脱细胞血管基质材料同种异体移植10个月研究*☆

背景：异体血管移植之所以至今未能在临床应用,关键是异体组织抗原性排斥反应的难题未能得到解决。 目的：制备动脉脱细胞血管基质,探讨脱细胞血管异体移植的可行性。 方法：采用不同去垢剂(1%Triton X-100、1%SDS)多步骤对犬血管进行脱细胞处理,通过组织学和力学观测,建立犬动脉血管脱细胞的方法;并进行脱细胞血管的异体移植。 结果与结论：经胰蛋白酶、低渗溶液和去垢剂Triton X-100、SDS等多步骤处理,犬颈总动脉血管的细胞基本脱除,细胞外基质保持完好,血管的弹性、韧性保存较好;用该法制备的犬颈总动脉(直径约4.0 mm)进行异体移植,经10个月观察,4/5通畅。提示经去垢剂Triton X-100、SDS加低渗溶液、胰蛋白酶和蛋白酶抑制剂处理的多步法,可以脱除血管的细胞成分,细胞外基质和力学特性保持完好,是一种较好的方法;用该法制备的犬颈总动脉可以直接进行异体移植,是一可选择的血管移植材料。 关键词：动脉血管;犬;脱细胞;同种异体移植;血管基质 doi:10.3969/j.issn.1673-8225.2012.12.014     

猪胆总管脱细胞支架的制备及组织学评价

目的 用不同的脱细胞方法对猪胆总管进行处理,比较脱细胞前后的组织学变化,筛选适宜的脱细胞方法,为组织工程胆管支架材料的应用提供理论依据。 方法 30例猪胆总管随机分为5组：对照组（A组）：0.05% 胰蛋白酶+核酸酶（B组）：0.1%十二烷基硫酸钠（SDS）+核酸酶（C组）：1.0% Triton X-100+核酸酶（D组）：1.0% Triton X-100+0.1% SDS +核酸酶（E组）。通过HE染色光学显微镜下观察脱细胞基质的组织结构和细胞残留情况;用紫外分光光度法检测脱细胞基质的DNA含量,计算脱细胞率。结果 脱细胞B组有少量细胞残留,纤维有损伤;脱细胞C组、D组、E组的细胞均被去除,纤维无明显损伤。A组的DNA含量为（71.24 ± 2.56）μg /100 mg。B、C、Ｄ、Ｅ4个脱细胞组的DNA含量与A组的差异有统计学意义（F =15.29, P<0.01）,均有明显的脱细胞效果（P<0.01）。Ｂ组的脱细胞率为77.03%,比C组、Ｄ组、Ｅ组的脱细胞效果稍差（P<0.05）,Ｅ组的脱细胞率高达99.03%。 结论 应用1.0% Triton X-100+0.1% SDS +核酸酶的脱细胞效果好,能更好地降低胆总管的免疫原性,是一种比较理想的猪胆总管脱细胞方法。     

猪胆总管脱细胞基质构建组织工程胆管的生物力学评价

目的　用不同的脱细胞方法对猪胆总管进行处理,比较脱细胞前后的生物力学变化,筛选适宜的脱细胞方法,为组织工程胆管支架材料的应用提供理论依据。 方法　30例猪胆总管,随机分为5组,A组：对照组, B组：0.05 % 胰蛋白酶+核酸酶,C组：0.1 % SDS+核酸酶,D组：1.0 % Triton X-100+核酸酶,E组：1.0 % Triton X-100+0.1 % SDS +核酸酶。在Test Resources生物力学试验机上进行加载一卸载试验和极限抗张强度试验。计算出生物力学材料常数（α1、β1、α2、β2）、弹性模量、极限抗张强度和断裂伸长率等指标。 结果　Ｄ组、Ｅ组的生物力学材料常数（α1、β1、α2、β2）与A组的差异无统计学意义（F = 12.21, P = 0.06）,Ｂ组、C组比A组、D组和Ｅ组的小（P < 0.01）;Ｄ组、Ｅ组的弹性模量比A组的稍增大,但差异不明显（P > 0.05）,Ｂ组、C组比A组的小（P < 0.05）;Ｄ组、Ｅ组的UTS值和SOF值与A组差异不明显（P > 0.05）;B组、C组的UTS值明显小于A组（P < 0.05）,SOF值明显大于A组（P < 0.05）。 结论 应用1.0 % Triton X-100+核酸酶和1.0 % Triton X-100+0.1 % SDS +核酸酶的脱细胞效果好,且不会影响猪胆总管的生物力学特性,是一种比较理想的猪胆总管脱细胞方法。     

人源脱细胞羊膜与脱细胞羊膜下层组织修复皮肤缺损

文题释义：脱细胞生物支架：其原理是将生物体原型组织中的实质细胞运用化学、酶解或机械方法给予去除后制成的构架,保留了以细胞间质为主的其他成分,维持了生物材料原有的机械性和可塑性,更加适合细胞黏附和增殖生长。 细胞毒性：是化学物质(药物)作用于细胞基本结构和/或生理过程,如细胞膜或细胞骨架结构,细胞的新陈代谢过程,细胞组分或产物的合成、降解或释放,离子调控及细胞分裂等过程,导致细胞存活、增殖和/或功能的紊乱,所引发的不良反应。背景：羊膜与羊膜下层具有同源性且均属于医疗废物,均具有较低的免疫原性与一定的韧性,羊膜作为组织工程支架材料已有较多报道,但羊膜下层作为组织工程支架的研究较少。 目的：对比脱细胞羊膜与脱细胞羊膜下层分别复合骨髓间充质干细胞用于皮肤修复的差异。 方法：采用十二烷基硫酸钠+核酸酶法制作脱细胞羊膜下层材料,采用TritonX-100+胰酶法制作脱细胞羊膜材料。采用两种脱细胞材料浸提液分别培养骨髓间充质干细胞,利用CCK-8法检测两种脱细胞材料的细胞毒性。将骨髓间充质干细胞接种于2种脱细胞材料表面,光学显微镜与扫描电镜下观察细胞的生长与黏附。在每只SD大鼠一侧脊柱背部制作深达真皮层的皮肤缺损,将36只造模大鼠随机分为3组处理,每组12只：空白组不植入任何材料,对照组植入脱细胞羊膜与骨髓间充质干细胞复合物,观察组植入脱细胞羊膜下层与骨髓间充质干细胞复合物,术后7,14,21 d分别进行创面愈合率、创面病理及创面基因与蛋白检测。动物实验获得江南大学动物伦理委员会批准。结果与结论：①在培养的3-7 d内,两种脱细胞材料浸提液均可促进骨髓间充质干细胞的增殖;②光学显微镜与扫描电镜显示,骨髓间充质干细胞在两种脱细胞材料表面黏附、生长良好,细胞形态与培养瓶中培养的相同;③观察组与对照组术后各时间点的创面愈合率均高于空白组(P < 0.05),且观察组高于对照组(P < 0.05);④术后14 d的病理观察显示,观察组与对照组的皮肤修复优于空白组,且观察组优于对照组;⑤观察组、对照组术后各时间点的Ⅲ型胶原、CK18、Ⅰ型胶原与血管内皮生长因子基因表达量均高于空白组(P < 0.05),且观察组高于对照组(P < 0.05);⑥观察组、对照组各时间点的CK18、血管内皮生长因子蛋白表达均高于空白组(P < 0.05),观察组高于对照组(P < 0.05);⑦相较于脱细胞羊膜,脱细胞羊膜下层可促进皮肤的修复。ORCID: 0000-0001-5543-5392(王丹) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

去细胞化全肝生物支架循环灌注培养条件下体外细胞再植*

背景：通过去细胞化技术制备全肝生物支架成为缓解供体短缺的新技术,优化肝脏组织的去细胞化流程成为新的课题。 目的：通过去细胞化技术建立完整保留肝脏三维结构和脉管系统的全肝生物支架,利用自主设计的循环灌注培养装置实现生物支架体外细胞再植。 方法：通过门静脉路径循环灌注去垢剂Triton X-100,十二烷基硫酸钠,并用磷酸盐缓冲液洗脱残留去垢剂。通过动态循环灌注培养装置进行支架与HepG2细胞的共培养,观察植入细胞在全肝生物支架内的功能表达。 结果与结论：经去垢剂灌注后,支架呈现保持肝脏三维结构的透明结构,苏木精-伊红染色以及扫描电镜结果显示细胞成分被完全移除,Masson’s Trichrome染色可见大量胶原纤维,免疫组化结果证实纤维连接蛋白和层粘连蛋白保存完整。循环灌注培养下细胞白蛋白表达量及尿素合成量较平板培养明显提高。说明去细胞化肝脏生物支架可作为体外肝脏组织重建的基础材料,动态循环灌注方法可实现支架中细胞再植。      

3D打印淫羊藿苷/脱钙骨基质修复兔股骨髁骨缺损

文题释义：脱钙骨基质：主要由93%胶原、5%可溶性蛋白及2%残余矿化基质组成的商业化生物材料，包含不同浓度的骨形态发生蛋白、生长因子及转化生长因子，具备良好的骨传导与骨诱导能力，可以单独或联合其他材料广泛用于骨折、骨不连、骨肿瘤、骨融合、股骨头坏死等治疗中。结构模型指数：是描述骨小梁组成结构中板层结构和杆状结构比例的参数。如果结构中骨小梁主要为板层结构，那么结构模型指数接近于0；如果结构中骨小梁主要为杆状结构，那么结构模型指数接近于3，该值越小骨小梁越成熟。背景：淫羊藿苷可促进骨髓间充质干细胞的增殖与成骨分化，具有良好的促成骨性能。脱钙骨基质具备良好的骨传导与骨诱导能力，可以单独或联合其他材料广泛用于骨修复中。 目的：观察3D打印淫羊藿苷/脱钙骨基质材料的缓释性能、细胞相容性与体内成骨性能。方法：利用3D打印技术制备淫羊藿苷/脱钙骨基质材料与脱钙骨基质材料，检测淫羊藿苷/脱钙骨基质材料的体外缓释性能。将骨髓间充质干细胞分别接种于两种材料表面，以单独培养的细胞为对照，于设定的时间点进行活/死染色、MTT、碱性磷酸酶活性与骨钙素含量检测。取新西兰大白兔30只，建立股骨髁骨缺损模型后分3组处理：对照组不植入任何材料，一组植入脱钙骨基质与同种异体兔骨髓间充质干细胞复合物，另一组植入淫羊藿苷/脱钙骨基质材料与同种异体兔骨髓间充质干细胞复合物，术后4，12周进行Micro-CT、组织学与力学性能检测。结果与结论：①3D打印淫羊藿苷/脱钙骨基质材料具有缓释性能，在28 d时淫羊藿苷释放量达总量的(54.9±7.9)%；②活/死染色显示，接种1 d后两组材料表面的细胞数量较少，随着培养时间的延长，细胞数量明显增多，至7 d时细胞形态良好，并且淫羊藿苷/脱钙骨基质材料表面的细胞更加均匀、数量更多；③MTT检测显示，淫羊藿苷/脱钙骨基质组培养7，10，14 d的细胞增殖快于其余两组(P < 0.05)；④淫羊藿苷/脱钙骨基质组培养7，10，14 d的碱性磷酸酶活性与骨钙素含量均高于其余两组(P < 0.05)；⑤术后12周Micro-CT显示，植入材料两组均可见大量的骨小梁，其中淫羊藿苷/脱钙骨基质组骨小梁数量更多、骨小梁厚度增加、骨小梁分离度更小；⑥术后12周组织学显示，植入材料两组可见大量骨组织形成，其中淫羊藿苷/脱钙骨基质组新生骨量最多；⑦淫羊藿苷/脱钙骨基质组抗压强度高于其余两组(P < 0.05)；⑧结果表明，3D打印淫羊藿苷/脱钙骨基质具有良好的缓释性能、细胞相容性与骨诱导性能。ORCID: 0000-0002-5272-812X(张虎雄) 中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程     

比较两种方法制备脱细胞小血管支架

目的:比较0.5%Triton X-100 0.05%NH4OH和酶法制备脱细胞小血管支架的效果.方法:分别用含0.5%Triton X-100 0.05%NH4OH处理3 d或1.0H Triton X-100 0.125%胰蛋白酶 Dnase Rnase孵育48 h,脱去犬股静脉中的细胞成分,50Co辐照消毒,血清浸泡24 h,将平滑肌细胞和内皮细胞接种到脱细胞支架中;进行H-E染色、胶原纤维和弹力纤维染色,扫描电镜观察及力学检测.结果:0.5%Triton X-100 0.05%NH4OH法完全地脱去了血管细胞;细胞外基质较完整地保留下来,其形态结构与脱细胞前无明显改变;见种植细胞在支架内生长良好,连成片,支架具有良好的生物相容性;力学结果显示其弹性回复率和最大断裂强度好于酶组.结论:两种方法比较,0.5%Triton X-100 0.05%NH4OH法简便易行,成本低,脱细胞效果好,组织相容性佳,对力学性状影响小,是比较理想的制备脱细胞小血管支架的方法.     

脱细胞、病毒灭活与灭菌组合工艺对猪真皮基质理化性能的影响

背景：脱细胞、去抗原、病毒灭活与终端灭菌工艺的组合优化是制备符合临床要求组织再生材料的关键技术。目的：系统比较分析两种脱细胞、去抗原、病毒灭活与灭菌组合工艺对猪真皮基质理化性能的影响。方法：取新鲜猪皮,分两组工艺制备猪真皮基质：(1)方法A：采用1%Triton X-100、DNase和RNase、1%磷酸三丁脂进行脱细胞处理,1%过氧乙酸+25%乙醇病毒灭活,伽马射线辐照终端灭菌;(2)方法B：采用中性蛋白酶、0.5%Triton X-100和DNase进行脱细胞处理,α-半乳糖苷酶去除抗原,0.1%过氧乙酸病毒灭活,伽马射线辐照终端灭菌。对两种方法制备的猪真皮基质进行表征。结果与结论：(1)A组胶原蛋白含量低于B组,弹性蛋白、糖含量高于B组,材料硬度值高于B组;A组基质材料的悬垂性较差,B组基质材料的柔韧性较好;(2)A组脱细胞处理不影响基质材料的热稳定性,B组脱细胞略降低基质材料的热稳定性;伽马射线灭菌后,A组基质材料的热稳定性大幅度下降,B组基质材料的热稳定性下降很小;(3)与B组相比,A组基质材料的拉伸强度和弹性较小;体外酶降解实验显示,A组基质材料对胶原酶降解具有很强的抗性...     

脱细胞异种生物外科补片的免疫原性

文题释义：免疫原性：指能够刺激免疫系统的细胞引起某种抗原特异性免疫应答。当动物源性材料植入人体后,其细胞表面的α-Gal抗原与人体内存在的天然抗α-Gal抗体结合,会激活补体系统引起严重的超急性排斥反应;还可通过抗体依赖细胞介导的细胞毒性作用,引起异种移植排斥反应。对于异种材料植入人体所产生的潜在风险,有必要对其进行免疫原性风险评估。脱细胞技术：指通过物理、化学、生物学等一系列的方法处理同种异体的组织、器官,使其细胞内基质除去完全而保留相应的细胞外基质的方法。脱细胞技术可降低甚至除去异体组织、器官的免疫源性(如α-Gal抗原)等,降低异体生物材料移植引起的排斥反应,为异体生物材料的临床运用提供基础。背景：脱细胞异种生物外科补片的免疫原性直接关系到其植入人体后的成功与否,因而评价材料的免疫原性至关重要。 目的：评价脱细胞异种生物外科补片的免疫原性。 方法：将20只Balb/c小鼠随机分4组,每组5只：实验组与对照组背部皮下分别植入脱细胞异种生物外科补片与心包膜原材料;阴性对照组行假手术操作;阳性对照组背部皮下注射弗氏佐剂和牛血清白蛋白等体积混合液。植入4周后,记录小鼠体质量,计算各组脾脏和胸腺的脏脑系数,检测血清总IgG和IgM水平、体外淋巴细胞增殖活性及脾脏淋巴细胞亚型分布,并进行植入部位皮肤组织及脾脏、胸腺组织病理学观察。实验方案经四川省食品药品检验检测院安全评价中心实验动物管理和使用委员会批准(IACUC-2018-KYYL-008)。结果与结论：①实验组与阴性对照组体质量比较差异无显著性意义(P > 0.05),对照组大于阴性对照组(P < 0.05);②实验组与阴性对照组脾脏脏脑系数、胸腺脏脑系数比较差异均无显著性意义(P > 0.05),对照组脾脏脏脑系数大于阴性对照组(P < 0.05);③实验组与阴性对照组淋巴细胞增殖活性比较差异无显著性意义(P > 0.05),对照组高于阴性对照组(P < 0.05);④与阴性对照组相比,实验组CD3⁺CD8⁺细胞百分比降低(P < 0.05);与阴性对照组相比,对照组CD3⁺细胞、CD3⁺CD4⁺细胞、CD3⁺CD8⁺细胞、CD45⁺SSClow细胞百分比下降(P < 0.05),CD3^-CD19⁺细胞百分比升高(P < 0.05);⑤实验组、对照组血清IgM和IgG抗体水平与阴性对照组相比差异均无显著性意义(P < 0.05);⑥组织学显示,实验组与对照组脾脏和胸腺无明显病理改变,实验组植入部位无明显炎性反应,对照组植入部位出现严重肉芽肿性炎及纤维组织增生包裹、植入物坏死崩解等;⑦结果表明与原材料相比,经脱细胞处理的异种生物外科补片可有效降低免疫原性反应。ORCID: 0000-0003-3480-6101(程祥) 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

肿瘤坏死因子调节的肥大细胞蛋白酶激活受体表达分析

目的 检测肿瘤坏死因子(TNF)引起的肥大细胞蛋白酶激活受体(PAR)-1，2，3，4表达。方法 肥大细胞P815培养后以不同浓度的TNF激发肥大细胞，在不同时间点收集细胞，给予聚乙二醇辛基苯基醚( Triton X-100)非Triton X-100处理后用流式细胞术和激光共聚焦方法检测肥大细胞蛋白酶激活受体的表达。结果 TNF激发肥大细胞2h、6h和16 h后，Triton X-100与非Triton X-100处理后，肥大细胞PAR-1，3表达均无明显变化；但TNF以浓度依赖方式上调P515肥大细胞PAR-2，4表达(P＜0.05)；上述各时间点检测PAR-1，2，3表达情况，Triton X-100处理组与非Triton X-100处理组检测结果无明显差异，但PAR-4表达结果显示Triton X-100处理组表达强于非Triton X-100处理组。结论 TNF可以上调P815肥大细胞PAR-2，4表达，但对PAR-1，3表达的调节作用不明显，Triton X-100处理和非Triton X-100处理不影响TNF调节的肥大细胞PARs表达的检测结果。     

Comparison of aortic valve allograft decellularization techniques in the rat

Rodent models have been essential to understanding the immune-mediated failure of aortic valve allografts (AVAs). Decellularization has been proposed to reduce the immunogenicity of AVAs. The objective of this study was to determine the most effective method to decellularize AVAs for use in a rat model. Three different decellularization techniques were compared in Lewis aortic valves. Detergent decellularization involved a series of hypotonic and hypertonic Tris buffers at 4 degrees C for 48 h/buffer containing 0.5% Triton X-100 followed by a 72 h washout in phosphate-buffered saline. Osmotic decellularization was performed in similar manner to the detergent-based technique except without the addition of Triton X-100. Enzymatic decellularization consisted of trypsin/EDTA at 37 degrees C for 48 h. Assessment was performed with light microscopy (H&E, Movat's pentachrome), immunohistochemistry for residual cellular elements, and hydroxyproline assays. Detergent-based methodology effected near-complete decellularization of both the leaflets and aortic wall in addition to preservation of the extracellular matrix (ECM). Osmotic lysis was associated with preservation of ECM and moderate decellularization. Enzymatic decellularization resulted in complete decellularization but extensive degeneration and fragmentation of the ECM. When implanted into the infrarenal aorta of allogeneic rats for 1 week, valves decellularized with detergent-based and osmotic methodology failed to stimulate an allogeneic immune response as evidenced by an absence of T cell infiltrates. Osmotic lysis protocols with low dose detergent appear to be most effective at both removing antigenic cellular elements and preserving ECM.     

On the Decellularization of Fresh or Frozen Human Umbilical Arteries: Implications for Small-Diameter Tissue Engineered Vascular Grafts

Most tissues, including those to be decellularized for tissue engineering applications, are frozen for long term preservation. Such conventional cryopreservation has been shown to alter the structure and mechanical properties of tissues. Little is known, however, how freezing affects decellularization of tissues. The purpose of this study was two-fold: to examine the effects of freezing on decellularization of human umbilical arteries (HUAs), which represent a potential scaffolding material for small-diameter tissue-engineered vascular grafts, and to examine how decellularization affects the mechanical properties of frozen HUAs. Among many decellularization methods, hypotonic sodium dodecyl sulfate solution was selected as the decellularizing agent and tested on fresh HUAs to optimize decellularization conditions. The efficiency of decellularization was evaluated by DNA assay and histology every 12 up to 48 h. The optimized decellularization protocol was then performed on frozen HUAs. The stiffness, burst pressure, and suture retention strength of fresh HUAs and frozen HUAs before and after decellularization were also examined. It appeared that freezing decreased the efficiency of decellularization, which may be attributed to the condensed extracellular matrix caused by freezing. While the stiffness of fresh HUAs did not change significantly after decellularization, decellularization reduced the compliance of frozen HUAs. Interestingly, the stiffness of decellularized frozen HUAs was similar to that of decellularized fresh HUAs. Although little difference in stiffness was observed, we suggest avoiding freezing if more efficient and complete decellularization is desired.     

脱细胞技术在组织工程血管中的应用进展

将天然血管经过脱细胞处理得到的脱细胞血管,被认为是一种具有广阔应用前景的组织工程血管支架材料.截至目前,细胞外基质(ECM)支架的制备方法仍缺乏统一标准.脱细胞方法的选择取决于组织来源和基质支架的用途,尤其对于脱细胞血管等需要长期承受血流冲击的基质支架材料来说,脱细胞方案的选择至关重要.细胞清除效率和细胞外基质支架的性...     

An overview of tissue and whole organ decellularization processes

Biologic scaffold materials composed of extracellular matrix (ECM) are typically derived by processes that involve decellularization of tissues or organs. Preservation of the complex composition and three-dimensional ultrastructure of the ECM is highly desirable but it is recognized that all methods of decellularization result in disruption of the architecture and potential loss of surface structure and composition. Physical methods and chemical and biologic agents are used in combination to lyse cells, followed by rinsing to remove cell remnants. Effective decellularization methodology is dictated by factors such as tissue density and organization, geometric and biologic properties desired for the end product, and the targeted clinical application. Tissue decellularization with preservation of ECM integrity and bioactivity can be optimized by making educated decisions regarding the agents and techniques utilized during processing. An overview of decellularization methods, their effect upon resulting ECM structure and composition, and recently described perfusion techniques for whole organ decellularization techniques are presented herein.     

不同保护剂对大鼠肾脏冻融法脱细胞的影响

目的:利用不同保护剂对大鼠肾脏进行预处理,辅助冻融法脱细胞获得脱细胞支架,优化冻融脱细胞工艺。方法:采用不同保护剂预处理大鼠肾脏,放入-20℃环境下进行冷冻,12 h后进行37℃水浴复温,然后利用Triton X-100灌注24 h、PBS灌注2 h。最后通过CT三维重构、染色切片、蛋白质定量以及力学性能分析等手段评估所得脱细胞支架。结果:未添加保护剂组血管网络损伤较大,洗脱效果一般。添加不同保护剂组的血管网络也均存在一定的损伤,但其中10%DMSO和5%海藻糖组血管网络保留得较为完整。但10%DMSO组DNA等物质残留过多,5%海藻糖组洗脱细胞效果良好。结论:加载保护剂能够对冻融法脱细胞起到一定的促进作用,且能保护大鼠肾脏内部血管网络等结构,但不同保护剂对冻融法脱细胞的作用效果不同。     

Consequences of ineffective decellularization of biologic scaffolds on the host response

Biologic scaffold materials composed of extracellular matrix (ECM) are routinely used for a variety of clinical applications. Despite known variations in tissue remodeling outcomes, quantitative criteria by which decellularization can be assessed were only recently described and as a result, the amount of retained cellular material varies widely among commercial products. The objective of this study was to evaluate the consequences of ineffective decellularization on the host response. Three different methods of decellularization were used to decellularize porcine small intestinal ECM (SIS-ECM). The amount of cell remnants was quantified by the amount and fragmentation of DNA within the scaffold materials. The M1/M2 phenotypic polarization profile of macrophages, activated in response to these ECM scaffolds, was assessed in vitro and in vivo using a rodent model of body wall repair. The results show that, in vitro, more aggressive decellularization is associated with a shift in macrophage phenotype predominance from M1 to M2. While this shift was not quantitatively apparent in vivo, notable differences were found in the distribution of M1 vs. M2 macrophages within the various scaffolds. A clear association between macrophage phenotype and remodeling outcome exists and effective decellularization remains an important component in the processing of ECM-based scaffolds.     

Preservation of aortic root architecture and properties using a detergent-enzymatic perfusion protocol

Aortic valve degeneration and dysfunction is one of the leading causes for morbidity and mortality. The conventional heart-valve prostheses have significant limitations with either life-long anticoagulation therapeutic associated bleeding complications (mechanical valves) or limited durability (biological valves). Tissue engineered valve replacement recently showed encouraging results, but the unpredictable outcome of tissue degeneration is likely associated to the extensive tissue processing methods. We believe that optimized decellularization procedures may provide aortic valve/root grafts improved durability. We present an improved/innovative decellularization approach using a detergent-enzymatic perfusion method, which is both quicker and has less exposure of matrix degenerating detergents, compared to previous protocols. The obtained graft was characterized for its architecture, extracellular matrix proteins, mechanical and immunological properties. We further analyzed the engineered aortic root for biocompatibility by cell adhesion and viability in vitro and heterotopic implantation in vivo. The developed decellularization protocol was substantially reduced in processing time whilst maintaining tissue integrity. Furthermore, the decellularized aortic root remained bioactive without eliciting any adverse immunological reaction. Cell adhesion and viability demonstrated the scaffold's biocompatibility. Our optimized decellularization protocol may be useful to develop the next generation of clinical valve prosthesis with a focus on improved mechanical properties and durability.     

Decellularization of submillimeter-diameter vascular scaffolds using peracetic acid

Journal of Artificial Organs - Various decellularization methods for allogenic and xenogenic bioscaffolds have been previously reported; however, decellularization methods for very thin...     

脑去细胞生物支架的制备与鉴定

通过化学萃取加震荡法制备脑去细胞外基质,并对其进行形态学分析和成分鉴定。健康成年SD大鼠20只,分成正常对照组和去细胞组。去细胞组大鼠经心脏灌流后取脑,通过化学萃取加震荡法,依次用3%的TritionX-100、1%的SDS、4%的脱氧胆酸钠震荡萃取,无菌蒸馏水漂洗制备脑去细胞外基质(dBECM)。通过扫描电镜观察dBECM微观形态,用HE染色和荧光DAPI染色分析其去细胞化的效果,进一步通过Masson染色和免疫荧光染色进行成分的鉴定。通过HE染色及荧光DAPI染色可以发现,该方法去细胞化程度完全(去细胞程度大于99%),仅保留大量细胞外基质,未见明显的细胞及细胞核成分残留;Masson染色和免疫荧光染色发现,dBECM保留弹性蛋白(4.0%±1.1%)、层粘连蛋白(19.0%±1.6%)、纤维连接蛋白(9.0%±2.1%)、IV型胶原蛋白(16.0%±1.9%)等成分。化学萃取加震荡法可有效地去除大鼠脑组织内的细胞成分,制备得到的脑去细胞外基质较好地维持宏观和微观的三维结构,保留细胞外基质支架的蛋白成分,是一种简便且理想的脑去细胞外基质制备技术。     

Mass transfer trends occurring in engineered ex vivo tissue scaffolds

In vivo the vasculature provides an effective delivery system for cellular nutrients; however, artificial scaffolds have no such mechanism, and the ensuing limitations in mass transfer result in limited regeneration. In these investigations, the regional mass transfer properties that occur through a model scaffold derived from the human umbilical vein (HUV) were assessed. Our aim was to define the heterogeneous behavior associated with these regional variations, and to establish if different decellularization technologies can modulate transport conditions to improve microenvironmental conditions that enhance cell integration. The effect of three decellularization methods [Triton X-100 (TX100), sodium dodecyl sulfate (SDS), and acetone/ethanol (ACE/EtOH)] on mass transfer, cellular migration, proliferation, and metabolic activity were assessed. Results show that regional variation in tissue structure and composition significantly affects both mass transfer and cell function. ACE/EtOH decellularization was shown to increase albumin mass flux through the intima and proximate-medial region (0-250 μm) when compared with sections decellularized with TX100 or SDS; although, mass flux remained constant over all regions of the full tissue thickness when using TX100. Scaffolds decellularized with TX100 were shown to promote cell migration up to 146% further relative to SDS decellularized samples. These results show that depending on scaffold derivation and expectations for cellular integration, specificities of the decellularization chemistry affect the scaffold molecular architecture resulting in variable effects on mass transfer and cellular response.