软骨组织工程种子细胞应用策略及耳廓软骨组织构建研究

发布时间：2018-06-17 22:36

本文选题：软骨组织工程 + 耳廓软骨　；参考：《北京协和医学院》2012年博士论文

【摘要】：研究背景及意义：耳廓软骨组织工程为耳软骨缺损的修复重建提供了新的治疗思路,其中种子细胞是限制其发展及临床应用的瓶颈之一。目前,构建组织工程软骨主要通过三种方式来实现：(1)单独应用各种来源的软骨细胞；(2)单独应用成软骨诱导后的间充质干细胞(Mesenchymal stem cells, MSCs);(3)将软骨细胞与MSCs混合共培养。上述三类种子细胞应用方案从细胞生物学基础与临床应用的角度上讲各存利弊,但目前尚缺乏关于此类问题的深入探讨；此外,针对先天性小耳畸形,自体残耳组织是外耳重建重要的种子细胞来源,目前对残耳组织来源细胞及其软骨构建系统化的研究较少,确立最佳的种子细胞应用策略能够为耳廓软骨组织工程突破目前困境及发展未来应用提供理论基础和技术支持。研究目的： 1、通过在组织学、基因表达及生物学功能等方面系统比较单纯应用软骨细胞、软骨细胞与BMSCs混合共培养、BMSCs成软骨诱导三类种子细胞应用方案,确立构建弹性软骨最佳的种子细胞应用策略并探讨其构建耳廓形态软骨的可行性。 2、通过分析残耳软骨细胞的体外增殖、表型变化、细胞组织的量效关系以及研究传代、诱导对其体内成软骨能力的影响,确立基于残耳软骨细胞的种子细胞应用策略及其构建耳廓形态软骨的可行性。研究内容： 1.不同种子细胞及应用方案构建组织工程软骨 1.1比较不同组织来源软骨细胞构建组织工程软骨的差异方法选择耳廓和关节两种不同组织来源的软骨细胞接种PGA/PLA支架构建组织工程软骨组织,在其体外和体内的不同构建阶段,通过组织学检测进行观察,比较两组构建物是否因细胞的组织来源不同而存在差异。结果耳廓和关节来源的软骨细胞构建的软骨组织在体外未发现组织学水平的差异,但植入皮下6周后均恢复了各自的生物学特性,关节软骨细胞组发生了部分骨化,耳廓软骨细胞组出现了弹性纤维的阳性着色。小结体外构建的组织工程软骨植入体内后能恢复软骨细胞组织来源的特性,可在皮下构建与软骨细胞来源类型相同的组织工程软骨。 1.2比较三类种子细胞应用方案构建组织工程软骨的差异方法系统性比较单独应用耳廓软骨细胞、单独应用骨髓间充质干细胞(Bone marrow mesenchymal stem cells, BMSCs)并对其进行成软骨诱导、以及耳廓软骨细胞与BMSCs混合共培养构建的组织工程软骨在组织学、基因表达及生物力学功能方面存在的差异。结果复合物在植入体内6周后,BMSCs诱导组不仅不能构建弹性软骨组织而且发生了明显的骨化,COL10A1、MMP13、ALPL的表达较其余两组出现显著升高；耳廓软骨细胞与BMSCs混合共培养组不仅可保持稳定的软骨表型,而且相较单纯耳廓软骨细胞组其弹性纤维更为均质、密集,弹性模量也更高且与生理耳廓软骨无统计学差异,COL9A1、COMP、DCN、LOXL2的表达也有统计学意义的升高。此外,共培养构建的软骨组织其D1k1与Ki67的表达也高于单纯耳廓软骨细胞组。小结耳廓软骨细胞与BMSCs混合共培养是目前构建弹性软骨组织最佳的种子细胞应用策略,其构建的弹性软骨具备更密集的弹性纤维和更高的弹性模量,并且可延续干细胞带来的增殖与分化能力。 1.3软骨细胞与骨髓间充质干细胞共培养构建耳廓软骨方法将猪自体耳廓软骨细胞与BMSCs以5：5的比例混合接种于预成型的耳廓形态PGA/PLA支架上,体外培养10周,植入猪耳后皮下20周后进行软骨相关检测。结果猪耳廓软骨细胞与BMSCs以5：5的比例混合共培养能够在体外构建良好形态及弹性的耳廓软骨,在植入大动物体内20周后仍然能够稳定存活,且其组织学与弹性相较生理耳廓软骨无明显差异,但是最终难以维持耳廓的精细形态。小结在大动物体内实验中,共培养构建的耳廓软骨能够稳定存活20周,但因不能对抗皮肤收缩力最终难以维持耳廓的精细形态。 2.先天性小耳残耳软骨细胞构建组织工程软骨 2.1残耳软骨细胞的增殖、表型变化及量效关系研究方法通过对大量残耳组织进行称重和细胞分离计数,计算残耳组织的初始细胞获得率；通过绘制生长曲线和细胞计数研究4代以内残耳软骨细胞在bFGF影响下的增殖能力及扩增倍数；在细胞和基因水平检测传代对残耳软骨细胞表型的影响;并通过大量软骨相关基因谱的筛查比对残耳软骨细胞与正常耳软骨细胞在基因水平的异同。结果残耳组织的初始细胞获得率为(3.90±1.27)×106/g；残耳软骨细胞在bFGF的刺激下增殖能力明显提高,4代以内增殖能力未见差别,且扩增至P4代能够达到(328.4±50.4)倍的增殖效率；但是,同样条件下扩增至P3代的残耳软骨细胞其番红O染色、Ⅱ型胶原的基因表达已较弱,至P4代基本检测不到；此外,残耳软骨细胞在基因水平除个别个体的COL2A1成熟型异构体COL2A1V2及COL9A1的表达偏低外,与正常耳软骨细胞相较未见明显差异。小结残耳组织来源细胞与正常耳廓软骨细胞相比在基因水平未见明显差异,在bFGF培养下增殖到P3代仍可维持一定程度的软骨表型且能达到构建常人体积耳廓软骨的细胞数量(以获得500mg残耳组织计算)。 2.2体外传代及诱导对残耳软骨细胞体内成软骨能力的影响方法将P3-P8代残耳软骨细胞各自接种PGA/PLA支架进行软骨组织构建,根据组织学、基因表达和生物力学结果分析体外传代及诱导对其最终体内成软骨能力的影响。结果在P3-P8各代次细胞材料复合物中,P4代以前在体外4周可较高表达SOX9和DLK1,并能在体内形成良好的弹性软骨组织；经过体外诱导,P3-P8代的残耳软骨细胞材料复合物均能在体外形成类软骨组织,但DLK1的表达却均显著低于非诱导组,且其体内8周后的成骨现象十分明显。小结P4代以前的残耳软骨细胞不经体外诱导虽然不能在体外构建耳廓软骨,但仍可保持良好的体内成软骨能力形成弹性软骨。P3-P8代细胞经体外诱导可以形成类软骨组织,但是其体内的成骨倾向较为严重,DLK1可能在体外成软骨诱导致体内成骨过程中扮演重要角色。 2.3单例先天性小耳残耳软骨细胞构建常人体积耳廓软骨方法将单例病人残耳组织分离的软骨细胞在体外传至P3或P4代,在生长因子、藻酸盐凝胶、旋转培养的体外再分化系统中培养10周后进行软骨相关检测,以体外普通培养(不诱导)作为对照。结果利用单例先天性小耳的残耳组织经过细胞扩增和再分化系统培养可以在体外构建常人体积的耳廓形态软骨,所形成的软骨组织结构较接近正常生理软骨组织。小结大量增殖的残耳软骨细胞在再分化诱导系统培养下能够在体外构建常人体积的耳廓软骨。综上所述,本研究以耳廓软骨构建为最终目的,探讨了几种软骨组织工程种子细胞及其应用策略的可行性。明确了耳廓软骨细胞与BMSCs共培养方案在构建弹性软骨方面的优势,首次应用其在体外构建耳廓形态组织工程软骨并进行大动物体内研究。同时,系统研究了残耳软骨细胞构建软骨组织的量效关系及应用方案,并在体外构建常人体积耳廓软骨。这些研究结果为耳廓软骨组织工程的临床应用提供了理论基础和技术支持。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.1

【参考文献】