组织工程牙根HA/TCP支架材料的载银抗菌策略研究
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摘要
牙列缺损与缺失是人类口颌系统的常见病与多发病，自17世纪起，人们就在尝试用人工材料“替代”缺牙来恢复牙列的完整，在400多年后的今天，虽然修复理念从单纯保持美观演变成兼顾美观与口颌系统功能，修复材料从兽骨、象牙、木头发展成树脂、金属、陶瓷，修复工艺也由计算机仿真设计与制作逐步替代传统的手工加工，使得修复精度更高，修复效果也更为自然、逼真，但从本质上来说，这还仅仅是对牙冠部分的恢复。上世纪八十年代，瑞典骨科医生Branemark教授创立的种植体理论与技术体系，开辟了口腔颌面部“重建”修复的新纪元，但由于种植体与牙槽骨之间的骨性结合相对于自然牙与牙槽骨之间的牙周膜连接而言，对咬合力的缓冲作用大大减弱，也不能对超出生理范围的破坏性咬合力做出保护反应，达不到生理意义上的完全重建。因此，能够使缺失的牙齿自然“再生”一直是人类的一个梦想。
干细胞和组织工程技术的发展为牙齿再生带来了曙光，完整牙齿包含了牙釉质、牙本质、牙骨质和牙髓等多种组织，受复杂调控因素影响，其再生稳定性和成功率还远远达不到临床治疗需求。近年来，基于三维组织工程方法开展的牙根再生研究取得了突破性进展，2006年，Sonoyama报道通过将根尖牙乳头干细胞（SCAP）与HA/TCP支架材料复合，并在HA/TCP支架外围包裹一层复合牙周膜干细胞的明胶海绵植入小型猪牙槽骨，得到了矿化的组织工程牙根，并初步实现了组织工程牙根与牙槽骨的牙周膜性结合。然而，当课题组在对牙根进行上部结构修复后不久，便发现修复体周围牙龈结合不紧密，形成较深的牙周袋，并有炎性组织存在，类似于种植体周围炎症状，严重影响了组织工程牙根的远期疗效。因此，作为影响组织工程牙根临床应用的一个关键问题，开展其与牙龈组织的结合研究十分必要。
自然牙齿与牙龈的结合包括上皮和固有层与牙齿贴附，其中上皮层主要通过半桥粒与牙釉质或牙骨质连接，而以胶原纤维为主体的固有层通过致密的结缔组织与牙槽骨和牙齿的颈部形成牢固结合，起到较强的防御作用，并抑制牙龈上皮向根向移动，由此可见固有层在牙颈部软组织封闭中起到重要的作用。从目前的研究进展来看，影响牙龈与组织工程牙根结合的最关键因素就是因细菌附着而导致的局部感染。探索一种既具备一定抗菌能力，又不影响牙龈细胞生物学活性的穿龈模式，将会有效促进牙龈与组织工程牙根的结合。银由于抗菌谱广、抗菌时间持久、不易引起耐药等优点，已经成为口腔医学领域的一种重要的抗菌剂，而且其特有的选择毒性，可以使其在对动物细胞相对安全的情况下，具备较强的抗菌能力。
基于此理念，本课题组在前期成功构建组织工程牙根的基础上，对其穿龈部位的HA/TCP支架载银抗菌策略进行研究，研究分为三部分：（1）HA/TCP材料对牙龈成纤维细胞（GFs）、牙髓干细胞（DPSCs）的生物学行为影响研究（；2）载银HA/TCP材料的构建与抗菌性能研究；（3）载银HA/TCP材料对GFs、DPSCs的生物学行为影响的研究。结果如下：
1、HA/TCP材料为无毒性材料。电镜及免疫荧光染色显示，接种于HA/TCP材料表面GFs和DPSCs，细胞伸展，胞浆饱满，生长情况良好。
2、用AgNO3溶液浸泡，并以紫外光照射法，可以制备载银HA/TCP材料，载银量可以通过调整浸泡的AgNO3溶液浓度来实现。载银HA/TCP的银释放在最初24h呈突释状态，但24h后呈缓慢释放，并逐渐趋于稳定，且前24h内银的突释量与载银浓度呈正相关。
3、载银HA/TCP材料可以有效抑制细菌的生长，其抗菌性能与载银量呈正相关关系，但材料的最低有效抑菌浓度还有待于进一步研究。
4、MTT法检测四种载银浓度的HA/TCP材料，0.5mol/L、1mol/L、1.5mol/L和2mol/LAgNO3溶液浸泡制备的载银HA/TCP的RGR值分别为83.22%，51.01%，38.67%和19.84%，根据细胞毒性分级，0.5mol/L组试样的细胞毒性分级为1级，为轻度毒性，生物安全性合格；1mol/L组试样的细胞毒性为2级，属于中度毒性，其余2组试样的细胞毒性分级为3级和4级，属于重度毒性，除0.5mol/L组外，其余三组载银试样的生物安全性均不合格。
5、免疫荧光染色显示，接种于0.5mol/L组试样的GFs生长状态基本良好，有少部分细胞溶解成碎片，接种于其余三组的GFs细胞出现大部分的溶解，提示0.5mol/L组试样与GFs具有较好的生物相容性；接种于0.5mol/L组试样的DPSCs生长情况与GFs相似，有个别细胞溶解呈团块，接种于其余三组的DPSCs大部分出现溶解，提示0.5mol/L组试样与DPSCs具有较好的生物相容性。
Defect of dentition is very common in humans. It is recorded that people hadtried to complete the defective dentition with artificial materials since1600s.Today, more than400years later, although dentists focus more and more on bothesthetic and function of gnathostomatic system, the materials have developedfrom animal bones, wood or ivory to metal, resin and porcelain, the processingtechnic has changed from hand-crafted to computer-aided-design andcomputer-aided-manufacture, it is still similar to the old technique of replacement,because it can only replace the crown. In1980s, Branemark,a Swedishorthopedist built the theoretical and technical system of dental implant, whichopen up a new epoch of dental and maxillofacial reconstruction. But thesynostosis between implant and alveolar bone can’t buffer the occlusal force asmuch as the natural combination between tooth root and alveolar bone bypericementum. It can also not protect the implant out of the destructive occlusalforce and keep the reconstruction physiological. So, how to regenerate teeth has become a dream of mankind for a long time.
The development of stem cells and tissue engineering technique bringsbright sunshine to the tooth regeneration. Tooth is a complex organ whichcontains enamel, dentin, cementum and pulp. The stability and success rate ofwhole tooth regeneration is far from the clinical therapy because of thecomplicated control mechanism. Tooth root regeneration, which based on the3D tissue engineering technique, makes a breakthrough in recent years. In2006, areport on tooth root regeneration was presented by Sonoyama. They used humanSCAP to regenerate dentin on a HA/TCP carrier, while using human PDLSCs toregenerate PDL on a Gelfoam which was covered around the HA/TCP carrier.CT examination revealed a HA/SCAP Gelfoam/PDLSC structure forming insidethe alveolar bone with mineralized root-like tissue formation and periodontalligament space. However, shortly after the root in conjunction with the dentalcrown, they found there was a deep periodontal pocket around the root, withsome inflammatory tissue inside the pocket, which looked likes the symptom ofperi-implantitis. In order to promote the long term effect of tissue engineeringtooth root, it is essential to research the combination between gingival and theroot.
The natural combination between tooth and gingiva contains two parts, theone is epidermis, while the other is gingival lamina propria. Epidermis attach theenamel or cementum of dental cervix by hemidesmosome, and the gingivallamina propria, which mainly consists of collagenous fibre, can adhere to thetooth tightly by compact connective tissue to protect the clearance betweengingival and tooth from the bacterial attack. It can also inhibit the epidermismove to the root apex direction. So, the gingival lamina propria may plays a moreimportant role in the soft tissue closure on dental cervix. According to current result, the inflammation on the periodontal pocket may be the most importantfactor of interrupting gingival tissue to adhere to the root. If the inflammationwas removed, the combination between gingival and the root may be better.Nowadays, silver has become a kind of very important antimicrobial in dentalcare, because of its wide antimicrobial spectrum, long-acting antibiotic activityand low drug resistance. Furthermore, the toxicity of silver is selective, it canresist the bacterial specially, while the cell is relatively safe.
So, the aim of this research is to explore an antibacterial strategy of theHA/TCP scaffold which is on the gingiva-crossing part of the tooth root. Basedon the pre-research by Sonoyama, our research work can be devided into threepart,(1) The biological behaviour of GFs and DPSCs when they are cultured onthe HA/TCP.(2) The preparation of Ag+-HA/TCP and the research of itsantibacterial activity.(3) How the Ag+-HA/TCP affect the biological behaviour ofGFs and DPSCs. The results is reported as follows.
1. HA/TCP is a kind of innoxious material. Both SEM andimmunofluorescence shows that the GFs and DPSCs which cultured on theHA/TCP extends well, and the cytoplasm is full.
2. Irradiate by UV-light after marinated in AgNO3is a new way ofpreparing Ag+-HA/TCP. The content of Ag+in Ag+–HA/TCP can be adjusted bychanging the viscosity of AgNO3。Ag+releases abruptly in the beginning24h,and goes slowly after24h, finally it tended towards stability. There is a positivecorrelation between the released Ag+and the viscosity of AgNO3in the first24h.
3. Ag+–HA/TCP can inhibit the growth of bacterial.0.5mol/L group showeda weaker antibacterial activity than the other3groups (1mol/L group,1.5mol/Lgroup and2mol/L group), but there is no remarkable difference between thelatter3groups. It is indicated that there maybe also a positive correlation between the antibacterial activity and the viscosity of AgNO3, but the minimalantibacterial viscosity still needs to be researched.
4. Use MTT method to investigate the safety of Ag+–HA/TCP. The RGR ofthe4groups were83.22%，51.01%，38.67%and19.84%. It means that the0.5mol/L group has a little cytotoxicity and is qualified for the biologicalexperiment, while the other3groups are not fit for the clinical use.
5. According to the immunofluorescence, the GFs cultured on the0.5mol/Lgroup grows much better than the other3groups, there is only a little cellsdissolved, while most cells cultured on the other3groups died. Similar situationoccurs in the DPSCs. So we conclude that0.5mol/L group has a goodbiocompatibility with both GFs and DPSCs.
干细胞和组织工程技术的发展为牙齿再生带来了曙光，完整牙齿包含了牙釉质、牙本质、牙骨质和牙髓等多种组织，受复杂调控因素影响，其再生稳定性和成功率还远远达不到临床治疗需求。近年来，基于三维组织工程方法开展的牙根再生研究取得了突破性进展，2006年，Sonoyama报道通过将根尖牙乳头干细胞（SCAP）与HA/TCP支架材料复合，并在HA/TCP支架外围包裹一层复合牙周膜干细胞的明胶海绵植入小型猪牙槽骨，得到了矿化的组织工程牙根，并初步实现了组织工程牙根与牙槽骨的牙周膜性结合。然而，当课题组在对牙根进行上部结构修复后不久，便发现修复体周围牙龈结合不紧密，形成较深的牙周袋，并有炎性组织存在，类似于种植体周围炎症状，严重影响了组织工程牙根的远期疗效。因此，作为影响组织工程牙根临床应用的一个关键问题，开展其与牙龈组织的结合研究十分必要。
自然牙齿与牙龈的结合包括上皮和固有层与牙齿贴附，其中上皮层主要通过半桥粒与牙釉质或牙骨质连接，而以胶原纤维为主体的固有层通过致密的结缔组织与牙槽骨和牙齿的颈部形成牢固结合，起到较强的防御作用，并抑制牙龈上皮向根向移动，由此可见固有层在牙颈部软组织封闭中起到重要的作用。从目前的研究进展来看，影响牙龈与组织工程牙根结合的最关键因素就是因细菌附着而导致的局部感染。探索一种既具备一定抗菌能力，又不影响牙龈细胞生物学活性的穿龈模式，将会有效促进牙龈与组织工程牙根的结合。银由于抗菌谱广、抗菌时间持久、不易引起耐药等优点，已经成为口腔医学领域的一种重要的抗菌剂，而且其特有的选择毒性，可以使其在对动物细胞相对安全的情况下，具备较强的抗菌能力。
基于此理念，本课题组在前期成功构建组织工程牙根的基础上，对其穿龈部位的HA/TCP支架载银抗菌策略进行研究，研究分为三部分：（1）HA/TCP材料对牙龈成纤维细胞（GFs）、牙髓干细胞（DPSCs）的生物学行为影响研究（；2）载银HA/TCP材料的构建与抗菌性能研究；（3）载银HA/TCP材料对GFs、DPSCs的生物学行为影响的研究。结果如下：
1、HA/TCP材料为无毒性材料。电镜及免疫荧光染色显示，接种于HA/TCP材料表面GFs和DPSCs，细胞伸展，胞浆饱满，生长情况良好。
2、用AgNO3溶液浸泡，并以紫外光照射法，可以制备载银HA/TCP材料，载银量可以通过调整浸泡的AgNO3溶液浓度来实现。载银HA/TCP的银释放在最初24h呈突释状态，但24h后呈缓慢释放，并逐渐趋于稳定，且前24h内银的突释量与载银浓度呈正相关。
3、载银HA/TCP材料可以有效抑制细菌的生长，其抗菌性能与载银量呈正相关关系，但材料的最低有效抑菌浓度还有待于进一步研究。
4、MTT法检测四种载银浓度的HA/TCP材料，0.5mol/L、1mol/L、1.5mol/L和2mol/LAgNO3溶液浸泡制备的载银HA/TCP的RGR值分别为83.22%，51.01%，38.67%和19.84%，根据细胞毒性分级，0.5mol/L组试样的细胞毒性分级为1级，为轻度毒性，生物安全性合格；1mol/L组试样的细胞毒性为2级，属于中度毒性，其余2组试样的细胞毒性分级为3级和4级，属于重度毒性，除0.5mol/L组外，其余三组载银试样的生物安全性均不合格。
5、免疫荧光染色显示，接种于0.5mol/L组试样的GFs生长状态基本良好，有少部分细胞溶解成碎片，接种于其余三组的GFs细胞出现大部分的溶解，提示0.5mol/L组试样与GFs具有较好的生物相容性；接种于0.5mol/L组试样的DPSCs生长情况与GFs相似，有个别细胞溶解呈团块，接种于其余三组的DPSCs大部分出现溶解，提示0.5mol/L组试样与DPSCs具有较好的生物相容性。
Defect of dentition is very common in humans. It is recorded that people hadtried to complete the defective dentition with artificial materials since1600s.Today, more than400years later, although dentists focus more and more on bothesthetic and function of gnathostomatic system, the materials have developedfrom animal bones, wood or ivory to metal, resin and porcelain, the processingtechnic has changed from hand-crafted to computer-aided-design andcomputer-aided-manufacture, it is still similar to the old technique of replacement,because it can only replace the crown. In1980s, Branemark,a Swedishorthopedist built the theoretical and technical system of dental implant, whichopen up a new epoch of dental and maxillofacial reconstruction. But thesynostosis between implant and alveolar bone can’t buffer the occlusal force asmuch as the natural combination between tooth root and alveolar bone bypericementum. It can also not protect the implant out of the destructive occlusalforce and keep the reconstruction physiological. So, how to regenerate teeth has become a dream of mankind for a long time.
The development of stem cells and tissue engineering technique bringsbright sunshine to the tooth regeneration. Tooth is a complex organ whichcontains enamel, dentin, cementum and pulp. The stability and success rate ofwhole tooth regeneration is far from the clinical therapy because of thecomplicated control mechanism. Tooth root regeneration, which based on the3D tissue engineering technique, makes a breakthrough in recent years. In2006, areport on tooth root regeneration was presented by Sonoyama. They used humanSCAP to regenerate dentin on a HA/TCP carrier, while using human PDLSCs toregenerate PDL on a Gelfoam which was covered around the HA/TCP carrier.CT examination revealed a HA/SCAP Gelfoam/PDLSC structure forming insidethe alveolar bone with mineralized root-like tissue formation and periodontalligament space. However, shortly after the root in conjunction with the dentalcrown, they found there was a deep periodontal pocket around the root, withsome inflammatory tissue inside the pocket, which looked likes the symptom ofperi-implantitis. In order to promote the long term effect of tissue engineeringtooth root, it is essential to research the combination between gingival and theroot.
The natural combination between tooth and gingiva contains two parts, theone is epidermis, while the other is gingival lamina propria. Epidermis attach theenamel or cementum of dental cervix by hemidesmosome, and the gingivallamina propria, which mainly consists of collagenous fibre, can adhere to thetooth tightly by compact connective tissue to protect the clearance betweengingival and tooth from the bacterial attack. It can also inhibit the epidermismove to the root apex direction. So, the gingival lamina propria may plays a moreimportant role in the soft tissue closure on dental cervix. According to current result, the inflammation on the periodontal pocket may be the most importantfactor of interrupting gingival tissue to adhere to the root. If the inflammationwas removed, the combination between gingival and the root may be better.Nowadays, silver has become a kind of very important antimicrobial in dentalcare, because of its wide antimicrobial spectrum, long-acting antibiotic activityand low drug resistance. Furthermore, the toxicity of silver is selective, it canresist the bacterial specially, while the cell is relatively safe.
So, the aim of this research is to explore an antibacterial strategy of theHA/TCP scaffold which is on the gingiva-crossing part of the tooth root. Basedon the pre-research by Sonoyama, our research work can be devided into threepart,(1) The biological behaviour of GFs and DPSCs when they are cultured onthe HA/TCP.(2) The preparation of Ag+-HA/TCP and the research of itsantibacterial activity.(3) How the Ag+-HA/TCP affect the biological behaviour ofGFs and DPSCs. The results is reported as follows.
1. HA/TCP is a kind of innoxious material. Both SEM andimmunofluorescence shows that the GFs and DPSCs which cultured on theHA/TCP extends well, and the cytoplasm is full.
2. Irradiate by UV-light after marinated in AgNO3is a new way ofpreparing Ag+-HA/TCP. The content of Ag+in Ag+–HA/TCP can be adjusted bychanging the viscosity of AgNO3。Ag+releases abruptly in the beginning24h,and goes slowly after24h, finally it tended towards stability. There is a positivecorrelation between the released Ag+and the viscosity of AgNO3in the first24h.
3. Ag+–HA/TCP can inhibit the growth of bacterial.0.5mol/L group showeda weaker antibacterial activity than the other3groups (1mol/L group,1.5mol/Lgroup and2mol/L group), but there is no remarkable difference between thelatter3groups. It is indicated that there maybe also a positive correlation between the antibacterial activity and the viscosity of AgNO3, but the minimalantibacterial viscosity still needs to be researched.
4. Use MTT method to investigate the safety of Ag+–HA/TCP. The RGR ofthe4groups were83.22%，51.01%，38.67%and19.84%. It means that the0.5mol/L group has a little cytotoxicity and is qualified for the biologicalexperiment, while the other3groups are not fit for the clinical use.
5. According to the immunofluorescence, the GFs cultured on the0.5mol/Lgroup grows much better than the other3groups, there is only a little cellsdissolved, while most cells cultured on the other3groups died. Similar situationoccurs in the DPSCs. So we conclude that0.5mol/L group has a goodbiocompatibility with both GFs and DPSCs.
引文
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