冷冻干燥牙本质在牙髓—牙本质复合体再生中的应用基础研究

发布时间：2018-07-07 21:04

本文选题：机械性能 + 干细胞　；参考：《第四军医大学》2014年博士论文

【摘要】：龋齿、牙周病、创伤等，是引起牙齿缺失最常见的一类疾病。现有牙齿缺失的治疗方法主要是传统义齿修复或种植体修复，但传统义齿修复是一种被动的、需要余留组织补偿的修复方式，不能完全恢复咀嚼效率；而种植修复虽然能克服缺牙数目的限制，但由于其与牙槽骨的刚性连接缺乏牙周膜结构的缓冲、感知功能及天然的细胞封闭作用，故存在感染或因牙合力过大导致骨吸收的风险，与天然牙齿相比仍有较大的差异。牙缺失治疗的研究热点之一是利用组织工程再生牙组织。现今牙再生的瓶颈之一是缺乏理想的组织工程支架材料以提供种子细胞合适的生长环境。支架材料除了应具有良好的机械性能、良好的生物相容性，还应含有丰富的成牙相关生物活性因子以诱导种子细胞向成牙的方向分化，并应易于保存和运输。牙本质来源广泛，具有的特殊组织结构可以促进种子细胞的成牙能力。牙本质基质成分十分复杂，包含成牙相关的所有蛋白和因子并具有潜在的免疫原性。冷冻干燥技术（简称冻干）能使物料在干燥的同时能够保持自身的结构、形状和性质，很多领域被广泛应用。本课题拟通过冷冻干燥技术对牙本质进行处理，以降低牙本质的免疫原性、保护其中的生物活性物质，方便牙本质支架材料的保存和运输，对制成的冻干牙本质进行机械力学性能检测，并通过体内、外实验对冻干牙本质的生物相容性和诱导成牙能力进行评估，以期获得适宜牙再生的冻干牙本质生物支架材料。第一部分冷冻干燥牙本质（FDDM）的制作及材料性能检测研究目的 1）制作冷冻干燥牙本质生物支架材料 2）检测FDDM的机械力学性能及胶原释放能力研究方法 1）在去除牙髓、牙周组织的基础上，将经深冷冻的牙本质进行冷冻干燥处理，辐照灭菌，真空包装保存。 2）通过压实验、三点弯曲实验以及维氏显微硬度实验检测冻干牙本质的机械力学性能；ELISA法检测材料的胶原释放能力;通过扫面电镜、HE染色观察FDDM的组织结构。实验结果 1）得到的冻干牙本质结构均一，在形状、大小、色泽上没有显著变化。 2）冻干牙本质与普通牙本质的抗压强度没有显著性差异，但这两个牙本质组的抗压强度与HA组相比具有统计学差异（P0.01），抗压强度远高于HA；抗压弹性模量、挠曲强度、挠曲弹性模量检测结果显示相同趋势；FDDM的I型胶原（COL-1）释放能力与普通牙本质无统计学差异；FDDM的维氏显微硬度略高于普通牙本质；冻干处理对牙本质结构的影响不显著。结论制作出的FDDM与普通牙本质相比具有相似的组织结构、机械力学强度、COL-1释放能力及略高的表面硬度。第二部分冷冻干燥牙本质生物相容性检测及对DPSCs的影响研究目的 1）体外分离培养并鉴定DPSCs的生物学特性 2）体外检测冻干牙本质（FDDM）的生物相容性及对牙髓干细胞（DPSCs）的影响研究方法 1）对从人牙髓组织中分离出的DPSCs进行克隆形成能力、多向分化能力及间充质干细胞表面标志物的流式细胞检测。 2）对FDDM表面的DPSCs进行细胞增殖；细胞周期；胶原分泌；ALP活性检测；Real-time PCR检测FDDM表面DPSCs成牙、成骨及相关细胞外基质分泌基因表达；细胞免疫荧光染色检测相关蛋白表达。实验结果 1）分离培养出的DPSCs具有克隆形成能力和多向分化潜能；流式细胞仪检测细胞特定抗原表达显示：STRO-1, CD29, CD90, CD105及CD44表达呈阳性，而造血干细胞表面标志CD34、CD45以及内皮细胞表面标志CD31表达呈阴性。结果符合牙髓间充质干细胞的表面标志物表达。 2）冻干牙本质同普通牙本质一样，对牙髓干细胞具有良好的生物相容性，在细胞形态、细胞活性、胶原分泌及成牙、细胞外基质分泌的相关基因、蛋白表达上均优于羟基磷灰石-磷酸三钙，但ALP活性及骨向分化相关的基因、蛋白表达均低于羟基磷灰石-磷酸三钙。结论 1)成功分离培养出DPSCs。 2) FDDM具有良好的生物相容性，与HA相比，能够促进DPSCs的活性及细胞外基质的分泌，促进DPSCs的牙向分化。但降低DPSCs的骨向分化能力。第三部分利用冷冻干燥牙本质进行牙髓牙本质复合体再生的体内实验研究研究目的 1）体内验证冻干牙本质（FDDM）的生物相容性和诱导DPSCs再生牙髓-牙本质样复合体的能力研究方法将DPSCs细胞膜片聚集体分别与FDDM、普通牙本质(D)和HA复合后进行裸鼠皮下移植，每只裸鼠背部皮下植入一枚牙髓干细胞膜片-支架复合体， SPF级别饲养8周后进行HE染色、马氏三色染色及相关蛋白的免疫组化染色。实验结果冻干牙本质/普通牙本质-牙髓干细胞复合物再生出牙髓-牙本质样组织，，极性的成牙本质样细胞沿着新生成的前期牙本质整齐排列，且没有观察到炎症反应。羟基磷灰石-磷酸三钙-牙髓干细胞复合物中没有观察到典型的牙髓-牙本质样结构形成，可见炎性细胞浸润。免疫组化染色显示普通牙本质与冻干牙本质阳性表达ALP、COL-3、DSP、fibronectin和人线粒体（mitochondria）。结论冻干牙本质与普通牙本质在裸鼠体内具有相同的诱导人来源牙髓干细胞向成牙本质样细胞分化的能力，并能够在体内再生牙髓-牙本质复合体，具有良好的生物相容性。
[Abstract]:Dental caries , periodontal disease , trauma , etc . are the most common diseases that cause the loss of teeth . The existing treatment methods of tooth loss are mainly traditional denture repair or implant repair , but traditional denture restoration is a kind of passive and requires the restoration of residual tissue compensation , and the chewing efficiency cannot be fully recovered ;
while the implant repair can overcome the limitation of the number of missing teeth , because the rigid connection with the alveolar bone lacks the buffering , sensing function and natural cell sealing effect of the periodontal membrane structure , there is a risk of infection or excessive occlusal force which leads to bone resorption , and has great difference compared with the natural tooth .

Abstract : One of the hot topics of tooth loss therapy is to use tissue engineering to regenerate the dental tissue . One of the bottlenecks in today ' s tooth regeneration is the lack of ideal tissue engineering scaffold material to provide proper growth environment for seed cells .

Preparation of the first part of freeze - dried dentin ( FDDM ) and detection of material properties

Purpose of study

1 ) preparing freeze - drying dentin biological scaffold material

2 ) Mechanical property and collagen releasing ability of FDDM were measured .

Research Methods

1 ) performing freeze drying treatment on the deeply frozen dentin on the basis of removing dental pulp and periodontal tissue , irradiating and sterilizing , and vacuum packaging and storing .

2 ) the mechanical and mechanical properties of the freeze - dried dentin are detected by three - point bending experiments and Vickers microhardness testing ;
The collagen release ability of the material was detected by ELISA . The structure of FDDM was observed by scanning electron microscope and HE staining .

experimental results

1 ) the obtained freeze - dried dentin has uniform structure , and has no obvious change in shape , size and color .

2 ) There was no significant difference in compressive strength between freeze - dried dentin and common dentin , but the compressive strength of these two dentine groups was significantly higher than that of HA group ( P0.01 ) , and the compressive strength was much higher than HA ;
The test results of compressive elastic modulus , flexural strength and flexural modulus of elasticity show the same trend ;
There was no statistical difference between the release ability of type I collagen ( COL - 1 ) and common dentin in FDDM ;
The Vickers microhardness of FDDM is slightly higher than that of ordinary dentin ;
The influence of freeze - drying treatment on dentin structure was not significant .

Conclusion

Compared with common dentin , the produced FDDM had similar structure , mechanical strength , COL - 1 release ability and slightly higher surface hardness .

Study on the biocompatibility of the second part of freeze - dried dentin and its effect on DPSCs

Purpose of study

1 ) In vitro isolation culture and identification of the biological characteristics of DPSCs

2 ) The biocompatibility of freeze - dried dentin ( FDDM ) in vitro and the influence on dental pulp stem cells ( DPSCs )

Research Methods

and 1 ) performing cloning and forming on the DPSCs isolated from the human dental pulp tissue , and performing flow - type cell detection of the multi - directional differentiation capability and the mesenchymal stem cell surface marker .

2 ) performing cell proliferation on the DPSCs on the surface of the FDDM ;
Cell cycle ;
Collagen secretion ;
ALP activity detection ;
Real - time PCR was used to detect the expression of FDDM surface DPSCs into teeth , adult bone and related extracellular matrix .
Cell immunofluorescence staining was used to detect the expression of related proteins .

experimental results

1 ) separating and culturing the DPSCs with clonal formation ability and multi - directional differentiation potential ;
The expression of CD29 , CD90 , CD105 and CD44 was positive in the expression of CD29 , CD90 , CD105 and CD44 , and the expression of CD31 on the surface of hematopoietic stem cells was negative . The results were in agreement with the expression of surface markers of mesenchymal stem cells .

2 ) The freeze - dried dentin , like the common dentin , has good biocompatibility to the dental pulp stem cells , and is superior to the hydroxyapatite - tricalcium phosphate in cell morphology , cell activity , collagen secretion and the related genes and protein expression secreted by the odontoblasts and extracellular matrix , but the expression of ALP activity and bone - to - differentiation related genes is lower than that of hydroxyapatite - tricalcium phosphate .

Conclusion

1 ) DPSCs were isolated and cultured successfully .

2 ) FDDM has good biocompatibility . Compared with HA , it can promote the activity of DPSCs and the secretion of extracellular matrix , and promote the differentiation of DPSCs .

In vivo experimental study on the regeneration of dental pulp dentin complex by freeze - drying dentin

Purpose of study

1 ) In vivo verification of the biocompatibility of the lyophilized dentin ( FDDM ) and the ability to induce DPSCs to regenerate the pulp - dentin - like complex

Research Methods

DPSCs were transplanted subcutaneously with FDDM , common dentine ( D ) and HA . One dental pulp stem cell membrane - stent complex was implanted subcutaneously in the back of each nude mouse , HE staining was performed at SPF level for 8 weeks , three - color staining and immunohistochemical staining of related proteins were performed .

experimental results

A typical dentin - dentin - like structure was regenerated from the freeze - dried dentin / dentin - pulp stem cell complex . The dentin - like cells of the polarity were orderly arranged along the dentine of the new tooth , and no inflammatory reaction was observed . The typical dentin - dentin - like structure was not observed in the hydroxyapatite - tricalcium phosphate - pulp stem cell complex . The inflammatory cell infiltration was seen . Immunohistochemical staining showed that the normal dentin was positively correlated with the lyophilized dentin , ALP , COL - 3 , DSP , fibronectin and human mitochondria .

Conclusion

The freeze - dried dentin has the same ability of inducing human dental pulp stem cells to differentiate into odontoblasts in nude mice , and can regenerate the pulp - dentin complex in vivo , and has good biocompatibility .
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R783.6

【参考文献】