用牙髓干细胞构建牙齿样结构的实验研究
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摘要
牙髓干细胞是牙齿组织工程中重要的种子细胞,自2000年发现以来已经取得了许多突破性的认识和发现,但在牙髓干细胞的进一步研究和实际应用中,还有许多问题有待认识和解决,例如今后怎样用它与其它一种或多种细胞构建复杂牙齿结构的问题,怎样在体外培养中提供合适的微环境来诱导牙髓干细胞向特定方向分化的问题,尤其是向成牙方向分化的问题。本研究首次尝试将人牙髓干细胞与异种牙胚上皮细胞构建异种嵌合体牙齿结构、检测异种牙胚细胞条件培养基对其有何诱导作用以及将牙髓干细胞与牙周膜干细胞进行复合来尝试构建工程化牙根样结构,以期为牙髓干细胞的进一步研究和应用奠定实验基础,为牙髓干细胞在今后牙齿组织工程的合理使用提供一些有益的提示和参考,并探索出用牙齿来源干细胞进行牙齿再生的一些新途径和新方法。所取得的主要结果如下:
一、用牙髓干细胞制备异种嵌合体牙齿
用酶消化法培养了牙髓干细胞,结果显示所培养的牙髓干细胞形态典型,表达间充质干细胞标志物STRO-1、CD106、CD29,有克隆形成能力,经诱导后有成骨和成脂肪细胞能力,表明我们培养的细胞是来源于间充质的干细胞。另外分离获得猪第三磨牙牙胚并培养牙胚细胞,用差速消化法纯化获得牙胚上皮细胞,免疫细胞化学鉴定显示其对CK14、AMBN阳性表达,而对Vimentin阴性表达。然后将人牙髓干细胞和猪的牙胚上皮细胞以不同方式复合植入到裸鼠皮下,6周后取材。结果发现大部分移植物没有形成任何有序的结构形态,但有一部分移植物中形成了含有类似牙本质、牙髓和牙釉质的牙齿样结构的组织,但是形成这种结构的概率较低。免疫组化结果证明我们形成的釉质样结构来源于猪牙胚上皮,而牙本质样结构源于人的牙髓干细胞。
本部分结果表明人的牙髓干细胞和猪的牙源性上皮细胞在体内,可以模拟类似牙胚发育时期的上皮-间充质相互作用方式,相互诱导,并有可能成功形成异种嵌合体结构。因而我们推断可见只要进一步优化条件,用人的牙齿间充质干细胞与异种的牙源性上皮细胞进行异种嵌合在牙齿再生中是有一定可行性的,并将可能成为解决牙釉质再生的一条新途径。
二、猪牙胚条件培养基诱导人牙髓干细胞牙向分化的实验研究
以前的实验已经证实人牙胚条件培养基可以为人牙髓干细胞提供成牙微环境,诱导牙髓干细胞向成牙方向分化。但由于实际中应用人牙胚条件培养基并不可行,所以本实验中我们验证了用异种牙胚条件培养基来诱导人牙髓干细胞可行性。我们培养发育期猪的第三磨牙牙胚,并收集获得牙胚条件培养基,同时培养人的牙胚并获得人牙胚条件培养基。将猪和人的牙胚条件培养基分别诱导人的牙髓干细胞,并从体内、体外比较了猪牙胚条件培养基和人牙胚条件培养基对人牙髓干细胞的诱导作用。结果表明:人牙髓干细胞经过一定时间的猪牙胚条件培养基诱导后,能在细胞形态上和细胞周期上发生改变,能具有更高的克隆形成能力和多向分化能力,能维持更好的干细胞特性,具有更强的增殖能力,表达更高水平的碱性磷酸酶,免疫组化和RT-PCR提示诱导后的牙髓干细胞能在蛋白水平和基因水平表达出一些牙向分化的标志,如DSP、DSPP、DMP1、BSP、OCN、OPN等。此外,经猪牙胚条件培养基诱导过的人牙髓干细胞植入裸鼠体内能获得形状更为规则的牙本质-牙髓复合体样结构,而且形成这种规则结构的概率从20%提高到超过70%。这些结果与人牙胚条件培养基诱导的牙髓干细胞表现类似,但常规培养的人牙髓干细胞没有表现出特别变化。
所得结果说明猪牙胚条件培养基拥有与人牙胚条件培养基一样的能力,诱导人牙髓干细胞向成牙方向分化。这提示我们异种牙胚条件培养基有可能作为一种牙齿再生的有用工具,我们在今后的牙组织工程领域,应用异种的牙胚条件培养基将有助于构建理想的工程化牙齿。
三、用人牙髓干细胞和牙周膜干细胞尝试构建工程化牙根的实验研究
牙髓干细胞和牙周膜干细胞是牙齿再生领域最有潜力的两个成体干细胞,那把这两种干细胞一起复合,能否在体内获得牙根样结构呢?本实验进行了这方面的初步尝试。我们体外培养了人牙周膜干细胞并对其性质进行了鉴定,结果显示牙周膜干细胞对Vimentin染色阳性,CK阴性,显示其为间充质来源, STRO-1呈阳性染色,说明培养的牙周膜干细胞具有间充质来源干细胞的表型特点。成脂、成骨诱导实验验证了牙周膜干细胞的多向分化能力与可塑性。本研究中将人的牙髓干细胞进行标记后,再与牙周膜干细胞以不同的方式复合并植入裸鼠体内,希望获得有一定结构的工程化牙根。结果显示:以牙根状CBB为支架的复合物取材后肉眼观呈外形良好的小牙根状,HE染色表明所有移植物中都有矿化发生,且能在一部分移植物中发现有典型Sharpey纤维样结构。在以CBB颗粒为支架的移植物中,肉眼观是形成了扁平状的结构,HE染色结果显示也能也有矿化现象发生,但矿化基质较牙根状CBB为支架的标本少。而单纯细胞团移植物除小部分出现了一定的矿化现象,大部分不能产生任何的有形结构。在三个实验组中都没有发现有典型牙髓牙本质复合体样结构形成。
本实验结果提示我们:在构建组织工程化牙根中,支架材料最好是有预成形状的,以引导相关干细胞沿着支架生长并形成大体结构良好的牙齿样组织;另外多种细胞构建一定结构时,不同的种子细胞最好是分别复合到支架上,而不应该简单混合到一起;细胞团移植不适合于用多种细胞复合构建有良好大体观的牙齿样结构。
Dental pulp stem cells (DPSCs) is an important candidate stem cells in tooth tissue engineering, and great progress have been made in identifying DPSC. Nevertheless, many problems have to be solved if we carry out further researches and application, for example,how to construct complex tooh like structure with DPSCs and other cells,and how to create appropriate microenvironment to guide DPSC to differentiate into specific direction, especially into odontogenic cell linage when DPSCs were cultured in vitro.This research tried to construct heterogeneous chimeric tooth structure with human DPSCs and xenogenic epithelial cells from tooth germ , to evaluate the effect of xenogenic tooth germ conditioned medium on human DPSCs, and to construct engineered bioroot by comination of human DPSC and periodontal ligament stem cells (PDLSC) .The aim of these studies was to establish experimental fondation for the further research and application of human DPSCs, to provide some useful clues for the future application of human DPSCs in tooth tissue engieering, and to explore some new methods and approaches for tooth regeneration with tooth derived stem cells.
The main achivements are as follows:
Section 1.Construction of heterogeneous chimeric tooth with human dental pulp stem cells
Human DPSC were cultured with enzyme digestion solution. Results showed cultured DPSCs had typical morphology, and expressed surface markers of mesenchymal stem cells STRO-1 ,CD06 and CD29. cultured DPSCs held the potential to form colony ,and to differentiate into osteoblats or adipocytes after induction. These data indicated the DPSCs we cultured were mesenchymal stem cells. Porcine dental germ of the third molar was isolated and dental germ cells were cultured. Epithelial cells of dental germ were obtained by speed-differentiated digestion solution. Immunocytochemeical staining showed tooth germ derived epithelial cells were positive for CK14 and AMBN, but negative for Vimentin. Then human DPSCs and porcine tooth germ derived epithelial cells were combined with different methods and transplanted into subcutaneous pockets of nude mice. Implants were harvested after 6 weeks. It was found most of the implants did not form any ordinate structure, whereas some implants contained dentin-pulp like structure and enamel like structure.The probability of forming this kind of structure was just 20%. Immunohistochemistry results provided evidence that the enamel like structure was originated from porcine tooth germ derived epithelial cells while dentin like structure was originated from human DPSCs.
The results of this part suggested that human DPSC and porcine tooth germ derived epithelial cells could mimic the interaction between epithelial cells and mesenchymal cells during tooth development, and induce each other and possessed the possibility to form heterogeneous chimeric tooth structure. So we could come to the conclusion that it is feasible to construct heterogeneous chimric tooth with human mesenchymal stem cells and xenogenic tooth germ derived epithelial cells, and this may be a solution to cope with ename regeneration.
Section 2.Odontogenic differentiation of human dental pulp stem cells induced by swine tooth germ cell conditioned medium
Previous studies showed that tooth germ cell conditioned medium (TGC-CM) hold the potential to induce dental pulp stem cells (DPSCs) to differentiate into odontogenic lineage. Nevertheless, human TGC-CM (hTGC-CM) is not feasible in practical application. We hypothesized that xenogenic TGC-CM may exert similar influence on DPSCs. In this study, we selected swine as a xenogenic origin and compared the effects of swine tooth germ cell conditioned medium (sTGC-CM) with hTGC-CM on human DPSCs. in vivo and in vitro. Results showed sTGC-CM, which is similar with the effect of hTGC-CM, was able to induce human DPSCs to make odontoblastic changes as indicated by remarkable morphological changes, increased proliferation ability, higher multipotential and colony forming capability, and expression of some odontogenic markers in gene and protein levels,such as DSP, DSPP, DMP1, BSP, OCN, OPN. Induced DPSC also maintained better stem-cell characteristics,. Besides, human DPSCs treated with sTGC-CM could form more regular dentine-pulp complex in vivo.The probability of forming this kind of structure was elevated from 20% to over 70%. These results was similar to DPSCs treated with hTGC-CM while quite different from that of control DPSCs.
Our data provided evidence that sTGC-CM held almost the same potential as hTGC-CM in inducing DPSCs to differentiate along odontogenic direction.. The observations suggested xenogenic TGC-CM may be a useful tool in tooth regeneration and the application of xenogenic TGC-CM may facilitate generating a bioengineered tooth from tooth derived stem cells in future.
Section 3 The trial of constructing engineered bioroot with human dental pulp stem cells and periodontal ligament stem cells
Undoubtedly, DPSC and PDLSC was the most promising postnatal stem cells in future tooth tissue engineering.Then, is it possible to get tooth root like structure in vivo if these two kinds of stem cells were combined and transplanted?We carried out this kind of trial. PDLSCs were cultured ex vivo and were characterizied. Results showed PDLSCs were positive for Vimentin while negative for CK , indicating they were of mesenchymal origin.They were also positive for STRO-1, which suggest cultured PDLSCs were endowed with the phenotype of mesenchymal stem cells.The experiments of calcification and adipogenesis induction confirmed the multipotentiality and plasticity of human PDLSCs. DPSCs were labled with PKH26 and then were combined with PDLSCs by different methods and transplanted into nude mice. Results revealed the implants with toot-shaped CBB as scaffold presented small tooth root like structure in gross appearance.HE staining showed all the implants formed calcified matrix, and Sharpey fiber like structure could be found in some implants. While the implants with CBB particles as scaffold formed thin and plat structure in gross appearance and HE staining demonstated calcified matrix were also formed, but less than implants with root-shaped CBB as scaffold. Most cell pellet implants did not form any corporeal structure with exception that only a few contained calcified matrix. No dentin-pulp like structure was found in all the three kinds of implants.
These data suggested: When constructing engineered tooth root, preshaped scalffold was the best choice to guide related stem cells grow along the scaffold and form good-shaped structure in gross appearance; it is recommended to seed stem cells onto the scaffold respectively instead of mixing them simply if more than one kinds of stem cells were used; it is not suitable to construct tooth like structure with good–shaped gross appearance by cell pellet transplantation method.
一、用牙髓干细胞制备异种嵌合体牙齿
用酶消化法培养了牙髓干细胞,结果显示所培养的牙髓干细胞形态典型,表达间充质干细胞标志物STRO-1、CD106、CD29,有克隆形成能力,经诱导后有成骨和成脂肪细胞能力,表明我们培养的细胞是来源于间充质的干细胞。另外分离获得猪第三磨牙牙胚并培养牙胚细胞,用差速消化法纯化获得牙胚上皮细胞,免疫细胞化学鉴定显示其对CK14、AMBN阳性表达,而对Vimentin阴性表达。然后将人牙髓干细胞和猪的牙胚上皮细胞以不同方式复合植入到裸鼠皮下,6周后取材。结果发现大部分移植物没有形成任何有序的结构形态,但有一部分移植物中形成了含有类似牙本质、牙髓和牙釉质的牙齿样结构的组织,但是形成这种结构的概率较低。免疫组化结果证明我们形成的釉质样结构来源于猪牙胚上皮,而牙本质样结构源于人的牙髓干细胞。
本部分结果表明人的牙髓干细胞和猪的牙源性上皮细胞在体内,可以模拟类似牙胚发育时期的上皮-间充质相互作用方式,相互诱导,并有可能成功形成异种嵌合体结构。因而我们推断可见只要进一步优化条件,用人的牙齿间充质干细胞与异种的牙源性上皮细胞进行异种嵌合在牙齿再生中是有一定可行性的,并将可能成为解决牙釉质再生的一条新途径。
二、猪牙胚条件培养基诱导人牙髓干细胞牙向分化的实验研究
以前的实验已经证实人牙胚条件培养基可以为人牙髓干细胞提供成牙微环境,诱导牙髓干细胞向成牙方向分化。但由于实际中应用人牙胚条件培养基并不可行,所以本实验中我们验证了用异种牙胚条件培养基来诱导人牙髓干细胞可行性。我们培养发育期猪的第三磨牙牙胚,并收集获得牙胚条件培养基,同时培养人的牙胚并获得人牙胚条件培养基。将猪和人的牙胚条件培养基分别诱导人的牙髓干细胞,并从体内、体外比较了猪牙胚条件培养基和人牙胚条件培养基对人牙髓干细胞的诱导作用。结果表明:人牙髓干细胞经过一定时间的猪牙胚条件培养基诱导后,能在细胞形态上和细胞周期上发生改变,能具有更高的克隆形成能力和多向分化能力,能维持更好的干细胞特性,具有更强的增殖能力,表达更高水平的碱性磷酸酶,免疫组化和RT-PCR提示诱导后的牙髓干细胞能在蛋白水平和基因水平表达出一些牙向分化的标志,如DSP、DSPP、DMP1、BSP、OCN、OPN等。此外,经猪牙胚条件培养基诱导过的人牙髓干细胞植入裸鼠体内能获得形状更为规则的牙本质-牙髓复合体样结构,而且形成这种规则结构的概率从20%提高到超过70%。这些结果与人牙胚条件培养基诱导的牙髓干细胞表现类似,但常规培养的人牙髓干细胞没有表现出特别变化。
所得结果说明猪牙胚条件培养基拥有与人牙胚条件培养基一样的能力,诱导人牙髓干细胞向成牙方向分化。这提示我们异种牙胚条件培养基有可能作为一种牙齿再生的有用工具,我们在今后的牙组织工程领域,应用异种的牙胚条件培养基将有助于构建理想的工程化牙齿。
三、用人牙髓干细胞和牙周膜干细胞尝试构建工程化牙根的实验研究
牙髓干细胞和牙周膜干细胞是牙齿再生领域最有潜力的两个成体干细胞,那把这两种干细胞一起复合,能否在体内获得牙根样结构呢?本实验进行了这方面的初步尝试。我们体外培养了人牙周膜干细胞并对其性质进行了鉴定,结果显示牙周膜干细胞对Vimentin染色阳性,CK阴性,显示其为间充质来源, STRO-1呈阳性染色,说明培养的牙周膜干细胞具有间充质来源干细胞的表型特点。成脂、成骨诱导实验验证了牙周膜干细胞的多向分化能力与可塑性。本研究中将人的牙髓干细胞进行标记后,再与牙周膜干细胞以不同的方式复合并植入裸鼠体内,希望获得有一定结构的工程化牙根。结果显示:以牙根状CBB为支架的复合物取材后肉眼观呈外形良好的小牙根状,HE染色表明所有移植物中都有矿化发生,且能在一部分移植物中发现有典型Sharpey纤维样结构。在以CBB颗粒为支架的移植物中,肉眼观是形成了扁平状的结构,HE染色结果显示也能也有矿化现象发生,但矿化基质较牙根状CBB为支架的标本少。而单纯细胞团移植物除小部分出现了一定的矿化现象,大部分不能产生任何的有形结构。在三个实验组中都没有发现有典型牙髓牙本质复合体样结构形成。
本实验结果提示我们:在构建组织工程化牙根中,支架材料最好是有预成形状的,以引导相关干细胞沿着支架生长并形成大体结构良好的牙齿样组织;另外多种细胞构建一定结构时,不同的种子细胞最好是分别复合到支架上,而不应该简单混合到一起;细胞团移植不适合于用多种细胞复合构建有良好大体观的牙齿样结构。
Dental pulp stem cells (DPSCs) is an important candidate stem cells in tooth tissue engineering, and great progress have been made in identifying DPSC. Nevertheless, many problems have to be solved if we carry out further researches and application, for example,how to construct complex tooh like structure with DPSCs and other cells,and how to create appropriate microenvironment to guide DPSC to differentiate into specific direction, especially into odontogenic cell linage when DPSCs were cultured in vitro.This research tried to construct heterogeneous chimeric tooth structure with human DPSCs and xenogenic epithelial cells from tooth germ , to evaluate the effect of xenogenic tooth germ conditioned medium on human DPSCs, and to construct engineered bioroot by comination of human DPSC and periodontal ligament stem cells (PDLSC) .The aim of these studies was to establish experimental fondation for the further research and application of human DPSCs, to provide some useful clues for the future application of human DPSCs in tooth tissue engieering, and to explore some new methods and approaches for tooth regeneration with tooth derived stem cells.
The main achivements are as follows:
Section 1.Construction of heterogeneous chimeric tooth with human dental pulp stem cells
Human DPSC were cultured with enzyme digestion solution. Results showed cultured DPSCs had typical morphology, and expressed surface markers of mesenchymal stem cells STRO-1 ,CD06 and CD29. cultured DPSCs held the potential to form colony ,and to differentiate into osteoblats or adipocytes after induction. These data indicated the DPSCs we cultured were mesenchymal stem cells. Porcine dental germ of the third molar was isolated and dental germ cells were cultured. Epithelial cells of dental germ were obtained by speed-differentiated digestion solution. Immunocytochemeical staining showed tooth germ derived epithelial cells were positive for CK14 and AMBN, but negative for Vimentin. Then human DPSCs and porcine tooth germ derived epithelial cells were combined with different methods and transplanted into subcutaneous pockets of nude mice. Implants were harvested after 6 weeks. It was found most of the implants did not form any ordinate structure, whereas some implants contained dentin-pulp like structure and enamel like structure.The probability of forming this kind of structure was just 20%. Immunohistochemistry results provided evidence that the enamel like structure was originated from porcine tooth germ derived epithelial cells while dentin like structure was originated from human DPSCs.
The results of this part suggested that human DPSC and porcine tooth germ derived epithelial cells could mimic the interaction between epithelial cells and mesenchymal cells during tooth development, and induce each other and possessed the possibility to form heterogeneous chimeric tooth structure. So we could come to the conclusion that it is feasible to construct heterogeneous chimric tooth with human mesenchymal stem cells and xenogenic tooth germ derived epithelial cells, and this may be a solution to cope with ename regeneration.
Section 2.Odontogenic differentiation of human dental pulp stem cells induced by swine tooth germ cell conditioned medium
Previous studies showed that tooth germ cell conditioned medium (TGC-CM) hold the potential to induce dental pulp stem cells (DPSCs) to differentiate into odontogenic lineage. Nevertheless, human TGC-CM (hTGC-CM) is not feasible in practical application. We hypothesized that xenogenic TGC-CM may exert similar influence on DPSCs. In this study, we selected swine as a xenogenic origin and compared the effects of swine tooth germ cell conditioned medium (sTGC-CM) with hTGC-CM on human DPSCs. in vivo and in vitro. Results showed sTGC-CM, which is similar with the effect of hTGC-CM, was able to induce human DPSCs to make odontoblastic changes as indicated by remarkable morphological changes, increased proliferation ability, higher multipotential and colony forming capability, and expression of some odontogenic markers in gene and protein levels,such as DSP, DSPP, DMP1, BSP, OCN, OPN. Induced DPSC also maintained better stem-cell characteristics,. Besides, human DPSCs treated with sTGC-CM could form more regular dentine-pulp complex in vivo.The probability of forming this kind of structure was elevated from 20% to over 70%. These results was similar to DPSCs treated with hTGC-CM while quite different from that of control DPSCs.
Our data provided evidence that sTGC-CM held almost the same potential as hTGC-CM in inducing DPSCs to differentiate along odontogenic direction.. The observations suggested xenogenic TGC-CM may be a useful tool in tooth regeneration and the application of xenogenic TGC-CM may facilitate generating a bioengineered tooth from tooth derived stem cells in future.
Section 3 The trial of constructing engineered bioroot with human dental pulp stem cells and periodontal ligament stem cells
Undoubtedly, DPSC and PDLSC was the most promising postnatal stem cells in future tooth tissue engineering.Then, is it possible to get tooth root like structure in vivo if these two kinds of stem cells were combined and transplanted?We carried out this kind of trial. PDLSCs were cultured ex vivo and were characterizied. Results showed PDLSCs were positive for Vimentin while negative for CK , indicating they were of mesenchymal origin.They were also positive for STRO-1, which suggest cultured PDLSCs were endowed with the phenotype of mesenchymal stem cells.The experiments of calcification and adipogenesis induction confirmed the multipotentiality and plasticity of human PDLSCs. DPSCs were labled with PKH26 and then were combined with PDLSCs by different methods and transplanted into nude mice. Results revealed the implants with toot-shaped CBB as scaffold presented small tooth root like structure in gross appearance.HE staining showed all the implants formed calcified matrix, and Sharpey fiber like structure could be found in some implants. While the implants with CBB particles as scaffold formed thin and plat structure in gross appearance and HE staining demonstated calcified matrix were also formed, but less than implants with root-shaped CBB as scaffold. Most cell pellet implants did not form any corporeal structure with exception that only a few contained calcified matrix. No dentin-pulp like structure was found in all the three kinds of implants.
These data suggested: When constructing engineered tooth root, preshaped scalffold was the best choice to guide related stem cells grow along the scaffold and form good-shaped structure in gross appearance; it is recommended to seed stem cells onto the scaffold respectively instead of mixing them simply if more than one kinds of stem cells were used; it is not suitable to construct tooth like structure with good–shaped gross appearance by cell pellet transplantation method.
引文
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