Effect of fluoride on protein and collagen biosynthesis in rabbit dental pulp in vitro

abstract – The time course for incorporation of ¹⁴C-proline into various fractions of rabbit dental pulp in vitro has been measured. In the TCA-soluble precursor pool a steady state level of activity was indicated upon incubation after 3 h, whereas incorporation into protien and ¹⁴C-hydroxyproling, i.e. collagen formation, increased lineraly for 9 h, leaving off upon further incubation. A lag period of about 3 h was indicated for the appearance of high molecular weight ¹⁴C-activity, including ¹⁴C-hydroxyproline, in the medium, increasing linearly from 3 h to the end of the incubaition period (22 H). in this system, fluoride exhibited a dose-dependent inhibitory effect. at 5.3 mM fluoride the uptake of ¹⁴C-proline into the TCA-soluble pool was inhibited by about 50%, and the incorporation into protein and the subsequent conversion to hydroxyproline by about 90 and 60%, respectively. Release of collogen, i.e. ¹⁴C-hydrozyproline-contaning material, seemed to be the process most sensitive to fluoride; it was inhibited by about 50% at the lowest concertration (1.3 mM) tested.     

Effect of some heavy metals on protein and collagen biosynthesis in rabbit dental pulp in vitro

abstract – Cadmium, zinc, copper, and mercury, being components of dental alloys, have been tested at concentrations from 20 to 120 μM for their effect on the incorporation of ¹⁴C-proline into various fractions of rabbit dental pulp incubated in vitro . With cadmium and zinc an increased amount of ¹⁴C-activity was recovered from the TCA-soluble pool, whereas the further incorporation into total protein and collagen, i.e. ¹⁴C-hydroxypraline, was markedly inhibited. Copper and mercury, however, reduced the amount of label in the TCA-soluble fraction. Both metals exhibited a dose-dependent inhibitory effect on the hydroxylation step in collagen synthesis, whereas for mercury only, an inhibition of the general protein synthesis was indicated. With all four metals the proportion of ¹⁴C-labeled protein and hydroxyproline recovered from the medium was reduced, the effect being most prominent with cadmium and zinc.     

PH and the effect of fluoride and zinc on protein and collagen biosynthesis in rabbit dental pulp in vitro.

The effect of pH in the range 6.6-8.2 on the incorporation of 14C-proline into the rabbit dental pulp incubated in vitro has been studied. The amount of label in the cold TCA-soluble pool, total protein, and hydroxyproline (i.e. collagen) of the pulp tissue increased linearly with pH in all three fractions. A similar increase was found in the amount of labeled total protein and collagen recovered from the incubation medium. The results indicated that the uptake of 14C-proline into the TCA-soluble pool was the pH-sensitive step, whereas the incorporation into protein, formation of hydroxyproline, and the release of labeled macromolecules into the medium were not affected to any measurable degree by the ambient pH. In this system, zinc (60 micrometer) had less effect on the incorporation of 14C-proline into the different fractions of the pulp tissue at pH 6.6 as compared with pH 7.4, whereas with fluoride (1.3 mM) an increased inhibition of the uptake of 14C-proline into the TCA-soluble pool and the incorporation into total protein was found upon lowering the pH to 6.8. The inhibitory effect of zinc and fluoride on the release of labeled total protein and collagen into the incubation medium was not affected when the pH was lowered.     

Effect of fluoride on protein and collagen biosynthesis in rabbit dental pulp in vitro.

The time course for incorporation of KC-proline into various fractions of rabbit dental pulp in vitro has been measured. In the TCA-soluble precursor pool a steady state level of activity was indicated upon incubation after 3 h, whereas incorporation into protein and 14C-hydroxyproline, i.e. collagen formation, increased linearly for 9 h, leveling off upon further incubation. A lag period of about 3 h was indicated for the appearance of high molecular weight 14C-activity, including 14C-hydroxyproline, in the medium, increasing linearly from 3 h to the end of the incubation period (22 h). In this system, fluoride exhibited a dose-dependent inhibitory effect. At 5.3 mM fluoride the uptake of 14C-proline into the TCA-soluble pool was inhibited by about 50%, and the incorporation into protein and the subsequent conversion to hydroxyproline by about 90 and 60%, respectively. Release of collagen, i.e. 14C-hydroxyproline-containing material, seemed to be the process most sensitive to fluoride; it was inhibited by about 50% at the lowest concentration (1.3 mM) tested.     

高频电流处理家兔牙髓的初步实验研究

目的 验证高频电流处理家兔牙髓的效果。方法 选取约3月龄的健康实验家兔55只,共220颗切牙,分成8个实验组和2个对照组。实验组暴露牙髓后分别采用5、10、15、20W输出功率下的高频电流处理家兔牙髓,对照组牙髓不作任何处理。实验完成后,即刻将两组家兔切牙连同牙槽骨一同切下,制成联合切片,通过组织学方法 将实验组和对照组的切片进行对比,观察实验组处理后的牙髓组织、根管壁及根尖周组织的变化,比较各实验组中各组织损伤的程度。结果 5、10W组高频电流处理后的牙髓,主要是出现局部点灶状的变性坏死区,变性坏死区的改变以凝固性坏死改变为主。15、20W实验组高频电流可令根髓出现变性坏死及断裂现象,可达到药物失活牙髓的组织学变化指标。各实验组根管壁均无异常变化,根尖周未发现副损伤。结论 采用20W输出功率下的高频电流处理兔牙髓,可使牙髓失活,且未发现根管壁和根尖周的副损伤。?     

血小板衍化牛长因子对人牙髓成纤维细胞DNA和胶原蛋白合成的影响

目的：观察血小板衍化生长因子（platelet-derived growth factor,PDGF）对人牙髓成纤维细胞（human dental pulp fibroblast,HDPF）DNA合成和胶原蛋白合成的影响。方法：应用^3H-TdR和^3H-脯氨酸掺入方法，观察PDGF对体外培养的HDPF DNA合成和胶原蛋白合成的情况。结果：20-60ng/mL PDGF可明显促进HDPF DNA的合成，40ng/mL浓度使细胞DNA合成在36h达最大值；对细胞的胶原蛋白合成无明显促进作用。结论：PDGF明显促进HDPF DNA的合成，可能在治疗牙髓病中起重要作用。     

F^—对牙髓细胞亚细胞结构的体外影响

目的：观察不同浓度Ｆ－对牙髓细胞亚细胞结构的直接影响。方法：鼠牙髓细胞体外培养，透射电镜观察。结果：１０ｍｇ／Ｌ、２５ｍｇ／ＬＦ－组随Ｆ－浓度增加，细胞内粗面内质网逐渐扩张，游离核蛋白体减少；５０ｍｇ／ＬＦ－组，细胞内部结构变化明显，染色质分布不均，细胞器萎缩，膜结构模糊。结论：高浓度Ｆ－（５０ｍｇ／Ｌ）对牙髓细胞结构、代谢和分裂增殖活动有抑制性影响     

年轻恒牙牙根发育过程中牙髓中Ⅲ型胶原的表达

目的：研究牙根发育不同阶段的人年轻恒牙牙髓中Ⅲ型胶原的表达变化：方法：根据牙根的发育情况，分为牙根刚开始发育、牙根发育中和根尖闭合共3组：采用SP免疫组化法。对各组牙髓标本的石蜡切片进行Ⅲ型胶原的免疫组化染色：结果：图像分析显示、3个组的冠髓中心染色强度之间有非常显著性差异，以牙根刚开始发育时染色最强，根尖闭合时染色最弱：在牙根刚开始发育组、牙髓中心区染色强于牙髓外层。结论：随着年轻恒牙牙根的逐渐发育，Ⅲ型胶原在牙髓中的表达逐渐减弱，因而可能参与牙根的发育。     

F^—对牙髓细胞影响的扫描电镜观察

目的：利用扫描电镜、光镜观察不同浓度Ｆ－对鼠牙髓细胞形态和表面结构的直接影响。方法：大鼠牙髓细胞培养，相差显微镜、扫描电镜观察。结果：１０ｍｇ／Ｌ、２５ｍｇ／Ｌ、５０ｍｇ／ＬＦ－使细胞表面嵴和囊泡样结构减少。浓度越高，改变越明显。结论：高浓度Ｆ－对牙髓细胞表面结构有抑制性伤害，局部用氟化物直接作用牙本质－牙髓复合体应控制Ｆ－浓度     

Effect of some heavy metals on protein and collagen biosynthesis in rabbit dental pulp in vitro.

Cadmium, zinc, copper, and mercury, being components of dental alloys, have been tests at concentrations from 20 to 120 micronM for their effect on the incorporation of 14C-proline into various fractions of rabbit dental pulp incubated in vitro. With cadmium and zinc and increased amount of 14C-activity was recovered from the TCA-soluble pool, whereas the further incorporation into total protein and collagen, i.e. 14C-hydroxyproline, was markedly inhivited. Copper and mercury, however, reduced the amount of label in the TCA-soluble fraction. Both metals exhibited a dose-dependent inhibitory effect on the hydroxylation step in collagen synthesis, whereas for mercury only, an inhibition of the general protein synthesis was indicated. With all four metals the proportion of 14C-labeled protein and hydroxyproline recovered from the medium was reduced, the effect being most prominent with cadmium and zinc.     

内毒素血症致牙髓组织病理学改变的实验研究

目的：观察家兔病理性氧供依赖性（ｐａｔｈｏｌｏｇｉｃａｌｏｘｙｇｅｎｓｕｐｐｌｙｄｅｐｅｎｄｅｎｃｅ，ＰＯＳＤ）模型中各组织，特别是牙髓组织的病理学改变，以及抗ｒｈＴＮＦ单克隆抗体对实验动物是否有保护作用。方法：２８只大耳白兔随机分为对照、内毒素、抗体保护和无关抗体４组，每组７只。以小剂量大肠杆菌内毒素分次注射复制ＰＯＳＤ模型。ＥＬＩＳＡ测定血中ＴＮＦ浓度。采用组织学、组织化学和免疫组化法评价牙髓形态学特征。结果：①内毒素组与无关抗体组的牙髓组织有充血、水肿、渗出、弥漫性血管内凝血等病理变化，而对照组和抗体保护组形态正常；②内毒素组的ＴＮＦ浓度明显高于对照组（Ｐ＜０．０１），抗体保护组的ＴＮＦ浓度则明显低于无关抗体组（Ｐ＜０．０１）。结论：内毒素血症可致牙髓组织的病理学改变，抗ｒｈＴＮＦ单抗可以阻断其病变，具有临床应用前景。     

TGF-β3作用下人牙髓细胞内Smad2、Smad3蛋白表达水平的变化

目的：探讨TGF—B3对人牙髓细胞内Smad2、Smad3蛋白表达的调控作用和差异。方法：取生长良好的第5代人牙髓细胞，分为实验组和对照组，实验组用5ng／mL TGF—β3刺激并培养6、12、24、48h，对照组不予刺激。提取各组总蛋白质，分别用Smad2、Smad3抗体进行Western印迹杂交。结果：与对照组相比，Smad3在TGF—β3刺激后6h、12h，其蛋白表达量无明显变化，而在刺激后24h表达量明显下降，刺激48h后表达十分微弱；Smad2在对照组以及刺激后各时间组，其蛋白表达量无显著变化。结论：人牙髓细胞内存在Smad2、Smad3蛋白的表达，TGF—β3对二者表达的调控方式有所不同，刺激后期Smad3表达水平的下调可能与TGF-β3负反馈调节自身的信号有关。     

Behaviour of rat‐cultured dental pulp cells in three‐dimensional collagen type‐1 gel in vitro and in vivo

The purpose of this study was to investigate the growth and differentiation potential of dental pulp cells (DPCs) in three‐dimensional (3‐D) collagen type‐1 scaffold in vitro and in vivo. Third passage DPCs were cultured in a 3‐D collagen and expression of both bone‐ or dentin‐related mRNA (alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteopontin (OPN)) and morphological changes evaluated in vitro. In the in vivo study, two types of grafts were transplanted into the rectus abdominus muscles of rats and harvested after 7 days: DPCs in α‐minimal essential medium and DPCs mixed with a collagen gel. ALP, BSP and OPN were used as primary antibodies for immunohistochemical study. Histological and immunohistochemical results showed that DPCs in collagen gel were spindle shaped and showed significantly greater expression of ALP, BSP and OPN in vitro than the controls. Transplanted DPCs in collagen type‐1 gel showed greater positive immunoreactivity for ALP, BSP and OPN than the controls. It was concluded that the collagen gel scaffold encouraged the differentiation of DPCs into osteoblastic cells.     

体外持续培养对人牙髓细胞分化能力的影响

目的：探讨人牙髓细胞在体外持续培养时，其分化能力的变化趋势。方法：体外持续培养人牙髓细胞，检测不同代次细胞的碱性磷酸酶活性、钙化能力及Ⅰ型胶原表达，并对比分析。结果：随着体外培养细胞代次的增多，在同等刺激分化条件下，人牙髓细胞的碱性磷酸酶活性整体呈下降趋势，钙化结节的数目和面积逐渐减少，Ⅰ型胶原表达合成逐渐减少。结论：人牙髓细胞在体外持续培养时，分化能力逐渐下降，可能与其含有的未分化细胞（人牙髓干细胞）数目逐渐减少有直接关系。     

Immunolocalization of bone extracellular matrix proteins (type I collagen,osteonectin and bone sialoprotein) in human dental pulp and cultured pulp cells

AIM: To simultaneously analyse the expression of type I collagen, osteonectin and bone sialoprotein (BSP) in human dental pulp of different ages. METHODOLOGY: Cultured dental pulp fibroblasts (FP1 cell line), pulps from dental germs with incomplete root formation (n = 4) and pulps of erupted teeth with total root formation (n = 4) were used. Bone proteins were searched by immunohistochemistry and immunofluorescence using polyclonal antibodies and compared among the three groups assessed. RESULTS: Immunohistochemistry detected the three proteins in dental pulp tissue, as it labelled extracellular matrix, predentine and odontoblasts. The BSP label was weaker, when compared to both type I collagen and osteonectin. The presence of type I collagen was more evident in pulps from erupted teeth, when compared to germ dental pulps. On the other hand, a strong expression of osteonectin in germ dental pulps was observed. CONCLUSIONS: Regardless of the degree of maturation, dental pulps present type I collagen, osteonectin and BSP in the extracellular matrix (ECM) and in the odontoblastic layer. Thus, the results suggest that these proteins are related to the production and mineralization of dentine.     

Effects of local anesthetics on the respiratory activity in vitro of cells in the dental pulp

abstract — The respiratory activity of isolated dental pulps from rat incisors was studied using a Gilson respirometer. The activity was compared with activities after administration of varying concentrations of commercial standard solutions of lidocaine with and without adrenaline and prilocaine with felypressin. Above a 2.5% concentration of the standard solution added to the respiratory medium a significant inhibition was registered.     

Structural studies on calcium fluoride formation and uptake of fluoride in surface enamel in vitro

abstract — The structural changes in human enamel during exposure to an acetate buffer, pH 4, containing 150 parts/10⁶ fluoride have been studied using scanning electron microscopy and polarized light microscopy. After exposure for 2 h the enamel surface was covered by a fine-granular layer of calcium fluoride. The underlying enamel was highly eroded with an increased pore volume in the outer layer. Following an equilibration period of 3 months the uptake of fluoride in the apatite lattice had resulted in a highly mineralized, 100-μm-thick surface layer rich in fluoride covering a subsurface porous zone with an unchanged fluoride content. The deeply located, caries-like porous zone may have provided calcium and phosphate for saturation of the liquid     

牙本质黏结剂诱导人牙髓细胞凋亡的体外研究

目的:比较不同固化时间牙本质黏结剂对牙髓细胞凋亡的诱导作用。方法:体外培养人牙髓细胞,与黏结剂模型共同孵育24h,经PI染色,采用流式细胞记数仪检测凋亡细胞数目。结果:经t检验,与对照组比较,光照0s、10s和20s对牙髓细胞凋亡都有不同程度的影响(P<0.05),光照时间延长可减少对牙髓细胞凋亡的诱导。结论:牙本质黏结剂可诱导牙髓细胞凋亡,适当延长固化时间可以减轻黏结剂对牙髓的刺激。