本文選題：細(xì)胞外基質(zhì) + 細(xì)胞膜片��； 參考：《第四軍醫(yī)大學(xué)》2016年博士論文

【摘要】：研究背景口腔頜面部骨、軟骨組織生物性再生修復(fù)是口腔醫(yī)學(xué)領(lǐng)域需要解決的棘手問題�？谇活M面部腫瘤、創(chuàng)傷以及先天性畸形等都會導(dǎo)致頜面部骨和軟骨組織的缺損,如下頜骨成釉細(xì)胞瘤、髁突發(fā)育不良、先天性上頜骨缺失等等。目前,臨床上治療骨缺損的金標(biāo)準(zhǔn)仍然是“自體骨移植”,但這種拆東墻補(bǔ)西墻的修復(fù)方式需要開辟第二戰(zhàn)場,給患者造成二次損傷,病人所受的傷害和所需的花費(fèi)都要增加很多。組織工程和再生醫(yī)學(xué)的出現(xiàn)以及蓬勃發(fā)展,為醫(yī)學(xué)上治療這類缺損提供了很好的方向。其中,支架材料作為組織工程領(lǐng)域的三大支柱之一,對組織工程的成功構(gòu)建起著非常重要的作用。現(xiàn)在常用的支架材料主要分為兩類:一是來源于天然組織,如膠原等;二是人工合成的高分子支架材料,如PCL等。兩種材料都各有其優(yōu)勢和不足。其中,細(xì)胞外基質(zhì)支架材料(ECM)由于其在組織形成和器官發(fā)育中的重要作用而被人們所熟悉并深入研究,而且,ECM對于細(xì)胞的生物學(xué)行為如遷移、增殖和分化等也有著直接的影響。因此ECM作為一種支架材料有著其獨(dú)特的優(yōu)勢。本實(shí)驗就是著眼于探討ECM支架材料在軟骨再生和骨組織工程中的應(yīng)用,為以后臨床應(yīng)用ECM修復(fù)組織缺損提供一些可靠的依據(jù)。第一部分軟骨細(xì)胞來源的細(xì)胞外基質(zhì)支架材料在軟骨再生中應(yīng)用的初步探討實(shí)驗一軟骨細(xì)胞膜片的構(gòu)建目的:探討軟骨細(xì)胞膜片的構(gòu)建方法,并分析其結(jié)構(gòu)特征。方法:分離4周齡幼兔的耳軟骨細(xì)胞,在高糖DMEM培養(yǎng)條件下連續(xù)培養(yǎng)2周;然后對獲得的膜片進(jìn)行大體學(xué)、組織學(xué)和超微結(jié)構(gòu)的觀察。結(jié)果:幼兔耳軟骨細(xì)胞分離后生長狀態(tài)良好,連續(xù)培養(yǎng)2周后即可形成膜片狀結(jié)構(gòu);HE和番紅O染色均證實(shí)了膜片是由軟骨細(xì)胞及其自身分泌的細(xì)胞外基質(zhì)(Extracellular Matrix,ECM)構(gòu)成,掃描電鏡(Scanning electronic microscope,SEM)和投射電鏡(Transmission electron microscope,TEM)也都證實(shí)了軟骨細(xì)胞膜片是由大量的ECM和鑲嵌在其中的軟骨細(xì)胞構(gòu)成。結(jié)論:軟骨細(xì)胞經(jīng)過適宜的培養(yǎng)后可以形成膜片狀結(jié)構(gòu),可用于以后的實(shí)驗。實(shí)驗二軟骨細(xì)胞來源的細(xì)胞外基質(zhì)支架材料的制備及其特征分析目的:探討軟骨細(xì)胞膜片最佳的脫細(xì)胞方法,并分析其脫細(xì)胞前后組織結(jié)構(gòu)和生物力學(xué)特征等有無變化。方法:取實(shí)驗一培養(yǎng)的軟骨細(xì)胞膜片,然后分別在1%、5%和10%SDS中脫細(xì)胞處理24h,然后均再經(jīng)過1%Triton X-100和脫氧核糖核酸酶中去除細(xì)胞碎片和殘余的DNA,在對其進(jìn)行組織學(xué)、超微結(jié)構(gòu)以及生物力學(xué)的檢測,分析其脫細(xì)胞前后特性有無變化。結(jié)果:1%、5%和10%SDS三組處理方法均可比較徹底地去除細(xì)胞,但10%SDS會對細(xì)胞膜片的結(jié)構(gòu)和特性造成很大的損害;而1%SDS既能比較徹底地脫細(xì)胞,又能在最大程度上維持細(xì)胞膜片原有的組織學(xué)結(jié)構(gòu)和生物力學(xué)特征。結(jié)論:1%SDS是本實(shí)驗中最佳的脫細(xì)胞方法,脫細(xì)胞后所獲得ECM可以用于下一步的實(shí)驗。實(shí)驗三軟骨細(xì)胞來源的細(xì)胞外基質(zhì)支架材料在兔膝關(guān)節(jié)骨軟骨缺損修復(fù)作用的初步探討目的:探討ECM在軟骨缺損中的修復(fù)作用,并比較1%、5%和10%SDS三種脫細(xì)胞方法的優(yōu)劣。方法:35只成年新西蘭大白兔隨機(jī)分為四組,分別為1%、5%、10%SDS和空白對照組,另外三只作為陽性對照;膝關(guān)節(jié)骨軟骨缺損模型建立后,分別植入已成功制備的ECM支架材料;在移植后的6周和12周時,分別安樂死犧牲掉實(shí)驗動物,取材進(jìn)行大體學(xué)觀察、Micro-CT掃描和組織學(xué)染色,并做大體學(xué)和組織學(xué)評分;比較各實(shí)驗組間有無差異。結(jié)果:三個實(shí)驗組均可在一定程度上修復(fù)骨軟骨缺損,但大體學(xué)和組織學(xué)分析均證實(shí)1%SDS制備的ECM具有最好的修復(fù)效果,在移植12周后,缺損處已基本被新生的透明軟骨組織所充填;而且Micro-CT掃描也表明1%DS組中,軟骨下骨板再生情況最好。結(jié)論:1%SDS是最佳的脫細(xì)胞方法,經(jīng)過1%SDS制備而來的ECM可以很好地修復(fù)骨軟骨缺損,該ECM支架材料可作為一種新型的生物材料用于組織工程和再生醫(yī)學(xué)。實(shí)驗四軟骨細(xì)胞來源的細(xì)胞外基質(zhì)支架材料修復(fù)作用機(jī)制的初步探討目的:初步探討軟骨細(xì)胞來源的ECM支架材料在骨軟骨缺損修復(fù)中的作用機(jī)理。方法:首先利用Transwell小室分析VEGF和BMSCs對內(nèi)皮細(xì)胞的遷移有無影響,并通過添加VEGF抑制劑V1來觀察VEGF被抵消后,各實(shí)驗組中內(nèi)皮細(xì)胞的遷移率有無變化;然后再分析ECM對BMSCs的遷移有無影響;最后再通過RT-PCR和Western Blot分析ECM對BMSCs的分化有無影響。結(jié)果:VEGF、BMSCs和軟骨細(xì)胞磚(Chondrocytes bricks,CB)等都對內(nèi)皮細(xì)胞的遷移具有促進(jìn)作用,而且BMSCs與最佳濃度的VEGF(10ng/ml)的促進(jìn)作用相當(dāng),但當(dāng)加入VEGF抑制劑V1后,內(nèi)皮細(xì)胞遷移率明顯降低,說明BMSCs可通過分泌VEGF來募集更多的內(nèi)皮細(xì)胞;同時ECM還可以促進(jìn)BMSCs的遷移,而且RT-PCR和Western Blot也證明了軟骨細(xì)胞來源的ECM可以降低BMSCs的骨向分化和增加其軟骨向分化的能力。結(jié)論:由于ECM或是富含ECM的CB具有很多生物活性成分,可以募集更多的干細(xì)胞達(dá)到缺損部位,啟動修復(fù)和再生過程;而募集而來的干細(xì)胞又可以促進(jìn)更多的血管內(nèi)皮細(xì)胞遷移而來,同時也會分泌更多的VEGF,從而有利于組織再生的形成和維持;而同時,ECM又可以促進(jìn)BMSCs的成軟骨向分化,抑制其骨向分化,最終達(dá)到了軟骨修復(fù)和再生。第二部分基于細(xì)胞外基質(zhì)的骨髓間充質(zhì)干細(xì)胞膜片在骨組織工程中應(yīng)用的初步探討實(shí)驗一骨髓間充質(zhì)干細(xì)胞膜片復(fù)合富血小板纖維蛋白在兔顱骨極限缺損修復(fù)作用的初步探討目的:我們前期實(shí)驗已經(jīng)在裸鼠體內(nèi)證實(shí)了富血小板纖維蛋白(Platelet-richfibrin,PRF)可以促進(jìn)骨髓間充質(zhì)干細(xì)胞(Bone marrow mesenchymal stem cells,BMSCs)膜片的成骨能力,本實(shí)驗旨在驗證BMSCs膜片-PRF復(fù)合體在骨缺損修復(fù)中是否有作用。方法:15只新西蘭大白兔隨進(jìn)分為三組:空白對照組(只做顱骨極限缺損而沒有任何治療);BMSCs膜片組(顱骨極限缺損+BMSCs膜片);BMSCs膜片-PRF復(fù)合組:(顱骨極限缺損+BMSCs膜片-PRF復(fù)合體)。移植8周后對其進(jìn)行Micro-CT掃描和組織學(xué)分析。結(jié)果:空白對照組中僅有少量纖維組織覆蓋缺損,而BMSCs膜片組中雖然有少量骨形成,但不足以修復(fù)缺損;但在BMSCs膜片-PRF復(fù)合組中,則可見大量新生的骨組織;新生骨體積/新生所有組織體積(Bone volume/Total volume,BV/TV)和組織學(xué)半定量分析也證實(shí)了BMSCs膜片-PRF復(fù)合組中缺損修復(fù)的效果最好。結(jié)論:BMSCs膜片-PRF復(fù)合體可以用于骨組織的缺損修復(fù),該方法可作為一種新型的組織工程骨的構(gòu)建方法,將來也許可以應(yīng)用于臨床。實(shí)驗二骨髓間充質(zhì)干細(xì)胞膜片復(fù)合脂肪來源干細(xì)胞構(gòu)建組織工程骨的實(shí)驗研究目的:探討脂肪來源的間充質(zhì)干細(xì)胞(Adipose-derived stem cells,ADSCs)能否促進(jìn)BMSCs膜片的成骨能力。方法:首先從同一供體身上分離培養(yǎng)ADSCs和BMSCs膜片,然后構(gòu)建BMSCs膜片-ADSCs復(fù)合體,8只裸鼠隨機(jī)分為兩組:BMSCs膜片組,BMSCs膜片-ADSCs復(fù)合組;移植到裸鼠的背部皮下,移植8周后對其進(jìn)行Micro-CT掃描和組織學(xué)分析。結(jié)果BMSCs膜片組中有少量散在分布的骨小梁和骨島形成,但新生骨組織量很少;但在BMSCs膜片-ADSCs復(fù)合組中,則可見大量新生的骨組織和未完全成熟鈣化的軟骨組織;BV/TV和組織學(xué)半定量分析也證實(shí)了BMSCs膜片-ADSCs復(fù)合組中新生骨量更多。結(jié)論:BMSCs膜片-ADSCs復(fù)合體可以用于組織工程骨的構(gòu)建。該方法可作為一種新型的組織工程骨的構(gòu)建方法,將來也許可以應(yīng)用于臨床中。實(shí)驗三骨髓間充質(zhì)干細(xì)胞膜片復(fù)合高溫煅燒鴕鳥骨支架材料構(gòu)建組織工程骨的實(shí)驗研究目的:探討B(tài)MSCs膜片和高溫煅燒鴕鳥骨(Ostrich true bone ceramic,OTBC)支架材料用于構(gòu)建組織工程骨的可能性。方法:首先制備OTBC支架材料,分析其理化特征、生物相容性和生物降解性,然后將預(yù)先培養(yǎng)的BMSCs膜片與OTBC復(fù)合后移植到裸鼠背部皮下觀察期能否成骨,單純OTBC材料作為對照組;移植8周后,組織學(xué)分析觀察其成骨情況。結(jié)果:OTBC材料作為一種由羥基磷灰石(HA)和β-磷酸三鈣(β-TCP)構(gòu)成的新型的支架材料,疏松多孔,具有很好地生物相容性和生物可降解性能;裸鼠體內(nèi)成骨實(shí)驗也證明了BMSCs膜片-OTBC聯(lián)合應(yīng)用具有很好地成骨潛能。結(jié)論:聯(lián)合應(yīng)用BMSCs膜片-OTBC支架材料具有很好的成骨潛能,該方法可作為一種新型的組織工程骨的構(gòu)建方法,也許將來可應(yīng)用于臨床。
[Abstract]:Background, oral and maxillofacial bone, and the biological regeneration of cartilage tissue is a difficult problem to be solved in the field of oral medicine. Oral and maxillofacial tumors, trauma, and congenital malformations will all lead to the defects of the maxillofacial bone and cartilage tissue, such as ameloblastoma, dysplasia of the condyle, congenital maxillary loss and so on. The gold standard for the clinical treatment of bone defects is still the "autologous bone graft", but the way to repair the wall needs to open up second battlefields, cause two damage to the patient, the damage to the patient and the cost required, and the emergence and flourishing of the tissue engineering and regenerative medicine and the medical treatment of this type. The defect provides a good direction. Among them, as one of the three pillars in the field of tissue engineering, the scaffold plays a very important role in the successful construction of tissue engineering. The commonly used scaffold materials are divided into two types: one is from natural tissue, such as collagen, and two is synthetic polymer scaffold materials, such as PCL and so on. Two All kinds of materials have their advantages and disadvantages. Among them, extracellular matrix scaffold (ECM) is well known and deeply studied because of its important role in tissue formation and organ development. Moreover, ECM has a direct effect on the biological behavior of cells such as migration, proliferation and differentiation. Therefore, ECM is used as a scaffold material. This experiment is aimed at exploring the application of ECM scaffold materials in cartilage regeneration and bone tissue engineering, and providing some reliable basis for the future clinical application of ECM repair tissue defects. Construction of cell diaphragm: To explore the construction of chondrocyte membrane and analyze its structural characteristics. Methods: the ear cartilage cells of 4 week old rabbit were isolated for 2 weeks under high glucose DMEM culture, and then the obtained diaphragm was observed in general, histology and ultrastructure. The diaphragm like structure could be formed after 2 weeks of continuous culture. HE and O staining confirmed that the diaphragm was composed of chondrocytes and their own extracellular matrix (Extracellular Matrix, ECM), and the scanning electron microscopy (Scanning electronic microscope, SEM) and projective electron microscopy (Transmission electron microscope) were also confirmed. The cartilage cell membrane is made up of a large number of ECM and the cartilage cells embedded in it. Conclusion: the cartilage cells can form a diaphragm like structure after proper culture and can be used for future experiments. The preparation and characteristic analysis of the extracellular matrix scaffold materials from two cartilage cells are used to investigate the best cartilage cell diaphragm. Methods: there are no changes in cell structure and biomechanical characteristics before and after cell removal. Methods: take a cultured chondrocyte membrane, and then deactivate 24h in 1%, 5% and 10%SDS, and then remove cell fragments and residual DNA through 1%Triton X-100 and deoxyribonuclease. Histology, ultrastructure and biomechanics test and analysis of the changes before and after cell removal. Results: 1%, 5% and 10%SDS three groups can completely remove the cells, but 10%SDS can cause great damage to the structure and characteristics of the cell diaphragm; and 1%SDS can be more thoroughly deactivated, and can be in the greatest degree. The original histological structure and biomechanical characteristics of the cell diaphragm. Conclusion: 1%SDS is the best method of cell removal in this experiment. ECM can be used in the next experiment after cell removal. Preliminary study on the effect of three chondrocyte derived extracellular matrix scaffold on the repair of osteochondral defect of the knee joint of rabbit: to explore ECM Repair of cartilage defects, and compare the advantages and disadvantages of three kinds of decellular methods of 1%, 5% and 10%SDS. Methods: 35 adult New Zealand white rabbits were randomly divided into four groups, 1%, 5%, 10%SDS and blank control group, and the other three were used as positive control. After the establishment of the osteochondral defect model of the knee joint, the successfully prepared ECM scaffold materials were implanted respectively. At the 6 and 12 weeks after transplantation, the experimental animals were sacrificed by euthanasia for general observation, Micro-CT scanning and histological staining, and the gross and histological scores were done. There was no difference between the experimental groups. Results: the three experimental groups could repair the osteochondral defect to a certain extent, but the general and histological analysis were all It was confirmed that the 1%SDS prepared ECM had the best repair effect. After 12 weeks of transplantation, the defect was basically filled with the newborn hyaline cartilage tissue, and Micro-CT scan also showed that the subchondral bone plate regeneration was best in the 1%DS group. Conclusion: 1%SDS is the best way to remove the cell, and the 1%SDS prepared ECM can repair bone soft well. Bone defect, the ECM scaffold material can be used as a new kind of biomaterial for tissue engineering and regenerative medicine. Experimental study on the mechanism of the repair of extracellular matrix scaffold materials derived from four chondrocytes: preliminary study on the mechanism of the ECM scaffold material derived from chondrocytes in the repair of osteochondral defects. Using the Transwell chamber to analyze the effect of VEGF and BMSCs on the migration of endothelial cells, and by adding the VEGF inhibitor V1 to observe the migration of the endothelial cells in each experiment group after the VEGF was offset, and then to analyze the effect of ECM on the migration of BMSCs, and finally to analyze the differentiation of ECM by RT-PCR and Western Blot. Results: VEGF, BMSCs and cartilage cell brick (Chondrocytes bricks, CB) all promote the migration of endothelial cells, and BMSCs and the best concentration of VEGF (10ng/ml) promote the same, but when the VEGF inhibitor V1 is added, the mobility of endothelial cells decreases obviously, indicating that BMSCs can raise more inside by secreting VEGF. ECM also promotes the migration of BMSCs, and RT-PCR and Western Blot also demonstrate that ECM derived from cartilage cells can reduce the osteogenic differentiation of BMSCs and increase the ability to differentiate into cartilage. Conclusion: as ECM or CB containing ECM rich CB has a lot of bioactive components, more stem cells can be raised to reach the defect site. The regenerative and regenerative processes are initiated, and the recruited stem cells can promote the migration of more vascular endothelial cells, and also secrete more VEGF, which is beneficial to the formation and maintenance of tissue regeneration. At the same time, ECM can also promote the differentiation of the cartilage of BMSCs, inhibit its bone differentiation, and eventually achieve cartilage repair and re Primary study on the application of bone marrow mesenchymal stem cell membrane based on extracellular matrix in bone tissue engineering second preliminary study on the effect of bone marrow mesenchymal stem cell membrane complex platelet rich fibrin in the repair of extreme defect of rabbit skull: our preliminary experiments have confirmed the platelet rich in the nude mice Platelet-richfibrin (PRF) can promote the osteogenesis of bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs). The purpose of this experiment is to verify the effect of the BMSCs diaphragm -PRF complex in the repair of bone defect. Method: 15 New Zealand white rabbits were divided into three groups: blank control group (only the skull pole) Limited defect without any treatment); BMSCs patch group (+BMSCs diaphragm for extreme defect of skull) and BMSCs diaphragm -PRF composite group (+BMSCs diaphragm -PRF complex of skull defect). After 8 weeks of transplantation, Micro-CT scan and histological analysis were carried out. Results: only a small amount of fibrous tissue covered defect was found in the blank control group, while the BMSCs diaphragm group had a small number of tissue defects. A small amount of bone was formed, but it was not sufficient to repair the defect; but in the BMSCs -PRF composite group, a large number of new bone tissues were seen; the volume of new bone volume / all tissue volume (Bone volume/Total volume, BV/TV) and histological semi quantitative analysis also confirmed the best effect of the defect repair in the BMSCs diaphragm -PRF complex group. Conclusion: BMSCs diaphragm -PRF. The complex can be used to repair bone tissue defects. This method can be used as a new construction method of tissue engineering bone, and may be used in the future. Experiment two bone marrow mesenchymal stem cell membrane combined with adipose derived stem cells to construct tissue engineering bone: the study of adipose derived mesenchymal stem cells (Adipos E-derived stem cells, ADSCs) could promote the osteogenesis of BMSCs film. Methods: first, ADSCs and BMSCs films were isolated and cultured from the same donor body, and then the BMSCs diaphragm -ADSCs complex was constructed. 8 nude mice were randomly divided into two groups: BMSCs diaphragm group, BMSCs diaphragm -ADSCs complex group, transplanted to the back of the nude mice and transplanted for 8 weeks. Results the icro-CT scan and histological analysis showed a small amount of scattered bone trabeculae and bone islands in the BMSCs patch group, but the new bone tissue was small, but a large number of new bone tissue and immature cartilage tissue were found in the BMSCs diaphragm -ADSCs composite group; BV/TV and histologic semi quantitative analysis also confirmed the BMSCs diaphragm -AD. The new bone mass of the SCs composite group is more. Conclusion: the BMSCs diaphragm -ADSCs complex can be used for the construction of tissue engineering bone. This method can be used as a new construction method of tissue engineering bone, and may be used in the future. Experiment three bone marrow mesenchymal stem cell membrane composite high temperature calcined ostrich bone scaffold material The purpose of the experimental study of Cheng bone: To explore the possibility of building tissue engineering bone with BMSCs film and high temperature calcined ostrich bone (Ostrich true bone ceramic, OTBC). Methods: firstly, the preparation of OTBC scaffold material was prepared, the physical and chemical characteristics, biocompatibility and biodegradability of the scaffold were analyzed, and the pre cultured BMSCs diaphragm was then mixed with OTBC. OTBC material was used as a control group, and the bone formation was observed after 8 weeks of transplantation. Results: OTBC was a new type of scaffold made up of hydroxyapatite (HA) and beta tricalcium phosphate (beta -TCP), porous, biocompatible and biodegradable. Performance; the bone formation test in nude mice also proved that the BMSCs film -OTBC combined with the bone potential of osteogenesis. Conclusion: the combined application of BMSCs film -OTBC scaffold material has good osteogenic potential. This method can be used as a new method of construction of tissue engineering bone, and may be applied to clinical.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R318.08;R687

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