【作者】 姚军；

【导师】 卢世璧； 郭全义； 彭江；

【作者基本信息】 中国人民解放军军医进修学院 ， 骨外科学， 2009， 博士

【摘要】 研究背景:因创伤、炎症、退变、肿瘤切除等原因导致的关节软骨损伤是临床上常见的疾病。关节软骨再生及修复能力极为有限,一旦损伤后,难以自身修复,病情继续发展,必然导致骨性关节炎。目前的治疗方法,如微骨折术、自体或异体组织（骨膜、软骨膜、骨软骨块）移植等,存在种种缺陷从而限制了其临床应用。自体软骨细胞移植技术（autologous chondrocyteimplantion ACI）和十九世纪早期出现的组织工程表明了软骨修复是可能的,从而出现了许多的修复或者替代损伤软骨的方法。在众多的方法中,将种子细胞培养在生物材料支架上然后移植到缺损部位,形成新的软骨组织,经过重塑形并与机体组织整合在一起,是软骨组织工程修复软骨损伤的基本方式,而将软骨细胞复合到一个三维的,多孔的支架上形成软骨组织是关键内容。组织工程支架的生物学、生物化学、力学以及结构特性都是非常重要的。支架材料除了提供工程化软骨样组织的三维几何形状,还为细胞提供了生存的微环境（人造临时细胞外基质）,支持细胞的粘附、增殖、分化并最终形成新的软骨组织。软骨组织工程支架的特性与天然关节软骨细胞外基质的特性相似程度越高,越有利于成功构建组织工程软骨。当然,完全具有软骨细胞外基质的各种特性的支架是不可能的也没有意义的。实际上,研究表明,天然的软骨组织并不是软骨组织工程应用的理想的支架,因为成熟的软骨组织没有高度的相互联通的孔隙结构以利于快速的、均匀的细胞分布。因此,理想的软骨组织工程支架仍是人工设计、具有天然软骨细胞外基质的某些特性的支架。我们前期研究工作将关节软骨湿法粉碎,获得天然软骨细胞外基质材料,其在生化组成上完全类似于天然关节软骨细胞外基质,再利用冷冻干燥技术,制备出适宜于软骨组织工程需要的多孔海绵样结构支架,体外实验及犬关节软骨损伤修复动物实验研究均取得了良好的效果。然后,在此基础上,我们再从天然关节软骨细胞外基质的各向异性的力学结构特性出发,拟从结构上仿生天然关节软骨细胞外基质,制备从生化组成及结构特性上仿生的软骨组织工程支架。另一方面,前期我们实验室的研究是利用人的关节软骨制备软骨细胞外基质材料,来源不算充分,考虑到猪与人的同源性高,且来源丰富,故利用猪的关节软骨作为材料来源进行本研究。本研究的目的是进一步完善提取天然软骨细胞外基质材料的工艺和流程,利用天然软骨细胞外基质材料制备软骨组织工程取向性支架、从生化组成及结构特性上仿生天然关节软骨组织的特点,对其理化性质进行检测,并探讨其对种子细胞生物学行为的影响以及与自体骨髓基质干细胞复合修复兔膝关节全层关节软骨缺损的效果。方法:（1）将天然猪软骨超微湿法粉碎,充分稀释后利用差速离心及有机溶剂沉淀的方法收集无细胞的软骨细胞外基质纳米纤维材料,用pH=3.2的稀盐酸配成2%溶液,然后利用定向结晶技术及冷冻干燥技术,采用物理/化学方法交联,得到软骨细胞外基质来源的三维多孔定向取向支架（orientedscaffold,OS）,传统冷冻干燥技术制备非取向三维多孔支架（nonorientedscaffold,NOS）,对支架材料进行组织学、生化分析,对取向和非取向支架的理化特性进行比较研究。（2）分离培养兔关节软骨细胞接种在支架上体外培养,评价两种支架对负载软骨细胞的生物学行为的影响。（3）将体外培养1w的兔软骨细胞复合关节软骨细胞外基质源性取向和非取向支架,植入裸鼠背部皮下,4w取材,进行组织学检测,评价两种支架异位成软骨的能力。（4）抽取自体骨髓分离、培养、扩增骨髓基质干细胞（BMSCs）,用含10ng/ml TGF-β1,10ng/ml bFGF,10^-7M地塞米松的条件培养基成软骨诱导1w后,观察诱导后细胞的形态和组织学特性变化,然后接种到关节软骨细胞外基质源性OS和NOS上,体外观察BMSCs在两种支架上的分布排列方式。在兔膝关节髌股关节沟造一直径4.0mm,深及软骨下骨的全层软骨缺损,分别植入取向支架加自体BMSCs（组1）,非取向支架加自体BMSCs（组2）,空白取向支架（组3）,空白非取向支架（组4）,旷置空白缺损（组5）;分别在3,6,12月取材,从修复组织的大体,组织学,生物化学等方面进行评估。结果:（1）组织学显示提取的关节软骨细胞外基质材料和制备的支架中无软骨细胞残留,甲苯胺蓝染色、阿新蓝、蕃红花“0”、Ⅱ型胶原免疫组织化学染色呈阳性;生化定量结果表明支架保留了大部分的细胞外基质成分;扫描电镜结果显示取向支架具有平行排列的柱状的孔道结构,非取向支架内孔洞呈海绵状;孔径（100～200μm）和孔隙率（≥95%）均满足软骨组织工程需要;吸水性能（吸水膨胀率≥96%）良好,取向支架的纵向压缩模量（2.02±0.02MPa）远大于横向上的压缩模量（0.36±0.02MPa）,非取向支架的压缩模量（1.07±0.05 MPa）介于取向支架的纵向和横向压缩模量之间,但远小于取向支架的纵向压缩模量。取向支架纵向上拉伸模量（22.10±0.67MPa）也远大于非取向支架的拉伸模量（5.104±0.45MPa）。差异具有统计学意义（p＜0.05）。（2）组织学、倒置显微镜、扫描电镜观察显示细胞在两种支架上均能良好粘附、增殖,用CCK-8试剂盒检测显示软骨细胞在取向支架上的生长情况,结果显示软骨细胞增殖明显比在非取向支架上快,差异具有统计学意义（p＜0.05）。细胞在取向支架内部的数量明显比非取向支架多。（3）大体观察及组织学结果显示兔软骨细胞复合取向支架及非取向支架在裸鼠皮下均能构建类软骨组织,在取向支架中,种子细胞沿着支架的孔道结构排列形成柱状排列的类软骨样组织,而在非取向支架中则呈无序的随机排列。（4）将BMSCs接种在取向和非取向两种支架上体外软骨诱导培养经PKH26荧光标记,可见BMSCs在取向支架内部也是沿着支架孔道结构分布排列,电镜结果也证实这一点,组织学结果表明蕃红花“0”、Ⅱ型胶原免疫组化染色阳性。在修复兔膝关节骨软骨缺损的实验中,组织学评分表明BMSCs复合取向支架和非取向支架组结果均优于空白支架和空白旷置组,差异具有统计学意义。细胞复合取向支架优于非取向支架组,差异具有统计学意义。结论:利用差速离心方法可以有效去除湿法粉碎后关节软骨细胞外基质浆料中的大颗粒及软骨细胞,利用有机溶剂沉淀可以收集到大部分的细胞外基质成分。利用定向结晶及冷冻干燥技术,制备了结构,力学和生物化学特性类似于自然软骨的组织工程支架,管道孔的结构和直径分布均匀,在一定程度上类似于自然关节软骨。支架细胞复合物在体外培养3天后就呈现出其排列类似于天然软骨表层的平行表面排列,深层的柱状排列。关节软骨细胞外基质源性支架有助于种子细胞的粘附和增值,相对于非取向支架,取向支架更有利于细胞的进入及增值。关节软骨细胞外基质源性取向支架复合自体骨髓基质细胞可用于关节软骨损伤的修复。更多还原

【Abstract】 BACKGROUND Degenerative and rheumatic diseases,traumatic events and excision of tumors are the main reasons for articular cartilage impairs.By itself,cartilage has very limited capabilities of regeneration,if not treated,they extend to be osteoarthritis.Nowadays,numerous strategys have been made to promote reparation of the cartilage defect such as drilling,microfracture,autograft of periosteum,perichondrium and osteochondral autogratt.However all of these generally leads to a mechanical and biological inferior repair tissue based on fibrocartilage.Moreover there is a shortage of autogenous donor sources for grafting.However,the development of the ACI technique and the progress in the tissue-engineering field in the early nineties,have suggested that the repair of cartilage actually might be feasible,leading to the development of numerous strategies to repair or replace the damaged surfaces.In many approaches,cells are grown on biomaterial scaffolds and then implanted into the defect,where new functional tissue is formed,remodelled and integrated into the body.In a typical cartilage tissue engineering approach,to control cartilage repair tissue formation in three dimensions(3D),a highly porous scaffold is critical.And the biological, biochemical,mechanical,and structural properties of scaffolds for cartilage tissue engineering are known to be of great importance.However most materials currently investigated lack a scaffold-structure similar to that of native cartilage and they strongly differ in their biological and biochemical composition.In addition to defining the 3D geometry for the tissue to be engineered,the scaffold provides the microenvironment(synthetic temporary extracellular matrix) for regenerative cells,supporting cell attachment,proliferation,differentiation,and neo tissue genesis.Therefore,the chemical composition,physical structure,and biologically functional are all important attributes to biomaterials for tissue engineering.To serve as the temporary extracellular matrix(ECM) for regenerative cells,it may be beneficial for the scaffold to emulate certain advantageous features of the natural cartilage ECM.However,it is likely unnecessary and impractical for a scaffold to entirely duplicate the cartilage ECM. A natural cartilage ECM may actually not be the ideal scaffold for cartilage tissue engineering applications because mature cartilage tissue matrix often does not possess the highly interconnected macro-or micro-pore structures to allow for quick and uniform cell population throughout,which is essential for a tissue engineering/repair process.Therefore optimal cartilage tissue engineering scaffold should have certain artificially designed scaffold features(such as porosity,pore size,interpore connectivity,etc.) Based on the above discussion,we previously shattered the natural cartilage and collected the nano-material derived cartilage extracellular matrix which biochemical composition mimic the native cartilage ECM,and by freeze-dry technique,we fabricated a porous scaffold,and the scaffold were proved to be sure for supporting the cartilage regenerating in vitro and in vivo.Base on our previous research,considered the structured feature of the native cartilage tissue,we attempt to fabricate the oriented scaffolds that characterized by a perpendicular pore channel structure and biomechanical properties similar to native cartilage tissue.And on the other hand,our previously cartilage materials were human cartilage,which were shortage of donor sources, so in this study we use the porcine cartiage to fabricate the scaffolds,because the porcine cartilage is economic and its homology is similar to human.OBJECTIVE This study aims to further promote the technology and procedure of collecting the native cartilage ECM material.To fabricate oriented scaffold and investigate the charactarization of the scaffolds.To investigate the effect of the oriented scaffold on the the attachment,proliferation,distribution and alignment of chondrocytes seeded in the scaffolds cultured in vitro.And to test the clinical applicability of the scaffolds,we repaired rabbit osteochondral defect using OS and NOS combined the autologous BMSCs.METHOD①Cartilage slices were shattered in sterile phosphate-buffered saline(PBS),and the suspension were differentially centrifugated untill the micro-fiber of the acellular articular cartilage extracellular matrix were divided from the residue cartilage fragments.At last the supernatant were centrifugated, the precipitation were collected and were made into 2～3%suspension.Using unidirectional solidification as a freezing process and freeze-dried method the articular cartilage extracellular matrix derived oriented scaffolds were fabricated. The scaffolds were then cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution.And the cartilage articular cartilage extracellular matrix scaffolds with random and spherical pores were prepared as a control at the same time.②Oriented cartilage extracellular matrix derived scaffolds were fabricated by using unidirectional solidification as a freezing process and freeze-dried method.After being labeled with PKH26,rabbits chondrocytes were seeded onto the scaffolds and incubated for 1,3,5 and 7 days. Attachment,proliferation and viability of the cells were measured by mitochondrial dehydrogenase activity(CCK-8 assay),the morphology, distribution and alignment of the chondrocytes in the scaffolds was analyzed by invert microscopy,fluorescent microscopy,scanning electron microscopy and H&E staining.Chondrocytes seeded into nonoriented cartilage extracellular matrix derived scaffolds were used as controls.③The chondrocytes/scaffold compounds were incubated for 1 week at 37℃,5%CO2 incubator,divided into two groups:chondrocytes/OS,chondrocytes/NOS.And then implanted respectively into subcutaneous pockets on the backs of 6 athymic nude mouse. The composites were harvested and examined with histology at 4weeks after implantation.④A full-thickness articular-cartilage defect(4ram in diameter) was created in the patellar grove of distal femur of rabbits.The rabbits were divided into five groups:(A) the defects were left empty.(B) filled with OS.(C) filled with NOS.(D) filled with BMSCs/OS.(E) filled with BMSCs/NOS.Specimens were harvested at 3,6 and 12 months postoperatively,and assessing the reconstituted defects grossly,histologically,biochemically and biomechanically.RESULTS:There were no cells in the cartilage extracellular matrix fabricated from the shattered cartilage slices;and the fiber size of the acellular articular cartilage extracellular matrix was nano scale.The histological staining showed that toluidine blue,safranin O,alcian blue and anti-collagenⅡimmunohistochemistry staining of acellular articular cartilage extracellular matrix fabricated from the shattered cartilage slices were positive.By SEM analysis a perpendicular pore-channel structures in the scaffolds were verified with channel diameters about 100μm.The porosity and the water uptake had no difference between the oriented scaffolds and the nonoriented scaffolds(p＞0.05).Mechanical testing showed that compression modulus and tension modulus of the oriented scaffolds were much higher than those of the nonoriented scaffolds(p＜0.05). Chondrocytes seeded in both scaffolds showed good attachment and viability from day 1 to day 7,most of the cells in both scaffolds were spheric morphology. However,proliferation of chondrocytes seeded in oriented scaffolds(OS) was higher than that of chondrocytes seeded in nonoriented scaffolds(NOS).Moreover, chondrocytes seeded in the OS widely distributed within the scaffolds and aligned themselves along the oriented pore mimic a columnar arrangement whereas chondrocytes seeded in the NOS randomly distributed within the scaffolds and its amount was less.In nude mice model,histological examination showed that both of OS and NOS support the chondrogenisis in that toluidine blue,safranin O, alcian blue and anti-collagenⅡimmunohistochemistry staining of the chondrocytes/scaffolds constructs were positive and there were large mount extracellular matrix around the cells.The difference of the two kinds of scaffolds was that chondrocytes seeded in OS and the collagen aligned mimic a columnar arrangement whereas chondrocytes seeded in the NOS and collagen randomly distributed within the scaffolds.In animal experimental,results indicated that every group have been repaired by fibrocartilage or hyaline cartilage in different degree.By histological and histochemical grading scale evaluation of area of defect,the group filled with BMSCs/OS or BMSCs/NOS was better to repair full-thicks cartilage defect than the others.And the score of BMSCs/OS groups were higher than that of the BMSCs/NOS groups.There was significance between the BMSCs/OS and BMSCs/NOS groups,especially in early stage after the implantation.CONCLUSION The ACECM derived oriented scaffolds have promising composition,and structural,mechanical properties similar to that of nature cartilage,can support the chondrocytes to accommodate and guide their growth into cartilagous tissue,and can repair articular cartilage defects when combined with BMSCs.This optimized scaffold might be a ideal cartilage tissue engineering scaffolds for cartilage defect regeneration.更多还原