犬牙周膜中神经纤维的形态特点及分析

目的通过免疫组织化学染色方法鉴定犬牙周膜中神经纤维的性质。方法获取健康成年比格犬第二前磨牙近中至第三前磨牙远中牙槽骨颊舌侧下牙槽神经管上方的完整牙槽骨块,制作硬组织切片后进行S100、神经丝蛋白（NFP）免疫组织化学染色,显微镜下观察犬牙周膜中的神经分布。结果犬牙周膜中分布的S100（+）组织呈以下几种形态：密集环形组织聚成不同直径的束状结构;条形纤维状伴行于管腔状结构;游离末梢状或椭圆形片层状深染结构。NFP（+）组织分布特点与S100（+）相似,但形态主要为不同直径条索状纤维结构,呈束状、游离末梢状或分枝状散布于牙周膜胶原纤维束中。结论S100免疫组织化学结果可初步判断犬牙周膜中神经纤维的结构,NFP免疫组织化学结果可比较神经轴突的粗细区分神经类型,从而有助于鉴定犬牙周膜中神经纤维的性质。     

Fine structure of adrenergic nerve fibers in human periodontal ligament

The presence of adrenergic nerve fibers was demonstrated ultrastructurally in the human periodontal ligament obtained from extracted premolar teeth from 8 young patients. The nerve endings were located close to arterioles. The results suggest that they seem to control blood flow in the human periodontal ligament.     

两种体外培养人牙周韧带成纤维细胞方法比较

目的探索一种在体外短时间内简便、可靠获取大量人牙周韧带成纤维细胞(human periodontal ligament fibroblast,HPLF)、建立稳定的体外培养体系的方法。方法采用酶消化法和组织贴块法进行HPLF体外原代培养及传代培养的对比研究。通过细胞形态学、超微结构观察及波形蛋白和角蛋白免疫组化染色等对细胞进行定性研究;测定细胞生长曲线了解细胞生长基本规律及其增殖能力。结果采用酶消化法和组织贴块法均可成功的进行HPLF连续传代培养。最高传代数为30代。培养的细胞具有成纤维细胞的典型形态,波形蛋白染色阳性,角蛋白染色阴性,生长稳定期倍增时间为48~72h。组织块培养法需培养时间长,原代培养获取的细胞量较少,较难传代。酶消化法可短时间内获取大量细胞,细胞产量高,但操作手续复杂易污染,细胞易受损伤。结论成功建立了一个稳定的HPLF体外培养体系。除常用的组织块法外,胰蛋白酶及胶原酶联合消化法不失为一种简便、快速、可靠的组织原代分离培养方法。     

Location of putative stem cells in human periodontal ligament

BACKGROUND AND OBJECTIVE: The origin of cells in the mature periodontium, and the location of their progenitors, are still unknown. It is also unknown whether inflammation influences the number and distribution of these cells within the periodontium. Molecules such as STRO-1, CD146 and CD44 have been identified on a variety of mesenchymal stem cells. The aim of this study was to identify and localize putative stem cells in diseased and healthy human periodontal ligament using cell-surface markers for mesenchymal stem cells. MATERIAL AND METHODS: Healthy and periodontitis-affected teeth were collected, fixed in 10% neutral-buffered formalin, decalcified and embedded in paraffin in preparation for immunohistochemistry. Antibodies against STRO-1, CD146 and CD44 were used to identify putative stem cells in the periodontal ligament. RESULTS: Putative stem cells were identified in both healthy and diseased periodontal ligament. They were mainly located in the paravascular region and small clusters of cells were also found in the extravascular region. Wider distributions of these cells were detected in sections of diseased ligament. CONCLUSION: Within the periodontal ligament of both healthy and diseased teeth, cells have been identified consistent with their identification as putative stem cells. The presence of an inflammatory reaction associated with periodontitis may enhance the number of these cells.     

Innervation of the periodontal ligament in the alligatorid Caiman crocodilius

The mode of development and structure of crocodilian teeth and periodontium parallels that of mammals, but the teeth are continuously replaced throughout the lifetime of those animals. In this report, the innervation and fibres of the crocodilian periodontal ligament were examined using histology, immunohistochemistry for S-100 protein and transmission electron microscopy. Crocodilian periodontal ligaments had the following characteristics: (1) horizontal fibres, which connect the alveolar bone to the root cementum and (2) longitudinal fibres, which ran parallel to the tooth axis, with nerves and blood vessels in the middle layer of the ligament. The apex of root and tooth germs were both embedded in thick circular fibres. S-100 protein was detected in neural elements including terminal portions which were densely distributed in the periodontal ligament and dental follicle. The S-100 positive neural elements formed a periodontal plexus. We found two types of nerve endings; free endings and simple encapsulated corpuscles as described in mammals. The presence of such nerve endings in caiman suggests that these teeth, in addition to having a biting function, may also act as highly sensitive sensory organs.     

Morphology of neural endings in the human periodontal ligament: An electron microscopic study

The ultrastructure of sensory nerve endings in the human periodontal ligament from 43 extracted teeth was studied using serial sections. Three types of nerve endings were found: free nerve endings (FNE), Ruffini-like endings and lamellated corpuscles. Free nerve endings stem from unmyelinated or from myelinated nerve fibers. The endings contain neurotubuli, neurofilaments and vesicles. Ruffini-like receptors were mostly found in the apical part of the periodontal ligament. In these Ruffini-like receptors a particularly abundant concentration of mitochondria appears. In some cases desmosome-like junctions are present between neurite and ensheathing cell. Lamellated corpuscles were also found in the periodontal ligament. The lamellae are extremely endocytotic and are in close contact with each other.     

Presence of oxytalan fibers in human regenerated periodontal ligament

The aim of the present study was to investigate whether oxytalan fibers are formed in the regenerated human periodontal ligament. 6 patients, each of them exhibiting an advanced intrabony defect, were treated with a bioresorbable membrane according to the GTR-principle. Following a healing period of 6 months, the teeth were extracted together with their surrounding soft and hard tissues and subsequently fixed in 10% buffered formalin. Following decalcification in EDTA, the specimens were embedded in paraffin and 8-microm histological sections were cut in the mesio-distal direction, parallel to the long axes of the teeth. The sections were stained with hematoxylin and eosin, or with the oxone-aldehyde-fuchsin-Halmi staining method and examined in the light microscope. A regenerated periodontal ligament containing newly-formed oxytalan fibers was observed in all specimens. Many of them inserted into the newly formed cementum on the root surface. It is concluded that oxytalan fibers are formed de novo in human regenerated periodontal ligament tissue.     

The periodontal ligament of teeth connected to osseointegrated implants

Abstract The aim of the present investigation was to analyze the periodontal tissues at immobilized teeth connected to osseointegrated implants. 10, 1-year old beagle dogs, were used. Bilaterally, the mandibular 2nd (₂P₂) and 3rd pre-molars (₃P₃) and 1st molars (₁M₁) were extracted. 2 titanium fixtures were installed in the edentulous segment of the right side of the mandible, one about 10 mm mesial and the other about 10 mm distal to 4P (test tooth). 3 months later, abutment connection was performed and healing allowed for one month. The dogs were randomly divided into 2 groups of 5 each, group A and group B. In group A, a fixed gold splint, rigidly connecting the tooth and the 2 implants, was installed on day 0 and ₄P was hereby immobilized. The controlateral 4th premolar (P4) served as the non-splinted control tooth. Plaque control measures continued until the end of the experiment (day 180). In group B, plaque control measures were abandoned 1 month after abutment connection and a 4-month period of experimental periodontal tissue breakdown was initiated. This was accomplished by placing cotton floss ligatures submarginally around ₄P and P₄. At the end of this 4-month period, the ligatures were removed, and an apically positioned flap procedure was performed. Healing was allowed for another 2 months. Plaque control measures were re-established and continued throughout the experiment. A given day was termed day 0 and ₄P was rigidly connected to the adjacent implants in the manner described for group A. At the end of a subsequent 6-month period, radiographs of ₄P and P₄ were taken and biopsies harvested from all the dogs. The results of measurements, made in histological sections, revealed that the splinting of mandibular premolars to osseointegrated implants failed to induce marked alterations (qualitative and quantitative) in the gingiva and periodontal tissues of the immobilized teeth. These findings offer a biological explanation for the fact that a fixed bridge, utilizing both teeth and implants as abutments, seems to function well in the rehabilitation of partially edentulous patients.     

NGF mRNA在人牙周膜成纤维细胞的表达

目的:研究牙周组织中是否存在神经生长因子(NGF)的表达。方法:采用RT-PCR方法研究培养的人牙周膜成纤维样细胞(hPDLF)是否表达NGF mRNA。结果:通过RT-PCR技术,在培养的hPDLF中扩增出NGF mRNA阳性产物。结论:通过离体研究初次显示培养的人牙周膜成纤维样细胞(hPDLF)表达NGF mRNA。     

Role of periodontal ligament receptors in the tactile function of teeth: a review

The tactile function of the human periodontal mechanoreceptors has mostly been studied by psychophysical approaches. It was concluded that periodontal mechanoreceptors play a major role in the tactile function of teeth. It must be noted however that the interocclusal tactile threshold is not solely determined by periodontal mechanoreceptors but also by pulpal, muscular or articular receptors. While temporomandibular joint receptors play a minor role, muscular receptors are important in the discriminatory ability for a mouth opening of 5 mm and more. To discriminate between the contribution of periodontal and other receptors in the oral tactile function, future studies should use appropriate psychophysical methodologies and well-defined stimulus parameters.     

黄芩苷对人牙周韧带细胞超微结构的影响

目的：观察黄芩苷对人牙周韧带超微结构的影响。方法：10ng/mL的黄芩苷孵育人牙周韧带细胞5d，采用透射电镜观察人牙周韧带细胞超微结构的变化。结果：与对照组比较，用药组细胞的内质网、线粒体等明显增多。结论：黄芩苷能促进细胞的代谢和蛋白质的合成，与细胞增殖实验和总蛋白测定结果一致。     

骨膜蛋白及其在牙周膜中的生物学作用

骨膜蛋白是一种细胞基质蛋白,是在鼠成骨细胞中发现的一种具有细胞黏附作用的蛋白质,其为细胞外基质功能完整的重要组成部分;骨膜蛋白表达于胚胎发育阶段和成年机体的多种组织中,如骨、心脏、肺、动静脉及牙周膜等.诸多研究揭示了骨膜蛋白参与维持牙周膜细胞功能及调节牙周膜胶原纤维形成过程,能够促进牙周膜细胞黏附、增殖、分化,促进牙周膜细胞成牙周膜样和成牙骨质样作用;在牙周膜胶原纤维形成过程中,骨膜蛋白可和胶原共存,在机械应力刺激下,可维持牙周膜纤维系统完整性.本文基于骨膜蛋白的发现和表达,对骨膜蛋白参与牙周膜生物学功能维持的研究进展作一综述.     

人釉原蛋白基因转染牙周膜细胞的实验研究

目的:构建含有人釉原蛋白(human amelogenin,hAm)基因的慢病毒载体质粒,用重组慢病毒感染人牙周膜细胞(Human periodontal ligament cells,hPDLCs),初步探讨釉原蛋白基因修饰种子细胞用于牙周组织工程修复的可行性.方法:采用RT-PCR方法获取hAm编码基因,构建慢病毒载体质粒FUAmW,重组质粒经双酶切及测序鉴定.聚乙烯亚胺(polytheylenimine,PEI)法三质粒共转染293T细胞,获取重组慢病毒FUAmW FUGW,感染hPDLCs,应用荧光显微镜观察绿色荧光蛋白(green fluoreseene protein,GFP)表达,流式细胞仪flow cvtometer,FCM)检测慢病毒载体转染效率.通过RT-PCR检测hPDLC中hAm基因的表达.结果:测序证实,RT-PCR产物序列与Genebank公布的Am编码序列一致,双酶切证实目的片段插入重组质粒.293T细胞及hPDLCs感染72h后.荧光显微镜下可见绿色荧光.FCM测得GFP感染2种细胞的阳性率分别为69.46%和33.99%.RT-PCR证实重组慢病毒FUAmW感染的细胞能表达hAm基因.结论:成功构建含有人釉原蛋白基因的慢病毒载体质粒,经293T细胞包装.得到重组慢病毒可感染牙周膜细胞.     

人牙周膜干细胞的分离培养及初步鉴定

目的 体处分离培养人类牙周膜干细胞并对其生物学性状进行初步鉴定.方法 采用有限稀释法进行牙周膜干细胞克隆筛选,获得单细胞克隆来源细胞,检测其克隆形成率,并采用免疫组织化学染色、碱性磷酸酶染色、免疫荧光染色等方法鉴定牙周膜干细胞的组织来源及生物学特性.结果 有限稀释法获得的牙周膜干细胞克隆株呈簇状生长,排列紧密,细胞呈圆形或椭圆形,细胞较小,排列形成紧密的团块状.免疫组化染色显示细胞波形蛋白阳性,角蛋白阴性;碱性磷酸酶染色阳性;早期间充质干细胞的标志物STRO-1荧光染色显示细胞发出明亮的红色荧光,表达阳性.结论 牙周膜中存在具有高增殖能力的干细胞,克隆化分离培养的人牙周膜干细胞具有强克隆形成能力,具有间充质干细胞表型和生物学特性.     

体外培养的人牙周膜细胞成骨样细胞表型特征的研究

目的 对体外培养的人牙周膜细胞成骨样细胞的表型特征进行研究探讨。方法 组织块 法培养人牙周膜细胞,放射免疫法检测条件培养基中碱性磷酸酶（ＡＬＰ）和骨钙素（ＯＣＮ）的分泌活性,免疫 组化法检测非分泌型的ＡＬＰ和ＯＣＮ的表达,原位杂交方法检测二者的ｍＲＮＡ水平的表达。结果 人牙 周膜细胞具有成骨细胞的部分表型,碱性磷酸酶在蛋白水平包括分泌和非分泌型及基因转录水平都有一 定的表达,随培养时间的变化不大;而不具有成熟的成骨细胞的表型特征——骨钙素的表达,其无论是在 基因和蛋白水平都没有表达二这一结果为进一步研究人牙周膜细胞在机械力作用下参与骨改建打下基础。     

Proliferative activity in the juxtaradicular human periodontal ligament

Abstract— The aim of the present study was to evaluate cell proliferation, assessed by MIB 1, with respect to the type and the distribution of proliferating cells in the healthy juxtaradicular periodontal ligament (PDL) from completely formed human teeth. Immunohistochemical markers against vimentin, CD68 and S-100 were used to characterize cell type. The applicability of the immunohistochemical method on explants of human PDL was also evaluated. The results indicated that under physiological conditions, the majority of the proliferating cells in the PDL were mesenchymal cells predominantly located paravascularly in the middle third of the PDL. Furthermore, MIB 1 reacting with the Ki-67 antigen together with the avidin-biotin-complex technique was proved to be an efficient marker of cell proliferation in explants of human PDL.     

金栀含漱液对人牙周膜成纤维细胞毒性的实验研究

目的：采用MTT法评价中药制剂金栀含漱液、氢氧化钙药尖及FC对人牙周膜成纤维细胞（HPDLF）的细胞毒性及毒性的可逆性。方法：体外培养HPDLF，选用金栀含漱液作为实验组（A组），氢氧化钙药尖浸提液为阴性对照组（B组），FC（C组）为阳性对照组，培养基作为空白对照组，分别与HPDLF接触1、3、5、7d时用Mr丌法测细胞增殖率及接触第1天的回复度。结果：A组及B组对HPDLF增殖无显著性抑制作用，细胞毒性可逆；C组对HPDLF增殖有显著的抑制作用，细胞毒性不可逆。FC组与金栀含漱液组和氢氧化钙药尖组相比抑制作用均有显著性差异，后两组的作用与培养基相当。结论：中药制剂金栀含漱液对HPDLF无明显细胞毒性作用。