,人工形成牙周骨缺损 ,国产e PTFE膜覆盖 ,分别在 2、4、8周刮取膜根间隙的再生组织进行细胞培养及稀释法克隆化 ,2 8d后进行钙化染色 ,对不同时间组的钙化数量进行分析 ;结果 :2周和 4周时钙化细胞的量基本相同 (P >0 .0 5 ) ;而 2周和 4周的钙化细胞数量远大于 8周 (P <0 .0 1)。结论 :不可吸收膜的取出时间和可吸收膜开始降解时间不应少于 6周 ,4周时将膜取出 ,可能会对组织再生产生一定的影响     

异种脱细胞真皮基质膜在引导牙周组织再生中应用的临床效果评价

目的：评价异种脱细胞真皮基质膜在引导牙周组织再生中应用的临床效果。方法：30例牙周缺损区患牙随机分两组：实验组20例,异种脱细胞真皮基质膜和羟基磷灰石修复;对照组10例仅羟基磷灰石修复。统计学比较6个月时两组各项临床指标和牙槽骨水平改变。结果：实验组牙周袋深度、临床附着丧失的减少量和牙槽骨水平的增加量均较对照组高（P〈0.05）,牙龈退缩量低于对照组（P〈0.05）。结论：异种脱细胞真皮基质膜应用于引导牙周组织再生有较好的临床效果,值得推广。     

纳米骨水泥修复根分叉病变的实验研究

目的:研究纳米骨水泥修复犬慢性牙周组织缺损.方法:将3只成年Beagle犬形成慢性Ⅱ度根分叉病变模型,按拉丁方设计方法分成3组,每组6颗牙,分别采用引导组织再生治疗术(Guided tissue regeneration,GTR)+纳米骨水泥(nano-calcium phosphate bone cement,CPC)、GTR+珊瑚羟基磷灰石人造骨(coralline hydroxyapatite por-ous,CHAP)、GTR进行移植修复治疗.12周观察牙周组织再生情况.结果:组织学观察GTR+CPC组牙槽骨、牙骨质和牙周组织的修复再生效果明显优于GTR组(P<0.05).X线片显示:GTR+CPC组骨缺损处骨质生长情况优于其他两组.结论:应用GTR技术结合纳米骨水泥移植可显著促进狗牙根分叉处牙周组织缺损的再生.     

应用细胞-支架构建方式的组织工程方法促进牙周组织再生的实验研究

目的:观察评价细胞 -支架构建方式的组织工程方法对牙周组织再生修复的影响和意义,探讨自体牙周膜细胞(PDLCs)和纳米羟基磷灰石材料 -纳米羟晶 -胶原仿生骨材料 (nHAC)分别用作牙周组织工程种子细胞和支架材料的可行性。方法:改良组织块法体外培养动物自体PDLCs,传代扩增后接种到nHAC三维支架上,再植入动物人工牙周组织缺损,表面覆盖聚四氟乙烯 (e-PTFE)膜,以单纯翻瓣组和单纯GTR组作为对照。术后 8周进行组织学观察和测量,分析评价其牙周组织的再生情况。结果:自体PDLCs-nHAC-膜复合植入组较单纯翻瓣组和单纯GTR组有更多的新生牙槽骨、新生牙周膜和新生牙骨质生成,且未见上皮长入。结论:应用细胞-支架构建方式的牙周组织工程方法能更有效地促进牙周组织再生和重建,而自体PDLCs可作为牙周组织工程的种子细胞,nHAC可作为牙周组织工程的支架材料。     

牙周组织引导再生术的研究及临床应用

一、牙周组织引导性再生的概念组织引导性再生(guided tissue regeneration,GTR)是近十年来在牙周治疗的研究和临床应用中的一个热门课题.由于牙龈上皮和结缔组织的再生修复速度快于牙周膜和牙槽骨,使得牙周治疗后牙周韧带附着的再建立和牙槽骨的再修复成为困难。牙周组织引导再生术就是在牙周手术中利用具有某种特性的膜状材料,覆盖在病变牙槽骨嵴以及     

Participation of periodontal ligament cells with regeneration of alveolar bone

BACKGROUND: It is important to clarify the participation of periodontal ligament (PDL) cells in the regeneration of alveolar bone to establish a reliable approach for obtaining periodontal regeneration. The aim of this study was to determine whether PDL cells play an important role in alveolar bone repair during the course of periodontal regeneration. METHODS: In an in vitro study, the expression of the osteoblast phenotype, such as alkaline phosphatase activity and parathyroid hormone-dependent 3',5'-cyclic adenosine monophosphate accumulation, was investigated in dog PDL cells (DPLC) and dog bone cells isolated from mandibles (DBC). In a related study, the roots of mandibular third premolars extracted from aged dogs were divided into a PDL(+) group, in which the PDL was preserved, and a PDL(-) group, in which the PDL was removed. These roots were respectively transplanted into surgically created bone cavities with buccal and interproximal bone defects in an edentulous area, prepared in advance by extraction of mandibular fourth premolars. These bone defects with the transplanted roots were completely covered with submerged physical barrier membranes. New bone formation and new connective tissue attachment, which require new cementum and insertion of functionally oriented new collagen fibers of periodontal ligament, were histomorphometrically assessed, and were compared between the PDL(+) and PDL(-) groups 6 weeks after transplantation. RESULTS: Both cultured DPLC and DBC exhibited the osteoblast phenotype. New connective tissue attachment was observed only in the PDL(+) group. However, alveolar bone was almost completely regenerated to the original bone height in both the PDL(+) and PDL(-) groups, and the amount of newly formed bone was not significantly different between the 2 groups. CONCLUSIONS: DPLC retain the capability to differentiate into an osteoblast lineage and may act in the regeneration of periodontal ligament with new cementum formation, whereas these cells may have a limited influence on alveolar bone formation during the course of periodontal regeneration.     

The study of calcification of autogenous bone marrow stem cell transplantation on alveolar bone defect in dogs]

OBJECTIVE: To evaluate the effect of calcification of autogenous bone marrow stem cell transplantation in periodontal tissue regeneration. METHODS: Bone marrow stem cells derived from the same dog were cultured with alpha-MEM. 1 x 10(7) cells of first passage were allowed to attach to the collagen membrane for 24 hours. The membrane-cells were transplanted into periodontal defect in the same dog. Then the defects were covered with e-pTFE membranes. The defects covered only with e-pTFE without membrane-cells were served as control. Eighteen teeth of 6 dogs for every group were studied. The dogs were sacrificed after 6 weeks. RESULTS: The results showed that new bone formation in test group was significantly higher than that of control group. The calcification of new bone in test group was better than control group. CONCLUSIONS: The results suggested that autogenous bone marrow stem cell transplantation with guided tissue regeneration technique could enhance periodontal tissue regeneration and could form new bone tissue fast and could shorten times of periodontal tissue regeneration in dogs.     

Application of periodontal ligament cell sheet for periodontal regeneration: a pilot study in beagle dogs

OBJECTIVE: The ultimate goal of periodontal treatment is to regenerate the damaged periodontal support. Although periodontal ligament (PDL) cells are essential for periodontal regeneration, few studies have reported the transplantation of periodontal ligament cells to periodontal defects. We developed a new method to apply periodontal ligament cells as a sheet to the defect. The aim of this study was to investigate the periodontal healing after application of the periodontal ligament cell sheet in beagle dogs. METHODS: Autologous periodontal ligament cells were obtained from extracted premolars of each beagle dog. Periodontal ligament cell sheets were fabricated using a temperature-responsive cell culture dish. Dehiscence defects were surgically created on the buccal surface of the mesial roots of bilateral mandibular first molars of each dog. In the experimental group (five defects), periodontal ligament cell sheet with reinforced hyaluronic acid carrier was applied to the defect. Only the hyaluronic acid carrier was applied to the contralateral side as a control (five defects). Eight weeks after surgery, the animals were sacrificed and decalcified specimens were prepared. Healing of the periodontal defects was evaluated histologically and histometrically. RESULTS: No clinical signs of inflammation or recession of gingiva were observed in both experimental and control groups. In the experimental group, periodontal tissue healing with bone, periodontal ligament and cementum formation was observed in three out of five defects. In the control group, such periodontal tissue formation was not observed except in one defect. Histometric analysis revealed that the formation of new cementum in the experimental group was significantly higher than that in the control group. CONCLUSION: The periodontal ligament cell sheet has a potential to regenerate periodontal tissue and may become a novel regenerative therapy.     

Root cementum modulates periodontal regeneration in Class III furcation defects treated by the guided tissue regeneration technique: a histometric study in dogs

BACKGROUND: Because the possibility of root cementum preservation as an alternative approach for the treatment of periodontal disease has been demonstrated, this study aimed to histometrically evaluate the effect of root cementum on periodontal regeneration. METHODS: Bilateral Class III furcation defects were created in dogs, and each dog was randomly assigned to receive one of the following treatments: control (group A): scaling and root planing with the removal of root cementum; or test (group B): removal of soft microbial deposits by polishing the root surface with rubber cups and polishing paste, aiming at maximum cementum preservation. Guided tissue regeneration (GTR) was applied to both groups. RESULTS: Four months after treatment, a superior length of new cementum (3.59 +/- 1.67 mm versus 6.20 +/- 2.26 mm; P = 0.008) and new bone (1.86 +/- 1.76 mm versus 4.62 +/- 3.01 mm; P = 0.002) and less soft tissue along the root surface (2.77 +/- 0.79 mm versus 1.10 +/- 1.48 mm; P = 0.020) was observed for group B. Additionally, group B presented a larger area of new bone (P = 0.004) and a smaller area of soft tissue (P = 0.008). CONCLUSION: Within the limits of this study, root cementum may modulate the healing pattern obtained by guided tissue regeneration in Class III furcation defects.     

仿生型改性胶原膜的动物学实验研究

目的初步探讨自行研制的新型引导组织再生膜材料(仿生型改性胶原膜)应用于动物引导牙周组织再生的能力。方法通过模拟牙周炎骨缺损形态在Beagle犬双侧下颌第三、四前磨牙颊侧制作3个大小为5mm×5mm的急性二壁骨袋的骨缺损模型,深达牙面。采用自体对照方法观察牙周组织再生情况,骨缺损区随机分为3组:胶原膜组、聚四氟乙烯(e-PTFE)膜组、空白对照组,共5只Beagle犬、14个实验部位。术后2、4、8、12周将分别进行大体标本观察、组织学观察、锥形束计算机体层摄影术(CBCT)扫描并重建、扫描电镜Ca、P元素微区定量测定。结果该仿生型改性胶原膜具有良好的骨引导和暴露后抗感染能力。术后12周,仿生型改性胶原膜组的新生骨高度、体积与e-PTFE膜组对比差异无统计学意义;其引导的新生骨钙磷比值(Ca/P值)高于空白组及e-PTFE膜组。结论动物实验显示,该新型仿生型改性胶原膜具有良好的提高牙周骨再生能力。     

Regeneration of periodontal tissues by basic fibroblast growth factor

Several growth factors (or cytokines) have recently received attention because of their ability to actively regulate various cellular functions of periodontal ligament (PDL) cells and the effects of topical application of such factor(s) on periodontal tissue regeneration has been evaluated. In this study, we examined the role of basic fibroblast growth factor (bFGF) in the wound healing and regeneration of periodontal tissues. Alveolar bone defects (such as 2-wall, 3-wall and furcation class II bone defects) were created surgically in beagle dogs and primates. Recombinant bFGF was topically applied to the artificial bony defects. Six or 8 wk after application, the periodontal regeneration was morphologically and histomorphometrically analyzed. In all sites where bFGF was applied, significant periodontal ligament formation with new cementum deposits and new bone formation was observed in amounts greater than in the control sites. We found it noteworthy that no instances of epithelial down growth, ankylosis or root resorption were observed in the bFGF sites. In vitro studies demonstrated that bFGF enhances the proliferative responses of human PDL cells, which express FGF receptor-1 and -2, but inhibits the induction of alkaline phosphatase activity and mineralized nodule formation by PDL cells. Interestingly, we observed that the mRNA level of laminin in PDL cells, which plays an important role in angiogenesis, was specifically upregulated by bFGF stimulation, but that of type I collagen was downregulated. The present study demonstrates that bFGF can be applied as one of the therapeutic modalities which actively induce periodontal tissue regeneration. The results of in vitro studies suggest that by suppressing the cytodifferentiation of PDL cells into mineralized tissue forming cells, bFGF may play important roles in wound healing by promoting angiogenesis and inducing the growth of immature PDL cells, and may in turn accelerate periodontal regeneration.     

The effects of a bioabsorbable barrier membrane containing safflower seed extracts on periodontal healing of 1-wall intrabony defects in beagle dogs

BACKGROUND: Recently, there has been much research done into the regenerative potential of materials used in oriental medicine. In several studies, evidence was found that these materials have an effect on bone regeneration. Among these materials, safflower seeds are of particular interest as they have been used for the treatment of blood stasis, bone fracture, and osteoporosis in traditional Korean medicine. In addition, they are known to have anti-inflammatory effects. The objective of this study is to evaluate the periodontal tissue regenerative effects of a bioabsorbable barrier membrane (polylactide glycolic acid electro-spun non-woven membrane) containing safflower seed extracts applied to surgically created 1-wall intrabony defects in beagle dogs. METHODS: One-wall intrabony defects were surgically created bilaterally at the mesial and distal sides of the mandibular second premolars and mesial side of the fourth premolars. These defects were randomly assigned either to the surgical control group which received a flap operation only or to one of two experimental groups consisting of defects which received a guided tissue regenerative procedure with either a bioabsorbable membrane (PLGA) or a bioabsorbable membrane containing safflower seed extracts (SSE/PLGA). The dogs were sacrificed 8 weeks after the operation, and a comparative histological examination was done. RESULTS: The new cementum formation was 2.49+/-0.41 mm in the surgical control group, 3.22+/-0.35 mm in the PLGA group, and 3.67+/-0.82 mm in the SSE/PLGA group. The extent of new cementum formation in barrier groups was significantly different from the surgical control group (P <0.05). The amount of intrabony cementum was 1.75+/-0.06 mm, 2.40+/-0.33 mm, and 2.70+/-0.81 mm for the surgical control group, the PLGA group, and the SSE/PLGA group, respectively; the amount of infrabony cementum in the barrier groups was significantly different from the surgical control group (P<0.05). The value of the suprabony cementum was 0.73+/-0.48 mm, 0.82+/-0.21 mm, and 0.97+/-0.09 mm for the surgical control group, the PLGA group, and the SSE/PLGA group, respectively, with no significant differences being observed among the treatments. The amount of new alveolar bone formation was 1.74+/-0.25 mm, 2.36+/-0.30 mm, and 2.64+/-0.74 mm for the surgical control group, the PLGA group, and the SSE/PLGA group, respectively, with a significant difference exhibited between the surgical control group and other groups (P <0.05). Superficial root resorption was often observed, but ankylosis was not present. CONCLUSION: Our results suggest that surgical application of polylactide glycolic acid non-woven membrane with or without safflower seed extract could promote the regeneration of alveolar bone and cementum in intrabony periodontal defects.     

Injectable calcium phosphate bone cement provides favorable space and a scaffold for periodontal regeneration in dogs

BACKGROUND: An earlier study showed that an injectable calcium phosphate cement (CPC) served as a stable scaffold for bone formation and promoted histocompatible healing of periodontal tissue in dogs. In this study, we evaluated the influence of CPC on regeneration of periodontal defects with experimental periodontitis in dogs. METHODS: Experimental periodontitis was induced by placing stainless-steel mesh on the mesial side of maxillary canines in six adult, healthy beagle dogs. Subsequently, intrabony defects were resized so as to be standard, and CPC was injected in the experimental bone defects. Non-grafted defects on the contralateral side served as controls. Twelve weeks after surgery, the animals were sacrificed and histologic specimens were prepared. Periodontal tissue healing was evaluated histologically and histometrically. RESULTS: Healing of periodontal tissues, in terms of bone and cementum formation, was consistently observed in the CPC-applied sites. CPC was partly replaced by new bone. New cementum and periodontal ligament-like tissue were observed between CPC and the root surface. New bone (P <0.05), new cementum (P <0.01), and new connective tissue attachment and adhesion (P <0.05) were significantly enhanced in the experimental sites. CONCLUSION: Calcium phosphate cement provides stable wound healing and enhanced periodontal regeneration in periodontal defects in dogs with experimental periodontitis.     

聚四氟乙烯膜和几丁质膜引导骨再生的基础实验研究

目的：探讨国产聚四氟乙烯膜和自制几丁质膜引导骨再生的效果以及骨缺损大小与修复程度的关系，为临床应用提供依据。方法：在犬胫骨上段内侧制作直径5mm和7mm的洞穿形骨缺损，分别覆盖几丁质膜、聚四氟乙烯膜、及钛网加强的聚四氟乙烯膜，不覆盖膜做空白对照。术后2、4、8、12周取材，通过组织学及新骨形成量观察骨再生情况，比较不同的膜材料引导骨再生效果。结果：术后第2周开始，直径5mm和7mm骨缺损区覆盖膜的三组均有骨再生，至12周时骨缺损区已完全为新骨充填，对照组骨缺损区均为纤维结缔组织充填，相差显著。直径5mm和7mm的骨缺损区在膜覆盖后骨再生量无显著差别。结论：几丁质膜、聚四氟乙烯膜及钛网加强的聚四氟乙烯膜均能引导骨再生。