This work was supported by NIH-NIDCR IRO1DE019355 (MJ Yost, PI), and NSF-EPSCoR (EPS-0903795).

1.确定物中的胶原蛋白批次的百分比<ol><li>提取继先前公布的程序13的胶原蛋白。解冻的最低20ml的胶原蛋白。确定以操纵在形成的水凝胶，胶原浓度在批处理胶原固体的初始百分比。 <ol><li>切三块铝箔（约6×6厘米）中，并通过用25毫升烧杯的底部形状每一个作为锅。记录每个锅的重量。 </li><li>胶原的少量添加到每个平移和记录铝锅和胶原的总重量。添加0.5 - 0.8克胶原每个铝盘。 注意:在此步骤之后，有三个铝箔盘和每一个应当有胶原的量小。确保记录每个（总共3）空铝盘的重量和锅的加成胶原后的重量。 </li><li>使用F计算胶原的重量在每一个锅ollowing公式: 胶原蛋白的重量=潘和胶原蛋白的重量 - 潘重</li><li>放置持有在100℃下在烘箱中胶原24小时三个铝盘中。 </li><li> 24小时后，记录每个铝盘的重量和脱水的胶原蛋白。 </li><li>计算使用下面的等式脱水胶原的重量: 脱水的胶原蛋白的重量=平移和脱水的胶原蛋白的重量 - 潘重</li><li>计算的固体的百分比为三个样品，使用以下公式确定胶原固体浓度: <img alt="公式（2）" src="/files/ftp_upload/53578/53578eq2.jpg" /></li><li>计算使用胶原固体为三个样本的百分比批次的平均胶原固体含量。 注意:将要使用的胶原是留下水合胶原蛋白。脱水的胶原蛋白没有将被使用。 </li><li>确定胶原固体（我的百分比后该批nitial胶原浓度），继续使用剩余的水合胶原。使用经过校准的pH计的胶原批次的pH调节至3。 <ol><li>加入少量（每次2-5微升）12 N盐酸（HCl）的。保持冰在任何时候。不直接添加盐酸以胶原 - 酸添加到该管的一侧。加酸后，用锅铲酸推到胶原蛋白，迅速搅拌混合。 </li></ol></li><li>一旦它的pH值达到3，让胶原坐O / N在4℃。 </li></ol></li></ol> 2. BSA橡胶的制备<ol><li>制备按照下面所列的步骤的牛血清白蛋白（BSA）溶液。 <ol><li>制备2倍磷酸盐缓冲盐水液（PBS）中。添加两个PBS片剂至100毫升的水，使0.02M的PBS溶液。 </li><li>结合BSA的2倍的PBS来创建使用下列顺序的30％BSA溶液。 <ol><li>例如，为了使30％的BSA用30ml 2×PBS中，使用12.9克的BSA。添加2倍的PBS（例如，10ml）中的1/3到有搅拌棒的烧瓶中。添加BSA（如，6.45克BSA）在烧瓶1/2和使用刮刀，润湿干溶质。 </li><li>重复加入PBS中，然后BSA的这个过程，直到所有的溶质和溶剂在烧瓶中。用锅铲湿所有的溶质。它看起来像一些液体和团块的混合物。让它坐大约30分钟。 </li><li>然后，打开一个小周期的搅拌器，并确保周围有搅拌棒无团块。它应该采取90分钟得到解决方案的所有或者也可以在4℃下搅拌O / N。溶质全部溶解后，放置在20毫升注射器的溶液，并用0.20微米的注射器过滤器盖上。按下柱塞通过过滤器排出液体，并收集在一个新的管中的灭菌溶液。保存在4℃。 </li></ol></li><li>准备3％戊二醛soluti通过稀释无菌过滤2X PBS 25％戊二醛溶液。例如，10毫升3％戊二醛溶液，使用2个ml等于25％的戊二醛和8毫升2×PBS中。保存在4℃。 </li></ol></li></ol> 3.治疗模具 注:本文中所描述的原型采用的是定制的不锈钢Ÿ防霉片。模具包含一个流入和分别为4和3mm两个流出通道。首先，清洁模具，喷洒与不饱和猪油和消毒他们。按照以下描述的步骤制备的模具。 <ol><li>使用超声波仪，在35千赫的频率干净的不锈钢模具。放置模具在超声波仪，用水和冰淹没它们。保持模具冷在任何时候都超声波仪运行时。运行超声波仪90分钟2​​期。 <ol><li>每一个时期后，用针，以确保没有在鲁尔无重大锁定不锈钢或bRASS连接器。用肥皂和水清洗模具双方的整个表面。验证是否有在渠道上没有障碍。 </li></ol></li><li>放置模具，螺丝，和鲁尔锁定连接​​器在高压釜中袋和高压灭菌它。 </li><li>填补重视与市售猪油（混合脂肪酸脱模剂）的空气喷雾器一半的瓶子。更换一个普通盖瓶帽。将其放置在高压釜中袋和高压灭菌它。 注意:猪油用于促进该材料，就可以注射以后反应（BSA橡胶）的释放。不要把喷雾器瓶盖在autoclave-它会损坏内部密封。 </li><li>热身猪油45秒，直到它的明确和液体微波炉。螺杆空气喷涂机盖猪油瓶。盖连接的喷雾器。附加喷雾器在实验台上的气源。打开空气阀，并打开喷雾器的喷嘴，直到它开始润湿表面的纸巾。 </li><li>喷猪油垂直于模具表面，直到表面被完全覆盖。每一块被喷后，将它们放置在培养皿和密封。放置模具在4℃下2小时。 </li><li>继续进行由表面向UV光暴露30分钟以消毒该模具。将它们放回在4°C，直到他们准备好要反应注射。 </li></ol> 4. BSA橡胶反应注射 注:所有的材料和解决方案应保持低温，直到准备使用，以防止BSA橡胶过早凝固在接下来的步骤。 <ol><li>准备分配器交付BSA橡胶以下步骤模具。 <ol><li>消毒所有混合组分（二O形环，注射器帽，双注射器，混合尖端，和4:1分配器）通过暴露于UV光在聚合酶链反应（PCR）罩30分钟。 注意:PCR罩被使用，因为Ť他的过程涉及固定剂。这些化学物质不能在细胞/组织培养橱中使用，由于曝光和毒性的细胞的风险。包含UV光的任何其它罩将是合适的。 </li><li>放置在溶液保持器的尖端帽。 </li><li> BSA的比为1:在一个4进行混合和注射戊二醛。 30％BSA加入双注射器腔，将提供的解决方案的最高金额（这将需要大约4毫升填写）。确保留有足够的空间，以O形圈放置，以防止相邻的腔室的溢流和污染。 </li><li>加入3％戊二醛溶液到另一个房间（这将需要约1毫升填写）。请务必留出足够的空间放置O形环，以防止相邻室四溢和污染。 </li><li>放置在分配器中的双注射器。垂直倾斜组件使得注射器帽是在顶部。更换混合头帽。 </li><li>钪REW两个不锈钢模具拼在一起。 </li><li>将装配高压釜袋里面。 </li><li>以除去任何空气在分配器，将其保持在直立位置，并迅速挤压手柄一次，以释放与戊二醛混合波塞少量。然后迅速装上不锈钢Ÿ模具的鲁尔锁连接器连接到注射器尖端。 </li><li>持用左手和右侧的BSA的戊二醛混合物分配器不锈钢ý模具。通过按排出到高压釜袋的侧面，以确保内部的空隙填充有溶液交替覆盖各流出通道的左侧和右侧排气。然后，水平放置的模具和再次注入。 </li><li>从分离饮水机和地点该模具在25毫米的培养皿。 </li><li>放置封口膜周围的培养皿，以防止橡胶的脱水。 </li><li>放置在4℃冰箱O / N模具。 </li></ol></li></ol>注意:胶原应在所有时间的过程中被保持在冰上。 <ol><li>修改使用胶原固体的百分比胶原浓度。 <ol><li>通过调整用冷水初始浓度胶原蛋白使10毫升1.75％的胶原蛋白。 </li><li>使用校准pH计，调节用12N盐酸将pH至3。不直接添加盐酸到胶原的酸添加到该管的一侧。加酸后，用锅铲酸推入的胶原蛋白，迅速搅拌混合。 </li><li>权衡4克胶原在一个单独的锥形管中。 </li><li>离心胶原在4℃和9,343×g离心20-30分钟以除去空气。 </li><li>紫外线消毒细胞培养罩30分钟，并添加14微升粘连到4毫升胶原。这将导致10微克/微升的最终层粘连蛋白的浓度。注:层粘连蛋白将提供关于Structural完整性，附着力，并促进各种细胞应答。 </li><li>打开PCR紫外灯上20-30分钟用消毒罩之前。 </li><li>放置路厄锁盖，附加到20ml的注射器，在乙醇中2小时。然后，使其干燥，并放置在UV光。 </li><li>伽玛辐照胶原蛋白8.6分钟达到1200 cGy的。 注意:时间将取决于铯源的衰减。调整以达到相同的剂量的时间。 </li></ol></li></ol>对BSA橡胶6.Casting胶原蛋白<ol><li>以聚合胶原，使用8:1:1的比例（胶原:HEPES:MEM）中。下面的过程是基于一个初始4克酸化胶原（来自步骤5.1.8）的。 <ol><li>使在水中的0.2N的HEPES溶液，并通过加入1-5 1M氢氧化氢氧化钠（NaOH）溶液少量（1-5微升）将pH调节至9。使用校准pH计，监测每次加入后的溶液的pH值。保存在4℃或科EP冰上。 </li><li>打开组织培养罩的UV光20-30分钟进行消毒罩。 </li><li>蒸压钳，铲，手术刀和消毒。 </li><li>拌1.5的0.2N的HEPES（pH为9）和1.5的10倍的MEM毫升利用组织培养罩毫升。请一定要保持它在冰上旋涡其5秒后才能使用。存放在4℃或保持在冰上。 </li><li>消毒的PCR罩，开启UV光30分钟。 </li><li>放置在-20℃的12孔平板和一个20毫升注射器10分钟，或直至准备继续。注意:将所有材料冷待用。温度的增加导致过早胶原原纤维。 </li><li>喷雾所有管和孔板那将是在用70％乙醇罩，并让他们干灭菌。置于冰上20毫升注射器冷却以备后用。 </li><li>在PCR罩，打开不锈钢模具释放BSA橡胶和使用手术刀，切开BSA R的排气通道ubber模具。 </li><li>下的PCR罩中，打开无菌胶原管和加入1毫升的HEPES-MEM溶液（确保提取的HEPES-MEM溶液，这是很好的混合，并且没有固体沉淀之前）。 </li><li>使用无菌刮刀，调匀胶原蛋白和缓冲溶液。 </li><li>关闭并涡旋它迅速，以确保充分混合的水凝胶。 </li><li>转移到冷20毫升注射器。 </li><li>用一只手握住的BSA橡胶阱的内部，并使用其他分配一半胶原凝胶溶液到井的底部。 </li><li>使用无菌镊子，确保橡胶流入和流出的两端接触的侧面很好。 </li><li>倒入橡胶的顶部胶原溶液直至完全覆盖。 </li><li>确保BSA橡胶胶原内暂停，不存在任何气泡，尤其是靠近橡胶的端部。 </li><li>将机盖和包装封口膜周围的阱的周边。 </li><li>把在培养箱中1小时，在37℃。保持对PCR紫外光。 </li></ol></li><li>胶原聚合后，紫外线交联通过以下过程中的水凝胶。 注意:胶原的交联将使用其中可以控制能量的量的UV交联剂装置来完成。 <ol><li>打开UV交联剂和使用的能源与环境μJ630000 /厘米2照射空室。 </li><li>从培养箱中取出凝胶。 </li><li>喷雾手用乙醇，而且，在室内，迅速取下盖越好。 </li><li>关闭室和紫外线通过选择能量设置和照射630000μJ/ cm 2的交联水凝胶。 </li><li>交联循环后，关掉紫外线灯的PCR罩</li><li>喷雾手用乙醇和打开密室，迅速将盖放回孔板。移动孔板到PCR罩。 </li><li>使用无菌刮刀，轻轻拧松并从井中取出凝胶。翻转引擎盖下的凝胶以交联水凝胶的底部。重复步骤6.2.3和6.2.4。 </li></ol></li></ol> 7. BSA橡胶酶切<ol><li>为了具有中空胶原支架，除去BSA橡胶中的方式，不影响嵌在水凝胶的尺寸。该过程描述如下。 <ol><li>打开紫外光20-30分钟来灭菌组织培养罩。 </li><li>使0.25％的胰蛋白酶溶液的pH值7.8。例如，15毫升水，在50毫升锥形管中添加0.0376克胰蛋白酶。通过加入1M NaCl的少量（2-5微升）调节pH值至7.8。放置在20毫升注射器的溶液，并用0.20微米的注射器过滤器盖上。按下柱塞通过过滤器排出液体，并收集在一个新的管中的无菌溶液。 </li><li>放水浴，将温度设定为30C。 </li><li> 30分钟后，关掉紫外线灯。喷洒在将在罩中使用的所有管和材料乙醇。 </li><li>在不同的锥形管引擎盖下发生转移的胶原凝胶。 </li><li>周围添加3-5毫升（刚好足以覆盖凝胶）的0.25％胰蛋白酶溶液，pH值为7.8至每个管。 </li><li>密封用封口膜和涡轻管约1分钟。 </li><li>发生在30℃的水浴中15-24小时。而在水浴，轻轻频繁涡凝胶直到BSA的橡胶已被消化或从水凝胶中移除。 注意:为了确定是否波塞橡胶已被去除，或者将橡胶漂浮在胰蛋白酶溶液或有破旧件。必须有水凝胶内没有视觉暗区。 </li></ol></li><li>为了确保所有的BSA的橡胶和胰蛋白酶已经从水凝胶取出，如下所述冲洗。 <ol><li>打开PCR呼ð紫外线20-30分钟。 </li><li>准备Mosconas解决方案。在水结合氯化钾（KCl，28.6毫摩尔），（ 碳酸氢钠 ，11.9毫摩尔），葡萄糖（9.4毫摩尔）和（的NaH 2 PO 4，0.08毫摩尔）。调节pH值至7.4，用1M的NaOH或12M的HCl溶液。放置在20毫升注射器的溶液和使用0.20微米的注射器过滤器进行消毒的溶液中。 </li><li>喷雾用乙醇一切之前将它们放置在引擎盖上，并允许乙醇干燥。 </li><li>打开管并转移5-10无菌Mosconas溶液中到一个新的50ml锥形管中。 </li><li>转移胶原水凝胶包含酶溶液的锥形管中。确保该水凝胶完全覆盖该溶液中。留在冰箱摇床管在4℃下30分钟。 </li><li>吸出Mosconas解决方案，并重复步骤7.2.5两次。 </li><li>保存在4℃。 </li></ol></li></ol>

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Today. 16, 496-504 (2013).</li><li>Billiet, T., Vandenhaute, M., Schelfhout, J., Van Vlierberghe, S., Dubruel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+trends+and+limitations+in+hydrogel-rapid+prototyping+for+tissue+engineering.">A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering.</a> Biomaterials. 33, 6020-6041 (2012).</li><li>Yost, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+tubular+scaffold+for+cardiovascular+tissue+engineering.">A novel tubular scaffold for cardiovascular tissue engineering.</a> Tissue Eng. 10, 273-284 (2004).</li><li>Habeeb, A. J., Hiramoto, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+of+proteins+with+glutaraldehyde.">Reaction of proteins with glutaraldehyde.</a> Arch Biochem Biophys. 126, 16-26 (1968).</li><li>Tanford, C., Buzzell, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Viscosity+of+Aqueous+Solutions+of+Bovine+Serum+Albumin+between+pH+4.3+and+10.5.">The Viscosity of Aqueous Solutions of Bovine Serum Albumin between pH 4.3 and 10.5.</a> J. Phys. Chem. 60, 225-231 (1956).</li><li>Yadav, S., Shire, S. J., Kalonia, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viscosity+analysis+of+high+concentration+bovine+serum+albumin+aqueous+solutions.">Viscosity analysis of high concentration bovine serum albumin aqueous solutions.</a> Pharm Res. 28, 1973-1983 (2011).</li><li>Tobitani, A., Ross-Murphy, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intrinsic+viscosity+of+polyelectrolytes+revisited.">The intrinsic viscosity of polyelectrolytes revisited.</a> Polym. Int. 44, 338-347 (1997).</li><li>Arakawa, T., Timasheff, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+protein+solubility.">Theory of protein solubility.</a> Methods Enzymol. 114, 49-77 (1985).</li><li>Migneault, I., Dartiguenave, C., Bertrand, M. J., Waldron, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutaraldehyde:+behavior+in+aqueous+solution,+reaction+with+proteins,+and+application+to+enzyme+crosslinking.">Glutaraldehyde: behavior in aqueous solution, reaction with proteins, and application to enzyme crosslinking.</a> Biotechniques. 37, 790-796 (2004).</li><li>Burmeister, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutaraldehyde+cross-linked+glutamate+oxidase+coated+microelectrode+arrays:+selectivity+and+resting+levels+of+glutamate+in+the+CNS.">Glutaraldehyde cross-linked glutamate oxidase coated microelectrode arrays: selectivity and resting levels of glutamate in the CNS.</a> ACS Chem Neurosci. 4, 721-728 (2013).</li><li>Chatterji, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelatin+with+hydrophilic/hydrophobic+grafts+and+glutaraldehyde+crosslinks.">Gelatin with hydrophilic/hydrophobic grafts and glutaraldehyde crosslinks.</a> J. Appl. Polym. Sci. 37, 2203-2212 (1989).</li><li>Freije, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+modified,+immobilized+trypsin+reactor+with+improved+digestion+efficiency.">Chemically modified, immobilized trypsin reactor with improved digestion efficiency.</a> J Proteome Res. 4, 1805-1813 (2005).</li><li>Cunha-Filho, M. S., Alvarez-Lorenzo, C., Martinez-Pacheco, R., Landin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-sensitive+gels+for+intratumoral+delivery+of+beta-lapachone:+effect+of+cyclodextrins+and+ethanol.">Temperature-sensitive gels for intratumoral delivery of beta-lapachone: effect of cyclodextrins and ethanol.</a> ScientificWorldJournal. 2012, 126723 (2012).</li><li>Basak, R., Bandyopadhyay, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+hydrophobic+drugs+in+Pluronic+F127+micelles:+effects+of+drug+hydrophobicity,+solution+temperature,+and+pH.">Encapsulation of hydrophobic drugs in Pluronic F127 micelles: effects of drug hydrophobicity, solution temperature, and pH.</a> Langmuir. 29, 4350-4356 (2013).</li></ol>

作者什么都没有透露。

生物制造是在生物学和工程学原理合并产生模仿自然组织复杂的材料高度多学科领域。为了达到这个目的，有必要开发使用从体内组织收集到的信息，并将其转换成一个在体外的支架技术。以这种方式，平台可改造成非常类似于体内组织的建筑，功能，和机械性能。最佳的支架材料应具有一定的性质，例如是生物相容的，模仿的感兴趣的组织的机械性能，能够受控降解的，能?...

在组织工程领域，制作组织支架的能力是至关重要的。合适的组织支架具有三维结构，是由生物相容的材料制成，并在体内组织架构模拟物，以促进细胞和组织的生长和重建。该支架必须允许营养物质的运输和清除废物1-4。一种在生产这些支架的主要障碍是概括特定几何特征成生物相容的材料的能力。几个生物制造技术已经报道，以控制这些支架的几何特征，实施例是静电5-8，溶剂浇铸9，立体光刻10，和3D印刷11，等等。这些技术达不到在提供相对容易的可控内部和外部的建筑特色转移，是昂贵的，由它们的分辨率和印刷性能（限<em&gt;例如，喷嘴计，材料限制），或需要制造后技术，这需要的时间，以产生可行的支架12长的时间。 在许多商业制造系统，内部空隙，通道和特征的创建是用砂或其他合适的可移动或牺牲材料来实现。的金属或塑料部件围绕砂模形成，并且一旦它被固化时，砂被去除。在大致相同的方式，下一代生物材料的所需要的生物沙等效。因此，波塞橡胶被开发作为用于生物沙的替代物。 BSA的橡胶是由具有戊二醛交联的牛血清白蛋白的一个新制定的材料。最终的目标是具体的建筑特色重建成可生物降解的胶原支架。维持二维保真度与原组织的模具牺牲biorubber的特性进行说明。 <p cl屁股="jove_content"&gt; BSA和戊二醛浓度的几种组合进行使用各种溶剂进行测试。该物质经BSA和戊二醛之间的反应产生。 BSA的橡胶可以是注入组织模具的复杂的几何形状的反应。交联的BSA的胰蛋白酶不稳定，并通过在温和的pH和温度条件下的酶容易地消化。相反，完整的I型胶原蛋白是胰蛋白酶消化非常有抗性。这些特征被资本以选择性地移除的BSA橡胶留下胶原后面。目前的工作包括确定获得不稳定的模具，可以到生物相容支架提供特定的建筑特色所需要的理想的参数。进行了评价，该具体特征包括混合性，酶消化，承载，和被注入阴模的反应能力。 30％BSA和3％戊二醛的组合满足这些要求。这个协议提供了necess元指导创建这些三维支架。所述原型包括表示与一个流入和直径分别为4-和3毫米，二流出通道的支链结构的胶原蛋白支架的。该技术具有模仿感兴趣的组织的宏观和微观环境的潜力。这种技术提供了一个可行的技术，以在以高的保真度，其可被调谐以模仿体内组织的弹性和感兴趣的组织的其他特性的相对容易且及时物质递送的特定几何启发可生物降解的材料。

<table><tbody><tr><td>Collagen type I</td><td></td><td></td><td>Collagen extracted from calf hide</td></tr><tr><td>Hydrocloric Acid (HCl)</td><td>Sigma-Aldrich</td><td>7647-01-0</td><td></td></tr><tr><td>Phosphate Buffer Solution (PBS Tablets)</td><td>MP Biomedical</td><td>U5378</td><td>1 tablet per 100 ml makes 1x PBS</td></tr><tr><td>Albumium from bovine serum (BSA)</td><td>Sigma-Aldrich</td><td>A9647</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Sigma -Aldrich</td><td>G5882</td><td>Toxic</td></tr><tr><td>Lard</td><td>Fields</td><td>3090</td><td></td></tr><tr><td>Stainless Steel Molds</td><td></td><td></td><td>Milled using Microlution Machine</td></tr><tr><td>Air Brush Kit</td><td>Central Pneumatic</td><td>47791</td><td></td></tr><tr><td>Mixing Tip for double syringe</td><td>Medmix</td><td>ML2.5-16-LLM</td><td>Mixer, DN2,5X16, 4:1 brown, med</td></tr><tr><td>Small O ring for double syringe</td><td>Medmix</td><td>PPB-X05-04-02SM</td><td>Piston B, 5 ml, 4:1, PE natural</td></tr><tr><td>Double Syringe cap&amp;nbsp;</td><td>Medmix</td><td>VLX002-SM</td><td>Cap, 4:1/10:1, PE brown, med</td></tr><tr><td>Big O ring for double syringe</td><td>Medmix</td><td>PPA-X05-04-02SM</td><td>Piston A, 5 ml, 4:1</td></tr><tr><td>Double Syringe</td><td>Medmix</td><td>SDL X05-04-50M</td><td>Double syringe, 5 ml, 4:1</td></tr><tr><td>Double Syringe Dispenser</td><td>Medmix</td><td>DL05-0400M</td><td>Dispenser, 5 ml, 4:1, med , plain</td></tr><tr><td>Laminim</td><td></td><td></td><td>3.6 mg/ml - extracted USC lab</td></tr><tr><td>20 ml Syringe Luer Lock Tip</td><td>BD</td><td>302830</td><td></td></tr><tr><td>Luer Lock Caps</td><td>Fisher</td><td>JGTCBLLX</td><td></td></tr><tr><td>HEPES</td><td>Sigma -Aldrich</td><td>H4034</td><td></td></tr><tr><td>Gibco Minimum Essential Media 10x (MEM)</td><td>Life Technologies</td><td>1143-030</td><td></td></tr><tr><td>Trypsin</td><td>Life Technologies</td><td>27250-018</td><td></td></tr><tr><td>UV Crosslinker&amp;nbsp;</td><td>Spectroline UV</td><td>XLE1000</td><td></td></tr><tr><td>Sodium Cloride (NaCl)</td><td>Fisher</td><td>S271-10</td><td>To prepare Mosconas</td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma -Aldrich</td><td>P5405-250</td><td>To prepare Mosconas</td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Fisher</td><td>BP328-500</td><td>To prepare Mosconas</td></tr><tr><td>Glucose</td><td>Sigma -Aldrich</td><td>G-8270</td><td>To prepare Mosconas</td></tr><tr><td>Sodium Phosphate didasic (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S-7907</td><td>To prepare Mosconas</td></tr><tr><td>Sterile Filter for syringes</td><td>Corning</td><td>431224</td><td></td></tr></tbody></table>

three-dimensional biomimetic technology: novel biorubber creates defined micro- and macro-scale architectures in collagen hydrogels

组织支架起到组织再生过程中的关键作用。理想的脚手架必须满足几个条件，如适当有组成，有针对性的模量，以及明确的建筑特色。该概括体内组织的内在结构的生物材料可用于研究疾病，以及便于丢失和畸形软组织的再生是至关重要的。一种新型的生物制造技术的开发，结合​​了先进的成像，三维（3D）打印，和选择性酶活性的国家创造了新一代​​生物材料的研究和临床应用。开发材料，牛血清白蛋白橡胶，是注入一个维护特定几何特征的模具的反应。这种牺牲材料允许建筑特色的充分转移到天然支架材料。所述原型包括一个三维胶原蛋白支架的带4和3mm渠道再版ESENT支架构。本文强调的自然结构的产生使用这种生物制造技术。这个协议利用计算机辅助软件（CAD）来制造固体模具，这将是用BSA橡胶接着橡胶的酶消化注入反应，而使支架材料内的建筑特色。

的结果表明，该生物制造技术是在产生三维支架，可以模仿体内组织中所看到的空间排列高效。建筑特色是用于组织工程应用的重要参数，打在组织的体内细胞相互作用和功能的关键作用。 BSA的橡胶的一致​​性和混合能力是在生产的BSA橡胶是均匀的，并能保持其预定形状的重要参数。蛋白质的溶解度是通过分?...

Watch this Scientific Journal Video about Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels at JoVE.com

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

three-dimensional-biomimetic-technology-novel-biorubber-creates

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.1K 次观看.  Medical University of South Carolina. An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

视频: Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

使用动态面膜投影光刻系统的神经文化Micropatterned水凝胶的制备

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

科研

生物工程

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

便于操作的体系结构基于蛋白质的凝胶中为单元格文化应用程序

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

生物化学

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. ...

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 ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

构建模块化水凝胶片的微模式化宏观缩放3D细胞结构

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

3D Microtissues 用于可注射再生疗法和高通量药物筛选

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Interlinked Macroporous 3D Scaffolds from Microgel Rods

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions without further additives. Continuous photoinitiated on-chip gelation enables variation of the microgel aspect ratio, which determines the building block properties for the obtained constructs. Glycidyl methacrylate (GMA) or 2-aminoethyl methacrylate (AMA) monomers are copolymerized into the microgel network based on polyethylene glycol (PEG) star-polymers to achieve either epoxy or amine functionality. A focusing oil flow is introduced into the microfluidic outlet structure to ensure continuous collection of the functionalized microgel rods. Based on a recent publication, microgel rod-based constructs result in larger pores of several hundred micrometers and, at the same time, lead to overall higher scaffold stability in comparison to a spherical-based model. In this way, it is possible to produce higher-volume constructs with more free volume while reducing the amount of material required. The interlinked macroporous scaffolds can be picked up and transported without damage or disintegration. Amine and epoxy groups not involved in interlinking remain active and can be used independently for post-modification. This protocol describes an optimized method for the fabrication of microgel rods to form macroporous interlinked scaffolds that can be utilized for subsequent cell experiments.

Microgel rods with complementary reactive groups are produced via microfluidics with the ability to interlink in aqueous solution. The anisometric microgels jam and interlink into stable constructs with larger pores compared to spherical-based systems. Microgels modified with GRGDS-PC form macroporous 3D constructs that can be used for cell culture.

来自微凝胶棒的互连大孔3D支架

A two-component system of functionalized microgels from microfluidics allows for fast interlinking into 3D macroporous constructs in aqueous solutions ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

具有长距离纤维排列的微工程3D胶原蛋白水凝胶

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...