笔者想衷心感谢詹妮弗Kadlowec博士的协助下在胶粘剂拉伸测试的协议的发展。 研究本出版物中报告是由关节肌肉骨骼及皮肤疾病研究所和生物医学成像和美国国立卫生研究院生物工程的下奖号1R15 AR 063920-01研究所的支持。内容完全是作者的责任，并不一定代表美国国立卫生研究院的官方意见。

1.聚（N-异丙基丙烯酰胺）-g-硫酸软骨素合成<ol><li>之前的生物粘附凝胶的合成，纯化N-异丙基（的NIPAAm）单体和甲基丙烯酸酯硫酸软骨素（CS）。 <ol><li>称出至少10g的NIPAAm和在60℃下溶解在400ml正己烷的单体。定期搅动容器，直到完全溶解。再结晶在-20℃冷冻该溶液24小时。 </li><li>从容器和真空过滤除去结晶单体使用布氏漏斗正己烷。置于在RT真空烘箱单体24小时以除去任何剩余的正己烷。储存在-20℃冰箱供以后使用单体（参见步骤1.2）。 </li><li>称出并溶解2.0克硫酸软骨素8.0毫升去离子水。轻轻加热溶液至60℃。 </li><li>使用大约10至40微升的50％（W / W）调节溶液的pH至10的NaOH。 </li><li>加入0.298毫升丙烯酸酐（MA）的。回流24小时，而在60℃下搅拌。 </li><li>倒入400毫升丙酮溶液成一个罐子，寒意O / N在-20℃的冰箱。 </li><li> 24小时已经过去后，慢慢倒入整个甲基丙烯酸酯化的CS（MCS）溶液分解成400毫升冷丙酮中，同时用磁搅拌棒搅拌。 </li><li>从丙酮中取出MCS和沉淀物放入一个真空炉中。快速执行此步骤来维护MCS完整和凝聚力的状态。允许残余的丙酮在真空下蒸发为至少24小时。 </li></ol></li><li>助溶解10克纯化的NIPAAm和232毫升去离子水2.209克MCS的。 </li><li>通过使用惰性氮气吹扫氧气的溶液。保持旺盛，但是较低的气体流速，防止起泡超过15分钟的解决方案。 </li><li>继续用氮气吹扫该溶液，并添加0.976毫升tetramethylet的hylenediamine（TEMED）。 </li><li>接着，发起通过混合97.6毫克过硫酸铵（APS）的聚合反应。 </li><li>混合该溶液约20秒，并迅速密封该溶液，以防止任何空气进入容器中。使该溶液在荧光灯下聚合24小时。 </li><li> 24小时后，将反应容器到37℃的烘箱。允许该水凝胶从液体转变为凝胶样状态。淹没在用于共7天1X磷酸盐缓冲盐水（PBS）的聚合的水凝胶，以允许未反应的单体的扩散出凝胶。 </li><li>使用剪刀，切割凝胶成若干小片，不超过5厘米，直径更大。以防止从一个凝胶状状态转变到液态的水凝胶，切块的37℃的PBS溶液中的聚合物。转移部到50ml锥形管中。 </li><li>通过包装的顶部制备用于冻干的样品管带的Kimwipes和橡皮筋。在-70℃下进行1至2小时冻结管。 </li><li>放置在冷冻干燥机的锥形管，从水凝胶中移除所有的水。操作前，冷冻干燥器需要达到大约一温度和压力下-40℃，0.04毫巴，分别。 7天至少需要完全干燥。 </li><li>研磨在4℃冰箱中以备后用（参见步骤3.1）干燥聚合物成细粉并存储的冻结。 </li></ol> 2.钙海藻酸钠交联微粒合成<ol><li>制备2％（重量/体积）通过在20毫升的去离子水溶解400毫克藻酸盐溶液。加热该溶液至约60℃以促进溶解。 </li><li>通过加入400毫克至20毫升去离子水制备的2％（重量/体积）氯化钙溶液中。 </li><li>通过组合百毫升菜籽油加入1.0ml吐温20，和20毫升创建在水混合物中的油的2％（重量/体积）藻酸盐。乳化10分钟，混合物用在15,000rpm匀化。此外，混合用磁力搅拌棒以200转或2000转分别创建或大或小的微粒。 </li><li>吸出2％（重量/体积）使用具有18G的针尖的注射器氯化钙溶液。慢慢加入氯化钙滴加到乳液。 </li><li>一旦20毫升氯化钙已经用尽，使乳剂中的微粒进行交联10分钟。 </li><li>均匀批料微粒分散到封50ml锥形管和离心机1400 xg的力2分钟以除去低芥酸菜子油的初始层。 </li><li>连续洗涤，涡旋，离心用异丙醇的微粒的共三次，在1400克2分钟以除去任何残留的低芥酸菜子油。丢弃每离心步骤后异丙醇上清液，用新鲜的异丙醇洗涤。 </li><li>一旦OI升已被去除，重复洗涤过程额外三次以去离子水以除去任何残留的异丙醇。丢弃每离心步骤后的去离子水上清液，用新鲜的去离子水洗净。在小瓶中取出约100毫克的湿微粒和商店。湿微粒将被用于通过光学显微镜来测量批料的平均直径。 <ol><li>使用光镜，分散在显微镜载玻片湿微粒在去离子水中的小样本。根据微粒的大小，使用10倍或20X物镜，并调整数值孔径直到颗粒成为焦点。确保该显微镜校准为正确的目标，并根据需要调节光源的亮度。使用行工具来测量的50个随机藻微粒最长直径和获得用于批次的平均尺寸。 </li></ol></li><li>准备冻干B中的微粒Ÿ包装他们的Kimwipes并用橡皮筋将其固定。在-70℃下1小时冻结微粒。 </li><li>放置在冻干机微粒和让样品干燥至少24小时。研冻干微粒成细粉用研钵和储存在4℃冰箱中供以后使用（见步骤3.3）。 </li></ol> 3.胶粘剂的制备<ol><li>使用冷冻干燥的聚合物粉末，制造5％（重量/体积）通过在1ml 1X PBS中溶解50mg的水凝胶粉末的PNIPAAm-G-CS溶液。 </li><li>混合使用的冰箱的涡流和寒冷的溶液在4℃下24小时。 </li><li>一旦粘稠溶液的形式，通过将25或50mg冷冻干燥的藻酸盐微粒的水凝胶溶液中，涡流形成均匀的复合材料。储存在冰箱复合在4℃以备后用（参见步骤4.3和4.8）。 </li></ol> 4.生物粘附机甲anical拉伸试验<ol><li>之前进行拉伸试验，从猪耳朵提取软骨衬底。 <ol><li>在温暖的0.9％除霜猪耳朵（重量/体积）NaCl溶液;这将有助于放松组织。 </li><li>使用手术刀，通过垂直地切割虽然表皮层，皮下组织和软骨分离和部分关闭的扁平，矩形区域。避免切割猪耳朵的弯曲脊。 </li><li>通过皮下组织切割平行从软骨中分离皮肤的表皮层。软骨是发现猪皮肤下一个坚硬的白色组织。 </li><li>仔细刮掉附加到软骨用手术刀侧的皮下组织。避免切入用手术刀刀片的软骨组织。 </li><li>确保只有一个软骨侧暴露和拉伸测试期间平。如果需要的话，平坦化通过切断皮肤不平上侧的软骨。 </li><li>切割软骨组织成几片以1cm×1cm的尺寸。存储在填充有0.9％（重量/体积）NaCl溶液在-20℃冷冻锥形管的软骨组织。 </li></ol></li><li>制备2.0升0.9％（重量/体积）NaCl溶液。预加热的盐水溶液至37℃。该溶液的温度将需要保持整个测试。 </li><li>解冻冷冻软骨。保留两个猪软骨和在用冰袋一个泡沫聚苯乙烯容器的水凝胶样品。 </li><li>安装测力计的试验台的手臂。放在力支架顶部加热板，并设置温度至50℃。 </li><li>贴上1厘米×1厘米长的软骨与氰基丙烯酸酯胶的不锈钢块和一对镊子的。胶到的Delrin圆柱体的附加片软骨和缸体连接到测力计。它是两个软骨表面彼此面对并接触水凝胶非常重要的。 </li><li>地点在热板上的顶部的树脂玻璃水浴，并插入同浴内的软骨基质的不锈钢块。 </li><li>降低试验台的臂并彼此对齐两个软骨基质。抬起手臂，并留下足够的空间来应用水凝胶。 </li><li>一个正排量吸管，取200微升生物粘附水凝胶的底部件软骨。 </li><li>在1毫米放下力架的臂/分钟，直到水凝胶接触上一块软骨和发挥0.001 N的预紧力</li><li>倒入37℃的生理盐水溶液入熔池，直至体积达到指定水位。检查有热电偶的溶液的温度。 </li><li>允许水凝胶，总共5分钟设定。一旦凝胶固化，以2mm / min的速度抬高手臂。测试完成后，一旦生物粘合剂由软骨基质脱落，当力显著下跌时，信号故障。 </li><li>收集记录的数据，提高了试验台的手臂。取下测力计的聚甲醛树脂圆柱体。倒入盐溶液到其先前的容器和再热至37℃。 </li><li>减去一个"空白"，该聚甲醛树脂附着没有软骨或粘合剂，以相同的速度升温的浮力通过聚合物施加的力，并通过粘结区域分割计算应力。 </li></ol> PNIPAAm的-G-CS与海藻酸钠微粒5肿胀研究<ol><li>准备干净的玻璃瓶和一个时间点，样品类型和样品编号正确标记他们。所述粘合剂也可被创建的，如先前所描述。对于每个时间点的每个样品类型制备总共五个样品（n = 5）。 </li><li>预权衡所有的玻璃小瓶中，在室温记录他们的初始质量。 </li><li>使用注射器，分配约为0.4毫升制备的粘合剂样本到每五个小瓶。重复此步骤，在所有测试的时间点，每个样品类型。 </li><li>称量和记录与在RT粘合剂样品类型各小瓶的质量。 </li><li>预温热的轨道培养箱和1L 1X PBS溶液至37℃。 </li><li>组织全体小瓶在机架上，然后插入机架进入孵化器。允许粘合剂从液体转变为凝胶约5至10分钟。 </li><li>添加5.0毫升1×PBS中的每个小瓶中。务必沿着小瓶的侧仔细添加PBS溶液，以防止水凝胶圆盘从断开。 </li><li>帽各小瓶，以防止PBS溶液蒸发并在37℃下保持培养箱的内部温度。每组的水凝胶盘，需要在PBS中以保持浸没的时间的指定长度。 </li><li>一旦7天的时间点已经达到，开盖设定小瓶并从每个小瓶PBS液。称量用在RT再次粘合剂样品的每个小瓶中。 </li></ol> &lt;p系列="jove_title"&gt; 6。质细胞活力使用活/死分析<ol><li>用的PNIPAAm-G-CS进行活/死法之前，培养人类胚胎肾293细胞（HEK-293），以80％汇合。 <ol><li>通过将离心管在37℃水浴中快速解冻冷冻的HEK-293细胞悬浮液，然后离心细胞以400 xg离心在4℃下5分钟在15ml锥形管中。 </li><li>为了使第一通道的细胞生长培养基，结合在高葡萄糖（4.5克/升）的Dulbecco氏改良的Eagle氏培养基（DMEM）与热灭活的小牛血清（FBS），以20％的终浓度，并在100×青霉素 - 链霉素溶液（笔链球菌）至100国际单位/毫升青霉素和100微克/毫升链霉素的终浓度。对于所有其他段落，切换到含10％FBS和1X青霉素 - 链霉素。 </li><li>重悬在10毫升生长培养基的细胞沉淀并将悬浮液转移到一个直径100毫米的陪替氏培养皿。均匀ð轻轻倾斜盘从前到后istribute细胞，从左到右几次每个方向。 </li><li>培养中的细胞在37℃培养箱，用5％ 的 CO 2。如果有必要，改变介质每两天。 </li><li>当细胞达到80％汇合，复制文化成多个菜肴。除去从板的介质。 </li><li> 1毫升0.5％胰蛋白酶的添加到板。将平板在37℃孵育10分钟。检查，看看是否细胞通过轻轻摇动该板从表面脱离。 </li><li>用于分离培养1:10，添加9毫升正常生长培养基（DMEM含10％FBS）的对胰蛋白酶的细胞并轻轻通过倾斜菜混合。 </li><li>分配将1ml稀释细胞悬浮液到每个新培养皿添加9毫升生长培养基。轻轻倾斜的菜混合。 </li><li>培养中的细胞在37℃培养箱，用5％ 的 CO 2。如果需要的话，永远改变介质Y两天，直到细胞达到80％汇合。 </li></ol></li><li>从生长至80％汇合两种培养菜取出介质。 </li><li> 1毫升0.5％胰蛋白酶添加到每个板。将平板在37℃孵育10分钟。检查，看看是否细胞通过轻轻摇动该板从表面脱离。 </li><li>洗上用3ml生长培养基的平板的HEK-293细胞两次，并都悬浮剂添加到相同的15毫升锥形管中。 </li><li>离心的15毫升锥形管中10分钟在4℃400 XG。 </li><li>除去上清液并通过上下抽吸悬浮在3ml培养基的细胞沉淀。 </li><li>移取10微升细胞悬浮液成血球和进行细胞计数，以确定细胞密度。增加介质的悬浮液体积，如果初始细胞浓度太高，反之降低音量，如果浓度过低，为了保全靶concent的1×10 6个细胞/ ml的配给。离心机的HEK-293细胞在400 xg离心，并相应地重悬细胞沉淀中的媒体的校正量。 </li><li>轻轻涡旋细胞悬液和细胞混合物均匀地分布到四个独立的15毫升锥形管中对以下测试条件:阳性对照单层，阴性杀控制的PNIPAAm-G-CS，用70％（V / V）甲醇，HEK-293包封在PNIPAAm的接枝的CS细胞，HEK-293细胞包封在PNIPAAm的接枝的CS与藻酸盐微粒。 </li><li>吹打细胞混合的多个300微升体积于24孔板，以产生4-6重复样品创建阳性对照单层。 </li><li>从有关被杀的控制，PNIPAAm的-G-CS和PNIPAAm的-G-CS用海藻酸钠微粒的锥形管中取出介质。 <ol><li>离心机锥形管在400 XG 5分钟，去除上清。 </li><li>通过再悬浮的C保持相同的细胞浓度厄尔在相同体积的PNIPAAm-G-CS作为除去介质。移液器聚合物电池混合上下使用血清吸管，直到均匀。 <ol><li>如果用海藻酸钠微粒播种，转移的PNIPAAm-G-CS-细胞悬液（为6.10.2描述获得）到35毫米的培养皿中，并引入颗粒进入组合。吸取细胞混合上下用血清吸管直至均匀。 </li></ol></li><li>使用血清吸管，分配多个300微升聚合物电池混合物到各个孔对被杀控制，PNIPAAm的-G-CS和PNIPAAm的接枝的CS与藻酸盐微粒，以产生4 - 6重复样品。 </li></ol></li><li>孵育所述板在37℃下进行10至15分钟，并允许的PNIPAAm-G-CS从液体转变为凝胶（聚合物形成不透明的，白色光盘）。 </li><li>定位在罩的滑动温暖及设定温度至37℃。使用幻灯片温暖维持孔板温度和吸移管600微升预热媒体到每个孔中。以进一步防止聚合物为液态的过渡，定位用荧光灯泡的菜上述预热空气在它上面的灯。 </li><li>孵育5天将细胞在37℃，5％的CO 2。 </li><li>五天过后，使用乙锭同型二聚体（EthD-1）和钙黄绿素AM制备活/死染料混合。这两种物质是光敏感和对使用当天制备。 <ol><li>从在RT试剂盒除霜钙黄绿素AM和EthD-1。 </li><li> 5毫升无菌1×PBS中的添加至锥形管，并在铝箔包裹。 </li><li>添加6.67微升2mM的EthD-1至锥形管中。彻底漩涡混合溶液。 </li><li>接下来，添加2.5微升4毫钙黄绿素AM到锥形管中。彻底VORTEX混合物。 </li></ol></li><li>从单层（阳性对照），PNIPAAm的接枝CS和PNIPAAm的接枝CS机智的孔中取出介质^ h海藻酸钠微粒。彻底，但轻轻地洗所有的井，用PBS和删除PBS。 </li><li>允许含有藻酸盐微粒，水凝胶盘，以在RT液化并加2ml 50mM柠檬酸钠到每个孔中。 </li><li>移除灭活细胞孔中的培养基（阴性对照）。允许水凝胶盘，以在RT液化，并添加300μl的70％（V / V）甲醇的每个孔中。 </li><li>在37℃下孵育45分钟的板。 </li><li>从孔中取出的柠檬酸钠和甲醇溶液和吸取各水凝胶悬浮液成单独的离心管中。离心试管在2000×g离心10分钟以将细胞从水凝胶悬浮液中分离出来。 </li><li>小心吹打从锥形管的溶液和离开细胞沉淀后面取出悬浮液。重悬在300μl的PBS中的沉淀和转移到原始孔板中。 </li><li>加入300微升的t他活/死染料混合到每个孔中。包裹在铝箔的孔板，孵育45分钟对在RT摇杆。 </li><li>图像全部用10X的目标倒置荧光显微镜下的样本。活细胞和杀死细胞会显示一个绿色和红色荧光，分别为。 </li></ol> 7.定量细胞活力使用XTT分析<ol><li>生长的HEK-293细胞以80％汇合。从两个培养皿除去生长培养基。 </li><li> 1毫升0.5％胰蛋白酶添加到每个板。将平板在37℃孵育10分钟。检查，看看是否细胞通过轻轻摇动该板从表面脱离。 </li><li>洗上用3ml介质板上的HEK-293细胞两次，并都悬浮剂添加到相同的15毫升锥形管中。 </li><li>离心的15毫升锥形管中10分钟在4℃400 XG。 </li><li>除去上清液和重悬细胞沉淀在3毫升的媒体通过上下吹吸。 </li><li>移取10微升细胞悬浮液成血球和进行细胞计数，以确定细胞密度。如前所述，保持为1×10 6个细胞/ ml的目标浓度。离心机的HEK-293细胞在400 xg离心，并相应地重悬细胞沉淀中的媒体的校正量。 </li><li>轻轻涡旋细胞悬液和细胞混合物均匀地分布到四个独立的15毫升锥形管中对以下测试条件:阳性对照单层，阴性杀控制的PNIPAAm-G-CS，用70％（V / V）甲醇，HEK-293包封在PNIPAAm的接枝的CS细胞，HEK-293细胞包封在PNIPAAm的接枝的CS与藻酸盐微粒。 </li><li> 6重复样品 - 吹打300微升细胞混合成24孔板产生4创造积极控制单层。 </li><li>从有关被杀控制锥形管取出纸张，PNIPAAm的-G-CS和PNIPAAm的-G-CS用海藻酸钠微粒。 <ol><li>离心锥形管在400 xg离心5分钟，去除上清。 </li><li>通过在相同体积的PNIPAAm-G-CS作为去除培养基重悬细胞保持相同的细胞浓度。吸取细胞混合上下用血清吸管直至均匀。 <ol><li>如果用海藻酸钠微粒播种，转移的PNIPAAm-G-CS-细胞悬液到35毫米的培养皿中，并引入颗粒进入组合。吸取细胞混合上下用血清吸管直至均匀。 </li></ol></li><li>使用血清吸管，取300微升聚合物电池混合物到各个孔对被杀控制，PNIPAAm的-G-CS和PNIPAAm的接枝的CS与藻酸盐微粒，以产生4 - 6重复样品。 </li></ol></li><li>一旦所有的含细胞的样品已被加入到24孔板，分配的PNIPAAm-G-CS和PNIPA多个300μl的体积上午-G-CS与没有在相邻井细胞藻酸盐微粒，以产生4 - 6重复样品。 </li><li>孵育24孔板在37℃下进行10至15分钟，并允许的PNIPAAm-G-CS从液体转变为凝胶（聚合物形成不透明的，白色光盘）。 </li><li>定位在罩的滑动温暖及设定温度至37℃。使用幻灯片温暖维持孔板的温度和吸600微升预热清晰的DMEM到每口井。以进一步防止聚合物为液态的过渡，定位用荧光灯泡的菜上述预热空气在它上面的灯。 </li><li>孵育5天将细胞在37℃在5％的CO 2。 </li><li>五天过后，从制造商制备2,3-双 - （2-甲氧基-4-硝基-5-磺苯基）-2H-四唑-5-羧酰（XTT）试剂。该试剂XTT对光敏感，并准备在使用的当天。 <ol><li>取下琥珀瓶含有ING从冰箱XTT粉末。使用无菌注射器和针头，注射5毫升明确的DMEM（无酚红）通过橡胶隔膜到琥珀色瓶。 </li><li>加热该试剂至56℃的水浴中10分钟，或直至溶解。 </li><li>在15ml锥形管包裹在铝箔进一步稀释XTT试剂至20％（体积/体积）。分装在离心管的过量XTT试剂和将它们放置在-20℃冷冻长期储存。 </li></ol></li><li>移除灭活细胞孔中的培养基（阴性对照），并添加300μl的70％（V / V）甲醇的每个孔中。孵育45分钟，在37℃和从孔中除去甲醇。 </li><li>从单层（阳性对照），PNIPAAm的接枝CS和PNIPAAm的接枝的CS与藻酸盐微粒的剩余孔中取出介质。彻底，但轻轻地洗所有的井，用PBS然后取出PBS。 </li><li>添加300微升的20％XTT试剂到每个孔中。包裹在铝箔中24孔板并孵育24小时就在RT摇杆。 </li><li> 24小时后，加入200微升50mM柠檬酸钠的所有孔中，并孵育在摇臂一个额外小时。 </li><li>在RT传输所有聚合物悬浮液至微量离心管并离心以2,000×g离心10分钟。 </li><li>转移200μl的上清液从每个离心管中，以一个96孔板中。在450nm和非特异性吸光度读数使用微量滴定板读数器，获得在690纳米的特定吸光率读数。 </li></ol>

有在合成水凝胶微粒复合材料和评价其粘合强度，溶胀能力和细胞生物相容性几个关键步骤。 PNIPAAm的接枝的CS的自由基聚合需要硫酸软骨素成功methacrylation，单体组分完全溶解，无氧的反应条件。被选择的NIPAAm单体甲基丙烯酸酯化的硫酸软骨素在反应混合物中的比率，因为它已被证明，在我们以前的工作，以产生具有机械性能类似于天然椎间盘组织29共聚物。在水凝胶增加硫酸软骨素的量将减少水的损失凝胶化过程中，从而减少了凝胶的压缩刚度。 methacrylation所选程度也已经证明通过18G的针，以产生与用于注射性良好处理性质的水凝胶溶解在水溶液中时&lt;SUP&gt; 29对CS的甲基丙烯酸酯替代将增加聚合物网络的交联密度，从而导致低于LCST增加溶液的粘度和减少的凝胶溶胀能力的增加的程度。在合成的PNIPAAm凝胶他人已经研究不同的方法如原子转移自由基聚合（ATRP）35或反向加成-断裂链转移（RAFT）36。通过ATRP聚合已通过改变单体与引发剂的比例导致与罚款调谐分子量狭窄，多分散水凝胶的发展。官能团可以在一个梳状的方式，使用RAFT聚合接枝，从而影响聚合物链缠结和相分离。 的均质的水包油混合物在创建均匀的，交联的藻酸盐微粒必要。的乳液技术简单进行，并且不需要使用有害的化学物质，但是既microparticle形状和分布是难以控制。微粒尺寸可以通过增加乳剂搅拌速度，从而创造更小的颗粒，反之亦然被改变。在这个特殊的研究中，海藻酸钠微粒的大小进行了调查作为可能影响肿胀，力学，或细胞活力的潜在性能。光散射也可以使用以替代测量平均微粒大小。其它乳液性能如表面活性剂类型和藻酸盐浓度将影响微粒的尺寸和聚集体形成37。降低藻酸盐浓度会降低微粒大小，但是聚集体更容易形成。在表面活性剂的亲水和疏水特性的变化会影响到油和水相之间的表面张力，从而改变该乳液中的微滴大小。藻酸盐微粒可替换地使用一个微流体装置，其允许均匀的粒子来制备e形和粒度分布窄38。该技术包括流过一个信道并进入氯化钙浴连续油相内的藻酸盐相分散。喷雾draying是产生非常小的微粒39的另一种技术。同样重要的是要注意，藻酸盐可加载与蛋白质或生长因子，并且可以是交联的其它二价离子如锌或钡。 在这项工作中，我们描述的粘合剂系统在组织工程的应用潜力。我们的拉伸试验专门旨在量化的复合材料的粘合强度与猪软骨接触时。这种类型的衬底的被选择以模仿到椎间盘软骨终板中的水凝胶的复合的粘合性。其它组织基材，如猪皮肤中，可以使用来替代量化的粘合剂40的强度，但组织类型将根据预期应用而变化。维持盐水水浴和预冷却该复合使用前的适当的温度控制是精确测量的机械性能是至关重要的。升高水浴的温度高于37℃会导致水凝胶迅速收缩并变硬，而下面37℃机械测试复合材料将表现出由于缺少相分离的减少拉伸强度。目前，我们正在开发的方法来研究各种注射胶粘剂配方的剪切粘接力。未来的工作将包括执行离体的猪体外诊断机械测试，以确定粘合剂是否通过一个环形缺陷抵抗挤压和还原的生物力学功能到脊柱运动节段。 高于32℃，PNIPAAm的接枝CS收缩并排出从水凝胶基质的水由于PNIPAAm的的LCST行为，从而导致降低的质量比在7天期间在PBS（ 图2）。相反，除了藻酸盐微粒的赋予以25毫克/米的微粒的浓度，甚至更因此在50毫克/毫升的溶胀效果和粘合剂在质量显著升高（p &lt;0.05）。在这个溶胀研究中，在水凝胶的湿质量变化很可能归因于与周围环境的水交换，由于PNIPAAm的接枝的CS已经被证明是在酶29的存在下选择性降解的。许多为肿胀研究开发的协议步骤，适用于基于PNIPAAm的-G-CS的thermoreversibility。水凝胶圆盘必须充分添加预热的PBS溶液之前在37℃下转换，否则凝胶会分散。肿胀一周后，将PBS溶液必须从小瓶迅速并小心除去水凝胶恢复为液态状态之前。应当指出，在本文中所描述的膨胀协议不完全模拟在天然椎间盘的体内环境。 体内支架的溶胀性能将依赖于周围组织41和凝胶的降解行为的渗透压。然而，在一般情况下，用藻酸盐微粒掺入增加溶胀能力被认为是有利的，因为这将有助于注射水凝胶在生理温度下留空间填充。如溶胀率和水含量的特性可以与附加干质量测量来计算。未来溶胀研究将包括浸渍针对聚乙二醇（MW = 20,000克/摩尔）浴，将适用的渗透压和模仿IVD 的体内环境的水凝胶的光盘。 除了机械和溶胀性能，粘合剂必须具有蜂窝的生物相容性，以被认为是合适的支架。以前的研究已经证明encapsula的生存特德细胞海藻酸钠42,43和基于PNIPAAm的材料44,45。同时从活/死和XTT测定法的结果与这些现有的研究结果一致，这表明适当细胞活力在为期5天（ 图3和4）。我们的实验室还在更长的时间场（21天）进行生存力的初步调查和观察到类似的行为。一般情况下，PNIPAAm的-G-CS和微粒的盈余应准备考虑中移液管传输的任何物质损失。再次，这是非常关键的，在37℃下保持适当的温度条件下保持支架结构。如果该支架网络从凝胶到液体状态，细胞附着和生活力恢复可能变得受损。至于对活/死测定法中，柠檬酸钠起着藻微粒的去除和解构了重要的作用。柠檬酸钠反转卡尔奇之间的交联作用微米和藻酸盐，并使浆料形成46。用PBS额外洗涤以除去的PNIPAAm-G-CS或可以执行藻酸盐以提高图像的质量。在XTT分析，在本孔板内产生不含有细胞的聚合物样品。这些重复用作空白标准，并需要计算一个准确的吸光度值。我们的初步可行性研究是严格基于使用HEK-293细胞。在将来的研究中，包封的脂肪来源的间充质干细胞的分化（AD-MSC）到髓核细胞将被证明。 AD-MSC向NP样表型分化已通过使用动态压缩47和缺氧48,49证明。 总体而言，所提出的粘合剂演示了在承载区域组织工程应用，例如椎间盘，其中该再生策略的成功将取决于支架的能力资源许运动和装载期间北京时间错位。这个系统是在它可能潜在地适于释放特定期望的应用外基质蛋白或生长因子非常灵活。重要的是，这里描述的方法可以适于与热胶凝聚合物的任何表征研究。

可注射生物材料是那些可方便地输送到身体中作为液体和原位固化。这样的材料已经在再生医学，在那里它们被用来封装细胞递送到受影响的部位1-4，并作为对电池单元5的三维临时的细胞外基质广泛应用。对于患者，因为用于植入的外科手术是微创和固相可以填充不规则形状的组织缺损，消除了对自定义尺寸的植入物的需要注射的生物材料是有利的。 注射能力可以通过各种机制来实现。外部因素，如pH值，已经研究作为用于封装细胞和生物活性分子6-8凝胶的形成的触发器。然而，pH值可能不是最方便的触发器中使用所有生理环境。另一个传统爱特纳略去对于实现可注射使用原位化学聚合或交联。 A组开发过硫酸铵和N，N，N&#39;，N"四甲基乙二胺组成的水溶性氧化还原体系，并用它进行反应的聚乙二醇和聚（丙烯）二醇9,10-组成的大分子单体。咱等人 11发达注射壳聚糖聚乙烯醇网络交联用戊二醛。在这样的系统中，反应性组分的细胞毒性，必须考虑，尤其是对涉及细胞封装的应用程序。另外，放热聚合可以产生足够高的温度妥协周围组织，这已被报道用于聚合的骨水泥12,13。 仍然其它可注射的聚合物系统已经开发显示出从液体到固体的状态的变化与温度作为触发。被称为热胶系统，这些都是aqueo我们不要求化学刺激，单体或交联剂原位形成14来实现聚合物溶液。更确切地说，通常接近生理温度中发生的相变诱导物理交联的三维网络的形成。泊洛沙姆例如Pluronic F127是热胶化药物递送15-17和电池封装18,19研究最广泛的聚合物之一。然而，可以很好地接受，这些凝胶缺乏在生理条件下的稳定性。研究使用的扩链剂20或化学交联剂21,22展示更高的稳定性。然而，使用这些试剂的可能会限制电池封装的材料的潜力。 聚（N-异丙基丙烯酰胺）是已经接收显著关注组织工程和药物输送14的合成热胶凝聚合物。聚的水溶液（N- isoprop基丙烯酰胺）（PNIPAAm的）表现出较低的临界溶液温度（LCST），通常发生约32 - 34°C 23,24。下面的LCST，水保湿PNIPAAm的链。高于转变温度时，聚合物变得疏水，从而导致剧烈的相分离25-27和地层的固体凝胶，无需使用有毒的单体或交联剂。然而，PNIPAAm的均聚物表现出较差的弹性性能，并在生理温度下，由于疏水28容纳少量的水。在这项工作中，我们选择共价结合的硫酸软骨素入PNIPAAm的网络，该网络提供了酶促降解29，抗炎活性30,31，并增加水和营养的吸收32的潜力。通过在甲基丙烯酸酯官能化的CS的存在下，单体的NIPAAm聚合形成接枝共聚物（PNIPAAm的-G-CS）在我们实验室中制备的CS PNIPAAm的共聚物。 BEC该共聚物的低交联密度的澳洲英语，PNIPAAm的接枝CS形成在RT在水中的粘性溶液中，在生理温度下的弹性凝胶由于LCST 29。聚合物溶液由于过渡的可逆性成为再次冷却时低于LCST悬浮剂。 我们已经证明，PNIPAAm的接枝的CS具有以用作组织工程支架，由于机械特性可定制，降解，和人类胚胎肾（HEK）细胞相容293细胞29的潜力。然而，在某些承载区域，如椎间盘，组织工程支架应该具有形成与周围椎间盘组织大幅接口消除错位33的风险的能力。这个接口也是必需的力在整个植入物和组织33之间的界面处的适当的传输。在我们的工作中，我们已经暂停了一在的PNIPAAm-G-CS的水溶液lginate微粒，发现凝胶化本地化的微粒，其提供与周围组织34的粘合性。在本文中，我们列出的步骤准备的热胶，粘性聚合物。生物材料表征，细胞成像的生存力的标准技术，并测定法适于考虑到该聚合物的温度灵敏度和相转变的可逆性。在本文中所述的可注射的聚合物具有那些本文中所描述的以外的用于药物递送和组织工程应用宽的电位。此外，这里所描述的表征方法可以适用于其它的热胶凝系统。

<table border="0" cellpadding="0" cellspacing="0" width="885">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">N-isopropylacrylamide, 99%, pure, stabilized</td>
			<td style="width:164px;">Acros Organics</td>
			<td style="width:120px;">2210-25-5</td>
			<td style="width:347px;">Refrigerate and remove stabilier with hexane</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Chondroitin sulfate A sodium salt (from bovine trachea)</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:120px;">39455-18-0</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Hexanes</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">H302-4</td>
			<td style="width:347px;">Store in a flammable cabinet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">50% (w/w) sodium hydroxide</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">SS254-1</td>
			<td style="width:347px;">Caustic in nature</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Methacrylic anhydride</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:120px;">276685</td>
			<td style="width:347px;">Strong fumes; use in a fume hood</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Acetone</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">A18-4</td>
			<td style="width:347px;">Chill in a refrigerator prior to use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Nitrogen Gas</td>
			<td style="width:164px;">Praxair</td>
			<td style="width:120px;">7727-37-9</td>
			<td style="width:347px;">Part Number: NI 4.8, cylinder style T, 99.998% pure nitrogen (Argon may be used as an alternative inert gas)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tetramethylethylenediamine, 99% extra pure</td>
			<td style="width:164px;">Acros Organics</td>
			<td style="width:120px;">110-18-9</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Ammonium persulfate</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">A3678</td>
			<td style="width:347px;">Hygroscopic and degrades in the presence of water</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Phosphate buffered saline tablets</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">BP2944</td>
			<td style="width:347px;">Keep dry</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Alginic acid, sodium salt</td>
			<td style="width:164px;">Acros Organics</td>
			<td style="width:120px;">177775000</td>
			<td style="width:347px;">Use heat to aid in dissolving</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Calcium chloride dihydrate</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">C79</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Canola oil</td>
			<td style="width:164px;">Local store</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Obtain from a local store</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tween 20</td>
			<td style="width:164px;">Sigma-Aldrich</td>
			<td style="width:120px;">93773</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">70% (v/v) Isopropoanol</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">A416-4</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Porcine ears</td>
			<td style="width:164px;">Haine&#39;s Pork Shop</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Obtain from a local butcher</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Sodium Chloride</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">S271-3</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Human embryonic kidney 293 cells</td>
			<td style="width:164px;">ATCC</td>
			<td style="width:120px;">ATCC CRL-1573</td>
			<td style="width:347px;">Store in liquid nitrogen for long-term use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">DMEM: 1X, high glucose, no pyruvate</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">11965126</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Fetal bovine serum</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">10082-147</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Penn Strep: 10,000 U/ml</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">15140-122</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Trypsin-EDTA: 0.5%, 10X</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">15400-054</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Methanol</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;">AAA44571-K7</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Live/Dead Cell viability kit</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">L3224</td>
			<td style="width:347px;">Light sensitive, keep frozen</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">XTT cell viability kit</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">TOX2-1KT</td>
			<td style="width:347px;">Light sensitive, keep frozen</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Clear DMEM: 1X, high glucose, no phenol</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">21063-029</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Dulbecco&#39;s PBS: 1X</td>
			<td style="width:164px;">Life Technologies</td>
			<td style="width:120px;">14190136</td>
			<td style="width:347px;">Refrigerate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Sodium citrate</td>
			<td style="width:164px;">EMD</td>
			<td style="width:120px;">SX0445-1</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Positive displacement pipette</td>
			<td style="width:164px;">BrandTech Scientific, INC</td>
			<td style="width:120px;">2702904</td>
			<td style="width:347px;">Dispenses 100 - 500 &#181;L and comes with attachable tips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">No 3. Stainless Steel scalpel handle</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">S2896</td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Miltex sterile surgical blades</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">12-460-440</td>
			<td style="width:347px;">Size 10</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Power gem homogenizer</td>
			<td style="width:164px;">Fisher Scientific</td>
			<td style="width:120px;">08-451-660</td>
			<td style="width:347px;">Model # 125</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Porcelain mortar and pestle</td>
			<td style="width:164px;">Sigma Aldrich</td>
			<td style="width:120px;">Z247464</td>
			<td style="width:347px;">Holds 50 mL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">FreeZone 1 L benchtop freeze dry system</td>
			<td style="width:164px;">Labconco</td>
			<td style="width:120px;">7740020</td>
			<td style="width:347px;">Freeze samples prior to use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Oil sealed rotary vane pump</td>
			<td style="width:164px;">Edwards</td>
			<td style="width:120px;">A65301906</td>
			<td style="width:347px;">Model # RV5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Incubating orbital shaker</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;">12620-946</td>
			<td style="width:347px;">Model # 980153</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Benchtop refrigerated centrifuge</td>
			<td style="width:164px;">Forma Scientific, INC</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model # 5682</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Heated ovens</td>
			<td style="width:164px;">VWR</td>
			<td></td>
			<td style="width:347px;">Model # 1235PC</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">2 N force gauge</td>
			<td style="width:164px;">Shimpo</td>
			<td style="width:120px;">FGV-0.5XY</td>
			<td style="width:347px;">Model # FGV-0.5XY</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">E-force test stand</td>
			<td style="width:164px;">Shimpo</td>
			<td style="width:120px;">FGS-200PV</td>
			<td style="width:347px;">Model # FGS-200PV</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tissue culture swinging bucket centrifuge</td>
			<td style="width:164px;">Beckman Coulter</td>
			<td style="width:120px;">366830</td>
			<td style="width:347px;">Model #6S-6KR</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Tissue culture microcentrifuge</td>
			<td style="width:164px;">Eppendorf</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model #5415C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Hemacytometer set</td>
			<td style="width:164px;">Hausser Scientific</td>
			<td style="width:120px;">3720</td>
			<td style="width:347px;">Requires replacement cover glass slips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Slide warmer</td>
			<td style="width:164px;">Lab Scientific</td>
			<td style="width:120px;">XH-2022</td>
			<td style="width:347px;">Model # XH-2002</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Portable heating lamp</td>
			<td style="width:164px;">Underwriters Laboratories</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Helps to maintain polymer temperature at 37&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Inverted fluorescent microscope</td>
			<td style="width:164px;">Zeiss</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model Axiovert 25 CFL</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Heated water bath</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model # 1235PC</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Rocking platform</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Series 100</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Multiskan FC microtiter plate reader</td>
			<td style="width:164px;">Thermo Scientific</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Type 357</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Cell culture incubator</td>
			<td style="width:164px;">VWR</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Model # 2350T</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:255px;">Purifier class II biosafety cabinet</td>
			<td style="width:164px;">Labconco</td>
			<td style="width:120px;"></td>
			<td style="width:347px;">Delta Series</td>
		</tr>
	</tbody>
</table>

synthesis of thermogelling poly(n-isopropylacrylamide)-graft-chondroitin sulfate composites with alginate microparticles for tissue engineering

可注射生物材料被定义为可被引入到体内的液体，并且在原位固化可植入材料。这种材料提供的植入微创侵入性，容易形成不规则形状的缺陷的空间填充物的临床优势。注射生物材料已被广泛研究作为组织工程支架。然而，对于某些承重区在体内的修复，如椎间盘，支架应该具有粘合性能。这将运动期间减少位错的风险，并确保与周围组织紧密接触，提供力的充分传输。在这里，我们描述了一个热敏感聚（N-异丙基）组成的支架的制备和表征接枝 - 硫酸软骨素（PNIPAAM-G-CS）和海藻酸钠微粒。所述的PNIPAAm-G-CS共聚物形成在RT在水中的粘性溶液，在其中alginatË颗粒悬浮以提高附着力。上面的低临界溶液温度（LCST），在30℃左右，该共聚物形成围绕微粒的固体凝胶。我们已经适应标准的生物材料的表征程序考虑到的PNIPAAm-G-CS的可逆相变。结果表明，50％或75毫克/毫升藻酸盐微粒中掺入5％（重量/体积）的PNIPAAm-G-CS溶液四倍的PNIPAAm-GCS的粘合抗拉强度单独（P &lt;0.05）。藻酸盐微粒的掺入也显著增加的PNIPAAm-G-CS的溶胀容量（P &lt;0.05），有助于保持组织缺损内的空间填充凝胶。最后， 体外毒理学测定试剂盒，2,3-双-的结果（2-甲氧基-4-硝基-5-磺苯基）-2H-四唑-5-羧酰（XTT）和活/死活力测定表明，该胶粘剂能够支持的生存和封装的人类胚胎肾细胞增殖（HEK）293ç厄尔超过5天。

一种热响应接枝共聚物已成功合成和表征其生物粘附强度，溶胀性能，并在体外细胞相容性。我们选择调查的褐藻胶因拥有完善粘膜粘附性。藻酸盐微粒，具有59.7±14.9微米的平均直径，用5％的共混（重量/体积）的PNIPAAm-G-CS在25，50，和75毫克/毫升的浓度。这些浓度是基于二分之一，等于，和在水溶液中的PNIPAAm-G-CS的等效干重的两倍。微粒浓度超过75毫克/毫升展出粘度过高，并没有影响。为5％的最大拉伸粘合强度（重量/体积）的PNIPAAm-G-CS具有不同的藻酸盐微粒的浓度比单独的水凝胶。的50％或75毫克加入/ ml的藻酸盐微粒的悬浮于5％（重量/体积）的PNIPAAm-G-CS导致了四倍增量rease在拉伸强度（P &lt;0.05），如在图1所示的25毫克/毫升的微粒浓度没有显示出比到5％（重量/体积）的PNIPAAm-G-CS（对抗拉强度显著增加&gt; 0.05）。有5％的拉伸强度没有显著变化（重量/体积）的PNIPAAm-G-CS时藻酸盐微粒的浓度为50至75毫克/毫升（P&gt; 0.05）增加。 通过在37℃下浸渍于PBS各种制剂的生物粘附水凝胶的溶胀性能进行表征。的25和50毫克/毫升藻微粒浓度进行测试，以确定在溶胀性能的任何差异。利用光学显微镜进行测定，有25.0±14.4微米，平均直径海藻酸钠微粒。 7天之后，水凝胶圆盘记录分别的最初和最后的湿重，并在质量变化计算为每粘合剂配方设计中的多个样本N（N = 5）。 图2示出在所有制剂的质量变化的急剧差异。含有25至50毫克/毫升藻微粒的粘合剂显示出在质量的积极变化，而5％（重量/体积）的PNIPAAm-G-CS以质量下降。所有样品被发现是从一个显著不同另一个（P &lt;0.05）用含有50毫克/毫升藻微粒表示的最大溶胀作用的粘合剂。 所提出的粘合剂的生物相容性是通过使用一个活/死和XTT细胞毒性测定的研究定性和定量。 HEK-293细胞在两个5％的包封（重量/体积）的PNIPAAm-G-CS和5％（重量/体积）的PNIPAAm-G-CS含有50毫克/毫升藻酸盐微粒与59.7±14.9微米的平均直径。用70％甲醇杀死的PNIPAAm-G-CS细胞和封装细胞的单层被认为是正和反齿刃分别略去对照。用细胞接种制剂后生长5天，在湿润的培养箱中在37℃下表征，用5％ 的 CO 2。每个测试表明对于两者的PNIPAAm-G-CS和PNIPAAm的接枝的CS与微粒良好的细胞生存力。从图3中的图像示出了活细胞荧光绿色在两个粘合剂配方（图A和B）和细胞单层上，作为阳性对照（图C）。杀死细胞（图D）荧光的红色，并显示细胞死亡。从XTT细胞毒性试验的结果证实了活/死测定法的结果。如在图4中所示相比的PNIPAAm-G-CS含有50毫克/毫升微粒的粘合剂的相对细胞活力降低1.3倍，但在降低无统计学显著（P&gt; 0.05）。既PNIPAAM-G-CS和PNIPAAM-G-CS与微粒也具有类似的存活力的细胞单层（P&gt; 0.05）。封装细胞在HYDRogel制剂和细胞单层被发现相比于阴性对照组（p &lt;0.05）是显著不同。总体而言，这些结果表明，含有微粒的粘接剂不仅表现出增加的粘合强度，但良好的生物相容性，以及。 <img alt="图1" src="/files/ftp_upload/53704/53704fig1.jpg" /> 图1.结合海藻酸钠微粒的不同浓度的入5％（重量/体积）的PNIPAAm-G-CS后上抗拉强度的影响 。 PNIPAAm的接枝CS的抗拉强度四倍与加入50或75毫克/毫升藻微粒（P &lt;0.5）的。误差棒代表一计算95％置信区间（N = 5）。海藻酸微粒有59.7±14.9微米的平均尺寸。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53704/53704fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> </一&gt; <img alt="图2" src="/files/ftp_upload/53704/53704fig2.jpg" /> 图2.藻酸盐微粒的不同浓度的影响的5％的溶胀容量（重量/体积）的PNIPAAm-G-CS，7天后浸渍于37℃的PBS，PNIPAAm的接枝的CS在湿物质由于减少PNIPAAm的的疏水性高于LCST。 PNIPAAm的接枝的CS用25或50mg / ml的表现出溶胀能力藻微粒。增加微粒的浓度显著升高（p &lt;0.05），超过7天在水凝胶的质量的变化，由于水的吸收。误差棒代表一计算95％置信区间。海藻酸钠微粒具有25.0±14.4微米的平均尺寸。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53704/53704fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> gether.within页="1"&gt; <img alt="图3" src="/files/ftp_upload/53704/53704fig3.jpg" /> 超过5天的时期培养HEK-293细胞的图3代表活/死荧光图像的细胞封装在（A）中的PNIPAAm-G-CS和（B）中的PNIPAAm-G-CS用50毫克/毫升藻酸盐的微粒。细胞也生长在的PNIPAAm-G-CS用甲醇杀来代表正和负的控制，分别一（C）的单层和（D）。的活/死生存能力实验用每个实验重复三次重复三次。刻度条表示100μm左右。海藻酸微粒有59.7±14.9微米的平均尺寸。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53704/53704fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> ftp_upload / 53704 / 53704fig4.jpg"/&gt; 图中的PNIPAAm-G-CS与 PNIPAAm的接枝CS内包封的HEK-293细胞使用和不使用藻酸盐微粒的藻酸盐微粒。存活的HEK-293细胞的4短期存活力评估，并在5天后使用XTT比较。用70％甲醇杀死的细胞单层和封装细胞中的PNIPAAm-G-CS分别用作阳性和阴性对照。两者的PNIPAAm-G-CS和PNIPAAm的接枝的CS与藻酸盐微粒表现出细胞存活率没有显著减少（P&gt; 0.05）相比，细胞单层，但表明在细胞活力显著减少（P &lt;0.05）相比，杀死细胞。在加入藻酸盐微粒的不显著妥协的PNIPAAm-G-CS（P&gt; 0.05）的细胞活力。误差棒代表一计算95％置信区间（n = 3时，每个实验重复六次）。藻酸盐微粒具有59.7±14.9微米的平均尺寸。REF ="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53704/53704fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering at JoVE.com

Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素组成的可注射的组织工程支架，制备（PNIPAAm的接枝CS）含藻酸盐微粒。粘合强度，溶胀性能和体外生物相容性在这项研究中进行分析。这里开发的表征技术可以适用于其他的热胶凝系统。

组织工程热胶凝聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素复合材料的海藻酸钠微粒的合成

Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer ...

synthesis-thermogelling-poly-n-isopropylacrylamide-graft-chondroitin

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Synthesis of Thermogelling Poly(N-isopropylacrylamide)-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

11.7K 次观看.  Rowan University. 聚（N-异丙基丙烯酰胺） - 接枝 - 硫酸软骨素组成的可注射的组织工程支架，制备（PNIPAAm的接枝CS）含藻酸盐微粒。粘合强度，溶胀性能和体外生物相容性在这项研究中进行分析。这里开发的表征技术可以适用于其他的热胶凝系统。 

视频: Synthesis of Thermogelling PolyN-isopropylacrylamide-graft-chondroitin Sulfate Composites with Alginate Microparticles for Tissue Engineering

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.

Methods for preparing an injectable matrix gel from decellularized tissue and injecting it into rat myocardium in vivo are described.

心肌组织工程生物衍生的注射材料的制备

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications.  ...

科研

生物工程

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

弹性PGS的动脉组织工程支架

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

毛细力光刻技术在心脏组织工程

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

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. ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

基于角蛋白纳米纤维生物医学工程合成

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

温敏纳米结构表面的组织工程准备

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

在组织工程由内在血管化<em&gt;在体内</em&gt;组织工程商会

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

水凝胶的合成与防污性能作为膜的净水

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...