Notch信号通路在牙髓和牙周细胞矿化及组织损伤修复中的调控作用研究

目的 探讨Notch信号通路在人牙髓细胞(dental pulp cells, DPCs)和牙周韧带细胞(periodontal ligament cells, PDLCs)向成牙本质/成骨样细胞分化及组织损伤修复中的调控作用.方法 酶消化法分离培养DPCs和PDLCs,实时荧光定量反转录聚合酶链反应(RT-PCR)检测Notch信号通路相关基因Notch1、Notch2、DLL1和DLL3 mRNA在DPCs和PDLCs矿化诱导过程中表达水平变化;建立大鼠牙髓和牙周联合损伤动物模型,观察4周后牙髓牙本质复合体及牙周附着装置修复情况,免疫组织荧光染色检测Notch1在牙髓和牙周联合损伤修复动物体内模型的表达和分布.结果 DPCs和PDLCs经矿化诱导,Notch1、Notch2、DLL1 mRNA表达均上调,与对照组间的差异有统计学意义(P<0.05);DLL3 mRNA表达上调,与对照组间差异无统计学意义(P>0.05).免疫荧光示Notch1蛋白在损伤处的新生牙髓及牙周韧带组织细胞浆强表达,而在正常牙髓及牙周韧带组织基本无表达.结论 在DPCs和PDLCs诱导成牙本质/成骨分化的过程中,Notch1、Notch2、DLL1呈现相似的表达变化趋势上调,Notch1信号在牙髓及牙周损伤修复的新生组织明显激活,提示Notch信号通路参与DPCs和PDLCs成牙本质/成骨分化,且在牙髓牙本质复合体和牙周附着装置的损伤修复中起重要的调控作用.     

细胞骨架及相关蛋白在人牙周韧带细胞成骨分化过程中的表达模式

目的探讨人牙周韧带细胞（PDLCs）成骨分化过程中细胞骨架及相关蛋白的表达模式及其可能的功能作用。方法酶消化法培养PDLCs．免疫细胞化学染色检测vimentin的表达：取诱导前（对照组）及矿化诱导7、14和21d的PDLCs。实时荧光定量RT—PCR检测vimentin、actin、caldeSIllon（CaD）、tropomyosin（Tm）和annexin A4 mRNA的表达变化并进行统计分析，Western blot检测蛋白表达。结果PDLCs表达丰富的vimentin：Vimentin、actin、CaD和Tm mRNA的表达在诱导7d组下调．诱导14d组和21d组逐渐上调：Annexin A4 mRNA的表达在诱导7d组表现为上调，诱导14d组和21d组逐渐下调：Vimentin的mRNA表达在各组间差异均有统计学意义（P〈0．05）：CaD和Tm在诱导7d组与14d组之间的mRNA表达差异无统计学意义（P〉0．05），其余各组间差异均有统计学意义（P〈0．05）：Actin和annexin A4在对照组与诱导21d组之间的mRNA表达差异无统计学意义（P〉0．05）．其余各组间差异均有统计学意义（P〈0．05）；Western blot检测蛋白表达显示类似的表达趋势。结论在PDLCs成骨分化过程中．一组细胞骨架及细胞骨架蛋白相关蛋白呈现相似的表达变化．提示其参与调节PDLCs成骨分化。     

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．     

碱性成纤维细胞生长因子对牙周细胞生物学活性的影响

目的：观察基因重组入碱性成纤维细胞生长因子（rh-bFGF)牙龈成纤维细胞（GF）、人牙周韧带成纤维细胞（PDLF）及人牙槽骨细胞（ABC）的增殖、碱性磷酸酶活性、总蛋白含量及对3种细胞矿化结节形成能力的影响。方法：采用细胞培养、MTT比色测定、碱性磷酸酶测定法、考马宙蓝法及茜素红染色法。结果bFGF能促进3种细胞的增殖,但对PDLF和ABC的ALP活性,蛋白含量及矿化结节的形成有抑制作用。结论bFGF可促进细胞的增殖,抑制细胞的分化成熟,从而促进牙周再生。     

骨形成蛋白-2和碱性成纤维细胞生长因子对人骨髓基质细胞的生物学作用

骨髓基质细胞具有多向分化潜能，在特定培养条件下可转化成多种间充质细胞。骨组织工程中为提高成骨效率，必须选择合适的培养条件，以促进骨髓基质细胞繁殖及定向分化为成骨细胞。本实验旨在观察重组人骨形成蛋白-2(recombinant human bone morphogenetic protein，rhBMP-2)和碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)分别和联合作用条件下，对人骨髓基质细胞增殖与分化的影响，以期建立一种理想的培养体系。     

骨形态发生蛋白-2与碱性成纤维细胞生长因子在异位和原位成骨中的作用

目的通过骨髓基质细胞（BMSCs）的体外增殖和分化、异位成骨和原位成骨实验来观察骨形态发生蛋白-2（BMP-2）和碱性成纤维细胞生长因子（bFGF）在成骨过程中的作用。方法分别用含BMP-2、bFGF和BMP-2+bFGF的培养液体外培养Beagle犬的BMSCs,通过甲基噻唑基四唑（MTT）比色法测定细胞增殖水平,通过测定碱性磷酸酶（ALP）活性观察细胞的分化情况。将BMSCs与多孔磷酸钙（CPC）分别在含BMP-2、bFGF和BMP-2+bFGF的培养液中复合培养,制成复合材料,一部分植入裸鼠皮下,观察异位成骨情况,另一部分植入Beagle犬的种植体周围骨缺损区,经过荧光标记观察原位成骨情况。结果含有BMP-2+bFGF的培养液促进BMSCs增殖和分化的能力最强。异位成骨情况：BMP-2+bFGF组的成骨量较其他组明显增加,其新骨形成百分比为48.79%±11.31%,高于单一BMP-2组（30.71%±10.85%）和bFGF组（27.33%±9.67%）以及对照组（10.65%±6.05%）。原位成骨术后12周,BMP-2+bFGF组的矿化沉积率高于其他组,其差异有统计学意义（P<0.01）。结论在促进成骨方面,BMP-2和bFGF共同作用优于单一因子。     

碱性成纤维细胞生长因子与骨再生修复

骨组织的再生修复一直是多年来的研究热点之一，而生长因子因其能够促进组织细胞的生长和分化也已引起了广泛的关注。骨组织工程学的发展把生长因子作为重要激活物与种子细胞、支架材料、立体培养等联系在一起，成为构建骨组织再生修复必不可少的要素。碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)是主要的生长因子之一，但由于其促进成骨与毛细血管生成的双重作用，使其在促进骨再生修复方面的前景日益受到人们的重视。另外，它还可通过细胞因子网络发挥广泛的生物学作用。现就bFGF在骨组织再生修复过程中的作用及机制，及其在细胞因子网络中与其他因子的相互作用作一综述。     

成牙本质细胞表达相关蛋白作用的研究进展

成牙本质细胞分泌的蛋白质、基质、生长因子等在牙齿发育和牙髓损伤修复中都起着重要的调节作用。牙本质基质蛋白(DMP1)、牙本质涎蛋白(DSP)、牙本质磷蛋白(DPP)、巢蛋白、clock蛋白、骨黏附蛋白聚糖(OSAD)等在牙本质形成、矿化及牙本质修复等过程中发挥着各自的作用。此外,其中某些蛋白对常染色体隐性低血磷性佝偻病、非矿化组织疾病发病机制的研究具有重要意义。本文就成牙本质细胞表达相关蛋白在牙发育及损伤修复中的作用机制做一综述。     

骨形成蛋白-2和碱性成纤维细胞生长因子对人骨髓基质细胞的生物学作用

目的：探讨重组人骨形成蛋白-2(rhBMP-2)和碱性成纤维细胞生长因子(b—FGF)单独或联合作用对人骨髓基质细胞(HBMSC)增殖和分化的影响。方法：利用四唑盐比色法(MTT)、碱性磷酸酶(ALP)测定法观察不同浓度的rhBMP-2和b—FGF单独或联合作用时HBMSC的增殖和分化情况。结果：rhBMP-2对HBMSC的增殖和ALP表达均有促进作用；b—FGF促进HBMSC增殖，但抑制ALP表达；rhBMP-2和b—FGF联合作用时HBMSC的增殖和ALP活性较单独作用有显著提高。结论：rhBMP-2和b—FGF联合应用时对HBMSC的增殖和向成骨细胞分化具有协同作用。     

骨形成蛋白和牙本质形成

骨形成蛋白(BMP)可存在于人及动物的牙本质中,其生物活性与骨BMP相同。免疫组化及核酸原位分子杂交研究显示,牙胚中造牙本质细胞在分化成熟过程中,BMP基因激活并表达,合成分泌BMP,BMP对造牙本质细胞的分化具有自分泌和旁分泌调节作用。BMP体外可促进牙髓细胞增殖分化,体内可诱导管状牙本质和骨样牙本质的早期形成。BMP还与牙源性肿瘤中的病理性牙本质形成有关。临床应用BMP盖髓研究表明,其有效率不低于氢氧化钙,BMP是一种生物相容性良好的蛋白质,在保存活髓治疗方面具有较大的应用前景。     

Effects of fibroblast growth factor 2 on osteoblastic proliferation and differentiation by regulating bone morphogenetic protein receptor expression

Fibroblast growth factors (FGFs) are known to play a critical role in bone growth and development, affecting both osteogenesis and chondrogenesis. Fibroblast growth factor 2 (FGF-2) is produced intracellularly by osteoblasts and secreted into the surrounding matrix in bone.The dose-dependent effects of FGF-2 were tested to examine the relationship between FGF-2 and osteoblast proliferation and differentiation. Tests used included a cell viability test, an alkaline phosphatase activity test, and a Western blot analysis.Cultures growing in the presence of FGF-2 showed an increased value for 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and a decreased value for alkaline phosphatase activity. Results of the Western blot analysis showed that the addition of FGF-2 seems to decrease osteocalcin and bone morphogenetic protein receptor IA.These data show that FGF-2 in the tested dosage within MC3T3-E1 cells seems to affect proliferation and differentiation. Results of the Western blot analysis may add some possible mechanisms, and it may be suggested that treatment of FGF-2 may have an influence on the expression of bone morphogenetic protein receptors in osteoprecursor cells. Further elucidation of the mechanisms related to this mechanism within the in vivo model may be necessary to ascertain greater detail.     

骨形成蛋白4对人牙髓细胞去分化基因表达的调控作用

目的研究外源性骨形成蛋白4(BMP-4)对体外连续传代培养人牙髓细胞(DPCs)生长和去分化基因表达的影响,探讨BMP-4对DPCs体外传代培养过程中细胞未分化性能的调控作用。方法重组人骨形成蛋白(rhBMP-4)诱导组和对照组体外培养4周的人牙髓组织中的细胞进行细胞计数;取P2和P7代rhBMP-4诱导组和对照组DPCs,免疫荧光染色和实时荧光定量聚合酶链反应检测Oct-4、Sox-2、c-Myc表达情况,统计学分析。结果体外培养4周的人牙髓组织经rhBMP-4诱导后,DPCs数量显著高于对照组(P<0.05)。免疫荧光示Oct-4、Sox-2、c-Myc在P2代rhBMP-4诱导组和对照组DPCs均为细胞核表达,在P7代诱导组DPCs保持细胞核表达,而在P7代对照组DPCs表现为细胞浆表达。实时荧光定量聚合酶链反应显示Oct-4、Sox-2、c-Myc在P2代诱导组和对照组DPCs表达差异无统计学意义(P>0.05),而在P7代诱导组DPCs表达显著高于对照组(P<0.05)。结论 BMP-4可促进DPCs生长,维持传代后期去分化基因Oct-4、Sox-2、c-Myc的表达。     

Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery

Objective: To evaluate the synergistic effect of bone morphogenetic protein 2 (BMP‐2) and vascular endothelial growth factor (VEGF) on the repair of bone defects around dental implants. Material and methods: Five groups of scaffold were fabricated by a freeze‐drying method, including pure chitosan/collagen scaffold; scaffold loaded with adenoviruses expressing BMP‐2, adenoviruses expressing VEGF, both adenoviruses expressing BMP‐2 and VEGF, VEGF protein and adenovirus expressing BMP‐2. In vitro studies examined whether bone marrow stromal cells were responsive to these scaffolds over time. Bone formation capacity, bone‐to‐implant contact, as well as removal torque values were investigated in vivo. Differences between the various groups were statistically analyzed using the one‐way analysis of variance test. Results: The in vitro study revealed a burst and rapid release of VEGF with a sustained high‐level expression of BMP‐2 in scaffold combined with VEGF protein and adenoviruses expressing BMP‐2. Histomorphometry demonstrated that scaffolds expressing BMP‐2 enhanced more bone formation compared with other groups; VEGF alone is insufficient to promote bone formation. New bone formation in the bone defects around dental implants, bone‐to‐implant contact and mean peak removal torque showed statistically significant difference for the adenoviral vector encoding human bone morphogenetic protein 2 (Ad‐BMP‐2) and VEGF protein and adenovirus expressing BMP‐2 groups. Furthermore, scaffold combined with VEGF protein and Ad‐BMP‐2 represented the best outcomes in this model. Conclusions: A combination of BMP‐2 gene and VEGF protein could have a synergistic effect in promoting bone healing. To cite this article:
Luo T, Zhang W, Shi B, Cheng X, Zhang Y. Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery.
Clin. Oral Impl. Res. 23 , 2012 467–474.
doi: 10.1111/j.1600‐0501.2011.02164.x     

bFGF和rhBMP对牙周膜细胞胶原酶水平的影响

目的:研究bFGF、rhBMP分别及联合作用对人牙周膜细胞(periodontalligamentcells ,PDLCs)胶原酶Ⅱ、Ⅳ(CollagenaseⅡ、Ⅳ)水平的影响并探讨其内在相关性;优选最佳显效浓度。方法:选取因正畸拔除的第一前磨牙,刮取根中1/3牙周膜组织作为标本,采用牙周膜细胞体外培养技术,免疫组化方法和图像分析技术动态观察bFGF、rhBMP梯度含量分别及联合作用对人PDLCs胶原酶Ⅱ、Ⅳ水平的影响。结果:①免疫组化定量分析,bFGF(10 )浓度组能显著抑制PDLCs胶原酶Ⅳ的表达(P <0 .0 1) ;rhBMP(2 0 0 )浓度组能诱导PDLCs胶原酶Ⅳ的表达(P<0 .0 5 ) ;bFGF(10 )、rhBMP(2 5 )浓度组均能显著诱导PDLCs胶原酶Ⅱ的表达(P <0 .0 1) ;动态观察bFGF(10 ) rhBMP(2 0 0 )连续7d对胶原酶Ⅱ、Ⅳ水平的抑制效应呈现连续的递增趋势(P <0 .0 5 )。②rhBMP与人PDLCs胶原酶Ⅱ水平呈显著负相关性(P <0 .0 1) ;rhBMP与人PDLCs胶原酶Ⅳ水平呈显著正相关性(P <0 .0 1)。结论:bFGF、rhBMP联合作用能更显著地抑制人PDLCs胶原酶Ⅱ、Ⅳ的表达,两者具有协同作用;其中bFGF(10 ) rhBMP(2 0 0 )为最佳显效浓度。     

表皮生长因子与碱性成纤维细胞生长因子对人牙髓干细胞分化的影响

目的探讨表皮生长因子(epidermal growth factor,EGF)和碱性成纤维细胞生长因子(basic fibroblastgrowth factor,bFGF)对人牙髓干细胞(human dental pulp stem cells,hDPSCs)碱性磷酸酶(alkaline phosphatase,ALP)活性的影响。方法确定EGF与bFGF最大效应浓度后,对细胞分组,根据EGF和bFGF单独或联合应用,分为4组:EGF组、bFGF组、EGF联合bFGF组、不加任何生长因子的对照组。作用1、3、5、7、14 d后,采用ALP活性检测法(酶动力法)检测hDPSCs细胞ALP活性。结果 1～14 d bFGF组ALP活性与对照组相比差异无统计学意义(P>0.05);在5、7、14 d,EGF组和EGF联合bFGF组ALP活性显著高于对照组(P<0.05);EGF联合bFGF组的ALP活性明显高于bFGF组(P<0.05),但EGF联合bFGF组ALP活性与EGF组相比差异无统计学意义(P>0.05)。结论 bFGF单独应用不能诱导hDPSCs分化,EGF在hDPSCs的分化中发挥作用,EGF和bFGF无明显协同作用。     

转化生长因子β1和骨形成蛋白2体外诱导成牙本质细胞分化

目的 观察转化生长因子β1（transforming growth factorβ1,TGF-β1）和骨形成蛋白2（bone morphogenetic protein2,BMP2)对体外培养的鼠牙乳头成牙本质细胞分化的影响。方法 取17d胎龄小鼠下颌第一磨牙牙胚，胰蛋白酶消化分离牙乳头，置半固态培养基培养6d，半固态培养基中加入重组TGF-β1或BMP2与肝素，组织学观察。结果 TGF-β1或BMP2加肝素可诱导牙乳头周边细胞发生极化，并分泌胞外基质。TGF-β1或BMP2单独加入时未见细胞极化，但基质分泌增加。结论 TGF-β1和BMP2均能诱导成牙本质细胞的细胞学分化和分泌功能。     

The effect of combination of recombinant human bone morphogenetic protein-2 and basic fibroblast growth factor or insulin-like growth factor-I on dental implant osseointegration by confocal laser scanning microscopy

BACKGROUND: The healing period of bone-implant osseointegration usually varies from 3 to 6 months or even longer. Failure may occur during this time. This study aimed to investigate whether osseointegration of dental implants can be enhanced by the combination of growth factors. METHODS: Sixty-four implants were coated with polylactic acid and divided into four groups. Group I was applied with 1.0 mg recombinant human bone morphogenetic protein-2 (rhBMP-2) and 200 microg recombinant human basic fibroblast growth factor (rhbFGF), group II with 1.0 mg rhBMP-2 and 250 mug recombinant human insulin-like growth factor-I (rhIGF-I), group III with 1.0 mg rhBMP-2, and group IV without growth factors as control. In total, 16 rabbits were used, and two osteotomies were drilled on each side of the femur, in which four different groups were randomly placed. Four weeks after implanting, 20 mg calcein green/kg body weight was administered intravenously, and 8 weeks after implanting, 20 mg alizarin/kg body weight was administered intravenously. Twelve weeks after implanting, the animals were sacrificed. The block of bone with implants was embedded in methylmethacrylate and sectioned, and the percentage of new bone surrounding the implant was analyzed by confocal laser scanning microscopy. RESULTS: There was a statistical difference in bone formation between rhBMP-2-applied groups and the non-applied group at 4 or 8 weeks, and no significant difference between groups I and II (although bone formation in group II was greater than that in group I at 4 weeks). The bone formation in group II was greater than that in group III at 4 or 8 weeks. The formed bone in group I was also greater than the one in group III at 8 weeks, but there was no difference at 4 weeks. CONCLUSIONS: rhBMP-2 could increase new bone formation, and it acted synergistically with rhbFGF and rhIGF-I to improve bone-implant osseointegration. The combination of rhBMP-2 and rhbFGF (group 1) showed faster growth of new bone than other groups at 8 months.