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...

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><tbody><tr><td>N-isopropylacrylamide, 99%, pure, stabilized</td><td>Acros Organics</td><td>2210-25-5</td><td>Refrigerate and remove stabilier with hexane</td></tr><tr><td>Chondroitin sulfate A sodium salt (from bovine trachea)</td><td>Sigma-Aldrich</td><td>39455-18-0</td><td>Refrigerate</td></tr><tr><td>Hexanes</td><td>Fisher Scientific</td><td>H302-4</td><td>Store in a flammable cabinet</td></tr><tr><td>50% (w/w) sodium hydroxide</td><td>Fisher Scientific</td><td>SS254-1</td><td>Caustic in nature</td></tr><tr><td>Methacrylic anhydride</td><td>Sigma-Aldrich</td><td>276685</td><td>Strong fumes; use in a fume hood</td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>A18-4</td><td>Chill in a refrigerator prior to use</td></tr><tr><td>Nitrogen Gas</td><td>Praxair</td><td>7727-37-9</td><td>Part Number: NI 4.8, cylinder style T, 99.998% pure nitrogen (Argon may be used as an alternative inert gas)</td></tr><tr><td>Tetramethylethylenediamine, 99% extra pure</td><td>Acros Organics</td><td>110-18-9</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Sigma Aldrich</td><td>A3678</td><td>Hygroscopic and degrades in the presence of water</td></tr><tr><td>Phosphate buffered saline tablets</td><td>Fisher Scientific</td><td>BP2944</td><td>Keep dry</td></tr><tr><td>Alginic acid, sodium salt</td><td>Acros Organics</td><td>177775000</td><td>Use heat to aid in dissolving</td></tr><tr><td>Calcium chloride dihydrate</td><td>Fisher Scientific</td><td>C79</td><td></td></tr><tr><td>Canola oil</td><td>Local store</td><td></td><td>Obtain from a local store</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>93773</td><td></td></tr><tr><td>70% (v/v) Isopropoanol</td><td>Fisher Scientific</td><td>A416-4</td><td></td></tr><tr><td>Porcine ears</td><td>Haine&#039;s Pork Shop</td><td></td><td>Obtain from a local butcher</td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S271-3</td><td></td></tr><tr><td>Human embryonic kidney 293 cells</td><td>ATCC</td><td>ATCC CRL-1573</td><td>Store in liquid nitrogen for long-term use</td></tr><tr><td>DMEM: 1x, high glucose, no pyruvate</td><td>Life Technologies</td><td>11965126</td><td>Refrigerate</td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>10082-147</td><td>Refrigerate</td></tr><tr><td>Penn Strep: 10,000 U/ml</td><td>Life Technologies</td><td>15140-122</td><td>Refrigerate</td></tr><tr><td>Trypsin-EDTA: 0.5%, 10x</td><td>Life Technologies</td><td>15400-054</td><td>Refrigerate</td></tr><tr><td>Methanol</td><td>VWR</td><td>AAA44571-K7</td><td></td></tr><tr><td>Live/Dead Cell viability kit</td><td>Life Technologies</td><td>L3224</td><td>Light sensitive, keep frozen</td></tr><tr><td>XTT cell viability kit</td><td>Sigma Aldrich</td><td>TOX2-1KT</td><td>Light sensitive, keep frozen</td></tr><tr><td>Clear DMEM: 1x, high glucose, no phenol</td><td>Life Technologies</td><td>21063-029</td><td>Refrigerate</td></tr><tr><td>Dulbecco&#039;s PBS: 1x</td><td>Life Technologies</td><td>14190136</td><td>Refrigerate</td></tr><tr><td>Sodium citrate</td><td>EMD</td><td>SX0445-1</td><td></td></tr><tr><td>Positive displacement pipette</td><td>BrandTech Scientific, INC</td><td>2702904</td><td>Dispenses 100 - 500 &amp;micro;l and comes with attachable tips</td></tr><tr><td>No 3. Stainless Steel scalpel handle</td><td>Sigma Aldrich</td><td>S2896</td><td></td></tr><tr><td>Miltex sterile surgical blades</td><td>Fisher Scientific</td><td>12-460-440</td><td>Size 10</td></tr><tr><td>Power gem homogenizer</td><td>Fisher Scientific</td><td>08-451-660</td><td>Model # 125</td></tr><tr><td>Porcelain mortar and pestle</td><td>Sigma Aldrich</td><td>Z247464</td><td>Holds 50 ml</td></tr><tr><td>FreeZone 1 L benchtop freeze dry system</td><td>Labconco</td><td>7740020</td><td>Freeze samples prior to use</td></tr><tr><td>Oil sealed rotary vane pump</td><td>Edwards</td><td>A65301906</td><td>Model # RV5</td></tr><tr><td>Incubating orbital shaker</td><td>VWR</td><td>12620-946</td><td>Model # 980153</td></tr><tr><td>Benchtop refrigerated centrifuge</td><td>Forma Scientific, INC</td><td></td><td>Model # 5682</td></tr><tr><td>Heated ovens</td><td>VWR</td><td></td><td>Model # 1235PC</td></tr><tr><td>2 N force gauge</td><td>Shimpo</td><td>FGV-0.5XY</td><td>Model # FGV-0.5XY</td></tr><tr><td>E-force test stand</td><td>Shimpo</td><td>FGS-200PV</td><td>Model # FGS-200PV</td></tr><tr><td>Tissue culture swinging bucket centrifuge</td><td>Beckman Coulter</td><td>366830</td><td>Model #6S-6KR</td></tr><tr><td>Tissue culture microcentrifuge</td><td>Eppendorf</td><td></td><td>Model #5415C</td></tr><tr><td>Hemacytometer set</td><td>Hausser Scientific</td><td>3720</td><td>Requires replacement cover glass slips</td></tr><tr><td>Slide warmer</td><td>Lab Scientific</td><td>XH-2022</td><td>Model # XH-2002</td></tr><tr><td>Portable heating lamp</td><td>Underwriters Laboratories</td><td></td><td>Helps to maintain polymer temperature at 37 &amp;deg;C</td></tr><tr><td>Inverted fluorescent microscope</td><td>Zeiss</td><td></td><td>Model Axiovert 25 CFL</td></tr><tr><td>Heated water bath</td><td>VWR</td><td></td><td>Model # 1235PC</td></tr><tr><td>Rocking platform</td><td>VWR</td><td></td><td>Series 100</td></tr><tr><td>Multiskan FC microtiter plate reader</td><td>Thermo Scientific</td><td></td><td>Type 357</td></tr><tr><td>Cell culture incubator</td><td>VWR</td><td></td><td>Model # 2350T</td></tr><tr><td>Purifier class II biosafety cabinet</td><td>Labconco</td><td></td><td>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...

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

Research

JoVE Journal

Bioengineering

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

11.8K 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

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

科研

医学

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

打印温敏反向模具图案创作的双组分水凝胶的三维细胞培养

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

免疫与感染

Composite Scaffolds of Interfacial Polyelectrolyte Fibers for Temporally Controlled Release of Biomolecules

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial polyelectrolyte complexation (IPC) fibers can be used for controlled delivery of various biological agents such as small molecule drugs, cells, proteins and growth factors. The simplicity of the methodology in making IPC fibers gives flexibility in its application for controlled biomolecule delivery. Here, we describe a method of incorporating IPC fibers into two different polymeric scaffolds, hydrophilic polysaccharide and hydrophobic polycaprolactone, to create a multi-component composite scaffold. We showed that IPC fibers can be easily embedded into these polymeric structures, enhancing the capability for sustained release and improved preservation of biomolecules. We also created a composite polymeric scaffold with topographical cues and sustained biochemical release that can have synergistic effects on cell behavior. Composite polymeric scaffolds with IPC fibers represent a novel and simple method of recreating the cellular niche.

Scaffolds for tissue engineering need to recapitulate the complex biochemical and biophysical microenvironment of the cellular niche. Here, we show the use of interfacial polyelectrolyte complexation fibers as a platform to create composite, multi-component polymeric scaffolds with sustained biochemical release.

界面聚电解质纤维的生物分子在时间控释复合支架

Various scaffolds used in tissue engineering require a controlled biochemical environment to mimic the physiological cell niche. Interfacial ...

生物工程

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D水凝胶支架的关节软骨细胞培养及软骨生成

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

分层海藻酸钠构造:一个异质细胞群的共培养平台

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

RGD功能化水凝胶作为治疗应用的工具合成

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Core/shell Printing Scaffolds For Tissue Engineering Of Tubular Structures

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the interface with a liquid core. The latter is removed post-printing, leaving behind a hollow tube. Integrating an additive manufacturing technique (like the one described here with tailor-made [bio]inks, which structurally and biochemically mimic the native extracellular matrix [ECM]) is an important step towards advanced tissue engineering. However, precise fabrication of well-defined structures requires tailored fabrication strategies optimized for the material in use. Therefore, it is sensible to begin with a set-up that is customizable, simple-to-use, and compatible with a broad spectrum of materials and applications. This work presents an easy-to-manufacture core/shell nozzle with luer-compatibility to explore core/shell printing of woodpile structures, tested with a well-defined, alginate-based scaffold material formulation.

Presented here is a simple-to-use, core/shell, three-dimensional bioprinting set-up for one-step fabrication of hollow scaffolds, suitable for tissue engineering of vascular and other tubular structures.

管状结构组织工程的核心/壳印刷支架

Three-dimensional (3D) printing of core/shell filaments allows direct fabrication of channel structures with a stable shell that is cross-linked at the ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

明胶美沙克里洛醇水凝胶生物油墨3D生物印刷方案

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

用于组织工程应用的尺寸控制和无乳化壳聚糖 -染料木酚微凝胶的制备

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that can form a structurally stable scaffold with cell-scale porosity in situ following a secondary light-based chemical crosslinking step (i.e.,&#160;annealing). MAP scaffold has shown success in a variety of regenerative medicine applications, including dermal wound healing, vocal fold augmentation, and stem cell delivery. This paper describes the methods for synthesis and characterization of poly(ethylene glycol) (PEG) microgels as the building blocks to form a MAP scaffold. These methods include the synthesis of a custom annealing macromer (MethMAL), determination of microgel precursor gelation kinetics, microfluidic device fabrication, microfluidic generation of microgels, microgel purification, and basic scaffold characterization, including microgel sizing and scaffold annealing. Specifically, the high-throughput microfluidic methods described herein can produce large volumes of microgels that can be used to generate MAP scaffolds for any desired application, especially in the field of regenerative medicine.

This protocol describes a set of methods for synthesizing the microgel building blocks for microporous annealed particle scaffold, which can be used for a variety of regenerative medicine applications.

微流体合成微孔退火粒子支架用微凝胶构建块

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that ...