The authors thank the Swedish Society for Medical Research, the Promobilia Foundation, the Crown Princess Lovisa Foundation, the Freemason Foundation for Children&#8217;s Welfare, the Swedish Society of Medicine, the Solstickan Foundation, Karolinska Institutet, and the Stockholm City Council for financial support.

所有动物方案都预先核准由斯德哥尔摩县委员会关于动物和所有程序符合动物用，以及相关的联邦法规的规定。 1.动物规程<ol><li>准备手术的动物<ol><li>与所需的无菌条件下的操作的所有材料和仪器制备手术台。只进行手术无菌条件下，以减少感染的风险，并优化在存活手术的条件。 </li><li>禁食12小时动物手术前并测量它的重量。管理二甲苯胺噻嗪的对术前用药肌内注射（2毫克/千克）。麻醉与替来他明次氯酸钠（2.5毫克/千克），唑拉西泮次氯酸钠（2.5毫克/千克），美托咪定（25微克/千克）和阿托品（25微克/千克）肌内注射动物。 </li><li>注入phenobarbiturate（15毫克/千克），以endotrache前人插管，并继续与0.8％-2％的异氟醚全身麻醉。插入外周静脉导管一只耳朵和静脉注射的过程中注入葡萄糖（25毫克/毫升），以保持动物的福祉。 </li><li>放置监测在耳朵上或尾部设备检查体温，血压，饱和度，和外周脉搏。由耳或蹄的疼痛刺激控制麻醉。适用软膏眼睛，防止干燥时的麻醉下。 </li></ol></li><li>膀胱活检标本切除术<ol><li>使用10法国硅氧烷导管通过引入窥器查看尿道和通过尿道插入导管并进入膀胱清空半无菌的条件下尿导尿膀胱。填用无菌生理盐水溶液膀胱20-25厘米的 H 2 0，约100-300毫升的压力，然后空至20厘米的 H 2 O（约8毫升/公斤体重）。 </li><li>随着日Ë猪仔细转向侧的位置，与葡萄糖酸氯己定的应用程序执行皮肤的基本术前消毒。与剃剪去掉皮毛后，应用一个透热板的肩膀。 </li><li>小心地将猪成仰卧位并删除使用剃剪腹毛色和做葡萄糖酸氯己定应用腹部皮肤的基本术前消毒。 <ol><li>软服装嵌入四肢以最小化过伸损坏在四肢关节的风险。消毒用葡萄糖酸洗必泰的连续应用动物的腹部皮肤，并放置无菌披盖周围的手术野。 </li></ol></li><li>之前切开皮肤，应用静脉镇痛在脐下中线选自丁丙诺啡（45微克/千克），卡洛芬（3毫克/千克）和利多卡因的局部注射。抓住SK检查疼痛反应在用钳子。通过使筋膜较低的正中切口，用透热为控制出血腹膜。本地化具有在猪完全腹膜内位置，并且可以通过手术伤口动员它被自由地暴露膀胱。 </li><li>以保持与镊子膀胱，并用消毒卷尺测量，并标志着一个椭圆形活检标本纵向约两厘米见方，1厘米横向，或更小，使用杀菌笔（猪容忍膀胱大小的四分之一减少很好）。切除标记的区域，使用解剖刀，并放置在DMEM膀胱活检标本在无菌条件下。 </li><li>与biotransplant执行自体移植或两层用5-0薇乔缝合它关闭膀胱。仔细2-0或3-0薇乔运行关闭腹部筋膜。关闭用3-0薇乔皮下组织和3-0 Ethilon皮肤。将包扎伤口，并仔细往往给动物，直到它已经从麻醉充分恢复和不表达的疼痛。 </li><li>移动动物的动物护理设施，以确保对术后护理情况。把动物在一个笼子里有取暖灯和照顾动物，直到从麻醉中完全恢复，然后让动物成对停滞。 </li><li>提供恢复顺利关于疼痛和福祉和日常管理丁丙诺啡（45微克/千克）肌注术后镇痛和甲氧苄氨嘧啶（4毫克/千克）和磺酰胺（20毫克/千克）的两倍了三天，每天一次五天以减少术后感染的风险。 </li></ol></li><li>皮肤活检标本切除术<ol><li>与所有必要的材料准备手术台。麻醉如先前在1.1中描述的动物。用蜡取出皮毛，洗净，用聚维酮碘和70％的酒精消毒切口区域，然后将消毒铺巾不要蜘蛛d是切口区域。 </li><li>使用皮收获为0.3毫米的厚度局部皮肤活检标本。放在DMEM皮肤样品切碎之前，如2.2所述。覆盖脂肪软膏和包扎伤口面积。 </li></ol></li></ol> 2.组织糜准备<ol><li>膀胱粘膜<ol><li>在DMEM洗膀胱穿刺活检的两倍。膀胱活检标本放置到朝上粘膜灭菌解剖板并用解剖针固定侧之一到板上。 </li><li>分开使用细剪刀和镊子（图3）逼尿肌粘膜组织并保持粘膜通过滴盐水或DMEM在它湿润。 </li><li>通过将其上的粘膜用切碎设备，然后从一端通过设备到另一垂直和水平，施加人工压力，以获得0.8毫米×0.8毫米切碎组织片（1.3毫米是rotatin之间的距离摹切割刀片）。 </li></ol></li><li>皮肤<ol><li>如果皮肤biopsis厚:皮肤放置到无菌解剖板，并使用手术剪刀从皮下脂肪及真皮表皮分离。表皮较薄和半透明（约0.3 MM），当它准备好切碎。 </li><li>通过将其上的表皮使用切碎设备。压力，从一端通过设备到另一垂直和水平，以获得0.8毫米×0.8毫米组织糜的碎片。 </li></ol></li></ol> 3.塑料压缩PCL /胶原自体移植的制备<ol><li>广场上冰的所有成分，保持冷。所需用具:猎鹰管，10X DMEM，1X DMEM，1N NaOH和鼠尾型胶原1。 <ol><li>混合料2毫升10×DMEM（小心，以避免气泡）的12型胶原毫升1.添加1N的NaOH，逐滴，使pH值达到7.4-8（在介质中颜色应可通过从激烈改变显示pH黄引脚K）。此外，使用pH条。 </li><li>小心加2ml 1X DMEM和混合解决方案。板约2毫升的钢制矩形模具（20x30x10毫米）的各孔的胶原蛋白，并在37孵化 °下在5％CO 2的10分钟。 注:胶原蛋白的浓度应为2.06毫克/毫升在0.6％乙酸和胶原以1ml /厘米2的量。 </li><li>一旦胶原设置到模具，放置在胶原凝胶（20毫米×30mm）的顶部上的生物材料（PCL）和倒在它上面的其余胶原（约6 ml）中。孵育在37℃下在5％CO 2 20分钟。 </li><li>放置组织糜（对于1:6扩展）对胶原凝胶的顶部。采用塑性压缩如下通过机械力按水从构建体（图3和4）。 </li><li>地方纱布上厚厚一层无菌表面。放置一个不锈钢筛网（400微米厚）上gauz顶部Ë垫，然后尼龙网（110微米厚）的片材。小心地将胶原凝胶/肉末组织转移到尼龙网，小心地取出矩形模具钢。 </li><li>放置尼龙网的一个新的层上的胶原凝胶/组织糜的顶部。放在尼龙网顶部上的第二钢丝网。在适当的位置放置压力或装载板称重最低为120克（ 即 ，玻璃板）5分钟。 </li><li>除去重量，尼龙和钢网格。自体移植的现在准备缝合到猪膀胱中的全层皮肤伤口或体外培养。 </li><li> 对于体外培养，切细构建成小片接头12孔板中。加入1 ml角质细胞培养基。放置在孵化所述板在37℃，5％CO 2和培养长达6周和改变介质每周3次。 </li></ol></li></ol> 4.自体移植的缝合<ol><li>自体缝合剁碎膀胱黏膜的猪膀胱<ol><li>保持自体的DMEM湿润时的等待时间。缝合用细单丝运行的缝线自体移植。使用非吸收5-0 Ethilon用于研究目的。 </li><li>检查，如果通过经由留置导尿管填充盐水膀胱不漏水。如果可能的话，请用大网膜一层的自体移植。如1.2.6所述关闭腹壁，皮下组织和皮肤。应用伤口敷料。 </li></ol></li><li>用剁碎的皮肤表皮自体移植的缝合全层伤口<ol><li>保持自体的DMEM湿润时的等待时间。 </li><li>缝合移植到由在弯道间断缝合，并在自体移植的中间卷绕保持紧密地附着在底层表面上的自体移植的皮肤全层的底部。 </li><li>盖上塑料敷料，保持伤口湿润的伤口。 </li></ol></li></ol><ol><li>稳重与唑拉西泮次氯酸钠肌肉注射（2.5毫克/千克）和美托咪定（25微克/千克）之前终止，并应用监控设备的耳朵或尾巴动物检查脉搏和血压。 </li><li>通过给予戊巴比妥钠（60〜140毫克/千克）静脉注射了致命剂量的安乐死的动物。检查脉搏和血压，直到发生死亡。 </li></ol> 6.在 体外培养 注:评价组织学在体外在PCL /胶原构建体的组织糜的进展，胶原/ PCL /剁碎贴剂使用角化细胞培养基的12孔板中培养。 <ol><li>角质形成细胞培养基的制备: <ol><li>消毒的500毫升玻璃瓶中。 </li><li>混合400毫升DMEM中，用100ml含的F12（4:1的混合物）。用10％胎牛血清补充，5微克/毫升胰岛素，0.4微克/ ml氢化可的松，21微克/毫升腺嘌呤，10 -10 mol / L的霍乱毒素，2×10 -9 mol / L的碘甲状腺原氨酸，5微克/毫升转铁蛋白，10ng / ml的表皮生长因子，50U / ml青霉素和50μg/ ml链霉素。 </li><li>通过一个0.2微米的过滤器过滤灭菌，并收集滤液在无菌一瓶500毫升。 </li></ol></li></ol> 7.免疫组化注:免疫组化协议一般分为以下步骤:（1）固定，石蜡包埋，（2）的微切片至5μm切片，在载玻片上，deparaffination和补液安置，（3）抗原修复，染色和安装。在启动免疫过程中的最后步骤之前，准备清洗缓冲区和抗原修复液（参见单独的材料详细说明）。使用前准备ABC复杂的解决方案至少30分钟。 <ol><li>固定术 没有TE:在体外培养的结束，修复补丁程序如下: <ol><li>准备Eppendorf管1毫升4％的缓冲甲醛（PFA）的（注意:甲醛是有毒的，请与本化学品之前，请先阅读材料安全数据表戴上手套和防护眼镜，并准备在通风橱内的溶液）。 </li><li>传送每个胶原补丁含有4％PFA的Eppendorf管中。在室温下固定过夜。 </li><li>在70％的乙醇代替样品长期储存在4℃。样品现已准备好脱水和切片之前石蜡包埋块。 </li></ol></li><li>补液<ol><li>放置载玻片在以X-TRA SOLV 15分钟染色罐子。通过使用X-TRA SOLV一个新的染色缸重复。放置载玻片与10分钟无水乙醇染色缸。通过使用无水乙醇新的染色缸重复。放置在用95％乙醇的染色缸幻灯片10分钟，此后与70％乙醇的染色缸10分钟。最后洗载玻片两次5分钟，用蒸馏水。 </li></ol></li><li>抗原修复<ol><li>放幻灯片的科普林缸用TE溶液，并把罐子在水浴中煮沸20分钟。就拿瓶子经过严格水浴。冷却该滑动到室温30分钟，并在Tris缓冲液5分钟洗涤两次。放置幻灯片中，用3％的过氧化氢为10分钟的染色缸。洗载玻片两次在Tris缓冲液中5分钟。绘制使用防水记号笔样品一圈。 </li><li>方框使用100-300微升的封闭溶液的抗体的非特异性结合。除去封闭溶液，并添加100-300微升溶解于Tris缓冲液中推荐的浓度初级抗体。孵育过夜。除去抗体溶液，并在Tris缓冲液洗部分两次5分钟。 </li><li>与二级抗体孵育在室温TEMPERAT 1小时URE。在Tris缓冲液5分钟洗涤两次。孵育使用ABC精英套件（按照制造商的说明）30分钟。在Tris缓冲液洗两次。 </li><li>发展，通过使用矢量贵宾套件，按照制造商的说明（1-7分钟温育通常产生明显的紫色强度）抗体反应。把载片于蒸馏水。染液用Mayer氏苏木30秒。 </li><li>洗5分钟自来水。放置幻灯片中，用70％乙醇的染色缸1分钟。通过使用具有70％的乙醇新染色缸重复。放置载玻片在用95％乙醇的染色缸1分钟。通过使用具有95％的乙醇新染色缸重复。 </li><li>放置在以X-TRA SOLV一个染色缸载玻片5分钟。除去，一次一个，以保持湿润。放置在每张幻灯片的顶部安装介质下降，并把顶部玻璃盖（请小心操作，避免气泡）。让幻灯片干通宵，并查看微下滑梯范围。 </li></ol></li></ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructs+of+electrospun+PLGA,+compressed+collagen+and+minced+urothelium+for+minimally+manipulated+autologous+bladder+tissue+expansion.">Constructs of electrospun PLGA, compressed collagen and minced urothelium for minimally manipulated autologous bladder tissue expansion.</a> Biomaterials. 35, 5741-5748 (2014).</li><li>Orabi, H., AbouShwareb, T., Zhang, Y., Yoo, J. 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The authors have nothing to disclose.

这项研究提出了一种易于使用的方法，以产生具有用于移植自体组织膀胱壁修补在手术台。贴片通过在中间和胶原有和没有外表面与塑性压缩组合组织糜可生物降解的聚合物针织的组合形成。塑性压缩是以前由其他作者描述的方法，并且可以被定义为从胶原凝胶12,13流体的迅速排出。膀胱粘膜或皮肤的组织糜接种到该支架和膀胱或皮肤上皮的形成可能在6周被遵循。免疫组织化学分析表明当量正常组织的特征的形成。这些结果允许不仅用于研究再上皮和伤口愈合的体外模型，而且还形成了自体组织移植的生物材料。最重要的是泌尿外科的目的，AUTografts可移植11之前用于重建泌尿外科作为工程自体组织修补组织工程的体内应用而无需在体外培养的要求。 相对于电池膨胀及体外培养技术，皮肤上皮和尿路上皮有着共同的特点。动机提出的技术中用于塑性压缩和切碎在本研究两个上皮组织是收获和体内研究皮肤上皮比为膀胱尿路上皮更容易。通过这些手段，生物材料和皮肤上皮可以研究作为第一步中泌尿生殖器官执行更侵入研究之前确认生物相容性和物理性能。 在体内研究中以前，使用组织糜的概念与膀胱粘膜导管组织再生，调查结果是第È小移植颗粒需要以承受机械创伤和移植剁碎粒子的初始取期间和愈合过程和组织再生6,7-期间留在原处的一些机械支撑。 为了克服这些缺点，该研究旨在开发一种混合有聚合物针织物和塑料压缩胶原11,14的可生物降解的芯构造。胶原蛋白为细胞的附着和生长有利的表面，并允许直接和快速整合组织糜入支架。然而，由于胶原的机械性能，甚至塑性压缩之后，仍弱，不会维持收缩和天然膀胱运动的扩展，加入支承芯支架的胶原蛋白。对于更复杂的结构，移植的组织可以绕预制模具中膨胀以产生三维EPithelialized结构具有中央内腔。 在这些研究中，我们使用的PCL作为稳定聚合物和biograft的核心。 PCL被广泛用于各种生物医学装置如牙齿填充物，可吸收的合成外科网格和缝合材料14。为了促使再生组织的成熟， 即神经支配，平滑肌组织和细胞外基质，我们选择了PCL聚合物与缓慢降解率超过几个月。然而，降解率可通过聚合物，厚度和密度11,16的选择进行调节。 胶原被选为由于众所周知的生物相容性，安全性和良好的愈合特性的复合结构的一个组成部分。胶原促进肉芽组织，重新建模和细胞外基质15的成熟的向内生长。由于胶原蛋白是退化，新生血管及肉芽组织支持移植ED切碎颗粒重组，迁移并扩大5-7,11,16。此外，胶原蛋白是无论在皮肤和在膀胱和已用于创建在体外和在体内 ，包括在泌尿生殖系统17,18伤口愈合研究和重建支架的细胞外基质的主要天然组分。 该协议中的关键步骤是容易管理。首先，活检标本有湿润和有利的条件下插入之前保持到移植，否则细胞的生长将被延迟或缺席。第二个陷阱可能涉及获取胶原蛋白的正确牢固。我们必须确保胶原蛋白通过避免胶原蛋白凝胶内的气泡编写自体移植的过程中变得僵硬。气泡通过小心移液避免和小气泡可以用针破裂。胶原蛋白有气泡应该被丢弃。 这是LSO重要的是得到组织糜的大小合适;切碎组织之前，请确保只有薄膀胱粘膜在膀胱扩张或仅在皮肤扩张的情况下，表皮的情况下使用。最后缺陷是当缝合:确保垂直移动到与针的移植，以避免分离不同的层。在自体移植的边缘也需要为了不胶原从聚合物中分离小心处理。缝合片材之​​后将很难分离。这些学习技术时可能出现的常见问题。 这种技术的一个限制是在切碎的颗粒细胞依靠营养和氧气的扩散。因此，在自体移植的厚度必须小于1毫米，并具有被放置在一个良好的血管化位点。这可能是一个不利因素，因为通过气囊的移植部分的尿液泄漏的风险。取决于PLASTIC压缩，不同渗透性常数可以实现。在我们的例子中透水性（k）的值，根据以往的研究19为0.034。在临床上，我们预计，我们需要保持膀胱空虚与导管约1-2周，以便给时间尿路上皮细胞，建立一个多层次的连续膀胱上皮，使补丁未渗透之前活跃使用。 另一个限制是一个健康的器官用于组织收获和扩张的假设。在当膀胱已丢失或不宜扩展越严重的情况下，例如，影响当由癌症，该技术可能不适合。 至于其他的组织工程细胞构建体，关于功能和形态学效果最后结论仅可在长期的体内研究回答。下一步，我们将分析在尊重VIAB我们在长期的动物实验结果ility和生理特性移植后。 所提出的技术的意义是只有一个单一的外科手术之后，以扩大在体内组织的可能性。目前正在评估的其它技术都依赖于移植前扩大细胞在体外。 体外程序有被成本高有关，需要先进的实验室工作人员，并成为耗时下行。它可能需要长达组织或细胞和组织工程自体移植的收获2之间个月;相对于小于1小时，根据本研究中所描述的技术。 在未来，在压缩的胶原技术组织糜可扩大和其他器官，如腹壁缺损，横膈疝气，并且其中一个缺陷是不容易与现有的组织重建的其他条件下使用。 在体内膨胀的移植说明。制备自体移植的过程是很容易做到，并且可以在无菌条件下在普通外科设置中使用。自体移植抗拒外科处理和可生物降解。在自体移植的核心可以根据患者的具体需要可以由根据关于降解和其它的特性，如弹性，厚度和孔隙率偏好不同的可生物降解的聚合物，和。在临床环境中，患者将经历组织收获，制备该复合体移植和自体移植作为单一阶段程序的。此外，该含细胞bioconstructs的所有部分都目前FDA批准。

上移植到皮肤和泌尿生殖道最组织工程研究包括来自于专门配备细胞培养设施1,2-健康组织和细胞扩张的自体细胞的收成。 细胞扩增后，将细胞通常存储供以后使用时制备的患者以接收自体移植。氮冷冻允许在-150℃或更低的低温长期贮存。冷冻的过程中一定要小心，为了不失去控制的细胞。细胞死亡的一个危险是在解冻过程中，这可导致细胞膜的破裂的细胞内的水的结晶。细胞冷冻通常通过缓慢和受控冷却（-1℃，每分钟）进行的，使用的细胞，胎牛血清和二甲亚砜的浓度高。解冻后，将细胞需要通过除去冷冻培养基并培养细胞培养塑料或再次处理移植前生物材料回给患者。 所有上述步骤是费时，费力，且昂贵3。此外，针对患者的移植细胞全部排出体外处理是高度管制的，需要训练有素和认可的人员和实验室4。总而言之，促使安全和可靠的制造工艺，这项技术只有在极少数技术先进中心成立，并在常见的外科疾病更广泛的使用是值得怀疑的。 为了克服在实验室环境的细胞培养的局限性，移栽组织糜用于体内细胞扩增的概念是通过使用体本身作为生物反应器引入的。为了这些目的，在自体移植将优先被上一个3D模具按照所需要对i的器官的最终重建的形状移植nterest 5-7。 原来，移栽剁碎上皮细胞的想法，在1958年提出由温顺，当他描述的上皮细胞从伤口的边缘如何生长。他表明，一小片皮肤的将增加其利润率并且由此其为细胞扩增潜力100％通过切割片两次在垂直的方向（图1）8。理论已被使用啮合部分厚度皮肤移植的皮肤移植9支撑 ，并在皮肤伤口愈合模型10。 <img alt="图1" src="/files/ftp_upload/53061/53061fig1.jpg" width="600" /> 图1:米克理论根据温顺的理论，上皮生长从伤口的边缘。通过增加切碎技术的曝光区域，组织糜处上皮许多景点的伤口。 本研究是基于低论文，相同的原理可以通过将剁碎上皮围绕模具施加到皮下组织。上皮细胞会从剁碎移植（重组），以便形成连续neoepithelium从内部主体覆盖伤口区域，并分离异物（模具）动员，覆盖伤口区域（迁移）和除（展开）（ 图2）。 <img alt="图2" src="/files/ftp_upload/53061/53061fig2.jpg" /> 图2:根据米克理论三维模具剁碎上皮用于体内intracorporal组织扩张的卡通通过使用放置在模具中，然后移植到皮下组织组织糜，所述假设是，上皮细胞从迁移组织糜的边缘，重组，和扩展，以便形成一个连续的neoepithelium覆盖伤口区域和异物（模具）从内体中分离。 尽管以前的体内研究表明可喜的成果，进一步改善可以通过加强自体移植来实现，使再生上皮细胞可以抵御机械损伤更好7。为了这些目的，一个成功的生物材料的重要先决条件被确定，如:容易扩散营养素和废物的，可能性模具在三维方式和手术操作容易。结论所做的这些需要可以通过添加的复合生物材料的组织糜得到满足。 目前的研究旨在开发一种在组织糜构成的支架塑性压缩胶原含有可生物降解的织物的增强芯。通过这些手段，活细胞能够从组织糜粒子迁移，并与原来的上皮细胞（皮肤或上皮）的形态特征特性增殖。使用塑料压缩，脚手架是减少d在1厘米大小至约420微米的切碎的颗粒在上层胶原进行了包裹。该芯部织物可以是任何聚合物，但需要用亲水表面修饰，以便与覆盖胶原层 11相互连接起来。 该方法通过使用其作为支架用于培养切碎膀胱粘膜或猪碎肉皮肤​​掺入两个塑料压缩胶原凝胶内的编织网由聚（ε - 己内酯）（PCL）提供了一种增强的支架的完整性。该构建物维持在细胞培养条件为长达6周的体外 ，证明成功地形成一个良好的固结杂交构建体的顶部分层，多层上皮或鳞状皮肤上皮。该构建是易于处理和来代替膀胱增加目的或皮肤缺陷的覆盖物被缝合。组织支架的所有部件都FDA批准和技术可以通过组织收获，切碎，塑性压缩，并移栽回病人作为一个单一阶干预可用于单级的程序。该程序可以在任何普通外科单位在无菌条件下组织扩张和重建进行。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Silicone catheter 10-French</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>DMEM 10X</td>
			<td>Gibco</td>
			<td>31885-023</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>Falcon</td>
			<td>08-772-1</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>3&#39;3,&#39;5-Triiodothyronine</td>
			<td>Sigma-Aldrich</td>
			<td>IRMM469</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>4% PFA</td>
			<td>Labmed Solutions</td>
			<td>200-001-8</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Histolab</td>
			<td></td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>ABC Elite kit: Biotin -Streptavidin detection kit</td>
			<td>Vector</td>
			<td>PK6102</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Absolute ethanol</td>
			<td>Histolab</td>
			<td>1399.01</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma-Aldrich</td>
			<td>A8626</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Atropine 25 &#956;g/kg&#160;</td>
			<td>Temgesic, RB Pharmaceuticals, Great Britain</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Azaperone 2&#8201;mg/kg&#160;</td>
			<td>Stresnil, Janssen-Cilag, Pharma, Austria</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Biosafety Level 2 hood&#160;</td>
			<td></td>
			<td></td>
			<td>Plastic compression; Section 4</td>
		</tr>
		<tr>
			<td>Blocking solution:&#160; Normal serum from the same species as the secondary secondary antibody was generated in.</td>
			<td>Vector</td>
			<td>The blocking solution depends of the&#160; origin of&#160; first antibody</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Buprenorphine 45 &#956;g/kg</td>
			<td>Atropin, Mylan Inc, Canonsburg, PA</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Carprofen 3&#8201;mg/kg&#160;&#160;&#160;</td>
			<td>Rimadyl, Orion Pharma, Sweden</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Chlorhexidine gluconate&#160;</td>
			<td>Hibiscrub 40&#8201;mg/mL, Regent Medical, England</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Cholera toxin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C8052</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Coplin jar: staining jar for boiling</td>
			<td>Histolab</td>
			<td>6150</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Stainless mold&#160; (33x22x10 mm) custom made</td>
			<td></td>
			<td></td>
			<td>Plastic compression; Section 4</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>3188-5023</td>
			<td>Plastic compression section 4. Keep on ice&#160; when using it in plastic compression</td>
		</tr>
		<tr>
			<td>Epidermal growth factor</td>
			<td>Sigma-Aldrich</td>
			<td>E9644</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Ethilon (non-absorbable monofilament for skin sutures)</td>
			<td>Ethicon</td>
			<td></td>
			<td>Surgery, Section 1</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10437-036</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>Forceps (Adison with tooth)</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Gauze (Gazin Mullkompresse)&#160;</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Ham&#180;s F12</td>
			<td>Gibco</td>
			<td>31765-027</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Histolab</td>
			<td>1820</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Humidity chamber</td>
			<td>DALAB</td>
			<td></td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide Solution 30%</td>
			<td>Sigma-Aldrich</td>
			<td>H1009</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I3536</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Isoflurane, Baxter, Deerfield, IL</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Lidocaine 5 mg/ml</td>
			<td>Xylocaine, AstraZeneca, Sweden</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Lucose 25&#8201;mg/mL&#160;</td>
			<td>Baxter, Deerfield, IL</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Marker pen pap pen</td>
			<td>Sigma-Aldrich</td>
			<td>Z377821-1EA</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Medetomidine 25 &#956;g/kg&#160;</td>
			<td>Domitor, Orion Pharma, Sweden</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Mincing device</td>
			<td>Applied Tissue Technologies LLC</td>
			<td></td>
			<td>&#160;Minced tissue preparation, section 2</td>
		</tr>
		<tr>
			<td>Monocryl (absorbable monofilament)</td>
			<td>Ethicon</td>
			<td></td>
			<td>Surgery, Section 1</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>NaOH 1N</td>
			<td>Merck Millipore</td>
			<td>106462</td>
			<td>Plastic compression section 4 and cell culture</td>
		</tr>
		<tr>
			<td>Nylon mesh, 110 uM thick pore size 0.04 sqmm</td>
			<td></td>
			<td></td>
			<td>Plastic compression; Section 4</td>
		</tr>
		<tr>
			<td>Oculentum simplex APL: ointment for eye protection</td>
			<td>APL</td>
			<td>Vnr 336164</td>
			<td>Surgery, Section 1</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Plastic compression section 4</td>
		</tr>
		<tr>
			<td>Phenobarbiturate 15&#8201;mg/kg&#160;</td>
			<td>Pentobarbital, APL, Sweden</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>PLGA Knitted fabric</td>
			<td></td>
			<td></td>
			<td>Plastic compression; Section 4</td>
		</tr>
		<tr>
			<td>Rat-tail collagen</td>
			<td>First LINK, Ltd, UK</td>
			<td>60-30-810</td>
			<td>Plastic compression section 4, keep on ice</td>
		</tr>
		<tr>
			<td>Scalpel blade - 15</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Shaving shears</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Stainless stell mesh, 400 uM thick pore size&#160;</td>
			<td></td>
			<td></td>
			<td>Plastic compression; Section 4</td>
		</tr>
		<tr>
			<td>Steril gloves</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Sterile gowns</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Sterile drapes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilium</td>
			<td>Bode Chemie HAMBURG</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Suture Thread Ethilon</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>TE-solution (antigen unmasking solution) consist of 10 mM Tris and 1 mM EDTA, pH 9.0</td>
			<td></td>
			<td></td>
			<td>10 mM Tris/1 mM EDTA,&#160; adjust pH to&#160; 9.0</td>
		</tr>
		<tr>
			<td>Tiletamine hypochloride 2,5&#8201;mg/kg</td>
			<td></td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Transferrin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T8158</td>
			<td>&#160; In vitro culture; Section 5</td>
		</tr>
		<tr>
			<td>Trizma Base, H2NC&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T6066</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Vector VIP kit: Enzyme&#160; peroxidase substrate&#160; kit</td>
			<td>Vector</td>
			<td>SK4600</td>
			<td>Immunocytochemistry; Section 6</td>
		</tr>
		<tr>
			<td>Vicryl (absorbable braded)</td>
			<td>Ethicon</td>
			<td></td>
			<td>Surgery, Section 1</td>
		</tr>
		<tr>
			<td>Tris buffer pH 7.6 (washing buffer)</td>
			<td></td>
			<td></td>
			<td>TE solution: Make 10X&#160; (0,5M Tris, 1,5M NaCl) by mixing: 60,6 g Tris (Trizma Base, H2NC(CH2OH)3, M=121.14 g/mol), add 800 ml&#160; distilled water adjust the pH till 7.6, add 87,7 g NaCl and fill to 1000 ml with&#160; distilled water. Dilute to 1X with distilled water.</td>
		</tr>
		<tr>
			<td>X-tra solv (solvent)</td>
			<td>DALAB</td>
			<td>41-5213-810</td>
			<td>Immunocytochemistry; Section 6. Use under fume hood</td>
		</tr>
		<tr>
			<td>Zolazepam hypochloride</td>
			<td>Zoletil, Virbac, France</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Depilatory wax strips</td>
			<td>Veet</td>
			<td></td>
			<td>Preparing the animal for surgery , Section 1</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium</td>
			<td>Lundbeck</td>
			<td></td>
			<td>Termination, Section 3</td>
		</tr>
	</tbody>
</table>

minced tissue in compressed collagen: a cell-containing biotransplant for single-staged reconstructive repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

本研究提出，显示了如何使用胶原蛋白和组织糜的塑料压缩生产用于移植的生物材料的方法。 膀胱粘膜和皮肤可以收获并然后机械剁碎成小颗粒（ 图3）。通过塑性压缩，碎粒子一个居中设置可生物降解的聚合物，胶原凝胶（图4）的外层内机械强度高的组成的复合支架内并入本文。组织糜颗粒可以被分离，以允许一个1:6膨胀率。通过压出胶原的水含量，自体重建手术一个biotransplant完毕。 Biotransplants可用于自体移植的动物用于体内研究或体外培养在通常的细胞培养环境进行进一步的体外研究 。 ŧ<html>他复合自体允许抓住和缝合和维持手术操作（图5）。从组织收获，准备重建手术的含细胞自体移植的方法，大约需要20分钟，目的是要作为单个阶的次序进行。 在体外研究细胞迁移，展开和重新组织的自体移植的表面成单细胞的连续层在两个星期。四周后，连续上皮约为4细胞层厚（图6）。在免疫组化时间在不同的点发现，细胞增殖，迁移和重新组织成一个连续的上皮细胞具有典型的细胞表型的微架构。同样的结果适用于用剁碎的皮肤上皮细胞作为肉末膀胱粘膜自体移植。 1 / 53061fig3.jpg"/&gt; 图3:组织糜的制备和塑性压缩膀胱粘膜（A）的用于切碎（C）和塑性压缩的制备（D - F）中，使用切碎装置在（B）和（D）中的模具，以产生一个420微米厚的移植（H）。注意，胶原本身就是一个很好的胞外基质，但难以机械处理（G）;通过将可生物降解的织物作为增强芯，移植变得容易掌握（H）。 <img alt="图4" src="/files/ftp_upload/53061/53061fig4.jpg" /> 图4:塑料压缩动画片展示塑料压缩碎颗粒的过程。 <img alt="图5" src="/files/ftp_upload/53061/53061fig5.jpg" /> 图5:在大鼠示范缝合塑料压缩生物材料与标记为T（自体移植），而标记为S（假）切碎的皮肤缝合生物材料自体移植皮剁碎处理手术全层皮肤创伤模型。在这种情况下，切碎的皮肤上皮移植为1:3膨胀率。 <img alt="图6" src="/files/ftp_upload/53061/53061fig6.jpg" /> 图6:免疫组化染色，以可视化的三维文化发展中的胶原蛋白生物材料切碎皮肤，膀胱粘膜 （A - D）（A）原生肌肤苏木/ HE染色，（B）原生膀胱，（C）剁碎。在培养和 （D）剁碎膀胱后5周皮肤在培养6周后。的上皮细胞和增殖标志物MNF116（E）和Ki 67抗体（表达<stro纳克&gt; F），分别为2周后在培养物（剁碎膀胱）。

Watch this Scientific Journal Video about Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair at JoVE.com

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

组织工程经常包括在体外扩增 ，以创建用于组织再生自体移植。在这项研究中用于组织扩张，再生， 和体内重建的方法，以便最小化在体外的细胞和生物材料的处理被开发。

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

minced-tissue-compressed-collagen-cell-containing-biotransplant-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.9K 次观看.  Karolinska Institutet. 组织工程经常包括在体外扩增 ，以创建用于组织再生自体移植。在这项研究中用于组织扩张，再生， 和体内重建的方法，以便最小化在体外的细胞和生物材料的处理被开发。 

视频: Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

科研

生物工程

Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two tissue edges1,2. These are then irradiated by a laser that is selectively absorbed by the RB. The resulting photochemical reactions supposedly crosslink the collagen fibers in the tissue with minimal heat production3. In this report, RB has been incorporated in thin chitosan films to fabricate a novel tissue adhesive that is laser-activated. Adhesive films, based on chitosan and containing ~0.1 wt% RB, are fabricated and bonded to calf intestine and rat tibial nerves by a solid state laser (&lambda;=532 nm, Fluence~110 J/cm2, spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, is used to test the bonding strength. The RB-chitosan adhesive bonds firmly to the intestine with a strength of 15 &plusmn; 6 kPa, (n=30). The adhesion strength drops to 2 &plusmn; 2 kPa (n=30) when the laser is not applied to the adhesive. The anastomosis of tibial nerves can be also completed without the use of sutures. A novel chitosan adhesive has been fabricated that bonds photochemically to tissue and does not require sutures.

Sutures are usually needed to repair tissue during surgical procedures. However, their application can be problematic as they are invasive and may damage tissue. The fabrication and application methods of a novel tissue adhesive are here reported. This adhesive film is laser-activated and does not require the use of sutures.

激光对生物组织的修复孟加拉玫瑰红壳聚糖膜的制备及应用

Photochemical tissue bonding (PTB) is a sutureless technique for tissue repair, which is achieved by applying a solution of rose bengal (RB) between two ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

对于I型胶原蛋白的制备皮肤方法的改进

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

成像变性的胶原股<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;通过光引发杂交笼胶原蛋白仿生肽

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

层与层胶原沉积在微流体装置的Microtissue稳定

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.