笔者想感谢加布里埃尔芳香化酶和迈克尔Yeranosian其早期这个项目的迭代技术贡献。该项目资金由临床医生OREF科学家培训格兰特（NL），HH李外科研究基金（NL），退伍军人管理局BLR＆D优异回顾1 I01 BX00012601（DM）和肌肉骨骼移植基金会青年科学家奖（FP）。

注：所有动物手术均经当地兽医人员和动物使用前开始试验委员会。 1.准备静电纺丝聚己内酯的支架<ol><li>在颗粒形式称重并溶解医用级酯封端的聚（ε - 己内酯）在1,1,1,3,3,3-六氟-2-丙醇以创建10％w / w的在PCL聚合物的溶液。让使用搅拌盘为至少3小时，以确保均匀的溶液的溶液搅拌。 </li><li>静电纺丝的PCL解决方案来创建高度一致PCL纤维支架制造的袖口。 <ol><li>制备在化学通风橱电纺丝设置与在任何时候都将风扇。这由一个大的丙烯酸框，将作为电纺丝过程的分离的真空介质，并具有入口点由一个电压源，该马达驱动的心轴收集和真空端口驱动源的PCL溶液。清洁acryliC箱彻底用乙醇和盖用Parafilm薄片所有的表面，以除去任何杂质，可能损害静电的产品的质量。 </li><li>截至18 G，1½英寸针负荷约3毫升上述溶液置于10 ml注射器用钝的。通过推动注射器向上除去任何气泡。封管溶液到可编程注射器泵。将通过一个小孔，放入丙烯酸盒针而离开约半针英寸的亚克力盒外面附着电压源线。 </li><li>使用30毫米的旋转车床心轴作为集电极的高度对齐的PCL纤维;覆盖心轴紧紧用薄铝箔带。锁心轴插入包装盒上的相对侧约15cm距离注射器针头的马达。 </li><li>将塑料软管进入真空口，并连接到通风柜真空源。打开真空源上并盖上盖子丙烯酸框。 </li><li> S等可编程注射器泵至2.5毫升/小时的输注速率。转动电机上操作的心轴在3450转，并附加电压源的正极引线到针尖使用鳄鱼夹框外。 </li><li>一旦输注在PCL溶液中已经开始，接通电压源并设置到20千伏的操作电压。 </li><li>注入的溶液12分钟，以0.5毫升的PCL溶液形成均匀箍。 注：平均来说，每个翻边有足够的电纺材料，以创建两个六薄带材，它可用于创建总共三个四层状支架。 </li></ol></li><li>激光切割的PCL箍上形成在低真空环境，10.0 keV的着陆电压，6.4毫米的工作距离，以及3.0探头直径操作的VersaLaser切纸机2.3多个小片。 注意：在本实施例中，计算机辅助设计被用于指示切割器，以产生1.5mm的多张纸×35毫米×150微米的支架与均匀分布的150微米直径的孔在15％的孔面积。 </li><li>等离子体蚀刻使用等离子体清洁器以诱导PCL表面与离子加速的亲水的PCL支架。将真空到450毫托和治疗支架30秒在高功率（29.6 W）。 </li><li>沐浴在70％的乙醇的支架在无菌环境中。 </li><li>外套与胶原各个支架，以促进体内细胞的粘附和增殖。 <ol><li>在4℃下9在1×PBS中：1：的Purecol胶原3毫克/毫升标准溶液，10倍的PBS和0.1N氢氧化钠1 2.5无菌溶液通过稀释8创建胶原涂布溶液。调匀，以确保解决方案同质性。 </li><li>外套个别1.5毫米×35毫米×150微米的支架与上述胶原溶液的薄层的膜。允许在无菌环境中晾干24小时。 </li></ol></li><li>使用5-0薇乔缝线，堆叠并加盖四个单项1.5毫米×35毫米×150微米支架使用Krackow缝合以创建最终0.6mm厚，多层，胶原包被的支架是准备用于植入。 </li></ol> 2.鼠手术协议<ol><li>通过将大鼠在吸入腔和递送2％异氟烷以2升/分钟的氧气诱导麻醉。确认大鼠被适当施加压力后足和评价为任何响应麻醉。 </li><li>在非无菌的表背，皮下注射25mg / kg的氨苄青霉素和0.03毫克/公斤丁丙诺啡。 </li><li>应用眼药膏眼睛。剪辑从手术后肢的毛皮和用洗必泰三个交替磨砂和70％的乙醇预习手术部位。 </li><li>大鼠转移到手术台，在加热垫，以防止体温过低。安全鼻锥，并通过在2升/分钟的氧气，用2％异氟醚的方法维持麻醉，递送鼻锥体。悬垂以无菌的方式，使手术肢体露出。 </li><li&gt;在开始手术前，先确认足够的麻醉由缺乏反应后足的压力。 </li><li>使2厘米长的纵切口内侧到膝盖，中心在髌骨的水平。收回的皮肤横向直到切口集中在膝盖。 </li><li>使用手术刀切只是内侧至髌骨和股四头肌和远侧到在胫骨结核杆菌髌腱插入水平的肌腱结的水平向近侧延伸，使髌旁内侧入关节切开术。小心不要削减髌骨或股四头肌肌腱。 </li><li>释放髌骨横向通过在膝关节囊刚横向于髌腱使1cm的垂直切口。 </li><li>确保膝关节被延长。取一对精致剪刀从侧面髌骨向内侧下通过。传播剪刀几次以便伸肌装置可以转化为任何一方。 </li><li>而弯曲吨他的膝盖，翻译髌骨横向以暴露膝关节的内侧。确保清晰髁间窝和股骨髁的可视化。使用手术刀，横切ACL和PCL的缺口。 </li><li>负载功率电钻用1.6毫米克氏针。放在ACL产地克氏针尖在髁间窝。钻superolaterally和可视化的出口点在股骨的外侧面，除去必要用手术刀的任何软组织。通过克氏针进出几次，以确保畅通无阻的移植。 </li><li>放置第k导线上的胫骨平台的ACL的足迹。钻前外侧和可视化的退出点前外侧胫骨近端。用手术刀以清除软组织作为必要的，这样，其中第k线离开胫骨的点被完全显现。 </li><li>经股骨隧道传递缩短基思针（理想地不大于2英寸长）。通过在K的眼睛线程从接枝的一端两个缝线端eith针。使用针拉接枝的一端穿过股骨隧道。 </li><li>重复先前步骤通过胫骨隧道传递接枝的另一端。 </li><li>使用4-0薇乔缝合贴上移植物的股骨端周围骨膜或其它软组织结合图-的八个针迹。手动张力的伸展膝关节移植。加盖移植物的胫骨端周围骨膜或其它软组织结合图-的八个针迹。 </li><li>用剪刀切断多余的接枝两端，留下1-2毫米每端过去花样的-8的线圈。 </li><li>伸膝关节，减少髌骨。使用4-0薇乔，将花样的八个单针关闭内侧关节囊，防止髌骨半脱位外侧。 </li><li>关闭皮肤与正在运行的皮内5-0 Monocryl或薇乔缝合，小心不要缝合下面的肌肉，或有任何可见缝合一旦皮肤被关闭。 </li><li>仁济克拉大鼠皮下丁丙诺啡，每12小时，共三天术后。检查手术部位为任何排水或伤口裂开在注射的时间。跛行有些肿胀是正常的手术后的最初几天，但及时解决与兽医人员配合术后的任何疑虑。动物可术后恢复社会住房在2周时，手术切口完全愈合。 </li></ol> 3.数据收集协议<ol><li>在处死时，单独窒息大鼠在密闭的 CO 2室中，随后通过开胸。 </li><li>通过在髋关节中分离收获两个手术重建和对侧肢体。 <ol><li>用于重构的四肢，删除所有的软组织，包括后交叉韧带和从手术扰乱本地ACL中的残留物，由细解剖分离仅股骨，胫骨和接枝。 </li> &lt;LI&gt;对于对侧肢体，删除除本地ACL所有的软组织及股骨和胫骨细清扫。 </li><li>使用旋转工具如的Dremel，到但¾1厘米骨从股骨 - 接枝 - 胫骨复杂的每个端部除去所有。 </li><li>在这个过程中和整个生物力学测试，定期和经常喷韧带地区生理盐水，以防止收获的膝盖可能错误地改变结果的干燥。 </li></ol></li><li>用于组织学分析，分别在4％多聚甲醛溶液固定每个膝关节在25℃下48小时。接着，淹没在Immunocal试剂完全脱钙的溶液膝盖;这个过程是依赖于样品的钙化内容和最多可能需要五天。检查个别样品的日常评估进展不完全脱钙可以降低样品的质量。根据需要完成后，进行切片，幻灯片安装和染色。 </li><li> Perfor米生物力学测试，以评估组织工程韧带的功能的能力。 <ol><li>通过围绕每个骨的骨骺包装纸28克镀锌钢丝分别固定在股骨和胫骨。这是为了防止从样品过早拉伸破坏不精确生物力学测试数据在骨，而不是在感兴趣的韧带。 </li><li>锅股骨成的聚甲基丙烯酸甲酯骨粘固剂的混合物。要做到这一点，混合两种粘固剂组分，并立即使用粘性混合物，以确保股骨进入金属，完全包住骨的骨干在胶合锅骨骺和附韧带自由突出。允许自发自由基聚合来逐步改变混合粘性成分为面团状材料，最终成固体硬化基质。 注意：此过程需要几分钟，并且可以通过手动评估从制成的丸剂的温度进行监测剩下的水泥;温度应在放热聚合反应过程中瞬时增加和材料固化后消退至RT。 </li><li>重复同样的过程上面固井胫骨，但同时保持膝盖韧带在20°屈曲理想的机械测试。 </li><li>装入烧结股骨 - 接枝 - 胫骨络合物上的拉伸试验装置，并准备记录载荷和位移作为时间从张力失败的开始的功能。在本实施例中，我们使用的Instron型号5564用1千牛顿负荷单元。 </li><li>预张力接枝到2当量在0.5N /分钟的升温速率，然后测试接枝到故障以0.5毫米/秒的应变速率。在这个过程中，可以肯定的是，韧带拜倒在中间物质和骨的股骨和胫骨是安全的，而不是过早地失败，这可能不准确评估测试韧带的生物力学性能。 </li><li>使用所产生的载荷 - 位移曲线来计算测试韧带的破坏载荷和刚度。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

ACL损伤是一种常见的疾病在骨科运动手术，与在目前有限的选择重建。为了制定合适的组织工程替代，将允许再生体内 ACL中，需要一个合适的动物模型。在这项研究中，可生物降解的工程化移植物的制造中被描述，因为这是它在体内使用ACL重建的一个可重复模型中的无胸腺大鼠注入。该模型可以用于评估各种生物材料，包括细胞移植物和那些与PJ内置的生长因子组成的不同组织工程...

前十字韧带（ACL）的破裂是膝关节1的最常见的韧带损伤之一。由于破裂ACL的无法无手术干预治疗，在日常生活和参与体育活动的局限性开车超过175000例患者，每年2到接受手术，以每年十亿美元3估计成本。目前，无论是自体或异体肌腱用于韧带重建。尽管较高的成功率可以与自体移植和更换均可以实现，严重的并发症与这些重建方案4有关。自体移植的组织与供区的发病率相关联，并且在供水有限的，特别是在重新破裂或多韧带损伤的实例。另一方面，同种异体移植组织被延迟接枝集成，不利的炎症反应，理论上的传染疾病的风险，和有限的增刊联LY 5。合成的非降解的移植开发了20世纪70年代和80年代，但过早嫁接破裂，异物反应，骨溶解和滑膜炎6受阻。由于这些严重的问题的结果，目前有可用的在美国的临床使用中没有合成移植物。 由于与现有的移植选项和最近的事态发展在生物学，工程和再生医学这些限制，出现了为ACL移植组织工程解决方案极大的兴趣。当前组织工程策略采用可降解的生物材料和合成材料，以允许宿主的组织向内生长，同时避免与永久合成材料植入7相关的限制。 聚己内酯（PCL）是可生物降解的聚合物，其是FDA批准用于许多医疗应用中，包括粘连屏障和伤口敷料8，即一直Úsed的在各种各样的应用，包括血管，骨，软骨，神经，皮肤，食管组织工程5,9-16。良好的生物相容性，相对较长的体内半衰期，足够的机械强度，以及高弹性有助于该聚合物在组织工程的普及。在伤口愈合的啮齿动物模型中，注入的电纺PCL被证明是无免疫原性，并整合到局部组织无不良反应13。静电PCL的SEM图像示于图1中 。 当前FDA监管标准，疗效和安全性在小型和大型动物模型将需要一个PCL或任何其他工程ACL移植迁入在美国的临床试验。另外， 在体内条件往往能增加在体外组织工程的ACL移植物的性质。自体韧带重建的大鼠模型的屈digitor微米长肌腱先前已经描述的，在其中天然的ACL被切断，股骨和胫骨隧道钻，和接枝物通过并固定就位用缝合17-22。在本文中，我们将描述这个模型的变形对工程化的ACL替换的评价，而不是用于自体移植的基于重建（ 图2）。 尽管许多动物模型韧带组织工程存在，相比于许多原因较大的模型大鼠是有利的。这些优势包括更容易饲养和处理，减少伦理方面的考虑，并降低成本17,23。此外，鼠模型已被广泛地用作模型矫形组织再生，包括软骨，腱和骨组织工程24。特别是，无胸腺裸鼠被选择，因为它们缺乏的细胞介导的免疫应答25，允许最终植入Ò˚F异种供体细胞在此模型中，以进一步增强在未来的工程化移植物。在此方法本文中，我们描述了在ACL重建的无胸腺大鼠模型的制作和无细胞的手术植入，可生物降解的聚合物的接枝。

<table border="0" cellpadding="0" cellspacing="0" width="888">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:147px;">Company</td>
			<td style="width:77px;">Catalog Number</td>
			<td style="width:317px;">Comments/Description</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Medical grade ester terminated poly (&#949;-caprolactone), granule form (MW = 110,000)</td>
			<td style="width:147px;">Lactel Absorbable Polymers</td>
			<td style="width:77px;"></td>
			<td style="width:317px;">Custom synthesized polymer to desired molecular weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">1,1,1,3,3,3-hexafluoro-2-propanol</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>105228</td>
			<td style="width:317px;">Solvent for PCL polymer</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">18G x 1 1/2&#34;bevel needle</td>
			<td style="width:147px;">BD Medical</td>
			<td>305196</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Remote Infuse/Withdraw Programmable Syringe Pump</td>
			<td style="width:147px;">Harvard Apparatus</td>
			<td style="width:77px;">702101</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">VersaLaser VLS2.30 Laser Engraver</td>
			<td style="width:147px;">Microgeo USA</td>
			<td style="width:77px;">VLS2.30</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Expanded Plasma Cleaner 115V</td>
			<td style="width:147px;">Harrick Plasma</td>
			<td style="width:77px;">PDC-001</td>
			<td style="width:317px;">Plasma etch just prior to collagen coating for surface modification</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">PureCol&#160; Collagen Standard Solution, 3 mg/ml</td>
			<td style="width:147px;">Advanced Biomatrix</td>
			<td style="width:77px;">5015-A</td>
			<td style="width:317px;">Mix 8:1:2.5 solution of PureCol, 10x PBS, 0.1N NaOH 1:9 in 1x PBS</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Suture, 5-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1086471</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Suture, 4-0 Vicryl</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">6540072</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Sharp-pointed Dissecting Scissors (Straight; 4.5 inch)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:77px;">8940</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Buphrenorphine hydrochloride</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:77px;">B9275</td>
			<td style="width:317px;">Use 0.03 mg/kg for both intra- and post-operatively for pain control</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Ampicillin, injectable</td>
			<td style="width:147px;">Henry Schein</td>
			<td style="width:77px;">1185678</td>
			<td style="width:317px;">Use 25 mg/kg subcutaneously during the procedure</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">K-wire, 1.6 mm</td>
			<td style="width:147px;">Spectrum Surgical</td>
			<td style="width:77px;">SI040062</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Keith Needle, Straight 1 1/2&#34;</td>
			<td style="width:147px;">Delasco Dermatology Lab &#38; Supply</td>
			<td style="width:77px;">KE-112</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:347px;">Immunocal Decalcifying Solution</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td>NC9491030</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:347px;">Opticryl Acrylic Resin Bone Cement (PMMA) (Monomer and polymer)</td>
			<td style="width:147px;">US Dental Depot</td>
			<td style="width:77px;">OPTICRYL 100410&#160;</td>
			<td style="width:317px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:347px;">Instron Model 5564 Tensile Testing Machine</td>
			<td style="width:147px;">Instron</td>
			<td style="width:77px;">5564</td>
			<td style="width:317px;">Any comparable tensile testing apparatus is suitable</td>
		</tr>
	</tbody>
</table>

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.

在我们的92大鼠手术由单个外科医生的经验，平均手术时间从切口到伤口完成为16.9分钟，用4.7分钟的标准偏差。在牺牲的时候，老鼠体重356±23克所有大鼠耐受手术顺利，经历了无并发症。立即手术后，将大鼠注意到承受重量的可操作端，但表现出轻微跛行。一个星期后操作，所有大鼠走动，无明显跛行。在研究过程中的所有动物体重增加稳步推进，在喂养，排尿，排便或习惯没有观察到异常。在...

Watch this Scientific Journal Video about Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts at JoVE.com

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

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 ...

athymic-rat-model-for-evaluation-engineered-anterior-cruciate

Research

JoVE Journal

Bioengineering

10.3K 次观看.  University of California Los Angeles. 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

无胸腺大鼠模型工程前交叉韧带移植评估