支持这项工作是由西悉尼大学研究资助。

1。壳聚糖黏胶剂<ol><li>脱乙酰壳多糖粉末溶解在乙酸溶液中，用于制备乙酸2％（体积/体积）的储备液，添加10毫升冰醋酸的490毫升去离子水（DI-H 2 O）。 </li><li>如果在孟加拉玫瑰红（RB）在乙酸（0.01％重量/体积）的储备液的制备中，称量5毫克的RB的小瓶中，用铝箔包裹，以避免光漂白。 DI-H 2 O中加入0.5 ml溶解的粉末，然后加49.5毫升的醋酸原液（详见第1.1节）。 </li><li>为了制备脱乙酰壳多糖溶液（1.7％w / v的），重0.85克脱乙酰壳多糖粉末（中等分子量，85％的乙酰化度），并加至50毫升的RB乙酸溶液。制备脱乙酰壳多糖溶解在乙酸溶液中没有RB的控制。 </li><li>加入磁力搅拌器的脱乙酰壳多糖的混合物，并在室温下搅拌。为了确保各组成部分的完全溶解，搅拌几丁聚糖乙酸+ RB两星期和壳聚糖在乙酸中5天（对照组）。当壳聚糖溶解，溶液的pH值增加〜2.6〜3.9。请注意，RB是难溶的，在酸性pH值，而且它需要在溶液的pH值的增加和扩展的搅拌时间以溶解。 </li><li>离心机的解决方案，在3270×g离心1小时，并收集上清液，使用，例如，一个无菌的10毫升注射器。确保不打扰杂质颗粒。在此步骤中，将溶液从宏观的杂质和不溶性物质清洗。 </li><li> （离心）壳聚糖溶液注入一个的平板perpex板使用无菌注射器。干燥后的膜的厚度取决于所分配的量的溶液，并超过该溶液扩散的表面积之间的比值。确保该解决方案是无气泡，均匀地涂在板。 〜13微米厚度的薄膜，注入1毫升溶液超过12厘米的表面面积（尺寸〜3×4厘米， 图1A）。 </li><li>量，制成铝箔例如，带有屏幕的有机玻璃板坯的解决方案，以避免光漂白。确保足够的空气流通，保证干燥的解决方案，在屏幕上。 </li><li>发布的溶液在室温下（25℃）和压力（1个大气压），直到干燥，将所得的膜是不溶于水和不膨胀宏观。这通常是在2个星期。 </li><li>谨慎使用一个薄和平面抹刀分离的影片，这些影片的有机玻璃板可以很容易地剥离而不损坏它们。 </li><li>测量的膜的厚度，使用测微计。 </li><li>长方形的胶粘剂用干净锋利的剪刀剪下，并把他们之间的无菌载玻片上，以保持其平面形状。包裹在铝箔滑动遮光，并将其储存在室温下。标签的粘合剂，带或不带包"玫瑰胶"或"壳聚糖adhes香港专业教育学院"。 </li><li>上升的粘接膜，可以用乙醇（90％）或伽马辐射（0.1千戈瑞/ h的24小时）4,5消毒。 </li></ol> 2。粘合型光学衰减<ol><li>石英比色皿内固定的玫瑰粘合剂。 </li><li>记录其衰减的频谱，在400 - 800nm ​​的使用双光束分光光度计4的范围内。 </li><li>假设的有效性Beer定律，计算的粘接剂的衰减长度如下:I = I 0 电子Ax的其中 I 0是入射光强度，1 / A是的衰减长度，和x是膜的厚度。 </li><li>重复步骤2.1至2.3的壳聚糖胶粘剂作为对照。 </li></ol> 3。 在体外应用程序:激光对生物组织焊接的小肠<ol><li>收获小牛肠后，立即动物安乐死并储存于-80°C。 </li><li>在使用之前，除霜和水合物的TIssue浸渍DI-H 2 O中的10分钟。 </li><li>平分组织成在手术显微镜（×10）下使用＃10叶片的部分（2×1厘米）。使用DI-H 2 O的部分保持湿润</li><li>带来的切口树桩，并拢（端到端）。擦拭多余的水分从表面采用纯棉提示。 </li><li>与microforceps与肠道内（ 图2）确保充分的接触，将玫瑰跨越切口浆膜层上的粘合带（10×6毫米）。 </li><li>玫瑰粘合剂的组织粘附性的被激活的二极管泵浦的固态激光器，它被耦合到多模光纤（芯直径200微米）4。 </li><li>设置到第180毫瓦的激光的功率电平，并照射的粘接剂〜6分钟〜5毫米的束斑大小，使用在表1中列出的参数。现货照射的粘合剂，确保〜5秒前束移动到相邻的点，每个点的照射。 Ţ他的程序保证，在激光束扫描的整个表面区域的粘接剂数次。 </li><li>为了评估的组织粘结强度，到一个经过校准的单柱张力（英斯特朗，MA，USA）使用机械夹具夹紧试样。将掌握在22毫米/分钟，直到两个组织的树桩单独的4。 </li><li>放置在步骤3.5中所描述的组织上的玫瑰粘接剂。不要照射样品，测量组织的接合强度，如在步骤3.8所述。样本作为对照。 </li></ol> （4） 在体内应用:胫骨神经吻合术<ol><li>稳重的Wistar大鼠异氟醚/ O 2混合物（4％，2％，此后在诱导）5。皮下注入Buprenex（0.03毫克/千克）提供手术镇痛活性，并减轻可能出现的疼痛或不适。 </li><li>应在手术显微镜（×10）下进行以下的外科手术。斜皮肤切开约3厘米长在后背成的右大腿外侧部和暴露胫骨神经与肌肉分离的办法，通过臀肌2。 </li><li>部分解剖和胫神经外膜采用直板微剪刀修剪，用无菌纱布或棉花提示，并吸收多余的水分。 </li><li>微型剪刀切割胫神经。 </li><li>放置一个无菌壳聚糖条（尺寸为5×4毫米）使用微镊子下方的胫骨神经。 </li><li>约神经树桩端至端的微型镊子在玫瑰粘合带。 </li><li>带钢完全附着到神经形成的轴环，它可以协助的带状激光照射期间与旋转的神经。 </li><li>激活玫瑰粘合剂用激光中描述的步骤3.6）和3.7）。 </li><li>修剪多余的玫瑰胶粘剂的衣领手术的神经。 </li><li>关闭的肌肉和皮肤与5 3-0缝合线5。 </lI&gt; <li>标准的手术后恢复应遵循的程序，包括日常检查，开裂或脓液形成和镇痛药，如果需要的话。 </li></ol> 5。代表性的成果所得到的薄膜是在色彩明亮的上升，超薄，具有光滑的表面（ 图1）。他们也很灵活，可以卷成小直径管，而不会造成破损或任何其他明显的损伤（ 图1B）。玫瑰粘合剂具有两个吸收峰，在​​530和562 nm的绿色激光，因此强烈地吸收在532 nm处的粘接剂和相应的衰减长度为〜12微米（ 图3）。相反，壳聚糖，薄膜没有RB衰减弱激光（1 / A〜162微米），可能是由于散射。从光谱图，没有显着的聚集RB​​出现在电影中。 玫瑰粘合剂粘合牢固激光照射后的肠道达到破坏时的一个典型的最大负载为0.9±0.4 N组（n = 30）（ 图2）。的粘合强度，估计作为除以由粘接剂的表面面积，即，15±6 kPa组（n = 30）的最大负载。非照射玫瑰粘合剂粘合显着少到组织（2±2千帕，n = 30的非配对t检验p &lt;10 6）。成功地实现了连同绿色激光的应用程序和玫瑰粘合剂粘接后的被检体内的胫神经吻合。术后1周，手术神经连续性和维修强度为17±9千帕（N = 10）。 <img alt="图1" src="/files/ftp_upload/4158/4158fig1.jpg" /> 图1:A）RB-壳聚糖溶液浇铸在平坦的有机玻璃板（楼板厚度约5毫米）。绘图纸被置于下面的板坯，以促进干铸工序。水不溶性fILM是两个星期后铸造。 B）玫瑰胶是灵活的，可以卷成小圆筒。 <img alt="图2" src="/files/ftp_upload/4158/4158fig2.jpg" /> 图2。玫瑰粘接剂粘接到激光照射后的小牛肠。上施加均匀的照射组织接合过程中的RB粘合剂，但是选择的点在这张图片中所示，来说明上的粘接剂的激光光漂白效果。 <img alt="图3" src="/files/ftp_upload/4158/4158fig3.jpg" /> 图3。玫瑰粘合剂的吸收光谱（在任意单位中）显示了在530和562 nm处的两个峰。从而强烈地吸收由激光曝光过程中的粘接剂的绿色激光器（λ= 532nm）的。壳聚糖粘合剂没有RB吸收不佳的激光束。 <table border="1"><tbody><tr><td></td><td> Ň </td><td><s豪华型&gt;面积（mm 2） </td><td> 功率（W） </td><td> 时间（s） </td><td> 能量密度（J /厘米2） </td><td> I（瓦/厘米2） </td><td> 最大负荷/面积（千帕） </td></tr><tr><td> 胶+激光 </td><td> 30 </td><td> 60±10 </td><td> 0.18±0.03 </td><td> 365±5 </td><td> 〜110 </td><td> 〜0.9 </td><td> 15±1 </td></tr><tr><td> 胶粘剂 </td><td> 30 </td><td> 60±10 </td><td> NA </td><td> NA </td><td> NA </td><td> NA </td><td> 2±2 </td></tr></tbody></table> 表1。传奇。 N，样本数;面积，玫瑰（平均±最大误差）粘合剂的表面面积;功率，激光功率（平均±最大误差）;时间，照射时间（平均±最大误差）;注量，平均激光能量密度，我，预计内部收益率adiance;最大负荷/地区，在故障修复组织的粘合剂表面面积除以最大负载，（平均值±SE）。

<ol>
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没有利益冲突的声明。

玫瑰粘合剂制造是基于干铸在一个简单的过程，但在酸性pH条件下，需要延长的RB的溶解的脱乙酰壳多糖溶液中搅拌。这是重要的，让溶液干涸，直到它成为一个水不溶性膜。这种情况发生时的重量的水的含量为〜10％，在干燥的膜6。不溶性的膜通常是获得干铸造后2个星期，在标准条件下的温度和压力（〜25℃，1个大气压）。组织接合的机理尚未完全清楚，但有人指出，RB扩散从进入相邻?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>孟加拉玫瑰红</td><td> Sigma-Aldrich公司</td><td> 632-69-9 </td><td></td></tr><tr><td>壳聚糖（MW中等） </td><td> Sigma-Aldrich公司</td><td> 10318AJ </td><td></td></tr><tr><td>冰醋酸</td><td> Sigma-Aldrich公司</td><td> 08050051 </td><td> 2％（体积/体积）在DI水</td></tr><tr><td>磁力搅拌器</td><td> Heidolph </td><td>陈喜混合小号</td><td></td></tr><tr><td>离心分离</td><td> Beckman Coulter公司</td><td> Allegra的X-12R </td><td></td></tr><tr><td>分光光度计</td><td>瓦里安</td><td>卡里4000紫外 - 可见</td><td></td></tr><tr><td>激光</td><td> CNI激光</td><td> MGL-532 </td><td></td></tr><tr><td>千分尺</td><td>三丰</td><td>系列227 </td><td></td></tr><tr><td>手术显微镜</td><td>蔡司</td><td> OPMI </td><td></td></tr></tbody></table>

fabrication and application of rose bengal-chitosan films in laser tissue repair

光化学组织结合（PTB）是一种无缝合的组织修复的技术，这是实现通过施加一个解决办法，玫瑰红（RB）之间的两个组织的边缘1,2。然后这些被选择性地吸收由RB通过激光照射。据说所得的光化学反应交联，在组织中的胶原纤维与最小的热量生产3。在这份报告中，RB已成立于薄的壳聚糖膜制造一个新的组织粘合剂，是激光激活。基于脱乙酰壳多糖和含有〜0.1％（重量）RB，粘接膜，制造和粘接小牛肠和大鼠胫神经由固态激光器（λ= 532nm的，注量〜110焦耳/厘米2，光斑大小〜0.5厘米） 。一个单一的列的张力计，与个人计算机连接，用于测试的接合强度。 RB-壳聚糖粘合剂粘合牢固，肠强度为15±6千帕（N = 30）。的粘接强度下降到2＆plusmn; 2千帕（= 30）时，激光不施加到粘合剂。胫神经的吻合，也可以不使用的缝合线的情况下完成。一种新型的壳聚糖胶粘剂已债券光化学组织制造的，不需要缝合。

Watch this Scientific Journal Video about Fabrication and Application of Rose Bengal-chitosan Films in Laser Tissue Repair at JoVE.com

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

缝线在手术过程中，通常需要修复组织。然而，它们的应用可能是有问题的，因为它们是侵入性的，并且可能会损坏组织。新的组织粘合剂的制备及应用方法，在这里报道。该粘合膜的激光激活的，并且不需要使用缝线。

激光对生物组织的修复孟加拉玫瑰红壳聚糖膜的制备及应用

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

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12.1K 次观看.  University of Western Sydney, NSW Australia. 缝线在手术过程中，通常需要修复组织。然而，它们的应用可能是有问题的，因为它们是侵入性的，并且可能会损坏组织。新的组织粘合剂的制备及应用方法，在这里报道。该粘合膜的激光激活的，并且不需要使用缝线。

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

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

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

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

心肌组织工程生物衍生的注射材料的制备

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

科研

生物工程

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

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

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

电场控制的二维和三维环境中的神经祖细胞定向迁移

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

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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

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

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

肿瘤微环境基质与应用癌细胞侵袭组织工程

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

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

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

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

三维全层皮肤的生成等效和自动伤人

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

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

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

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

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

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

影像引导下，血管源性微流控网络的基于激光的研制

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...