细胞外基质支架材料在软骨组织再生和骨组织工程中应用的初步探讨

发布时间：2018-04-20 21:13

本文选题：细胞外基质 + 细胞膜片　；参考：《第四军医大学》2016年博士论文

【摘要】：研究背景口腔颌面部骨、软骨组织生物性再生修复是口腔医学领域需要解决的棘手问题。口腔颌面部肿瘤、创伤以及先天性畸形等都会导致颌面部骨和软骨组织的缺损,如下颌骨成釉细胞瘤、髁突发育不良、先天性上颌骨缺失等等。目前,临床上治疗骨缺损的金标准仍然是“自体骨移植”,但这种拆东墙补西墙的修复方式需要开辟第二战场,给患者造成二次损伤,病人所受的伤害和所需的花费都要增加很多。组织工程和再生医学的出现以及蓬勃发展,为医学上治疗这类缺损提供了很好的方向。其中,支架材料作为组织工程领域的三大支柱之一,对组织工程的成功构建起着非常重要的作用。现在常用的支架材料主要分为两类:一是来源于天然组织,如胶原等;二是人工合成的高分子支架材料,如PCL等。两种材料都各有其优势和不足。其中,细胞外基质支架材料(ECM)由于其在组织形成和器官发育中的重要作用而被人们所熟悉并深入研究,而且,ECM对于细胞的生物学行为如迁移、增殖和分化等也有着直接的影响。因此ECM作为一种支架材料有着其独特的优势。本实验就是着眼于探讨ECM支架材料在软骨再生和骨组织工程中的应用,为以后临床应用ECM修复组织缺损提供一些可靠的依据。第一部分软骨细胞来源的细胞外基质支架材料在软骨再生中应用的初步探讨实验一软骨细胞膜片的构建目的:探讨软骨细胞膜片的构建方法,并分析其结构特征。方法:分离4周龄幼兔的耳软骨细胞,在高糖DMEM培养条件下连续培养2周;然后对获得的膜片进行大体学、组织学和超微结构的观察。结果:幼兔耳软骨细胞分离后生长状态良好,连续培养2周后即可形成膜片状结构;HE和番红O染色均证实了膜片是由软骨细胞及其自身分泌的细胞外基质(Extracellular Matrix,ECM)构成,扫描电镜(Scanning electronic microscope,SEM)和投射电镜(Transmission electron microscope,TEM)也都证实了软骨细胞膜片是由大量的ECM和镶嵌在其中的软骨细胞构成。结论:软骨细胞经过适宜的培养后可以形成膜片状结构,可用于以后的实验。实验二软骨细胞来源的细胞外基质支架材料的制备及其特征分析目的:探讨软骨细胞膜片最佳的脱细胞方法,并分析其脱细胞前后组织结构和生物力学特征等有无变化。方法:取实验一培养的软骨细胞膜片,然后分别在1%、5%和10%SDS中脱细胞处理24h,然后均再经过1%Triton X-100和脱氧核糖核酸酶中去除细胞碎片和残余的DNA,在对其进行组织学、超微结构以及生物力学的检测,分析其脱细胞前后特性有无变化。结果:1%、5%和10%SDS三组处理方法均可比较彻底地去除细胞,但10%SDS会对细胞膜片的结构和特性造成很大的损害;而1%SDS既能比较彻底地脱细胞,又能在最大程度上维持细胞膜片原有的组织学结构和生物力学特征。结论:1%SDS是本实验中最佳的脱细胞方法,脱细胞后所获得ECM可以用于下一步的实验。实验三软骨细胞来源的细胞外基质支架材料在兔膝关节骨软骨缺损修复作用的初步探讨目的:探讨ECM在软骨缺损中的修复作用,并比较1%、5%和10%SDS三种脱细胞方法的优劣。方法:35只成年新西兰大白兔随机分为四组,分别为1%、5%、10%SDS和空白对照组,另外三只作为阳性对照;膝关节骨软骨缺损模型建立后,分别植入已成功制备的ECM支架材料;在移植后的6周和12周时,分别安乐死牺牲掉实验动物,取材进行大体学观察、Micro-CT扫描和组织学染色,并做大体学和组织学评分;比较各实验组间有无差异。结果:三个实验组均可在一定程度上修复骨软骨缺损,但大体学和组织学分析均证实1%SDS制备的ECM具有最好的修复效果,在移植12周后,缺损处已基本被新生的透明软骨组织所充填;而且Micro-CT扫描也表明1%DS组中,软骨下骨板再生情况最好。结论:1%SDS是最佳的脱细胞方法,经过1%SDS制备而来的ECM可以很好地修复骨软骨缺损,该ECM支架材料可作为一种新型的生物材料用于组织工程和再生医学。实验四软骨细胞来源的细胞外基质支架材料修复作用机制的初步探讨目的:初步探讨软骨细胞来源的ECM支架材料在骨软骨缺损修复中的作用机理。方法:首先利用Transwell小室分析VEGF和BMSCs对内皮细胞的迁移有无影响,并通过添加VEGF抑制剂V1来观察VEGF被抵消后,各实验组中内皮细胞的迁移率有无变化;然后再分析ECM对BMSCs的迁移有无影响;最后再通过RT-PCR和Western Blot分析ECM对BMSCs的分化有无影响。结果:VEGF、BMSCs和软骨细胞砖(Chondrocytes bricks,CB)等都对内皮细胞的迁移具有促进作用,而且BMSCs与最佳浓度的VEGF(10ng/ml)的促进作用相当,但当加入VEGF抑制剂V1后,内皮细胞迁移率明显降低,说明BMSCs可通过分泌VEGF来募集更多的内皮细胞;同时ECM还可以促进BMSCs的迁移,而且RT-PCR和Western Blot也证明了软骨细胞来源的ECM可以降低BMSCs的骨向分化和增加其软骨向分化的能力。结论:由于ECM或是富含ECM的CB具有很多生物活性成分,可以募集更多的干细胞达到缺损部位,启动修复和再生过程;而募集而来的干细胞又可以促进更多的血管内皮细胞迁移而来,同时也会分泌更多的VEGF,从而有利于组织再生的形成和维持;而同时,ECM又可以促进BMSCs的成软骨向分化,抑制其骨向分化,最终达到了软骨修复和再生。第二部分基于细胞外基质的骨髓间充质干细胞膜片在骨组织工程中应用的初步探讨实验一骨髓间充质干细胞膜片复合富血小板纤维蛋白在兔颅骨极限缺损修复作用的初步探讨目的:我们前期实验已经在裸鼠体内证实了富血小板纤维蛋白(Platelet-richfibrin,PRF)可以促进骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)膜片的成骨能力,本实验旨在验证BMSCs膜片-PRF复合体在骨缺损修复中是否有作用。方法:15只新西兰大白兔随进分为三组:空白对照组(只做颅骨极限缺损而没有任何治疗);BMSCs膜片组(颅骨极限缺损+BMSCs膜片);BMSCs膜片-PRF复合组:(颅骨极限缺损+BMSCs膜片-PRF复合体)。移植8周后对其进行Micro-CT扫描和组织学分析。结果:空白对照组中仅有少量纤维组织覆盖缺损,而BMSCs膜片组中虽然有少量骨形成,但不足以修复缺损;但在BMSCs膜片-PRF复合组中,则可见大量新生的骨组织;新生骨体积/新生所有组织体积(Bone volume/Total volume,BV/TV)和组织学半定量分析也证实了BMSCs膜片-PRF复合组中缺损修复的效果最好。结论:BMSCs膜片-PRF复合体可以用于骨组织的缺损修复,该方法可作为一种新型的组织工程骨的构建方法,将来也许可以应用于临床。实验二骨髓间充质干细胞膜片复合脂肪来源干细胞构建组织工程骨的实验研究目的:探讨脂肪来源的间充质干细胞(Adipose-derived stem cells,ADSCs)能否促进BMSCs膜片的成骨能力。方法:首先从同一供体身上分离培养ADSCs和BMSCs膜片,然后构建BMSCs膜片-ADSCs复合体,8只裸鼠随机分为两组:BMSCs膜片组,BMSCs膜片-ADSCs复合组;移植到裸鼠的背部皮下,移植8周后对其进行Micro-CT扫描和组织学分析。结果BMSCs膜片组中有少量散在分布的骨小梁和骨岛形成,但新生骨组织量很少;但在BMSCs膜片-ADSCs复合组中,则可见大量新生的骨组织和未完全成熟钙化的软骨组织;BV/TV和组织学半定量分析也证实了BMSCs膜片-ADSCs复合组中新生骨量更多。结论:BMSCs膜片-ADSCs复合体可以用于组织工程骨的构建。该方法可作为一种新型的组织工程骨的构建方法,将来也许可以应用于临床中。实验三骨髓间充质干细胞膜片复合高温煅烧鸵鸟骨支架材料构建组织工程骨的实验研究目的:探讨BMSCs膜片和高温煅烧鸵鸟骨(Ostrich true bone ceramic,OTBC)支架材料用于构建组织工程骨的可能性。方法:首先制备OTBC支架材料,分析其理化特征、生物相容性和生物降解性,然后将预先培养的BMSCs膜片与OTBC复合后移植到裸鼠背部皮下观察期能否成骨,单纯OTBC材料作为对照组;移植8周后,组织学分析观察其成骨情况。结果:OTBC材料作为一种由羟基磷灰石(HA)和β-磷酸三钙(β-TCP)构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;裸鼠体内成骨实验也证明了BMSCs膜片-OTBC联合应用具有很好地成骨潜能。结论:联合应用BMSCs膜片-OTBC支架材料具有很好的成骨潜能,该方法可作为一种新型的组织工程骨的构建方法,也许将来可应用于临床。
[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.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R318.08;R687

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