牙龈卟啉单胞菌对牙髓成纤维细胞胞内感染的研究

目的研究牙龈卟啉单胞菌活菌细胞内感染牙髓成纤维细胞的体外模型。方法将牙龈卟啉单胞菌和牙髓细胞以100∶1比例共同孵育0.5、1.0、2.0 h,倒置显微镜观察牙髓细胞形态。加入双抗和甲硝唑杀死胞外细菌,牙髓细胞用蒸馏水裂解后厌氧培养细胞裂解液,观察活细菌是否进入牙髓细胞胞内。将牙龈卟啉单胞菌和牙髓细胞以多重感染比（MOI）100、50共同孵育1.0 h,采用MTT法检测感染牙龈卟啉单胞菌后牙髓细胞存活率。结果倒置显微镜下显示,被胞内感染的牙髓细胞培养2.0 h未见胞膜破裂,形态完整。采用双抗能彻底杀灭胞外培养基中的细菌而对胞内细菌无影响,共同孵育1.0 h和2.0 h活细菌能进入牙髓细胞。MTT法显示细菌感染后牙髓细胞仍有一定存活率,其存活率分别是MOI 100组74.43%、MOI 50组99.07%。结论成功建立了牙龈卟啉单胞菌对牙髓成纤维细胞胞内感染的模型。     

4种牙本质粘结剂对牙髓成纤维细胞毒性作用的比较

目的:对比研究4种第七代牙本质粘结剂对人牙髓成纤维细胞的毒性作用,初步探讨其生物安全性。方法:组织块培养法原代培养人牙髓成纤维细胞,免疫组织化学染色SP法鉴定细胞来源,采用四甲基偶氮唑盐比色法和浸提液法,双盲观察4种新一代牙本质粘结剂(G-Bond、i-Bond、xeno V、Clearfil S3Bond)的不同体积分数浸提液(12.5%、25%、50%、100%)作用不同时间(24、48、72 h)对人牙髓成纤维细胞的毒性作用。结果:4种牙本质粘结剂不同体积分数浸提液的作用下,人牙髓成纤维细胞形态均发生不同程度的变化。24、48 h时,xeno V细胞毒性影响较小,i-Bond细胞毒性影响较大,G-Bond与Clearfil S3 Bond细胞毒性无明显差异;72 h时,4种牙本质粘结剂对细胞毒性影响无明显差异。结论:4种牙本质粘结剂毒性趋于0～2级,随着作用时间延长,细胞毒性无明显差异。     

碱性成纤维细胞生长因子对体外培养人牙髓细胞增殖和迁移的影响

目的研究碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）对体外培养人牙髓细胞增殖、迁移活性的影响。方法以常规组织块培养法体外获得人牙髓细胞,实验组分别加入终浓度为1.0、5.0、10.0和50.0ng/ml的bFGF,对照组仅加入等量的空白培养基,采用CCK-8法分别测定450nm下各组光吸收度A值,研究bFGF对牙髓细胞增殖活性的影响;应用Transwell小室培养法,研究bFGF对牙髓细胞迁移活性的影响。结果与对照组相比,bFGF在1.0～50.0ng/ml浓度下可促进牙髓细胞的增殖（P〈0.05）,bFGF最低显效浓度为1.0ng/ml,最佳显效浓度为10.0ng/ml。bFGF在1.0～50.0ng/ml浓度下诱导细胞迁移（P〈0.05）。结论 bFGF可诱导体外培养人牙髓细胞的增殖和迁移,在牙髓牙本质复合体修复中可能发挥重要作用。     

Modification of the proteoglycans of rat incisor dentin – predentin during in vivo fluorosis

Proteoglycans (PGs) were isolated from the dentin–predentin of fluorotic and control rat incisor teeth using demineralization in EDTA, followed by extraction with 4 M guanidinium chloride in the presence of protease inhibitors. Differences in the behaviour of fluorotic and control PG were evident during purification by anion exchange chromatography on Q-Sepharose and MONO-Q interfaced with fast protein liquid chromatography. The PG from fluorotic teeth exhibited a more anionic profile, due to changes in glycosaminoglycan characteristics, since no apparent differences were evident between the respective core proteins, both of which were 45 kDa. The constituent glycosaminoglycan chains of fluorotic dentin were of lower molecular size and showed the additional presence of dermatan sulfate and heparan sulfate by comparison to non-fluorotic controls, where only chondroitin-4-sulfate was detected.     

In vitro immunoexpression of extracellular matrix proteins in dental pulpal and gingival human fibroblasts

AIM: To compare the expression of extracellular matrix (ECM) components in human pulpal and gingival fibroblasts in vitro. METHODOLOGY: Cultured dental pulp fibroblasts and gingival mucosa fibroblasts were used. Tenascin (TN), fibronectin (FN), type I (col I) and III collagen (col III) and osteonectin (ONEC) were detected by immunofluorescence. Main morphological characteristics were also analysed by light microscopy (LM) and transmission electron microscopy. RESULTS: The results revealed different expression patterns of the proteins. TN and ONEC were only immunoexpressed by pulpal fibroblast cells, suggesting a role of these glycoproteins in formation of mineralized tissues. FN and col I were present in the cytoplasms of both cell types. No expression of col III was detected. Different morphological characteristics were visualized under LM, in which pulpal fibroblasts were spindle-shaped with a wide cytoplasm, while gingival fibroblast cells exhibited stellate/pyramidal configuration, with rounded nuclei. However, ultrastructurally, both cell lineages showed very well developed rough endoplasmatic reticulum and Golgi complex. CONCLUSIONS: Due to the immunodetection of TN and ONEC on pulpal fibroblasts, the present findings demonstrated that a pulpal fibroblast cell is similar to an osteoblastic cell rather than an undifferentiated mesenchymal cell, such as a gingival fibroblast cell. Functional differences between the two cell lines may then be suggested.     

FGF-2对体外培养人牙髓细胞FGFR和OPN表达活性的影响

目的:研究成纤维细胞生长因子-2(fibroblast growth factor-2,FGF-2)对体外培养的人牙髓细胞表面成纤维细胞生长因子受体(fibroblast growth factor receptor,FGFR)及骨桥蛋白(osteopontin,OPN)表达的影响。方法:以改良组织块培养法体外获得人牙髓细胞,采用Western blotting法检测不同浓度FGF-2作用下牙髓细胞FGFR的表达情况;应用Real Time-PCR法,检测不同浓度FGF-2作用下牙髓细胞OPN mRNA的表达。结果:与对照组相比,FGF-2在1～50 ng/mL浓度下均可促进牙髓细胞FGFR的表达(P<0.05),最佳显效浓度为10 ng/mL。FGF-2在1～50 ng/mL浓度下均可诱导牙髓细胞OPN mRNA表达(P<0.05),OPN mRNA的表达在10 ng/mL的FGF-2作用下达到高峰。结论:FGF-2可促进体外培养人牙髓细胞FGFR和OPN的表达,在牙髓牙本质复合体修复中可能发挥重要作用。     

碱性成纤维细胞生长因子对人牙髓细胞增殖和分化的作用

目的 探讨碱笥成纤维细胞生长因子（ｂａｓｉｃ ｆｉｂｒｏｂｌａｓｔ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ｂＦＧＦ）对培养人牙髓细胞增殖和分化的效应。方法 采用四唑盐法、^3Ｈ－ＴｄＲ掺入法、图象分析,检测重组人ｂＦＧＦ（ｈｂＦＧＦ）对细胞ＤＮＡ、胶原蛋白、纤维为连蛋白、碱笥磷酸酶、骨形成蛋白合成和凝集素表达的影响。结果 ｈｂＦＧＦ浓度在１～１０μｇ／Ｌ时显著促进细胞增殖,浓度为１～１００μｇ／Ｌ）,Ⅰ型胶原     

自酸蚀牙本质粘接剂对人牙髓成纤维细胞的毒性作用

目的比较和评价自酸蚀粘接剂XenoⅢ（XO）和Adper Prompt（AP）以及全酸蚀粘接剂Single bond2（SB）三者的细胞毒性大小。方法将3种牙本质粘接剂XO、AP和SB涂布于直径为5.0 mm、厚度为0.5 mm的牙本质圆片的两面,置于DMEM培养液中获得材料的浸提液,然后将培养液稀释成100.0%、50.0%、25.0%和12.5%四种体积分数。选用组织块法体外原代培养人牙髓成纤维细胞,并将不同体积分数的材料浸提液与第5代人牙髓成纤维细胞共同培养,通过MTT法评价材料24、72、120 h的细胞毒性。结果牙本质粘接系统XO、AP和SB在体外对人牙髓成纤维细胞均有一定程度的细胞毒性,两种自酸蚀粘接剂XO和AP的细胞毒性明显低于全酸蚀粘接剂SB,其差异有统计学意义（P<0.05）。结论自酸蚀牙本质粘接剂XO和AP的细胞毒性小于全酸蚀牙本质粘接剂SB。     

Calcium- and hydroxyapatite-binding properties of glucuronic acid-rich and iduronic acid-rich glycosaminoglycans and proteoglycans

This study describes the interaction of a small chondroitin sulphate proteoglycan and the glycosaminoglycans chondroitin 4-sulphate, dermatan sulphate and heparan sulphate with hydroxyapatite. All macromolecules possessed a high affinity, with the iduronic acid-rich dermatan sulphate and heparan sulphate displaying higher adsorption maxima than the glucuronic acid-rich chondroitin 4-sulphate. At similar concentrations, dermatan sulphate produced a 30% inhibition of hydroxyapatite-induced crystal growth, whilst chondroitin 4-sulphate yielded 50% inhibition. Estimation of the calcium binding capacity of these glycosaminoglycans using equilibrium dialysis indicated that chondroitin 4-sulphate bound five times more calcium than dermatan sulphate at a calcium concentration similar to that of serum. The data indicate a possible important role for chondroitin 4-sulphate in dentinogenesis where it is the dominant glycosaminoglycan, since it could act as a capture point for calcium ions during mineralisation, with the leucine-rich domain of its parent proteoglycan acting as anchor points to type I collagen.     

碱性成纤维细胞生长因子对体外培养人牙髓细胞迁移和碱性磷酸酶活性的影响

目的:研究碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对体外培养人牙髓细胞迁移和碱性磷酸酶(ALP)活性的影响。方法:以组织块培养法体外获得人牙髓细胞,采用Transwell培养法和ALP活性检测法,观察10 ng/mL bFGF对体外培养人牙髓细胞迁移和分化能力的影响。结果:bFGF可显著诱导体外培养牙髓细胞迁移,与对照组相比差异有统计学意义(P<0.05),并能抑制细胞ALP活性(P<0.05),随着培养时间延长,该抑制作用更加显著(P<0.05)。结论:bFGF能促进牙髓细胞迁移,抑制ALP活性,在牙本质牙髓复合体修复中可能发挥重要作用。     

Isolation, identification, and quantitation of glycosaminoglycans synthesized by human gingival fibroblasts in vitro

The glycosaminoglycans synthesized by diploid fibroblasts obtained from healthy human gingivae of three donors were isolated, identified, and quantified. Degradation with specific enzymes identified the glycosaminoglycans as hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparan sulfate; hyaluronic acid predominating. The distribution of the sulfated glycosaminoglycans in the cell layer and the medium was not the same. The cells contained mainly heparan sulfate (48.3%) and the medium mainly dermatan sulfate (47%).     

Donor variability in the proliferation of human dental pulp fibroblasts

Fibroblasts were isolated from human dental pulps of healthy third molars from 49 donors of ages ranging from 17 to 68. Significant variability was noted in the success of obtaining primary cultures from these pulps. Variability between the various cultures was also observed in the reliability of maintaining subcultures of the primary cultures as well as recovery from frozen stocks of established cell lines. Of the original 49 explant cultures studied, only three survived long-term passage and freezing. In addition to difficulties and variability in establishing cell lines, the human pulp fibroblasts also showed great variability in proliferative activity which could not be accounted for by donor age, source, or passage number. These findings highlight significant difficulties in establishing reliable human pulp fibroblast cultures and the need for great care in interpreting any in vitro data.     

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。     

冻融前后人牙髓等成纤维细胞生物学特征的比较

本研究对体外培养的第5代人牙髓牙周牙龈成纤维细胞进行了冷冻和复苏,三种细胞复苏的存活率分别为14%、19%和24%,复苏后细胞的生长曲线和倍增时间等生物学特征与冻存前基本一致,提示冻融后的人牙髓牙周牙龈成纤维细胞基本保持冻融前的生物学特征.     

Effects of interleukin-4 on proteoglycan accumulation in human gingival fibroblasts

In inflammatory gingival diseases, cytokines have been demonstrated to play critical roles by coordinating the stimulation of immunological and connective tissue cells. The activities of these cells, degrading and remodeling extracellular matrices, constitute the major pathological and repair processes. Thus, elucidating cellular and molecular events occurring in inflamed connective tissues is crucial for the understanding and treatment of inflammation. In order to test a hypothesis that proinflammatory cytokines affect metabolism of major extracellular matrix molecules, we studied metabolism of proteoglycans (PGs) by human gingival fibroblasts (HGF) under the influence of interleukin-4 (IL-4) as a model of gingivitis. HGF in cell culture were metabolically radiolabeled using [3H]glucosamine and [35S]sulfate in the presence or absence of IL-4, and the labeled PGs were analyzed by chromatographic techniques. The incorporation of 35S into PGs increased with IL-4 both in media and cell layer. At 100 ng/ml of IL-4, the increment of 35S incorporation over control culture was 16-39% (p<0.001) in media and 12-35% (p=0.01) in cell layer. The 35S-labeled macromolecules were PGs containing heparan sulfate (HS) and chondroitin sulfate (CS) chains. From the molecular weight and glycosaminoglycan composition analyses, versican and perlecan-type and biglycan and decorin-type were very likely to be the major PG constituents both in media and cell layer. IL-4 stimulated synthesis of versican and perlecan-type more potently than biglycan and decorin-type. With IL-4 treatment, the ratio of CSPG/HSPG decreased in media and increased in cell layer. This ratio suggested that syndecan family HSPGs were also present in HGF. In conclusion, IL-4 stimulated accumulation of CS/HSPGs in human gingival fibroblasts.     

Partial characterization of proteoglycans synthesized by human gingival epithelial cells in Culture

Proteoglycans (PGs) were extracted from the [³⁵S]-sulfate labelled medium and cell layer of proliferating human ginigival epithelial cells and anlyzed by ion exchanged and molecular sieve chromatorgraphy, and by SDS-PAGE. The majority of the incorporated radioactivity secreted into the medium eluted from a DEAE Sephacel ion exchange column as a single peak at 0.44 M NaCl with a small shoulder at 0.52 M NaCl. This material, when chromatographed on Sephartose CL-6B contained two spieces – a quantitatively major peak at K_av= 0.30 (M_t? 235 000 on SDS-PAGE) and a qantitatively major peak at K_av= 0.39. The major peak was sensitive to alkaline borohydride, shifting to K_av= 0.45, and nitrous acid degradtion, indicating the presence of heparan sulfate PG with glyscosaminoglycan chins with M_t? 26 000. The minor peak is chondroitin/dermatan sulfateP with glycpsminoglycan chains of M_t= 22 200 as indicted by sensitivity to alkaline borohydride (shifting to K_av= 0.48) and chondroitin ABC lyase digestion. The [³⁵S]-sulfate labelled material from the cell layer eluted in a broad peak between 0–0.50 M NaCL from DEAE Sephacel. Chromatography of this material on Sepharose CL-6B revealed the presence of three peaks at K_av=0.20, 0.31, and 0.75. The largest peak (K_av=0.20 and M_r? 245 000 on SDS-PAGE) shifted elution position to nitrous acid degradation. These results indicate that this peak contains heparan sulfate PG with glycosaminoglycan chains of M_t? 20000. Two peaks containing [³⁵S]-sulfate labelled glycosaminoglycan chains were detected by chromatography of the cell layer extract over Sepharose CL-6B with K_avs=0.42 (M_r? 30 500) and 0.75 (M_r? 5300). The larger peak was predominately chondroitin/dermatan glycosaminoglycan as indicated by susceptibility to chondroitin ABC lyase susceptibility to nitrous acid. These results indicate that cultured human gingival epithelial cells synthesize and secrete principally heparan sulfate PGs with small amounts of chondroitin/dermatan sulfate PGs. This work will serve as a basis for future studies designed to examine those factors involved in regulation of PG synthesis by these cells.     

Expression of fibrillins and tropoelastin by human gingival and periodontal ligament fibroblasts in vitro

The elastic system fibers consist of three different types, oxytalan, elaunin and elastic fibers, which differ in the relative content of microfibrils and elastin. In periodontal tissues, oxytalan fibers are known to be distributed in the periodontal ligament and gingiva, while elaunin and elastic fibers are present only in the gingiva. We examined the in vitro synthesis of microfibrils and elastin by human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (HPLF). The two kinds of HGF and HPLF were cultured in MEM containing 10% newborn calf serum for 30 days. Since fibrillin-1 and fibrillin-2 are the major components of microfibrils involved in elastogenesis, we investigated the synthesis of fibrillins and tropoelastin in the conditioned medium of HGF and HPLF. Western blot analysis revealed fibrillin-1 and fibrillin-2 to occur in the HGF and HPLF culture medium, HGF exhibiting a higher level of synthesis than HPLF. Tropoelastin, on the other hand, was detected only in the medium of HGF after day 24. In addition, analysis of RNA extracted from HGF and HPLF on day 30 showed that only HGF expressed mRNA encoding tropoelastin. Immunohistochemically, accumulation of tropoelastin in the perinuclear area was found only in HGF. These results show that HGF expressed microfibrils and elastin, while HPLF expressed only microfibrils for the experimental period, and suggest a biochemical basis for the different distribution of elastic system fibers of the gingiva and periodontal ligament in vivo.     

Isolation and characterization of dental pulp stem cells from a supernumerary tooth

Background: Dental pulp stem cells (DPSCs) were primarily derived from the pulp tissues of primary incisors and permanent third molar teeth, whereas no report to our knowledge has yet been documented on deriving DPSCs from the other tooth types. The aim of this study is to present a novel approach of harvesting stem cells from a supernumerary tooth (a mesiodens). Materials and methods: The pulp tissues from a mesiodens of a 20‐year‐old healthy male patient and the left lower deciduous canine of a healthy 10‐year‐old boy (the positive control) were extracted and cultured for DPSCs, which were examined with stem cells (Oct‐4, Nanog and Rex‐1) and differentiation (Osteonectin and Nestin) markers. Furthermore, DPSCs were directionally differentiated to osteogenic and adipogenic cell lineages. Results: Dental pulp stem cells derived from the mesiodens were capable of differentiating into adipogenic and osteogenic lineages. The mesioden’s DPSCs also expressed stem cell and differentiation markers, which suggested their stem cell origin and differentiation capability. All the aforementioned results for the mesiodens were consistent with those of the DPSCs derived from the positive control. Conclusion: We have demonstrated the feasibility of deriving DPSCs from a usually discarded tissue such as a supernumerary tooth.