【作者】 王锐；

【导师】 解慧琪；

【作者基本信息】 四川大学 ， 生物医学工程， 2021， 硕士

【摘要】 背景:因先天性或外伤性因素所致的外耳畸形或损伤是整形外科临床中常见的问题。小耳畸形的治疗方法主要有佩戴耳廓假体、植入不可吸收性耳廓支架材料或自体肋软骨移植。上述方法的治疗效果有限,且存在明显不足。组织工程技术的发展为耳软骨修复与再生提供了新的策略。以软骨细胞为基础的软骨组织工程,通常需要对软骨细胞进行大量的体外扩增培养,然而常规培养过程中,软骨细胞容易出现去分化,倾向于失去原有的形态、表型和生物学功能。目前,软骨细胞去分化现象已有的报道主要集中在关节软骨细胞,而耳廓软骨细胞的去分化现象及机制研究仍然较少。如何解决软骨细胞去分化这一瓶颈问题是研究人员关注的热点。软骨组织工程中三维支架材料可维持软骨细胞表型的稳定性,是最有望解决去分化问题并修复软骨缺损的有效手段。实验室前期结合生物源性的猪小肠黏膜下层材料(Small intestinal submucosa,SIS)和合成的高分子材料聚氨酯(Polyurethane,PU)的优点成功制备了具有良好生物相容性和优异回弹性能的弹性体材料——聚氨酯/小肠黏膜下层复合材料(Polyurethane/small intestinal submucosa,PU/SIS)。PU/SIS支架材料具有的表面形貌、结构特征以及力学性能,可能影响软骨细胞表型的维持,有望成为耳廓软骨组织工程支架材料的新选择。已有研究表明应用弹性软骨来源的软骨细胞更利于组织工程耳廓软骨的构建。本实验建立兔耳软骨缺损修复模型,选择兔耳来源的软骨细胞(Rabbit auricular chondrocytes,r ACs)作为种子细胞,与PU/SIS支架材料复合培养构建组织工程兔耳软骨组织。但由于软骨细胞来源十分有限,寻找新的且来源广泛的软骨组织工程种子细胞成为新的问题。骨髓间充质干细胞(Bone marrow stromal cells,BMSCs)和脂肪来源间充质干细胞(Adipose-derived stem cells,ADSCs)已有应用于组织工程软骨的研究,但取材方式均为有创,且来源有限。尿源性干细胞(Urine-derived stem cells,USCs)具有来源丰富、可无创获取以及增殖能力强等优点,逐渐成为组织工程再生修复领域可靠的种子细胞来源。本实验室分离培养的USCs经诱导分化实验证明其可以向软骨方向分化,但能否用于构建弹性软骨需进一步探索验证。目的:评价PU/SIS材料与r ACs及USCs的细胞相容性。观察r ACs在常规传代培养过程中软骨表型的变化情况,并利用PU/SIS支架材料培养r ACs,探究其对软骨细胞表型稳定性的影响。在动物实验部分,建立兔耳软骨全层缺损修复模型,探究PU/SIS材料分别与r ACs及USCs复合构建组织工程弹性软骨的可行性,以及单独应用PU/SIS材料,修复兔耳软骨缺损,评价其再生修复效果。进一步,通过普通转录组测序实验对PU/SIS材料的修复机制进行初探。方法:参照实验室已建立的方法制备PU/SIS支架材料。分别采用酶消化法和离心法分离培养原代r ACs和USCs,并进行传代培养。评价PU/SIS支架材料与r ACs和USCs的细胞相容性。在体外细胞毒性试验中,制备一系列浓度的PU/SIS材料培养基浸提液,对r ACs和USCs进行培养,采用CCK-8法对1天、3天、5天和7天的细胞活力进行检测,计算r ACs和USCs的相对增殖率,并对材料的细胞毒性进行评级。在细胞活死染色实验中,采用钙黄绿素和碘化丙啶分别对PU/SIS材料上的活细胞和死细胞进行染色,观察r ACs和USCs在材料上的存活情况。采用带有荧光探针的鬼笔环肽对细胞骨架进行染色,观察r ACs和USCs在PU/SIS材料表面的铺展形态。进一步,采用扫描电镜观察r ACs和USCs培养在PU/SIS材料上的形貌变化。在常规培养瓶中对r ACs进行传代培养时,倒置显微镜下观察不同代次r ACs的形态变化。采用免疫荧光染色,观察II型胶原和I型胶原表达水平的变化。对不同代次r ACs进行成球培养,并对软骨小球进行甲苯胺蓝染色,评价r ACs的成软骨能力。将不同代次r ACs分别接种在孔板和PU/SIS材料上培养,采用PCR技术检测软骨表型相关基因(COL2A1、COL1A1、ACAN、ELASTIN和SOX9)的m RNA表达水平变化情况,评价PU/SIS材料对软骨细胞表型维持的影响。选择新西兰大白兔建立兔耳软骨全层缺损模型。设置空白对照组、单纯PU/SIS材料修复组,以及PU/SIS材料复合细胞组,即PU/SIS+r ACs组和PU/SIS+USCs组。在术后8W和12W进行取材,采用相机对软骨缺损区域的整体修复情况进行观察、采图。对样本进行石蜡包埋及切片,行H&E、Masson染色对修复后的组织结构及材料降解情况进行观察。采用甲苯胺蓝染色观察修复区域新生软骨情况;采用弹性纤维染色观察软骨弹性纤维成分的合成。在PU/SIS材料修复兔耳软骨缺损的机制探索方面,增加样本数量,分别于三个时间点(6W、9W和12W)取材,通过大体观察和组织学染色方法(H&E、Masson、甲苯胺蓝以及弹性纤维染色)明确PU/SIS材料的修复效果。进一步,将兔耳正常软骨组织、空白对照组以及PU/SIS组修复术后12W取材的组织样本,进行普通转录组测序,结合生物信息学手段,分析差异表达基因,并对其进行基于GO数据库和KEGG数据库的注释,了解差异表达基因的生物学功能,筛选出可能的信号通路。结果:首先,酶消化法分离培养的原代r ACs,呈扁平形态,以多角形或三角形居多。体外细胞毒性实验结果显示,r ACs和USCs在PU/SIS材料浸提液培养1天、3天、5天和7天后相对增殖率均≥70%,材料毒性评级为0级、1级或2级,表明PU/SIS材料没有间接细胞毒性。活死染色结果显示,r ACs和USCs均能在PU/SIS材料上粘附生长,表现出良好的增殖能力,说明PU/SIS材料没有细胞毒性。细胞骨架染色和扫描电镜观察结果显示,随着培养时间的增加,r ACs呈聚集生长,覆盖在材料表面,伸出较多伪足,特别是向材料孔内生长,形态以多角形为主;而USCs则主要粘附在材料孔径的“嵴”上生长,围绕孔径边缘铺展,细胞形态以长梭形为主。r ACs接种在培养瓶中进行常规传代培养,P1～P3的软骨细胞形态基本维持不变,以三角形或多角形为主;P4～P5的软骨细胞开始向长梭形状态转变,并逐渐增多,提示r ACs出现去分化。免疫荧光染色结果显示,随着培养代次的增加,II型胶原表达水平逐渐降低,I型胶原表达水平逐渐升高,符合软骨细胞去分化特征。r ACs软骨小球甲苯胺蓝染色结果显示,P1～P3的r ACs小球可见大量蓝染的细胞外基质,而P4的r ACs小球仅可见少量蓝染的细胞外基质,表明软骨细胞已基本丧失分泌糖胺聚糖的能力。PCR的结果显示,r ACs随着传代次数的增加,基因表达谱发生明显变化,COL1A1基因表达上调,COL2A1、ACAN基因表达下降,SOX9、ELASTIN基因基本保持不变。与孔板培养相比,r ACs与PU/SIS支架材料复合培养的方式促进了COL2A1、SOX9和ELASTIN基因的表达上调,且对COL1A1基因的表达也有一定抑制作用。COL2A1与COL1A1表达水平的比值也表现出升高的趋势。在PU/SIS支架材料复合r ACs及USCs修复兔耳软骨缺损的实验研究中,组织学染色结果显示,空白对照组和PU/SIS+r ACs组修复区始终未见新生软骨细胞,甲苯胺蓝和弹性纤维染色均为阴性。而PU/SIS组随着修复时间的延长,修复区的新生软骨细胞数量逐渐增多并向成熟软骨细胞转变,产生了糖胺聚糖和弹性纤维等细胞外基质成分,具备弹性软骨形成的能力。PU/SIS+USCs组在术后8W和12W时均在交界处观察到新生软骨细胞团的出现,甲苯胺蓝和弹性纤维染色均呈阳性。在PU/SIS支架材料修复兔耳软骨缺损机制初探的实验研究中,增大样本量及增加时间点观察,PU/SIS支架材料的修复作用得到进一步确认。在对普通转录组测序结果进行分析时,差异表达基因个数的统计结果显示:空白对照组vs正常组和PU/SIS组vs正常组的差异表达基因个数无明显差距。聚类分析结果显示:(1)空白对照组vs正常组,软骨特异性ECM成分相关基因、软骨生长发育调节的生长因子和转录因子相关基因、软骨表型变化相关基因以及弹性纤维相关基因表达下调;软骨基质重塑相关基因以及炎症反应相关基因表达上调。这可能是空白对照组未有软骨再生的原因。(2)PU/SIS组vs正常组,与免疫调节相关的基因表达上调;与软骨特异性ECM成分和软骨表型变化相关的基因下调;与软骨基质重塑相关基因的部分上调,部分下调;软骨生长发育调节的生长因子和转录因子相关基因也部分上调,部分下调。(3)PU/SIS组vs空白对照组,与免疫调节相关的基因表达上调。GO富集分析结果显示:(1)空白对照组vs正常组和PU/SIS组vs正常组,均显著富集到蛋白多糖生物合成过程的正向调节。(2)PU/SIS组vs空白对照组,主要富集到免疫相关的生物学功能,同时也富集到整合素复合物、胶原蛋白代谢过程、参与细胞黏附的蛋白质复合物和整合素介导的信号通路等。KEGG富集分析结果显示:(1)空白对照组vs正常组和PU/SIS组vs正常组,富集到代谢通路的基因数量最多,但从q-value的角度来看,不具有显著性意义。(2)PU/SIS组vs空白对照组富集到细胞因子-细胞因子受体的相互作用的基因数量最多,且具有显著性意义。结论:PU/SIS支架材料具有良好的细胞相容性,r ACs与USCs均可在材料表面进行良好粘附、生长及增殖。PU/SIS三维支架材料与软骨细胞构建的复合培养体系,有利于兔耳软骨细胞表型稳定性的维持。在兔耳软骨缺损修复实验中,PU/SIS材料促进了软骨修复,有望成为弹性软骨组织工程支架材料的新选择。同时,USCs与PU/SIS材料复合构建组织工程弹性软骨,具有一定的可行性,USCs可作为耳廓软骨组织工程的种子细胞来源。普通转录组测序结果显示,PU/SIS组的软骨生长发育调节生长因子和转录因子的相关基因以及软骨基质重塑的相关基因部分上调,部分下调,综合来看,可能起到了一个正向和负向的相互调控作用。同时,这些差异表达基因富集到的信号通路有整合素复合物、整合素介导的信号通路和细胞因子-细胞因子受体的相互作用等,初步认为作用机制可能与PU/SIS材料的力学特性有关。更多还原

【Abstract】 Background:Congenital malformation of the external ear and auricular trauma are common problems in plastic surgery clinics.The main treatment options for microtia are wearing an auricular prosthesis,the implantation of non-absorbable auricular scaffold material or autologous rib cartilage grafts.These methods are limited in their effectiveness and have significant shortcomings.The development of tissue engineering technique has provided new strategies for the repair and regeneration of ear cartilage.Chondrocyte-based cartilage tissue engineering usually requires extensive expansion of chondrocytes in vitro.However,during the standard cell culture process,chondrocytes are prone to dedifferentiation and lose their original morphology,phenotypes and biological functions.At present,chondrocyte dedifferentiation has been reported mainly in articular chondrocytes,while the dedifferentiation of auricular chondrocytes and its mechanisms are still less studied.How to solve the bottleneck problem of chondrocyte dedifferentiation is a hot topic of interest for researchers.Three-dimensional scaffold materials in cartilage tissue engineering can maintain the stability of chondrocyte phenotypes and are the most promising means to address the problem of dedifferentiation and repair cartilage defects.The laboratory has successfully combined the advantages of a biologically derived porcine small intestinal submucosa(SIS)and a synthetic polymer,polyurethane(PU),to produce an elastomeric material(PU/SIS composites)with good biocompatibility and excellent resilience.The surface morphology,structural characteristics and mechanical properties of the PU/SIS scaffold material may influence the maintenance of the chondrocyte phenotypes and therefore PU/SIS is expected to be a new choice of scaffold material for auricular cartilage tissue engineering.It has been shown that the application of chondrocytes with elastic cartilage origin is more conducive to the construction of tissue-engineered auricular cartilage.In this experiment,a rabbit ear cartilage defect repair model was established,and rabbit earderived chondrocytes(Rabbit auricular chondrocytes,r ACs)were selected as seed cells and cultured in combination with PU/SIS scaffold material to construct tissueengineered rabbit ear cartilage tissue.As the sources of chondrocytes are very limited,the search for new and widely available seed cells for cartilage tissue engineering has become a new problem.Bone marrow stromal cells(BMSCs)and adipose-derived stem cells(ADSCs)have been used but are both invasive and from relatively limited sources.Urine-derived stem cells(USCs)are becoming a reliable source of seed cells for tissue engineering regenerative repair because of their abundant source,non-invasive acquisition and high proliferation capacity.The USCs that isolated and cultured in our laboratory have shown the ability to differentiate towards cartilage by induction differentiation experiments,but further exploration is needed to verify whether they can be used to construct elastic cartilage.Objective:In this study,we evaluated the cytocompatibility of PU/SIS with r ACs and USCs.In order to understand the dedifferentiation phenomenon of r ACs,we observed the changes of chondrocytes phenotypes during the standard passaging culture of r ACs and cultured r ACs with PU/SIS scaffold material to investigate its effect on the stability of chondrocyte phenotypes.In the animal experimental part,a rabbit ear cartilage whole-layer defect repair model was established to investigate the feasibility of PU/SIS materials to construct tissue-engineered elastic cartilage in composite with r ACs and USCs respectively,as well as to apply PU/SIS materials alone to repair rabbit ear cartilage defects and evaluate their regenerative repair effects.Furthermore,a transcriptome sequencing study was performed to investigate the repair mechanism of PU/SIS materials.In summary,this study hopes to provide the new scaffold materials and seed cell sources for auricular cartilage tissue engineering.Materials and Methods:The PU/SIS scaffold material was prepared with reference to the previous method in the laboratory.Primary r ACs and USCs were isolated and cultured by enzymatic digestion and centrifugation respectively,and passaged.The cytocompatibility of the PU/SIS scaffold materials with r ACs and USCs was evaluated.In the cytotoxicity assay in vitro,r ACs and USCs was cultured in a range of concentrations medium extraction of PU/SIS material,respectively.After being cultured for 1,3,5 and 7 days,the relative growth rate(RGR)of r ACs and USCs and the toxicity degrees were determined by CCK-8 method.As to the Live/Dead staining assay,r ACs and USCs was seeded on the surface of PU/SIS scaffold materials,respectively.The state of r ACs and USCs on PU/SIS materials was determined by the calcein and propidium iodide staining.The cytoskeleton was stained using the phalloidin with fluorescent probe to observe the spreading pattern of r ACs and USCs on the surface of PU/SIS material.Further,scanning electron microscopy was used to observe the morphological changes of r ACs and USCs cultured on PU/SIS material.Morphological changes of r ACs in the different generations were observed under inverted microscope when r ACs were cultured in conventional culture flasks for passaging.Immunofluorescence staining was used to observe changes in the expression levels of type II collagen and type I collagen.After the pellet culture of the different generations of r ACs,the chondrogenic ability of r ACs was evaluated by toluidine blue staining.Further,the different generations of r ACs were cultured on well plates and PU/SIS materials,respectively.The effect of PU/SIS materials on the maintenance of chondrocyte phenotype was determined by detecting the changes in m RNA relative expression levels of cartilage phenotypes-related genes(COL2A1,COL1A1,ACAN,ELASTIN and SOX9)with quantitative RT-q PCR.In the animal experimental part,we established a rabbit ear cartilage whole-layer defect repair model in New Zealand White rabbits and treated the defected cartilage as groups: A blank control group,a PU/SIS material repair group,and a PU/SIS material composite cell group,i.e.PU/SIS + r ACs and PU/SIS + USCs groups.The cartilage tissues were harvested at 8-week and 12-week postoperatively,and photographed by digital camera to observe the overall repair of the cartilage defect area.Subsequently,the samples were embedded in paraffin and sectioned into slices followed by hematoxylin and eosin(H&E)staining and Masson staining to evaluate the structure of cartilage tissue and the degradation of PU/SIS material.Toluidine blue staining was used to observe the new cartilage formation in the repaired area,and elastic fibre staining was used to observe the synthesis of the elastic fibre component of the cartilage.In the mechanism exploring,the number of samples was increased and samples were retrieved at three time point(6-week,9-week and 12-week).The repair effect of PU/SIS materials was clarified by gross observation and histological staining methods(H&E,Masson,toluidine blue and elastic fibre staining).In addition,the tissue specimens harvested from normal cartilage tissue of rabbit ear,blank control group and PU/SIS group after 12-week repair were subjected to a transcriptome sequencing study.Combined with bioinformatics,we analyze differentially expressed genes and annotate them based on GO database and KEGG database to understand the biological functions of them,then screen out possible signalling pathways.Results:Firstly,primary r ACs isolated and cultured by enzymatic digestion had a flat morphology,with polygonal or triangular shapes predominating.CCK-8 results presented that the RGR of r ACs and USCs was more than 70% after 1,3,5 and 7 days culture and the material toxicity grade was grade 0,1 or 2,indicating that PU/SIS material has no indirect cytotoxicity.According to the Live/Dead staining,r ACs and USCs were able to grow adherently on the PU/SIS material and showed good proliferation ability,indicating that the PU/SIS material was not cytotoxic.The results of cytoskeleton staining and scanning electron microscopy showed that,with the increase of culture time,r ACs grew in aggregation,spread well with a flattened morphology,covering the surface of the material,especially into the pores of the material,and their morphology was mainly polygonal,whereas USCs mainly adhered to the “crest” of the pores of the material,spread around the edges of the pores,and their cell morphology was mainly long spindle-shaped.The chondrocyte morphology of P1 to P3 remained the same,mainly triangular or polygonal in shape,while the chondrocytes of P4 to P5 started to change to a long shuttle shape and gradually increased,suggesting a dedifferentiation of r ACs.The toluidine blue staining of the chondrocytes from P1 to P3 showed a large amount of blue-stained extracellular matrix,while only a small amount of blue-stained extracellular matrix could be seen in the chondrocytes from P4,indicating that the chondrocytes had basically lost their ability to secrete glycosaminoglycans.The results of PCR showed that the gene expression profile of r ACs changed significantly with the increase in the number of passages,with COL1A1 gene expression being up-regulated,COL2A1 and ACAN gene expression decreasing,and SOX9 and ELASTIN genes has no significant fluctuation.Compared with the plate culture,r ACs cultured in combination with PU/SIS scaffold material promoted the up-regulation of COL2A1,SOX9 and ELASTIN gene expression,and also had some inhibitory effect on COL1A1 gene expression.The ratio of COL2A1 to COL1A1 expression level also showed a tendency to increase.In an experimental study on the repair of cartilage defects in rabbit ears with PU/SIS scaffold material compounded with r ACs and USCs,the histological staining results showed that no new chondrocytes appeared in the repair area in the blank control and PU/SIS + r ACs groups at all time,and the toluidine blue and elastic fibre staining were negative.While in PU/SIS group,the number of new chondrocytes increased over time and they gradually transformed to mature cell.Moreover,extracellular matrix such as glycosaminoglycans and elastic fibre were observed indicating the ability to form elastic cartilage.The PU/SIS + USCs group showed new chondrocytes at the junction at 8-week and 12-week postoperatively,and toluidine blue and elastic fibre staining was positive.The repairing effect of PU/SIS scaffold material was further confirmed in an experimental study to investigate the mechanism of repairing cartilage defects in rabbit ears by increasing the sample amount and observing time point.In the analysis of a general transcriptome sequencing results,the statistical results of the number of differentially expressed genes showed that there was no significant difference in the number of differentially expressed genes between the blank control group vs normal group and the PU/SIS group vs normal group.The clustering analysis showed that:(1)the expression of genes related to cartilage-specific ECM components,genes related to growth factors and transcription factors regulating cartilage growth and development,genes related to changes in cartilage phenotypes and genes related to elastic fibres were down-regulated in the blank control group vs normal group.The expression of genes related to cartilage matrix remodelling and genes related to inflammatory response were up-regulated.This may be the reason for the absence of cartilage regeneration in the blank control group.(2)In the PU/SIS group vs normal group,the expression of genes related to immune regulation was upregulated;genes related to cartilage-specific ECM components and cartilage phenotypic changes were down-regulated;genes related to cartilage matrix remodelling were partially upregulated and partially down-regulated;genes related to growth factors and transcription factors regulating cartilage growth and development were also partially up-regulated and partially down-regulated.(3)Gene expression associated with immune regulation was up-regulated in the PU/SIS group vs the blank control group.The GO enrichment analysis showed that:(1)Blank control vs normal and PU/SIS vs normal groups were significantly enriched for the positive regulation of proteoglycan biosynthesis processes.(2)The PU/SIS group vs the blank control group was mainly enriched for immune-related biological functions,but also for integrin complexes,collagen metabolic processes,protein complexes involved in cell adhesion and integrin-mediated signalling pathways.The KEGG enrichment analysis showed that(1)the blank control group vs normal group and PU/SIS group vs normal group were enriched to the highest number of genes in metabolic pathways,but were not significant in terms of q-value.(2)The PU/SIS group vs blank control group had the highest number of genes enriched to cytokine-cytokine receptor interactions with significance.Conclusion:USCs and r ACs can adhere,grow and proliferate well on the surface of PU/SIS scaffold material with good cytocompatibility.The composite culture system constructed by PU/SIS scaffold material and chondrocytes can promote the maintenance of phenotype stability of rabbit ear chondrocytes.In rabbit ear cartilage defect repair experiments,the PU/SIS material promoted cartilage repair and is expected to be a new choice of scaffold material for elastic cartilage tissue engineering.Meanwhile,the composite of USCs and PU/SIS materials to construct tissueengineered elastic cartilage is feasible,and USCs can be used as a seed cell source for auricular cartilage tissue engineering.The transcriptome sequencing results showed that genes related to growth factors and transcription factors regulating cartilage growth and development and genes related to cartilage matrix remodelling were partially up-regulated and partially down-regulated in the PU/SIS group,which may play a positive and negative regulatory role.Meanwhile,these differentially expressed genes were enriched to signal pathways such as integrin complexes,integrin-mediated signalling pathways and cytokine-cytokine receptor interactions.It is tentatively suggested that the mechanism may be related to the mechanical properties of PU/SIS materials.更多还原