牙髓牙本质复合体纤维粘连蛋白的免疫组织化学研究与定量分析

目的　研究纤维粘连蛋白在牙髓牙本质复合体内的表达 ,探讨其分布与功能间的关系。　方法　免疫组织化学染色与图像定量分析。结果　纤维粘连蛋白免疫反应 (- IR)普遍存在于牙髓、前期牙本质和成熟牙本质的牙本质小管与成牙本质细胞突起 ,以成牙本质细胞层 ,牙髓血管与神经周围最为丰富。冠髓和冠部牙本质纤维粘连蛋白的积分光密度分别为 :0 .49± 0 .33,0 .5 0± 0 .2 9;线密度分别为 :38.44± 2 0 .7,19.34± 18.72 ;体密度分别为 :2 4.5 6± 10 .8,44 .5 2± 2 8.5。　结论　纤维粘连蛋白 - IR不仅见于牙髓与前期牙本质 ,而且也见于成熟牙本质的牙本质小管与成牙质细胞突起 ,它对维持牙髓牙本质复合体各结构的正常位置 ,形态和防止炎症扩散可能发挥了重要作用     

人前磨牙牙髓牙本质复合体Ⅰ型和Ⅲ型胶原的来源及分布

目的 探讨牙髓牙本质复合体Ⅰ型和Ⅲ型胶原的来源与分布。方法 收集人前磨牙，固定、脱钙、石蜡包埋、切片、用SABC法进行免疫组织化学染色，显示Ⅰ型和Ⅲ型胶原蛋白。结果Ⅰ型胶原在牙髓内十分丰富，聚集成束，根髓中致密，以粗纤维束沿神经血管纵行分布；冠髓中纤维束较细、大量分支、变细，随意分布，交织成疏松的纤维网。从根尖至牙冠，从牙髓中央至周边，纤维束由粗变细，沿途分支，大部分终止于牙髓基质，少部分经成牙本质细胞间进入前期牙本质。此外成牙本质细胞、成纤维细胞与血管内皮细胞亦见Ⅰ型胶原蛋白表达。前期牙本质Ⅰ型胶原染色深，与牙髓基质和成牙本质细胞突起内者相延续，成熟牙本质近髓1／3可见Ⅰ型胶原阳性免疫反应物。Ⅲ型胶原在牙髓牙本质复合体的表达基本与Ⅰ型原类似，仅数量和密度略少。结论 牙髓牙本质复合体内的Ⅰ型和Ⅲ型胶原可能由成牙本质细胞、成纤维细胞和血管内皮细胞产生；不仅牙髓内存在Ⅰ型和Ⅲ型胶原，而且前期牙本质也有Ⅰ型和Ⅲ型胶原。     

牙髓牙本质复合体中VIP阳性神经纤维的研究

目的:研究血管活性肠肽(VIP)在牙髓牙本质复合体中的表达,探讨其功能。方法:收集人前磨牙,石蜡包埋切片,作免疫组织化学和图象定量分析。结果:VIP阳性神经纤维自根尖孔呈束状进入牙髓,至颈部扇形分开,在冠髓大量分支,部分围绕在血管周围,部分终止于牙髓基质,部分参与形成成牙本质细胞层下Raschkow神经丛,然后发出分支伸入成牙本质细胞层和前期牙本质,但不进入成熟牙本质。VIP阳性神经纤维在冠髓的积分光密度为12.74±1.807,体密度为0.0192±0.0127,线密度为0.0046±0.0029,在前期牙本质的积分光密度为13.07±1.927,线段长度为(19.60±8.597)mm。结论:VIP阳性神经纤维存在于人牙髓牙本质复合体,部分纤维围绕血管,部分纤维止于牙髓基质和前期牙本质,这种分布提示该纤维除与血管运动有关外,可能还与感觉有关,在痛觉传导、血管调节等方面发挥重要作用。     

三氧矿合物复合纤维粘连蛋白盖髓的实验研究

目的:观察三氧矿合物复合纤维粘连蛋白的盖髓效果, 探讨牙髓细胞分化和修复性牙本质形成的机制, 为临床筛选理想的盖髓剂提供实验依据。方法:将16只猫192颗牙的唇侧开髓, 每只猫的A、B、C、D区选取3颗实验牙, 分别用三氧矿合物、三氧矿合物与100 mg/L纤维粘连蛋白、氢氧化钙、100 mg/L纤维粘连蛋白与医用淀粉盖髓, 运用组织学方法观察修复性牙本质的形成及牙髓组织学改变。结果:术后1周, 4个盖髓组均无修复性牙本质桥的形成;术后4周, 与氢氧化钙、三氧矿合物、纤维粘连蛋白/医用淀粉盖髓比较, 三氧矿合物复合纤维粘连蛋白能形成完整的管样牙本质桥, 牙髓组织炎症反应轻。结论:三氧矿合物复合纤维粘连蛋白盖髓可诱导形成完整的牙本质桥, 牙髓组织反应轻微, 作为生物活性盖髓剂, 起到良好的活髓保存效果, 值得临床推广应用。     

三氧矿合物复合纤维粘连蛋白盖髓的扫描电镜观察

目的:采用扫描电子显微镜技术观察三氧矿合物复合纤维粘连蛋白作为盖髓剂形成修复性牙本质桥的超微结构特点并对其疗效进行评价。 方法:以4只猫为实验对象,每只猫分为A、B、C、D 4个区,每区选取3颗实验牙,机械暴露牙髓后,分别用三氧矿合物、三氧矿合物复合纤维粘连蛋白、氢氧化钙、纤维粘连蛋白与医用淀粉盖髓,盖髓12周后应用扫描电镜观察新形成的牙本质桥。 结果: 氢氧化钙组、三氧矿合物组可见不规则的牙本质桥结构。三氧矿合物复合纤维粘连蛋白组可见完整的牙本质桥。在纤维粘连蛋白和医用淀粉组,未见牙本质桥形成。结论: 三氧矿合物复合纤维粘连蛋白能刺激修复性牙本质形成,形成的牙本质桥完整,值得临床推广应用。     

重组人骨形成蛋白-2复合胶原I盖髓的实验研究

目的:观察重组人骨形成蛋白-2复合胶原I的盖髓效果和对牙髓组织骨钙素含量的影响，探讨牙髓细胞分化和修复性牙本质形成的机制，为临床筛选理想的盖髓材料提供实验依据。方法:以30只猫为实验对象，机械暴露牙髓后，分别用重组人骨形成蛋白-2复合胶原I、氢氧化钙、胶原及对照的牛血清白蛋白盖髓，运用组织学方法观察牙髓修复性牙本质的形成及牙髓组织学改变，采用放射免疫分析技术检测盖髓后1周、2周、4周时牙髓组织骨钙素的含量。结果:与氢氧化钙、胶原和牛血清白蛋白盖髓比较，用重组人骨形成蛋白-2复合胶原I盖髓，能形成完整的管样牙本质桥，牙髓组织骨钙素的含量显著多于正常牙髓。结论:重组人骨形成蛋白-2复合胶原I盖髓可诱导形成完整的牙本质桥，显著增加牙髓组织骨钙素的含量。重组人骨形成蛋白-2复合胶原I作为生物活性盖髓剂，起到良好的活髓保存效果，值得临床推广应用。     

低浓度β-甘油磷酸钠培养牙髓干细胞向成牙本质细胞分化及相关因子的表达

背景:以往研究用来诱导成牙本质细胞分化的培养基普遍借鉴成骨细胞的诱导浓度,其他浓度的诱导情况尚无相关研究。目的:观察在低浓度β-甘油磷酸钠条件培养下,牙髓干细胞向成牙本质细胞分化过程中,牙本质基质蛋白1、牙本质涎蛋白和细胞外基质磷酸糖蛋白的表达情况。方法:分离培养人牙髓干细胞,用不同浓度的诱导液诱导牙髓干细胞分别向脂肪细胞,成骨细胞分化,证实其多向分化能力。用5 mmol/Lβ-甘油磷酸钠诱导培养牙髓干细胞向牙本质细胞分化,分别在培养第7,14,21,28天提取各组细胞RNA,反转录PCR检测牙本质基质蛋白1、牙本质涎蛋白和细胞外基质磷酸糖蛋白的表达情况;诱导牙髓干细胞向成牙本质细胞分化形成的矿化结节用Alizarin Red S法检测。结果与结论:人牙髓干细胞经过诱导可证实其成功分化为脂肪细胞和成骨细胞;反转录PCR结果显示,5 mmol/Lβ-甘油磷酸钠培养牙髓干细胞在培养7,14,21 d,各组细胞牙本质基质蛋白1、牙本质涎蛋白的mR NA表达增加,伴随细胞外基质磷酸糖蛋白表达下调;培养至28 d,行矿化结节检测发现牙髓干细胞向成牙本质细胞成功矿化,可见红染的矿化结节。结果证实,5 mmol/Lβ-甘油磷酸钠诱导培养的牙髓干细胞可成功分化为成牙本质细胞,并下调细胞外基质磷酸糖蛋白mR NA的表达,同时上调牙本质基质蛋白1及牙本质涎蛋白mR NA的表达。     

牙髓干细胞的研究进展及应用(综述)

牙齿的再生现在已是国内外专家、学者研究的热门课题,而牙髓干细胞作为牙齿再生的主要种子细胞,对其进行研究有着重要的意义。笔者就牙髓干细胞的研究进展及应用做一综述。牙髓干细胞的提出Gouble用β-磷酸甘油诱导牙髓细胞分化成牙本质细胞,证实了牙髓组织中成牙本质细胞前体的存在。2000年Gronthos利用collagenase type I和dispase消化法,将人的第三磨牙牙髓组织制成单细胞悬液并培养,获得了具有细胞克隆能力和高度增殖能力的细胞,与骨髓基质干细胞相比较有许多相似的地方,将其与羟基磷灰石-磷酸三钙粉末混合并接种于免疫耐受小鼠背部皮下形成了牙髓牙本质复合体样结构,因此提出了牙髓干细胞(dental pulp stem cell,DPSCs)的概念。     

骨形态发生蛋白2基因转染犬牙髓细胞

背景：研究发现骨形态发生蛋白2具有诱导间充质细胞向成骨细胞表型分化以及诱导新骨及修复性牙本质形成的能力。 目的：通过对细胞超微结构的分析,探讨骨形态发生蛋白2基因转染的犬牙髓细胞,向成牙本质细胞转化的过程。 方法：将培养的性状稳定的第4代犬牙髓细胞分为3组：基因转染组转染pEGFP-N1-BMP-2基因,空白载体转染组转染EGFP-N1空白荧光载体,未转染组不转染。 结果与结论：成功构建pEGFP-N1-BMP-2真核表达质粒,并成功将其转染犬牙髓细胞, pEGFP-N1-BMP-2质粒转染促进犬牙髓细胞分泌骨形态发生蛋白2。pEGFP-N1-BMP-2质粒转染有促进牙髓细胞碱性磷酸酶活性的作用。透射电镜观察结果显示：转染后的犬牙髓细胞具有成牙本质细胞的形态特点。说明pEGFP-N1-BMP-2 真核表达质粒转染后的牙髓细胞,具有成牙本质细胞的特征。     

大鼠三叉神经脊束核尾侧亚核和脊髓向丘脑和臂旁核的谷氨酸能投射

本研究用荧光金逆行追踪与免疫荧光组比技术相结合的方法,对大鼠三叉神经脊束核尾侧亚核和脊髓向丘脑和臂旁核的谷氨酸能投射进行了观察。磷酸激活的谷氨酸胺酶（PAG）是谷氨酸能神经元的特异性标识物。PAG样阳性胞体主要位于三叉神经脊束核尾侧亚核和颈髓背角的Ⅰ层,少量PAG样阳性胞体也见于它们的Ⅱ层外侧部及外侧网状核。将荧光金注入丘脑腹基底复合体后．荧光金逆标神经元主要见于对侧三叉神经脊束核尾侧亚核和颈髓背角的Ⅰ层及外侧网状核;将荧光金注入臂旁核后,荧光金逆标神经元也主要见于对侧三叉神经脊束核尾侧亚核和颈髓背角的Ⅰ层及外侧网状核。三叉神经脊束核尾侧亚核向丘脑腹基底复合体投射神经元的12．4％,向臂旁核投射神经元的13．2％呈PAG样阳性;颈髓背角浅层向丘脑瓜基底复合体投射神经元的12．7％,向臂旁核投射神经元的14．3％呈PAG样阳性。向丘脑腹基底复合体和臂旁核投射的PAG／荧光金双标神经元分别占三叉神经脊束核尾侧亚核浅层内PAG样阳性神经元总数的13％和24．6％,向丘脑腹基底复合体和臂旁核投射的PAG／荧光金双标神经元分别占颈髓背角浅层内PAG样阳性神经元总数的11．6％和30．1％。外侧网状核内的部分PAG样阳性神经元也向丘脑腹基底复合体或臂旁核投射。Ⅰ层内的双?     

Expression of galanin receptor-1 (GALR1) in the rat trigeminal ganglia and molar teeth

The expression of galanin receptor-1 (GALR1) was investigated in the rat trigeminal ganglion by using immunocytochemistry and in situ hybridization. In addition, the regional distribution of GALR1-immunoreactive pulpal nerves and their ultrastructure were examined in the molar teeth. In the trigeminal ganglion, the immunoreactivity for GALR1 was recognizable in about 30% of the total number of neurons. Most of the cell bodies were small to medium in size. Analysis of serially cut sections alternately stained with GALR1 and galanin antisera demonstrated that some GALR1-positive cells displayed immunoreactivity for galanin. In situ hybridization analysis, expression of GALR1 mRNA was detected in trigeminal ganglion cells. The cell size distribution was similar to that of GALR1-immunoreactive cells. In the dental pulp, a small number of nerve fibers displayed immunoreactivity for GALR1. The labeled fibers formed terminal arbors in the coronal pulp around and within the odontoblast cell layer, but never penetrated into the predentin and dentin. Ultrastructurally, GALR1 immunoreactivity in the dental pulp was confined to the axoplasm of unmyelinated nerve fibers. The present study provided new evidence that unmyelinated primary afferents innervating dental pulp possessed galanin receptor, and suggests the existence of nociceptive primary afferents functioning as autocrine cells.     

Quantitative analysis of the calcium and phosphorus content of developing and permanent human teeth.

It was the aim of this study to investigate the distribution of Ca, P and C in predentin, dentin and enamel in human tooth buds and permanent teeth by EDX element analysis. The mandible of a 16-week-old human fetus containing eight mineralizing tooth buds and three human permanent molars were fixed in formaldehyde and embedded in Technovit 9100. Serial sections of 80 microm thickness of the mandible were cut in the frontal-dorsal direction, and polarized light micrographs were taken of these sections. The permanent teeth were cut in mesio-distal direction. The sections were investigated with scanning electron microscopy and EDX element analysis with a Philips XL 30 FEG scanning microscope and an EDAX energy-dispersive X-ray system using spot measurements, EDX line-scans and element mapping. Quantitative measurements were made in predentin, mineralizing dentin adjacent to predentin, mature dentin, mineralizing enamel and young enamel of developing teeth and mature enamel of permanent teeth. In developing teeth the Ca and P content increased rapidly from outer predentin towards mineralizing dentin. In enamel prisms of developing teeth the Ca and P content increased linearly from the surface towards the enamel-dentin junction. In permanent teeth only a small layer of predentin was found. The Ca and P content in enamel and circumpulpal dentin of permanent teeth was higher than in developing teeth. The Ca/P ratio differed between predentin and dentin areas reflecting different calcium phosphate compositions, but it was the same in mineralizing and young enamel. The differences in the distribution of Ca and P reflect different mineralizing patterns of the enamel and dentin matrices.     

Combination of aligned PLGA/Gelatin electrospun sheets,native dental pulp extracellular matrix and treated dentin matrix as substrates for tooth root regeneration

In tissue engineering, scaffold materials provide effective structural support to promote the repair of damaged tissues or organs through simulating the extracellular matrix (ECM) microenvironments for stem cells. This study hypothesized that simulating the ECM microenvironments of periodontium and dental pulp/dentin complexes would contribute to the regeneration of tooth root. Here, aligned PLGA/Gelatin electrospun sheet (APES), treated dentin matrix (TDM) and native dental pulp extracellular matrix (DPEM) were fabricated and combined into APES/TDM and DPEM/TDM for periodontium and dental pulp regeneration, respectively. This study firstly examined the physicochemical properties and biocompatibilities of both APES and DPEM in vitro, and further investigated the degradation of APES and revascularization of DPEM in vivo. Then, the potency of APES/TDM and DPEM/TDM in odontogenic induction was evaluated via co-culture with dental stem cells. Finally, we verified the periodontium and dental pulp/dentin complex regeneration in the jaw of miniature swine. Results showed that APES possessed aligned fiber orientation which guided cell proliferation while DPEM preserved the intrinsic fiber structure and ECM proteins. Importantly, both APES/TDM and DPEM/TDM facilitated the odontogenic differentiation of dental stem cells in vitro. Seeded with stem cells, the sandwich composites (APES/TDM/DPEM) generated tooth root-like tissues after being transplanted in porcine jaws for 12 w. In dental pulp/dentin complex-like tissues, columnar odontoblasts-like layer arranged along the interface between newly-formed predentin matrix and dental pulp-like tissues in which blood vessels could be found; in periodontium complex-like tissues, cellular cementum and periodontal ligament (PDL)-like tissues were generated on the TDM surface. Thus, above results suggest that APES and DPEM exhibiting appropriate physicochemical properties and well biocompatibilities, in accompany with TDM, could make up an ECM microenvironment for tooth root regeneration, which also offers a strategy for complex tissue or organ regeneration.     

Comparative Qualitative and Quantitative Assessment of Biomineralization of Tooth Development in Man and Zebrafish (Danio rerio)

It was the aim of this study to investigate the distribution of Ca, P, and C in predentin, mineralizing dentin, and mature dentin of human tooth buds and compare these results with those of zebrafish (Danio rerio) teeth using energy dispersive X‐ray analysis (EDX) element analysis. The mandible of a 16‐week‐old human fetus containing 6 mineralizing tooth buds and three complete heads of zebrafish were fixed in formaldehyde and embedded in Technovit 9100. Serial sections of 80‐μm thickness were cut in frontal‐dorsal direction, and from these sections, polarized light micrographs were taken. The sections with tooth buds were then investigated with scanning electron microscopy, and EDX element analysis was performed with a Philips XL 30 FEG scanning microscope and an EDAX energy‐dispersive X‐ray system using spot measurements, EDX line‐scans and element mapping. Quantitative measurements were made in predentin, mineralizing dentin adjacent to predentin, and mature dentin. The Ca and P content increased rapidly from outer predentin toward mineralizing dentin in human tooth buds and in zebrafish teeth. The Ca/P ratio was different for predentin and dentin areas, reflecting different calcium phosphate compositions in predentin and fully mineralized dentin. Because of the similarities between human tooth buds and zebrafish teeth, it can be concluded that the zebrafish tooth development may be an excellent model for studying biomineralization processes during odontogenesis. Anat Rec, 291:571–576, 2008. © 2008 Wiley‐Liss, Inc.     

Effects of alginate hydrogels and TGF-beta 1 on human dental pulp repair in vitro

The objective of this study was to investigate the use of alginate hydrogels to present either exogenous or endogenous transforming growth factor (TGF)-beta 1 to the dentin-pulp complex to signal reparative processes. Hydrogels were prepared, applied to cultured human tooth slices and the effects on tertiary dentinogenesis examined histologically. Both TGF-beta 1-containing and acid-treated alginate hydrogels, but not untreated hydrogels, upregulated dentin matrix secretion and induced odontoblast-like cell differentiation with subsequent secretion of regular tubular dentin matrix on cut pulpal surfaces. It is concluded that TGF-beta 1 can signal both induction of odontoblast-like cell differentiation and upregulation of their matrix secretion in the human dentin-pulp complex. Alginate hydrogels provide an appropriate matrix in which dental regeneration can take place and may also be useful for delivery of growth factors, including TGF-beta s, to enhance the natural regenerative capacity of the dental pulp.     

Immunohistochemical localization of endothelin-like immunoreactivity in human tooth germ and mature dental pulp

Summary The distribution in oral tissues of endothelin, a multifunctional peptide originally identified within endothelial cells, and subsequently in some epithelial cells, neurons and neuroendocrine cells, has not been investigated yet. We have studied the localization of endothelin-like immunoreactivity in human tooth germ and mature dental pulp by immunohistochemical techniques. Such immunoreactivity was detected only within endothelial cells in both mature dental pulp and developing tooth. Arteries and veins of various sizes as well as small thin vessels displayed endothelin-like immunoreactivity. In the tooth germ, the cells of the enamel organ or the precursors of the odontoblasts were found unreactive. In the mature pulp, no cells of the stroma or nerves displayed endothelin-like immunoreactivity. These findings suggest that vascular endothelium may be the only source of endothelin in human dental tissues. It is tentatively proposed that endothelin released in mature tooth pulp may participate in the regulation of the pulpal blood flow. Although the possible role of endothelin in developing tissues is far from being clear, the mitogenic effects and the proto-oncogenes expression induced by endothelin in some cells raise the possibility that this peptide might also play a role during tooth development.     

Collagen fibrils in the odontoblast layer of the rat incisor by scanning electron microscopy using the maceration method

Background: There is not universal agreement on the existence of the extracellular pathway from the pulp along the odontoblast layer to the predentin. Method: To confirm this pathway, the architecture of collagen fibrils in the rat incisor dentin and pulp, especially in the odontoblast layer of the lateral (periodontal ligament) sides of the tooth, was demonstrated in the present investigation using scanning electron microscopy of the maceration method for collagen networks. Results: Numerous collagen bundles were observed in the odontoblast layer in the mature odontoblast region which, except for the young odontoblast region, comprises the major portion of the incisor. The collagen bundles went from the pulp, through the odontoblast layer, and were woven into the collagen network of the predentin. The meshwork structure was composed of fine secondary fibrils among these collagen bundles. The surface of the predentin contained many oval-shaped holes which were surrounded by collagen fibrils. Fracturing the dentin longitudinally relative to the dentinal tubules revealed that the arrangement of the collagen fibrils at the surface of the tubules was either circular or oblique. In the young odontoblast region, i.e., the thin portion from the apical end of the incisor where the mineralization of the dentin does not occur and where the height of the odontoblasts was less than 30 μm, many thick bundles composed of thick collagen fibrils ran straight from the pulp to the predentin through the odontoblast layer and fanned out into the collagen network of the predentin. These thick bundles might correspond to the so-called “von Korff fibers.” The distribution of collagen fibrils in the pulp was random except on the surface of the blood vessels where the fibrils comprised two sheets of collagen: the inner sheet which coursed longitudinally to the long axis of the vessel, and the outer sheet which ran transversely. Conclusion: It was considered that the fluid in the pulp could flow to the predentin along the collagen fibrils through the tight junction between the odontoblasts. © 1994 Wiley-Liss, Inc.     

Effects of Alginate Hydrogels and TGF-β1 on Human Dental Pulp Repair In Vitro

The objective of this study was to investigate the use of alginate hydrogels to present either exogenous or endogenous transforming growth factor (TGF)- &#103 1 to the dentin-pulp complex to signal reparative processes. Hydrogels were prepared, applied to cultured human tooth slices and the effects on tertiary dentinogenesis examined histologically. Both TGF- &#103 1-containing and acid-treated alginate hydrogels, but not untreated hydrogels, upregulated dentin matrix secretion and induced odontoblast-like cell differentiation with subsequent secretion of regular tubular dentin matrix on cut pulpal surfaces. It is concluded that TGF- &#103 1 can signal both induction of odontoblast-like cell differentiation and upregulation of their matrix secretion in the human dentin-pulp complex. Alginate hydrogels provide an appropriate matrix in which dental regeneration can take place and may also be useful for delivery of growth factors, including TGF- &#103 s, to enhance the natural regenerative capacity of the dental pulp.