Name: athymic rat model for evaluation of engineered anterior cruciate ligament grafts
Uploaded: 2015-03-26T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts at JoVE.com

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的ACL移植。 在这项研究中，我们测试的无细胞，静电聚己内酯移植对齐纤维。组织工程韧带重建以前的研究已经植入编织或制成的各种材料，包括丝绸，PLLA挤出移植和聚氨酯27-29。没有这些材料导致两个成功的访问控制列表7的机械性能的接枝和概括的集成。虽然许多聚合物在韧带重建使用的潜力，本研究调查了PCL，因为它是生物惰性的，无毒，降解慢慢在体内 ，并且容易加工成所期望的构象30。 PCL是也机械坚固和示出的机械应力下30小的塑性变形。静电纺丝31时，其使用已经确立了在骨组织工程文学作为一个可靠的水库矿化和I型胶原的沉积，由于其排列纳米纤维结构。另外，已经表明，在高的表面与体积的静电PCL垫的小直径纤维的短扩散长度尺度是有利的受控的药物递送和组织工程31使用。 我们发现植入移植物生物相容性的基础上，缺乏临床不良反应，并协助看到组织学在16周术后原生细胞浸润。我们用在这项研究中的裸鼠的动物模型，因为它是一个两阶段的项目，将最终利用植入的移植物的人细胞的第一阶段，和关于抑制人类细胞的无胸腺的模型将降低的担忧。在静电聚合物支架促进了再生ACL两种细胞和基质对齐。正如在移植物的横截面，大部分细胞在纤维的方向上被对准。植入的移植物表现出随时间增加的机械性能。负荷相比，重构的ACL立即术后一倍聚合物移植物的失败。而在PCL接枝可能看起来低相比，天然ACL的高峰负荷和刚度，它是要记住，这些结果应在光的F的被视为是非常重要的作用，即使是目前的黄金标准，自体或同种异体移植物，不能够达到健康ACL的机械强度16周术后。例如，Xu 等人报告了一个ACL移植在兔模型，其中高峰负荷为20％-35％，和硬度为23％-36％，即由术后6个月32名健康天然的ACL。另外，在犬模型中的同种异体移植物研究30周术后33展示了大约30％的高峰负荷和40％的天然ACL的刚度。 虽然这是本文的范围之外，许多其他的分析，可以执行使用该动物模型的后评估接枝质量。这包括但不限于:生物发光成像或X射线和体内 CT扫描，和许多污渍和测定法如免疫组织化学对胶原标记或炎症标志物的。举例来说，我们之前公布的结果上对齐协量化用天狼星红染色植入动物模型26静电PCL支架植入后llagen纤维。 本研究的潜在的局限性，包括动物模型本身的选择。在解剖和步态的四足鼠相比，双足人类固有的差异意味着ACL的生物力学确实有所不同，而翻译模型之间的临床参数应与这些限制的知识来完成。然而，这个问题是在动物研究中常见的，并不否定该研究的重要性或翻译的潜力。 一直存在对使用可被应用到我们的模型设计的ACL替换在将来移植大鼠韧带重建手术后的协议有趣的研究。外部固定装置已被用于以固定大鼠术后，以允许改进的腱 - 骨愈合的腱移植模型19，另外，它已经表明，延迟循环加载，如与由Stasiak 等人 34所述的曲伸装置，可以进一步提高自体掺入20。然而，它也已显示，持续时间短的低幅度循环载荷也可以引起增加的炎症和降低在骨-腱接口35骨形成。进一步调查必须进行的这些研究结果的适用性评估到一个静电，基于聚合物的ACL更换，因为这样的接枝将具有较弱的初始机械性能比肌腱移植。 目前的研究已经发展ACL重建模型，在无胸腺大鼠脱细胞移植静电，基于掀起了前面描述的大鼠移植模型17-22修改。我们展示了密集排列的胶原制定整个移植与负载的同时改善故障的接枝随时间。这项研究还提供了概念证明采用这种模式在未来评估各种组织工程移植物的韧带重建。特别是，无胸腺大鼠允许异种供体细胞的播种。

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><tbody><tr><td>Medical grade ester terminated poly (&amp;epsilon;-caprolactone), granule form (MW = 110,000)</td><td><a href="http://www.absorbables.com" target="_blank"><img src="/img/new_window.png" /> Lactel Absorbable Polymers</a></td><td></td><td>Custom synthesized polymer to desired molecular weight</td></tr><tr><td>1,1,1,3,3,3-hexafluoro-2-propanol</td><td>Sigma-Aldrich</td><td>105228</td><td>Solvent for PCL polymer</td></tr><tr><td>18 G x 1&amp;#189;&quot; bevel needle</td><td>BD Medical</td><td>305196</td><td></td></tr><tr><td>Remote Infuse/Withdraw Programmable Syringe Pump</td><td>Harvard Apparatus</td><td>702101</td><td></td></tr><tr><td>VersaLaser VLS2.30 Laser Engraver</td><td>Microgeo USA</td><td>VLS2.30</td><td></td></tr><tr><td>Expanded Plasma Cleaner 115&amp;nbsp;V</td><td>Harrick Plasma</td><td>PDC-001</td><td>Plasma etch just prior to collagen coating for surface modification</td></tr><tr><td>PureCol Collagen Standard Solution, 3&amp;nbsp;mg/ml</td><td>Advanced Biomatrix</td><td>5015-A</td><td>Mix 8:1:2.5 solution of PureCol, 10x PBS, 0.1&amp;nbsp;N NaOH 1:9 in 1x PBS</td></tr><tr><td>Suture, 5-0 Vicryl</td><td>Henry Schein</td><td>1086471</td><td></td></tr><tr><td>Suture, 4-0 Vicryl</td><td>Henry Schein</td><td>6540072</td><td></td></tr><tr><td>Sharp-pointed Dissecting Scissors (Straight; 4.5&amp;nbsp;inch)</td><td>Fisher Scientific</td><td>8940</td><td></td></tr><tr><td>Buphrenorphine hydrochloride</td><td>Sigma-Aldrich</td><td>B9275</td><td>Use 0.03 mg/kg for both intra- and post-operatively for pain control</td></tr><tr><td>Ampicillin, injectable</td><td>Henry Schein</td><td>1185678</td><td>Use 25 mg/kg subcutaneously during the procedure</td></tr><tr><td>K-wire, 1.6 mm</td><td>Spectrum Surgical</td><td>SI040062</td><td></td></tr><tr><td>Keith Needle, Straight 1&amp;#189;&quot;</td><td>Delasco Dermatology Lab &amp;amp; Supply</td><td>KE-112</td><td></td></tr><tr><td>Immunocal Decalcifying Solution</td><td>Fisher Scientific</td><td>NC9491030</td><td></td></tr><tr><td>Opticryl Acrylic Resin Bone Cement (PMMA) (Monomer and polymer)</td><td>US Dental Depot</td><td>OPTICRYL 100410&amp;nbsp;</td><td></td></tr><tr><td>Instron Model 5564 Tensile Testing Machine</td><td>Instron</td><td>5564</td><td>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。在临床上，无毛伤口裂开，出现红斑，肿胀，积液，或引流术后观察。 没有进行上述92大鼠手术主要用于该方法稿件的目的。相反，它们被用来测试各种工程接枝的条件。而detaileð机械测试和组织学结果是本文的范围之外，更多的细节可以在一份文件中梁等 26发现。简言之，在下面的重建16周的剖膝盖的组织学分析表明，该支架基质变得很大程度上渗透由成纤维细胞分泌的嗜酸性胶原具有良好的整合到骨隧道（ 图3）。此时，支架已经被完全再吸收并没有证据聚合物被可视化。此外，免疫组织化学对巨噬细胞标记物CD68的证明在16周后的操作性（ 图4）的最低限度的炎症反应。 生物力学性能的牺牲后，立即进行评估。所有测试样品没有在中间物质（ 图5）。使用从拉伸试验产生的载荷-位移曲线（ 图6），破坏载荷和刚度计算了各组。在16周植入后，所述静电聚合物接枝有测试立即植入后的移植物的大约两倍的峰值负荷和刚度，但是这些值比天然的ACL 26低。 <img alt="图1" src="/files/ftp_upload/52797/52797fig1.jpg" /> 图1.静电聚己内酯支架与排列的纤维SEM图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52797/52797fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52797/52797fig2.jpg" /> ACL重建图2.无胸腺大鼠模型。（A）通过内侧髌旁皮肤切口髌腱隔离。股tunne的（B）钻孔l采用1.6毫米克氏针。 （C）胫骨钻隧道。 1.2毫米基思针穿过股骨隧道（D）放置，通过拉移植。 （E）电纺聚己内酯移植到股骨隧道拉。 （F）接枝通过双方股骨和胫骨隧道拉，两端被修剪和缝合前骨膜和层状封闭进行。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52797/52797fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52797/52797fig3.jpg" /> 图3.苏木和静电聚合物接枝（顶部）在胫骨隧道（左）midsubstance（中心），和股骨隧道（右）伊红染色。为了比较，本地ACL示（下部），在胫骨插入（左），midsubstance（CENTER）和股原点（右），10X。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52797/52797fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52797/52797fig4.jpg" /> 图4.比色法免疫组织化学染色对CD68，一个标记为巨噬细胞。定性地，似乎存在于骨隧道稍多阳性染色8周比在所述移植物的关节内的区域或16周后的运算。似乎有在移植最小炎症。所有图像放大20倍。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52797/52797fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52797/52797fig5.jpg" /><br/&gt; 图机械测试植入静电移植，在移植midsubstance证明失败5.形象。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52797/52797fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52797/52797fig6.jpg" /> 为组织工程ACL移植物在植入裸鼠模型大鼠16周后图6.示例荷载位移曲线。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52797/52797fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

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.

视频: Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

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

Injury to the ACL is a commonly encountered problem in active individuals. Even partial tears of this intra-articular knee ligament lead to biomechanical deficiencies that impair function and stability. Current options for the treatment of partial ACL tears range from nonoperative, conservative management to multiple surgical options, such as: thermal modification, single-bundle repair, complete reconstruction, and reconstruction of the damaged portion of the native ligament. Few studies, if any, have demonstrated any single method for management to be consistently superior, and in many cases patients continue to demonstrate persistent instability and other comorbidities.
The goal of this study is to identify a potential cell source for utilization in the development of a tissue engineered patch that could be implemented in the repair of a partially torn ACL. A novel protocol was developed for the expansion of cells derived from patients undergoing ACL reconstruction. To isolate the cells, minced hACL tissue obtained during ACL reconstruction was digested in a Collagenase solution. Expansion was performed using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). The cells were then stored at -80 &#186;C or in liquid nitrogen in a freezing medium consisting of DMSO, FBS and the expansion medium. After thawing, the hACL derived cells were then seeded onto a tissue engineered scaffold, PLAGA (Poly lactic-co-glycolic acid) and control Tissue culture polystyrene (TCPS). After 7 days, SEM was performed to compare cellular adhesion to the PLAGA versus the control TCPS. Cellular morphology was evaluated using immunofluorescence staining. SEM (Scanning Electron Microscope) micrographs demonstrated that cells grew and adhered on both PLAGA and TCPS surfaces and were confluent over the entire surfaces by day 7. Immunofluorescence staining showed normal, non-stressed morphological patterns on both surfaces. This technique is promising for applications in ACL regeneration and reconstruction.

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

For future applications as a patch to repair partial tears of the Anterior Cruciate Ligament (ACL), human ACL derived cells were isolated from tissue obtained during reconstructive procedures, expanded in vitro and grown on tissue engineered scaffolds. Cellular adhesion and morphology was then performed to confirm biocompatibility on scaffold surface.

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 ...

科研

生物工程

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

用运动调节血清治疗韧带结构:转化组织工程模型

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

A Reliable and Reproducible Critical-Sized Segmental Femoral Defect Model in Rats Stabilized with a Custom External Fixator

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. Critical-sized segmental defects are challenging to treat clinically, and research efforts could benefit from a reliable, ambulatory small animal model of a segmental femoral defect. In this study, we present an optimized surgical protocol for the consistent and reproducible creation of a 5 mm critical diaphyseal defect in a rat femur stabilized with an external fixator. The diaphyseal ostectomy was performed using a custom jig to place 4 Kirschner wires bicortically, which were stabilized with an adapted external fixator device. An oscillating bone saw was used to create the defect. Either a collagen sponge alone or a collagen sponge soaked in rhBMP-2 was implanted into the defect, and the bone healing was monitored over 12 weeks using radiographs. After 12 weeks, rats were sacrificed, and histological analysis was performed on the excised control and treated femurs. Bone defects containing only collagen sponge resulted in non-union, while rhBMP-2 treatment yielded the formation of a periosteal callous and new bone remodeling. Animals recovered well after implantation, and external fixation proved successful in stabilizing the femoral defects over 12 weeks. This streamlined surgical model could be readily applied to study bone healing and test new orthopedic biomaterials and regenerative therapies in vivo.

In vivo mammalian models of critical-sized bone defects are essential for researchers studying healing mechanisms and orthopedic therapies. Here, we introduce a protocol for the creation of reproducible, segmental, femoral defects in rats stabilized using external fixation.

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

Orthopedic research relies heavily on animal models to study mechanisms of bone healing in vivo as well as investigate the new treatment techniques. ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

大鼠双侧髌腱损伤模型中干细胞治疗的评价

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

医学

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

一种生成周期负荷诱导的大鼠膝关节内软骨病变模型的非侵入性方法

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Development and Evaluation of a Rat Model of Full-Thickness Cartilage Defects

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired movement, and eventually, knee osteoarthritis (kOA). However, there is little effective treatment for cartilage defects or even kOA. Animal models are important for developing therapeutic drugs, but the existing models for cartilage defects are unsatisfactory. This work established a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats, and the subsequent pain behavior and histopathological changes were used as readout experiments. After surgery, the mechanical withdrawal threshold was decreased, chondrocytes at the injured site were lost, matrix metalloproteinase MMP13 expression was increased, and type II collagen expression decreased, consistent with the pathological changes observed in human cartilage defects. This methodology is easy and simple to perform and enables gross observation immediately after the injury. Furthermore, this model can successfully mimic clinical cartilage defects, thus providing a platform for studying the pathological process of cartilage defects and developing corresponding therapeutic drugs.

This protocol establishes a full-thickness cartilage defects (FTCD) model by drilling holes in the femoral trochlear groove of rats and measuring the subsequent pain behavior and histopathological changes.

全层软骨缺损大鼠模型的开发与评价

Cartilage defects of the knee joint caused by trauma are a common sports joint injury in the clinic, and these defects result in joint pain, impaired ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

优化结构静电纺丝血管移植在大鼠模型中的植入

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...

生物学

非侵入性 ACL 损伤和手术对小鼠蛋白酶活性的影响

Non-Invasive Compression-Induced Anterior Cruciate Ligament (ACL) Injury and In Vivo Imaging of Protease Activity in Mice

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within 10-20 years following injury. Understanding the early biological processes initiated by joint injuries (e.g., inflammation, matrix metalloproteinases (MMPs), cathepsin proteases, bone resorption) is crucial for understanding the etiology of PTOA. However, there are few options for in vivo measurement of these biological processes, and the early biological responses may be confounded if invasive surgical techniques or injections are used to initiate OA. In our studies of PTOA, we have used commercially available near-infrared protease activatable probes combined with fluorescence reflectance imaging (FRI) to quantify protease activity in vivo following non-invasive compression-induced ACL injury in mice. This non-invasive ACL injury method closely recapitulates clinically relevant injury conditions and is completely aseptic since it does not involve disrupting the skin or the joint capsule. The combination of these injury and imaging methods allows us to study the time course of protease activity at multiple time points following a traumatic joint injury.

作者聚焦：研究 ACL 损伤对骨关节炎进展的早期事件和长期影响

Non-invasive ACL injury is a reliable and clinically relevant method for initiating post-traumatic osteoarthritis (PTOA) in mice. This injury method also allows for in vivo quantification of protease activity in the joint at early time points post-injury using protease-activatable near infrared probes and fluorescence reflectance imaging.

非侵入性压缩诱导的前交叉韧带 （ACL） 损伤和小鼠蛋白酶活性 的体内 成像

Traumatic joint injuries such as anterior cruciate ligament (ACL) rupture or meniscus tears commonly lead to post-traumatic osteoarthritis (PTOA) within ...

无胸腺大鼠模型工程前交叉韧带移植评估

摘要

探索更多视频

此视频中的章节

手术取出，分离和

用运动调节血清治疗韧带结构:转化组织工程模型

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

大鼠双侧髌腱损伤模型中干细胞治疗的评价

一种生成周期负荷诱导的大鼠膝关节内软骨病变模型的非侵入性方法

全层软骨缺损大鼠模型的开发与评价

优化结构静电纺丝血管移植在大鼠模型中的植入

非侵入性压缩诱导的前交叉韧带（ACL）损伤和小鼠蛋白酶活性的体内成像

非侵入性压缩诱导的前交叉韧带（ACL）损伤和小鼠蛋白酶活性的体内成像

非侵入性压缩诱导的前交叉韧带（ACL）损伤和小鼠蛋白酶活性的体内成像

无胸​​腺大鼠模型工程前交叉韧带移植评估

摘要

探索更多视频

此视频中的章节

手术取出，分离和

用运动调节血清治疗韧带结构:转化组织工程模型

一种可靠、可重复的可重复的大鼠部分股骨缺陷模型

大鼠双侧髌腱损伤模型中干细胞治疗的评价

一种生成周期负荷诱导的大鼠膝关节内软骨病变模型的非侵入性方法

全层软骨缺损大鼠模型的开发与评价

优化结构静电纺丝血管移植在大鼠模型中的植入

非侵入性压缩诱导的前交叉韧带 （ACL） 损伤和小鼠蛋白酶活性 的体内 成像

非侵入性压缩诱导的前交叉韧带 （ACL） 损伤和小鼠蛋白酶活性 的体内 成像

非侵入性压缩诱导的前交叉韧带 （ACL） 损伤和小鼠蛋白酶活性 的体内 成像

无胸腺大鼠模型工程前交叉韧带移植评估

非侵入性压缩诱导的前交叉韧带（ACL）损伤和小鼠蛋白酶活性的体内成像

非侵入性压缩诱导的前交叉韧带（ACL）损伤和小鼠蛋白酶活性的体内成像

非侵入性压缩诱导的前交叉韧带（ACL）损伤和小鼠蛋白酶活性的体内成像