凝胶样支架材料对三维培养的人牙髓干细胞增殖的影响

目的　评价牙髓组织工程中不同浓度的两种可注射性凝胶样支架材料对牙髓干细胞增殖的影响。方法：配制不同浓度的I型胶原、肽段水凝胶（Puramatrix）支架材料,将牙髓干细胞分别接种于各组支架材料上;CCK-8方法检测细胞增殖情况;活死细胞染色观察细胞数量及形态。 结果　接种在各浓度支架材料上的牙髓干细胞均生长良好,其中1%的胶原支架和0.25%的水凝胶支架对牙髓干细胞的增殖有明显的促进作用;活死细胞染色观察牙髓干细胞在各浓度的两种支架材料中均生长良好,细胞数量随培养时间的延长呈递增趋势;细胞充分伸展,呈纺锤型。     

五倍子水提取物对人牙髓细胞Ⅰ型胶原合成的影响

目的:观察五倍子水提取物对人牙髓细胞Ⅰ型胶原合成的影响。方法:取第6代体外培养的人牙髓细胞分别与终末浓度为5、10μg/m L的五倍子水提取物共同培养3 d后,采用免疫组化染色法观察人牙髓细胞中Ⅰ型胶原的表达情况,并用HPIAS-1000图像分析系统进行定量分析。结果:对照组和不同浓度五倍子水提取物作用的实验组人牙髓细胞中均有Ⅰ型胶原的表达,其免疫反应物质多定位于胞浆。人牙髓细胞经5、10μg/m L浓度的五倍子水提取物作用后,其Ⅰ型胶原的表达水平均明显增高,其中以10μg/m L组的增高程度最大,各组间两两相比差异均有统计学意义(P<0.05)。结论:5、10μg/m L五倍子水提取物均能明显促进人牙髓细胞中Ⅰ型胶原的表达;提示其具有促进牙髓细胞向成牙本质细胞分化的作用。     

辛伐他汀对大鼠成骨细胞增殖和分化功能的实验研究

目的 :研究辛伐他汀对大鼠成骨细胞增殖和分化功能的影响 ,探讨其刺激成骨的作用机制。方法 :体外培养4周龄大鼠骨髓基质细胞 ,在条件培养液诱导下 ,分化为成骨细胞。以不同浓度辛伐他汀 (0.062μmol/l ,0.125μmol/l,0.25μmol/l ,0.5μmol/l,1.0μmol/l)作用于成骨细胞 ,用MTT法测定细胞增殖情况 ;PNPP法测定细胞碱性磷酸酶 (ALP)活性 ;检测培养液中羟脯氨酸 (Hpro)含量反映细胞I型胶原的分泌情况 ;通过矿化结节计数反映细胞的矿化能力。结果 :辛伐他汀抑制细胞增殖 ,在10 -7mol/l浓度组与对照组比较有统计学意义 (P<0.05) ;各浓度组的ALP活性均增加 ,以0.5μmol/l对酶活性的影响最显著 (P<0.05) ;羟脯氨酸含量增加 ,以10-7mol/l浓度组与对照组比较有统计学意义 (P<0.05) ;辛伐他汀显著提高细胞的矿化能力 ,0.5μmol/l浓度作用最显著 ( +43.2 %,P<0.05)。结论 :辛伐他汀抑制细胞增殖 ,促进细胞ALP活性增加 ,I型胶原分泌增强 ,提高成骨细胞的矿化能力     

降钙素基因相关肽对人牙髓细胞的矿化影响

目的：分析降钙素基因相关肽（calcitonin gene-related peptide,CGRP)对人牙髓细胞促矿化功能的影响，探讨CGRP在牙髓炎症状态下表达量增多的生物学意义。方法：人牙髓细胞分实验组和对照组培养，实验组加入适量的CGRP。分别测定细胞的碱性磷酸酶活性、骨钙素含量、I型胶原mRNA表达量变化。结果：培养3、6、9、12、15d,5个时间点实验组比对照组细胞碱性磷酸酶活性增强、骨钙素含量增加，且有显著性差异（P＜0．01）。培养6d的实验组的细胞I型胶原mRNA表达比对照组明显高。结论：CGRP刺激牙髓细胞分化，促进矿化形成，CGRP表达增高将有助于修复性牙本质形成。     

牛血浆粘连蛋白对培养的人牙髓成纤维细胞FN和Ⅲ型胶原表达的影响

观察了不同浓度牛血浆纤维粘连蛋白（ＦＮ）对培养的人牙髓成纤维细胞Ⅲ型胶原、ＦＮ表达的影响；用图象分析仪对染色强弱进行相对定量分析，ＦＮ（１０、２０μｇ／ｍｌ）组的人牙髓细胞Ⅲ型胶原表达最强，ＦＮ（４０，８０μｇ／ｍｌ）组的人牙髓细胞ＦＮ表达最强，说明ＦＮ的不同浓度影响了培养的人牙髓成纤维细胞的功能，ＥＬＩＳＡ测定结果可做为图象分析的参考与补充。     

I型和Ⅲ型胶原在修复性牙本质形成中的免疫定位

目的 :研究 I型和 型胶原在修复性牙本质形成中的免疫定位和分布特征。方法 :在大鼠第一磨牙制备单面洞 ,观察修复性牙本质形成。免疫组化 SABC法检测 I型胶原和 型胶原的免疫反应。结果 :在术后 3d,修复性牙本质尚未形成。I型胶原和 型胶原均分布于牙髓内 ,前期牙本质为弱阳性。术后 15 d,I型胶原和 型胶原在牙髓细胞内呈阳性染色。I型胶原在前期牙本质中呈弱阳性。术后 30 d,I型胶原和 型胶原的强阳性染色集中于已形成的修复性牙本质下的牙髓内 ,成牙本质细胞样细胞染色呈强阳性。结论 :形成修复性牙本质的成牙本质细胞样细胞合成、分泌 I型胶原和 型胶原。 I型胶原是修复性牙本质的主要基质成分     

大鼠牙髓细胞复合β-磷酸三钙多孔陶瓷体外培养的实验研究

目的:研究大鼠牙髓细胞与β-TCP复合培养后的生长情况及细胞活性,探讨β-TCP作为大鼠牙髓细胞支架材料的可行性。方法:实验分两组,实验组为大鼠牙髓细胞与β-TCP复合培养,对照组为大鼠牙髓细胞常规培养。采用相差显微镜、电子显微镜观察细胞生长、贴附情况;采用MTT、细胞蛋白检测、碱性磷酸酶检测的方法评价大鼠牙髓细胞生长情况及细胞活性。结果:大鼠牙髓细胞复合β-TCP后生长良好,贴附于材料表面及孔隙内壁。MTT及细胞蛋白检测复合培养组和对照组无差异,6 d、9 d时复合β-TCP培养的大鼠牙髓细胞碱性磷酸酶活性明显高于对照组。结论:复合β-TCP进行培养的大鼠牙髓细胞生长良好,没有出现生长抑制现象,其细胞活性更明显增强,表明β-TCP完全符合牙髓细胞支架材料的要求,是一个具有很好应用前景的牙髓组织工程支架材料。     

釉基质蛋白对人牙周膜细胞形成I型胶原、Ⅲ型胶原的影响

目的：观察釉基质蛋白对体外培养的人牙周膜细胞形成I型胶原、Ⅲ型胶原的影响。方法：改良组织块法培养人牙周膜细胞，免疫细胞化学方法和图像分析方法观察细胞形成I型胶原、Ⅲ型胶原的能力。结果：50、100、200mg/L的釉基质蛋白可以促进牙周膜细胞合成I、Ⅲ型胶原，其中，以100mg/L釉基质蛋白的促I型胶原合成作用最明显，50mg/L釉基质蛋白的促Ⅲ型胶原合成作用最明显。但是，这种促进作用有一定的时间性。结论：一定浓度的釉基质蛋白可以促进牙周膜细胞形成I型胶原、Ⅲ型胶原。     

人牙本质涎蛋白对狗牙髓损伤修复影响的实验研究

目的 探讨人牙本质涎蛋白对狗牙髓损伤修复的影响。方法 实验共分3组，实验组(hDSP组)；对照l组(无hDSP组)；对照2组(PBS组)；选择10Kg、6～12月龄的杂种狗3只，上、下尖牙、上、下磨牙和下前磨牙为实验牙，每只狗选10个牙，随机分配实验组和对照组，实验组共18个牙，对照l组和2组各6个牙。于所选牙齿颈部，用l／2球钻在灭菌生理盐水滴注下穿通髓腔、露髓处置上述实验组和对照组胶原膜，光固化封闭窝洞。2、4、8周处死动物，经4％多聚甲醛灌注后，分离实验牙，再固定48h后，脱钙3～4周，脱水，石蜡包埋，制备5～8μm厚连续切片，HE染色。结果 术后2周：对照组与实验组无明显差异，无牙本质桥形成。术后4周：对照组无牙本质桥形成，实验组有部分骨样牙本质桥形成。术后8周：对照组牙髓组织基本正常，无牙本质桥形成，实验组有完整的骨样牙本质桥形成，其下方有排列整齐的成牙本质细胞样细胞形成。结论 首次发现hDSP在体内能够诱导牙髓细胞分化，形成修复性牙本质。     

人牙髓干细胞在不同HA含量的PLGA/HA复合支架材料上的粘附研究

目的:构建不同质量构成比的PLGA/HA复合支架并评价材料机械性能;探讨不同比例PLGA/HA复合生物学支架对牙髓干细胞粘附能力的影响。方法:采用溶液浇筑/颗粒沥析技术构建出分别含有10%HA、20%HA及30%HA的PLGA/HA复合支架,对照组采用单纯PLGA支架。应用电子万能测验机检测支架的拉伸强度,扫描电镜观察表面结构。将牙髓干细胞与不同比例的PLGA/HA支架复合培养,应用DAPI染色细胞计数法检测细胞粘附能力。结果:在对3组实验组的拉伸强度测试中,10% HA组拉伸强度最高,20% 组次之,两组均高于对照组,而30% HA组拉伸强度低于对照组(P<0.05)。扫描电镜观察支架表面,显示支架孔径在100~250 μm之间,孔隙间 连通性良好,孔隙率较高。细胞接种于支架2、6、12 h后,荧光染色显示细胞与3种支架紧密贴合,粘附良好,细胞计数结果显示各实验组细胞粘附数量均多于对照组,其中在30%HA组中细胞粘附性能最佳(P<0.05)。结论:3种不同构成比的HA/PLGA复合支架均为良好的组织工程支架材料,其中10%HA组具有良好的机械性能,30%HA组细胞粘附性能更佳。     

Effects of TGF-beta s on the growth, collagen synthesis and collagen lattice contraction of human dental pulp fibroblasts in vitro

Transforming growth factor-beta (TGF-beta) is important in regulating the repair and regeneration of damaged dental pulp. For further elucidating the roles of different isoforms of TGF-beta in the healing and inflammatory processes of human dental pulp, we found that TGF-beta1, TGF-beta2 and TGF-beta3 inhibited the growth of two human dental pulp cell strains in vitro by 19-29, 18-25 and 23-26%, respectively, at a concentration of 0.5 ng/ml. TGF-beta also differentially stimulated the collagen synthesis of pulp cells. Collagen synthesis increased by 1 ng/ml of TGF-beta1 and TGF-beta2 by 42 and 51%, respectively. TGF-beta3 (0.1-1 ng/ml) lacked of stimulatory effect on collagen synthesis of pulp cells. Pulp cells have the intrinsic capacity to contract collagen lattice, leading to decreasing of lattice diameter. An 8 h exposure to TGF-beta1 and TGF-beta2 enhanced the pulp cell-populated collagen lattice contraction at concentrations ranging from 0.2 to 3 ng/ml. At similar concentrations, TGF-beta3 lacked of this stimulatory effect. When collagen lattice were detached after 24 h of exposure, TGF-beta1 and TGF-beta2 (0.6-3 ng/ml) induced the pulp cells-populated collagen lattice contraction within 4-8h of gel detachment. These results indicate that TGF-beta-induced collagen lattice contraction is a late cellular event. These in vitro results indicate that effects of TGF-beta isoforms on the growth, collagen synthesis and collagen lattice contraction of pulp cells may play crucial roles in the pathobiological processes of dental pulp.     

干细胞和支架与牙髓再生及其血运重建

在牙髓再生中,获取干细胞的方法包括干细胞移植、细胞归巢和诱导出血。干细胞移植可产生异位的牙髓样组织,可控制移植细胞的数量并选择对牙髓再生潜在效能最佳的细胞亚种。细胞归巢是指利用信号分子招募宿主内源性干细胞至需治疗的牙体根管中增殖和分化,形成牙髓-牙本质样组织。诱导根尖出血进入根管为年轻恒牙牙髓再生的一个重要步骤。支架是细胞在合成组织时的支撑结构,可促进细胞黏附,为牙髓再生提供有利的环境。牙髓再生离不开血运重建或者血管再生,感染控制、根管预处理、冠方封闭等操作,可为牙髓再生包括其血运重建提供适宜的环境。总之,组织工程技术在牙髓领域的应用发展为牙髓再生带来了新的希望。     

富血小板血浆支架诱导牙髓再生的体内研究

目的：探讨不同浓度的富血小板血浆（PRP）支架在体内诱导牙髓组织再生的能力。方法将小型猪乳牙牙根段进行化学预备,采用二次离心法制备PRP,根据注入根管中的成分不同将研究分为4组：（1）阴性对照组,即全血组;（2）100％ PRP组;（3）50％ PRP组;（4）空白组,即空的牙根段;每组5个样本,分别植入裸鼠背部皮下,于术后5周处死动物,取出样本进行组织学观察。结果植入5周后,100％ PRP组根管内充满了炎性细胞,50％ PRP组根管内有少量牙髓样的组织生成。结论合适浓度的PRP作为生物支架在体内再生牙髓样的组织是可行的。     

Proliferation of mature ex vivo human dental pulp using tissue engineering scaffolds

Introduction

The purpose of this study was to measure and compare the proliferation of mature human dental pulp tissue using three types of tissue engineering scaffolds.

Methods

Mature human teeth were collected immediately after extraction for routine dental treatment reasons. Three types of tissue engineering scaffolds were investigated (1) open-polylactic acid (polymer) scaffolds, (2) bovine collagen (collagen) scaffolds, and (3) calcium phosphate bioceramic (calcium phosphate) scaffolds. The scaffolds were placed in direct contact with the dental pulp of the tooth slices from 7 to 30 days. Neutral-red dye was added to the culture media to stain metabolically active cells. The specimens were processed for histology. The numbers of proliferating cells were counted per unit area of scaffold according to ISO criteria.

Results

The proliferating dental pulp cells had a fibroblast phenotype, no cells of other phenotypes were observed, and none of the cells appeared to be mineralizing. The average rate of mature vital dental cell proliferation was 1.305 cells per day in the calcium phosphate scaffolds compared with 7.195 (a rate increase of 551%) in the collagen scaffolds and 13.885 (a rate increase of 1,064%) in the polymer scaffolds.

Conclusions

Tissue engineering scaffolds can enhance the proliferation of mature dental pulp tissue. The rate of dental pulp proliferation is dependent on the chemical composition of the scaffold. Within the limitations of this study, the polymer scaffolds were more optimal than collagen or calcium phosphate scaffolds for mature dental pulp proliferation.     

血管内皮生长因子和骨形成蛋白2诱导犬恒牙原位牙髓再生

目的通过选择犬根尖孔发育完成的恒牙建立根尖周炎模型,探索血管内皮生长因子（VEGF）和骨形态生成蛋白2（BMP2）诱导原位牙髓再生的可能性。 方法2只10~ 12月龄的杂种犬,选择根尖孔发育完成的14颗恒前牙建立根尖周炎模型,分别将VEGF（VEGF组）、BMP2（BMP2组）单独和VEGF+BMP2联合（VEGF+BMP2组）与水凝胶复合植入感染控制后的根管腔内,对照组仅植入水凝胶。8周后组织学观察根管内组织再生情况。 结果植入8周后,VEGF组和VEGF+BMP2组根管腔内可见含有大量成纤维样细胞和血管的新生组织形成;而BMP2组和对照组根管腔内见均质状物质,未见细胞、血管形成。 结论VEGF或VEGF+BMP2复合水凝胶支架可以诱导犬根尖发育成熟的根尖周炎患牙在根管腔内生成含有血管的疏松结缔组织。     

老年人弯曲钙化根管的显微超声治疗的临床观察

目的:评价手术显微镜及超声技术处理老年人弯曲钙化根管的临床效果。方法:弯曲钙化根管137个,牙科手术显微镜下超声去除髓腔及根管中上段钙化组织,C型先锋锉结合EDTA凝胶探查并疏通根管。结果:疏通弯曲钙化根管91个,疏通率66.4%。上下颌间及不同牙位间疏通率差异无统计学意义,但钙化部位的不同导致根管疏通率有统计学差异。结论:显微镜下超声技术对于疏通老年人弯曲钙化根管具有积极有效的作用。     

内皮素1对人牙髓细胞胶原合成的影响

目的 探讨内皮素１地人牙髓细胞胶原合成的影响。方法 采用细胞培养、酶联免疫吸附分析及免疫组化染色等方法,就ＥＴ－１对ＨＤＰＣ胶原合成的影响进行了研究。结果 ＥＴ－１较对照组除２４小时时相外,均有不同程度的促ＨＤＰＣ可溶性Ｉ型胶原合成作用,但对Ⅲ型胶原作用不明显;经免疫组化染色证实;ＥＴ－１尚可同时促进细胞非溶解性Ｉ型胶原合成。结论 ＥＴ－１促进ＨＤＰＣＩ型胶原合成增加,可能对牙髓组织的自身修复起作     

The Role of Small Extracellular Vesicles Derived from Lipopolysaccharide-preconditioned Human Dental Pulp Stem Cells in Dental Pulp Regeneration

IntroductionRegenerative endodontics has created a desirable shift in the treatment paradigm despite current limitations of regenerative outcomes. Mesenchymal stem cells (MSCs) facilitate tissue regeneration and repair in a mild inflammatory environment. Small extracellular vesicles (sEVs) derived from MSCs play an imperative role in the paracrine modulation of regenerative responses modulated by MSCs. However, it remains unknown whether MSCs enhance dental pulp regeneration or whether this enhancement is mediated by sEVs in a mild inflammatory environment. The present study aimed to elucidate the effects of sEVs originated from lipopolysaccharide (LPS)-preconditioned human dental pulp stem cells (hDPSCs) on dental pulp regeneration.MethodsAll sEVs were isolated from hDPSCs cultured with or without LPS (ie, N-sEVs and L-sEVs, respectively). The effect of N-sEVs and L-sEVs on proliferation, migration, angiogenesis, and differentiation of rat bone marrow MSCs was identified in vitro. Moreover, N-sEVs or L-sEVs were implanted into rat pulpless root canal models, and the regenerated tissue in root canals was assessed via hematoxylin-eosin staining, Masson staining, and immunohistochemistry after 30 days of transplantation.ResultsBoth N-sEVs and L-sEVs could modulate BMSC proliferation, migration, angiogenesis, and differentiation. Both kinds of sEVs enhanced the structure of the regenerated tissue closer to that of a normal dental pulp in vivo. L-sEVs had a more significant effect than N-sEVs.ConclusionssEVs released by hDPSCs in a mild inflammatory microenvironment are capable of facilitating the regeneration of dental pulp through functional healing instead of scar healing, which has potential applications in regenerative endodontics.     

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.