本文選題：軟骨組織工程 + 耳廓軟骨��； 參考：《北京協(xié)和醫(yī)學院》2012年博士論文

【摘要】：研究背景及意義： 耳廓軟骨組織工程為耳軟骨缺損的修復重建提供了新的治療思路,其中種子細胞是限制其發(fā)展及臨床應用的瓶頸之一。目前,構(gòu)建組織工程軟骨主要通過三種方式來實現(xiàn)：(1)單獨應用各種來源的軟骨細胞；(2)單獨應用成軟骨誘導后的間充質(zhì)干細胞(Mesenchymal stem cells, MSCs);(3)將軟骨細胞與MSCs混合共培養(yǎng)。上述三類種子細胞應用方案從細胞生物學基礎與臨床應用的角度上講各存利弊,但目前尚缺乏關(guān)于此類問題的深入探討；此外,針對先天性小耳畸形,自體殘耳組織是外耳重建重要的種子細胞來源,目前對殘耳組織來源細胞及其軟骨構(gòu)建系統(tǒng)化的研究較少,確立最佳的種子細胞應用策略能夠為耳廓軟骨組織工程突破目前困境及發(fā)展未來應用提供理論基礎和技術(shù)支持。 研究目的： 1、通過在組織學、基因表達及生物學功能等方面系統(tǒng)比較單純應用軟骨細胞、軟骨細胞與BMSCs混合共培養(yǎng)、BMSCs成軟骨誘導三類種子細胞應用方案,確立構(gòu)建彈性軟骨最佳的種子細胞應用策略并探討其構(gòu)建耳廓形態(tài)軟骨的可行性。 2、通過分析殘耳軟骨細胞的體外增殖、表型變化、細胞組織的量效關(guān)系以及研究傳代、誘導對其體內(nèi)成軟骨能力的影響,確立基于殘耳軟骨細胞的種子細胞應用策略及其構(gòu)建耳廓形態(tài)軟骨的可行性。 研究內(nèi)容： 1.不同種子細胞及應用方案構(gòu)建組織工程軟骨 1.1比較不同組織來源軟骨細胞構(gòu)建組織工程軟骨的差異 方法選擇耳廓和關(guān)節(jié)兩種不同組織來源的軟骨細胞接種PGA/PLA支架構(gòu)建組織工程軟骨組織,在其體外和體內(nèi)的不同構(gòu)建階段,通過組織學檢測進行觀察,比較兩組構(gòu)建物是否因細胞的組織來源不同而存在差異。 結(jié)果耳廓和關(guān)節(jié)來源的軟骨細胞構(gòu)建的軟骨組織在體外未發(fā)現(xiàn)組織學水平的差異,但植入皮下6周后均恢復了各自的生物學特性,關(guān)節(jié)軟骨細胞組發(fā)生了部分骨化,耳廓軟骨細胞組出現(xiàn)了彈性纖維的陽性著色。 小結(jié)體外構(gòu)建的組織工程軟骨植入體內(nèi)后能恢復軟骨細胞組織來源的特性,可在皮下構(gòu)建與軟骨細胞來源類型相同的組織工程軟骨。 1.2比較三類種子細胞應用方案構(gòu)建組織工程軟骨的差異 方法系統(tǒng)性比較單獨應用耳廓軟骨細胞、單獨應用骨髓間充質(zhì)干細胞(Bone marrow mesenchymal stem cells, BMSCs)并對其進行成軟骨誘導、以及耳廓軟骨細胞與BMSCs混合共培養(yǎng)構(gòu)建的組織工程軟骨在組織學、基因表達及生物力學功能方面存在的差異。 結(jié)果復合物在植入體內(nèi)6周后,BMSCs誘導組不僅不能構(gòu)建彈性軟骨組織而且發(fā)生了明顯的骨化,COL10A1、MMP13、ALPL的表達較其余兩組出現(xiàn)顯著升高；耳廓軟骨細胞與BMSCs混合共培養(yǎng)組不僅可保持穩(wěn)定的軟骨表型,而且相較單純耳廓軟骨細胞組其彈性纖維更為均質(zhì)、密集,彈性模量也更高且與生理耳廓軟骨無統(tǒng)計學差異,COL9A1、COMP、DCN、LOXL2的表達也有統(tǒng)計學意義的升高。此外,共培養(yǎng)構(gòu)建的軟骨組織其D1k1與Ki67的表達也高于單純耳廓軟骨細胞組。 小結(jié)耳廓軟骨細胞與BMSCs混合共培養(yǎng)是目前構(gòu)建彈性軟骨組織最佳的種子細胞應用策略,其構(gòu)建的彈性軟骨具備更密集的彈性纖維和更高的彈性模量,并且可延續(xù)干細胞帶來的增殖與分化能力。 1.3軟骨細胞與骨髓間充質(zhì)干細胞共培養(yǎng)構(gòu)建耳廓軟骨 方法將豬自體耳廓軟骨細胞與BMSCs以5：5的比例混合接種于預成型的耳廓形態(tài)PGA/PLA支架上,體外培養(yǎng)10周,植入豬耳后皮下20周后進行軟骨相關(guān)檢測。 結(jié)果豬耳廓軟骨細胞與BMSCs以5：5的比例混合共培養(yǎng)能夠在體外構(gòu)建良好形態(tài)及彈性的耳廓軟骨,在植入大動物體內(nèi)20周后仍然能夠穩(wěn)定存活,且其組織學與彈性相較生理耳廓軟骨無明顯差異,但是最終難以維持耳廓的精細形態(tài)。 小結(jié)在大動物體內(nèi)實驗中,共培養(yǎng)構(gòu)建的耳廓軟骨能夠穩(wěn)定存活20周,但因不能對抗皮膚收縮力最終難以維持耳廓的精細形態(tài)。 2.先天性小耳殘耳軟骨細胞構(gòu)建組織工程軟骨 2.1殘耳軟骨細胞的增殖、表型變化及量效關(guān)系研究 方法通過對大量殘耳組織進行稱重和細胞分離計數(shù),計算殘耳組織的初始細胞獲得率；通過繪制生長曲線和細胞計數(shù)研究4代以內(nèi)殘耳軟骨細胞在bFGF影響下的增殖能力及擴增倍數(shù)；在細胞和基因水平檢測傳代對殘耳軟骨細胞表型的影響;并通過大量軟骨相關(guān)基因譜的篩查比對殘耳軟骨細胞與正常耳軟骨細胞在基因水平的異同。 結(jié)果殘耳組織的初始細胞獲得率為(3.90±1.27)×106/g；殘耳軟骨細胞在bFGF的刺激下增殖能力明顯提高,4代以內(nèi)增殖能力未見差別,且擴增至P4代能夠達到(328.4±50.4)倍的增殖效率；但是,同樣條件下擴增至P3代的殘耳軟骨細胞其番紅O染色、Ⅱ型膠原的基因表達已較弱,至P4代基本檢測不到；此外,殘耳軟骨細胞在基因水平除個別個體的COL2A1成熟型異構(gòu)體COL2A1V2及COL9A1的表達偏低外,與正常耳軟骨細胞相較未見明顯差異。 小結(jié)殘耳組織來源細胞與正常耳廓軟骨細胞相比在基因水平未見明顯差異,在bFGF培養(yǎng)下增殖到P3代仍可維持一定程度的軟骨表型且能達到構(gòu)建常人體積耳廓軟骨的細胞數(shù)量(以獲得500mg殘耳組織計算)。 2.2體外傳代及誘導對殘耳軟骨細胞體內(nèi)成軟骨能力的影響 方法將P3-P8代殘耳軟骨細胞各自接種PGA/PLA支架進行軟骨組織構(gòu)建,根據(jù)組織學、基因表達和生物力學結(jié)果分析體外傳代及誘導對其最終體內(nèi)成軟骨能力的影響。 結(jié)果在P3-P8各代次細胞材料復合物中,P4代以前在體外4周可較高表達SOX9和DLK1,并能在體內(nèi)形成良好的彈性軟骨組織；經(jīng)過體外誘導,P3-P8代的殘耳軟骨細胞材料復合物均能在體外形成類軟骨組織,但DLK1的表達卻均顯著低于非誘導組,且其體內(nèi)8周后的成骨現(xiàn)象十分明顯。 小結(jié)P4代以前的殘耳軟骨細胞不經(jīng)體外誘導雖然不能在體外構(gòu)建耳廓軟骨,但仍可保持良好的體內(nèi)成軟骨能力形成彈性軟骨。P3-P8代細胞經(jīng)體外誘導可以形成類軟骨組織,但是其體內(nèi)的成骨傾向較為嚴重,DLK1可能在體外成軟骨誘導致體內(nèi)成骨過程中扮演重要角色。 2.3單例先天性小耳殘耳軟骨細胞構(gòu)建常人體積耳廓軟骨 方法將單例病人殘耳組織分離的軟骨細胞在體外傳至P3或P4代,在生長因子、藻酸鹽凝膠、旋轉(zhuǎn)培養(yǎng)的體外再分化系統(tǒng)中培養(yǎng)10周后進行軟骨相關(guān)檢測,以體外普通培養(yǎng)(不誘導)作為對照。 結(jié)果利用單例先天性小耳的殘耳組織經(jīng)過細胞擴增和再分化系統(tǒng)培養(yǎng)可以在體外構(gòu)建常人體積的耳廓形態(tài)軟骨,所形成的軟骨組織結(jié)構(gòu)較接近正常生理軟骨組織。 小結(jié)大量增殖的殘耳軟骨細胞在再分化誘導系統(tǒng)培養(yǎng)下能夠在體外構(gòu)建常人體積的耳廓軟骨。 綜上所述,本研究以耳廓軟骨構(gòu)建為最終目的,探討了幾種軟骨組織工程種子細胞及其應用策略的可行性。明確了耳廓軟骨細胞與BMSCs共培養(yǎng)方案在構(gòu)建彈性軟骨方面的優(yōu)勢,首次應用其在體外構(gòu)建耳廓形態(tài)組織工程軟骨并進行大動物體內(nèi)研究。同時,系統(tǒng)研究了殘耳軟骨細胞構(gòu)建軟骨組織的量效關(guān)系及應用方案,并在體外構(gòu)建常人體積耳廓軟骨。這些研究結(jié)果為耳廓軟骨組織工程的臨床應用提供了理論基礎和技術(shù)支持。
[Abstract]:Research background and significance:
The cartilage tissue engineering of the auricle provides a new treatment idea for the repair and reconstruction of ear cartilage defects, in which seed cells are one of the bottlenecks to restrict its development and clinical application. At present, the construction of tissue engineered cartilage is realized mainly through three ways: (1) the chondrocytes of various sources are applied alone; (2) the cartilage is induced alone after induction of cartilage. Mesenchymal stem cells (MSCs); (3) co culture of chondrocytes and MSCs. The above three kinds of seed cell application programs have the advantages and disadvantages from the angle of cell biology and clinical application, but there is still a lack of in-depth discussion on such problems. In addition, in view of congenital microtia, autologous residual ear Tissue is an important source of seed cells for the external ear reconstruction. At present, few studies have been made on the systematic construction of the stem cells and cartilage of the residual ear tissue. The establishment of the best application strategy of seed cells can provide theoretical basis and technical support for the breakthrough and future application of auricular cartilage tissue engineering.
The purpose of the study is:
1, through the systematic comparison of histology, gene expression and biological function, the combination of chondrocytes, chondrocytes and BMSCs co culture, BMSCs cartilage induction to induce three kinds of seed cell application scheme, establish the best seed cell application strategy for constructing elastic cartilage and explore the feasibility of the construction of auricular morphologic cartilage.
2, by analyzing the proliferation in vitro, phenotypic changes, the quantitative effect relationship of cell tissue and the study of the influence of subculture on the chondrogenic ability of the residual ear cartilage cells, the application strategy of the seed cells based on the residual ear cartilage cells and the feasibility of the construction of the auricular morphologic cartilage were established.
Research content:
1. construction of tissue-engineered cartilage with different seed cells and application protocols
1.1 comparison of tissue-engineered cartilage with chondrocytes derived from different tissue sources
Methods chondrocytes of two different tissue sources of the auricle and joint were selected to construct tissue engineering cartilage tissue with PGA/PLA scaffold. In the different construction stages of the cartilage and in vivo, the two groups were observed by histological examination, and the difference between the two groups was compared with the different tissue sources of the cells.
Results the cartilage tissue of the cartilage cells from the origin of the auricle and joint had not found the difference in the histological level in vitro, but after 6 weeks of subcutaneous implantation, the biological characteristics of the cartilage cells were restored. The cartilage cell group of the articular cartilage was partially ossified, and the positive coloring of the elastic fiber in the auricle cartilage cell group was found.
The tissue engineered cartilage constructed in vitro can restore the tissue origin of chondrocytes after implantation in vivo, and the tissue engineering cartilage which is the same as the source of chondrocytes can be constructed subcutaneously.
1.2 comparison of three types of seed cell application schemes to construct tissue-engineered cartilage
Methods a systematic comparison of the auricle cartilage cells was used to separate the bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BMSCs) and to induce cartilage, as well as the histology, gene expression and biomechanical functions of the tissue engineered cartilage constructed by co culture of the auricle cartilage cells and BMSCs. Difference.
Results after 6 weeks of implantation, the BMSCs induced group was not only unable to construct elastic cartilage tissue but also had obvious ossification. The expression of COL10A1, MMP13 and ALPL increased significantly compared with the other two groups, and the mixed culture group of auricular cartilage cells and BMSCs not only maintained stable cartilage phenotype, but also compared with simple auricular cartilage cells. The elastic fibers in the group were more homogeneous, denser, and more elastic, and had higher modulus and no statistical difference from the physiological auricle cartilage. The expression of COL9A1, COMP, DCN, LOXL2 also increased statistically. Moreover, the expression of D1k1 and Ki67 in the co cultured cartilage tissue was also higher than that of the Dan Chuner cartilage cell group.
The co culture of auricular cartilage cells and BMSCs is the best seed cell application strategy for constructing elastic cartilage tissue at present. Its elastic cartilage has more dense elastic fiber and higher modulus of elasticity, and can continue the proliferation and differentiation ability of stem cells.
1.3 co culture of bone marrow mesenchymal stem cells and chondrocytes to construct auricle cartilage
Methods the porcine autologous auricle cartilage cells were inoculated with BMSCs with the proportion of 5:5 in the preformed auricle morphology PGA/PLA scaffold. It was cultured in vitro for 10 weeks, and the cartilage was detected after 20 weeks after the implantation of the pig ear.
Results the co culture of porcine auricle cartilage cells and BMSCs can construct good morphological and elastic auricular cartilage in vitro. It can still survive for 20 weeks after implantation in large animals, and there is no obvious difference between the histology and the elastic phase of the auricle cartilage, but it is difficult to maintain the fine shape of the auricle.
The cocultivated auricle cartilage can survive for 20 weeks in a large animal experiment, but it is difficult to maintain the fine shape of the auricle because of the inability to resist the skin contractile force.
2. congenital ear ear cartilage cells construct tissue-engineered cartilage.
Proliferation, phenotypic changes and dose effect relationship of 2.1 residual ear cartilage cells
Methods by weighing and counting the large number of residual ear tissues, the initial cell acquisition rate of the residual ear tissue was calculated. The proliferation and amplification factors of the residual ear cartilage cells under the influence of bFGF were studied by drawing growth curve and cell count, and the phenotype of the residual ear cartilage cells was detected at the cell and gene level. The difference in gene level between residual ear cartilage cells and normal ear cartilage cells was compared through a large number of screening of cartilage related gene spectrum.
Results the initial cell acquisition rate of the residual ear tissue was (3.90 + 1.27) x 106/g, the proliferation ability of the residual ear cartilage cells increased obviously under the stimulation of bFGF, and the proliferation ability within 4 generations was not different, and the proliferation of the P4 generation could reach (328.4 + 50.4) times the proliferation efficiency. However, the red O dyeing of the residual ear cartilage cells of the P3 generation under the same condition Color, the gene expression of type II collagen has been weak, and the basic detection of P4 generation can not be found. In addition, there is no significant difference between the residual ear chondrocytes at the gene level and the low expression of the COL2A1 mature isomer COL2A1V2 and COL9A1 at the level of individual individual, compared with the normal ear cartilage cells.
Compared with normal auricle cartilage cells, the source cells of the residual ear tissue did not differ significantly at the gene level. The proliferation to P3 generation under bFGF culture can maintain a certain degree of cartilage phenotype and can reach the number of cells that construct the human auricle cartilage (to obtain the 500mg residual ear tissue calculation).
Effects of 2.2 generation and induction on chondrogenesis of residual ear cartilage cells in vivo
Methods P3-P8 generation of residual ear cartilage cells were respectively inoculated with PGA/PLA scaffold for cartilage tissue construction. The effects of transmission and induction on the chondrogenic ability in the final body were analyzed according to histology, gene expression and biomechanical results.
Results in the P3-P8 subcellular material complex, SOX9 and DLK1 were highly expressed in 4 weeks before the P4 generation, and a good elastic cartilage tissue could be formed in the body. After the induction in vitro, the P3-P8 generation of the residual ear cartilage cell material complex could form the cartilage like tissue in vitro, but the expression of DLK1 was significantly lower than that of the non induced group. The osteogenesis is very obvious after 8 weeks in the body.
Although the P4 generation of residual ear cartilage cells can not construct auricular cartilage in vitro, it can still maintain a good cartilaginous capacity in vivo and form elastic cartilage.P3-P8 cells to form cartilage like tissue in vitro, but the osteogenic tendency in the body is more serious and DLK1 may induce cartilage in vitro. The body plays an important role in the process of osteogenesis.
2.3 single case of congenital auricular ear cartilage cells construct normal human ear cartilage.
Methods the chondrocytes isolated from the single patient's residual ear tissue were transferred from body to P3 or P4 generation, and the cartilage related detection was carried out in the growth factor, alginate gel and the rotated culture in vitro for 10 weeks, and the normal culture in vitro (no induction) was used as control.
Results the normal human auricle morphologic cartilage can be constructed in vitro by cell amplification and regeneration system of the single ear of single congenital small ear. The tissue structure of the cartilage is closer to the normal cartilage tissue.
Conclusion: a large number of residual ear cartilage cells can be constructed in vitro with normal human ear cartilage under the induction of re differentiation.
To sum up, this study aims at the construction of auricular cartilage. The feasibility of several cartilage tissue engineering seed cells and their application strategies is discussed. The advantage of the co culture scheme of auricle cartilage cells and BMSCs in the construction of elastic cartilage is made clear. At the same time, the quantitative relationship and application of the cartilaginous tissue constructed by the residual ear cartilage cells were systematically studied and the human volume auricle cartilage was constructed in vitro. These results provide the theoretical basis and technical support for the clinical application of the auricular cartilage tissue engineering.
【學位授予單位】：北京協(xié)和醫(yī)學院
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R318.1

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