DOZ的日内瓦基金会颁发的赠款支持。支持博士后奖学金资助从匹兹堡组织工程​​倡议的SN。

1。分离脂肪干细胞（ASC） 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>分离大鼠肾周和附睾脂肪用无菌汉克的缓冲盐溶液，含1％胎牛血清（FBS），如先前所述6（HBSS中）。 </li><li>百果组织和转移到25毫升管离心8分钟在室温和500克50毫升含1％胎牛血清的HBSS 1-2克。 </li><li>收集的自由浮动的脂肪层，并转移到125毫升三角瓶和治疗II型胶原酶（200 U / ml的）25毫升在45分钟的HBSS 37°，轨道摇床（125转） </li><li>小心地取出液体部分（油和脂肪层以下），并依次通过100μm和70μm的尼龙网过滤器过​​滤。离心滤液在500克，在室温下10分钟，吸出supernatant和25毫升的HBSS洗两次沉淀。 </li><li>悬浮细胞沉淀在50毫升的培养基中补充MesenPRO RS生长添加物，抗生素，抗霉菌（100单位/毫升100微克/毫升硫酸链霉素，青霉素G，和0.25微克/毫升两性霉素B）（MesenPRO RS基础培养基） ，2毫米到2的T75瓶（25毫升/瓶）L-谷氨酰胺和吸管细胞。 </li><li>文化ASC在37°C（使用2-4升序所有实验）在5％CO 2培养箱孵化器。 </li></ol> 2。制备壳聚糖微球（CSM） 注：所有的程序，在室温下进行，除非另有说明。 <ol><li> CSM的准备由水油乳化过程中，随着离子的凝聚技术，用我们以前的协议6。乳化在油相混合的豆浆100毫升的壳聚糖水溶液（6毫升3％W / V壳聚糖在0.5 M醋酸）油，正辛醇（1:2 V / V）和5％山梨醇单油酸酯（跨度80）乳化剂，使用开销（1700转）和磁力搅拌（1000转）同时在相反的方向。这种混合确保早期形成之前，在交联胶束可以留在溶液中，并没有解决的底部双方法。此外，磁力搅拌棒的艾滋病在聚合过程中胶束的形成和刚化壳聚糖。 </li><li>不断搅拌约1小时，直到获得一个稳定的中水乳化油的混合物。离子交联开始增加1.5毫升1％W / V正辛醇15分钟，每4小时（共24毫升）的氢氧化钾。 </li><li>交联反应完成后，慢慢地倾倒出油的混合物含有的CSM阶段，马上加入100 mL丙酮球。用丙酮清洗球，直到完全除去油相。 </li><li>干燥在真空干燥器和肛门恢复领域YZE没有进一步的处理。使用粒度分析仪测定平均CSM粒径，表面积每毫克和单位立方体体积。 </li><li>对于随后的实验中，用无菌水的CSM三次，以除去残留的盐和无水酒精消毒。 </li></ol> 3。在CSM数确定的游离氨基酸组 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>免费使用的硝基苯磺酸（TNBS）的Bubins和Ofner 7的酸法目前CSM的离子交联后的氨基酸组确定的数量。用1 mL 0.5％三硝基苯磺酸溶液50毫升的玻璃试管中4小时在40°C孵育5毫克的微球，并在60°C 3毫升6N盐酸水解时间为2h。 </li><li>样品冷却至室温，加入5毫升的去离子水和10毫升乙酸乙酯提取免费硝基苯磺酸乙醚。 </li><li> 5毫升的水相等分预热至40°C的水浴蒸发冷却至室温，任何残余的乙醚，15分钟和15毫升水稀释。 </li><li>与壳聚糖不使用三硝基苯磺酸溶液作为空白和壳聚糖为CSM准备用于确定氨基酸组总数分光光度计在345 nm处测量吸光度。估计CSM的相对壳聚糖的游离氨基酸组的数量。 </li></ol> 4。在CSM加载升序 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>平衡在一夜之间无菌的HBSS 2.5节从5毫克和消毒的CSM添加到8微米孔径的膜培养板插入（24孔板）。 </li><li>后落户到膜的CSM，小心吸的HBSS中，添加300μL培养基中生长介质的插入和700微升内的THE境外插入。 </li><li>升序重悬在适当的浓度（1×10 4 4×10 4）200μL生长介质和种子在培养板内插入的CSM。 ，播种后，内插入的文化语言的最终体积为500μL。 </li><li> CSM升序种子孵育24小时在5％CO 2培养箱孵化器在37°C。 </li></ol> 5。在CSM确定ASC的装载和细胞活力的百分比 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>孵化后，收集，而不会干扰细胞已迁移到插入膜ASC装在无菌的1.5 ml离心管的CSM。 </li><li>去除残留的培养基，并添加250μL新鲜生长培养基管。 </li><li>每管加入25微升MTT法[3 - （4,5 - dimethylthiozole -2 - 基）-2,5 - 二苯基溴化]解决方案（5毫克/毫升）和5％CO 2培养箱孵化器孵化为4小时，在37°C。 </li><li>孵化后，取出介质，添加250μL二甲基亚砜，涡2-5分钟，以溶解甲臜的复杂混合物。 </li><li>在2700 xĞCSM的离心5分钟，并确定在570作为参考使用630纳米的纳米测量上清液的吸光度。 </li><li>确定细胞数量与CSM的相对吸光度值获得从可行升序知名的关联。 </li></ol> 6。表征的ASC-CSM-嵌入式胶原蛋白凝胶 注：所有的程序，在室温下进行，除非另有说明。 <ol><li>混合ASC装CSM（含5毫克≈2×10 4细胞）1型从鼠尾腱胶原蛋白的提取，使用2列印氢氧化钠调节pH值至6.8后，根据伯恩斯坦8和fibrillate方法（7.5毫克/毫升）。 </l我&gt; <li>纤胶原蛋白ASC-CSM的混合物加入12孔板，孵育30分钟在5％CO 2培养箱孵化器在37°C。 </li><li>完成后房颤，在5％CO 2培养箱孵化至14天孵化，在37°C胶原蛋白ASC-CSM的凝胶</li><li>使用标准的显微镜技术观察从CSM的细胞到凝胶的释放和迁移。 </li></ol> 7。代表结果在本研究中，我们已经开发提供成胶原蛋白凝胶支架壳聚糖微球（CSM）的干细胞在体外战略。大小均匀（直径在175-225微米）和组成的多孔CSM的准备，并作为细胞载体（ 图2）。孵化与CSM的ASC，细胞附着在一个2×10 4 CSM的cells/5mg的浓度。这些细胞能在微蔓延，同时延长丝状伪足微多孔缝隙（ 图3）。一旦细胞加载的CSM与胶原凝胶混合，细胞立刻开始迁移到凝胶（ 图4）。 <img alt="表1" src="/files/ftp_upload/3624/3624table1.jpg" /> 表1。在干细胞输送系统使用壳聚糖和胶原蛋白的生物优势。 <img alt="图1" src="/files/ftp_upload/3624/3624fig1.jpg" /> 图1。示意图描绘两用的ASC-CSM装胶原支架的整体战略。 图2，3，4内的示意图，以帮助解释图像注释。 <img alt="图2" src="/files/ftp_upload/3624/3624fig2.jpg" /> 图2示意图描绘的干细胞接种于壳聚糖微球的过程。在proc的ESS涉及共培养与CSM在8升序 - 微米孔径的膜培养板插入。 24小时后，球从插入删除，并嵌入到生物材料的基体的准备。 <img alt="图3" src="/files/ftp_upload/3624/3624fig3.jpg" /> 图3。CSM的加载与ASC的形态特征。 A组描绘了一个ASC装的CSM光显微镜，而B组显示了同一领域的共聚焦荧光显微镜获得的图像叠加的观点。升序预装了钙黄绿素AM（绿色）。 C组描绘了一个卸载微球的SEM图像的图像，而面板D显示加载到微（星号）的细胞。 TEM图像显示在面板E卸载微球的截面。众多的孔隙和裂缝遍布微球。 F显示面板的连接并延伸到综合招聘考试的丝状伪足与细胞的横截面微球（箭头）恶习。原放大倍率：A和B = 70X，C = 500X，丁= 2000×，E及F = 2,500 X。 <img alt="图4" src="/files/ftp_upload/3624/3624fig4.jpg" /> 图4。迁移到三维胶原支架的CSM的ASC。板A和B细胞迁移从微球和胶原基质进入第3天（一个箭头）描绘的CSM。后12天，B组显示一个类似的文化。透射电子显微镜（TEM）影像描绘在C，D和E在C和D星号显示，一直与细胞迁移从微球（箭头）的横截面的微球。描述了更高的放大倍率，面板D面板E，并显示出细胞伪足的胶原纤维（插图）。原放大倍率：＆，B = 100X，C＆D = 6000 X;Ë= 20000，插图= 150,000 X。 <img alt="图5" src="/files/ftp_upload/3624/3624fig5.jpg" /> 图5。示意图描绘的CSM在再生医学和药物输送广阔的用途。

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没有竞争的金融利益存在。 免责声明所载的意见或主张是作者的私人意见，是不被理解为官方或反映国防部或美国政府部门的意见。作者是美国政府的雇员，以及作为其执行公务的一部分准备工作。所有的工作是由美国陆军医学研究和装备司令部的支持。这项研究是根据美国陆军医学研究和装备司令部机构审查委员会，并按照批准的协议，审查和批准了一项协议。

干细胞为基础的治疗的一个主要障碍是细胞运送到指定维修地区发展的有效方法。由于病人到病人的变异，组织类型，损伤的大小和深度;提供干细胞的方法，必须逐案的基础上确定。虽然嵌入矩阵内的干细胞，并提供他们的伤口，似乎是组织工程的下一个合乎逻辑的做法，一些技术上的障碍仍然存在。这包括嵌入细胞的能力附加到基体，并提供周围的生物相容性，可以附加在细胞增殖和失巢凋亡...

<table border="1" ><tbody><tr><td> 试剂/设备名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>汉克斯BalancedSalt溶液（HBSS中） </td><td> Gibco公司</td><td> 14175 </td><td>耗材</td></tr><tr><td>胎牛血清</td><td>胎牛血清</td><td> SH30071.03 </td><td>耗材</td></tr><tr><td>二型胶原酶</td><td> Sigma-Aldrich公司</td><td> C6685 </td><td>耗材</td></tr><tr><td> 70微米尼龙网过滤器</td><td> BD公司</td><td> 352350 </td><td>耗材</td></tr><tr><td> 100μm的尼龙网过滤器</td><td> BD公司</td><td> 352360 </td><td>耗材</td></tr><tr><td> MesenPRO生长介质系统</td><td> Invitrogen公司</td><td> 12746-012 </td><td>耗材</td></tr><tr><td> L-谷氨酰胺</td><td> Gibco公司</td><td> 25030 </td><td>耗材</td></tr><tr><td>的T75组织培养瓶</td><td> BD公司</td><td> 137787 </td><td>耗材</td></tr><tr><td>壳聚糖</td><td> Sigma-Aldrich公司</td><td> 448869 </td><td>耗材</td></tr><tr><td>醋酸</td><td> Sigma-Aldrich公司</td><td> 320099 </td><td>耗材</td></tr><tr><td>正辛醇</td><td> Acros Organics公司</td><td> 150630025 </td><td>耗材</td></tr><tr><td>山梨醇单声道，油酸</td><td> Sigma-Aldrich公司</td><td> S6760 </td><td>耗材</td></tr><tr><td>氢氧化钾</td><td> Sigma-Aldrich公司</td><td> P1767 </td><td>耗材</td></tr><tr><td>丙酮</td><td> Fisher Scientific则</td><td> L - 4859 </td><td>耗材</td></tr><tr><td>乙醇</td><td> Sigma-Aldrich公司</td><td> 270741 </td><td>耗材</td></tr><tr><td>硝基苯酸</td><td> Sigma-Aldrich公司</td><td> P2297 </td><td>耗材</td></tr><tr><td>盐酸</td><td> Sigma-Aldrich公司</td><td> 320331 </td><td>耗材</td></tr><tr><td>乙醚</td><td> Sigma-Aldrich公司</td><td> 472-484 </td><td>耗材</td></tr><tr><td> 8微米的细胞培养板插入</td><td> BD公司</td><td> 353097 </td><td>耗材</td></tr><tr><td> 1.5毫升离心管</td><td>费舍尔</td><td> 05-408-129 </td><td>耗材</td></tr><tr><td> MTT法试剂</td><td> Invitrogen公司</td><td> M6494 </td><td>耗材</td></tr><tr><td>二甲基亚砜</td><td> Sigma-Aldrich公司</td><td> D8779 </td><td>耗材</td></tr><tr><td> qtracker细胞标记试剂盒（问跟踪655） </td><td>分子探针</td><td> Q2502PMP </td><td>耗材</td></tr><tr><td> 1型胶原</td><td> travigen </td><td> 3447-020-01 </td><td>耗材</td></tr><tr><td>氢氧化钠</td><td> Sigma-Aldrich公司</td><td> S8045 </td><td>耗材</td></tr><tr><td> 12孔细胞培养板</td><td> BD公司</td><td> 353043 </td><td>耗材</td></tr><tr><td>离心分离</td><td> Eppendorf公司</td><td> 5417R </td><td>设备</td></tr><tr><td>轨道摇床</td><td>新不伦瑞克省Scienctific </td><td> C24的</td><td>设备</tD&gt; </tr><tr><td>湿润的孵化器与空气，5％的CO 2 </td><td> Thermo Scientific的</td><td> 370型</td><td>设备</td></tr><tr><td>架空搅拌器</td><td>伊嘉</td><td> visc6000 </td><td>设备</td></tr><tr><td>磁力搅拌器</td><td>康宁</td><td> PC-210 </td><td>设备</td></tr><tr><td>真空干燥器</td><td> - </td><td> - </td><td>设备</td></tr><tr><td>粒度分析仪</td><td>马尔文</td><td> STP2000 Spraytec </td><td>设备</td></tr><tr><td>水浴</td><td> Fisher Scientific则</td><td> isotemp210 </td><td>设备</TD&gt; </tr><tr><td>分光光度计</td><td>贝克曼</td><td>贝克曼DU800UV/Visible分光光度计</td><td>设备</td></tr><tr><td>涡旋</td><td> diagger </td><td> 3030A </td><td>设备</td></tr><tr><td>酶标仪</td><td>分子器件</td><td> SpectraMax M2的</td><td>设备</td></tr><tr><td>光/荧光显微镜</td><td>奥林巴斯</td><td> IX71 </td><td>设备</td></tr><tr><td>共聚焦显微镜</td><td>奥林巴斯</td><td> FV-500激光共聚焦显微镜</td><td>设备</td></tr><tr><td>扫描电子显微镜</td><td>卡尔·蔡司麦克风roImaging </td><td>狮子座435副总裁</td><td>设备</td></tr><tr><td>透射电子显微镜</td><td> JEOL </td><td> JEOL 1230 </td><td>设备</td></tr></tbody></table>

constructing a collagen hydrogel for the delivery of stem cell-loaded chitosan microspheres

多能干细胞已被证明是非常有用的在再生医学领域的1-3。然而，为了有效使用这些细胞的组织再生，变量的数目必须考虑。这些变量包括：植入网站的总体积和表面面积，该组织的力学性能和组织的微环境，其中包括血管的数量和细胞外基质的组成部分。因此，被用来传递这些细胞的材料必须与定义的化学成分的生物相容性，同时保持了机械强度，模仿宿主组织。这些材料还必须渗透到细胞附着和增殖提供了有利的微环境的氧气和营养物质。壳聚糖阳离子多糖，具有优良的生物相容性，可以很容易地化学改性，并具有较高的亲和力体内 MAC绑定romolecules 4-5。壳聚糖模仿外基质糖胺部分，使其能够正常细胞黏附，迁移和增殖的基质。在这项研究中，我们利用壳聚糖微球的形式提供基于胶原蛋白的三维支架6脂肪干细胞（ASC）。一个理想的细胞微球比确定培养时间和细胞密度达到最大数量的细胞可以加载。一旦升序接种到壳聚糖微球（CSM），它们被嵌入在胶原支架和可长时间保持文化。总之，这项研究提供了一种方法，准确地提供三维生物材料支架内的干细胞。

Watch this Scientific Journal Video about Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres at JoVE.com

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

在目前的干细胞疗法的一个主要障碍是确定最有效的方法来提供这些宿主组织细胞。在这里，我们描述了一个基于壳聚糖的交付方法，该方法是有效的方法简单，同时让脂肪干细胞维持其多能。

构建交付的干细胞壳聚糖微球的胶原凝胶

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

constructing-collagen-hydrogel-for-delivery-stem-cell-loaded-chitosan

Research

JoVE Journal

Bioengineering

16.7K 次观看.  United States Army Institute of Surgical Research. 在目前的干细胞疗法的一个主要障碍是确定最有效的方法来提供这些宿主组织细胞。在这里，我们描述了一个基于壳聚糖的交付方法，该方法是有效的方法简单，同时让脂肪干细胞维持其多能。

视频: Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

科研

生物工程

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

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

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

对于I型胶原蛋白的制备皮肤方法的改进

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

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

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

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

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

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

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

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

釉原蛋白 - 壳聚糖水凝胶的发展<em&gt;体外</em&gt;搪瓷愈合了密集接口

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

微型涡辅助电穿孔的顺序传递分子

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

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

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

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

重组胶原蛋白 i. 肽载体在生物反应器中的应用及其作为细胞传递系统的潜力

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Doxycycline Loaded Collagen-Chitosan Composite Scaffold for the Accelerated Healing of Diabetic Wounds

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an injury leading to delayed wound healing. We selected doxycycline (DOX), as a potential drug of choice, due to its anti-bacterial properties along with its reported anti-inflammatory properties. The current study aims to formulate DOX loaded collagen-chitosan non-crosslinked (NCL) &amp; crosslinked (CL) scaffolds and evaluate their healing ability in diabetic conditions. The characterization result of scaffolds reveals that the DOX-CL scaffold holds ideal porosity, a low swelling &amp; degradation rate, and a sustained release of DOX compared to the DOX-NCL scaffold. The in vitro studies reveal that the DOX-CL scaffold was biocompatible and enhanced cell growth compared with CL scaffold treated and control groups. The anti-bacterial studies have shown that the DOX-CL scaffold was more effective than the CL scaffold against the most common bacteria found in DW. Using the streptozotocin and high-fat diet-induced DW model, a significantly (p&#8804;0.05) faster rate of wound contraction in the DOX-CL scaffold treated group was observed compared to those in CL scaffold treated and control groups. The use of the DOX-CL scaffold can prove to be a promising approach for local treatment for DWs.

The prepared DOX-CL scaffold satisfied the prerequisites of an ideal DW dressing in mechanical strength, porosity, water absorption, degradation rate, sustained release, anti-bacterial, biocompatibility, and anti-inflammatory properties, which are considered to be essential for the recovery of damaged tissue in DWs.

多西环素加载胶原蛋白-奇托桑复合支架，用于加速治疗糖尿病伤口

One major complication of diabetes mellitus is diabetic wounds (DW). The prolonged phase of inflammation in diabetes obstructs the further stages of an ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

用于外周神经工程的石墨烯基3D生物杂化水凝胶生物墨水的制备与表征

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...

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, ...