本文選題：機(jī)械性能 + 干細(xì)胞��； 參考：《第四軍醫(yī)大學(xué)》2014年博士論文

【摘要】：齲齒、牙周病、創(chuàng)傷等，是引起牙齒缺失最常見的一類疾病�，F(xiàn)有牙齒缺失的治療方法主要是傳統(tǒng)義齒修復(fù)或種植體修復(fù)，但傳統(tǒng)義齒修復(fù)是一種被動(dòng)的、需要余留組織補(bǔ)償?shù)男迯?fù)方式，不能完全恢復(fù)咀嚼效率；而種植修復(fù)雖然能克服缺牙數(shù)目的限制，但由于其與牙槽骨的剛性連接缺乏牙周膜結(jié)構(gòu)的緩沖、感知功能及天然的細(xì)胞封閉作用，故存在感染或因牙合力過(guò)大導(dǎo)致骨吸收的風(fēng)險(xiǎn)，與天然牙齒相比仍有較大的差異。 牙缺失治療的研究熱點(diǎn)之一是利用組織工程再生牙組織�，F(xiàn)今牙再生的瓶頸之一是缺乏理想的組織工程支架材料以提供種子細(xì)胞合適的生長(zhǎng)環(huán)境。支架材料除了應(yīng)具有良好的機(jī)械性能、良好的生物相容性，還應(yīng)含有豐富的成牙相關(guān)生物活性因子以誘導(dǎo)種子細(xì)胞向成牙的方向分化，并應(yīng)易于保存和運(yùn)輸。牙本質(zhì)來(lái)源廣泛，具有的特殊組織結(jié)構(gòu)可以促進(jìn)種子細(xì)胞的成牙能力。牙本質(zhì)基質(zhì)成分十分復(fù)雜，包含成牙相關(guān)的所有蛋白和因子并具有潛在的免疫原性。冷凍干燥技術(shù)（簡(jiǎn)稱凍干）能使物料在干燥的同時(shí)能夠保持自身的結(jié)構(gòu)、形狀和性質(zhì)，很多領(lǐng)域被廣泛應(yīng)用。本課題擬通過(guò)冷凍干燥技術(shù)對(duì)牙本質(zhì)進(jìn)行處理，以降低牙本質(zhì)的免疫原性、保護(hù)其中的生物活性物質(zhì)，方便牙本質(zhì)支架材料的保存和運(yùn)輸，對(duì)制成的凍干牙本質(zhì)進(jìn)行機(jī)械力學(xué)性能檢測(cè)，并通過(guò)體內(nèi)、外實(shí)驗(yàn)對(duì)凍干牙本質(zhì)的生物相容性和誘導(dǎo)成牙能力進(jìn)行評(píng)估，以期獲得適宜牙再生的凍干牙本質(zhì)生物支架材料。 第一部分冷凍干燥牙本質(zhì)（FDDM）的制作及材料性能檢測(cè) 研究目的 1）制作冷凍干燥牙本質(zhì)生物支架材料 2）檢測(cè)FDDM的機(jī)械力學(xué)性能及膠原釋放能力 研究方法 1）在去除牙髓、牙周組織的基礎(chǔ)上，將經(jīng)深冷凍的牙本質(zhì)進(jìn)行冷凍干燥處理，輻照滅菌，真空包裝保存。 2）通過(guò)壓實(shí)驗(yàn)、三點(diǎn)彎曲實(shí)驗(yàn)以及維氏顯微硬度實(shí)驗(yàn)檢測(cè)凍干牙本質(zhì)的機(jī)械力學(xué)性能；ELISA法檢測(cè)材料的膠原釋放能力;通過(guò)掃面電鏡、HE染色觀察FDDM的組織結(jié)構(gòu)。 實(shí)驗(yàn)結(jié)果 1）得到的凍干牙本質(zhì)結(jié)構(gòu)均一，在形狀、大小、色澤上沒(méi)有顯著變化。 2）凍干牙本質(zhì)與普通牙本質(zhì)的抗壓強(qiáng)度沒(méi)有顯著性差異，但這兩個(gè)牙本質(zhì)組的抗壓強(qiáng)度與HA組相比具有統(tǒng)計(jì)學(xué)差異（P0.01），抗壓強(qiáng)度遠(yuǎn)高于HA；抗壓彈性模量、撓曲強(qiáng)度、撓曲彈性模量檢測(cè)結(jié)果顯示相同趨勢(shì)；FDDM的I型膠原（COL-1）釋放能力與普通牙本質(zhì)無(wú)統(tǒng)計(jì)學(xué)差異；FDDM的維氏顯微硬度略高于普通牙本質(zhì)；凍干處理對(duì)牙本質(zhì)結(jié)構(gòu)的影響不顯著。 結(jié)論 制作出的FDDM與普通牙本質(zhì)相比具有相似的組織結(jié)構(gòu)、機(jī)械力學(xué)強(qiáng)度、COL-1釋放能力及略高的表面硬度。 第二部分冷凍干燥牙本質(zhì)生物相容性檢測(cè)及對(duì)DPSCs的影響 研究目的 1）體外分離培養(yǎng)并鑒定DPSCs的生物學(xué)特性 2）體外檢測(cè)凍干牙本質(zhì)（FDDM）的生物相容性及對(duì)牙髓干細(xì)胞（DPSCs）的影響 研究方法 1）對(duì)從人牙髓組織中分離出的DPSCs進(jìn)行克隆形成能力、多向分化能力及間充質(zhì)干細(xì)胞表面標(biāo)志物的流式細(xì)胞檢測(cè)。 2）對(duì)FDDM表面的DPSCs進(jìn)行細(xì)胞增殖；細(xì)胞周期；膠原分泌；ALP活性檢測(cè)；Real-time PCR檢測(cè)FDDM表面DPSCs成牙、成骨及相關(guān)細(xì)胞外基質(zhì)分泌基因表達(dá)；細(xì)胞免疫熒光染色檢測(cè)相關(guān)蛋白表達(dá)。 實(shí)驗(yàn)結(jié)果 1）分離培養(yǎng)出的DPSCs具有克隆形成能力和多向分化潛能；流式細(xì)胞儀檢測(cè)細(xì)胞特定抗原表達(dá)顯示：STRO-1, CD29, CD90, CD105及CD44表達(dá)呈陽(yáng)性，而造血干細(xì)胞表面標(biāo)志CD34、CD45以及內(nèi)皮細(xì)胞表面標(biāo)志CD31表達(dá)呈陰性。結(jié)果符合牙髓間充質(zhì)干細(xì)胞的表面標(biāo)志物表達(dá)。 2）凍干牙本質(zhì)同普通牙本質(zhì)一樣，對(duì)牙髓干細(xì)胞具有良好的生物相容性，在細(xì)胞形態(tài)、細(xì)胞活性、膠原分泌及成牙、細(xì)胞外基質(zhì)分泌的相關(guān)基因、蛋白表達(dá)上均優(yōu)于羥基磷灰石-磷酸三鈣，但ALP活性及骨向分化相關(guān)的基因、蛋白表達(dá)均低于羥基磷灰石-磷酸三鈣。 結(jié)論 1)成功分離培養(yǎng)出DPSCs。 2) FDDM具有良好的生物相容性，與HA相比，能夠促進(jìn)DPSCs的活性及細(xì)胞外基質(zhì)的分泌，促進(jìn)DPSCs的牙向分化。但降低DPSCs的骨向分化能力。 第三部分利用冷凍干燥牙本質(zhì)進(jìn)行牙髓牙本質(zhì)復(fù)合體再生的體內(nèi)實(shí)驗(yàn)研究 研究目的 1）體內(nèi)驗(yàn)證凍干牙本質(zhì)（FDDM）的生物相容性和誘導(dǎo)DPSCs再生牙髓-牙本質(zhì)樣復(fù)合體的能力 研究方法 將DPSCs細(xì)胞膜片聚集體分別與FDDM、普通牙本質(zhì)(D)和HA復(fù)合后進(jìn)行裸鼠皮下移植，每只裸鼠背部皮下植入一枚牙髓干細(xì)胞膜片-支架復(fù)合體， SPF級(jí)別飼養(yǎng)8周后進(jìn)行HE染色、馬氏三色染色及相關(guān)蛋白的免疫組化染色。 實(shí)驗(yàn)結(jié)果 凍干牙本質(zhì)/普通牙本質(zhì)-牙髓干細(xì)胞復(fù)合物再生出牙髓-牙本質(zhì)樣組織，，極性的成牙本質(zhì)樣細(xì)胞沿著新生成的前期牙本質(zhì)整齊排列，且沒(méi)有觀察到炎癥反應(yīng)。羥基磷灰石-磷酸三鈣-牙髓干細(xì)胞復(fù)合物中沒(méi)有觀察到典型的牙髓-牙本質(zhì)樣結(jié)構(gòu)形成，可見炎性細(xì)胞浸潤(rùn)。免疫組化染色顯示普通牙本質(zhì)與凍干牙本質(zhì)陽(yáng)性表達(dá)ALP、COL-3、DSP、fibronectin和人線粒體（mitochondria）。 結(jié)論 凍干牙本質(zhì)與普通牙本質(zhì)在裸鼠體內(nèi)具有相同的誘導(dǎo)人來(lái)源牙髓干細(xì)胞向成牙本質(zhì)樣細(xì)胞分化的能力，并能夠在體內(nèi)再生牙髓-牙本質(zhì)復(fù)合體，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 .
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R783.6

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相關(guān)期刊論文 前3條

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2 洪法廉;李穎超;史俊南;楊連甲;王志良;李耀君;劉守智;;牙齒的顯微硬度和元素成分[J];口腔醫(yī)學(xué)縱橫;1991年01期

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本文編號(hào)：2106267