【作者】 杨亚军；

【导师】 朱庆生；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 硕士

【摘要】 关节软骨是一种允许可动关节进行光滑无摩擦运动的特殊的狭窄层连组织。它由数量相对较少的埋植在胞外基质里的软骨细胞组成。胞外基质主要包含II型胶原,蛋白聚糖和水,伴随着数量更少的其它类型胶原和非胶原蛋白。创伤、骨性关节炎(OA)以及关节软骨病都能造成软骨组织的损伤,严重的影响了患者的生活质量,关节置换手术仍然是缓解患者疼痛的最终选择。软骨组织的损伤可在多个关节发病,但多数发生在髋关节和膝关节,国人多累及膝关节,尤其多见于绝经后女性。关节软骨一旦被破坏,病变将逐步发展,可导致关节软骨下骨的硬化、囊性变,关节囊增厚、纤维化等,最终关节变形,功能丧失。而关节软骨的损伤是不易修复的。损伤的关节软骨自我修复的能力极其有限。关节面过度损伤的治疗效果是不乐观的,通常容易发展为骨性关节炎。由于软骨自发修复能力的局限性,轻度关节软骨损伤会使损伤发展甚至退变。关节软骨的退变是临床的挑战之一,它缺乏令人信服的治疗策略[1]。近年来,组织工程倍受学者的青睐。它的核心是活的细胞、可供细胞进行生命活动的支架材料以及细胞与支架材料的相互作用,这是组织工程学研究的主要科学问题。种子细胞、可降解的支架材料与细胞生长调节因子并称为组织工程的三大基本要素。组织工程学的发展使人们开始寻找具有强大的体外增殖力和多向系分化的干细胞。但种子细胞的来源和细胞生长调节因子是组织工程的难题之一。近年有学者注意到,在某些病理情况下脂肪中有异位骨形成,这表明脂肪组织中可能存在某些细胞可分化为其他组织细胞。脂肪组织抽取后残留于供区的脂肪组织仍可扩增,且肥胖人群也越来越多,许多人主动要求抽吸脂肪,所以容易大量获得,对供区的损伤也小,这就使其自然成为干细胞生物工程研究的理想来源。CDMP1现已了解骨形态发生蛋白(bone morphogenetic protein。BMP)家族中一类特异生长因子——软骨源性形态发生蛋白(cartilage-derived morphogenetic protein CDMP)在软骨发育过程中发挥重要的调控作用。在目前所发现的BMP全部家族成中,CDMP1是最为特异的与软骨形态发生与发育相关的一类生长因子,它在出生后可继续维持正常软骨组织的生长并促进关节软骨的损伤修复过程。CDMP主要通过调节间质前体细胞的分化、参与了软骨组织的发生、生长与损伤修复的几乎全部生物学过程,是重要的调节软骨细胞分化的生长因子。本试验在体外和体内二个水平研究了CDMP1诱导大鼠脂肪干细胞(ADSCs)成软骨的研究。首先在体外利用不同浓度剂量的CDMP1诱导大鼠脂肪干细胞(ADSCs),利用MTT法测定CDMP1对ADSCs增殖分化的影响;然后复合自制牛松质骨支架植入裸鼠腋窝下,定期观察裸鼠体内成软骨的情况;最后用CDMP1诱导的ADSCs复合牛松质骨支架,修复兔膝关节软骨缺损,探讨探讨异种软骨细胞作为软骨组织工程种子细胞的可行性。本研究的内容和结果如下。1. MTT法测定CDMP1对ADSCs增殖分化的影响取第二代ADSCs细胞,以1X103个/孔密度接种于5块平底96孔培养板里。每板分ABCD四组,对照组(D组)加基础培养液,诱导组(A,B,C组)加相应的诱导培养液。每隔1 d检测1块,分析CDMP1对ADSCs的增殖能力的影响。结果表明,发现诱导组A ,诱导组B,诱导组C的细胞比对照组D同期增殖力强,尤以C组为最。10 d后,A,B,C三组细胞仍有增殖趋势,D组细胞则已成下降趋势。各组细胞在第10 d的吸光值,经方差分析及LSD- t检验,A,B,C三组分别与D组比较有统计学意义(P <0.05 );但A,B,C三组之间并无统计学意义(P >0.05)故提示CDMP1诱导的最佳浓度约为50 ng/mL,可用于进一步实验。2. CDMP1诱导ADSCs体外成软骨细胞的作用取生长良好的第二代ADSCs细胞以1X103个/孔的密度接种于24孔的培养板里,孔底放置由多聚赖氨酸包被的盖玻片。每板分ABCD四组:对照组(D组)加基础培养液(10 mL/L新生牛血清-DMEM)培养,诱导组(A,B,C组)于细胞爬片12 h后,分别加入相应的诱导液(A组:50 ng/mL CDMP1 +基础培养液;B组:100 ng/mL CDMP1 +基础培养液; C组:150 ng/mL +基础培养液)。每2 d换液一次。37℃CO2箱里培养14 d。分别行细胞形态变化学观察、Ⅱ型胶原免疫组织化学、GAG定性检测。结果表明,CDMP1诱导大鼠脂肪干细胞可以分泌软骨特异性基质糖胺聚糖(GAG)和Ⅱ型胶原,并使其向软骨方向增殖分化。3.扫描电镜下自制牛松质骨支架和诱导细胞-支架复合物的形态观察扫描电镜示:软骨细胞和经CDMP1诱导的ADSCs在自制牛松质骨支架上均生长良好。自制牛松质骨支架孔隙率适中,平均孔径约为382 um。为细胞提供了空间三维培养条件,这种培养方式有利于细胞获得足够的营养物质和保持相互间的接触和通信,使其按预制形态的三维支架生长。4. CDMP1诱导ADSCs裸鼠体内成软骨的作用将第二代ADSCs消化离心制成细胞悬液,以1X104/cm2密度用移液器复合于预湿的松质骨上,4 h后加入诱导液(10 mL/L新生牛血清-DMEM培养液+ 50 ng/mL CDMP1)继续体外培养2 w。20只裸小鼠,左侧腋窝皮下均埋入实验组细胞-支架复合物(经CDMP1诱导的ADSCs-支架复合物),右侧腋窝皮下均埋入对照组细胞-支架复合物(软骨细胞-支架复合物)。手术严格无菌操作。定期行组织学观察,结果:8 w时,甲苯胺蓝染色示试验组阳性,细胞呈球形,可见软骨陷窝;对照组阴性,细胞呈梭形。但两组支架都已降解。CDMP1体外诱导SD仔鼠ADSCs,复合自制牛松质骨支架,裸鼠体内可成软骨。5. CDMP1诱导ADSCs复合牛松质骨支架修复兔膝关节软骨缺损的实验20只新西兰大白兔,戊巴比妥钠1 ml/kg麻醉兔,行双侧膝关节内侧切口,掀开髌骨,于膝关节面用磨钻钻直径约4.0 mm,深达髓腔的缺损,于左侧膝关节植入松质骨支架-CDMP1诱导的ADSCs复合物,作为试验组,植入物与膝关节表面相齐。于右侧膝关节植入单纯松质骨支架为对照组。在8 w、16 w、24 w,分别行番红O和甲苯胺蓝染色。结果24w时,试验组修复良好,修复区新生软骨细胞与周围正常软骨细胞形态相近,呈球形,可见软骨陷窝,番红O、甲苯胺蓝染色阳性;对照组缺损处与周边界限清楚,缺损中央处凹陷空洞,新生细胞呈长梭形,番红O、甲苯胺蓝染色阴性。CDMP1诱导SD仔鼠ADSCs复合自制牛松质骨支架可成功修复兔膝关节软骨缺损,异种软骨细胞有望成为软骨组织工程的种子细胞。更多还原

【Abstract】 -6-Articular cartilage is a narrow layer of specialized connective tissue that permits smooth, frictionless movement of diarthrodial joints. It is comprised of a relatively small number of cells (chondrocytes) embedded in an abundant extracellular matrix. The latter consists predominantly of type-II collagen, proteoglycans andwater, along with smaller amounts of other collagen types and non-collagenous proteins.Damaged articular cartilage has a limited capacity for self-repair. Joint surface defects that exceed a critical size heal poorly and usually lead to osteoarthritis.Since cartilage is an avascular tissue, spontaneous repopulation of the defects with cells forming repair cartilage is commonly not observed. In the end, joint replacement is still the only therapeutic option for many patients to regain mobility and relieve chronic pain.Multiple joints can be affected in osteoarthritis, but mostly in hip and knee joints. Osteoarthritis is characterised by destruction of cartilage, subchondral bone eburnation, cyst formation, fibrilation of capsule, and eventually, joint deformation and function loss.It is well known that the capacity of articular cartilage for repair is limited. Injuries of the articular cartilage that do not penetrate the subchondral bone do not heal and usually progress onto the degeneration of the articular surface.Cartilage injuries are a common clinical problem that, if left untreated, could lead to osteoarthritis. Degeneration of articular cartilage is one of the great clinical challenges that still lack a convincing therapeutic solution.Recently, tissue engineering has emerged as a new method in which a combination of cells, scaffold, and bioactive agents is used to fabricate functional new tissue to replace damaged cartilage.The damage and loss of organs and tissues lead to metabolic and structural changes that can cause significant morbidity and decrease the quality of life. Currently employed therapies for the treatments of joint tissues loss or disease are unsatisfactory as they rely on metal joints prosthesis which offer structural replacement albeit limited functionality. Furthermore, artificial implants lack tissue’s physiological activities and often do not provide the lifelong solution for the patient. The field of tissue engineering (TE) has emerged over the past decades to improve the treatments for tissue and organ failure.Adipose-derived mesenchymal cells (AMCs) are an attractive source of multipotent mesenchymal cells (MSCs) for use in tissue engineering and clinical applications .Their relative abundance and easy access in adult tissues make them ideal candidates for cell-based therapies, and much current research is devoted to further elucidate the pathways involved in these cells’differentiation to bone,cartilage, and other tissues. Cartilage-derived morphogenetic protein-1 (CDMP-1: also known as growth differentiation factor-5, GDF-5) is a key regulatory factor in regulation of the development of the appendicular skeleton, particularly at the early stages of chondrogenesis of limb Growth/differentiation factor 5 (GDF5) is a member of the bone morphogenetic protein (BMP) family, which has been implicated in several skeletogenic events including cartilage and bone formation. CDMP-1 promotes differentiation of chondrocytes into hypertrophy and enhances commitment of mesenchymal cells into the chondrocytic lineage.This research work was designed to investigate the feasibility of diferentiation of Sprague-Dawley rat adipose-derived stem cells(ADSCs) in vitro into chondrogenic phenotype with induction of cartilage-derived morphogenetic protein(CDMP1)growth factor at both in vitro and in vivo levels. Firstly, ADSCs was induced by different dose of CDMP1 and the proliferation and differentiation of ADSCs after induced by CDMP1 was analyzed with MTT ; Sencondly , to investigate the feasibility of using Sprague-Dawley rat adipose-derived stem cells which was induced by cartilage-derived morphogenetic protein(CDMP1)growth factor and combined with the scaffold that was made of spongy bone of cattle forming into cartilage in vivo of nude mouse ; Finaly , to investigate the feasibility of heterogeneity cartilage to be the seeds cell of cartilage tissue engineering by repairing knee joints defect in rabbits with the cell induced by CDMP1 of Sprague-Dawley rat. The contents and results of this work were as follows.1. Effects of the proliferation and differentiation of ADSCs after induced by CDMP1 was analyzed with MTTThe second generation ADSCs were cultured with the density of 1X103/well in 96-well plate in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) +10 ml/L neonatal bovine serum (NBS) and induced with CDMP1 50 ng/mL (group A),CDMP1 100 ng/mL (group B),CDMP1 150ng/mL (group C) and CDMP1 0 ng/mL (group D) after 12 h . To analyzed the proliferation and differentiation of ADSCs after induced by CDMP1 . As a result , CDMP1 had contribution to the proliferation and differentiation of ADSCs and the optimal concentration of CDMP1 should be 50ng/mL since there was no statistically significant difference in proliferation and differention of ADSCs between groups A,B,C.2. The fuction of the diferentiation of Sprague-Dawley rat adipose-derived stem cells(ADSCs) in vitro into chondrogenic phenotype with induction of cartilage-derived morphogenetic protein(CDMP1)growth factor The second generation ADSCs were cultured with the density of 1X104/well in 24-well plate in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) +10 ml/L neonatal bovine serum (NBS) and induced with CDMP1 50 ng/mL (group A),CDMP1 100 ng/mL (group B),CDMP1 150ng/mL (group C) and CDMP1 0 ng/mL (group D) after 12 h .)The changes of the cell shape were observed with inverted phase contrast microscope, and the proliferation and differentiation of ADSCs after induced by CDMP1 was analyzed with MTT. Expression of collogenⅡwas detected by immunohistochemistry and the expression of GAG was detected by toluidine blue O. ADSCs from SD rat, can differentiate into chondrogenic phenotype with CDMP1 induction in vitro. And it supplies us great hopes in sources of seed cell for tissue engineering.3. To observe the shape of the scaffold of spongy bone of cattle with scanning electron microscope (SEM)The SEM showed: cartilage and ADSCs induced by CDMP1 which both were combined into the scaffolds of spongy bone of cattle growed well .The interval porosity of the scaffold of spongy bone of cattle was modicus , the average aperture was 382 um . It gived a very good three-dimensional scaffolds for the growth of cells. 4. The fuction of rat adipose-derived stem cells forming into cartilage with induction of cartilage-derived morphogenetic protein (CDMP1) growth factor in vivo of nude mouseThe second generation cartilage cells of SD rat were cultured with elementary nutrient liquid which contains DMEM+10 mL/L NBCS only with the scaffold for another two weeks . There were twenty nude mice which were imbedded the composite of cells-scaffold of the experimental group in their left armpits and the composite of cells-scaffold of the control group in their right armpits. After eight weeks, toluidine blue staining showed the slices of the experimental group was positive and the shape of cells became globular , and there were apparent lacunas of cartilage while the control group was negative and there were no lacunas of cartilage. But the scaffolds of both the groups were degradation entirely. It’s possible to form into cartilage in vivo of nude mouse by using ADSCs of SD rat which was induced by CDMP1 and combined with the scaffold of spongy bone of cattle.5. Study of reparing knee joints defection in rabbit with the ADSCs induced by CDMP1 which were combined with the scaffold made of the spongy bone of cattleTwenty adult New Zealand rabbits were included in this study. To make the models of knee joints defect of rabbits and embed the composites of ADSCs-scaffold into the defect of the left legs of the rabbits as the experimental group while embedding the scaffold only into the defect of the right legs of the rabbits as the control group. Every six rabbits were killed after eight, sixteen and twenty-four weeks and to make the slices of safranine o and toluidine blue staining .The repairing of the experimental group were satisfactory and the cells of the area of repairing and the normal circumjacent area were very similar in morphous .But the control group were still cavitates and the boundary between the repairing area and the normal circumjacent area was visible . Toluidine blue and safranine o staining showed the slices of the experimental group was positive and the shape of cells were globular , and there were apparent lacunas of cartilage while the control group was negative and there were no lacunas of cartilage. The knee joints defect of rabbits were repaired successfully by using cartilage of SD rat combining with the scaffold of spongy bone of cattle and it’s possible that heterogeneity cartilage to be the seeds cell of cartilage tissue engineering .更多还原