Name: evaluation of stem cell therapies in a bilateral patellar tendon injury model in rats
Uploaded: 2018-03-30T14:00:00.000+00:00
Duration: 9 min 31 s
Description: Watch this Scientific Journal Video about Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats at JoVE.com

作者要感谢博士, 博士, 她对数据的统计分析。作者还感谢麦克卢尔博士, DVM, 博士 DACLAM, 她的意见, 在麻醉和疼痛管理协议中使用的研究。该项目得到了西方卫生科学大学研究副会长 (12678v) 和美国农业部1433基金 (2090) 的资助。

这里描述的所有方法都已被西方健康大学机构动物护理和使用委员会 (IACUC) 批准。
1. 马脐基质中 MSCs 的分离和扩增
<ol><li>从成年母马 (怀孕) 中获得胎盘, 观察 foaling, 灌装从胎盘中分离脐带。将脐在磷酸盐缓冲盐水 (PBS) 与1% 青霉素-链霉素 (P/秒) 在4°c 在转移, 直到处理。</li>
	<li>用室温 PBS 在50毫升管中清洗脐带两次, 1% P/秒。将脐带分为2英寸长的碎片在150毫米板和洗涤室温 PBS 与 1% P/秒在50毫升管两个或三次, 直到大部分的血液被冲洗出来。</li>
	<li>纵向切断脐带以暴露血管。用镊子和剪刀从脐带上取出血管, 包括2动脉、静脉和尿囊茎。收集脐带基质 (沃顿的果冻), 这是类似果冻的基质围绕容器, 用手术刀刮上150毫米的盘子, 将果冻切成细块。</li>
	<li>将沃顿的果冻从2–3脐带片段 (大约 12–15 g) 放置在50毫升管中, 15 毫升 0.1% (瓦特/v) 胶原酶 IA 溶液在 PBS。孵育在37°c 与柔和的震动为3-4 小时直到溶化。</li>
	<li>离心机消化组织在 300 x g 15 分钟和吸入上清。并用重悬消化组织15毫升室温 PBS 与 1% P/秒, 混合吹打, 离心机在 150 x g 5 分钟, 并吸入上清 (洗涤)。重复洗两次。</li>
	<li>并用重悬洗涤细胞15毫升室温 PBS 与 1% P/秒, 混合吹打, 应变100µm 细胞过滤器, 离心机在 150 x g 为5分钟, 并吸入上清。</li>
	<li>将低葡萄糖 Dulbecco 的改良鹰培养基 (LG-DMEM) 与胎牛血清 () 和 1% P/s 混合, 通过过滤来制备培养基 (CM)。</li>
	<li>并用重悬紧张的细胞在10毫升 CM 前预热到37°c, 混合吹打, 转移到 25CM 2 组织培养瓶, 并且孵化在 5%CO 2 在90% 湿气和37.0 °c。改变 CM 每3天, 直到文化达到70–80% 汇合, 当文化将传代。</li>
	<li>通过培养, 从烧瓶中抽出厘米, 用5毫升室温 PBS 冲洗两次, 并与3毫升0.25% 胰蛋白酶/乙二胺 (EDTA) 分离, 37 摄氏度, 5 分钟。</li>
	<li>中和胰蛋白酶/EDTA 与6毫升 CM 预热到37.0 °c, 混合吹打, 转移到15毫升管, 离心机在 150 x g 为5分钟, 并吸入上清。并用重悬分离细胞在1毫升厘米, 混合吹打。用台盼蓝和 hemocytometer 计数活细胞。重新种子成25厘米2组织培养瓶在5000细胞/cm2, 并孵化在 5% CO2 90% 湿度和37.0 °c。</li>
</ol>2. 用壳聚糖膜培养 UCM-MSCs 制备球体
<ol><li>要准备100毫升 1% (w/v) 壳聚糖溶液, 添加1克壳聚糖在99毫升蒸馏水 (dH2O), 并与磁性搅拌器混合良好。</li>
	<li>加入670µL 的冰乙酸, 继续搅拌直到壳聚糖溶解并变得粘性。这通常需要 3–4 h。</li>
	<li>在12井组织培养板的每个井中加入500µL 壳聚糖溶液, 并将其旋转, 均匀分布壳聚糖溶液, 覆盖所有的底面。</li>
	<li>将壳聚糖溶液涂覆板置于层流柜下, 无盖过夜24小时。一旦干燥, 薄膜就会形成并粘附在板材上。</li>
	<li>将壳聚糖膜加入1毫升0.5 氮氢氧化钠 (氢氧化钠) 溶液, 在室温下孵育2小时。从各井中抽出氢氧化钠, 用1毫升 dH2O 清洗每一个井三次。一次用1毫升70% 乙醇 (乙醇) 冲洗每一个井。</li>
	<li>为壳聚糖膜消毒, 每井加1毫升70% 乙醇, 并在层流柜内一夜间孵化。第二天从每个井中抽吸剩余的乙醇。用1毫升的无菌 PBS 冲洗每一个井三次。在层流柜内无遮盖的情况下, 用紫外光消毒壳聚糖膜。</li>
	<li>要形成球体的 UCM-MSCs, 种子扩展和传代细胞到每个井在5000细胞/cm2, 并孵化 5% CO2在90% 湿度和37.0 °c。 注意: 细胞可以在缺氧或 normoxia 下孵化, 这取决于研究者的兴趣。</li>
</ol>3. 流式细胞仪分析表面标记物的表达
<ol><li>
单细胞悬浮液的制备
	<ol>
<li>标准板材<ol>
<li>从每个井中取出介质, 用室温 PBS 清洗两次。</li>
			<li>分离细胞与500µL 细胞离解试剂入每井12井板为5–10分钟在室温下。</li>
			<li>孵化后, 添加1毫升的染色缓冲 (PBS 包含 0.5% BSA) 到每个井和混合通过吹打分离细胞。</li>
			<li>将电池悬浮液转移到15毫升锥形管中, 离心机在 150 x g 处进行5分钟。</li>
		</ol>
</li>
		<li>壳聚糖板材<ol>
<li>使用1000µL 尖的吸管吸吸培养基收集所有球体。将收集到的介质转化为15毫升锥形管。收集介质后, 通过加入1毫升的 pbs 进行冲洗, 并将冲洗过的 pbs 转换成相同的15毫升圆锥管。</li>
			<li>离心机球体为 150 x 克, 5 分钟, 清除上清液。</li>
			<li>添加500µL 细胞离解试剂 (例如, accutase), 并在室温下孵育5–10分钟。混合吹打使用1毫升尖端, 直到球体离解, 不再可见。</li>
			<li>在单细胞悬浮液中加入1毫升的染色缓冲液, 离心机在 150 x g 处为5分钟。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
洗涤单元
	<ol>
<li>从锥形管中取出上清, 并在3毫升的冷染色缓冲器中重新悬浮细胞。从这一步, 在实验中保持细胞在冰上。</li>
		<li>离心机在 300 x g 5 分钟 (洗涤)。</li>
		<li>洗两次。</li>
	</ol>
</li>
	<li>
染色细胞
	<ol>
<li>离心机在 300 x g 5 分钟, 并清除上清。</li>
		<li>使用台盼蓝和 hemocytometer 计数可行细胞。在50µL 阻塞缓冲区中重新挂起 1 x 106单元格 (PBS 含有10% 匹马血清), 并在冰上孵化30分钟。</li>
		<li>添加10µL 荧光素异硫氰酸酯 (FITC) 共轭抗体 (CD44, CD90, CD105, CD34, 主要组织相容性复合物 (MHC) II 类, 或同种控制每个抗体) 和40µL 的染色缓冲器, 然后孵化保护从光1小时冰。</li>
		<li>添加3毫升的冷染色缓冲和混合, 离心机在 300 x g 5 分钟, 并吸入上清 (洗涤)。</li>
		<li>重复洗涤两次。</li>
		<li>在0.5 毫升的染色缓冲器中重新悬浮细胞颗粒</li>
		<li>添加5µL 7-反式 (活性染料) 和孵育30分钟冰</li>
		<li>用流式细胞仪分析染色样品。通过其较小的 SSC 和 FSC 排除碎片, 并识别出低吸收 7-反的可行细胞。分别在 y 和 x 轴上绘制 FL1 和 FL2。使用同种控件在对角线上方创建一个门。测量该区域阳性染色细胞的百分比。计数至少2万个事件/示例 (辅助图 1)。</li>
		<li>用门控细胞和自动荧光测量抗体染色细胞的百分比, 并减去同种控制的细胞百分比。</li>
	</ol>
</li>
</ol>4. 大鼠双侧髌腱缺损模型的建立
<ol><li>选择大大鼠 (成年男性, 第4-5 月大, 体重 350–375 g)。注意这个模型所用的大鼠的体积相对较大。</li>
	<li>麻醉和应用人工眼润滑剂的大鼠与8% 七氟醚在2升/分100% 氧气通过面罩提供, 直到消失的捏脚趾反射在感应室。</li>
	<li>用肌肉注射 Meloxicam (1 毫克/千克) 作为先发制人镇痛。</li>
	<li>将老鼠放在装有温水的两个0.5 升水瓶之间, 用布覆盖以保持体温和位置, 同时防止皮肤损伤。把每一个肢体带到桌子上。为了降低体温过低的风险, 请用气泡包装覆盖身体 (图 1)。</li>
	<li>保持麻醉, 连续5% 七氟醚在1升100% 氧混合物通过鼻锥, 与动物在背卧床上的水加热垫。</li>
	<li>为避免皮肤外伤, 请勿夹住手术部位。相反, 在两个窒息中应用脱毛膏。使用舌抑制剂去除奶油和头发。</li>
	<li>用洗必泰 digluconate 擦洗手术部位, 用70% 乙醇冲洗3次。</li>
	<li>用无菌 #15 手术刀刀片在近端向远端切开皮肤, 在窒息的 craniomedial 方面。开始切口约1厘米近的髌骨水平, 并延长约5毫米远端胫骨结节。</li>
	<li>通过 #15 手术刀刀片释放底层皮下组织, 反映皮肤暴露髌骨肌腱。</li>
	<li>
使用 #15 手术刀刀片, 把每个髌骨肌腱 (1 毫米) 的中心第三个从髌骨远端到胫骨股骨。
	<ol>
<li>将0.99 毫米直径的克氏针线与肌腱对齐, 作为模板, 以规范每个肢体缺陷的大小 (图 2)。</li>
		<li>用 #15 手术刀刀片在克氏针的两侧进行2个全厚切口, 以隔离肌腱的中心部分 (图 3)。切除中心部分下部和远端的细虹膜剪刀 (图 4)。</li>
		<li>在关闭窒息的筋膜前, 将血栓 (混合细胞悬浮和非加太) 插入适当治疗组, 如5.5 所述。使用 5-0 polyglactin 910 缝线 (图 5), 用交叉图案和皮肤与皮图案闭合筋膜。</li>
	</ol>
</li>
	<li>重复对侧窒息的程序。 注意: 一个缺陷被随机分配给治疗 (在壳聚糖或标准板材上培养的干细胞)。对侧缺损留空, 作为内部控制。</li>
	<li>术后, 4 粒的恩诺沙星 (2 毫克/片, 口服, 一次每日) 和一片剂的 meloxicam (2 毫克/片剂, 口服, 每天一次) 7 天。这些剂量是由实验动物兽医推荐并在 IACUC 协议中批准的。</li>
</ol>5. 骨髓间充质干细胞在髌骨肌腱缺损中的传递
<ol><li>麻醉一只不用于髌骨肌腱缺损的健康大鼠, 8% 七氟醚在2升/分100% 氧气通过面罩传递, 直到在感应腔内的夹趾反射消失。</li>
	<li>收集5毫升血液的心脏穿刺从麻醉的大鼠 (成年男性, 第4-5 月, 体重 350–375 g), 在5毫升注射器与20克针含有1毫升酸柠檬酸-葡萄糖 (5:1 v/v)。</li>
	<li>弄死大鼠心脏穿刺和采血后, 心内注射戊巴比妥 (100 毫克/千克), 同时在同一麻醉下。</li>
	<li>在室温下离心样品在 350 x g 15 分钟。转移上清和存储整除数 (120 µL 每) 在-20 °c, 直到使用。</li>
	<li>在体内植入之前, 立即解冻上述整除, 并将20µL 与 0.5 x 106 MSCs (从1.9 或 3.1.2.1) 分离出来, 使用胰蛋白酶/EDTA, 或通过冲洗 (与 PBS/培养基) 从壳聚糖板中收集。</li>
	<li>将6µL 10% 氯化钙 (CaCl2) 添加到96井板井中解冻血浆的其余100µL, 以激活血浆并诱发血栓形成 (活化条件血浆: 非加太)。</li>
	<li>混合细胞悬浮 (20 µL) 和非加太 (100 µL) 形成血栓 (图 6)。在关闭窒息的筋膜前, 将血栓放置在髌骨肌腱缺损中 (见步骤 4.10.3)。</li>
</ol>6. 功能成果
<ol><li>监测老鼠每天两次的疼痛迹象, 根据大鼠的鬼脸比例 (RGS)37,38和肿胀在植入点。 注: 建立评分系统 (0–6), 以评估动态功能的基础上3活动, 包括定时后肢站立的前肢支持 (0-3), 定时无辅助后肢站立 (0–2), 并有能力爬上17厘米塑料笼壁 (0–1) (表 1)。</li>
	<li>评价大鼠的两个后肢站立活动在早上和晚上的每个时间点计算平均得分。</li>
	<li>评估大鼠的能力, 成功地爬上一个17厘米的塑料笼壁与两个后肢到达顶端。</li>
</ol>7. 髌腱的毛外观和组织病理学
<ol><li>弄死大鼠在7天 (评估炎症) 或28天 (评估组织愈合) 后治疗的心内注射戊巴比妥 (100 毫克/千克) 麻醉下, 8% 七氟醚和2升100% 氧气通过口罩提供。</li>
	<li>在安乐死后用手术刀和剪刀收割髌骨-肌腱-胫骨股骨单位。除髌腱外, 除去窒息周围的软组织和韧带。</li>
	<li>检查每个标本的总外观和加厚的肌腱 (图 7)。</li>
	<li>将标本放置在肌腱的近端和侧向处, 将 5.0 Polydioxanone 的外科医生的结放在一起。将每个标本固定在10% 中性缓冲福尔马林溶液中, 从每根肌腱的中部获得横断面 (5 µm)。</li>
	<li>用苏木精和三色染色切片, 并按照标准规程对马尾松进行染色。</li>
	<li>检查部分, 以检测是否存在血肿内的缺陷, 以及病理变化, 如炎症。</li>
	<li>根据胶原分级、血管生成程度和软骨形成 (表 2)39, 使用先前发布的评分系统评估各节的组织学评分。</li>
</ol>

作者没有披露利益冲突。

为这个项目选择了马细胞, 因为我们最终打算测试在马的自然 tendinopathies 管理的候选方法。事实上, 由于马表面数字屈肌和跟腱在人类中的生物学相似性, 马匹肌腱损伤是肌腱的自然模型 (41。根据国际细胞治疗学会建议的标准, CD44、CD90、CD105、CD34 和 MHC II 的细胞表面标记物为免疫分型细胞选择了42。在最近的一项研究中, 无论培养条件如何, eqUCM-MSCs 对 CD34 和 MHC II 的 CD44 和阴性均呈阳性。在标准条件下培养的细胞对 CD90 和 CD105 也有积极作用, 从而满足了 MSCs 在表面标记表达方面的标准。此外, 这项研究是第一次报告的球体的形成时, eqUCM-MSCs 在壳聚糖膜培养, 确认以前的报告, 人类 mscs19,20。3D 细胞培养技术在几十年来一直被广泛探索, 旨在创造一种类似于生理生态位的环境。事实上, 本研究表明, 改进的 stemness 基因表达水平的Oct4, Sox2和北美,和改进的分化潜力, 这是一致的其他报告19,20。另一些报告则使用 MSCs 球体改进免疫调节特性43和透明软骨再生44 。在各种3D 种栽培技术中, 如微调瓶、旋转细胞生物反应器、非粘附板、天然和合成基质, 壳聚糖薄膜似乎具有吸引力, 原因有多种。这种方法不需要昂贵的设备, 而且与合成矩阵的非粘附板相比成本效益高, 因为壳聚糖是海产品行业的副产品。在壳聚糖薄膜上培养细胞在技术上很容易。结合起来, 这些优势将使临床应用在大规模的45。
这里提出的双侧髌腱模型在发病率方面似乎是可以接受的。在最近的一项研究中没有发现并发症。我们尤其关注缺陷引起的压力效应的风险, 因为它足够大, 可以检测到生物力学和组织学变化。这些关注促使了对小鼠的选择, 这项研究的基础上的研究与肌腱的大小和成熟度的临床前损伤模型。在兔子46和大鼠30,31中描述了 "窗缺损" 或中第三髌腱缺损,以研究肌腱愈合和隆增。用髌腱 "窗缺损" 模型研究组织学31、39、生物力学试验30、31和前体荧光法在大鼠肌腱修复中的重现性映像31。在本研究中, 缺陷的大小相当于髌骨肌腱的中央第三, 并以0.99 毫米口径的克氏针为模板进行标准化。这些维度足以研究肌腱愈合而不导致术后破裂。大鼠的解剖尺寸与小鼠相比, 可适应较大的缺陷, 改善损伤的重现性, 治疗分娩, 以及预后的措施。
本研究中对大鼠实施的镇痛协议有效控制术后疼痛, 如 RGS3738所评估。这个评分系统是在这里选择的, 因为它以前被验证为检测术后行为变化, 反映疼痛的老鼠37,38。RGS 是基于标志相对简单的识别, 如眼眶收紧, 鼻子/脸颊扁平, 耳/晶须变化。在整个研究中, 老鼠也保持正常的食物摄取量, 这进一步支持了这种镇痛方案的有效性。事实上, 以前的报告发现, 在非甾体抗炎治疗大鼠术后疼痛和体重变化之间的相关性, 如 meloxicam47。此项研究中选择了此代理, 而抢先管理则是从老鼠37、48和狗49的研究中获得的, 在那里, 在手术前立即对抗炎剂进行了管理, 发现减轻手术引起的疼痛。功能被评估为手术后疼痛的附加指标, 使用评分系统, 评估使用两个骨盆四肢。在这里选择的三项活动提供了对骨盆四肢的运动范围和承受重量的意愿的一般评估。这个评分系统来自于老鼠的静态和动态功能恢复研究,50,51 , 旨在比较观察到的功能结局的时间过程。评分表明, 双侧肌腱缺损在7天内导致功能暂时下降, 28 天内恢复正常。与 RGS 相比, 在骨科条件下的疼痛检测中, 功能评分显得更为敏感。类似的功能测试被用来评估肌腱愈合, 如阿基里斯功能指数28, 爪子和步幅措施51, 或其他宏观评分系统52。
该模型旨在减少在研究中注册的动物数量, 同时区分未治疗肌腱和两种干细胞治疗的结果。本研究中测试的治疗效果在另一出版物36中讨论。这里使用的模型允许检测的治疗差异, 通过使用内部控制的每一个鼠, 结合非破坏性生物力学测试肌腱在28天 (这里没有提供的数据)。
被选择的被激活的被适应的血浆由于它的共同的用途作为载体为干细胞、生物相容性、辅助功能和当前临床应用在管理 tendinopathies53,54。它可能有助于治疗肌腱愈合, 但允许局部保留干细胞, 它不会干扰治疗组的比较, 因为两者都包括相同数量的活化条件血浆。未经治疗的肌腱的组织学特征与以前的肌腱愈合描述一致, 在炎症阶段, 巨噬细胞吞噬和分解的酶会去除坏死组织和破坏的胶原束。持续10天后受伤54,55,56。在增生期, 修复部位的细胞增殖和血供在肌腱修复后28天内最大, 即57,58。我们发现了肌腱的表观增厚、横截面积和试样弹性模量的相关性。根据这些结果, 组织学评分似乎反映了可能不可取的组织变化, 并不会导致生物力学强度, 如胶原纤维的解体, 血管生成和软骨。在未来, 通过结合肌腱细胞、细胞外基质组织、蛋白多糖含量等附加标准, 可以改善该模型对基于组织学特征的治疗方法的鉴别能力, 并弹力纤维的分布52,59。

肌腱损伤是一个最常见的原因, 严重疼痛和肌肉萎缩跨物种1。在兽医中, 肌腱和韧带损伤对马匹有特殊的兴趣, 因为赛马中82% 的受伤都涉及肌肉骨骼系统, 46% 的人会影响肌腱和韧带2,3。瘢痕组织形成影响愈合肌腱的生物力学特性, 解释屈肌腱损伤后恢复运动的保护预后;再伤害发生在2年之内在67% 匹马保守地对待了4。再生医学提供了新的替代条件, 挑战传统治疗。自体干细胞治疗产生了一些令人鼓舞的结果5,6 , 但受发病率与组织收集, 延迟管理, 由于处理/重新编程的细胞, 和影响患者的健康状况 (如年龄) 对干细胞的属性7,8。这些限制为研究同种异体干细胞作为一种现成的替代方法提供了理论依据。胎儿附件细胞是吸引候选者, 因为他们规避的伦理关注和风险的畸胎瘤形成与胚胎干细胞。在胎儿附件, 脐带基质 (UCM), 也命名沃顿的果冻, 是丰富和容易收集。
无论细胞来源如何, 加强 stemness 是建立同种异体再生医学细胞库的必要条件。从功能的角度来看, stemness 可以定义为自我更新和多谱系分化的可能性9。stemness 的证据依赖于增殖和分化化验, 以及基因标记Oct4、 Sox2、和北美9的表达。增强 stemness 的一个策略是依靠使用生物材料作为空隙填料和载体, 从而增强 UCM-MSCs 的增殖和分化。这种方法消除了对转录因子的操纵, 将成熟细胞重新编程为诱导的多潜能细胞的担忧。在被认为是干细胞潜在载体的生物材料中, 壳聚糖对其生物相容性和降解能力有吸引力10。这种天然 aminopolysaccharide 是由碱性脱乙酰甲壳素, 第二种最丰富的天然多糖, 主要获得作为 subproduct 的贝类10。我们以前研究过 MSCs 和壳聚糖支架之间的相互作用, 观察到球体的形成11,12,13,14,15,16. 我们还报告了软骨在壳聚糖矩阵的优越性12,13,14,15,16,17, 18。最近, 两项独立研究描述了在壳聚糖薄膜上培养的脂肪组织和胎盘组织衍生 MSCs 的球体形成19,20。这种球体的形成不仅增强了 stemness, 而且还改善了体内植入20后干细胞的保留。
肌腱的患病率和发病率促使实验模型的发展, 以研究 tendinopathies 的病理生理学和试验新的治疗方法, 如干细胞注射。在马匹中, 胶原酶诱导的炎是一种常用的模型, 用于证明在肌腱修复中使用 MSCs 的有效性21。这种方法的相关性是有限的, 因为注射引起急性炎症变化, 而临床 tendinopathies 通常是由慢性致使22,23造成的。此外, 化学诱导肌腱疾病诱发愈合反应, 并没有复制受损愈合过程中存在的临床病例22,23。切除的表面数字屈肌腱部分已被描述为一个外科模型炎在马24。最近, 一种微创的方法被用来限制外伤损伤的中心核心的表面数字屈肌腱25。手术模型不模拟可能导致自然肌腱疾病的疲劳机制, 而且在造成的损害程度上往往缺乏重现性25。无论模型, 与马模型肌腱疾病的发病率和成本是额外的限制, 这证明了一个兴趣的啮齿动物模型作为在体内评估新的疗法的第一步。
啮齿类动物实验模型的主要优点之一是控制个体间变异的成本和能力。啮齿类动物可以标准化的各种生理因素, 由于其快速生长速度和相对较短的寿命, 限制变异的来源, 因此减少了检测差异所需的动物数量。在啮齿动物中诱导肌腱疾病的策略依赖于化学诱导, 同时也用于部分肌腱缺损的外科创建21。手术模型可以模拟自然 tendinopathies 比化学模型更好, 但可能导致更高的发病率和灾难性破坏肌腱。在这方面, 老鼠似乎比老鼠更适合这些模型, 因为它们的大小允许产生更大的缺陷, 从而有助于评价组织愈合。大大鼠已被用于四主要肌腱组的 tendinopathies 实验研究: 肩袖、屈肌、阿基里斯和髌腱26。其中, 涉及髌骨肌腱的模型特别吸引人, 因为这种肌腱的体积较大, 并且易于访问27。髌腱将股四头肌与胫骨股骨。在这个伸肌机制中, 髌骨是一种籽骨, 它引导四头肌的作用, 并勾勒出髌腱的近端程度。髌腱近端和远端骨锚的存在有利于生物力学试验。涉及髌骨肌腱的模型通常依赖于单边外科缺损, 而对侧完整肌腱作为控制28,29。最常见的髌骨肌腱缺损模型包括切除髌骨远端的髌腱中央部分 (1 毫米) 至胫骨股骨的插入, 而对侧髌腱保持完好。结果的措施包括组织学, 无损生物力学测试或对失败的生物力学测试, 超声成像,活体荧光成像, 总观察和功能测试28,30 ,31。单边模型不允许比较建议的治疗与保守的管理类似的伤害在同一动物。同样, 几种治疗方法之间的比较需要单独的动物。双边模式将消除个体间的变异, 减少研究所需的动物数量32。然而, 双边伤害可能增加发病率, 双侧跛行可能阻碍治疗评估。几项研究简要报告了双侧髌腱缺损在大鼠中的应用, 但侧重于治疗的效果而非围手术期管理和模型3334的发病率。
这项研究的长期目标是制定一个战略, 以改善 stemness 和在体内生存的 UCM-MSCs, 注定到同种异体移植。为了实现这一目标, 我们最近报告了在缺氧环境下, 球体在壳聚糖膜上的形成和孵化的 stemness 改善了 UCM-MSCs 的结构35。这些体外属性与改善的髌骨肌腱缺损的生物力学性能相关. 基于这些结果, 大鼠双侧髌腱缺损模型似乎适合于考生肌腱损伤的治疗方法36。本研究报告的目的是为 UCM-MSCs 的分离和鉴定提供详细的协议, 为干细胞制备生物递质系统, 建立和治疗双侧髌骨肌腱缺损, 以及术后缺损内组织愈合的恢复和评估。

<table><tbody><tr><td>PBS 10x</td><td>Hyclone</td><td>SH30258.01</td><td>Consumable</td></tr><tr><td>Collagenase type IA</td><td>Worthington</td><td>LS004197</td><td>Consumable</td></tr><tr><td>DMEM low glucose</td><td>Hyclone</td><td>SH30021.FS</td><td>Consumable</td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30910.03</td><td>Consumable</td></tr><tr><td>Penicillin/Streptomycin 100x</td><td>Hyclone</td><td>SV30010</td><td>Consumable</td></tr><tr><td>Trypsin 0.25%</td><td>Hyclone</td><td>SH30042.01</td><td>Consumable</td></tr><tr><td>Accutase</td><td>Innovative Cell Technologies</td><td>AT104</td><td>Consumable</td></tr><tr><td>Trypan blue</td><td>Hyclone</td><td>SV30084.01</td><td>Consumable</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma</td><td>D2650</td><td>Consumable</td></tr><tr><td>Chitosan</td><td>Sigma</td><td>C3646</td><td>Consumable</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>S8045</td><td>Consumable</td></tr><tr><td>Bovine Serum Albumin</td><td>Hyclone</td><td>SH30574.01</td><td>Consumable</td></tr><tr><td>Round bottom polystyrene tube</td><td>Corning</td><td>149591A</td><td>Consumable</td></tr><tr><td>Mouse anti-horse CD44 (FITC)</td><td>AbD serotec</td><td>MCA1082F</td><td>Consumable</td></tr><tr><td>Mouse anti-rat CD90 (FITC)</td><td>AbD serotec</td><td>MCA47FT</td><td>Consumable</td></tr><tr><td>Mouse anti-horse MHC-II (FITC)</td><td>AbD serotec</td><td>MCA1085F</td><td>Consumable</td></tr><tr><td>Mouse IgG1 (FITC) - Isotype Control</td><td>AbD serotec</td><td>MCA928F</td><td>Consumable</td></tr><tr><td>Mouse monoclonal [SN6] to CD105 (FITC)</td><td>abcam</td><td>ab11415</td><td>Consumable</td></tr><tr><td>Mouse IgG1 (FITC) - Isotype Control</td><td>abcam</td><td>ab91356</td><td>Consumable</td></tr><tr><td>Mouse anti-human CD34 (FITC)</td><td>BD</td><td>BDB560942</td><td>Consumable</td></tr><tr><td>Mouse IdG1 kappa (FITC)</td><td>BD</td><td>BDB555748</td><td>Consumable</td></tr><tr><td>7-AAD</td><td>BD</td><td>BDB559925</td><td>Consumable</td></tr><tr><td>BD Accuri C6 Flow Cytometer</td><td>BD</td><td></td><td>Equipment</td></tr><tr><td>Vacutainer 5 mL</td><td>Med Vet International</td><td>RED5.0</td><td>Consumable</td></tr><tr><td>Acid-citrate-dextrose</td><td>Sigma</td><td>C3821</td><td>Consumable</td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>C5670</td><td>Consumable</td></tr><tr><td>Sevoflurane</td><td>JD Medical</td><td>60307-320-25</td><td>Consumable</td></tr><tr><td>Rats</td><td>Charles River</td><td>Strain code: 400</td><td>Experimental animal</td></tr><tr><td>Rat surgical kit</td><td>Harvard apparatus</td><td>728942</td><td>Equipment</td></tr><tr><td>Surgical Blade #15</td><td>MEDLINE</td><td>MDS15115</td><td>Consumable</td></tr><tr><td>Rat MD&#039;s Baytril (2 mg/Tablet),&lt;br/&gt; Rimadyl (2 mg/Tablet)</td><td>Bio Serv</td><td>F06801</td><td>Consumable</td></tr><tr><td>Polyglactin 910, 5-0</td><td>Ethicon</td><td>J436G</td><td>Consumable</td></tr><tr><td>Eosin alchol shandon</td><td>Thermo scientific</td><td>6766007</td><td>Consumable</td></tr><tr><td>Harris Hematoxylin</td><td>Thermo scientific</td><td>143907</td><td>Consumable</td></tr></tbody></table>

evaluation of stem cell therapies in a bilateral patellar tendon injury model in rats

再生医学提供了新的替代条件, 挑战传统治疗。肌腱的发病率和发病率, 加上该组织的愈合特性有限, 促使寻找细胞治疗, 推动实验模型的发展, 以研究其功效。脐带基质源性干细胞 (UCM MSC) 是吸引候选者, 因为它们丰富, 易于收集, 规避了对畸胎瘤形成的伦理关注和风险, 但与原始胚胎干细胞相比, 更接近于成人组织衍生的骨髓间充质干细胞. 对壳聚糖作为一种通过球形形成增强 MSCs 性能的策略, 具有重要的研究价值。本文详细介绍了 UCM-MSCs 的分离技术, 制备了壳聚糖膜球体, 并分析了球形形成对表面标记表达的影响。因此, 本文介绍了在壳聚糖膜上形成的 UCM-MSC 球体的体内植入的大鼠双侧髌腱损伤模型的建立。在研究中没有发现并发症的发病率, 压力上升的影响, 或组织感染。7天大鼠的总功能评分低于正常大鼠, 术后28天内恢复正常。组织愈合的组织学评分证实在治疗缺陷中存在血栓, 7 天, 没有异物反应, 并在28天的进展愈合。这种双侧髌骨肌腱缺损模型通过在每只老鼠体内建立内部控制来控制个体间的变异, 与可接受的发病率相关, 并允许检测未治疗肌腱与治疗之间的差异。

在目前的研究中, 结果被显示为平均值, 即标准差。细胞从6匹母马的脐带中分离出来, 在标准或壳聚糖条件下表达每个细胞表面标记物的分离细胞系的百分比与弗里德曼试验比较, 作为非参数分析的方差与重复措施.对于肌腱缺损模型的建立, 8 只大鼠进行7天术后评估, 12 只大鼠用于28天的评估。将功能结局的结果显示为均值电子扫描电镜 (平均标准误差), 并与 t 检验进行比较。UCM 被选为一种细胞源, 原因是它的丰度、易于采集, 以及相对于其他胎儿附件源的优异增殖40。在标准板上培养的 UCM 中分离出来的细胞在整个扩张过程中保持了成纤维样的形状。在壳聚糖板上培养的细胞形成球体 (图 8)。
在标准条件下培养的大多数细胞表达 CD44 (98.6 1.62%), CD90 (88.7 @ 3.04%), CD105 (77.9 @ 6.84%), 而 CD34 (0.07 @ 0.17%) 和 MHC II (1.33 @ 1.12%) 的表达非常低。在条件文化下, CD44 (97.0 2.12%) 和 CD34 (1.13) 的表达水平与标准文化不同, 而 CD90 (33.3 @ 37.1%) 和 CD105 (54.0 @ 19.0%) 的表达水平下降, MHC II (2.84 @ 0.98%) 增加了35。
通过对 UCM-MSCs体外的分离和表征, 将条件细胞的生物学行为与标准条件下培养的大鼠双侧髌腱缺损模型进行比较。未处理的缺陷在每只老鼠担当内部控制。术后肿胀在所有大鼠中最小, 并在48小时内解决。根据 RGS, 没有一个老鼠显示疼痛或窘迫的迹象, 如眼眶收紧, 鼻子/脸颊扁平, 或耳晶须变化。在整个研究过程中, 所有的老鼠每天消耗正常范围的3–4丸或15–20克饲料。正常大鼠与7天 (n = 8, p < 0.0001,表 3) 的总功能评分, 包括辅助和无帮助的后肢站立以及攀爬评分均较高。然而, 在手术后28天内, 总功能评分恢复正常 (n = 12, p = 0.78)。因此, 大鼠双侧髌腱缺损模型已被证明是有效的限制个体间的变化和减少必要数量的动物, 而不造成严重的发病率 (视频 1-5)。
约37% 的肌腱收集28天出现明显加厚 (图 7)。这种增稠在空的和处理过的缺陷之间同样分布。总组织学评分随着时间的增加而未处理的缺陷 (n = 20, p = 0.0034)。当治疗评分的每个组分独立地被评估时, 唯一的参量改变以时间包括软骨形成, 增加在7和28天之间 (n = 20, p < 0.0001)。血管生成倾向于随时间增加 (p = 0.3), 但胶原形成没有不同 (p = 0.69)。经组织学检查, 在手术后7天检查的所有肌腱组织切片中均观察到炎症, 无论是否有治疗。所有治疗缺陷均存在血肿。组织学血管生成评分 (表 2, 图 9) 从0到 1, 建议适度的动脉和毛细血管浸润, 而胶原评分评分 (表 2, 图 10) 从2到3不等, 这表明中等至广泛的胶原形成。软骨形成评分 (表 2, 图 11) 不等于0到 2, 建议没有软骨形成, 以中等软骨形成25% 到50% 的肌腱区。所有组织学图均以左侧为前部, 顶端为肌腱中段横断面的内侧部分。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56810/56810fig1.jpg"> 图 1: 大鼠的定位和无菌手术的准备.每只老鼠放在两个装有温水的0.5 升水瓶之间, 用布覆盖以保持体温和位置, 同时防止皮肤损伤。每只老鼠的四肢都被贴在桌子上, 它的身体布满了气泡包裹。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/56810fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56810/56810fig2.jpg"> 图 2: 髌骨肌腱缺损的手术模型 (对齐).一个0.99 毫米直径的克氏针线是对腱作为一个模板, 以规范的缺陷大小的每一个肢体。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56810/56810fig3.jpg"> 图 3: 髌骨肌腱缺损 (切口) 的手术模型.在克氏针的每一侧都进行两个全厚度切口, 以隔离肌腱的中心部分。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56810/56810fig4.jpg"> 图 4: 髌骨肌腱缺损的手术模型 (切除).中心部分切除下部和远端, 造成一个缺陷, 与克氏丝的大小匹配。切除区域显示在虚线框内。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56810/56810fig5.jpg"> 图 5: 操作的窒息的手术后外观.筋膜闭合的交叉模式和皮肤被关闭的皮模式。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/56810fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56810/56810fig6.jpg"> 图 6: 用于提供 UCM-MSCs 的活性条件等离子体的制备.在激活前, 干细胞在条件血浆中悬浮。结果血栓入髌骨肌腱缺损。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/56810fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56810/56810fig7.jpg"> 图 7:正常外观 (C、 D) 和加厚 (A、 B) 肌腱。(A, C)后景色;(B, D)横向观点;块箭头识别胫骨和细箭识别肌腱。
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56810/56810fig8.jpg"> 图 8: 在19% 氧水平下 (标准组: A) 和壳聚糖膜在5% 氧水平下培养的细胞形态学 (条件组: B).独立单元格是标准组 (A) 上的主轴形状, 并形成条件组上的球体 (B)。缩放条 = 400 µm<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/56810fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56810/56810fig9.jpg"> 图 9: 用于血管生成分级的代表性光显微照片.经过7天的愈合, 没有治疗的肌腱接收到0级 (a、 D), 而具有条件的单元格 (C、 F) 或标准单元 (B、 E) 的级别分别获得了0.5 和0级。28天后, 未经处理的肌腱 (G、 J) 的级别为 0, 而具有条件的单元格 (I、L) 或标准单元 (H、K) 的分数分别为1.0 和0.5。箭头识别肌腱样本中存在的血管。箭头指向血管在100X 放大倍数对应于同一箭头指向血管在400x 放大倍数。正常肌腱组织 (M, N)。马尾松的三色染色 (A、B、C、G、H、I、M);苏木精和伊红染色 (D、E、F、J、K、L、N);缩放条 = 100 µm (-n)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/56810fig9large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56810/56810fig10.jpg"> 图 10: 用于胶原分级的代表性光显微照片.经过7天的愈合, 没有治疗肌腱获得2.5 级 (a, D), 而那些有治疗 (B, C, E, F) 获得3.0 级。28天后, 未经治疗的肌腱 (G、J) 获得3.0 级, 而有条件的细胞 (I、L) 或标准细胞 (H、K) 则分别获得1.0 和3.0 的分数。肌腱纤维的解体可以在400X 插入 (H, K) 中得到赞赏。正常肌腱组织 (M, N)。马尾松的三色染色 (A、B、C、G、H、I、M);苏木精和伊红染色 (D、E、F、J、K、L、N);缩放条 = 100 µm (-n)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/56810fig10large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56810/56810fig11.jpg"> 图 11: 用于软骨分级的代表性光显微照片.经过7天的愈合, 肌腱没有治疗 (A, D), 那些有条件的细胞 (C, F) 或标准细胞 (B, E), 都得到了0级。28天后, 未经治疗的肌腱 (G、J) 获得1.0 级, 而有条件的细胞 (I、L) 或标准细胞 (H、K) 则分别获得0和2.0 的分数。箭头指向肌腱样本中的软骨细胞。箭头指向软骨细胞在100X 放大倍数对应于同一箭头指向软骨细胞在400X 放大倍数。正常肌腱组织 (M, N)。马尾松的三色染色 (A、B、C、G、H、I、M);苏木精和伊红染色 (D、E、F、J、K、L、N);缩放条 = 100 µm (-n)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/56810fig11large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
视频 1: 对大鼠动态功能的评估。定时无腿的后肢站立 (1–5: 评分 1) 观察4小时后手术。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/Video_1.mov" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
视频 2: 对大鼠动态功能的评估.定时后肢站立与前肢辅助 (0–5 s: 评分 0) 被指出 4 h 后手术。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/Video_2.mov" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
视频 3: 对大鼠动态功能的评估。定时无辅助后肢站立 (1–5 s: 评分 1), 定时后肢站立与前肢协助 (5–10: 评分 1) 被指出14天后手术。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/Video_3.mov" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
视频 4: 对大鼠动态功能的评估.定时后肢站立与前肢协助 (10–15 s: 比分 2), 没有能力攀登 17 cm 塑料笼子墙壁 (没有攀登: 比分 0) 被注意了27天后手术。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/Video_4.mov" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
视频 5: 对大鼠动态功能的评估.定时后肢站立与前肢协助 (5–10 s: 比分 1), 以能力攀登 17 cm 塑料笼子墙壁 (是: 比分 1) 被注意了14天后手术。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56810/Video_5.mov" target="_blank">请单击此处查看此视频。(右键单击可下载)</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>功能测试</td>
			<td>0</td>
			<td>1</td>
			<td>2</td>
			<td>3</td>
			<td>得分</td>
		</tr>
<tr>
<td>后肢支架辅助</td>
			<td>0–5 s</td>
			<td>> 5–10 s</td>
			<td>> 10–15 s</td>
			<td>> 十五年代</td>
			<td></td>
		</tr>
<tr>
<td>后肢独立站立</td>
			<td>0–1 s</td>
			<td>> 1–5 s</td>
			<td>> 5 s</td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>攀登能力</td>
			<td>不</td>
			<td>是的</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td>总</td>
			<td></td>
		</tr>
</tbody></table>表 1: 评估大鼠动态功能的评分系统。建立了评分系统 (0–6), 以评估动态功能的基础上3活动, 包括定时后肢站立与前肢支持 (0–3), 定时无辅助后肢站立 (0–2), 并有能力爬上17厘米的塑料笼壁 (0–1)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="6">胶原分级</td>
		</tr>
<tr>
<td>0</td>
			<td colspan="5">正常胶原定向切向</td>
		</tr>
<tr>
<td>1</td>
			<td colspan="5">轻度变化, 胶原纤维少于25% 无序</td>
		</tr>
<tr>
<td>2</td>
			<td colspan="5">25% 和50% 之间的胶原纤维适度变化无序</td>
		</tr>
<tr>
<td>3</td>
			<td colspan="5">显着的变化与胶原蛋白超过50% 无序</td>
		</tr>
<tr>
<td colspan="6">血管生成程度</td>
		</tr>
<tr>
<td>0</td>
			<td colspan="5">组织与微动脉的适度浸润</td>
		</tr>
<tr>
<td>1</td>
			<td colspan="5">毛细血管的存在</td>
		</tr>
<tr>
<td>2</td>
			<td colspan="5">无血管浸润</td>
		</tr>
<tr>
<td colspan="6">软骨形成</td>
		</tr>
<tr>
<td>0</td>
			<td colspan="5">无软骨形成</td>
		</tr>
<tr>
<td>1</td>
			<td colspan="5">孤立透明软骨结节</td>
		</tr>
<tr>
<td>2</td>
			<td colspan="5">适度软骨形成25% 到50%</td>
		</tr>
<tr>
<td>3</td>
			<td colspan="5">广泛的软骨形成, 超过50% 的领域涉及</td>
		</tr>
</tbody></table>表 2:组织学评分等级.根据胶原分级、血管生成程度和软骨形成 (从鲍姆等) 对切片的组织学评分进行分级。39)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>组</td>
			<td>平均电子扫描电镜</td>
		</tr>
<tr>
<td>正常 (n = 3)</td>
			<td>6.00 @ 0.48</td>
		</tr>
<tr>
<td>手术后7天 (n = 8)</td>
			<td>2.25 @ 0.30</td>
		</tr>
<tr>
<td>手术后28天 (n = 12)</td>
			<td>5.83 @ 0.24</td>
		</tr>
</tbody></table>表 3:正常大鼠和未治疗大鼠的功能评分.对正常大鼠和未治疗大鼠术后7、28天的功能评分进行分级。n = 老鼠的数量。
补充图 1: 浇口策略和表面标记表达式示例:碎片是根据小型 FSC 和 SSC (A) 排除的。根据 FL3 (B) 检测到的 7-反色阴性染色, 选择活细胞。同种匹配控件 (C) 用作负控制, 以创建 CD44-FITC 正单元格 (D) 的门。

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

大鼠双侧髌腱损伤模型中干细胞治疗的评价

本文介绍了大鼠双侧髌腱缺损模型球体脐基质干细胞的制备及评价。该模型与可接受的发病率相关, 并发现检测到未治疗和治疗肌腱之间的差异, 以及两种治疗方法之间的区别。

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

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11.1K 次观看.  Western University of Health Sciences. 本文介绍了大鼠双侧髌腱缺损模型球体脐基质干细胞的制备及评价。该模型与可接受的发病率相关, 并发现检测到未治疗和治疗肌腱之间的差异, 以及两种治疗方法之间的区别。

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

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

科研

医学

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 ...

生物工程

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 ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

A Rat Tibial Growth Plate Injury Model to Characterize Repair Mechanisms and Evaluate Growth Plate Regeneration Strategies

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found at the end of all long bones in children that is responsible for longitudinal bone growth. Once damaged, cartilage tissue within the growth plate can undergo premature ossification and lead to unwanted bony repair tissue, which forms a "bony bar." In some cases, this bony bar can result in bone growth deformities, such as angular deformities, or it can completely halt longitudinal bone growth. There is currently no clinical treatment that can fully repair an injured growth plate. Using an animal model of growth plate injury to better understand the mechanisms underlying bony bar formation and to identify ways to inhibit it is a great opportunity to develop better treatments for growth plate injuries. This protocol describes how to disrupt the rat proximal tibial growth plate using a drill-hole defect. This small animal model reliably produces a bony bar and can result in growth deformities similar to those seen in children. This model allows for investigation into the molecular mechanisms of bony bar formation and serves as a means to test potential treatment options for growth plate injuries.

The growth plate is a cartilaginous region in children's long bones where longitudinal growth occurs. When injured, bony tissue can form and impair growth. We describe a rat model of growth plate injury that leads to bony repair tissue, allowing the study of repair mechanisms and growth plate regeneration strategies.

大鼠胫骨生长板损伤模型，用于表征修复机制并评估生长板再生策略

A third of all pediatric fractures involve the growth plate and can result in impaired bone growth. The growth plate (or physis) is cartilage tissue found ...

Effects of Allogeneic Platelet-Rich Plasma (PRP) on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a lesion in the Achilles tendon; prepare PRP and inject it (or the saline solution); remove the tendon; and perform biomechanical, molecular, and histological evaluations. At each step, all the procedures and methods are described in detail, so they can be reproduced easily.
Achilles tendons have been surgically sectioned (removal of a 5-mm long section). Afterwards, PRP or saline solution was injected to study whether PRP has a positive effect on the healing of the tendon. Three groups of 40 animals (a total of 120 rats were used in this study) were subdivided into 2 subgroups: PRP injection group and a saline injection control group. Rats were sacrificed at increasing time points (Group A: 5 days; Group B: 15 days; Group C: 30 days) and tendons were removed. 90 tendons underwent biomechanical testing before performing transcriptomic analysis and the 30 remaining tendons were submitted to histological analysis.

Effects of Allogeneic Platelet-Rich Plasma PRP on the Healing Process of Sectioned Achilles Tendons of Rats: A Methodological Description

This protocol describes the evaluation process of healing tendons in rats that have been injected with allogeneic platelet rich plasma (PRP) or saline solution after removing part of the Achilles tendon. The progress of tendon healing is evaluated at several time points using different types of analyses.

同种异体富血小板血浆 (PRP) 对大鼠分段腱愈合过程的影响: 方法学描述

This article describes the experimental procedures used to observe if PRP can positively affect tendon healing. There are 4 main steps to follow: induce a ...

了解肌腱撞击 – 用于研究机械生物学的小鼠外植体模型

Murine Hind Limb Explant Model for Studying the Mechanobiology of Achilles Tendon Impingement

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a localized fibrocartilage phenotype characterized by accumulation of glycosaminoglycan (GAG)-rich matrix and remodeling of the collagen network. While fibrocartilage is a normal feature in impinged regions of healthy tendons, excess GAG deposition and disorganization of the collagen network are hallmark features of tendinopathy. Accordingly, impingement is clinically recognized as an important extrinsic factor in the initiation and progression of tendinopathy. Nevertheless, the mechanobiology underlying tendon impingement remains understudied. Prior efforts to elucidate the cellular response to tendon impingement have applied uniaxial compression to cells and excised tendon explants in vitro. However, isolated cells lack a three-dimensional extracellular environment crucial to mechanoresponse, and both in vitro and excised explant studies fail to recapitulate the multiaxial strain environment generated by tendon impingement in vivo, which depends on anatomical features of the impinged region. Moreover, in vivo models of tendon impingement lack control over the mechanical strain environment. To overcome these limitations, we present a novel murine hind limb explant model suitable for studying the mechanobiology of Achilles tendon impingement. This model maintains the Achilles tendon in situ to preserve local anatomy and reproduces the multiaxial strain environment generated by impingement of the Achilles tendon insertion upon the calcaneus during passively applied ankle dorsiflexion while retaining cells within their native environment. We describe a tissue culture protocol integral to this model and present data establishing sustained explant viability over 7 days. The representative results demonstrate enhanced histological GAG staining and decreased collagen fiber alignment secondary to impingement, suggesting elevated fibrocartilage formation. This model can easily be adapted to investigate different mechanical loading regimens and allows for the manipulation of molecular pathways of interest to identify mechanisms mediating phenotypic change in the Achilles tendon in response to impingement.

作者聚焦：揭开肌腱撞击的力学生物学 – 多轴鼠后肢外植体模型 

We present a custom experimental platform and tissue culture protocol that recreates fibrocartilaginous change driven by impingement of the Achilles tendon insertion in murine hind limb explants with sustained cell viability, providing a model suitable for exploring the mechanobiology of tendon impingement.

小鼠后肢外植体研究跟腱撞击力学生物学的模型

Tendon impingement upon bone generates a multiaxial mechanical strain environment with markedly elevated transverse compressive strain, which elicits a ...

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients&#8217; life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

大鼠双侧髌腱损伤模型中干细胞治疗的评价

摘要

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此视频中的章节

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

无胸腺大鼠模型工程前交叉韧带移植评估

中的应用

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

大鼠胫骨生长板损伤模型，用于表征修复机制并评估生长板再生策略

同种异体富血小板血浆 (PRP) 对大鼠分段腱愈合过程的影响: 方法学描述

小鼠后肢外植体研究跟腱撞击力学生物学的模型

小鼠后肢外植体研究跟腱撞击力学生物学的模型

小鼠后肢外植体研究跟腱撞击力学生物学的模型

应用三维单轴机械刺激生物反应器系统诱导肌生衍生干细胞的畸因分化

大鼠双侧髌腱损伤模型中干细胞治疗的评价

摘要

探索更多视频

此视频中的章节

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

无胸​​腺大鼠模型工程前交叉韧带移植评估

中的应用

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

大鼠胫骨生长板损伤模型，用于表征修复机制并评估生长板再生策略

同种异体富血小板血浆 (PRP) 对大鼠分段腱愈合过程的影响: 方法学描述

小鼠后肢外植体研究跟腱撞击力学生物学的模型

小鼠后肢外植体研究跟腱撞击力学生物学的模型

小鼠后肢外植体研究跟腱撞击力学生物学的模型

应用三维单轴机械刺激生物反应器系统诱导肌生衍生干细胞的畸因分化

无胸腺大鼠模型工程前交叉韧带移植评估