非负荷期牙种植体结合上皮形成的动物实验
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摘要
研究背景 种植义齿是由种植体和种植体支持的上部结构组成的特殊修复体。对于采用常规义齿修复难以得到足够支持、固位和稳定的患者，种植义齿有明显的优越性。
自二十世纪六十年代起，钛作为一种较理想的牙种植生物材料应用于临床。为了获得种植体在体内长期有效的存留，人们不断从种植材料的种类、性能、生物学特性、种植方案、操作技术，以及术后护理等各个环节进行了大量研究、总结和改进，使人们对种植体的认识不断加深，大大提高了种植义齿临床应用的成功率。
Inoue等在临床上发现种植体周围常常会形成盲袋，种植体界面上不能获得有效牙龈粘膜封闭，长结合上皮移行进入种植体骨界面，破坏骨整合，最终导致种植体松动、脱落。牙种植体贯穿口腔环境及软组织、骨内环境，欲使种植体在口腔环境中行使功能而不损害粘膜下骨组织，就必须保证种植体—牙龈界面的健康，并能防止口腔内细菌等破坏因素侵蚀到颌骨内环境。因此，牙种植体成功的先决条件之一是能够获得附着于种植体颈部表面的口腔粘膜生物屏障。建立并保持这种屏障，主要依赖于口腔再生软组织对种植体体颈部的附着和封闭。
由于牙种植体表面没有牙骨质及牙周膜纤维，结合上皮的生物屏障作用显得
浙江大学硕士学位论文20()4
特别重要。如果种植体颈部不能形成或保持这种屏障，细菌及其它致炎因子就会
侵入组织内环境，引起炎症;并导致附着上皮向根端迁移进入种植体与骨的界面。
一旦种植体与骨的界面有上皮组织迁移进入，就会破坏骨整合，导致种植体松动，
使种植修复失败。因此，形成并保持穿粘膜处种植体颈部的牙靓上皮屏障完整，
对保护骨组织及提高种植成功率极其重要[3，“}。
Jamesl’嗜先证实了结合上皮能通过半桥粒一基板结构附着于钻铬合金种植
体表面。以后，更多学者对钦、复合树脂、陶瓷种植体的牙跟上皮界面进行了研
究，均发现了半桥粒一基板复合物结构。还有一些学者利用组织化学方法对种植
体一上皮组织界面进行了进一步研究，发现结合上皮与种植体之间存在一层含有
酸性粘多糖和糖蛋白的无定形物质，与基底膜的组化成分一致。这都证实了在种
植体颈部表面能够形成并存在上皮界面16. 71。本课题组也曾经通过透射电镜在动物
实验中发现，种植术后15天的纯钦种植体颈部表面有上皮袖口结构，种植体表面
上皮细胞可见伪足、半桥粒和基底板结构。但并未作种植术后上皮屏障形成的连
续观察[“l。
steflik等101在超微结构水平上，提出了结合上皮形成的动态过程。他认为结
合上皮细胞通过分泌性小囊泡，将氨基葡聚糖排出在种植体表面形成基底膜，通
过半桥粒一基底板结构使结合上皮粘附在种植体表面形成上皮屏障。
种植术后，半桥粒一基底板复合体是种植体一上皮屏障形成过程中的重要结
构。此结构的形成、根向迁移与术后上皮细胞的再生、功能密切相关。上皮屏障
形成过程中，上皮细胞附着复合体超微结构及动态形成过程的研究相当明了。但
在此过程中，关于上皮细胞再生、增殖状况的研究却不多。
增殖细胞核抗原(prol iferating Cell noelear antigen，PCNA)可作为评
价细胞增殖状态的重要指标，它的细胞浓度与细胞再生状态密切有关。检测尸CNA
的表达水平能较好地反映细胞进入增殖周期的状况，代表细胞的增殖潜能。
本实验利用动物模型，观察了种植术后种植体颈部牙跟上皮再生、根向迁移、
形成结合上皮的连续组织学过程以及在此过程中上皮细胞PCNA的表达变化，探讨
种植体一上皮界面形成中上皮细胞的增殖、迁移。
浙江大学硕士学位论文2004
研究目的(1)观察二段式非埋植型牙种植体非负荷期结合上皮形成过程。(2)
观察结合上皮形成过程中，PCNA的表达变化。
研究方法(l)建立二段式非埋植型牙种植体动物模型。(2)HE染色观察结合上
皮形成过程。(3)超敏S一P免疫组织化学检测PCNA。
研究结果(l)种植术后12d见结合上皮开始形成，PCNA呈强阳性表达。(2)1
sd上皮细胞增生趋于平稳，PCNA表达显著减弱。(3) 20d形成类似天然牙的上皮
附着，维持低水平表达。
研究结论(1)纯钦牙种植体周在非负荷期能形成类似天然牙的上皮附着。(2)
结合上皮形成过程中，PCNA表达与上皮形成的组织学变化密切相关。
Background Dental implants are used widely to restore function of lost
teeth. The variety of devices available is purportedly because modifi cations in design or materials will enable better performance of the i mplant. Events leading to integration of implant into bone, and hence determining the performance of the device, take place largely at the t issue-implant interface.
Although percutaneous implants are widely used, their long-term succ ess is compromised by problems such as infection, mechanical avulsion and epithelial lose or downgrowth. Because of the aggressive migratory
behaviour of epithelium on percutaneous implants, in the absence of h eroic measures most experimental implants are likely to fail within a few weeks. A successful experimental implant should be integrated with in the surrounding tissue and have sufficient stability to perform des ignated functional tasks.
Accordingly, one of the preconditions of successful implants is th e biological seal, mucosa barrier around the neck of implant.
Objective To detect the histological change and the expression of prol if'erating cell nuclear antigen(PCNA) in junctional epithelializationof dental implants.
Methods The junctional epithelium was detected by HE staining and immu nohistochmical staining of PCNA on the 1st, 3rd, 6th, 9th, 12th, 15th, 20th and 30th day after implantation.
Results 12 days after implantation , junctional epithelium began to fo rm and PCNA expressed positively. 15 days after implantation, epitheli al hyperplasia tended to stabilization and the expression of PCNA weak ened drastically. 20 days later, epithelial attachment similar to natu ral teeth was shown.
Conclusion The gingiva epithelium can attach to titanium implants forming junctional epithelium of peri-implant similar to natural teeth.
自二十世纪六十年代起，钛作为一种较理想的牙种植生物材料应用于临床。为了获得种植体在体内长期有效的存留，人们不断从种植材料的种类、性能、生物学特性、种植方案、操作技术，以及术后护理等各个环节进行了大量研究、总结和改进，使人们对种植体的认识不断加深，大大提高了种植义齿临床应用的成功率。
Inoue等在临床上发现种植体周围常常会形成盲袋，种植体界面上不能获得有效牙龈粘膜封闭，长结合上皮移行进入种植体骨界面，破坏骨整合，最终导致种植体松动、脱落。牙种植体贯穿口腔环境及软组织、骨内环境，欲使种植体在口腔环境中行使功能而不损害粘膜下骨组织，就必须保证种植体—牙龈界面的健康，并能防止口腔内细菌等破坏因素侵蚀到颌骨内环境。因此，牙种植体成功的先决条件之一是能够获得附着于种植体颈部表面的口腔粘膜生物屏障。建立并保持这种屏障，主要依赖于口腔再生软组织对种植体体颈部的附着和封闭。
由于牙种植体表面没有牙骨质及牙周膜纤维，结合上皮的生物屏障作用显得
浙江大学硕士学位论文20()4
特别重要。如果种植体颈部不能形成或保持这种屏障，细菌及其它致炎因子就会
侵入组织内环境，引起炎症;并导致附着上皮向根端迁移进入种植体与骨的界面。
一旦种植体与骨的界面有上皮组织迁移进入，就会破坏骨整合，导致种植体松动，
使种植修复失败。因此，形成并保持穿粘膜处种植体颈部的牙靓上皮屏障完整，
对保护骨组织及提高种植成功率极其重要[3，“}。
Jamesl’嗜先证实了结合上皮能通过半桥粒一基板结构附着于钻铬合金种植
体表面。以后，更多学者对钦、复合树脂、陶瓷种植体的牙跟上皮界面进行了研
究，均发现了半桥粒一基板复合物结构。还有一些学者利用组织化学方法对种植
体一上皮组织界面进行了进一步研究，发现结合上皮与种植体之间存在一层含有
酸性粘多糖和糖蛋白的无定形物质，与基底膜的组化成分一致。这都证实了在种
植体颈部表面能够形成并存在上皮界面16. 71。本课题组也曾经通过透射电镜在动物
实验中发现，种植术后15天的纯钦种植体颈部表面有上皮袖口结构，种植体表面
上皮细胞可见伪足、半桥粒和基底板结构。但并未作种植术后上皮屏障形成的连
续观察[“l。
steflik等101在超微结构水平上，提出了结合上皮形成的动态过程。他认为结
合上皮细胞通过分泌性小囊泡，将氨基葡聚糖排出在种植体表面形成基底膜，通
过半桥粒一基底板结构使结合上皮粘附在种植体表面形成上皮屏障。
种植术后，半桥粒一基底板复合体是种植体一上皮屏障形成过程中的重要结
构。此结构的形成、根向迁移与术后上皮细胞的再生、功能密切相关。上皮屏障
形成过程中，上皮细胞附着复合体超微结构及动态形成过程的研究相当明了。但
在此过程中，关于上皮细胞再生、增殖状况的研究却不多。
增殖细胞核抗原(prol iferating Cell noelear antigen，PCNA)可作为评
价细胞增殖状态的重要指标，它的细胞浓度与细胞再生状态密切有关。检测尸CNA
的表达水平能较好地反映细胞进入增殖周期的状况，代表细胞的增殖潜能。
本实验利用动物模型，观察了种植术后种植体颈部牙跟上皮再生、根向迁移、
形成结合上皮的连续组织学过程以及在此过程中上皮细胞PCNA的表达变化，探讨
种植体一上皮界面形成中上皮细胞的增殖、迁移。
浙江大学硕士学位论文2004
研究目的(1)观察二段式非埋植型牙种植体非负荷期结合上皮形成过程。(2)
观察结合上皮形成过程中，PCNA的表达变化。
研究方法(l)建立二段式非埋植型牙种植体动物模型。(2)HE染色观察结合上
皮形成过程。(3)超敏S一P免疫组织化学检测PCNA。
研究结果(l)种植术后12d见结合上皮开始形成，PCNA呈强阳性表达。(2)1
sd上皮细胞增生趋于平稳，PCNA表达显著减弱。(3) 20d形成类似天然牙的上皮
附着，维持低水平表达。
研究结论(1)纯钦牙种植体周在非负荷期能形成类似天然牙的上皮附着。(2)
结合上皮形成过程中，PCNA表达与上皮形成的组织学变化密切相关。
Background Dental implants are used widely to restore function of lost
teeth. The variety of devices available is purportedly because modifi cations in design or materials will enable better performance of the i mplant. Events leading to integration of implant into bone, and hence determining the performance of the device, take place largely at the t issue-implant interface.
Although percutaneous implants are widely used, their long-term succ ess is compromised by problems such as infection, mechanical avulsion and epithelial lose or downgrowth. Because of the aggressive migratory
behaviour of epithelium on percutaneous implants, in the absence of h eroic measures most experimental implants are likely to fail within a few weeks. A successful experimental implant should be integrated with in the surrounding tissue and have sufficient stability to perform des ignated functional tasks.
Accordingly, one of the preconditions of successful implants is th e biological seal, mucosa barrier around the neck of implant.
Objective To detect the histological change and the expression of prol if'erating cell nuclear antigen(PCNA) in junctional epithelializationof dental implants.
Methods The junctional epithelium was detected by HE staining and immu nohistochmical staining of PCNA on the 1st, 3rd, 6th, 9th, 12th, 15th, 20th and 30th day after implantation.
Results 12 days after implantation , junctional epithelium began to fo rm and PCNA expressed positively. 15 days after implantation, epitheli al hyperplasia tended to stabilization and the expression of PCNA weak ened drastically. 20 days later, epithelial attachment similar to natu ral teeth was shown.
Conclusion The gingiva epithelium can attach to titanium implants forming junctional epithelium of peri-implant similar to natural teeth.
引文
[1] Inoue M, Nakamura T, Shigeno K, et al. Regeneration of the junctional epithelium and connective tissue after transplantation of detergent-processed allo-teeth. Int J Artif Organs, 2000; 23(12): 845.
[2] Chavrier CA, Couble ML. Ultrastructural immunohistochemical study of interstitial collagenous components of the healthy human keratinized mucosa surrounding implants.Int J Oral Maxillofac Implants, 1999; 14(1): 108.
[3] Albrektsson T, Branemark PI, Hansson HA, et al. Osseointegrated titanium implants. Acta Orthop Scand, 1981; 52: 155.
[4] Schroder A, Van Der Zypen E, Sticg H, et al. The reactions of bone connective tissue, and epithelium to endosteal implants with titaniumsprayed surfaces. J Maxillofac Surg, 1981; 9: 15.
[5] James RA, Schultz RL. Hemidesmosomes and the adhesion of junctional epithelial cells to metal implants. J oral Implantol, 1973; 4: 294.
[6] Worthington P, Branemark PI. Advanced osseointegration surgery: Application in the maxillofacial region illirois. Quintessence Publishing Co, 1992: 56.
[7] Meffert RM. How to treat ailing and failing implants. Implant Dent, 1992; 1(1):25
[8] 何福明，刘丽，张凯等．表面为金红石型纯钛种植体与口腔粘膜界面的组织学观察．浙江大学学报(医学版)，2002；31(6)：444．
[9] Steflik DE, Mckinney RV, Koth DL. Ultrastructural comparisons of ceramic and titanium dental implants in vivo: A scanning electron microscopic study. J Biomed Mter Res, 1989; 23: 895.
[10] 巢永烈，梁星．种植义齿学．北京北京医科大学、中国协和医科大学联合出版社，1999，第1版：24．
[11] Branemark PI, Breine U, Adell R, et al. Intra-osseous anchorage of dental prosthesis. Experimental studies. Scand J Plast Res Hand Surg, 1969; 3: 81.
[12] Nociti FH Jr, Caffesse RG, Sallum EA, et al. Evaluation of guided bone regeneration and/or bone grafts in the treatment of ligature-induced peri-implantitis defects: a morphometric study in dogs. J Oral Implantol, 2000; 26(4): 244.
[13] Rutar A, Lang NP, Buser D, et al. Retrospective assessment of clinical and microbiological factors affecting periimplant tissue conditions. Clin Oral Implants Res, 2001; 12(3): 189.
[14] E Chaar ES, Jalbout ZN. Regeneration of an osseous peri-implantitis lesion. Periodontal Clin Investig, 2002; 24(1): 5.
[15] Listgarten, MA. Phase-contrast and electron microscopic study of the junction between reduced enamel epithelium and enamel in unerupted human teeth. Arch Oral Biol, 1966; 11(10): 999.
[16] McKinney RV, Steflik DE, Koth KL. Evidence for junctional epithelial attachment to ceramic dental implants, a transmission electron microscopic study. J Periodontol, 1985;56: 579.
[17] Listgareen MA, Lai CH. Ultrastructure of the intact interface between an endosseous Ipoxy resin dental implant and the host tissres. J Biol Buccale, 1975; 3:13.
[18] Moon IS, Berglundh T, Abrahamsson I, et al. The barrier between the keratinized mucosa and the dental implant. An experimental study in the dog. J Clin Periodontol, 1999; 26(10): 658.
[19] Kido MA, Inoue T, Shimono M, et al. Ultrastructural and immunoelectron microscopic studies of the peri-implant epithelium-implant (Ti-6Al-4V) interface of rat maxilla. J Periodontol, 2000; 71(6): 961.
[20] Keda H, Shiraiwa M, Yamaza T. Difference in penetration of horseradish peroxidase tracer as a foreign substance into the peri-implant or junctional epithelium of rat gingivae. Clin Oral Implants Res, 2002; 13(3): 243.
[21] 李琼英，仝毅，肖邦良．EDTA脱钙法在硬组织化学中的应用．华西口腔医学杂志，1988；6(1)：68．
[22] 于洪藻，李玉林，张丽红等．三种脱钙液对狗牙齿及牙槽骨脱钙效果的比较．口腔医学纵横杂志，1994；10(3)：175．
[23] 孙庆妹，刘军，金岩等．三种脱钙液对免疫组化染色效果的比较．华西口腔医学杂志，1997；15(1)：80．
[24] Tenenbaum H, Schaaf JF, Cuisinier FJ. Histological analysis of the Ankylos
peri-implant soft tissues in a dog model. Implant Dent, 2003; 12(3): 259.
[25] Arvidson K, Fartash B, Hilliges M, et al. Histological characteristics of peri-implant mucosa atound Branemark and single-crystal sapphire implants. Clin Oral Implants Res, 1996; 7: 1.
[26] Dawson AE, Norton JA, Weinberg DS. Comparetive assessment of proliferation and DNA content in breast carcinoma by image analysis and flow cytometry. Am J Pathol, 1990; 136(5): 1115.
[27] Inoue T, Takeda T, Lee CY, et al. Immunolocalization of proliferating cell nuclear antigen in the peri-implant epithelium. Bull Tokyo Dent Coll. 1997; 38: 187.
[2] Chavrier CA, Couble ML. Ultrastructural immunohistochemical study of interstitial collagenous components of the healthy human keratinized mucosa surrounding implants.Int J Oral Maxillofac Implants, 1999; 14(1): 108.
[3] Albrektsson T, Branemark PI, Hansson HA, et al. Osseointegrated titanium implants. Acta Orthop Scand, 1981; 52: 155.
[4] Schroder A, Van Der Zypen E, Sticg H, et al. The reactions of bone connective tissue, and epithelium to endosteal implants with titaniumsprayed surfaces. J Maxillofac Surg, 1981; 9: 15.
[5] James RA, Schultz RL. Hemidesmosomes and the adhesion of junctional epithelial cells to metal implants. J oral Implantol, 1973; 4: 294.
[6] Worthington P, Branemark PI. Advanced osseointegration surgery: Application in the maxillofacial region illirois. Quintessence Publishing Co, 1992: 56.
[7] Meffert RM. How to treat ailing and failing implants. Implant Dent, 1992; 1(1):25
[8] 何福明，刘丽，张凯等．表面为金红石型纯钛种植体与口腔粘膜界面的组织学观察．浙江大学学报(医学版)，2002；31(6)：444．
[9] Steflik DE, Mckinney RV, Koth DL. Ultrastructural comparisons of ceramic and titanium dental implants in vivo: A scanning electron microscopic study. J Biomed Mter Res, 1989; 23: 895.
[10] 巢永烈，梁星．种植义齿学．北京北京医科大学、中国协和医科大学联合出版社，1999，第1版：24．
[11] Branemark PI, Breine U, Adell R, et al. Intra-osseous anchorage of dental prosthesis. Experimental studies. Scand J Plast Res Hand Surg, 1969; 3: 81.
[12] Nociti FH Jr, Caffesse RG, Sallum EA, et al. Evaluation of guided bone regeneration and/or bone grafts in the treatment of ligature-induced peri-implantitis defects: a morphometric study in dogs. J Oral Implantol, 2000; 26(4): 244.
[13] Rutar A, Lang NP, Buser D, et al. Retrospective assessment of clinical and microbiological factors affecting periimplant tissue conditions. Clin Oral Implants Res, 2001; 12(3): 189.
[14] E Chaar ES, Jalbout ZN. Regeneration of an osseous peri-implantitis lesion. Periodontal Clin Investig, 2002; 24(1): 5.
[15] Listgarten, MA. Phase-contrast and electron microscopic study of the junction between reduced enamel epithelium and enamel in unerupted human teeth. Arch Oral Biol, 1966; 11(10): 999.
[16] McKinney RV, Steflik DE, Koth KL. Evidence for junctional epithelial attachment to ceramic dental implants, a transmission electron microscopic study. J Periodontol, 1985;56: 579.
[17] Listgareen MA, Lai CH. Ultrastructure of the intact interface between an endosseous Ipoxy resin dental implant and the host tissres. J Biol Buccale, 1975; 3:13.
[18] Moon IS, Berglundh T, Abrahamsson I, et al. The barrier between the keratinized mucosa and the dental implant. An experimental study in the dog. J Clin Periodontol, 1999; 26(10): 658.
[19] Kido MA, Inoue T, Shimono M, et al. Ultrastructural and immunoelectron microscopic studies of the peri-implant epithelium-implant (Ti-6Al-4V) interface of rat maxilla. J Periodontol, 2000; 71(6): 961.
[20] Keda H, Shiraiwa M, Yamaza T. Difference in penetration of horseradish peroxidase tracer as a foreign substance into the peri-implant or junctional epithelium of rat gingivae. Clin Oral Implants Res, 2002; 13(3): 243.
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