bFGF在恒牙牙根发育不同阶段的牙髓中表达的免疫组化研究
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摘要
前言
在牙齿发育期间，牙乳头间充质细胞分化为成牙本质细胞，形成原发性牙本质；在牙齿萌出后，无论年轻恒牙，还是成熟恒牙，受损伤后均有修复性牙本质的形成，表明牙髓中存在有分化潜力的祖细胞群(progenitor cells)，后期可能分化为成牙本质细胞样细胞(odontoblast-like cells)。目前认为一些生长因子可能参与调节牙髓组织中的细胞增殖、分化和基质合成。碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)是一种多肽调节因子，对绝大多数来自中胚层和神经外胚层的细胞均具有刺激增殖活性，在胚胎形成、血管新生、神经营养及创伤愈合等方面起重要作用。迄今国内外学者对bFGF在人、鼠牙胚发育过程中的表达进行了免疫组化研究，认为bFGF与成釉细胞、成牙本质细胞的分化成熟及牙本质基质的沉积有关。本实验首次运用SP免疫组化技术和图像分析手段，显示bFGF在恒牙牙根不同发育阶段的牙髓中的表达及分布特征，探寻其在牙髓发育成熟中的作用。
材料与方法
牙髓标本取自多生牙、因阻生而拔除的第三磨牙或因正畸而拔除的健康无龋牙齿。所有研究对象均无全身系统性疾病，近三个月未曾服用免疫制剂和抗菌药物。将研究对象分成三组，即牙根刚开始发育(第一组)、牙根发育中(第二组)、牙根发育完成(第三组)。牙齿拔除后立即将牙齿纵向劈开，完整取出牙髓组织，放入4％的多聚甲醛液中，4℃下固定。梯度酒精脱水，二甲苯透明，石蜡包埋。每个标本于牙髓组织纵向最大面积处连续切片，厚
5卜m，做 SP免疫组化染色。光学显微镜下观察阳性染色的分布及
强度。
利用图像分析仪和Meta Morph／Cool snap仇AX70软件系统
对阳性染色部位的透光强度进行定量分析问灰度值的测定人
随机选取8个视野，对每张切片的冠髓与根髓组织的外层部分和
中心区分别进行测量，结果取其平均值。用SPSS软件做统计学分
析。
实验结 果
在牙髓组织中，bFGF主要在成纤维细胞、成牙本质细胞及血
管壁内皮细胞的胞浆中呈阳性着色，同时在细胞外基质中呈线网
状分布。年轻恒牙第一组牙髓的bFGF呈强阳性表达，第H组次
之，第三组成熟恒牙牙髓的bFGF呈弱阳性表达。随着牙根的发
育成熟，牙髓中阳性表达的成纤维细胞数逐渐减少。第一组牙髓
外层的胜CF灰度值大于中心区（P＜O．001八第二组冠髓中心区
的m＊F灰度值大于冠髓外层评＜0．05八成熟恒牙的冠髓外层灰
度值大于年轻恒牙河＜O．001人各实验组的冠髓中心区灰度值
两两之间比较，有非常显著性差异泽＜O．00八年轻恒牙的根髓
中心区灰度值低于外层（P＜0．05厂且低于成熟恒牙的根髓中心
区灰度值（P＜O．00）。
讨 论
国内外学者对牙髓细胞的体外培养实验研究表明，bFGF能
促进牙髓细胞DNA的合成，是牙髓细胞的潜在有丝分裂原；加人
bFGF使牙髓细胞的碱性磷酸酶（Alkaline PhosPhatase，ALPase）合
成受到抑制，而Aryase诱导牙髓细胞的终末分化和钙化。成牙本
·2·
质细胞为终末细胞，失去增殖活性，bFGF对牙髓细胞的终末分化
和钙化的抑制作用对于牙髓发育和修复中牙髓细胞的募集（re－
cmiboent）是必备的先决条件。
近年来的研究表明bFGF参与牙胚的形态发生和牙齿形成细
胞的分化。本实验采用免疫组化技术，发现牙髓中的成纤维细胞
表达bFGF，而且随着牙根的发育，牙髓中表达bFGF的成纤维细
胞数目逐渐减少，其染色强度逐渐减弱。因此，我们认为bFGF对
牙髓细胞的发育和成熟起着重要作用。实验结果显示年轻恒牙牙
根刚开始发育时，牙髓中心区的bFGF阳性表达强于牙髓外层，这
可能是造成年轻恒牙牙髓中心区ALPase活力减弱，髓腔壁和壁附
近的成牙本质细胞中ALPase活力最强的原因之一。有学者认为，
牙髓细胞在分化为成牙本质细胞之前，需要经过一个去分化的过
程，即ALPase活力下降，细胞进人分化潜力大的中胚层细胞，进而
酶活力升高，分化为成牙本质细胞。因为ALPase的活性与牙髓的
形成功能密切相关，所以bFGF可能通过控制ALPase的活性而参
与了牙髓细胞的去分化过程，从而调节牙髓细胞向成牙本质细胞
的分化。bFGF的阳性表达在年轻恒牙的冠髓外层强于冠髓中心
区，根髓中心区强于根髓外层。由此我们推测年轻恒牙冠髓的成
牙本质细胞附近的牙髓细胞以及根髓中心的牙髓细胞具有较强地
分化为成牙本质细胞的能力。bFGF表达于牙髓细胞外基质kx－
racellular matriX，ECM）中，表明成纤维细胞分泌的 bFGF可能被
贮存在ECM中，从而刺激成纤维细胞的生长和分化。细胞的分化
是由牙髓ECM来调节的。bFGF能与IV型胶原，层粘连蛋白
（laminin，LN）及纤维粘连蛋白（ibronechn，FN）等ECM结合，这
可以用来解释牙本质基质沉积过程中成牙本质细胞层和牙髓
ECM中bFGF的强染现象，同11寸揭示了 bFGF在与ECM成分的相
互作用以及伴随牙根发育的牙本质沉积中的重要作用。概括而
言，bFGF释放后，一部分与周围细胞或自身胞膜受体结合，促进
Preface
Dental papilla mesenchymal cells differentiate into primary odon-toblasts and form primary dentin during tooth development. After e-ruption, a certain fraction of pulp cells differentiate into odontoblasts and form secondary dentin. Investigations suggested that some growth factors might modulate proliferation and matrix synthesis in pulp. However the precise mechanisms remain unknown. Scholars have described the spatial - temporal expression of basic fibroblast growth factor (bFGF) during odontogenesis of mouse and human embryo as revealed by immunohistology. They believed that bFGF was involved in the odontoblast differentiation and dentin matrix deposition. But study on the expression and distribution of bFGF in pulps of permanent teeth during root development has not been reported. The purpose of this study was to discuss the significance of bFGF in the development and maturation of dental pulp.
For the first time, by use of immunohistochemical and image a-nalysis techniques, we presented the expression and distribution pattern of bFGF in pulps of permanent teeth at different root development stage.
Materials and Methods
Dental pulps were obtained from healthy caries - free permanent teeth in need of extraction. All subjects had no systematic disease and didn' t take any immune - inhibited drugs within three months. Based on the root development status, the pulp tissue was classified into three groups; root just starting development, root being in development and root finishing development. After extracted, the dental pulp was rapidly removed, then fixed in 0. 4% paraformaladehyde ( pH 7.4) at4C for 48 hours. Tissue blocks were dehydrated in ascending series of ethanol, cleared in xylene and embedded in paraffin. Serial sections were cut 5um thick. Anti - human poly clone antibody was used as the primary antibody and SP immunohistochemistry method was used for staining. The samples were observed under light microscope. Meta Morph/cool snapfx/AX70 image analysis software was used to detect the gray value of bFGF positive stained in dental pulp. The data were statistically analyzed by use of SPSS.
Results
Staining of immunohistochemistry for bFGF in dental pulps revealed that there were positive reactions in the cytoplasm of fibroblast, odontoblast, and epithelial cells in blood vessel wall and in extracellular matrix as well. Staining was strongly positive in immature permanent teeth, especially at the stage of root just starting development. The number of fibroblasts in positive expression was the most in them, and the least in the mature permanent teeth.
Image analysis indicated that with the development of root formation , the gray value of the positive reaction in three groups were higher and higher and were significantly different ( P <0. 001) . For the first group, the gray value of the outer part was higher than that of the pulp core. For the second group, the pulp core has a higher gray value in the coronal pulp, while a lower value in root pulp compared to the outer parts. For the third group, there was no statistical difference between gray values in different pairts.
Discussion
Studies of pulp cells in cultures indicated that adding bFGF enhanced DNA synthesis, whereas decreased ALPase level. ALPase may induce the terminal differentiation and calcification of pulp cells. Because terminal cells lose proliferate activity, the inhibition of terminal differentiation by bFGF may be a prerequisite for the recruitment of pulp cells during tooth development and repair.
Recent investigations suggested that bFGF might be involved in tooth morphogenesis and differentiation of tooth - forming cells. By immunohistology we observed bFGF staining in pulp fibroblasts. With development of the root, the cell number expressed bFGF becomes less and the staining intensity weaker. It appears that bFGF may play a role in the development and maturation of pulp cells. The results indicate that when the young permanent teeth initially develop, bFGF positive expression in pulp core is strong
在牙齿发育期间，牙乳头间充质细胞分化为成牙本质细胞，形成原发性牙本质；在牙齿萌出后，无论年轻恒牙，还是成熟恒牙，受损伤后均有修复性牙本质的形成，表明牙髓中存在有分化潜力的祖细胞群(progenitor cells)，后期可能分化为成牙本质细胞样细胞(odontoblast-like cells)。目前认为一些生长因子可能参与调节牙髓组织中的细胞增殖、分化和基质合成。碱性成纤维细胞生长因子(basic fibroblast growth factor，bFGF)是一种多肽调节因子，对绝大多数来自中胚层和神经外胚层的细胞均具有刺激增殖活性，在胚胎形成、血管新生、神经营养及创伤愈合等方面起重要作用。迄今国内外学者对bFGF在人、鼠牙胚发育过程中的表达进行了免疫组化研究，认为bFGF与成釉细胞、成牙本质细胞的分化成熟及牙本质基质的沉积有关。本实验首次运用SP免疫组化技术和图像分析手段，显示bFGF在恒牙牙根不同发育阶段的牙髓中的表达及分布特征，探寻其在牙髓发育成熟中的作用。
材料与方法
牙髓标本取自多生牙、因阻生而拔除的第三磨牙或因正畸而拔除的健康无龋牙齿。所有研究对象均无全身系统性疾病，近三个月未曾服用免疫制剂和抗菌药物。将研究对象分成三组，即牙根刚开始发育(第一组)、牙根发育中(第二组)、牙根发育完成(第三组)。牙齿拔除后立即将牙齿纵向劈开，完整取出牙髓组织，放入4％的多聚甲醛液中，4℃下固定。梯度酒精脱水，二甲苯透明，石蜡包埋。每个标本于牙髓组织纵向最大面积处连续切片，厚
5卜m，做 SP免疫组化染色。光学显微镜下观察阳性染色的分布及
强度。
利用图像分析仪和Meta Morph／Cool snap仇AX70软件系统
对阳性染色部位的透光强度进行定量分析问灰度值的测定人
随机选取8个视野，对每张切片的冠髓与根髓组织的外层部分和
中心区分别进行测量，结果取其平均值。用SPSS软件做统计学分
析。
实验结 果
在牙髓组织中，bFGF主要在成纤维细胞、成牙本质细胞及血
管壁内皮细胞的胞浆中呈阳性着色，同时在细胞外基质中呈线网
状分布。年轻恒牙第一组牙髓的bFGF呈强阳性表达，第H组次
之，第三组成熟恒牙牙髓的bFGF呈弱阳性表达。随着牙根的发
育成熟，牙髓中阳性表达的成纤维细胞数逐渐减少。第一组牙髓
外层的胜CF灰度值大于中心区（P＜O．001八第二组冠髓中心区
的m＊F灰度值大于冠髓外层评＜0．05八成熟恒牙的冠髓外层灰
度值大于年轻恒牙河＜O．001人各实验组的冠髓中心区灰度值
两两之间比较，有非常显著性差异泽＜O．00八年轻恒牙的根髓
中心区灰度值低于外层（P＜0．05厂且低于成熟恒牙的根髓中心
区灰度值（P＜O．00）。
讨 论
国内外学者对牙髓细胞的体外培养实验研究表明，bFGF能
促进牙髓细胞DNA的合成，是牙髓细胞的潜在有丝分裂原；加人
bFGF使牙髓细胞的碱性磷酸酶（Alkaline PhosPhatase，ALPase）合
成受到抑制，而Aryase诱导牙髓细胞的终末分化和钙化。成牙本
·2·
质细胞为终末细胞，失去增殖活性，bFGF对牙髓细胞的终末分化
和钙化的抑制作用对于牙髓发育和修复中牙髓细胞的募集（re－
cmiboent）是必备的先决条件。
近年来的研究表明bFGF参与牙胚的形态发生和牙齿形成细
胞的分化。本实验采用免疫组化技术，发现牙髓中的成纤维细胞
表达bFGF，而且随着牙根的发育，牙髓中表达bFGF的成纤维细
胞数目逐渐减少，其染色强度逐渐减弱。因此，我们认为bFGF对
牙髓细胞的发育和成熟起着重要作用。实验结果显示年轻恒牙牙
根刚开始发育时，牙髓中心区的bFGF阳性表达强于牙髓外层，这
可能是造成年轻恒牙牙髓中心区ALPase活力减弱，髓腔壁和壁附
近的成牙本质细胞中ALPase活力最强的原因之一。有学者认为，
牙髓细胞在分化为成牙本质细胞之前，需要经过一个去分化的过
程，即ALPase活力下降，细胞进人分化潜力大的中胚层细胞，进而
酶活力升高，分化为成牙本质细胞。因为ALPase的活性与牙髓的
形成功能密切相关，所以bFGF可能通过控制ALPase的活性而参
与了牙髓细胞的去分化过程，从而调节牙髓细胞向成牙本质细胞
的分化。bFGF的阳性表达在年轻恒牙的冠髓外层强于冠髓中心
区，根髓中心区强于根髓外层。由此我们推测年轻恒牙冠髓的成
牙本质细胞附近的牙髓细胞以及根髓中心的牙髓细胞具有较强地
分化为成牙本质细胞的能力。bFGF表达于牙髓细胞外基质kx－
racellular matriX，ECM）中，表明成纤维细胞分泌的 bFGF可能被
贮存在ECM中，从而刺激成纤维细胞的生长和分化。细胞的分化
是由牙髓ECM来调节的。bFGF能与IV型胶原，层粘连蛋白
（laminin，LN）及纤维粘连蛋白（ibronechn，FN）等ECM结合，这
可以用来解释牙本质基质沉积过程中成牙本质细胞层和牙髓
ECM中bFGF的强染现象，同11寸揭示了 bFGF在与ECM成分的相
互作用以及伴随牙根发育的牙本质沉积中的重要作用。概括而
言，bFGF释放后，一部分与周围细胞或自身胞膜受体结合，促进
Preface
Dental papilla mesenchymal cells differentiate into primary odon-toblasts and form primary dentin during tooth development. After e-ruption, a certain fraction of pulp cells differentiate into odontoblasts and form secondary dentin. Investigations suggested that some growth factors might modulate proliferation and matrix synthesis in pulp. However the precise mechanisms remain unknown. Scholars have described the spatial - temporal expression of basic fibroblast growth factor (bFGF) during odontogenesis of mouse and human embryo as revealed by immunohistology. They believed that bFGF was involved in the odontoblast differentiation and dentin matrix deposition. But study on the expression and distribution of bFGF in pulps of permanent teeth during root development has not been reported. The purpose of this study was to discuss the significance of bFGF in the development and maturation of dental pulp.
For the first time, by use of immunohistochemical and image a-nalysis techniques, we presented the expression and distribution pattern of bFGF in pulps of permanent teeth at different root development stage.
Materials and Methods
Dental pulps were obtained from healthy caries - free permanent teeth in need of extraction. All subjects had no systematic disease and didn' t take any immune - inhibited drugs within three months. Based on the root development status, the pulp tissue was classified into three groups; root just starting development, root being in development and root finishing development. After extracted, the dental pulp was rapidly removed, then fixed in 0. 4% paraformaladehyde ( pH 7.4) at4C for 48 hours. Tissue blocks were dehydrated in ascending series of ethanol, cleared in xylene and embedded in paraffin. Serial sections were cut 5um thick. Anti - human poly clone antibody was used as the primary antibody and SP immunohistochemistry method was used for staining. The samples were observed under light microscope. Meta Morph/cool snapfx/AX70 image analysis software was used to detect the gray value of bFGF positive stained in dental pulp. The data were statistically analyzed by use of SPSS.
Results
Staining of immunohistochemistry for bFGF in dental pulps revealed that there were positive reactions in the cytoplasm of fibroblast, odontoblast, and epithelial cells in blood vessel wall and in extracellular matrix as well. Staining was strongly positive in immature permanent teeth, especially at the stage of root just starting development. The number of fibroblasts in positive expression was the most in them, and the least in the mature permanent teeth.
Image analysis indicated that with the development of root formation , the gray value of the positive reaction in three groups were higher and higher and were significantly different ( P <0. 001) . For the first group, the gray value of the outer part was higher than that of the pulp core. For the second group, the pulp core has a higher gray value in the coronal pulp, while a lower value in root pulp compared to the outer parts. For the third group, there was no statistical difference between gray values in different pairts.
Discussion
Studies of pulp cells in cultures indicated that adding bFGF enhanced DNA synthesis, whereas decreased ALPase level. ALPase may induce the terminal differentiation and calcification of pulp cells. Because terminal cells lose proliferate activity, the inhibition of terminal differentiation by bFGF may be a prerequisite for the recruitment of pulp cells during tooth development and repair.
Recent investigations suggested that bFGF might be involved in tooth morphogenesis and differentiation of tooth - forming cells. By immunohistology we observed bFGF staining in pulp fibroblasts. With development of the root, the cell number expressed bFGF becomes less and the staining intensity weaker. It appears that bFGF may play a role in the development and maturation of pulp cells. The results indicate that when the young permanent teeth initially develop, bFGF positive expression in pulp core is strong
引文
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2. Goldberg M, Lasfargues JJ. Pulp - dental complex revisited. J Dent Res, 1995, 23(1): 15-20
3．郝建军，史俊南．成纤维细胞生长因子家族的研究进展．牙体牙髓牙周病学杂志，1994，4(2)：111-113
4. Cam Y, Neumann MR, Oliver L, et al. Immunolocalization of acidic and basic fibroblast growth factors during mouse odontogenesis. Int J Der Biol, 1992, 36: 381-389
5．郝建军，史俊南，牛忠英，等．碱性成纤维细胞生长因子在人牙胚中表达的免疫组化定位．牙体牙髓牙周病学杂志，1997，7(3)：183-185
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7. Haralabakis N B, Yiagtzis S Ch, Toutountzakis N M. Premature or delayed exfoliation of deciduous teeth and root resorption and formation. Angle Orthodontist, 1994, 64(1): 151-157
8．施作熔．链霉素亲生物素蛋白-生物素酶标免疫组化染色方法的初步研究．中国免疫杂志，1986，2(2)：169
9．文玲英，吴海珍，樊淑梅．乳牙年轻恒牙牙髓组织碱性磷酸酶的分布特征．华西口腔医学杂志，1994，12(4)：235-237
10. Nakashima M. The effects of growth factors on DNA synthesis, proteoglyean synthesis and alkaline phosphatase activity in bovine
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11. Shiba H, Fujita T, Doi N, et al. Differential effects of various growth factors and cytokines on the synthesis of DNA, type Ⅰ collagen, laminin, fibronectin, osteonectin/secrected protein, acidic and rich in cystein (SPARC), and alkaline phosphatase by human pulp cells in culture. J Cell Physiol, 1998 Feb, 174(2): 194-205
12．郝建军，汪平，史俊南，等．碱性成纤维细胞生长因子对人牙髓细胞增殖和分化的作用．中华口腔医学杂志，1999，34(4)：239-241
13. Shiba H, Nakamura S, Shirakawa M, et al. Effects of basic fibroblast growth factor on proliferation, the expression of osteonectin(SPARC) and alkaline phosphatase and calcification in cultures of human pulp cells. Dev Biol, 1995 Aug, 170(2): 457-466
14．于世凤，主编．口腔组织病理学．第4版．北京：人民卫生出版社，2001．19
15. James K. Avery. Oral development and histology. Baltimore, USA: Waverly Press, 1987. 167
16．郝建军，史俊南，牛忠英，等．碱性成纤维细胞生长因子对体外大鼠牙胚分化的影响．牙体牙髓牙周病学杂志，1997，7(3)：180-182
17. Yoshiki S. A light and electron microscopic study of alkaline phosphatase activity in early atage of dentinogenesis in the young rat. Arch Oral Biol, 1971, 16(12): 1143
18. Alliot-Licht B, Jean A, Gregoire M. Comparative effect of calcium and hydroxyapatite on the cellular activity of human pulp fibroblasts in vitro. Arch Oral Biol, 1994, 39(6): 481-489
19．王忠英，文玲英．牙髓自身修复潜能的研究进展．国外医学口腔医学分册，1996，23(2)：65-69
20．徐劲军，雷芳，何红英．碱性成纤维细胞生长因子在正常和炎性牙髓中的免疫组化研究．广东牙病防治，1999，7(3)：170-171
21. Nakashima M. Establishment of primary cultures of pulp cells from bovine permanent incisors. Arch Oral Boil, 1991, 36(9):655-663
22. Ruch JV. Odontoblast differentiation and the formation of the odontoblast layer. J Dent Res, 1985 Apr, 64(spe): 489-498
23. Tziafas D, Alvanou A, Kaidoglou K. Dentinogenic activity of allogenic plasma fibronectin on dog dental pulp. J Dent Res, 1992 May, 71(5): 1189-1195
24. Yoshida S, Ohshima M. Distribution and organization of peripheral capillaries in dental pulp and their relationship to odontoblasts. Anat Record, 1996 Jun, 245(2): 313-326
25. Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy. J Dent, 2000, 28(1): 77-92
26. Martin A, Unda F-J, Bègue-Kirn C, et al. Effects of aFGF, bFGF, TGF-β1 and IGF-Ⅰ on odontoblast differentiation in vitro. Eur J Oral Sci, 1998, 106(suppl1): 117-121
27．李国华，朱南洲，王学英，等．牙髓治疗剂牛血小板衍化生长因子盖髓的临床研究．牙体牙髓牙周病学杂志，1998，8(3)：196-197
28. Tziafas D, Alvanou A, Papadimitriou S, et al. Effects of recombinant basic fibroblast growth factor, insulin -like growth factor-Ⅱ and transforming growth factor-β1 on dog dental pulp cells in vivo. Arch Oral Biol, 1998, 43(6): 431-444
29. Tziafas D, Papadimitriou S. The role of exogenous TGF -β in induction of reparative dentinogenesis in vivo. Eur J Oral Sci, 1998, 106(Suppl1): 192-196
30．轩昆，杨富生．牙根发育的研究进展．牙体牙髓牙周病学杂志，2001，11(1)：57-59
2. Goldberg M, Lasfargues JJ. Pulp - dental complex revisited. J Dent Res, 1995, 23(1): 15-20
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4. Cam Y, Neumann MR, Oliver L, et al. Immunolocalization of acidic and basic fibroblast growth factors during mouse odontogenesis. Int J Der Biol, 1992, 36: 381-389
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6. Russo LG, Maharajan P, Maharajan V. Basic fibroblast growth factor (FGF-2) in mouse tooth morphogenesis. Growth Factors, 1998, 15(2): 125-133
7. Haralabakis N B, Yiagtzis S Ch, Toutountzakis N M. Premature or delayed exfoliation of deciduous teeth and root resorption and formation. Angle Orthodontist, 1994, 64(1): 151-157
8．施作熔．链霉素亲生物素蛋白-生物素酶标免疫组化染色方法的初步研究．中国免疫杂志，1986，2(2)：169
9．文玲英，吴海珍，樊淑梅．乳牙年轻恒牙牙髓组织碱性磷酸酶的分布特征．华西口腔医学杂志，1994，12(4)：235-237
10. Nakashima M. The effects of growth factors on DNA synthesis, proteoglyean synthesis and alkaline phosphatase activity in bovine
dental pulp cells. Arch Oral Biol, 1992, 37(3): 231-236
11. Shiba H, Fujita T, Doi N, et al. Differential effects of various growth factors and cytokines on the synthesis of DNA, type Ⅰ collagen, laminin, fibronectin, osteonectin/secrected protein, acidic and rich in cystein (SPARC), and alkaline phosphatase by human pulp cells in culture. J Cell Physiol, 1998 Feb, 174(2): 194-205
12．郝建军，汪平，史俊南，等．碱性成纤维细胞生长因子对人牙髓细胞增殖和分化的作用．中华口腔医学杂志，1999，34(4)：239-241
13. Shiba H, Nakamura S, Shirakawa M, et al. Effects of basic fibroblast growth factor on proliferation, the expression of osteonectin(SPARC) and alkaline phosphatase and calcification in cultures of human pulp cells. Dev Biol, 1995 Aug, 170(2): 457-466
14．于世凤，主编．口腔组织病理学．第4版．北京：人民卫生出版社，2001．19
15. James K. Avery. Oral development and histology. Baltimore, USA: Waverly Press, 1987. 167
16．郝建军，史俊南，牛忠英，等．碱性成纤维细胞生长因子对体外大鼠牙胚分化的影响．牙体牙髓牙周病学杂志，1997，7(3)：180-182
17. Yoshiki S. A light and electron microscopic study of alkaline phosphatase activity in early atage of dentinogenesis in the young rat. Arch Oral Biol, 1971, 16(12): 1143
18. Alliot-Licht B, Jean A, Gregoire M. Comparative effect of calcium and hydroxyapatite on the cellular activity of human pulp fibroblasts in vitro. Arch Oral Biol, 1994, 39(6): 481-489
19．王忠英，文玲英．牙髓自身修复潜能的研究进展．国外医学口腔医学分册，1996，23(2)：65-69
20．徐劲军，雷芳，何红英．碱性成纤维细胞生长因子在正常和炎性牙髓中的免疫组化研究．广东牙病防治，1999，7(3)：170-171
21. Nakashima M. Establishment of primary cultures of pulp cells from bovine permanent incisors. Arch Oral Boil, 1991, 36(9):655-663
22. Ruch JV. Odontoblast differentiation and the formation of the odontoblast layer. J Dent Res, 1985 Apr, 64(spe): 489-498
23. Tziafas D, Alvanou A, Kaidoglou K. Dentinogenic activity of allogenic plasma fibronectin on dog dental pulp. J Dent Res, 1992 May, 71(5): 1189-1195
24. Yoshida S, Ohshima M. Distribution and organization of peripheral capillaries in dental pulp and their relationship to odontoblasts. Anat Record, 1996 Jun, 245(2): 313-326
25. Tziafas D, Smith AJ, Lesot H. Designing new treatment strategies in vital pulp therapy. J Dent, 2000, 28(1): 77-92
26. Martin A, Unda F-J, Bègue-Kirn C, et al. Effects of aFGF, bFGF, TGF-β1 and IGF-Ⅰ on odontoblast differentiation in vitro. Eur J Oral Sci, 1998, 106(suppl1): 117-121
27．李国华，朱南洲，王学英，等．牙髓治疗剂牛血小板衍化生长因子盖髓的临床研究．牙体牙髓牙周病学杂志，1998，8(3)：196-197
28. Tziafas D, Alvanou A, Papadimitriou S, et al. Effects of recombinant basic fibroblast growth factor, insulin -like growth factor-Ⅱ and transforming growth factor-β1 on dog dental pulp cells in vivo. Arch Oral Biol, 1998, 43(6): 431-444
29. Tziafas D, Papadimitriou S. The role of exogenous TGF -β in induction of reparative dentinogenesis in vivo. Eur J Oral Sci, 1998, 106(Suppl1): 192-196
30．轩昆，杨富生．牙根发育的研究进展．牙体牙髓牙周病学杂志，2001，11(1)：57-59