骨形成蛋白在狗牙周组织中的分布及其意义

本研究首次应用bBMP-McAb免疫组化染色法，观察BMP在狗的牙周组织中的分布。结果显示：BMP主要分布于牙周韧带中，尤其是牙槽骨和牙骨质附近染色更深，并发现牙槽骨外侧BMP阳性区经过牙槽嵴与牙周韧带BMP阳性区相连续。而牙龈组织染色阴性．表明牙周韧带细胞与牙龈成纤维细胞不同，前者其有骨母样细胞的特性，并可参与牙周硬组织的形成；未分化细胞是否含有BMP，可作为评价该细胞是否具有骨母样细胞特性的参考标准之一．     

骨形成蛋白在正常兔牙周组织中的表达及定量分析

目的 进一步了解骨形成蛋白（ＢＭＰ）在牙齿及牙周组织中的表达，为研究ＢＭＰ在牙周组织的修复再生及骨改建中的作用提供实验数据。方法 用ＢＰＭ单克隆抗体免疫组化染色及计算机图像分析的方法对正常兔牙周组织ＢＭＰ的表达进行了定性、定量分析。结果 在牙周膜中，ＢＭＰ的分布不同，在靠近牙槽骨和牙骨质侧染色较深，成骨细胞、成牙骨质细胞、成纤维细胞染色均呈强阳性，牙槽骨、牙龈染色阴性。近中侧ＢＭＰ表达量明显低于远     

牙周膜牵张成骨牙齿快速移动和传统正畸牙齿移动两种牙移动方式牙周组织中骨形成蛋白表达变化的研究

目的 :探讨不同性质 ,不同程度的牙周改建活动中BMP的分布情况 ,初步探讨牙周膜牵张成骨的生物学机制。方法 :本实验利用骨形成蛋白 (bonemorphogeneticprotein ,BMP)的单克隆抗体 (BMP McAb)对实验犬牙周膜牵张成骨牙齿快速移动和传统正畸牙齿移动这两种不同牙齿移动方式下的牙周组织进行免疫组化染色 ,并通过计算机图像分析的方法 ,对牙周膜中BMP的表达变化进行了定性 ,定量分析。结果 :BMP在犬牙周组织中主要分布在牙周膜区域 ,尤其是在邻近牙槽骨及牙骨质区域染色呈强阳性 :成骨细胞、破骨细胞、成纤维细胞染色均呈强阳性 :在牙槽骨及牙骨质中染色阴性 ,牙槽骨中骨细胞陷窝壁染色弱阳性 :染色强度张力侧强于压力侧 ,骨改建尤其是成骨活跃区域染色相应也呈强阳性 ,新生骨质和牙骨质层染色弱阳性 :牙周血管和骨髓腔周围的未分化间充质细胞胞浆染色较深。结论 :牙周膜细胞在骨形成蛋白的诱导下向成骨样细胞分化 ,在牙槽骨的改建 ,尤其是在成骨方面起了重要的作用。骨形成蛋白的表达与局部的骨改建活动相一致 ,因此可以作为骨改建活动 ,尤其是成骨活动的标志之一。     

牙周韧带细胞体外矿化的研究——骨唾蛋白与骨桥素在矿化过程中的表达

目的：研究骨唾蛋白与骨桥素在体外培养的人牙周韧带细胞矿化过程中的表达。方法：体外培养的人牙周韧带细胞，经地塞米松矿化液诱导，分别于不同时间在相差显微镜下观察其矿化结节的形成，采用免疫细胞化学的方法观察骨唾蛋白与骨桥素的表达情况。结果：矿化培养1周细胞呈复层生长，骨桥素呈阳性染色；两周细胞密集呈结节状，骨唾蛋白与骨桥素均为阳性，胞浆染成黄褐色；3周出现矿化结节，矿化以后骨唾蛋白与骨桥素保持稳定表达。结论：牙周韧带细胞在适当的条件下可以向成骨细胞表型分化，表达矿化相关蛋白，形成矿化结节。     

核心结合因子a1、骨形成蛋白及骨桥素在小鼠牙周组织发育中的免疫组化定位研究

目的探讨核心结合因子a1、骨形成蛋白和骨桥素3者在小鼠牙周组织发育过程中的时间和空间分布特点、表达意义及它们之间的相互作用。方法用SABC免疫组化法检测3者在出生后不同时期小鼠牙周组织发育中的表达及特点。结果牙齿发育早期,仅骨形成蛋白在牙囊细胞中表达;当牙根开始发育后,3者皆在牙周膜细胞、成牙骨质细胞中表达,但骨形成蛋白在无细胞牙骨质和细胞牙骨质中皆不表达,核心结合因子a1在细胞牙骨质中表达,而骨桥素则在无细胞牙骨质、细胞牙骨质和牙槽骨表面皆呈强阳性表达。结论核心结合因子a1、骨形成蛋白、骨桥素3者皆参与牙周组织的发育,对矿化和生长发育发挥重要作用。     

牙周韧带细胞体外矿化的研究——骨唾蛋白与骨桥素在矿化过程中的表达

目的 :研究骨唾蛋白与骨桥素在体外培养的人牙周韧带细胞矿化过程中的表达。方法 :体外培养的人牙周韧带细胞 ,经地塞米松矿化液诱导 ,分别于不同的时间在相差显微镜下观察其矿化结节的形成 ,采用免疫细胞化学的方法观察骨唾蛋白与骨桥素的表达情况。结果 :矿化培养 1周细胞呈复层生长 ,骨桥素呈阳性染色 ;两周细胞密集呈结节状 ,骨唾蛋白与骨桥素均为阳性 ,胞浆染成黄褐色 ;3周出现矿化结节 ,矿化以后骨唾蛋白与骨桥素保持稳定表达。结论 :牙周韧带细胞在适当的条件下可以向成骨细胞表型分化 ,表达矿化相关蛋白 ,形成矿化结节。     

Ⅰ、Ⅲ型胶原在牙龈成纤维细胞和牙周韧带细胞中表达的比较研究

目的比较人牙龈成纤维细胞与牙周韧带细胞在胶原基质合成方面的差别,并探讨其意义。方法采用组织块法体外培养人牙龈成纤维细胞与牙周韧带细胞,利用免疫细胞化学技术检测Ⅰ、Ⅲ型胶原在两种细胞中的表达,通过图像分析探讨其表达的差异。结果Ⅰ、Ⅲ型胶原在牙周韧带细胞中表达阳性,在牙龈成纤维细胞中染色较弱。统计学分析显示,Ⅰ、Ⅲ型胶原在牙龈成纤维细胞与牙周韧带细胞的免疫细胞化学染色结果具有显著性差异(P<0.01)。结论牙龈成纤维细胞与牙周韧带细胞胶原基质的合成或分泌存在差异,这种差异可作为鉴别两种细胞的标志之一。     

大鼠成牙骨质细胞的体外培养及其生物学特性的实验研究

目的 :探讨体外培养源于大鼠磨牙牙骨质的成牙骨质细胞 (cementoblast ,CMB)的可行性 ,以期建立能排除牙周其它细胞干扰的CMB体外实验模型。方法 :使用组织块结合酶消化的二步法 ,分别培养来自牙骨质的CMB ,来自牙槽骨的成骨细胞 (osteoblast ,OB)和来自牙周韧带 ( periodontalligament ,PDL)的成纤维细胞。然后对所培养的细胞分别进行骨钙素免疫组化染色和碱性磷酸酶 (ALP)活力测定 ,并利用茜素红S ,检测在条件培养基下细胞形成矿化结节的情况。结果 :3种牙周细胞都可从约 12周大鼠第一磨牙根周获得进行原代培养。骨钙素在体外培养的CMB和成骨细胞中被强烈表达 ,在PDL成纤维细胞 (PDLC)中几乎不表达。成骨细胞ALP活性最强 ,在成纤维细胞中适中 ,在CMB中几乎无活性。CMB形成矿化结节茜素红S染色阳性。结论 :CMB可以在体外成功培养 ,并可与牙槽骨OB、PDLC相区别。CMB在形态和蛋白表达等很多地方与成骨细胞相似 ,如表达矿化相关蛋白等 ,但它几乎没有ALP活性 ;矿化条件下 ,能在体外形成矿化结节     

富含亮氨酸的间质蛋白多糖在小鼠牙周组织发育中的组织学定位

目的研究富含亮氨酸的蛋白多糖类物质：纤维调节素（FMN）、核心蛋白聚糖（DCN）及双糖链蛋白聚糖（BGN）在小鼠牙周组织不同发育时期的分布特点，探讨其在牙周组织发育中的作用。方法取出生后第5、10、15、25天的BALB／c小鼠36只，引颈处死，解剖并分离含下颌第一磨牙区的下颌骨，新鲜配制4％多聚甲醛固定24h，甲酸一甲酸钠复合脱钙液脱钙1～3周，常规石蜡包埋，近远中向5μm连续切片。免疫组织化学Power Vision^TM二步法，观察各发育时期第一磨牙牙周组织中3种间质蛋白多糖的组织学分布特点。结果FMN在牙龈结缔组织和牙周膜中呈强阳性表达，且在牙周膜与牙骨质及牙槽骨界面有较强表达；DCN强阳性表达于牙龈结缔组织、靠近牙槽骨面的牙周膜及牙槽骨表面的成骨细胞中；BGN则在各期牙龈结缔组织及牙周膜中表达，在牙槽骨表面及成骨细胞中为阴性。结论FMN可能与DCN及BGN相互作用，调节牙龈结缔组织及牙周膜胶原纤维的网络形成，并可能参与牙槽骨和牙骨质的矿化过程。     

α-平滑肌肌动蛋白在牙龈成纤维细胞和牙周韧带细胞中的表达及意义

目的 研究α—平滑肌肌动蛋白在体外培养的牙龈成纤维细胞与牙周韧带细胞中的表达并探讨其意义。方法 利用细胞培养和免疫细胞化学技术检测α—平滑肌肌动蛋白在体外培养的牙龈成纤维细胞与牙周韧带细胞中的表达。结果 α—平滑肌肌动蛋白在两种成纤维细胞中的免疫细胞化学染色均为阳性。结论 α—平滑肌肌动蛋白在牙龈成纤维细胞与牙周韧带细胞中稳定表达，可能在细胞收缩与基质改建中起重要作用。     

Emdogain regulation of cellular differentiation in wounded rat periodontium

Emdogain is an enamel matrix derivative that may promote periodontal regeneration by recapitulating critical events in tooth morphogenesis. We hypothesized that Emdogain enhances periodontal regeneration by promoting the differentiation of cells required for the synthesis of periodontal ligament, bone and cementum. Cell differentiation was examined in rat periodontal window wounds in which there is no microbial biofilm or epithelial downgrowth, thereby simplifying the model system. Defects were filled with vehicle control or Emdogain (3 mg/ml or 30 mg/ml). Rats were sacrificed at 7, 14 and 21 d after wounding. Specimens of periodontium were immunostained for osteopontin, bone sialoprotein, osteocalcin as markers of osteogenic differentiation and for alpha-smooth muscle actin, a myofibroblastic marker. Morphometry and 3H-proline radioautography were used for assessment of tissue homeostasis and matrix production. Rats treated with Emdogain (only at 30 mg/ml) showed widening of the periodontal ligament at 7 d; by 14 and 21 d, periodontal ligament width was restored to normal values for all groups. Emdogain exerted no effect on cementum thickness, bone volume, osteoid deposition rates, or extracellular staining for osteopontin, bone sialoprotein or osteocalcin. Further, the percentage of cells with intracellular staining for osteopontin, osteocalcin or bone sialoprotein was unaffected by Emdogain. Staining for alpha-smooth muscle actin was abundant in the repopulating wound but was also unaffected by Emdogain. In conclusion, Emdogain does not apparently affect the expression of differentiation markers or bone matrix protein synthesis in the repopulation response of wounded rat molar periodontium. Therefore the effect of Emdogain on wound healing in the periodontium may be independent of differentiation in the cell populations examined in this model.     

Expression of bone matrix protein mRNAs by primary and cloned cultures of the regenerative phenotype of human periodontal fibroblasts

The successful regeneration of periodontal tissues is dependent, in part, on the ability of cells to reconstitute the mineralized tissues of cementum and bone. The aim of the present study was to characterize regeneration-associated cells in terms of their ability to express mineralized tissue macromolecules. Following guided tissue regeneration, cell cultures were established from regenerating tissue, periodontal ligament, and gingiva. Additionally, these cells were transfected, and single-cell-derived clones were established. Following treatment with platelet-derived growth factor-BB and insulin-derived growth factor-1, the presence of mRNA for alkaline phosphatase, osteocalcin, bone sialoprotein, osteopontin, and bone morphogenetic proteins-2 and -4 was assessed. The three cell types expressed similar mRNA levels for alkaline phosphatase, bone morphogenetic protein-2, and bone morphogenetic protein-4, whereas the expression of osteopontin, osteocalcin, and bone sialoprotein was greater in the periodontal ligament and regenerating tissue fibroblasts compared with the gingival fibroblasts. The two growth factors did not affect the expression of any of the genes. This study has identified markers that correlate with the known ability of periodontal ligament and regenerating tissue-derived fibroblasts to facilitate regeneration of the mineralized tissues of the periodontium.     

Immunohistochemical study of bone sialoprotein and osteopontin in healthy and diseased root surfaces

BACKGROUND: Periodontal disease is marked by inflammation and damage to tooth-supporting tissues. In particular, damage occurs to factors present in cementum that are thought to have the ability to influence the regeneration of surrounding tissues. Bone sialoprotein and osteopontin are major non-collagenous proteins in mineralized connective tissues associated with precementoblast chemo-attraction, adhesion to the root surface, and cell differentiation. The purpose of this investigation was to determine whether the expression and distribution of bone sialoprotein and osteopontin on root surfaces affected by periodontitis are altered compared to healthy, non-diseased root surfaces. METHODS: Thirty healthy and 30 periodontitis-affected teeth were collected. Following fixation and demineralization, specimens were embedded in paraffin, sectioned, and exposed to antibodies against bone sialoprotein and osteopontin. Stained sections were assessed using light microscopy. RESULTS: Bone sialoprotein was not detected in the exposed cementum (absence of overlying periodontal ligament) of diseased teeth. In most areas where the periodontal ligament was intact, bone sialoprotein was detected for healthy and diseased teeth. For teeth reactive for bone sialoprotein, the matrix of the cementum just below the periodontal ligament was moderately stained. A similar immunoreactivity pattern for osteopontin was observed. CONCLUSIONS: The absence of bone sialoprotein and osteopontin staining along exposed cementum surfaces may be due to structural and compositional changes in matrix components associated with periodontal disease. This may influence the ability for regeneration and new connective tissue attachment onto previously diseased root surfaces.     

Collagen implants do not preserve periodontal ligament homeostasis in periodontal wounds

An improved understanding of the differentiation of periodontal ligament cells could facilitate the development of new treatment approaches for overcoming the loss of specialized cell types caused by periodontitis. To study healing of wounded periodontal tissues and the differentiation of mineralizing connective tissue cells in periodontal ligament, we have examined the influence of wound size and collagen implantation on the regeneration of periodontium and on immunohistochemical staining for osteopontin and bone sialoprotein. Four groups of Wistar rats were wounded by drilling through the alveolar bone and by extirpation of the periodontal ligament. Wounds were 0.6 or 1.8 mm in diameter and defects were either implanted with collagen gels or were treated without implants. Rats were killed at 1 wk or 2 months after wounding and tissue sections were stained with monoclonal antibodies against rat osteopontin and bone sialoprotein. Collagen implants strongly increased staining for osteopontin and bone sialoprotein in defects at 1 wk. By 2 months alveolar bone healed completely regardless of the wound size but in large defects, periodontal ligament width was significantly reduced with or without implants. In large wounds at 2 months, collagen implants inhibited bone regeneration and there was stronger staining for osteopontin and bone sialoprotein in the bone replacing the implant, indicating that collagen prolonged bone remodelling. We conclude that implantation of exogenous collagen affects alveolar bone healing but does not preserve the width of the regenerated periodontal ligament. Therefore collagen does not appear to contribute to homeostasis in the periodontium following wounding.     

Immunohistochemical localisation of extracellular matrix proteins in the periodontium during cementogenesis in the rat molar

OBJECTIVE: The development of the periodontium involves the coordinated expression of numerous extracellular matrix (ECM) macromolecules and their receptors (integrins). The aim of this study was to determine the expression of selected hard and soft tissue matrix molecules and the integrin alpha5beta1 in the periodontal tissues, during cementogenesis in the rat molar. METHODS: Using immunohistochemical methods, the distribution of the extracellular matrix proteins, fibronectin, tenascin, and bone sialoprotein (BSP), as well as the integrin subunits alpha5 and beta1 were studied in rats aged 3, 5 and 8 weeks. RESULTS: Fibronectin was widely distributed in the gingival epithelium, gingival connective tissue and in the periodontal ligament. Tenascin expression was less marked compared with fibronectin, but was more distinctly associated with cells and peri-cellular areas of the epithelial-connective tissue interface, the gingiva and within the periodontal ligament. The fibronectin-receptor alpha5beta1 integrins were expressed by epithelial cells, periodontal ligament cells and gingival fibroblasts. A notable finding was the increased staining intensity of fibronectin, tenascin and alpha5beta1 integrin in all 5-week old molar sections in the periodontal ligament matrix and cells, apical to the cemento-enamel junction (CEJ) along the alveolar crest (AC) ridge height. Bone sialoprotein was distinctly associated with the hard tissues of the periodontium as acellular cementum and alveolar bone matrix expressed bone sialoprotein throughout all sections, in all age groups. CONCLUSIONS: In conclusion, this study has demonstrated the selective distribution of several hard and soft tissue matrix molecules during periodontogenesis. The results highlight the complex nature of interactions of various proteins and molecules during development. The interactions between these molecules and their specific roles in development and regeneration await further investigation.     

Mesenchymal stem cells acquire characteristics of cells in the periodontal ligament in vitro

Mesenchymal stem cells differentiate into multiple types of cells derived from mesenchyme. Periodontal ligament cells are primarily derived from mesenchyme; thus, we expected mesenchymal stem cells to differentiate into periodontal ligament. Using a combination of immunohistochemistry and in situ hybridization on co-cultures of mesenchymal stem cells and periodontal ligament, we observed a significant increase in mesenchymal stem cells' expression of osteocalcin and osteopontin and a significant decrease in expression of bone sialoprotein, characteristics of periodontal ligament in vivo. Increased osteopontin and osteocalcin and decreased bone sialoprotein expression was detected within 7 days and maintained through 21 days of co-culture. We conclude that contact or factors from periodontal ligament induced mesenchymal stem cells to obtain periodontal-ligament-like characteristics. Importantly, analysis of the data suggests the feasibility of utilizing mesenchymal stem cells in clinical applications for repairing and/or regenerating periodontal tissue.     

Enhanced proliferation, attachment and osteopontin expression by porcine periodontal cells exposed to Emdogain

Emdogain^® (EMD) is an enamel matrix derivative extracted from developing porcine teeth with demonstrated periodontal regenerative potential. EMD has been shown to influence a number of properties of periodontal ligament cells including proliferation, cell attachment and matrix synthesis. To date, the effect of EMD on the epithelial cell rests of Malassez (ERM) is unknown.

In this study, periodontal ligament fibroblasts, ERM, alveolar bone cells and gingival fibroblasts were obtained from porcine periodontal ligament, alveolar bone and gingiva. This study investigated, in vitro, the effect of EMD at three concentrations on proliferation, cell attachment and expression of mRNA for two mineralised tissue-related proteins (osteopontin and bone sialoprotein).

As for other periodontal cells, the ERM proliferative response was enhanced by EMD. Attachment assays revealed a highly significant increase for ERM and gingival fibroblasts after EMD treatment at all concentrations. This study has also shown that EMD stimulated expression of osteopontin mRNA by ERM and alveolar bone cells.

The results from this study provide evidence that EMD enhanced cellular events related with proliferation, attachment and osteopontin mRNA expression by porcine periodontal cells, in a manner consistent with its role in periodontal regenerative therapy.