滨珊瑚用于组织工程骨构建的实验研究
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摘要
组织工程是应用细胞生物学和工程学的原理，对病损组织结构和功能的修复与重建进行研究开发的一门新兴科学。与胚胎干细胞、基因工程一起被认为是生命科学发展史上的又一里程碑，标志着人类将走出单纯器官移植领域，步入组织、器官再造的新领域。
骨是最早在实验室获得组织工程化的组织之一，是继皮肤、软骨之后最有希望进入临床应用的组织。但目前组织工程骨应用亟需解决的问题有①具有强的增殖和分化功能的多能干细胞；②组织工程骨的免疫及成骨细胞库的建立；③支架材料的强度及和周围骨床的结合。基于以上要求，本实验进行了以下三部分实验的研究。
第一部分 骨髓间充质干细胞的生物学特性的研究
实验一 成人骨髓间充质干细胞培养与鉴定
目的：建立体外分离培养人骨髓间充质干细胞(human mesenchymal stem cells HMSCs)的方法。方法：穿刺抽取成人骨髓约20ml，通过离心法获取HMSCs，体外培养并传代，观察原代及传代细胞的细胞形态及生长规律，第4代细胞用矿化液连续培养30天，在倒置显微镜下观察矿化结节形成情况。结果：体外利用骨髓基质细胞培养液成功培养了HMSCs，并连续传代5～7代。利用第4代细胞用矿化液连续培养30天，矿化结节形成，在细胞间有钙盐沉积。结论：HMSCs可以在体外培养成功，并可定向诱导为成骨细胞。
实验二 成骨细胞接种于珊瑚体外培养的扫描电镜观察
目的：研究家兔成骨细胞在珊瑚上的贴附与生长。方法：珊瑚处理后，将骨髓间充质干细胞来源的成骨细胞与其复合，体外培养2天、4天、7天、14天、28天后，扫描电镜下观察成骨细胞在珊瑚表面贴附数量和形态，分裂增殖能力及功能。结果：从2天开始，珊瑚表面及孔内即有细胞粘附生长，随时间延长，细胞数量急剧增加，到14天～28天时，全部长满，并分泌大量胶原纤维和形成钙化结节。结论：西沙群岛滨珊瑚作为组织工程骨的载体，在体外与成骨细胞具有良好的相容性。
实验三 bFGF和BMP联合使用对骨髓间充质干细胞的生物学效应
目的：研究不同剂量的碱性成纤维细胞因子(bFGF)和重组人骨形成蛋白-2(rhBMP2)单独或交互作用对成人骨髓间充质干细胞(BMSCs)增殖、碱性磷酸酶(ALP)和总蛋白含量合成影响。方法：将培养的BMSCs接种于96孔板，暴
后 庆 第回旱区大学俗士学位蛤文 第3 页
露于不同浓度的bFG或（和）thBMPZ中，观察BMSCS增殖、lP活性、蛋白
质含量的变化。结果：ie’n－可明显僻BMSCs的增殖，但却抑制Al，和总蛋白
含量；rhBMIQ对BMSCS的减性磷酸酶活性、蛋白质含量均有明显的促进作用，
且呈剂量依赖性，rilnMtu对BMSCs的增殖也有—定促进作用；而二者交互联合
应用对BMSCs的增殖和分化有协同作用。结论：二者交互联合使用，可提高BMSCs
向成骨细胞转化和增殖并提高骨形成的生物效应。
第二部分 组织工程骨的构建及免疫学研究
实铬一 高孔隙串滨瞩瑚用于组织工程骨的构建
目的：探讨西抄群岛蔚惯辉瞒珊晚眸为骨组织工程皮韶刺料的可行性。方法：取
4周龄兔骨髓，分离骨髓间充质干细胞，懈僻，经诱导狐为成骨细胞，欲
至滨珊瑚材料，植入裸赃一中。对照组单纯植入摈珊猢。分另于旱、8周取
材，行大体观察、XHN，观察新骨形成倩况。结果：4周时X线
片有店碎密度阴身伯以象，HE染名…u闪宅所骨形咸 吕周时X线阴旧拈敦像密度更高，
HE染色可见大量新骨形成并相互连接依滑，J择绢贫胸，骨细胞位于陷窝中。结论：
高孔隙率滨珊瑚可以作为骨组织口口涅的支架材料。
实验二 同种异体成骨细胞移桓的免疫学研究
目的：孤－一，探讨组织工程骨组织植
人体内后可能的免疫排斥机制，并为应用于工程骨的种子细胞寻找可靠的种系来
源。施行成骨细胞同胎、同脓体、同种异棚及自脚－内雕，
分另于术后1周、2周、4周、8周行免疫学及组织沮态u。结果：各组动物在
浴时直后玉仑周内均司耘觑9笋怵杉衬扫亢体，同种异基因移眶泪狂茨卖时间最久，异基因
组隧拽扫岗昏际高于同阑哆纠驷组。结论：自体、同胎及同种异体或同阳咕契塞因细胞均
可作为组织江程骨移植可靠的细胞来源。
实验三 以羹巨为活休生物反厦吕构建可移植组织工程骨的可 研究
目的：研究以裸鼠为活体生物反应氓奸朗彭组织D用群罚茨厅自体私阳拍饭刃厅性。方法
常城胰窍日舟固眨间夭洞定R翊胞后，选取第四脚胞向成骨细胞诱导分化。与珊瑚复
4iR 崔pV课即内，巴周后取出形沥凌1罪剁织，放人自林家旧炙炎抑叽肉组织间隙
中，分别于14、28、56 XNll：silllirff中棚鼠扶中陀瓤竟及2、7、14、28、56天检
测thZ水平，8周标本行免疫组化滦色，判定局部免疫反应水平。结果：整个实
验过程
Tissue engineering is an interdisciplinary science of co-development and combination of modem cell biology, biomaterial science and engineering. The establishment of tissue engineering is a breakthrough over conventional organ transplantation to restore the function and structure of tissues lost to the injury and disease. Tissue engineering, embryonic stem cell and genetic engineering together are regarded as the three marvels in life science development history.
Bone tissue has been successfully reconstructed in lab. In the light of present knowledge, the formation and repair of bone with tissue engineering require study on: (1)a source of pluripotentional mesenchymal stem cells with strongly ability of proliferation and differentiation; (2)immunology of tissue engineered bone and establishment of osteoblast bank; (3)introduce the metal rack to bone tissue engineering. The present study is based on these requirements and contains three main parts.
Parti Biok>g^cal characteristic research of marnnvmesencfavnial stem ceOs l.Cuttrvation and identification of human mesenchymal stem cells Objective To create the model of human mesenchymal stem cells (HMSCs) separation and culture in vitro. Methods Adult bone marrow about 20ml were drawed by puncture. After centrifuging, HMSCs were harvested, cultivated aoi transferred The growm and the characteristics of HMSCs were observed through cell morphobgical surey, cell growth and proKferationand ALP activity. The cells of fourth passage were cultured continuously in mineral condition for 30 days, the mineral nodules were observed by Von-Kossa staining. Results HMSCs were cultured in vitro and could be transferred for 5-7 passages steadily. The cells of fourth passage were ALP positive. When cultured up to 30 days, \bn-Kossa staining showed there was calcium deposition in the nodules. Conclusion The HMSCs were cultured successfully in vitro and can be induced to osteoblast cells. It wfll be a kind of excellent seeding-cells for bone tissue engineering.
ZJScanning electron microscope study of osteoblast cultivated with coral in vitro Objective To investigate osteoblasts growth and adhesion to natural coral Methods Ten
cuboid porous Xisha Islands coral ( 5mm X 5mm X 5mm) were processed Then they were cultivated in vitro simultaneously with osteoblast derived from mesenchymal stem cells of rabbit for 2 days, 4 days, 7 days, 14 days and 28days.The morphogenetic pattern and number of anchored cells, capability of cleavage and proliferation, and osteoblasts function were observed under scanning electron microscope. Results From 2 days, cells grew into the porous coral The number of cells enhanced greatly with time lasting. To 14-28 days, cells overgrew and many secretory collagen fiber bundles and mineralized nods were observed Coadusioo Xisha Islands natural coral have good histocompatibility with the osterblasts as carrier of tissue engineering bone in vitro.
3.The biological effects of combination of BMP and bPGF to bone mesenchymal stems cells
Objective To investigate the effects of BMP and bFGF on bone mesenchymal stems cells transformation and protiferatioa Methods Subcuturing the 4th passage of BMSCs into 96 pore plate and exposed to rhBMP2 and (or) bFGF of different concentratioa Cell proliferation, alkaline phosphate activity (ALP) activity and total cellular protein were measured Results The results clearly demonstrated that bFGF could promote the proliferation of BMSCs, but the content of ALP and total celhuar protein were suppressed RhBMP2 could enhance total cellular protein of BMSCs significantly at bw dose in a dose responsible manner, and increase cell proliferation. Synergistic action to the proliferation and differentiation of BMSCs was found Conclusion BFGF and BMP can enhance the BMSCs differentiated into osteoblast It indicates that BMSCs accompanied with cooperation of them might be an applicable method for bone defect repair. Part II Construction of tissue engineered bone and immunological study l.Tissue engineering bone with higher porosity po
骨是最早在实验室获得组织工程化的组织之一，是继皮肤、软骨之后最有希望进入临床应用的组织。但目前组织工程骨应用亟需解决的问题有①具有强的增殖和分化功能的多能干细胞；②组织工程骨的免疫及成骨细胞库的建立；③支架材料的强度及和周围骨床的结合。基于以上要求，本实验进行了以下三部分实验的研究。
第一部分 骨髓间充质干细胞的生物学特性的研究
实验一 成人骨髓间充质干细胞培养与鉴定
目的：建立体外分离培养人骨髓间充质干细胞(human mesenchymal stem cells HMSCs)的方法。方法：穿刺抽取成人骨髓约20ml，通过离心法获取HMSCs，体外培养并传代，观察原代及传代细胞的细胞形态及生长规律，第4代细胞用矿化液连续培养30天，在倒置显微镜下观察矿化结节形成情况。结果：体外利用骨髓基质细胞培养液成功培养了HMSCs，并连续传代5～7代。利用第4代细胞用矿化液连续培养30天，矿化结节形成，在细胞间有钙盐沉积。结论：HMSCs可以在体外培养成功，并可定向诱导为成骨细胞。
实验二 成骨细胞接种于珊瑚体外培养的扫描电镜观察
目的：研究家兔成骨细胞在珊瑚上的贴附与生长。方法：珊瑚处理后，将骨髓间充质干细胞来源的成骨细胞与其复合，体外培养2天、4天、7天、14天、28天后，扫描电镜下观察成骨细胞在珊瑚表面贴附数量和形态，分裂增殖能力及功能。结果：从2天开始，珊瑚表面及孔内即有细胞粘附生长，随时间延长，细胞数量急剧增加，到14天～28天时，全部长满，并分泌大量胶原纤维和形成钙化结节。结论：西沙群岛滨珊瑚作为组织工程骨的载体，在体外与成骨细胞具有良好的相容性。
实验三 bFGF和BMP联合使用对骨髓间充质干细胞的生物学效应
目的：研究不同剂量的碱性成纤维细胞因子(bFGF)和重组人骨形成蛋白-2(rhBMP2)单独或交互作用对成人骨髓间充质干细胞(BMSCs)增殖、碱性磷酸酶(ALP)和总蛋白含量合成影响。方法：将培养的BMSCs接种于96孔板，暴
后 庆 第回旱区大学俗士学位蛤文 第3 页
露于不同浓度的bFG或（和）thBMPZ中，观察BMSCS增殖、lP活性、蛋白
质含量的变化。结果：ie’n－可明显僻BMSCs的增殖，但却抑制Al，和总蛋白
含量；rhBMIQ对BMSCS的减性磷酸酶活性、蛋白质含量均有明显的促进作用，
且呈剂量依赖性，rilnMtu对BMSCs的增殖也有—定促进作用；而二者交互联合
应用对BMSCs的增殖和分化有协同作用。结论：二者交互联合使用，可提高BMSCs
向成骨细胞转化和增殖并提高骨形成的生物效应。
第二部分 组织工程骨的构建及免疫学研究
实铬一 高孔隙串滨瞩瑚用于组织工程骨的构建
目的：探讨西抄群岛蔚惯辉瞒珊晚眸为骨组织工程皮韶刺料的可行性。方法：取
4周龄兔骨髓，分离骨髓间充质干细胞，懈僻，经诱导狐为成骨细胞，欲
至滨珊瑚材料，植入裸赃一中。对照组单纯植入摈珊猢。分另于旱、8周取
材，行大体观察、XHN，观察新骨形成倩况。结果：4周时X线
片有店碎密度阴身伯以象，HE染名…u闪宅所骨形咸 吕周时X线阴旧拈敦像密度更高，
HE染色可见大量新骨形成并相互连接依滑，J择绢贫胸，骨细胞位于陷窝中。结论：
高孔隙率滨珊瑚可以作为骨组织口口涅的支架材料。
实验二 同种异体成骨细胞移桓的免疫学研究
目的：孤－一，探讨组织工程骨组织植
人体内后可能的免疫排斥机制，并为应用于工程骨的种子细胞寻找可靠的种系来
源。施行成骨细胞同胎、同脓体、同种异棚及自脚－内雕，
分另于术后1周、2周、4周、8周行免疫学及组织沮态u。结果：各组动物在
浴时直后玉仑周内均司耘觑9笋怵杉衬扫亢体，同种异基因移眶泪狂茨卖时间最久，异基因
组隧拽扫岗昏际高于同阑哆纠驷组。结论：自体、同胎及同种异体或同阳咕契塞因细胞均
可作为组织江程骨移植可靠的细胞来源。
实验三 以羹巨为活休生物反厦吕构建可移植组织工程骨的可 研究
目的：研究以裸鼠为活体生物反应氓奸朗彭组织D用群罚茨厅自体私阳拍饭刃厅性。方法
常城胰窍日舟固眨间夭洞定R翊胞后，选取第四脚胞向成骨细胞诱导分化。与珊瑚复
4iR 崔pV课即内，巴周后取出形沥凌1罪剁织，放人自林家旧炙炎抑叽肉组织间隙
中，分别于14、28、56 XNll：silllirff中棚鼠扶中陀瓤竟及2、7、14、28、56天检
测thZ水平，8周标本行免疫组化滦色，判定局部免疫反应水平。结果：整个实
验过程
Tissue engineering is an interdisciplinary science of co-development and combination of modem cell biology, biomaterial science and engineering. The establishment of tissue engineering is a breakthrough over conventional organ transplantation to restore the function and structure of tissues lost to the injury and disease. Tissue engineering, embryonic stem cell and genetic engineering together are regarded as the three marvels in life science development history.
Bone tissue has been successfully reconstructed in lab. In the light of present knowledge, the formation and repair of bone with tissue engineering require study on: (1)a source of pluripotentional mesenchymal stem cells with strongly ability of proliferation and differentiation; (2)immunology of tissue engineered bone and establishment of osteoblast bank; (3)introduce the metal rack to bone tissue engineering. The present study is based on these requirements and contains three main parts.
Parti Biok>g^cal characteristic research of marnnvmesencfavnial stem ceOs l.Cuttrvation and identification of human mesenchymal stem cells Objective To create the model of human mesenchymal stem cells (HMSCs) separation and culture in vitro. Methods Adult bone marrow about 20ml were drawed by puncture. After centrifuging, HMSCs were harvested, cultivated aoi transferred The growm and the characteristics of HMSCs were observed through cell morphobgical surey, cell growth and proKferationand ALP activity. The cells of fourth passage were cultured continuously in mineral condition for 30 days, the mineral nodules were observed by Von-Kossa staining. Results HMSCs were cultured in vitro and could be transferred for 5-7 passages steadily. The cells of fourth passage were ALP positive. When cultured up to 30 days, \bn-Kossa staining showed there was calcium deposition in the nodules. Conclusion The HMSCs were cultured successfully in vitro and can be induced to osteoblast cells. It wfll be a kind of excellent seeding-cells for bone tissue engineering.
ZJScanning electron microscope study of osteoblast cultivated with coral in vitro Objective To investigate osteoblasts growth and adhesion to natural coral Methods Ten
cuboid porous Xisha Islands coral ( 5mm X 5mm X 5mm) were processed Then they were cultivated in vitro simultaneously with osteoblast derived from mesenchymal stem cells of rabbit for 2 days, 4 days, 7 days, 14 days and 28days.The morphogenetic pattern and number of anchored cells, capability of cleavage and proliferation, and osteoblasts function were observed under scanning electron microscope. Results From 2 days, cells grew into the porous coral The number of cells enhanced greatly with time lasting. To 14-28 days, cells overgrew and many secretory collagen fiber bundles and mineralized nods were observed Coadusioo Xisha Islands natural coral have good histocompatibility with the osterblasts as carrier of tissue engineering bone in vitro.
3.The biological effects of combination of BMP and bPGF to bone mesenchymal stems cells
Objective To investigate the effects of BMP and bFGF on bone mesenchymal stems cells transformation and protiferatioa Methods Subcuturing the 4th passage of BMSCs into 96 pore plate and exposed to rhBMP2 and (or) bFGF of different concentratioa Cell proliferation, alkaline phosphate activity (ALP) activity and total cellular protein were measured Results The results clearly demonstrated that bFGF could promote the proliferation of BMSCs, but the content of ALP and total celhuar protein were suppressed RhBMP2 could enhance total cellular protein of BMSCs significantly at bw dose in a dose responsible manner, and increase cell proliferation. Synergistic action to the proliferation and differentiation of BMSCs was found Conclusion BFGF and BMP can enhance the BMSCs differentiated into osteoblast It indicates that BMSCs accompanied with cooperation of them might be an applicable method for bone defect repair. Part II Construction of tissue engineered bone and immunological study l.Tissue engineering bone with higher porosity po
引文
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