【作者】 刘冬梅；

【导师】 董福生；

【作者基本信息】 河北医科大学 ， 外科学， 2008， 博士

【摘要】 组织工程学的发展为组织缺损的修复以及器官的重建再造开辟了一条崭新的道路,构建组织工程化组织的研究是目前口腔颌面整形领域关注的热点之一。由于牙齿对于颌面骨的生长发育,对于维持颌面部的美观,对于语言、咀嚼功能有着重要的作用,许多国家试图利用组织工程学技术构建组织工程化牙齿。牙本质是构成牙齿的主体,在组织工程化牙齿的形成过程中牙本质的构建是一个关键。本研究对构建组织工程化牙本质的种子细胞、生物支架材料、组织工程化牙本质构建等方面进行了探讨。目的:利用基因转染技术对来源广泛易于获得和培养的口腔粘膜成纤维细胞进行牙本质基质蛋白1(dentin matrix protein1, DMP1)基因转染,获得牙本质基质蛋白1基因转染的口腔粘膜成纤维细胞(dentin matrix protein1-porcine oral mucosa fibroblasts, DMP1-POMF),通过对牙本质基质蛋白1基因转染的口腔粘膜成纤维细胞生物学性能、成牙本质细胞特异性基因蛋白表达的观察,探讨构建组织工程化牙本质的种子细胞。利用建立的组织工程化牙本质种子细胞(牙本质基质蛋白1基因转染的口腔粘膜成纤维细胞,DMP1-POMF)复合脱细胞真皮基质进行培养,通过HE染色光镜观察、扫描电镜观察、流式细胞检测探讨DMP1-POMF种子细胞与脱细胞真皮基质的相容性,评价脱细胞真皮基质作为构建组织工程化牙本质支架材料的可行性;通过裸鼠肌肉内植入DMP1-POMF复合脱细胞真皮基质支架材料,观察其在动物体内的生长、组织学结构的变化、牙本质特异性基因蛋白的表达,探讨组织工程化牙本质的构建。对建立的组织工程化牙本质种子细胞(牙本质基质蛋白1基因转染的口腔粘膜成纤维细胞,DMP1-POMF)进行细胞团三维立体培养,通过细胞形态学观察、蛋白表达定量分析,评价细胞团内DMP1-POMF细胞的生物学特性和成牙本质细胞特异性基因蛋白的表达;通过DMP1-POMF细胞团体内盖髓的组织学及免疫组织化学检测,评价组织工程化牙本质种子细胞(牙本质基质蛋白1基因转染的口腔粘膜成纤维细胞,DMP1-POMF)细胞团三维立体培养在修复牙本质缺损中的作用。方法:1 pEGFP-DMP1基因真核表达质粒的构建。参照2006年3月Kim J等发表的猪DMP1全基因序列,委托上海捷瑞生物工程有限公司合成并构建了重组绿色荧光融合蛋白pEGFP-DMP1质粒。重组质粒经扩增提取后用XhoI和EcoRI双酶切,产物进行琼脂糖凝胶电泳。确定后对全基因序列进行测序检测。2细胞培养及牙本质基质蛋白1(DMP1)基因转染2.1猪口腔粘膜成纤维细胞的原代培养及来源鉴定中国农业大学实验动物研究所购买10月龄中国实验小型猪,3%戊巴比妥钠耳后肌肉麻醉,无菌条件下留取颊粘膜固有层结缔组织标本,置含抗生素预冷DMEM培养液中,剪成约1mm3的小块,在含10% FBS的DMEM,37℃5%CO2饱和湿度条件下培养猪口腔粘膜成纤维细胞(porcine oral mucosa fibroblasts, POMF)。免疫组化ABC法进行波形丝蛋白和细胞角蛋白抗体染色,作细胞来源鉴定。第四代细胞用于基因转染。2.2骨髓间质干细胞(mesenchymal stem cells, MSC)体外分离与培养实验猪经麻醉后,用16号穿刺针于股骨大转子处穿刺,抽取骨髓4ml,置于肝素化的25ml无菌培养液中,1000r/min离心10min,弃上清,在含10%FBS的DMEM,37℃5%CO2饱和湿度条件下培养。5天后全量换液,以后每周2次换液。第三代细胞用于基因转染,作为口腔粘膜成纤维细胞(POMF)的对照组。2.3 DMP1基因转染及筛选转染前用含10%FBS的DMEM调整细胞浓度至1×105接种于6孔板中,CO2孵箱培养至60%汇合。将细胞分为三组:A组:为牙本质基质蛋白1基因转染猪口腔粘膜成纤维细胞组,对口腔粘膜成纤维细胞进行牙本质基质蛋白1基因转染,本研究将其命名为牙本质基质蛋白1基因转染的猪口腔粘膜成纤维细胞(dentin matrix protein1- porcine oral mucosa fibroblasts, DMP1-POMF)。B组:为载体基因转染口腔粘膜成纤维细胞组,对口腔粘膜成纤维细胞转染空载体pEGFP-C1基因,本研究将其命名为载体基因转染的口腔粘膜成纤维细胞(pEGFP-C1-porcine oral mucosa fibroblasts,C1-POMF)。C组:为牙本质基质蛋白1基因转染骨髓间质干细胞组,对骨髓间质干细胞进行牙本质基质蛋白1基因转染,本研究将其命名为牙本质基质蛋白1基因转染的骨髓间质干细胞(dentin matrix protein1- mesenchymal stem cells,DMP1-MSC)。细胞转染48h后,按1︰10稀传到大培养瓶中,加入G418 500ug/ml进行筛选,每4天换液1次。14天后未转染的细胞死亡,转染成功的细胞克隆生长,扩大培养备用。3 DMP1基因转染对成纤维细胞生物学行为的影响。3.1 RT-PCR检测DMP1、Ambn及DSP表达取转染后48h及G418筛选备用的DMP1-POMF细胞、C1-POMF细胞及DMP1-MSC细胞,TRIzol裂解法提取总RNA。一步法RT-PCR进行基因扩增,检测DMP1、釉鞘蛋白(amelin and ameloblastin ,Ambn)及牙本质涎蛋白(dentin sialoprotein,DSP)RNA水平的表达。3.2细胞周期检测及DNA倍体分析收集转染后筛选备用的DMP1-POMF细胞与C1-POMF细胞,同时检测未转染细胞作为对照,预冷70%乙醇制备1×106单细胞悬液100μl,加入10%鸡红细胞作为内参标准,流式细胞仪上机检测,以增殖指数表示细胞分裂增殖情况,用DNA指数表示细胞DNA含量,DNA指数为0.85～1.15时为二倍体,在此范围之外则为异倍体。3.3矿化诱导后Von Kossa染色观察取转染后筛选备用的DMP1-POMF细胞、C1-POMF细胞及DMP1-MSC细胞,用矿化诱导培养液培养(含10%FBS、10mmol/Lβ-GP、100mg/L维生素C、10nmol/L地塞米松的DMEM),每4d换液1次。培养10-31d后分别固定细胞进行Von Kossa染色。3.4透射电镜观察细胞超微结构收集转染后筛选备用的DMP1-POMF细胞、C1-POMF细胞及DMP1-MSC细胞,分别于100ml培养瓶中培养21天,胰酶充分消化后收集于离心管内,4℃预冷的0.01M PBS(pH7.4)1000rpm离心洗涤2次。4℃预冷的3%戊二醛固定细胞3h,系列丙酮脱水,真空干燥,Epon812环氧树脂包埋,超薄切片,经醋酸双氧轴和枸橼酸铅双重染色后,透射电镜观察DMP1基因转染对细胞超微结构的影响。4 DMP1基因转染成纤维细胞复合脱细胞真皮基质构建组织工程化牙本质的实验研究。4.1细胞复合ADM支架材料体外培养4.1.1脱细胞真皮基质(ADM)支架的制备取5×5mm大小的脱细胞真皮基质(acellular dermal matrix,ADM),用眼科剪将其制备成多裂隙ADM细胞支架以利细胞充分附着生长。4.1.2细胞的培养A组:DMP1-POMF细胞组体外分离培养口腔粘膜成纤维细胞(porcine oral mucosa fibroblasts,POMF),取第四代细胞行DMP1基因转染,G-418筛选获得DMP1-POMF细胞。B组:POMF细胞组体外分离培养口腔粘膜成纤维细胞(porcine oral mucosa fibroblasts,POMF)稳定传代备用。4.1.3细胞复合ADM支架培养A组:DMP1-POMF细胞复合ADM支架培养组将ADM支架用PBS冲洗3次,DMEM培养液浸泡,收集2×107牙本质基质蛋白1基因转染的猪口腔粘膜成纤维细胞(dentin matrix protein1-porcine oral mucosa fibroblasts, DMP1-POMF)单细胞悬液1ml接种于制备好的ADM上,首先在37℃5% CO2饱和湿度培养3h后,再缓慢加入培养液至5ml继续培养3d。B组:POMF细胞复合ADM支架培养组将ADM支架用PBS冲洗3次,DMEM培养液浸泡,收集2×107POMF单细胞悬液1ml接种于制备好的ADM上,首先在37℃5% CO2饱和湿度培养3h后,再缓慢加入培养液至5ml继续培养3d。4.1.4组织学观察DMP1-POMF及POMF细胞复合脱细胞真皮基质培养3d,标本常规固定、包埋、切片行HE染色,光镜下观察细胞复合支架材料的生长情况。4.1.5扫描电镜观察DMP1-POMF及POMF细胞复合脱细胞真皮基质培养3d,标本4℃条件下经2.5%戊二醛磷酸缓冲液固定1h后,PBS漂洗2次,每次10min;再经1%锇酸固定2h;梯度乙醇逐级脱水,醋酸正戊酯置换,CO2临界点干燥,离子喷射仪喷金,扫描电镜观察DMP1-POMF细胞复合脱细胞真皮基质的生长情况。4.1.6细胞增殖周期检测分别收集DMP1-POMF细胞复合ADM培养以及DMP1-POMF细胞单层贴壁培养3,6,10,14d的细胞,预冷70%乙醇制备1×106单细胞悬液100ul,加碘化丙啶染色1ml,在4℃冰箱染色30min,500目铜网过滤,流式细胞仪上机检测细胞增殖指数。4.1.7蛋白表达定量分析检测分别收集DMP1-POMF细胞复合ADM培养以及DMP1-POMF细胞单层贴壁培养3,6,10,14d的细胞,使用DMP1,牙本质涎蛋白(dentin sialoprotein, DSP),I型胶原蛋白抗体,于流式细胞仪进行细胞的免疫荧光标记,以免疫荧光指数表示其蛋白表达水平,同时检测同型对照。(荧光指数=转染细胞蛋白表达的平均荧光强度/同型对照细胞平均荧光强度),>1.0为阳性表达,<=1.0为阴性表达。4.2细胞复合ADM裸鼠肌肉内种植4.2.1细胞复合ADM裸鼠肌肉内种植动物分组实验组:收集2×107DMP1-POMF细胞复合ADM体外培养3d,无菌条件下种植于9只裸鼠(BALB/c, SCXK/Jing/2005/0013)股部肌肉内,每只裸鼠植入2个复合体。分别于术后7、14、21d处死3只动物,取材、福尔马林固定。对照组:无菌条件下在9只裸鼠股部肌肉内分别植入相同量的POMF复合ADM支架的复合物及DMP1-POMF单细胞悬液作为对照,分别于术后7、14、21d处死3只动物,取材、福尔马林固定。4.2.2 HE染色组织学观察取7、14、21d标本进行包埋、切片、HE染色。4.2.3免疫组织化学观察取7d的标本用ABC免疫组织化学方法检测DMP1、DSP及I型胶原蛋白的表达,抗体滴度为1:50。5 DMP1基因转染成纤维细胞团对牙本质缺损的修复作用5.1细胞培养实验方法5.1.1细胞团三维立体培养分组及过程A组:牙本质基质蛋白1基因转染的口腔粘膜成纤维细胞(DMP1-POMF)细胞团三维立体培养取2×107个DMP1-POMF细胞,置于15ml锥形聚丙烯试管中1000r/min离心5min,置于含10%FBS的DMEM培养液中进行培养。每周换液2次。B组:牙本质基质蛋白1基因转染的口腔粘膜成纤维细胞(DMP1-POMF)单层贴壁培养取2×107个DMP1-POMF细胞,置于含10%FBS的DMEM培养液中进行培养。每周换液2次。C组:口腔粘膜成纤维细胞(POMF)细胞团三维立体培养取2×107个POMF细胞,置于15ml锥形聚丙烯试管中1000r/min离心5min,置于含10%FBS的DMEM培养液中进行培养。每周换液2次。5.1.2 HE染色光镜观察细胞团培养的细胞培养7d,14d,21d后10%甲醇固定24h,石蜡包埋,4.5μm断层切片,HE染色,光镜观察。5.1.3光镜下细胞计数对细胞团三维立体培养及单层贴壁培养的DMP1-POMF细胞,培养6,10,14,21d, 0.25%胰酶消化制成单细胞悬液,与光镜下进行细胞计数。5.1.4流式细胞仪检测细胞增殖周期分别收集单层贴壁及细胞团三维立体培养的DMP1-POMF和POMF,培养3,6,10,14,21d,预冷70%乙醇制备1×106单细胞悬液100μl,加碘化丙啶染色1ml ,在4℃冰箱染色30min,500目铜网过滤,流式细胞仪上机检测细胞增殖指数。5.1.5流式细胞仪进行蛋白表达定量分析分别收集单层贴壁及细胞团三维立体培养的DMP1-POMF和POMF,使用DMP1,DSP, I型胶原蛋白抗体,于流式细胞仪进行细胞的免疫荧光标记,以免疫荧光指数表示其蛋白表达水平,同时检测同型对照。(荧光指数=细胞蛋白表达的平均荧光强度/同型对照细胞平均荧光强度)。5.2细胞团体内修复牙本质缺损实验方法5.2.1实验动物分组中国实验用小型猪4只,A组5颗牙、B组4颗牙、C组3颗牙,实验采用自身对照比较,每只动物选取完整恒牙12颗,按随机数字法分为3组。A组为牙本质基质蛋白1基因转染口腔粘膜成纤维细胞(DMP1 -POMF)细胞团修复组B组为口腔粘膜成纤维细胞(POMF)细胞团修复组C组为氢氧化钙修复组分别于一个月处死2只动物,三个月处死2只动物。5.2.2实验过程实验动物用3%戊巴比妥钠耳后肌注麻醉(1ml/kg),开口器固定颌骨,充分清洁口周及口腔,2%碘酊消毒实验牙及牙周组织,用高速涡轮机制备　面洞,制作1×1mm2牙本质缺损并与牙髓相通,生理盐水冲洗充分止血。按分组分别使用体外培养14d的DMP1-POMF细胞团、POMF细胞团以及氢氧化钙覆盖牙本质缺损处,其上用氧化锌丁香油糊剂垫底,玻璃离子充填。术中严格无菌操作。分别于术后1个月及3个月处死动物,整体分离上下颌骨,再细分出牙齿,用10%缓冲甲醛液固定1周。将固定充分的标本置于脱钙液中,2-3d换液一次,随时观察标本脱钙情况。5.2.3脱钙牙齿组织学观察脱钙的牙齿标本梯度酒精脱水,石蜡包埋制成4μm连续切片,切片经HE染色后观察牙本质缺损处修复性牙本质形成情况。5.2.4脱钙牙齿免疫组织化学观察脱钙牙齿标本梯度酒精脱水,用ABC免疫组织化学染色检测DMP1表达,抗体滴度1:50,光镜下观察。结果:1 DMP1真核表达载体pEGFP-DMP1的构建及鉴定对构建的DMP1真核重组质粒酶切产物电泳可见4.7kb和1.5kb处两条特异条带,进行全基因序列测序,报告100%符合,证明pEGFP-DMP1重组质粒构建成功。2口腔粘膜成纤维细胞来源鉴定免疫细胞化学染色波形丝蛋白阳性,角蛋白阴性,证明所培养细胞为来自中胚层的成纤维细胞。3脂质体介导的pEGFP-DMP1基因转染口腔粘膜成纤维细胞脂质体介导基因转染细胞5h后在DMP1-POMF细胞、C1-POMF细胞及DMP1-MSC细胞中即可见发绿色荧光的细胞,16h可见大量发绿色荧光的细胞。转染pEGFP-C1的口腔粘膜成纤维细胞内可见荧光均匀的分布在整个细胞,而在转染pEGFP-DMP1的口腔粘膜成纤维细胞及骨髓间质干细胞内荧光仅分布在胞浆及细胞膜中。4 DMP1基因转染对口腔粘膜成纤维细胞生物学行为的变化4.1 DMP1基因转染对细胞增殖指数及染色体的影响流式细胞仪检测显示,DMP1-POMF细胞、C1-POMF细胞及POMF细胞之间增殖指数无差异(P>0.0 5)。流式细胞仪DNA倍体分析显示筛选备用DMP1-POMF细胞及C1-POMF细胞DNA指数分别为0.95、0.98,均为二倍体,无染色体突变。4.2 RT-PCR检测结果牙本质基质蛋白1基因转染的猪口腔粘膜成纤维细胞(DMP1-POMF)及牙本质基质蛋白1基因转染的骨髓间质干细胞(DMP1-MSC)RT-PCR检测结果相同:转染48h后可检测到DMP 1和Ambn基因表达,转染后G418筛选30d可检测到DMP 1、Ambn及DSP基因表达。转染空载体的C1-POMF细胞RT-PCR检测未见DMP1、Ambn及DSP基因表达。4.3矿化诱导Von Kossa染色结果A组:DMP1-POMF矿化诱导10d后细胞局部密度增高,开始形成细胞结节,31d时形成较多且直径较大钙结节,单位面积矿化结节数量为16.8。B组:载体基因转染的口腔粘膜成纤维细胞(C1-POMF)矿化诱导16d后可见小结节形成, 31d时单位面积矿化结节数量为6.8。C组:牙本质基质蛋白1基因转染的骨髓间质干细胞(DMP1-MSC)矿化诱导7d后细胞局部密度增高,开始形成细胞结节,31d时形成较多且直径较大钙结节,单位面积矿化结节数量为19.0。A组单位面积矿化结节数量高于B组,差异有统计学意义(p<0.05);C组单位面积矿化结节数量稍高于A组,差异无显著性(p<0.05)。4.4透射电镜观察结果DMP1-POMF细胞与C1-POMF细胞相比较,细胞突起增多,细胞内质网池普遍扩张,电子密度较低,其内大量蛋白分泌物,细胞内见大量呈同心圆或板层状排列的髓鞘样结构。DMP1-MSC细胞透射电镜下的超微结构呈现与DMP1-POMF细胞相类似的特点。5细胞复合ADM支架材料实验结果5.1细胞复合ADM支架材料体外培养观察结果5.1.1 HE染色组织学观察结果牙本质基质蛋白1基因转染的猪口腔粘膜成纤维细胞(dentin matrix protein1- porcine oral mucosa fibroblasts, DMP1-POMF)复合脱细胞真皮基质(ADM)体外培养组织切片HE染色观察可见,圆形或多边形细胞均匀分布于ADM胶原纤维基质中。5.1.2扫描电镜观察结果DMP1-POMF复合脱细胞真皮基质(ADM)体外培养扫描电镜观察可见大量细胞附着于材料外表面及孔隙内,细胞形态多样,细胞外大量颗粒样分泌物,细胞间以细胞突起相连。5.1.3增殖状况检测结果流式细胞仪检测显示DMP1-POMF细胞复合ADM体外培养14天,增殖指数与单层贴壁培养无显著差异(P>0.05);5.1.4流式细胞仪蛋白表达定量分析检测结果蛋白表达定量分析结果显示DMP1-POMF细胞复合ADM培养DMP1、DSP、I型胶原蛋白均为阳性表达,且复合ADM培养14天蛋白表达水平高于单层贴壁培养细胞(P<0.05)。5.2细胞复合ADM裸鼠肌肉内种植观察结果5.2.1 HE染色组织学观察结果DMP1-POMF细胞复合ADM培养种植物组织学切片HE染色7d未见钙化结节,14d可见少量牙本质样小钙化结节,21d可见数量较多小钙化结节及少量较大含有牙本质小管的牙本质样钙化团块。POMF复合ADM支架及DMP1-POMF单细胞悬液种植物的对照组中均未见钙化结节形成。5.2.2免疫组织化学观察结果DMP1-POMF细胞复合ADM培养7d种植物组织学切片免疫组织化学DMP1、DSP及I型胶原蛋白染色阳性,胞质中均可见大量棕色阳性颗粒。POMF细胞复合ADM的对照组裸鼠内种植组织DMP1、DSP及I型胶原蛋白染色阴性。6 DMP1基因转染成纤维细胞团对牙本质缺损的修复作用6.1细胞培养观察结果6.1.1 HE染色光镜观察DMP1-POMF细胞团三维立体培养组织切片可见:细胞团外形呈球状,外周由细胞外基质包绕,细胞呈园形或多边形,细胞核内可见少量圆形或卵圆形核仁。培养21d,DMP1-POMF细胞团中可见多个大小不等的钙化结节(Fig 1)。POMF细胞团三维立体培养组织切片可见:细胞团外形呈球状,外周由细胞外基质包绕,细胞呈椭园形或多边形,细胞核内可见少量圆形或卵圆形核仁。培养21d,POMF细胞团中无钙化结节形成(Fig 2),6.1.2光镜下细胞计数DMP1-POMF细胞单层贴壁培养6d至21d,细胞数目增加。DMP1-POMF细胞团培养从14天开始细胞数目明显减少(Fig 3),细胞密度降低。故在本实验中选择体外培养14d的细胞团进行体内修复牙本质缺损的实验研究。6.1.3流式细胞仪检测细胞增殖周期DMP1-POMF细胞团的细胞增殖指数低于单层贴壁DMP1-POMF细胞(Fig 4)。DMP1-POMF细胞团细胞增殖指数与POMF细胞团细胞增殖指数无显著差异(P>0.05)。6.1.4流式细胞仪进行蛋白表达定量分析DMP1-POMF细胞团培养14天DMP1、DSP、I型胶原蛋白表达水平高于单层贴壁培养DMP1-POMF细胞,差异有显著性(P<0.05)(Fig 5)。6.2细胞团体内修复牙本质缺损观察结果6.2.1组织学观察结果6.2.1.1牙本质缺损修复术后1个月组织学观察结果DMP1-POMF细胞团修复组未见牙髓组织坏死,可见大量修复性牙本质团块,基质均匀致密(Fig 6)。部分团块可见埋入的少量细胞,呈骨样牙本质;部分团块可见前期牙本质及外层的管样牙本质形成(Fig 7)。POMF细胞团修复组同样未见牙髓组织坏死,但修复性牙本质形成明显少于转基因细胞团组,并未见管样牙本质形成。氢氧化钙修复组牙髓组织表面可见薄层坏死,毛细血管扩张充血,少量炎细胞浸润,修复性牙本质形成的厚度及密度均低于细胞团修复组。6.2.1.2牙本质缺损修复术后3个月组织学观察结果DMP1-POMF细胞团修复组大部分标本均有完整的牙本质桥形成,结构致密呈管状牙本质状,将牙本质缺损封闭(Fig 8),桥下方可见排列整齐的造牙本质细胞,牙髓组织正常,偶见轻度毛细血管扩张。POMF细胞团修复组形成牙本质桥薄于DMP1-POMF细胞团修复组。氢氧化钙组可见牙本质桥形成,但大部分标本修复性牙本质较细胞团组薄且形态不规则,部分标本髓腔内可见少量不规则钙化团块。6.2.2免疫组织化学观察DMP1-POMF细胞团修复组标本免疫组织化学检测显示,修复性牙本质中埋入的细胞DMP1染色呈阳性表达,胞浆内可见特异性黄褐色颗粒(Fig 9,10)。POMF细胞团修复组修复性牙本质中的细胞DMP1染色阴性。结论:1牙本质基质蛋白1 (DMP1)基因转染的口腔粘膜成纤维细胞(POMF)(DMP1-POMF细胞)可以稳定表达成牙本质细胞的特异性基因DMP1、Ambn及DSP,对细胞增殖和染色体无影响。2 DMP1-POMF细胞具有矿化的生物学特性。3 DMP1-POMF细胞可以作为组织工程化牙本质的种子细胞。4 DMP1-POMF细胞与脱细胞真皮基质(ADM)支架有良好的相容性。5 DMP1-POMF细胞复合脱细胞真皮基质支架裸鼠内种植成功构建牙本质结构形成。6脱细胞真皮基质(ADM)支架可以作为组织工程化牙本质的支架材料。7 DMP1-POMF细胞细胞团三维立体培养体内盖髓,可以促进牙本质缺损的修复。更多还原

【Abstract】 The development of tissue engineering enhances the possibility and feasibility for damaged oral tissue reparation and regeneration. Currently, the research on tissue engineering is one of the most fascinating topics in the field of oral and maxillofacial plastic surgery. Many scientists pay more attentions on regenerating tooth by using tissue engineering technology worldwide, since teeth play the important role in the development of jaws, the maintenance of maxillofacial profile, the function of language and chewing. As the main ingredient of the tooth, dentin constructs the outlook of tooth and its regeneration is the key point in the research of tissue engineered tooth. Therefore, in this study the construction of seed cells, biological scaffold and tissue engineered dentin were investigated.Objectives:Oral mucosa fibroblasts are easier to be obtained and cultured. To establish the dentin matrix protein1-porcine oral mucosa fibroblasts (DMP1-POMF) as seed cells for tissue engineered dentin, the oral mucosa fibroblasts were transfected by dentin matrix protein 1( DMP1). To evaluate the feasibility of using DMP1-POMF as seed cells for tissue engineered dentin, the biological function of DMP1-POMF and the expression of odontoblasts specific protein was detected.DMP1-POMF cells which were established as seed cells were seeded onto acellular dermal matrix (ADM) scaffold. In order to evaluate the feasibility of using ADM as scaffold for tissue engineered dentin, the biocompatibility between the seed cells (DMP1-POMF) and scaffold was detected by HE staining, scanning electron microscopy and flow-cytometric analysis. To investigate the construction of tissue engineered dentin, the ectopic dentinogenesis of seed cells (DMP1-POMF) compound ADM was evaluated by the observation of growing of the compound in vivo, the change of histologic structure and the expression of dentin specific protein.DMP1-POMF cells which were established as seed cells were cultured as three-dimensional pellet. To evaluate the accumulation of extracellular matrix protein in three-dimensional pellet, the cells morphologic characteristics was observed and the flow-cytometric analysis was carried out. The histological and immunohistochemistric detection was performed after the pulp capping with DMP1-POMF cells pellet to investigate the function of DMP1-POMF pellet on dentin regeneration in vivo.Methods:1 Construction of pEGFP-DMP1 PlasmidThe full length of dentin matrix protein 1( DMP1) cDNA was linked into an eukaryotic expression vector pEGFP-Cl . The recombinant plasmid pEGFP -DMP1 was then amplified and tested by an enzyme cutting technique in vitro.2 Cell Culture, Dentin Matrix Protein 1 Transfection2.1 Porcine Oral Mucosa Fibroblasts (POMF) Primary CulturePrimary fibroblast cell cultures were established from porcine oral mucosa biopsies. The samples of lamina propria connective tissue of oral mucosa were subsequently rinse three times with l ml ice-cold PBS supplemented with penicillin (100IU/ml)and streptomycin(100ug/ml)(Sigma, USA). Then connective tissue fragments were transferred to 60-cm2 cell culture bottles containing 2ml Dulbecco,s modified Eagle,s medium(DMEM) (Gibco BRL),10% fetal calf serum (FCS) (Gibco BRL), penicillin (100IU/ml)and streptomycin (100ug/ml; Sigma). Cells were cultured at 37°C and 5% CO2 until reaching 60% confluence. Growing cells were trypsinized and transferred to new cell culture bottles without the original connective tissue fragments.2.2 Mesenchymal Stem Cells (MSC) CultureMSC were isolated from bone marrow and purified by centrifuge in vitro.The proliferation and growth characteristics were observed in primary and passage culture.2.3 Dentin Matrix Protein 1 TransfectionGroup A: Porcine oral mucosa fibroblasts (POMF) were transfected by dentin matrix protein1(DMP1) and were named dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) in this research.Group B: Porcine oral mucosa fibroblasts (POMF) were transfected by empty vector pEGFP-C1 and were named pEGFP-C1-porcine oral mucosa fibroblasts(C1-POMF) in this research.Group C: Mesenchymal stem cells (MSC) were transfected by dentin matrix protein1(DMP1) and were named dentin matrix protein1- mesenchymal stem cells(DMP1-MSC) in this research.POMF,and MSC named group A and C between the 4th and 6th passages, were plated at 1×105 into 6-well culture dishes and transfected with 2μg pEGFP-DMP1 and Lipofectamine 5μl/ml (Invitrogen, USA). Solutions consisted of 150μl DMEM with no FCS were added to the plasmid vector in a concentration of 2μg/ml, mixed 150μl DMEM containing Lipofectamine for a concentration of 5μl/ml. After 20 min at 22°C, the DNA/lipofectamine complexes in a final volume of 300μl were added to the cell culture wells (3.5cm in diameter) containing 1×105 fibroblasts and cultured at 37°C and 5%CO2. After 5h, the DNA/lipofectamine complexes were removed, and a fresh medium with 10% FCS was added to stop transfection. The expression of recombinant plasmid pEGFP -DMP1 and transfer efficiency were evaluated by fluorescent microscope. Transfected cells achieved subconfluence after 7 days. At the 3rd passage, the cells were diluted 1:10, cultured with G418 sulfate at 0.8μg/ml of medium. Individual colonies were isolated and expanded by plating cells at low density. POMF named group B were mock transfected with empty vector pEGFP-C1 and selected identically. Cells were harvested for further analysis.3 The biological influence on fibroblasts of gene transfection3.1 RT-PCRTotal RNA was isolated from cultured cells during transfection on 24h, 48h and G418-resistant with the use of Trizol reagent (Invitrogen, USA). First-strand cDNA syntheses were performed by reverse transcription with the SuperScript pre-amplification system.The expression of DMP1, ameloblastin (Ambn) and dentin sialoprotein (DSP)was detected.3.2 Flow-cytometric Analysis of Cell-cycle and DNA PloidCell-cycle phases and DNA ploid were determied by flow-cytometric analysis based on expression of the ki-67 antigen (G1, G2/M, S) and the S-phase-specific proliferating cell nuclear antigen (PCNA) (Landberg et al., 1990). DMP1-POMF and C1-POMF were digested with trypsin/ethylene -dianmine tetraacetic acid (EDTA) into single cell suspensions (2×105 cells). Then the cells were fixed with cold ethanol (70%) on ice and incubated with the monoclonal antibodies specific to the cell cyucle-associated antigens ki-67 conjugated to FITC and PCNA conjugated to PE(1/10) (Dako corp) respectively. After being washed twice in PBS, the cells were analyzed with the use of a FACSCalibur flow cytometer.3.3 In vitro Assay for lnducement of Mineralized Nodule Formation and von Kossa StainingThe mineralization microenvironment was created by treating the DMP1-POMF , C1-POMF and DMP1-MSC (80-90% confluent) with 10mMβ-glycerophosphate and 100μg/ml ascorbic acid along with 10nM dexamethasone (Sigma, USA). The cells undergoing mineralization (40 days in culture) were fixed with 10% formalin in neutral buffer (Sigma) for 15min. The slides were washed with distilled water and then treated with 1% AgNO3 for 1h, washed again with distilled water, and treated with 2.5% sodium thiosulfate for 5 min. The specimens were counterstained and then examined under a light microscope.3.4 Transmission Electron Microscope (TEM)The DMP1-POMF , C1-POMF and DMP1-MSC cultured 21d were digested into single cell suspensions. Then the cells were washed twice, fixed in cold glutaraldehyde, post-fixed in osmium tetroxide, dehydrated in an ethanol series, embedded in epoxy resin, and then observed under transmission electron microscope.4 An experimental study on fibroblasts transfected by DMP1 loading on acellular dermal matrix4.1 Acellular dermal matrix (ADM) scaffold CultureFor loading of cells into transplant vehices, multi-fracture ADM cut by ophthalmologic scissors were prewetted in complete medium.Group A: The dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) were seeded directly onto ADM scaffold.Group B: Porcine oral mucosa fibroblasts (POMF) were seeded directly onto ADM scaffold.Approximately 2×105 cells were loaded per 0.2×0.2cm2 ADM by capillary action and then incubated at 37°C for 7 days.4.2 Histological ObservationThe transplants were fixed in 4% paraformal dehyde overnight and processed for paraffin-embedding. The sections of 4.5μm thickness were stained in hematoxylin and eosin.4.3Scanning Electron Microscopy (SEM)To evaluate the characteristics of the cellular proliferation and cell-material attachment electron microscopy imagine was conducted on carbon-coated, polished block faces of poly-methyl-methacrylate (PMMA) embedded ADM scaffold with DMP1-POMF, the POMF as control. The samples were imaged in a Zeiss DSM 962 digital scanning electron microscope operated at 20 or 30 KV in the back scattered electron mode with the use of a KE (Toft, cambs, UK) solid-state back-scatter electron detector. 4.4 Flow-cytometric Analysis of Cell-cycle and Protein Quantification of DMP1, DSP and Collagen Type ICell-cycle phases were determied by flow-cytometric analysis based on expression of the ki-67 antigen (G1, G2/M, S) and the S-phase-specific proliferating cell nuclear antigen (PCNA) (Landberg et al., 1990). The adherent monolayer DMP1-POMF and the DMP1-POMF transplanted in ADM scaffold were digested with trypsin/ethylene-dianmine tetraacetic acid (EDTA) into single cell suspensions (2×105 cells). Then the cells were fixed with cold ethanol (70%) on ice and incubated with the monoclonal antibodies specific to the cell cyucle-associated antigens ki-67 conjugated to FITC and PCNA conjugated to PE(1/10) (Dako corp) respectively. DMP1, DSP and collagen Type I polyclonal antibodies was added directly to the above-mentioned 2×105 cells for l hr on ice. The cells were then incubated with goat anti-mouse IgM conjugated to FITC (1/50 dilution, DAKO Corp.) for 45min on ice. After being washed twice in PBS, the cells were analyzed with the use of a FACSCalibur flow cytometer. Positive expression was defined as the level of fluorerscence greater than 99% of the corresponding isotype-matched control antibodies.4.5 Subcutaneous Transplantation ProcedureThe DMP1-POMF with ADM scaffold were transplanted into 10-week-old immuncompromised beige mice. Nine immuncompromised beige mice were used. Muscle of thigh incisions were made on each mouse and sarcous pockets were made by blunt dissection. A single transplant was placed in each pocket with up to 2 transplants per animal. The transplants were recovered at 7, 14 and 21 days post-transplantation, fixed with 4% formalin, decalcified with buffered 10% EDTA (PH8.0), and then embedded in paraffin. The sections of 4.5-μm thickness were stained in hematoxylin and eosin .The avidin-biotin peroxidase complex method was used for immunohistochemistry. The POMF with ADM scaffold and The DMP1-POMF single cell suspensions were as control.5 The dentin formation by dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) pellete5.1 Cell Pellet CultureGroup A: The dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) pellet culture.Group B: The adherent monolayer DMP1-POMF culture.Group C: Porcine oral mucosa fibroblasts (POMF) pellet culture.The pellet culture of the cells was performed. Briefly l-ml aliquots containing 2×105 cells were centrifuged in a 15ml conical polypropylene tube at 1000 rpm for 5 min. Pellets were maintained in DMEM supplemented with 10% heat-inactivated bovine calf serum and 50μg/ml L-ascorbic acid phosphate and penicillin-streptomycin. The medium was changed two times per wk.5.2 Histological ObservationPellets on days 7,14 and 21 were fixed in 4% paraformal dehyde overnight and processed for paraffin-embedding. The sections of 4.5μm thickness were stained in hematoxylin and eosin.5.3 Cells CountedThe cells were dispersed by trypsin and counted at each time point during pellet culture and monolayer culture.5.4 Flow-cytometric Analysis of Cell-cycle and Protein Quantification of DMP1, DSP and Collagen Type ICell-cycle phases were determied by flow-cytometric analysis based on expression of the ki-67 antigen and the S-phase-specific proliferating cell nuclear antigen. The adherent monolayer DMP1-POMF and the DMP1-POMF pellet were digested with trypsin/ethylene-dianmine tetraacetic acid (EDTA) into single cell suspensions (2×105 cells). The adherent monolayer POMF and the POMF pellet were as control. Then the cells were fixed with cold ethanol (70%) on ice and incubated with the monoclonal antibodies specific to the cell cyucle-associated antigens ki-67 conjugated to FITC and PCNA conjugated to PE(1/10) (Dako corp) respectively. DMP1, DSP and collagen Type I polyclonal antibodies was added directly to the above-mentioned 2×105 cells for l hr on ice. The cells were then incubated with goat anti-mouse IgM conjugated to FITC (1/50 dilution, DAKO Corp.) for 45min on ice. After being washed twice in PBS, the cells were analyzed with the use of a FACSCalibur flow cytometer. Positive expression was defined as the level of fluorerscence greater than 99% of the corresponding isotype-matched control antibodies.5.5 The dentin formation by DMP1-POMF pellete in vivo Group A: The dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) pellet reparationGroup B: Porcine oral mucosa fibroblasts (POMF) pellet reparationGroup C: Ca(OH)2 reparationA total of 48 permanent teeth were used from 4 adult miniature pigs with a mean age of 1 year. Animals were randomly divided into two groups.Observation time was 1 month group (n=2),3 months group (n=2).The animals were anesthetized with an injection of 3% pentobarbital sodium (lml/kg).The viable pulp tissue was exposed through a central cavity prepared with a sterie,round steel bur,2mm in diameter. Bleeding after pulpotomy was controlled with sterile cotton pellets.After the bleeding had stopped, the test materials, transfected POMF pellets (group A), non-transfected POMF pellets (group B) or calcium hy droxide.(group C),were applied directly onto the exposed pulp tissue respectively. After a 1-minute waiting period, the cavities were sealed with a glass-ionomer cement.At 1 or 3 months after surgery, animals were killed. Experimental teeth were removed in toto and fixed in cold 4% neutral buffered formaldehyde for 24 hours. The teeth were then demineralized in 12.5% ethylenediaminetetraacetic acid (EDTA) and subsequently embedded in paraffin. Following longitudinal serial sectioning, the section was stained with hematoxylin and eosin. Unstained sections were submitted to immunohistochemistry.Results:1 Plasmid Identification The constructed pEGFP-DMP1 could produced 1.5kb and 4.7kb fragments by an enzyme cutting technique at Xho I and EcoR I sites and their size were same as those of DMP1 and pEGFP–Cl. This indicated that DMP1 cDNA was successfully subcloned into pEGFP–Cl.2 DMP1 TransfectionThe fluorescence microscopy showed that fluorescence light was spread over a broader cellular distribution both in the nucleus and cytoplasm in all of POMF transfected with pEGFP–Cl, but fluorescence light was focused on the cellular membrane and cytoplasm of POMF and MSC transfected with pEGFP -DMP1. The transfection showed an efficiency of 36.5% at day 2 in porcine fibroblasts transfected DMP1. Cells transfected with pEGFP -DMP1 exhibited profound changes in their morphology. These cells were columnar and polarized tended to align themselves in straight parallel lines and had long dendrite-like processes. In contrast, cells transfecting mock pEGFP–Cl did not exhibit any characteristic morphological change with a silm spindle pattern.3 The variation of fibroblasts by gene transfectionRT-PCR analysis demonstrated that transfection of DMP1 triggered the differentiation of DMP1-POMF into odontoblast-like cells, with expression of Ambn corresponding to early stage markers and DSP corresponding to the terminally differentiated state. It was the most unexpected finding in this study that the expression of odontoblast-specific markers Ambn and DSP during the cellular differentiation process. As expected the mock fibroblasts (expressing the vector alone) did not express any of the odontoblast-specific genes. 4 In vitro Assay for lnducement of Mineralized Nodule Formation and von Kossa StainingAn in vitro nodule formation assay was carried out to determine whether the up-regulation of odontoblast-specific gene transcription resulted in a mineralized matrix formation by the von Kossa staining. Nodule formation due to secretion of extracellular matrix proteins in the presence of phosphate ions and ascorbic acid has been considered to be an important feature for mineralization and precedes mineralization. It was observed that transfection of DMP1 enhanced the onset of mineralization in fibroblasts, the size of the nodule formed was significantly large and the overall kinetics of nodule formation was favored by at least 4- to 5- fold in transgenic cell lines when compared with the mock cells.5 The Result of Transmission Electron Microscope (TEM) Discovery The ultrastructural features of pEGFP-DMP1 transfected fibroblast cells showed that there was massive expansive rough endoplasmic reticulum and incremental myelin sheath-like figures and matrix vesicles in the cytoplasm. The cisternae were dilated to varying degrees and were full of abundant proteinoid substances. The collagenous fibrillae was distributed widely intercellular.6 The effect of DMP1 on generation of ectopic osteodentin in transplantation Transfected cells adhered on the fiber of ADM for 24 hours began to proliferate and grow in the framework of three-dimension. The results of this transplantation demonstrated that the fibroblasts transplants had not yet generated mineralized tissues at 7 days. At 14 days ADM post-transplantation with transfected POMF generated osteodentin on the surface of ADM in all transplants. At 21 days ADM post-transplantation, abundant and large osteodentin formation was formed. Immunohistochemical staining showed that DMP1, DSP and collagen type I was positive in the connective tissue compartment prior to the generation of osteodentin formation in fibroblasts transplants. These proteins were not expressed in the mock fibroblasts transplants.5 The dentin formation induced by transfected fibroblasts pellet The dentin matrix protein1-porcine oral mucosa fibroblasts(DMP1-POMF) in the pellet were oval or polygonal, and the nuclei contained a few round or ovoid nucleoli. The pellet progressively became spherical on days 14 and the extracellular matrix accumulated. The histopathological results revealed that the restorative dentin bridge was observed a month later and the bridge was mainly tubular dentin in 3 month. There were well developed odontoblasts under the bridge . The cases of dentine bridge formation of non-transfected POMF pellet and Ca(OH)2 group were less than that of DMP1 transfected pellct .Conclusion:1 DMP1、Ambn and DSP gene could be expressed stably in Porcine oral mucosa fibroblasts (POME) transfected by dentin matrix protein1 (DMP1) (DMP1-POMF cells).2 The DMP1-POMF cells have biological characteristics of mineralization.3 DMP1-POMF cells could be used as seed cells for dentin formation.4 DMP1-POMF could be attached on the surface of acellular dermal matrix(ADM) and proliferated actively.5 DMP1-POMF cells attached on the surface of acellular dermal matrix could be successfully constructed to the dentin formation by implanted subcutaneously into nude mice.6 ADM could be used as scaffold material for dentin formation.7 DMP1-POMF pellet could induce the formation of reparative dentin in vivo.更多还原