【作者】 杨志高；

【导师】 吴立东；

【作者基本信息】 浙江大学 ， 外科学（专业学位）， 2013， 博士

【摘要】 前言软骨损伤在临床上十分常见,其治疗长期以来一直是骨科领域的热点、难点问题。成年人发生软骨损伤后,由于自然修复的软骨组织为纤维软骨而非功能性的透明软骨,因而不可避免出现渐进性退变,最终导致骨关节炎的发生。目前对于软骨缺损的治疗措施主要是疼痛控制、手术干预和软骨移植,但这些治疗方法的远期疗效并不理想。在这种情况下,作为一种可选择的替代疗法,以干细胞为基础的组织工程方法为软骨缺损的修复开辟了一条全新的途径。一般来讲,利用组织工程方法进行软骨缺损的修复需要具备三个主要条件：第一,足够数量、功能正常、具有成软骨分化能力的种子细胞；第二、具有良好生物相容性、可无毒降解、合适空隙大小和孔隙率的三维支架；第三、调节种子细胞增殖、分化,并维持其表型特征的细胞因子。脂肪间充质干细胞(adipose-derived mesenchymal stem cells, AMSCs)是来自中胚层的成体干细胞,由于其相比其他成体干细胞来源充足、取材方便、提取时对机体损伤小,与骨髓间充质干细胞(bone-marrow mesenchymal stem cells, BMSCs)具有相似的生物学特征和分化潜能,以及优于BMSCs的增殖能力,现已成为一种理想的种子细胞来源,也是当前研究的热点。聚乳酸-聚羟乙酸共聚物(poly-lactic-co-glycolic acid, PLGA)作为被美国食品和药品管理局(Food And Drug Administration, FDA)批准可以用于人体的组织工程支架材料,以其良好的生物相容性,来源广泛、容易获得且可大批量生产,其空间结构、大体形态、机械强度、降解时间等都能预先设计和调控等优点,已成为在组织工程中应用最广泛的支架材料。转化生长因子-β (transforming growth factor β, TGF-β)是一种细胞生长和细胞外基质合成的多功能调节器,它可以促进软骨细胞增殖,增加蛋白多糖和二型胶原合成,而且还能减少软骨胶原酶的表达和分泌。有研究认为TGF-β3相比于TGF-β1具有更强的诱导间充质干细胞向软骨分化的能力,而且TGF-β3同时还有抑制炎症反应和促进金属蛋白酶组织抑制剂表达的作用。虽然AMSCs已经被证实具有修复软骨缺损的作用,但应用非诱导的AMSCs进行软骨缺损修复的研究尚显不足,而且非诱导的AMSCs在植入体内后是否能长期存活以及是否通过直接分化为软骨细胞对缺损部位进行修复,这些问题都尚不明确。目前尚没有应用AMSCs联合TGF-β3负载的双层PLGA进行全层软骨缺损修复的研究报道。在本研究中,我们在体外实验的相关研究结果基础上,用超顺磁氧化铁(superparamagnetic iron oxide, SPIO)作为示踪剂,用SPIO标记的AMSCs联合TGF-β3负载的双层PLGA三维支架构筑AMSCs/TGF-β3/PLGA复合体,观察其对兔全层关节软骨缺损修复的效果,并用核磁共振成像(magnetic resonance imaging, MRI)对植入的AMSCs进行活体内追踪,观察其存活和分布情况,同时用普鲁士蓝染色检测新生的软骨细胞是否由植入的AMSCs直接分化而来。本研究主要分三部分：(1)首先进行兔AMSCs的分离、培养和SPIO标记；体外检测MRI对SPIO标记的AMSCs探测的敏感性。同时利用致孔剂浸出技术制作双层PLGA三维支架。结果显示SPIO可以高效的标记AMSCs;在被标记的AMSCs达到足够的浓度的时候,可以被MRI敏感的追踪到。电镜扫描(scanning electron microscopy, SEM)结果证实：利用致孔剂浸出技术可以成功制作满足组织工程需要,具有适合孔径大小的双层PLGA三维支架。(2)检测TGF-β3对体外培养的AMSCs增殖、分化的影响。根据实验结果可以认为TGF-β3浓度在10ng/ml时,短期内对体外单层培养的AMSCs的增殖没有影响。用TGF-β3对体外单层和三维培养的AMSCs进行诱导可以增加二型胶原(Collagen type Ⅱ),聚糖蛋白(Aggrecan)和SOX-9(SRY-related HMG box9, SOX-9)在基因和蛋白水平的表达,这种作用呈时间依赖性,但三维培养条件下相比单层培养更有利于促进它们在基因和蛋白水平的表达。(3)在前两部分的基础上,用AMSCs/TGF-β3/PLGA复合体对兔子全层软骨缺损模型进行修复,观察其修复效果,AMSCs在体内的存活情况以及新生软骨细胞是否直接由其分化而来。结果表明AMSCs/TGF-β3/PLGA复合体可有效修复关节软骨缺损；AMSCs在植入体内12周后仍然有部分存活,但新生的软骨细胞并非由植入体内的AMSCs直接分化而来。第一部分AMSCs的超顺磁氧化铁标记和双层PLGA支架的制作目的：观察MRI对SPIO标记的AMSCs探测的敏感性以及致孔剂浸出技术能否成功制作符合软骨组织工程需要的合适孔径大小的作双层PLGA三维支架。方法：用含SPIO为25μg/ml的培养基培养AMSCs,24h后进行普鲁士蓝染色,光镜下观察AMSCs被SPIO标记的情况,胰酶消化收集细胞,用PBS重悬细胞,分别检测细胞浓度在5×104,1x105,5×105,1×106/ml时MRI对被标记细胞的敏感性。用明胶颗粒作为致孔剂,利用致孔剂浸出技术制作双层PLGA支架。结果：用含SPIO为25μg/ml的培养基对AMSCs进行培养,AMSCs可以被高效标记,标记率接近100%。在被标记的AMSCs浓度达到1×105/ml时,MRI即可探测信号的改变,1×106/ml时T2加权像上呈现黑色。SEM扫描结果表明利用致孔剂浸出技术可成功制作具有大、小两层微孔,孔径分别在2001×m和400μm左右的符合组织工程需要的双层PLGA三维支架。结论：SPIO可以高效标记AMSCs并被MRI追踪到。以明胶作为致孔剂利用致孔剂浸出技术可成功制作符合组织工程需要的双层PLGA三维支架。第二部分TGF-P3对体外培养的AMSCs增殖、分化的影响目的：观察TGF-β3对体外培养的AMSCs增殖、分化的影响。方法：取成年新西兰兔腹股沟部皮下脂肪,用酶消化、梯度离心、贴壁培养法对兔AMSCs进行分离、培养。取第三代AMSCs,用含不同浓度的TGF-P3(0,1,5,10,20,50ng/ml)培养基进行培养,CCK-8方法检测不同浓度TGF-β3在第1、3、7天对AMSCs增殖的影响。用10ng/ml TGF-β3诱导AMSCs,分别于诱导后7天和14天,免疫荧光观察Collagen type Ⅱ的表达情况；实时定量聚合酶链反应(real-time quantitative polymerase chain reaction, RT-qPCR)法检测细胞中Aggrecan, Collagen type Ⅱ, SOX-9基因的表达,western blot法检测细胞中Aggrecan, Collagen type Ⅱ, SOX-9蛋白的表达。结果：用含不同浓度TGF-β3的培养基培养1、3天后,均对AMSCs的增殖均没有影响,7天后,除10ng/ml浓度组对AMSCs的增殖没有影响外,其余浓度均明显抑制了AMSCs的增殖。免疫荧光结果显示用10ng/ml的TGF-β3诱导7天后,AMSCs表达Collagen type Ⅱ,第14天荧光信号强度显著高于第7天,单纯低血清培养基培养AMSCs14天后也有少量的Collagen type Ⅱ表达。RT-qPCR和western blot结果表明用TGF-P3对体外单层和三维培养的AMSCs进行诱导可以增加Collagen type Ⅱ, Aggrecan和SOX-9在基因和蛋白水平的表达,这种作用呈时间依赖性,三维培养条件下三者在基因和蛋白水平的表达显著高于单层培养。结论：10ng/ml的TGF-β3短期内对体外培养的AMSCs的增殖没有影响。TGF-β3可诱导AMSCs向软骨分化,呈时间依赖性,三维培养优于单层培养。第三部分AMSCs/TGF-β3/PLGA复合体修复关节软骨缺损的动物实验研究目的：观察AMSCs/TGF-β3/PLGA复合体对兔子全层关节软骨缺损模型的修复作用及AMSCs在这一过程中的作用和机制。方法：取64只新西兰大白兔,在股骨髁髌股关节部位制作关节软骨缺损模型。随机分为4组,每组16只,分别为缺损组(Defect组),PLGA组,TGF-β3/PLGA组,AMSCs/TGF-β3/PLGA组。各组分别于术后第6周和第12周对32只兔的膝关节进行MRI扫描后处死,并收取包含修复组织的股骨髁,进行形态学、组织学观察。并用RT-qPCR和western法检测修复组织中Aggrecan, SOX-9, Collagen type Ⅰ, Ⅱ and X在基因和蛋白水平的表达。结果：MRI扫描结果提示在AMSCs/TGF-p3/PLGA复合体植入部位,术后第6周和第12周都可探测到明显的低信号改变。普鲁士蓝染色显示术后第6周时在新生软骨层和软骨下骨层均有蓝染颗粒,而在第12周只有软骨下骨层有蓝染颗粒,而新生软骨层未见蓝染颗粒。AMSCs、TGF-03均可促进软骨缺损的修复,这种作用是通过在基因和蛋白水平调节Aggrecan, SOX-9, Collagen type Ⅰ, Ⅱ and X的表达呈现,具有时间依赖性。PLGA自身对软骨缺损修复的效果并不明显。结论：AMSCs/TGF-p3/PLGA复合体可有效修复全层关节软骨缺损。AMSCs在植入缺损部位后可以长期存活并促进软骨修复,但新生的软骨细胞并非由植入的AMSCs直接分化而来,AMSCs可能通过释放某些细胞生长因子促进软骨缺损的修复。更多还原

【Abstract】 IntroductionCartilage injury is very common in clinical practice, all along, the treatment for it is a the hot and difficult problem in the field of orthopaedics. Self-healing cartilage tissue after injury in adult is fibrocartilage, but not hyaline cartilage which has function, thus inevitably appears progressive degeneration, eventually leading to the occurrence of osteoarthritis. The current treatments for cartilage defects are mainly focus on pain control, surgical intervention, and cartilage transplantation, but these can not yield satisfactory long-term efficacy. In this case, as an alternative treatment, mesenchymal stem cells based tissue engineering has emerged as a promising approach for cartilage defect repair. Three main conditions are needed for repair cartilage defect:First, sufficient number of properly functioning seed cells and chondrogenic capacity. Second, a three-dimensional scaffold with biocompatible, non-toxic degradation, suitable gap size and porosity. Third, cytokines which could adjust the seed cell differentiation and proliferation, and maintain their phenotypic. Adipose-derived mesenchymal stem cells (AMSCs) are adult stem cells originating from the mesoderm, because AMSCs are convenient of obtaining, adequate sources and small injury on body compared with others adult stem cells, similar biological characteristics and differentiation potential with bone-marrow mesenchymal stem cells (BMSCs), better proliferative capacity than BMSCs, AMSCs have become an ideal source of seed cells and research hotspot. As a three-dimensional scaffold which has been approved for human tissue engineering by the Food And Drug Administration (FDA) of American, poly-lactic-co-glycolic acid (PLGA) due to its advantages at good biocompatibility, wide source, easy to obtain and capable of mass production, more over, the space structure, general form, mechanical strength and degradation time of its could be design and control in advance, so it has become the most widely used in tissue engineering extracellular matrix material. Transforming growth factor-β (TGF-β) is a multifunctional regulator of a cell growth and extracellular matrix synthesis, it can promote the proliferation of chondrocytes increased proteoglycan and type Ⅱ collagen synthesis, but also to reduce the expression and secretion of the cartilage collagenase. The effect of TGF-β3inducing mesenchymal stem cells (mesenchymal stem cell, MSCs) differentiate to chondrocytes is better than TGF-β1, more over, TGF-β3could inhibit the inflammatory response and promote tissue inhibitor of metalloproteinase expression.Although AMSCs have been shown to have effects in the repair of cartilage defects, however, the studies which using non-induced AMSCs to repair full-thickness cartilage defects are still insufficient, whether non-induced AMSCs could be long-term survival after being implanted in the body and whether AMSCs repair cartilage defect by differentiating to chondrocytes derectly, these issues are unclear. Currently, no author has reported using AMSCs combined with TGF-β3-loaded PLGA scaffolds for the restoration of cartilage defects. In the present study, we based on the related research results of experiments in vitro, used superparamagnetic iron oxide (SPIO) labeled AMSCs combined with TGF-β3-loaded PLGA to design a AMSCs/TGF-β3/PLGA construct to repair the full-thickness cartilage defect model of rabbit and magnetic resonance imaging(MRI) to track the SPIO labeled AMSCs in vivo, observe their survival and distribution, at the same time, prussian blue staining to detect whether the new neo-chondrocytes were differentiated from implanted AMSCs directly.The study is mainly divided into three parts:(1) rabbit AMSCs isolation, culture, labeled by SPIO, then, to detect the sensitivity of MRI tracking SPIO-labeled AMSCs. Using a porogen leaching technology to fabricate bilayer PLGA scaffold. The results showed that AMSCs could be efficiently labeled by SPIO, MRI could track SPIO-labeled AMSCs sensitively when the concentration of cells was sufficient. The study confirmed the bilayer PLGA scaffold could be fabricate by porogen leaching technology. A bilayer PLGA scaffold which with suitable pore size for tissue engineering could be successfully produced by porogen leaching techniques.(2) investigation the effects of TGF-P3on the proliferation and differentiation of AMSCs in vitro. The results of present study showed that when the concentration of TGF-β3at lOng/ml, it had no effect on the proliferation of AMSCs in a short term. Treating AMSCs with TGF-β3in vitro could increase the espression of Collagen type II, Aggrecan and SRY-related of HMG box9(SOX-9) at gene and protein levels whether monolayer culture or three-dimensional culture in a time-dependent manner, but three-dimensional culture could promote the expression of them more efficient when compared to monolayer culture.(3) on the basis of the previous two parts, the AMSCs/TGF-β3/PLGA constructs were used to repair the full-thickness cartilage defects of rabbit models, investigation the regeneration effects of them and the survival and distribution of implanted AMSCs as well as whether the neo-chondrocytes were differentiated from implanted AMSCs directly. The results showed that the AMSCs/TGF-β3/PLGA constructs could effective repair the articular cartilage defects. The AMSCs were still survival partly after12weeks being implanted in vivo, but the neo-chondrocytes were not differentiated from the AMSCs directly. Part1SPIO-labeling of AMSCs and fabrication of bilayer PLGA scaffoldObjective:To observe the sensitivity of MRI on SPIO-labeled AMSCs and whether the porogen leaching technology could produced a three-dimensional bilayer PLGA scaffold with suitable pore sizes for cartilage tissue engineering.Methods:Culturing AMSCs with medium containing25μg/ml SPIO for24hour, then, did prussian blue staining and observed the mark effect by light microscopy. The AMSCs were trypsinized, collected, resuspended in PBS, the sensitivity of MRI were observed when the concentrations SPIO-labeled AMSCs were5x104,1x105,5x105,1x106/ml. A bilayer PLGA scaffold with different pore size was fabricated by a porogen-leaching technique with gelatin particles as the porogen.Results:Culturing AMSCs with medium containing25μg/ml SPIO, the AMSCs could be marked efficiently, the rate of labeled AMSCs was nearly100%. When the cells concentration at1x105/ml, MRI could track the signal change, when the cells concentration at1x106/ml, presented a significant black signal on T2-weighted images. SEM photographs revealed that the bilayer PLGA scaffold possessed macropores and micropores. The average pore sizes of the micropores and macropores in the scaffold were about200μm and400μm, which are suitable for tissue engineering needs.Conclusions:The AMSCs could be marked efficiently by SPIO and tracked by MRI in vitro. A bilayer PLGA scaffold which is suitable for tissue engineering could be successfully produced using gelatin as porogen by porogen leaching technology. Part2The effects of TGF-β3on the proliferation and differentiation of AMSCs in vitroObjective:Observation of the effects of TGF-β3on the proliferation, differentiation of AMSCs in vitro.Methods:Adipose tissue was isolated from the groin area of adult New Zealand rabbits by enzyme digestion gradient centrifugation and adhesive culture methods. The third generation of AMSCs were used, the method of CCK-8was used to detect the effect of different concentrations (0,1,5,10,20,50ng/ml) of TGF-β3on the proliferation AMSCs at the1,3,7day respectively. lOng/ml TGF-β3was used to induce the differentiation of AMSCs, at the7and14days after induction, immunofluorescence staining was used to observe the expression of Collagen type Ⅱ. Real-time quantitative polymerase chain reaction (RT-qPCR) and western-blot methods were used to the expression of Aggrecan, Collagen type Ⅱ, SOX-9at gene and protein levels respectively.Results:After one or three days induced by different concentrations of TGF-β3, each concentration had no effect on the proliferation of AMSCs, but at7th day, all the concentrations of TGF-P3inhibited the proliferation of AMSCs, except lOng/ml. The immunofluorescence showed that after induction of TGF-β3seven days, the expression of Collagen type Ⅱ could be detected by immunofluorescence,14th day of the fluorescence signal intensity was significantly higher than7th day. Further, it can be seen that the AMSCs cultured by low serum culture medium also had a small amount of Collagen type II expression. RT-qPCR and western blot results showed that inducing by TGF-β3could increase the espression of Collagen type Ⅱ, Aggrecan and SOX-9at gene and protein levels in a time-dependent manner, the genes and proteins expression of the three were significantly higher in three-dimensional culture than that in monolayer culture.Conclusions:When the concentration of TGF-β3was10ng/ml, it had no effect on the proliferation of AMSCs in a short term. TGF-β3could promote AMSCs differentiating to chondrocytes in a time-dependent manner, three-dimensional culture was more efficient than monolayer culture.Part3The restore of articular cartilage defects using AMSCs/TGF-p3/PLGA constructs in animal modelsObjective:Observation the repair effects of AMSCs/TGF-β3/PLGA constructs on rabbit full-thickness articular cartilage defects and the effects and mechanisms of AMSCs in this process.Methods:Full-thickness defects were created surgically on the femoropatellar groove of the knee joints of64rabbits. The rabbits were randomly divided into four groups: Defect group, PLGA group, TGF-β3/PLGAgroup and AMSCs/TGF-β3/PLGA group.32rabbits took SEM scanning, then sacrificed at6or12weeks after surgery and the restored tissues were retrieved for morphology, histological observation. RT-qPCR and western-blot methods were used to the expression of Aggrecan, SOX-9, Collagen type I, Ⅱ and X at gene and protein levels respectively.Results:MRI confirmed that there still had survival AMSCs in the repair tissue at6weeks and12weeks after surgery, low signal change can be detected in T2-weight images. Prussian blue staining showed that the neo-cartilage layer and subchondral bone layer all had blue particles at6th week, but at12th week the blue particles existed only in the subchondral bone layer, while the neo-cartilage layer no blue particles. TGF-β3and AMSCs could promote the restore, these effects of them were exerted by regulating the expression of Aggrecan, SOX-9, Collagen type Ⅰ, Ⅱ and X at gene and protein levels in a time-dependent manner. The effect of PLGA itself in the process was no obvious.Conclusions:The AMSCs/TGF-β3/PLGA constructs could repair the full-thickness articular cartilage defects effectively. AMSCs could be survival after implanted in the defect by a long-term and promote cartilage repair effects, but the neo-chondrocytes were not differentiated from the AMSCs directly, AMSCs maybe promote cartilage defect repair by releasing some cell growth factors.更多还原