Chinese Journal of Tissue Engineering Research ›› 2022, Vol. 26 ›› Issue (4): 591-596.doi: 10.12307/2022.097
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Chen Xiaoxu, Luo Yaxin, Bi Haoran, Yang Kun
Received:
2020-10-09
Revised:
2020-10-12
Accepted:
2020-11-11
Online:
2022-02-08
Published:
2021-12-06
Contact:
Yang Kun, Associate professor, Department of Periodontology, Hospital of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
About author:
Chen Xiaoxu, Master candidate, Department of Periodontology, Hospital of Stomatology, Zunyi Medical University, Zunyi 563000, Guizhou Province, China
Supported by:
CLC Number:
Chen Xiaoxu, Luo Yaxin, Bi Haoran, Yang Kun. Preparation and application of acellular scaffold in tissue engineering and regenerative medicine[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(4): 591-596.
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2.1 脱细胞支架制备方法 脱细胞支架也被称为合成细胞外基质,通过物理、化学、生物方法对组织器官进行脱细胞后最终获得的结构和功能蛋白复合体,是一种临时的物理支撑,有助于在组织发育阶段容纳细胞并支持其三维生长[4]。理想的脱细胞方案应该成功清除组织器官的所有细胞成分,同时保留细胞外基质的结构和机械性能,细胞外基质的保护成分,如胶原、弹性蛋白、糖胺聚糖和纤维连接蛋白应最大限度地保留。该部分总结了几种常用的脱细胞方法。 2.1.1 物理方法 去细胞的物理途径是指调节任何组织器官的物理特性,如温度、力和压力,以促进细胞膜的破坏,促进细胞溶解。物理方法包括冻融、溶液搅拌、压力梯度和使用超临界流体等。 冻融:冻融过程是通过低温(-80 ℃)形成细胞内冰晶破坏细胞膜,释放细胞内容物,然后再升至室温(37 ℃) 融解,多次反复冻融使细胞结构破碎,从而达到脱细胞的目的。在脱细胞过程中可以使用多个冻融循环[5],也不会显著增加组织中细胞外基质蛋白的损失。冻融循环在组织超微结构中产生较小的破坏,为了使对组织结构的不利影响最小化而又不阻止细胞裂解,有人建议使用冷冻保护剂(如5%海藻糖)[6],但是冻融无法完全去除细胞,基质的免疫原性无法去除,需联合其他方法来进一步去除组织中细胞的残余成分。 高静水压力:高静水压力施加大于600 MPa 的压力来破坏细胞膜,可以消除或减少组织脱细胞中刺激性去污剂的暴露时间。在控制温度下,FUNAMOTO等[7]通过浸入盐水中并随后暴露于高达980 MPa不断增加的压力中使猪血管脱细胞。SASAKI等[8]类似地在1 000 MPa上使用高静水压力对猪角膜进行了脱细胞处理。两项研究均表明,与在正常大气条件下基于洗涤剂的方法相比,使用高压可以更有效去除细胞成分。为了保持天然细胞外基质结构,在使用高静水压力时有必要避免冻结阶段。高静水压力脱细胞过程不添加化学剂,避免了溶剂毒性作用对组织的损伤,但是加压作用会破坏胶原蛋白和弹性蛋白纤维,并改变其机械性能[7]。 超临界流体:超临界流体使用超临界二氧化碳以类似于临界点干燥的受控速度通过组织时,可以去除细胞残留物。 GIL-RAMIREZ等[9]先用加压二氧化碳-柠檬烯处理组织,然后再用酶处理,可以有效去除DNA。超临界流体脱细胞的优点包括使用惰性物质(如二氧化碳)来去除细胞,对细胞外基质机械性能的影响最小,以及消除冷冻干燥作为细胞外基质加工和储存的准备步骤;缺点是超临界二氧化碳对高分子和极性物质的溶解力较差,虽通过加入夹带剂来提高溶解能力,但又会引进新的杂质[10]。 2.1.2 化学方法 化学方法包括使用酸和碱、洗涤剂、醇和其他溶剂,最常用的化学方法是洗涤剂脱细胞。洗涤剂可以分为离子型、非离子型或两性离子型。 离子洗涤剂:离子型洗涤剂是强力去污剂,可完全破坏细胞膜并使蛋白质完全变性。十二烷基硫酸钠是最常用的离子型脱细胞剂,因为它可有效溶解细胞质膜,脂质和DNA。基于离子型洗涤剂的方案,由于其具有使蛋白质完全变性及溶解细胞和核膜的功能,比非离子洗涤剂更多地破坏细胞外基质[11-12],这通常会导致胶原完整性被破坏和糖胺聚糖的含量丧失。十二烷基硫酸钠去污能力强,但有细胞毒性,必须彻底清洗脱细胞组织以确保接种细胞的活性,但十二烷基硫酸钠很难完全从剩余基质中去除,并且会对细胞相容性产生不利影响。 非离子洗涤剂:非离子型洗涤剂是温和型洗涤剂,能有效去除细胞残留物,对糖胺聚糖、生长因子和胶原等其他成分的破坏最小,维持细胞外基质的超微结构,保留了更大的细胞外基质生物活性,并减少了不良免疫反应。Triton X-100是最常用的非离子型洗涤剂,通过破坏脂质与脂质和脂质与蛋白质的相互作用来去除细胞,同时保持蛋白质与蛋白质的相互作用不变,对组织结构损害较小,这样就可以在不干扰细胞外基质蛋白功能构象的情况下去除细胞,但Triton X-100对角膜、尿道、心脏和肾脏等许多组织和器官的细胞去除能力很差[13-14]。然而,它没有十二烷基硫酸钠那么强的去污力,最终对组织结构完整性的破坏也更小[15]。 两性离子洗涤剂:两性离子洗涤剂的亲水基团上的净零电荷在去细胞过程中保护蛋白质的天然状态。两性离子去污剂的包括CHAPS、SB-10和SB-16。与非离子洗涤剂相比,SB-10 和SB-16表现出更好的细胞外基质保存性和更好的细胞去除性;与离子型去污剂相比,CHAPS保留了更多的胶原蛋白、糖胺聚糖和弹性蛋白[16-17],同时去除了95%的核物质。研究表明,CHAPS主要用于神经、血管或肺等薄层组织的脱细胞,不会损害细胞外基质中的胶原蛋白和弹性蛋白,但是对较厚的组织无效[18-20]。 2.1.3 生物方法 生物脱细胞方案主要涉及酶反应,使用的酶通常是蛋白酶和核酸酶。胰蛋白酶选择性裂解精氨酸或赖氨酸的羧基侧的细胞黏附蛋白,以使细胞从组织表面分离,可以破坏胶原纤维周围的细胞外基质,产生微小的通道,并促进随后脱细胞剂的渗透。胰蛋白酶对细胞和细胞外基质成分的去除具有时间依赖性,24 h的暴露足以对细胞外基质造成不可修复的损害[21]。脱氧核糖核酸酶和核糖核酸酶分别是水解脱氧核糖核酸链和核糖核酸链的内切酶,如果仅用洗涤剂无法实现有效脱细胞,可在洗涤剂处理中添加这些酶制剂,以帮助去除残留的DNA[22-23]。酶反应具有高度特异性,但是单独使用酶制剂很难彻底去除细胞,并且残留在支架上的酶可能会引起不良反应,影响组织再细胞化。除蛋白酶和核酸酶外,还可以在脱细胞溶液中添加选择性蛋白酶抑制剂,以保护细胞外基质免受外源性蛋白酶和裂解细胞泄漏的蛋白酶的伤害,常用的蛋白酶抑制剂包括苯甲基磺酰氟和抑肽酶。 2.1.4 灌注脱细胞法 灌注脱细胞法利用内源性血管通道将脱细胞溶液输送到具有高度结构的组织中,重要的是,这允许从整个器官和复合组织生成细胞外基质支架[24-25]。IONELA等[26]将6例猪肺动脉瓣接受浸泡脱细胞,5例以20-25 mmHg(1 mmHg= 0.133 kPa)的压力梯度循环灌注,组织学评估两组中细胞外基质的完整性,浸泡脱细胞瓣膜显示DAPI阳性结构,被鉴定为潜在的残留核物质;DNA提取和定量结果证实,灌注脱细胞瓣膜缺乏上述结构。 2.1.5 组合方法 脱细胞支架制备方法各有优缺点,一般采用两种或两种以上的方法联合脱细胞,在最大化脱细胞的前提下更多地保存细胞外基质的生理结构和生物活性,减少不良反应。例如,LIN等[27]将处理后的皮肤组织用PBS洗涤,用0.3%Triton X-100、0.25%脱氧胆酸钠和0.02%胰蛋白酶-EDTA浸泡,在 37 ℃连续摇动48 h 以去除基质中的细胞成分。 2.1.6 脱细胞支架的灭菌 由细胞外基质组成的脱细胞支架在植入或体外使用之前必须消毒灭菌,以消除可能存在的病毒和细菌DNA。环氧乙烷、γ射线辐照和电子束辐照是比较常用的灭菌方法,但是可能会改变细胞外基质超微结构和机械性能[28]。其中,环氧乙烷处理可能会导致不良的宿主免疫反应,从而损害植入后生物支架的正常功能[29]。γ射线照射会导致残留的脂质造成细胞毒性,并加速细胞外基质的酶促降解[30]。与其他灭菌方法相比,超临界二氧化碳灭菌后的脱细胞猪主动脉瓣具有优良的无菌性和完整性[31],但这种方法相对较新,需要进一步研究。无论使用哪种方法都应该注意浓度和剂量的选择,最后得到的脱细胞支架应无毒,且保留细胞外基质的超微结构和生物学性能。 2.2 脱细胞支架的作用 2.2.1 脱细胞支架保留了源组织、器官的结构 脱细胞支架以非常高的分辨率保存了源器官的结构。MA等[32]以巴沙鱼皮为原料制备脱细胞真皮基质,巴沙鱼皮肤用无菌PBS完全清洗,在10 g/L NaCl中孵育24 h,用10 g/L NaOH清洗24 h,连续摇晃去除脂肪;脱脂皮肤用10 g/L KMnO4清洗0.5 h,用20 g/L NaHSO3处理1 h,直至变白;然后,皮肤在40 g/L NaOH中间歇超声孵育18 h。皮肤经过3次冻融循环,然后冷冻干燥和灭菌以获得脱细胞真皮基质。通过检测,脱细胞真皮基质中的大部分细胞和DNA被去除,未检测到内毒素,脱细胞真皮基质的结构也没有被破坏。YUN等[33]通过脱细胞、酶消化、粉碎等步骤制备消化软骨脱细胞基质,将软骨脱细胞基质的理化性质与天然软骨组织进行比较,发现软骨脱细胞基质和天然软骨组织一样含有大量的胶原、糖胺聚糖和抗血管生成分子。 2.2.2 脱细胞支架保留了源细胞外基质生物学功能 脱细胞支架保留了源细胞外基质许多理想的生物学功能,例如:适当的脱细胞支架保留了许多细胞信号分子的特定配体,这些信号分子通过传递存活信号来支持细胞植入支架的能力,这是成功的组织衍生支架再细胞化的基础。FINDEISEN等[34]证明瓣膜用十二烷基硫酸钠/脱氧胆酸钠、Triton X-100+十二烷基硫酸钠或胰蛋白酶+Triton X-100脱细胞,然后用肽-N-糖苷酶F、内切糖苷酶或O-糖苷酶和神经氨酸酶联合酶解,脱细胞的瓣膜会降低免疫原性并保持机械稳定性。 2.2.3 脱细胞支架促进组织、器官的修复与再生 脱细胞生物支架可以激活内源性组织修复过程[35]。生物支架是生长因子、基质细胞蛋白和生物活性囊泡的储存库[36-37]。JI等[38]采用脱细胞技术制备神经脱细胞支架,神经脱细胞支架中的活性成分层粘连蛋白被阻断或上调,将预处理的神经脱细胞支架用于神经损伤小鼠模型的神经修复,上调组新生轴突数量和管径增加、有髓纤维增多、髓鞘增厚、髓鞘碱性蛋白表达增加,神经功能恢复明显改善。LIU等[39]用猪中厚皮肤通过去除细胞成分而衍生的猪脱细胞真皮基质与明胶混合制成支架,对骨骼肌细胞和人脐静脉内皮细胞的体外实验表明,培养后的细胞可以附着在支架上并保持较高的活性,证明了该材料的无毒性;体内实验结果表明,修复后的伤口光滑,术后2周内所有动物均顺利康复,无伤口裂开、感染或疝复发的临床迹象,移植物周围有新形成的结缔组织,重建面上形成血管化的网状结构,表明支架具有良好的促修复性。 2.3 脱细胞支架在组织工程的应用 脱细胞支架可以与自体细胞一起再细胞,并在生理上合适的生物反应器中培养,从而在体外创建一个功能器官。支架形式的脱细胞组织、器官在组织工程和再生医学中有着广泛的应用,许多组织、器官(包括心脏[40-41]、肝脏[42]、肺[43]、肾脏[44]、气管和皮肤[45-46])已经在实验室环境中进行了工程改造,移植到动物的受体体内并显示出再生功能。下面简要讨论组织工程应用的一些关键领域。 2.3.1 心脏组织工程 心血管疾病是世界范围内主要的死亡原因之一[47],而心肌梗死是心血管疾病死亡的主要原因。鉴于缺乏合适的供体器官,以及与排斥风险和终身免疫抑制治疗相关的困难,迫切需要改进当前的临床策略[48]。为了增强干细胞的再生能力,它们与合适的支架相结合可能代表着一种改进的治疗方法。脱细胞的心脏组织被用作心脏再生的潜在支架[49]。AKBAY等[50]研究了大鼠腹膜后植入脱细胞支架的心肌细胞存活和迁移能力,应用离子型和非离子型去污剂获得脱细胞心肌组织支架,并将标记的分化细胞接种到这些脱细胞支架上,用多种技术检测无细胞支架和细胞支架复合材料的异位迁移能力和支架上种植细胞之间的相互作用,观察到心肌细胞特异性基因在第1,2和4周的表达,显示每段时间的潜在性增加;反转录聚合酶链反应结果显示,种植在支架上细胞之间的相互作用增加,但无细胞支架实验组在基因表达方面没有显著差异。GAO等[51]通过去除患者主动脉组织中的细胞制备了有利于细胞传递和组织修复的脱细胞主动脉支架,脱细胞主动脉支架在体内可以促进细胞黏附和存活,减轻炎症反应,抑制骨髓来源的前体细胞的凋亡,促进新生血管形成。这项临床前研究表明,使用具有抗凋亡和抗炎作用的去细胞化主动脉支架可能是缺血性损伤后心血管修复的一种很有前途的策略。因此,脱细胞支架在心脏再生应用中有重要意义。 2.3.2 骨组织工程 发育畸形、肿瘤、感染、创伤等原因造成的骨缺损修复一直是医生们的追求,利用自体骨是“黄金标准”,但这种方法也有限制,如可用性、供骨部位发病率、感染、伤口引流时间延长、术后疼痛和神经血管损伤[52]。为了克服这些限制,同种异体移植被广泛使用,但免疫反应和病原体转移的风险促使人们研究脱细胞骨等组织替代品[1]。SLADKOVA等[53]提出了一种先在0.1%EDTA缓冲液中孵育,然后用洗涤剂和酶溶液去细胞的方案,支架用成骨介质处理,接种人诱导多能干细胞来源的间充质祖细胞,然后转移到灌流生物反应器,5周后支架在支持细胞活性和成骨分化及骨特异性基质沉积方面表现出充分性。LIN等[54]应用脱细胞骨膜支架,发现脱细胞支架本身可促进临界骨缺损再生和裸鼠异位成骨。脱细胞骨膜的天然胶原基质可以作为钙磷核启动的三维结构模板,控制骨样矿物晶体的大小和取向。天然交联、高度有序的脱细胞骨膜三维纤维网络不仅为体外和体内模拟矿化提供了模型,阐明了细胞外基质在骨形成和再生中的重要作用,而且有望成为骨组织工程和临床应用的生物材料。 2.3.3 气管组织工程 脱细胞支架因其简单的结构和功能特性被广泛应用于气管修复。GRAY等[55]用自体羊膜间充质干细胞重组的异种脱细胞气管段构建了组织工程支架,并与脱细胞支架进行了比较,动物研究结果证实,尽管胶原蛋白和糖胺聚糖正常,但植入后弹性蛋白水平增加的工程化支架具有完全上皮化的最大存活率。此外,许多来源于种植干细胞的脱细胞组织、器官气管植入物已经进入临床水平,从而证实了干细胞工程气道在气道修复和再生方面的潜力。 2.3.4 肺组织工程 在肺组织工程的进一步发展中,OHATA等[56]总结之前的实验发现,来自小动物和大动物的无细胞肺支架保存了原始的血管网、支气管树和大部分细胞外基质成分,是通过用洗涤剂溶液灌注肺动脉而产生的。将得到的肺支架在生物反应器中重新细胞化,并通过肺灌注培养获得成功的细胞生长,由此产生的保留了血管和气道结构的生物人工肺将用于移植研究。在大鼠和猪移植模型中,移植的生物人工肺促进了植入后的气体交换,移植失败是由于血管屏障功能不足和血栓形成增加所致。因此,有必要在优化移植物的再细胞化和成熟化方面取得进一步的进展,以提高移植物的可持续性和功能性。 2.3.5 肝组织工程 研究人员探索了不适合移植的人体肝脏的替代用途,主要焦点是获得可以重新植入正常肝细胞的无细胞肝细胞外基质,以便在体外重建具有功能的肝替代物[57]。有些研究人员已经在整个器官[57-58]、唯一的左叶或肝脏碎片的情况下从事了肝脏的去细胞研究[57,59]。研究表明,脱细胞支架可以为不同类型的细胞提供最佳的生长平台,无论是整个器官还是小组织活检,都要对肝细胞外基质碎片进行再细胞检验。种子细胞群包括人肝星状细胞系[57,60] 、肝细胞癌HepG2细胞[57]、在静态条件下培养的人腺癌细胞Sk-Hep-1内皮细胞[57],以及通过动态灌注系统在支架上培养的原代人肝细胞[60],所有4种类型的细胞都能通过肝基质移植和迁移,并具有高增殖率[57,60]。此外,人肝星状细胞系细胞在窦腔内定植获得了明确的肌成纤维细胞样细胞形态[60]。人脐静脉内皮细胞也被考虑用于肝支架的再内皮化,因为它们位于去细胞血管附近,用于血管的再生长[60]。 2.3.6 皮肤组织工程 修复烧伤、创伤或肿瘤手术引起的大面积皮肤缺损是一个临床挑战,自体皮肤移植被认为是黄金标准,对于皮肤缺损较小的健康患者,常采用自体皮肤移植和自体皮肤修复的方法;然而,对于皮肤缺损较大的患者,伤口不能通过自体修复。缺乏自体皮肤会导致伤口修复困难,影响治疗,甚至导致死亡,因此临床上迫切需要一种有效的皮肤替代品[61]。人脱细胞羊膜是一种很有前途的皮肤替代物,LIU等[62]研究了大鼠毛囊干细胞复合人脱细胞羊膜修复裸鼠全层皮肤缺损的能力,结果显示大鼠毛囊干细胞-人脱细胞羊膜复合物可促进裸鼠创面愈合;此外,大鼠毛囊干细胞-人脱细胞羊膜复合物中的细胞直接参与毛囊的形成和毛囊周围组织的血管生成。这些结果为皮肤组织工程提供了一条新的途径。 2.3.7 脱细胞技术与口腔医学 在口腔医学领域,脱细胞组织工程学被认为是一种有前途的方法。SANGKERT等[63-64]通过胶原酶和分散酶使牙髓脱细胞,该过程成功之后获得了一种溶液,该溶液使用0.1 g/L 的0.1%NaClO并与胶原蛋白或纤连蛋白相结合以覆盖丝载体,增加了成骨样细胞的碱性磷酸酶活性、钙合成、矿化作用,提示了支架生物功能的增加;同时,较高的应力值和杨氏模量证明了支架机械性能得到了改善。FARAG等[65]将牙周膜干细胞片负载到聚己内酯支架上,然后脱细胞,评估脱细胞构建物对同种异体牙周膜干细胞和骨髓间充质干细胞体外分化的影响,免疫组织化学染色检测间充质干细胞标志物Stro-1的表达,在大鼠牙周缺损模型中评估脱细胞构建物的生物相容性和组织整合,结果显示,脱细胞的牙周膜细胞薄片构建体促进了牙周膜和间充质干细胞的分化;特别是与单独的聚己内酯支架相比,接种在脱细胞基质上的牙周膜干细胞矿化组织标志物的表达表达上调,并且脱细胞的细胞片构建体在大鼠牙周缺损模型中支持牙周附着。SON等[66]旨在构建人牙片脱细胞牙周膜支架并检测其再生能力,结果显示,经SDS和Triton X-100脱细胞处理后的细胞外基质支架保持了完整的结构和组成,具有较好的去核效果;此外,脱细胞支架可以支持牙骨质附近的牙周膜干细胞再繁殖,并表达牙骨质和牙周膜相关基因。结果表明,人牙脱细胞牙周膜支架可诱导间充质干细胞增殖和分化,具有再生潜能,可用于未来牙周再生组织工程。"
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