作者要感谢的启动资金，并从南伊利诺伊大学医学院外科研究资助部门;和纪念医学基金会资助。

1，手术取出注意:一个IRB的批准，获得在ACL重建手术来收集的ACL残端。 IRB豁免给出所使用的ACL残端一般都弃之如手术废弃物。 20例患者用于收集的ACL残端。 <ol><li>管理全身麻醉和手术前抗生素作为每机构的协议给患者。 </li><li>应用止血带充气100毫克以上的收缩压。 </li><li>不要将病人仰卧位。做一个横向切口前外侧膝在30-45°屈曲位为5毫米的门户网站，并插入关节镜套管和相机进入髌上囊，在30℃。 </li><li>执行常规诊断关节镜检查。 </li><li>输入一个缺口，并清创膜状滑膜过十字韧带撕裂。 </li><li>使用4.5毫米剃须刀和消融仪来确定ACL残端。 </li><li>使用鸭嘴直苦收集多个SPEcimens从剩余的ACL残端。 </li><li>保持在生理盐水中的样品在无菌容器中。 </li><li>送样病理解码和最终交付给实验室设施。 </li><li>恢复病人和执行术后护理按机构的协议。 </li></ol> 2，组织消化和HACL隔离<ol><li>传输从病理接收人的ACL组织至培养皿用无菌生理盐水/ PBS（磷酸盐缓冲盐水）。 </li><li>剁碎，用无菌剪刀组织成1-2毫米3条并用盐水冲洗组织糜的至少3倍。 </li><li>消化组织糜在DMEM/F-12 0.4％胶原酶在37℃下在补充有10％胎牛血清（FBS）和1％青霉素/链霉素为4-6小时（50/50，1）培养基</li><li>离心将细胞在1000×g离心5分钟，然后重悬细胞于培养基中。 </li><li>与介质洗2〜3次的细胞，则sEED /文化T-25瓶2-3天。 </li></ol> 3，细胞膨胀，冻融<ol><li>使用光学显微镜来可视化培养细胞2-3天以T-25烧瓶中。 </li><li>保持在DMEM/F-12的细胞在37℃下在补充有10％胎牛血清（FBS）和1％青霉素/链霉素（P / S）（50/50，1×）培养基中。 注:更改介质时，细胞显示出坚持和标准形态。用光学显微镜观察细胞。 </li><li>用PBS在第7天（〜1.3×10 6个细胞/瓶）洗涤汇合细胞。添加胰蛋白酶（0.5克/升），并于室温孵育在37℃下5分钟。添加媒体到悬浮细胞中和胰蛋白酶。分三路T-25瓶之间的介质的细胞混合物中。 </li><li>用PBS洗涤汇合细胞，添加胰蛋白酶（0.5克/升），并悬浮在其中含有DMSO，FBS和完全培养基冻存培养基（DMEM/F-12培养基+10％FBS +1％P / S）中的比1:2:7。 </li><li>店该低温小瓶在-80℃下，如果细胞将在一个月内使用，并在液氮中，如果细胞将被存储为更长的持续时间。 </li><li>解冻小瓶在37℃水浴中再利用。 注:通常90-95％的细胞存活。 </li></ol> 4，组织工程化的2-D聚乳酸 - 乙醇酸共聚物（PLAGA）支架的制备<ol><li>溶解1g PLAGA的在12毫升二氯甲烷中，在20ml的闪烁瓶中，并旋涡振荡8小时的溶液以恒定的速度（每分钟800转）。 注:详细描述，Gupta 等人15。 </li><li>转移的溶解液，以玻璃培养皿内衬含氟保护纸。 </li><li>将培养皿真空罩30分钟以下。留在培养皿在-20℃下过夜，然后在室温下进行。将培养皿在冻干机，以确保溶剂完全蒸发，得到的薄膜的聚合物。 </li><li>将聚合物网络连接LMS在干燥器中放置24小时。 </li><li>切聚合物薄膜成12毫米直径的圆盘，并直到需要它们存储在干燥器中。 </li></ol> 5，扫描电子显微镜（SEM）观察<ol><li>洗涤细胞（50,000个细胞/盘）接种在控制TCPS和PLAGA支架用PBS7天之后播种。 </li><li>用在0.1M二甲胂酸盐缓冲液的1.5％戊二醛固定细胞和2.5％的OsO 4在0.1M二甲胂酸盐缓冲液后固定。 </li><li>用0.1M二甲胂酸盐缓冲液洗涤固定的细胞。采用串行乙醇脱水（50％，70％，80％，90％和100％），每次15分钟，并进一步干燥以六甲基二硅氮烷（HMDS）中过夜干燥固定的细胞。 </li><li>发泄SEM溅射涂覆系统的腔与按钮阀，提高顶板。 </li><li>将干燥的样品在室中。降低顶板。 </li><li>切换控制旋钮，水泵开始抽真空后。 </li><li>打开氩气泄漏阀。等待用于真空下降至0.05毫巴。 </li><li>大衣干燥的样品用薄金/钯层（0.13纳米）。 </li><li>返回氩气泄漏阀到它的关闭位置。 </li><li>控制旋钮切换到关闭。 </li><li>发泄室与按钮阀，提高顶板。 </li><li>取出样品，并将其储存在干燥器中放置24小时。 </li><li>观察样品的扫描电子显微镜下。 </li></ol> 6，免疫荧光染色<ol><li>洗涤细胞（50,000个细胞/盘）接种在控制TCPS和PLAGA支架用PBS7天之后播种。 </li><li>用70％乙醇（冷的）10分钟，固定细胞。 </li><li>孵育固定的细胞在室温下用1％牛血清白蛋白（BSA）的PBS中具有0.05％的Triton X-100的20分钟。 </li><li>浸没在样品中1％吐温在室温下20分钟。 </li><li>添加单克隆小鼠抗β-肌动蛋白抗体（1:400）孵育细胞持有者172吨，在4°C。 </li><li>用0.05％吐温洗细胞。二抗（山羊抗小鼠的F（ab&#39;）的IgG 2片段结合有荧光探针，1:400）添加到细胞中，并孵育1小时，在室温下进行。 </li><li>用PBS洗涤细胞。染色的细胞核染色，用80％甘油安装。 </li><li>观察共聚焦显微镜下的细胞。 </li></ol>

<ol>
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作者宣称，他们有没有竞争的财务权益。

这hACL/2D支架研究的主要目的是利用所获得的细胞修补，以增加局部ACL撕裂初级修复。部分ACL撕裂非手术治疗可能包括固定，支撑，循序渐进的康复计划，并定期随访评估13,16,17的一个短周期。然而，许多研究表明，在运动员的保守治疗已经效果差和故障有关。巴克利等人对25例局部ACL的眼泪在中间随访发现，只有44％的患者在受伤前的水平恢复运动，72％的活性相关的症状8。</sup...

前交叉韧带（ACL）是膝关节的常见的受伤关节内韧带。约200,000（ACL）损伤是在美国每年报告。超过75％的患者经历ACL损伤齐齐整形重建手术1,2,3,4。手术治疗往往是由于固有的愈合不良潜力3所示。 ACL重建通常是通过自体移植或异体移植腱的方法。自体移植和异体移植代表的金标准重建，因为他们拥有较高的成功率，并缝合修复，其他的治疗方案，已经显示出高达94％的5,6,7故障率。 该ACL部分撕裂占10％的所有ACL的眼泪8 28％。在一项前瞻性研究，诺伊斯等人估计，患者的影响超过了ACL半部分撕裂50％进展到完成的ACL功能不全后非手术治疗9。其他的研究报告持续不稳定，并与患者能够恢复到受伤前的活动水平9,10,11,12,13少于30％下降功能。治疗选择很有限，其中包括保守的方式，其余的ACL的热收缩，或ACL重建。最近，出现了增加在增强初级修复的兴趣。这些技术使用生物制剂，以加强基层缝合修复14。最近的研究已尝试收获的间充质样HACL来源的干细胞，以规避接枝限制，31,32，但这些细胞的效力和疗效尚属未知。理想的细胞来源，组织工程应用似乎非mesechymal HACL衍生的成纤维细胞。 目前的研究重点是确定一个合适的基质材料和细胞源工程支架。这是标准程序十字韧带撕裂被丢弃苏在重建手术rgical浪费，但是，这受损的韧带可能是一个质量的来源，收购，开发及提升工程的ACL替换一个理想的组织所需要的细胞。我们实验室已经开发出一种协议，用于体外扩增这些收获HACL来源的细胞。使用一个精心设计的二维矩阵注入HACL来源的细胞，我们设计了一个修补程序，可能会增加部分ACL修复和加强韧带撕裂。

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			<td style="width:143px;">Catalog Number</td>
			<td style="width:507px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri-dish</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">08-757-103C</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP3994</td>
			<td></td>
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			<td height="25" style="height:25px;width:216px;">Collagenase</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:143px;">17018-029</td>
			<td>Store at 40C</td>
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			<td height="25" style="height:25px;width:216px;">DMEM/F-12</td>
			<td style="width:139px;">Cellgro</td>
			<td style="width:143px;">10-092-CV</td>
			<td>Store at 40C. Warm in 370C water bath before use.</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:139px;">Gibco</td>
			<td style="width:143px;">10082</td>
			<td>Store at -800C</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:143px;">17-602E</td>
			<td>Store at 40C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge Tubes- 15 ml</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430790</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T-25 flasks</td>
			<td style="width:139px;">BD Falcon</td>
			<td style="width:143px;">3013</td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Trypsin-Versene mixture</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:143px;">17-161E</td>
			<td>Store at 40C. Warm in 370C water bath before use.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP231-100</td>
			<td>Combustible liquid. Can cause skin, eye and respiratory tract irritation.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryogenic vials</td>
			<td style="width:139px;">Corning</td>
			<td style="width:143px;">430489</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">PLAGA</td>
			<td style="width:139px;">Purac Biomaterials</td>
			<td style="width:143px;">Purasorb PLG8523</td>
			<td>Store at -800C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TCPS disks</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">12-545-82</td>
			<td></td>
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		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Dichloromethane</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">AC36423-0010</td>
			<td>Possible cancer hazard. Store in a dry, cool place.</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Bytac paper</td>
			<td style="width:139px;">Saint gobin performance plastics</td>
			<td style="width:143px;">1420652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scintillation vial</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td>03-339-21G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">A7906</td>
			<td>Store at 40C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tween</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP337-500</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">mouse Anti-&#946;-actin antibody (10 Ab)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">A5441</td>
			<td>Store at -200C</td>
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		<tr height="46">
			<td height="46" style="height:46px;width:216px;">goat-anti-mouse antibody (20 Ab)</td>
			<td style="width:139px;">Cell Signaling</td>
			<td style="width:143px;">4408</td>
			<td>Store at -200C</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Hoechst dye</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">14530</td>
			<td>Store at -200C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP229-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutaraldehyde</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">BP2547-1</td>
			<td>Toxic by inhalation and if swallowed. Causes burns by all exposure routes.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hexamethyldisilazane</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:143px;">AC43085-1000</td>
			<td>Flammable liquid and vapor. Causes burns by all exposure routes.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile Scissors</td>
			<td style="width:139px;">McKesson</td>
			<td style="width:143px;">25-716</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:139px;">Eppendorf</td>
			<td style="width:143px;">5804R</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Light Microscope</td>
			<td style="width:139px;">Olympus</td>
			<td style="width:143px;">CK40</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water Bath</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:143px;">2845</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:139px;">Labnet&#160;</td>
			<td style="width:143px;">VX-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Petri plates</td>
			<td style="width:139px;">fisher Scientific</td>
			<td style="width:143px;">S31473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acu-Punch</td>
			<td style="width:139px;">Acuderm Inc.</td>
			<td style="width:143px;">P1225</td>
			<td>Acu-Punch was used to cut 12mm disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cacodylate buffer</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">97068</td>
			<td>Flammable liquid, carcinogen and irritant.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Osmium tetraoxide</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">201030</td>
			<td>Highly toxic</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:143px;">T8787</td>
			<td>Harmful if swallowed</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:139px;">Decon Labs</td>
			<td style="width:143px;">2705</td>
			<td>Keep away from heat, sparks, flame and other form of ignition.</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">General/regional Anesthesia</td>
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			<td>1% Lidocaine, 0.25% Bupivacaine,Anesthetic agents for induction and maintenance</td>
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			<td>Ancef 1g i.v.</td>
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			<td height="21" style="height:21px;width:216px;">Arthroscopy trocar</td>
			<td style="width:139px;">Smith and Nephew</td>
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			<td height="21" style="height:21px;width:216px;">Arthroscopy Camera</td>
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			<td></td>
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			<td height="21" style="height:21px;width:216px;">Arthroscopic grasper and bitter</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4.5mm shaver</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Interference Screws</td>
			<td style="width:139px;">Arthrex&#160;</td>
			<td style="width:143px;"></td>
			<td>stainless steel screws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sputter Coater</td>
			<td style="width:139px;">Polaron</td>
			<td style="width:143px;">E5400</td>
			<td></td>
		</tr>
	</tbody>
</table>

surgical retrieval, isolation and in vitro expansion of human anterior cruciate ligament-derived cells for tissue engineering applications

伤到ACL是在活跃的人经常遇到的问题。即使是部分的这个关节膝关节韧带导致的损害的功能和生物力学稳定性不足的眼泪。当前选择的部分ACL撕裂的治疗范围从非手术治疗，保守管理多个手术方案，如:热改性，单束修，彻底重建，重建原生韧带的受损部分。一些研究，如果有的话，已经证明管理是一贯出众任何单一的方法，而且在许多情况下，患者继续表现出持续的不稳定等合并症。 本研究的目的是确定可能在部分十字韧带撕裂的修复来实现一个组织工程补丁的发展潜能细胞源的利用率。一种新的协议是从不稳定型心绞痛患者的细胞膨胀发展NTS进行ACL重建。为了分离细胞，ACL重建过程中获得剁碎HACL组织被消化的胶原酶溶液。用补充有10％胎牛血清（FBS）和1％青霉素/链霉素（P / S）的DMEM/F12培养基中进行扩展。然后将细胞保存在-80°C或在液氮中包括DMSO，FBS和膨胀介质的冷冻介质。融化后，将HACL衍生细胞，然后接种到组织工程支架，PLAGA（聚乳酸 - 共 - 乙醇酸）和控制组织培养聚苯乙烯（TCPS）。 7天后，扫描电镜进行到细胞粘附比拟的PLAGA与控制TCPS。采用免疫荧光染色细胞形态进行了评估。 SEM（扫描电子显微镜）显微照片表明，细胞生长和粘附这两种PLAGA和TCPS表面上，并分别由7天汇合，在整个表面上。免疫荧光染色显示正常，非胁迫形态学人图案两面上。这种技术是有前途的ACL中再生和重建应用。

的工作模式为手术取出，组织消化和人类前十字韧带（HACL）衍生细胞的分离示于图1，从外植体迁移和粘附到T-25培养瓶中的细胞。这些细胞培养3天，然后用光学显微镜（ 图2）下进行了可视化。通过7天而得到的汇合单层。健康，活细胞的存在表明HACL来源的细胞的成功的检索和文化。 细胞附着到PLAGA和TCPS的表面通过扫描电子显微镜定性测定图</s...

Watch this Scientific Journal Video about Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications at JoVE.com

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

对于未来的应用作为一个补丁来修复前交叉韧带（ACL）的部分撕裂，人类的ACL的细胞从内重建手术， 体外培养扩增和生长在组织工程支架材料获得的组织分离。然后进行细胞粘附和形态学进行确认的生物相容性支架上表面。

手术取出，分离和<em&gt;在体外</em&gt;扩展人类前十字韧带衍生细胞组织工程的应用

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical ...

surgical-retrieval-isolation-vitro-expansion-human-anterior-cruciate

Research

JoVE Journal

Bioengineering

Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

11.4K 次观看.  Southern Illinois University School of Medicine. 对于未来的应用作为一个补丁来修复前交叉韧带（ACL）的部分撕裂，人类的ACL的细胞从内重建手术， 体外培养扩增和生长在组织工程支架材料获得的组织分离。然后进行细胞粘附和形态学进行确认的生物相容性支架上表面。 

视频: Surgical Retrieval, Isolation and In vitro Expansion of Human Anterior Cruciate Ligament-derived Cells for Tissue Engineering Applications

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

科研

生物工程

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

无胸​​腺大鼠模型工程前交叉韧带移植评估

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

组织工程应用中植物组织去细胞的两种方法

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

跟踪开放全球伤害和治疗性能的前段器官文化平台

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

使用Y-27632的原发性人类金吉瓦尔表皮细胞的隔离与培养

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

山羊窒息关节髌下脂肪垫间充质干细胞的分离、扩增和分化

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...