这项工作得到了美国 （U.S.） 的 VA Merit Review #I01 BX000760 （RFK） 的支持。退伍军人事务部 BLR&D 和美国退伍军人事务部拨款 #I01 BX004146 （TAB）。我们也非常感谢明尼苏达大学利勒海心脏研究所的支持。本作品内容不代表美国政府退伍军人事务部的观点。

明尼阿波利斯退伍军人医疗中心和明尼苏达大学的机构动物护理和使用委员会（IACUC）已经批准了所有的动物研究。遵循了美国国立卫生研究院（NIH）关于使用和护理实验动物的现行指南。
1. 间充质干细胞的分离及外泌体的制备和表征
<ol><li>骨髓间充质干细胞（MSCs）的分离<ol><li>从 20 周龄雌性约克郡长白猪的胸骨或胫骨获取 30-50 mL 无菌骨髓。为此，将 25 mm 15G 骨间针插入胸骨或胫骨，并将样本吸入装有 10 mL 肝素的 60 mL 注射器中。 注意：有关骨髓收集的更多详细信息，请参阅Pittenger等人和Hocum-Stone等人12,16。</li>
		<li>简而言之，将骨髓标本通过含有肝素的Vacutainer CPT管以1800×g的速度持续30分钟。</li>
		<li>去除含有单核细胞的血沉棕黄层，并用Hank的平衡盐溶液洗涤。通过离心沉淀单核细胞并重悬于生长培养基（10%胎牛血清[FBS]）中。</li>
		<li>将单核细胞转移到细胞培养瓶中进行贴壁生长。通过其贴壁性将MSC从单核部分中分离出来。</li>
		<li>在24小时内洗涤所有非MSC，在组织培养瓶中留下单层MSC。通过流式细胞术确认 MSC 表型，确保 CD45（造血标志物）呈阴性，CD90 和 CD105（MSC 标志物）呈阳性。</li>
	</ol></li>
	<li>猪间充质干细胞外泌体的制备和表征<ol><li>将 1 x 104 个 H9C2 大鼠心肌细胞接种并在 1x DMEM+ 10% FBS 和 1x Pen/链球菌中培养。在高级 DMEM + 5% FBS 和 1x Pen/链球菌中播种 2 x 104 个猪 MSC。</li>
		<li>一旦两种细胞系至少 80% 汇合，将培养基更换为外泌体耗尽的 H9C2 和 MSC 培养基。</li>
		<li>将H9C2心肌细胞暴露于轻度缺氧（1%O2 持续24小时）。24小时后从缺氧中取出烧瓶，移出H9 C 2培养基。</li>
		<li>从 MSC 烧瓶中取出并丢弃 MSC 培养基。将纯化的 H9C2 培养基加入 MSC 烧瓶中。在常氧条件下（5%CO 2,20%O2和37°C）孵育烧瓶6小时。</li>
		<li>按照制造商的说明，使用总外泌体分离试剂从共培养的条件培养基中提取外泌体。</li>
		<li>通过使用抗 CD-63 抗体 （1：1000） 17 的常见外泌体蛋白进行蛋白质印迹检测来验证外泌体的鉴定。</li>
		<li>进行纳米颗粒跟踪分析（NTA）以量化外泌体并评估纳米颗粒大小及其分布。为此，将外泌体的总蛋白（50μg）溶解在500μL PBS中，以使用纳米颗粒跟踪分析仪确定外泌体的浓度和大小分布。</li>
		<li>使用纳米颗粒跟踪软件分析数据。</li>
	</ol></li>
</ol>2.离体外循环冠状动脉旁路移植手术
<ol><li>动物准备<ol><li>在计划手术前 3 天称量动物（16 周龄雌性约克郡长白猪）。手术前禁食动物12小时，同时在禁食期间喝水。</li>
		<li>手术前 2-4 小时通过肌内注射途径给予丁丙诺啡 0.18 mg/kg。</li>
	</ol></li>
	<li>动物的诱导<ol><li>通过肌肉注射 6.6 mg/kg 替他明-唑拉西泮/甲苯噻嗪来镇静动物。</li>
		<li>等待 15 分钟，通过评估下颌张力，然后在中央耳静脉中放置 22G 导管，以确保充分镇静。 注意：如果耳静脉不足，可以考虑另一种外周静脉（即头静脉）。</li>
		<li>将眼药膏局部施用于每只眼睛。通过静脉注射途径给予 1-2 mg/kg 异丙酚以诱导全身麻醉。下颌张力最可靠地反映了麻醉的深度，应在整个手术过程中进行评估。</li>
		<li>用适当尺寸的气管插管插管动物。</li>
	</ol></li>
	<li>手术<ol><li>剃掉动物的胸骨和腹股沟，为外科手术做准备。</li>
		<li>根据需要将机械通气设置为每分钟 10-15 次呼吸，氧气 1-4 L/min，异氟醚 1.0-3.0%，以维持手术深度麻醉。检查眼部或下颌反射是否消失，以确认深度麻醉。</li>
		<li>动物的位置监测设备（心电图、潮气末 CO2、心率、血氧饱和度、血压和体温）。</li>
		<li>将静脉导管连接到一袋生理盐水或乳酸林格溶液上，以持续给予维持液。</li>
		<li>使用无菌技术准备皮肤，包括聚维酮碘磨砂膏和 3 次溶液，以获得充分的无菌效果，并将手术部位感染的风险降至最低。</li>
		<li>通过血管内途径给予利多卡因（负荷剂量为 2 mg/kg 或以 50 mcg/kg/min 的剂量连续输注）以预防心律失常。</li>
		<li>将动物放在背部并用无菌毛巾覆盖。</li>
		<li>通过 Seldinger 技术切开左股动脉或右股动脉以放置动脉管路，然后将导管连接到换能器，以便在手术时进行连续血压监测。</li>
		<li>使用单极电烙术做一个 20 cm 的切口，从胸骨切迹近端向下延伸到远端的木突，并将肌肉、皮下脂肪和结缔组织层切开至胸骨。</li>
		<li>使用摆动锯进行正中胸骨切开术。 注意：重复胸骨切开术应避免使用标准锯，因为先前为放置 LAD 收缩器而进行的左开胸手术导致心包粘连的风险更高。</li>
		<li>用一把剪刀分开胸骨后板。使用专门的胸牵开器来充分观察纵隔。</li>
		<li>使用单极电烙术或 Metzenbaum 剪刀解剖粘连。仔细解剖胸骨周围肌肉和脂肪，以暴露左乳内动脉 （LIMA）。</li>
		<li>一旦 LIMA 暴露在胸骨边缘的外侧，使用带有电烙尖端的钝解剖将其与胸壁轻轻分开。将 LIMA 用作镂空移植物。</li>
		<li>在第 3肋 间隙水平开始解剖。轻轻抬高胸骨左缘以获得最佳可视化效果。</li>
		<li>对外膜使用轻柔的牵引，以暴露LIMA的动脉和静脉分支。使用血唇夹住树枝的LIMA侧，并烧灼树枝的胸壁侧。 注意： 必须注意不要烧灼 LIMA 上的夹子，因为这可能会导致导管变窄。</li>
		<li>一旦 LIMA 的初始节段被动员，继续向锁骨下静脉水平近端和远端解剖，直到 LIMA 分叉。</li>
		<li>解剖完成后，通过静脉注射途径给予肝素，剂量为100-300U / kg。给予肝素后等待3分钟。</li>
		<li>3分钟后，夹住LIMA的远端，就在LIMA分叉的水平之前，并分开导管。用免费的 2-0 真丝缝合带缝合远端。</li>
		<li>准备近端进行移植。通过让移植物出血几秒钟来目视检查流体质量。</li>
		<li>用无创伤的斗牛犬夹轻轻夹住 LIMA 导管的远端，以避免出血。用倒 T 形打开心包，做一个大约 5-6 厘米的切口。在心包上放置 3-0 尺寸的缝合线，以便在狭缝两侧进行牵引。</li>
		<li>用硅胶回缩胶带和组织稳定器稳定 LAD，该稳定器固定在胸骨牵开器上。用 11 刀片在狭窄远端（由收缩带引起）的 LAD 动脉进行动脉切开术，并用虹膜剪刀伸展。</li>
		<li>在 LAD 中放置适当大小的冠状动脉分流器。使用非泵旁路技术使用 7-0 运行不可吸收缝合线进行 LIMA 至 LAD 吻合术。松开 LIMA 上的斗牛犬封堵器并确认止血。</li>
	</ol></li>
	<li>间充质干细胞（MSC）来源的外泌体贴片的制备<ol><li>从间充质干细胞中成功分离外泌体后，将大约 3 x 108 个外泌体悬浮在 3 mL 生理盐水中并添加到胶原海绵中。</li>
		<li>将3mL外泌体悬浮液在22°C左右的室温下加热10分钟。将 2 块可吸收的拼贴海绵（每块 1.27 厘米 x 2.54 厘米）放入培养皿中。</li>
		<li>使用 5 mL 注射器和 18G 针头轻轻混合外泌体悬浮液。缓慢地将 1.5 mL 悬浮液移液到每个胶原海绵上，等待 5 分钟以完全吸收。</li>
	</ol></li>
	<li>外泌体贴片的放置<ol><li>将含有外泌体的海绵倒置在心脏的冬眠区域，该区域是LAD分布中前隔区域的心外膜。</li>
		<li>轻轻地放置两块海绵以覆盖心脏的冬眠区域。使用一个 3.5 cm x 1.0 cm 的聚胶乳蛋白网覆盖每个胶原海绵。</li>
		<li>用 7-0 间断缝合线将网状物缝合到心外膜上。</li>
	</ol></li>
	<li>胸管置入<ol><li>通过单独的刺伤切口放置胸管，靠近胸骨切开切口的下侧。小心地将胸管放在心脏的前侧。</li>
		<li>管子就位后，使用水平床垫缝线放置带有 3-0 缝合线的钱包绳缝合线，以便在取出管子时闭合伤口。</li>
		<li>胸管一直维持到胸部完全闭合。</li>
	</ol></li>
	<li>胸部闭合<ol><li>使用八字形图案使用不可吸收的缝合线近似胸骨。沿切口的整个长度通过肌内途径给予 1 mg/kg 布比卡因。 注意： 使用缝合线而不是电线，以避免干扰 MRI 成像。</li>
		<li>分别使用 2-0 和 3-0 可吸收缝合线以标准方式闭合肌肉和皮肤层。</li>
		<li>进行屏气和吸气，将所有空气排出胸腔。谨慎监测呼吸机上的气道压力，将压力保持在 15-22 mmHg 之间，完成后释放。</li>
		<li>排空所有空气后，拔出胸管，同时使用钱包线缝合伤口。局部涂抹胶水覆盖胸骨切口。</li>
	</ol></li>
	<li>术后护理<ol><li>当皮肤切口闭合时，逐渐让动物脱离呼吸机。在断开动物与麻醉设备的连接之前，确保动物能够自主呼吸并保护反射。</li>
		<li>确认动物能够保护其气道后拔出气管插管。用嵌入抗生素软膏的无菌和非粘附敷料覆盖皮肤切口，以尽量减少手术部位感染。</li>
		<li>每 15 分钟继续监测生命体征，包括心率、呼吸频率、体温，直到动物能够在没有帮助的情况下保持姿势。</li>
		<li>确保动物在能够抬起并抬起头并可以在没有帮助的情况下站立之前不会无人看管。在将动物运送到恢复病房之前，通过皮下途径以 0.2 mg/kg 的剂量给予美洛昔康。</li>
		<li>当动物稳定时，将动物运送到恢复单位。将手术部位敷料保留在切口上，直到术后第 3 天。如果敷料变脏，请更换敷料。</li>
		<li>在手术后的前 5 天内继续监测动物的疼痛程度、皮肤切口和整体健康状况。根据需要给予半剂量的美洛昔康（0.1 mg/kg），每日一次，用于突破性疼痛。</li>
		<li>手术后的前 5 天，在切口愈合时将动物单独饲养，以降低手术部位被另一只动物感染的风险。5天后将动物送回集体饲养。</li>
		<li>向兽医或适当的工作人员报告动物状况的任何并发症或变化（发烧、腹水、体重减轻、食欲不振等）。</li>
	</ol></li>
</ol>3. 使用股骨通路的冠状动脉造影
<ol><li>将动物固定在手术台上的背卧位上。以每分钟 10-15 次呼吸的速度开始机械通气。根据需要将氧气设置为 2-4 L/min，将异氟醚设置为 1% 和 4%，以维持深度麻醉。</li>
	<li>将心电图导联放在动物的肢体上以监测心律。评估动物的麻醉深度。考虑在没有眼睛或下颌反射时深度麻醉的动物。</li>
	<li>用聚维酮碘磨砂膏清洁胸部和颈部区域，然后用毛巾覆盖动物。</li>
	<li>通过手术切除进入股动脉，暴露股动脉和静脉。用 11 号刀片在股动脉中做一个 1-2 毫米的纵向切口，并使用血管腔中的 11 Fr 引入器鞘插管动脉。</li>
	<li>获得通路后，推进导管进行冠状动脉造影，以评估 LIMA-LAD 移植物的解剖通畅性。</li>
</ol>

辅助外泌体治疗：促进 CABG 手术后的心肌恢复

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+and+their+potential+as+cardiac+therapeutics.">Mesenchymal stem cells and their potential as cardiac therapeutics.</a> Circulation Research. 95 (1), 9-20 (2004).</li><li>Campos-Silva, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+detection+of+extracellular+vesicles+immune-captured+from+urine+by+conventional+flow+cytometry.">High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry.</a> Scientific Reports. 9 (1), 2042 (2019).</li><li>Hocum Stone, L. 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作者没有什么可透露的。

本研究提出了第一个慢性缺血性心肌的猪模型，其中表明，在手术血运重建期间，用 MSC 衍生的外泌体负载胶原蛋白贴片治疗可在正性肌力刺激下恢复舒张和收缩功能，可能通过靶向线粒体恢复。先前，研究表明，在HIB的大型动物模型中，通过心脏MRI测量的舒张和收缩功能仍然受损，并且仅随着血运重建而略有改善，而没有完全恢复6,19。尽管左心室射?...

在全球范围内，重症冠状动脉疾病影响超过一亿患者，尽管死亡率有所下降，但它仍然是导致死亡的主要原因之一1,2。冠状动脉疾病的临床范围很广，从心肌梗死 （MI） 到缺血，但仍能存活。大多数临床前研究都集中在心肌梗死上，其特征是存在梗死组织，因为可以在小型和大型动物模型中进行研究。然而，该模型并不能解决保留活力和适合血运重建的患者。大多数接受 CABG 的患者血液供应减少和功能受限，同时保持收缩储备和活力的可变性 3.如果不进行治疗，这些患者可能会进展为晚期心力衰竭和猝死，尤其是在工作量增加的情况下 4.在这些患者中，冠状动脉旁路移植术 （CABG） 是一种有效的治疗方法，但可能无法完全恢复功能5。重要的是，舒张功能障碍是较差临床结果的标志，在血运重建后无法恢复，这表明在 CABG 6,7 期间需要新的辅助治疗。目前，尚无临床上可用的辅助干预措施与 CABG 一起使用，以将心肌细胞恢复到全部功能能力。这是一个重大的治疗差距，因为尽管进行了适当的血运重建，但许多患者仍会发展为晚期心力衰竭8。
创建了一种适合 CABG 的创新猪慢性心肌缺血模型，以模拟临床 CAD 经验9。与其他大型动物相比，猪提供了良好的心脏病模型，因为它们没有心外膜桥接侧支，因此仅 LAD 狭窄会导致局部缺血10。在这项研究中，使用了 16 周龄的雌性约克郡长白猪。在该模型中，使用左乳内动脉 （LIMA） 移植物通过非体外循环 CABG 对 LAD 进行血运重建（补充表 1）。经皮冠状动脉介入治疗 （PCI） 无法打开狭窄，因为收缩器是一种刚性装置。心脏磁共振成像 （MRI） 用于评估整体和区域功能、冠状动脉解剖结构和组织活力。心脏 MRI 分析显示舒张功能，其特征是峰值充盈率 （PFR），尽管进行了6 次 CABG，但舒张功能仍然受损。舒张功能障碍的机制可能与 CABG11 后持续存在的 HIB 中线粒体生物能量学和胶原形成受损有关。
间充质干细胞 （MSC） 在 CABG 期间应用时通过外泌体提供治疗信号传导，以改善心肌恢复。在该猪模型和平行 体外 研究中，表明在 CABG 期间放置心外膜 MSC vicryl 贴片可恢复收缩功能，增加关键线粒体蛋白，即 PGC-1α12，线粒体能量代谢的重要调节剂13。 体外 模型使我们能够研究间充质干细胞对线粒体功能受损的信号传导机制。外泌体是分泌的稳定微囊泡 （50-150 nm），含有蛋白质或核酸，包括 microRNA （miRNA）14。最近的 体外 数据表明，MSC衍生的外泌体是恢复线粒体呼吸所必需的重要信号转导机制。
干细胞衍生的外泌体是有前途的辅助疗法，因为它们很容易获得，可以商业化生产，并且没有伦理冲突。考虑到临床转化，创建了一种嵌入 MSC 衍生外泌体的胶原蛋白贴片，可以通过手术缝合到心肌的冬眠区域。结果证明，使用该贴片可以持续递送外泌体，并提供一种具有旁分泌信号转导机制的无细胞再生疗法，该机制针对线粒体恢复并增强线粒体生物发生15。该程序提供了临床前模型，用于研究间充质干细胞衍生疗法的影响，通过增强线粒体功能和减少血运重建时的炎症来改善心脏功能，并逆转肌细胞对慢性缺血的适应。
在这项研究中，展示了一种使用 LIMA 到 LAD 吻合术的离体外循环 CABG 手术方法，以绕过近端 LAD 狭窄区域，模拟 CAD 患者的标准治疗。作为 CABG 的辅助治疗，证明了 MSC 来源的外泌体包埋胶原贴片在心肌缺血区的手术应用。该手术模型可用于研究使用外泌体贴片对旁分泌效应的生理反应以及恢复的分子机制。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5 Ethibond</td>
			<td>Ethicon</td>
			<td>MG46G</td>
			<td>Suture</td>
		</tr>
		<tr>
			<td># 40 clipper blade</td>
			<td>Oster</td>
			<td>078919-016-701</td>
			<td>Remove hair from surgery sites</td>
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		<tr>
			<td>0 Vicryl</td>
			<td>Ethicon</td>
			<td>J208H</td>
			<td>Suture</td>
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		<tr>
			<td>1 mL Syringe</td>
			<td>Medtronic/Covidien</td>
			<td>1188100777</td>
			<td>Administer injectable agents</td>
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		<tr>
			<td>1&#34; medical tape</td>
			<td>Medline</td>
			<td>MMM15271Z</td>
			<td>Secure wound dressing and IV catheters</td>
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		<tr>
			<td>1000mL 0.9% Sodium chloride</td>
			<td>Baxter</td>
			<td>2B1324X</td>
			<td>IV replacement fluid</td>
		</tr>
		<tr>
			<td>12 mL Syringe</td>
			<td>Medtronic/Covidien</td>
			<td>8881512878</td>
			<td>Administer injectable agents</td>
		</tr>
		<tr>
			<td>18 ga needles</td>
			<td>BD</td>
			<td>305185</td>
			<td>Administration of injectable agents</td>
		</tr>
		<tr>
			<td>20 ga needles</td>
			<td>BD</td>
			<td>305175</td>
			<td>Administration of injectable agents</td>
		</tr>
		<tr>
			<td>20 mL Syringe</td>
			<td>Medtronic/Covidien</td>
			<td>8881520657</td>
			<td>Administer injectable agents</td>
		</tr>
		<tr>
			<td>2-0 Vicryl</td>
			<td>Ethicon</td>
			<td>J317H</td>
			<td>Suture</td>
		</tr>
		<tr>
			<td>250 mL 0.9% saline</td>
			<td>Baxter</td>
			<td>&#160;UE1322D</td>
			<td>Replacement IV Fluid</td>
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		<tr>
			<td>3 mL Syinge</td>
			<td>Medtronic/Covidien</td>
			<td>1180300555</td>
			<td>Administer injectable agents</td>
		</tr>
		<tr>
			<td>3-0 Vicryl</td>
			<td>Ethicon</td>
			<td>VCP824G</td>
			<td>Suture</td>
		</tr>
		<tr>
			<td>36&#8221; Pressure monitoring tubing</td>
			<td>Smith&#8217;s Medical</td>
			<td>MX563</td>
			<td>Connect art. Line&#160; to transducer</td>
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		<tr>
			<td>4.0 mm ID endotracheal tube</td>
			<td>Medline</td>
			<td>DYND43040</td>
			<td>Establish airway for Hibernation</td>
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		<tr>
			<td>4-0 Tevdek II Strands</td>
			<td>Deknatel</td>
			<td>7-922</td>
			<td>Suture to secure constrictor around LAD</td>
		</tr>
		<tr>
			<td>48&#8221; Pressure monitoring tubing</td>
			<td>Smith&#8217;s Medical</td>
			<td>MX564</td>
			<td>Connect art. Line&#160; to transducer</td>
		</tr>
		<tr>
			<td>500mL 0.9% Sodium chloride</td>
			<td>Baxter</td>
			<td>2B1323Q</td>
			<td>Drug delivery, Provide mist for Blower Mister</td>
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		<tr>
			<td>6&#160; mL Syringe</td>
			<td>Medtronic/Covidien</td>
			<td>1180600777</td>
			<td>Administer injectable agents</td>
		</tr>
		<tr>
			<td>6.0 mm ID endotracheal tube</td>
			<td>Mallinckrodt</td>
			<td>86049</td>
			<td>Establish airway for Revasc,MRI and Termination</td>
		</tr>
		<tr>
			<td>6.5 mm ID endotracheal tube</td>
			<td>Medline</td>
			<td>DYND43065</td>
			<td>Establish airway for Revasc,MRI and Termination</td>
		</tr>
		<tr>
			<td>6&#8221; pressure tubing line</td>
			<td>Smith&#8217;s Medical</td>
			<td>MX560</td>
			<td>Collect bone marrow</td>
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		<tr>
			<td>60 mL Syringe</td>
			<td>Medtronic/Covidien</td>
			<td>8881560125</td>
			<td>Administer injectable agents</td>
		</tr>
		<tr>
			<td>7.0 mm ID endotracheal tube</td>
			<td>Medline</td>
			<td>DYND43070</td>
			<td>Establish airway for Revasc,MRI and Termination</td>
		</tr>
		<tr>
			<td>7-0 Prolene</td>
			<td>Ethicon</td>
			<td>M8702</td>
			<td>Suture</td>
		</tr>
		<tr>
			<td>Advanced DMEM (1X)</td>
			<td>ThermoFisher Scientific</td>
			<td>12491023</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Prep pads</td>
			<td>MedSource</td>
			<td>MS-17402</td>
			<td>Skin disinfectant</td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal Filter Unit</td>
			<td>Millipore Sigma</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr>
			<td>Anesthesia Machine</td>
			<td>Drager</td>
			<td>Fabious Trio</td>
			<td>maintains general anesthesia</td>
		</tr>
		<tr>
			<td>Anesthesia Machine + ventilator</td>
			<td>DRE Drager- Fabius Tiro</td>
			<td>DRE0603FT</td>
			<td>Deliver Oxygen and inhalant to patient</td>
		</tr>
		<tr>
			<td>Anesthesia Monitor</td>
			<td>Phillips&#160; Intellivue</td>
			<td>MP70</td>
			<td>Multiparameter for patient safety</td>
		</tr>
		<tr>
			<td>Arterial Line Kit</td>
			<td>Arrow</td>
			<td>ASK-04510-HF</td>
			<td>Femoral catheter for blood pressure monitoring</td>
		</tr>
		<tr>
			<td>Artificial Tears</td>
			<td>Rugby</td>
			<td>0536-1086-91</td>
			<td>Lubricate eyes to prevent corneal drying</td>
		</tr>
		<tr>
			<td>Bair Hugger</td>
			<td>3M</td>
			<td>Model 505</td>
			<td>Patient Warming system</td>
		</tr>
		<tr>
			<td>Basic pack</td>
			<td>Medline</td>
			<td>DYNJP1000</td>
			<td>Sterile drapes and table cover</td>
		</tr>
		<tr>
			<td>Blood Collection Tubes- green top</td>
			<td>Fisher Scientific</td>
			<td>02-689-7</td>
			<td>Collect microsphere blood samples</td>
		</tr>
		<tr>
			<td>Blower Mister Kit</td>
			<td>Medtronic/Covidien</td>
			<td>22120</td>
			<td>Clears surgical field for vessel anastomosis</td>
		</tr>
		<tr>
			<td>BODIPY TR Ceramide</td>
			<td>ThermoFisher Scientific</td>
			<td>D7540</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone marrow needle- 25mm 15 ga IO needle</td>
			<td>Vidacare</td>
			<td>9001-VC-005</td>
			<td>Collect bone marrow</td>
		</tr>
		<tr>
			<td>Bone Wax</td>
			<td>Medline</td>
			<td>ETHW31G</td>
			<td>Hemostasis of cut bone</td>
		</tr>
		<tr>
			<td>Bovie Cautery hand piece</td>
			<td>Covidien</td>
			<td>E2516</td>
			<td>Hemostasis</td>
		</tr>
		<tr>
			<td>Bupivicaine</td>
			<td>Pfizer</td>
			<td>00409-1161-01</td>
			<td>Local Anesthetic</td>
		</tr>
		<tr>
			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Sigma Aldrich</td>
			<td>B9275</td>
			<td>Pre operative Analgesic for survivial procedures</td>
		</tr>
		<tr>
			<td>Cell Scrapers</td>
			<td>Corning</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Cephazolin 1 gr</td>
			<td>Pfizer</td>
			<td>00409-0805-01</td>
			<td>Antibiotic</td>
		</tr>
		<tr>
			<td>Chest Tube</td>
			<td>Covidien</td>
			<td>8888561043</td>
			<td>Evacuates air from chest cavity</td>
		</tr>
		<tr>
			<td>Cloroprep</td>
			<td>Becton Dickenson</td>
			<td>260815</td>
			<td>Surgical skin prep</td>
		</tr>
		<tr>
			<td>Corning bottle-top vacuum Filter System (500mL)</td>
			<td>Millipore Sigma</td>
			<td>430758</td>
			<td></td>
		</tr>
		<tr>
			<td>CPT tube</td>
			<td>BD</td>
			<td>362753</td>
			<td>MSC isolation from bone marrow</td>
		</tr>
		<tr>
			<td>Delrin Constrictor</td>
			<td>U of MN</td>
			<td>Custom made</td>
			<td>Creates stenosis of LAD</td>
		</tr>
		<tr>
			<td>Dermabond</td>
			<td>Ethicon</td>
			<td>DNX12</td>
			<td>Skin adhesive</td>
		</tr>
		<tr>
			<td>DMEM (1X) Dulbecco&#39;s Modified Eagle Medium, HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>12430062</td>
			<td></td>
		</tr>
		<tr>
			<td>Dobutamine 12.5 mg/mL</td>
			<td>Pfizer</td>
			<td>00409-2344-01</td>
			<td>Increases blood pressure and heart rate during the second microsphere blood collection</td>
		</tr>
		<tr>
			<td>ECG Pads</td>
			<td>DRE</td>
			<td>1496</td>
			<td>Monitor heart rhythm</td>
		</tr>
		<tr>
			<td>Exosome-Depleted FBS</td>
			<td>ThermoFisher Scientific</td>
			<td>A2720801</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Disposable Polystyrene Serological Pipets, Sterile, 10mL</td>
			<td>Fisher Scientific</td>
			<td>13-675-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Femoral and carotid introducer</td>
			<td>Cordis- J&#38;J</td>
			<td>504606P</td>
			<td>femoral and carotis cannulas</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Heat Inactivated, Gibco FBS</td>
			<td>ThermoFisher Scientific</td>
			<td>16140089</td>
			<td></td>
		</tr>
		<tr>
			<td>Flo-thru 1.0</td>
			<td>Baxter</td>
			<td>FT-12100</td>
			<td>used to anastomos LIMA to L</td>
		</tr>
		<tr>
			<td>Flo-thru 1.25</td>
			<td>Baxter</td>
			<td>FT-12125</td>
			<td>FT-12125</td>
		</tr>
		<tr>
			<td>Flo-thru 1.5</td>
			<td>Baxter</td>
			<td>FT-12150</td>
			<td>FT-12150</td>
		</tr>
		<tr>
			<td>Flo-thru 2.0</td>
			<td>Baxter</td>
			<td>FT-12200</td>
			<td>FT-12200</td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Clipper</td>
			<td>Oster</td>
			<td>078566-011-002</td>
			<td>Remove hair from surgery sites</td>
		</tr>
		<tr>
			<td>Helistat collagen sponge</td>
			<td>McKesson</td>
			<td>570973 1690ZZ</td>
			<td>Sponge for embedding exosomes</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Pfizer</td>
			<td>&#160;0409-2720-03</td>
			<td>anticoaggulant</td>
		</tr>
		<tr>
			<td>Histology Jars</td>
			<td>Fisher Scientific</td>
			<td>316-154</td>
			<td>Formalin for tissue samples</td>
		</tr>
		<tr>
			<td>HyClone Characterized Fetal Bovine Serum (FBS)</td>
			<td>Cytiva</td>
			<td>SH30071.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypafix</td>
			<td>BSN Medical</td>
			<td>4210</td>
			<td>Secure wound dressing and IV catheters</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Sigma Aldrich</td>
			<td>CDS019936</td>
			<td>General Anesthestic- Inhalant</td>
		</tr>
		<tr>
			<td>IV Tubing for Blower Mister</td>
			<td>Carefusion</td>
			<td>42493E</td>
			<td>Adapts to IV Fluids for Blower/Mister</td>
		</tr>
		<tr>
			<td>Jelco 18 ga IV catheter</td>
			<td>Smiths medical</td>
			<td>4054</td>
			<td>IV access in Revasc, MRI and Term</td>
		</tr>
		<tr>
			<td>Lidocaine 2%</td>
			<td>Pfizer</td>
			<td>00409-4277-01</td>
			<td>Local Anesthetic/ antiarrthymic</td>
		</tr>
		<tr>
			<td>Ligaclips</td>
			<td>Ethicon</td>
			<td>MSC20</td>
			<td>Surgical Staples for LIMA takedown</td>
		</tr>
		<tr>
			<td>Long blade for laryngoscope</td>
			<td>DRE</td>
			<td>12521</td>
			<td>Allows for visualization of trachea for intubation</td>
		</tr>
		<tr>
			<td>Meloxicam 5 mg/mL</td>
			<td>Boehringer Ingelheim</td>
			<td>141-219</td>
			<td>Post operative Analgesic</td>
		</tr>
		<tr>
			<td>Microsphere pump</td>
			<td></td>
			<td></td>
			<td>Collect blood samples from femoral introducer</td>
		</tr>
		<tr>
			<td>Monopolar Cautery</td>
			<td>Covidien</td>
			<td>Valleylab&#8482; FT10</td>
			<td>Hemostasis</td>
		</tr>
		<tr>
			<td>Nanosight NS 300</td>
			<td>Malvern Panalytical</td>
			<td>MAN0541-03-EN</td>
			<td></td>
		</tr>
		<tr>
			<td>NTA 3.1.54 software</td>
			<td>Malvern Panalytical</td>
			<td>MAN0520-01-EN-00</td>
			<td></td>
		</tr>
		<tr>
			<td>OPVAC Synergy II</td>
			<td>Terumo Cardiovascular System</td>
			<td>401-230</td>
			<td>Heart positioner and Stabilizer</td>
		</tr>
		<tr>
			<td>Oxygen Tank E cylinder</td>
			<td>various</td>
			<td>various</td>
			<td>Used for Blower Mister if anesthesia machine doesn&#39;t have auxiliary flow meter</td>
		</tr>
		<tr>
			<td>PBS, pH 7.2</td>
			<td>ThermoFisher Scientific</td>
			<td>20012050</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin-Neomycin (PSN) Antibiotic Mixture</td>
			<td>ThermoFisher Scientific</td>
			<td>15640055</td>
			<td></td>
		</tr>
		<tr>
			<td>Pigtail 145 catheter 6 French</td>
			<td>Boston Scientific</td>
			<td>08641-41</td>
			<td>Measure LV pressures</td>
		</tr>
		<tr>
			<td>Pressure Transducer</td>
			<td>various</td>
			<td>Must adapt to anesthesia monitor</td>
			<td>Monitor direct arterial pressures</td>
		</tr>
		<tr>
			<td>Propofol</td>
			<td>Diprivan</td>
			<td>269-29</td>
			<td>Induction agent</td>
		</tr>
		<tr>
			<td>Roncuronium</td>
			<td>Mylan</td>
			<td>67457-228-05</td>
			<td>Neuromuscular blocking agent</td>
		</tr>
		<tr>
			<td>SR Buprenorphine 10 mg/mL</td>
			<td>Abbott Labs</td>
			<td>NADA 141-434</td>
			<td>Post operative Analgesic</td>
		</tr>
		<tr>
			<td>Sterile Saline 20 mL</td>
			<td>Fisher Scientific</td>
			<td>20T700220</td>
			<td>Flush for IV catheters</td>
		</tr>
		<tr>
			<td>Sternal Saw/ Necropsy Saw</td>
			<td>Thermo Fisher</td>
			<td>812822</td>
			<td>Used to open chest cavity</td>
		</tr>
		<tr>
			<td>Stop Cocks</td>
			<td>Smith Medical</td>
			<td>MX5311L</td>
			<td>2 to connect to pig tail</td>
		</tr>
		<tr>
			<td>Succinylcholine 20 mg/mL</td>
			<td>Pfizer</td>
			<td>00409-6629-02</td>
			<td>Neuromuscular blocking agent</td>
		</tr>
		<tr>
			<td>Suction&#160; tubing</td>
			<td>Medline</td>
			<td>DYND50223</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction Container</td>
			<td>Medline</td>
			<td>DYNDCL03000</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgery pack with chest retractor</td>
			<td>various</td>
			<td>See pack list</td>
			<td>Femoral cut down and median sternotomy</td>
		</tr>
		<tr>
			<td>Surgical Instruments</td>
			<td>various</td>
			<td>See pack list</td>
			<td>Femoral and carotid cutdowns and sternotomy</td>
		</tr>
		<tr>
			<td>Surgical Spring Clip</td>
			<td>Applied Medical</td>
			<td>A1801</td>
			<td>Clamp end of LIMA after takedown</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard</td>
			<td></td>
			<td>Delivers IV Dobutamine infusion</td>
		</tr>
		<tr>
			<td>SYTO RNASelect Green Fluorescent cell Stain - 5 mM Solution in DMSO</td>
			<td>Millipore Sigma</td>
			<td>S32703</td>
			<td></td>
		</tr>
		<tr>
			<td>Telazol 100 mg/mL</td>
			<td>Fort Dodge</td>
			<td>01L60030</td>
			<td>Pre operative Sedative</td>
		</tr>
		<tr>
			<td>Telpha pad</td>
			<td>Covidien</td>
			<td>2132</td>
			<td>Sterile wound dressing</td>
		</tr>
		<tr>
			<td>Timer</td>
			<td></td>
			<td></td>
			<td>Time collection of blood samples</td>
		</tr>
		<tr>
			<td>Total Exosome Isolation Reagent (from cell culture media)</td>
			<td>ThermoFisher Scientific</td>
			<td>4478359</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP Tissue Culture Flask, T75, Filter Cap w/ 0.22uM PTFE</td>
			<td>ThermoFisher Scientific</td>
			<td>TP90076</td>
			<td></td>
		</tr>
		<tr>
			<td>Triple Antibiotic Ointment</td>
			<td>Johnson &#38; Johnson</td>
			<td>23734</td>
			<td>Topical over wound</td>
		</tr>
		<tr>
			<td>Vicryl mesh</td>
			<td>Ethicon</td>
			<td>VKML</td>
			<td>Patch for epicardial cell application</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td>Mix microspheres</td>
		</tr>
		<tr>
			<td>Xylazine 100 mg/mL</td>
			<td>Vedco</td>
			<td>468RX</td>
			<td>Pre operative Sedative/ analgesic</td>
		</tr>
	</tbody>
</table>

surgical porcine model of chronic myocardial ischemia treated by exosome-laden collagen patch and off-pump coronary artery bypass graft

进行性冠状动脉狭窄导致的慢性心肌缺血导致冬眠心肌 （HIB），HIB 定义为通过降低代谢活动来适应氧气供应减少的心肌，从而防止不可逆的心肌细胞损伤和梗死。这与心肌梗死不同，因为 HIB 具有通过血运重建恢复的潜力。患有严重冠状动脉疾病 （CAD） 的患者会出现慢性缺血，这使他们面临心力衰竭和猝死的风险。重度冠状动脉旁路移植手术 （CABG） 是重度冠状动脉旁路移植手术 （CABG），但它已被证明是一种不完美的治疗方法，但尚无辅助疗法来恢复适应慢性缺血的肌细胞。 为了解决这一差距，使用适合 CABG 并模拟临床场景的猪 HIB 手术模型。该模型涉及两次手术。第一次手术涉及在左前降支 （LAD） 上植入 1.5 mm 的刚性收缩器。随着动物的生长，蟒蛇逐渐引起明显的狭窄，导致区域收缩功能下降。一旦狭窄达到 80%，心肌血流和功能就会受损，从而产生 HIB。然后使用左乳内动脉 （LIMA） 进行非体外循环 CABG，以使缺血区域血运重建。动物恢复一个月，以便在牺牲前获得最佳的心肌改善。这允许对不同的治疗组进行生理和组织研究。该动物模型表明，尽管进行了 CABG，但心脏功能仍然受损，这表明需要新的辅助干预。在这项研究中，开发了一种嵌入间充质干细胞 （MSC） 衍生外泌体的胶原贴片，该贴片可以通过手术应用于 LIMA 吻合口远端的心外膜表面。该材料符合心外膜，可吸收，并为信号因子的持续释放提供支架。这种再生疗法可以刺激心肌恢复，而仅对血运重建没有反应。该模型通过提供有关 HIB 恢复的生理和机制探索手段，转化为临床领域。

Globally, severe CAD affects over a hundred million patients, and although the mortality rate has decreased, it remains one of the leading causes of death1,2. CAD has a wide clinical spectrum from myocardial infarction (MI) to ischemia with preserved viability. Most pre-clinical research focuses on MI, characterized by the presence of infarcted tissue as it is possible to study in small and large animal models. However, that model does not address patients with preserved viability and amenable to revascularization. Most patients undergoing CABG have decreased blood supply and limited function while maintaining variability in contractile reserve and viability3. Without treatment, these patients can progress to advanced heart failure and sudden death, especially during increased workload4. Among these patients, coronary artery bypass graft (CABG) is an effective therapy but may not result in complete functional recovery5. Importantly, diastolic dysfunction, which is a marker for worse clinical outcomes, fails to recover after revascularization suggesting the need for novel adjuvant therapies during CABG6,7. Currently, there are no clinically available adjuvant interventions used with CABG to restore cardiomyocytes to full functional capacity. This is a major therapeutic gap given that many patients progress to advanced heart failure despite appropriate revascularization8.
An innovative porcine model of chronic myocardial ischemia that is amenable to CABG, to mimic clinical CAD experience was created9. Swine provide a good model of heart disease over other large animals as they do not have epicardial bridging collaterals so stenosis of the LAD alone results in regional ischemia10. In this study, 16-week-old female Yorkshire-Landrace pigs were used. In this model, the LAD was revascularized with off-pump CABG using the left internal mammary artery (LIMA) graft (Supplementary Table 1). Percutaneous coronary intervention (PCI) is not possible to open the stenosis as the constrictor is a rigid device. Cardiac magnetic resonance imaging (MRI) is used to assess global and regional function, coronary anatomy, and tissue viability. Cardiac MRI analysis showed diastolic function, characterized by peak filling rate (PFR) remains impaired despite CABG6. The mechanism of diastolic dysfunction likely relates to impaired mitochondrial bioenergetics and collagen formation in HIB that persist following CABG11.
Mesenchymal stem cells (MSC) provide therapeutic signaling through exosomes to improve myocardial recovery when applied during CABG. In this swine model and parallel in vitro studies, it was shown that placement of an epicardial MSC vicryl patch during CABG recovers contractile function with increase in key mitochondrial proteins namely PGC-1&#945;12, an important regulator of mitochondrial energy metabolism13. The in vitro model allowed us to investigate the signaling mechanism of MSCs on impaired mitochondrial function. Exosomes are secreted stable microvesicles (50-150 nm) that contain protein or nucleic acids including microRNA (miRNA)14. Recent in vitro data suggest that MSC-derived exosomes are an important signaling mechanism necessary for recovery of mitochondrial respiration.
Stem cell derived exosomes are promising adjunctive therapeutics as they are readily accessible, can be commercially produced, and lack ethical conflicts. In consideration of clinical translation, a collagen patch embedded with MSC-derived exosomes was created that can be surgically sutured to the hibernating region of myocardium. It was demonstrated that there is sustained delivery of exosomes using this patch and it provides a cell-free regenerative therapy with paracrine signaling mechanism that targets mitochondrial recovery and enhance mitochondrial biogenesis15. This procedure provides the pre-clinical model to study the impact of MSC-derived therapies to improve cardiac function by means of enhancing mitochondrial function and reducing inflammation at the time of revascularization and reverse the myocyte adaptations to chronic ischemia.
In this study, a surgical method of off-pump CABG using LIMA to LAD anastomosis to bypass the area of proximal LAD stenosis mimicking the standard treatment for patients with CAD is shown. As an adjunctive therapy with CABG, the surgical application of MSC-derived exosome embedded collagen patch on the ischemic region of the myocardium was demonstrated. This surgical model can be used to study the physiologic responses to the paracrine effect seen with use of an exosome patch as well as the molecular mechanisms of recovery.

作者聚焦：增强冠状动脉血运重建

外泌体胶原贴片和体外循环冠状动脉旁路移植术治疗慢性心肌缺血的外科猪模型

血运重建后，进行冠状动脉造影以评估LAD狭窄（大于80%）和LIMA-LAD移植物的通畅性（图1）。血运重建手术和放置富含外泌体的胶原蛋白贴片 4 周后，使用低剂量多巴酚丁胺输注以 5μg/kg/min 的速度进行心脏 MRI 以评估心脏在静息和压力下的收缩和舒张功能。通过测量壁厚百分比（收缩期末壁厚 - 舒张期末壁厚）来分析收缩功能。通过测量舒张末期容量的峰值充盈率（PFR/EDV; <str...

Watch this Scientific Journal Video about Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft at JoVE.com

本研究提出了进行性冠状动脉狭窄导致的慢性心肌缺血的外科猪模型，导致心脏功能受损而无梗死。缺血后，动物接受非体外循环冠状动脉旁路移植术，心外膜放置干细胞来源的富含外泌体的胶原蛋白贴片。这种辅助治疗可改善心肌功能和恢复。

Chronic myocardial ischemia resulting from progressive coronary artery stenosis leads to hibernating myocardium (HIB), defined as myocardium that adapts ...

该外科手术的总体目标是使用 LIMA 到 LAD 搭桥创建冠状动脉血运重建的手术模型，并使用富含外泌体的胶原蛋白贴片作为辅助手段来证明心脏功能的生理改善。我们的体内模型是单血管模型，不包括任何合并症，这不能代表接受多血管疾病治疗的患者的典型临床情况。然而，尽管存在这种局限性，我们的模型始终如一地产生了可靠的结果，并保持了较低的死亡率。我们开发了一种临床相关的辅助疗法，用于 CABG，旨在了解驱动慢性心肌缺血心肌功能改善的细胞机制。缺血组织的完全恢复似乎取决于线粒体能力的恢复和炎症的减少;代表了一个新的研究领域。

surgical-porcine-model-chronic-myocardial-ischemia-treated-exosome

Research

JoVE Journal

Medicine

Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft

530 次观看.  University of Minnesota. 该外科手术的总体目标是使用 LIMA 到 LAD 搭桥创建冠状动脉血运重建的手术模型，并使用富含外泌体的胶原蛋白贴片作为辅助手段来证明心脏功能的生理改善。我们的体内模型是单血管模型，不包括任何合并症，这不能代表接受多血管疾病治疗的患者的典型临床情况。然而，尽管存在这种局限性，我们的模型始终如一地产生了可靠的结果，并保持了较低的死亡率。我们?...

视频: 作者聚焦：增强冠状动脉血运重建

Chronic myocardial ischemia resulting from progressive coronary artery stenosis leads to hibernating myocardium (HIB), defined as myocardium that adapts to reduced oxygen availability by reducing metabolic activity, thereby preventing irreversible cardiomyocyte injury and infarction. This is distinct from myocardial infarction, as HIB has the potential for recovery with revascularization. Patients with significant coronary artery disease (CAD) experience chronic ischemia, which puts them at risk for heart failure and sudden death. The standard surgical intervention for severe CAD is coronary artery bypass graft surgery (CABG), but it has been shown to be an imperfect therapy, yet no adjunctive therapies exist to recover myocytes adapted to chronic ischemia. To address this gap, a surgical model of HIB using porcine that is amenable to CABG and mimics the clinical scenario was used. The model involves two surgeries. The first operation involves implanting a 1.5 mm rigid constrictor on the left anterior descending (LAD) artery. As the animal grows, the constrictor gradually causes significant stenosis resulting in reduced regional systolic function. Once the stenosis reaches 80%, the myocardial flow and function are impaired, creating HIB. An off-pump CABG is then performed with the left internal mammary artery (LIMA) to revascularize the ischemic region. The animal recovers for one month to allow for optimal myocardial improvement prior to sacrifice. This allows for physiologic and tissue studies of different treatment groups. This animal model demonstrates that cardiac function remains impaired despite CABG, suggesting the need for novel adjunctive interventions. In this study, a collagen patch embedded with mesenchymal stem cell (MSC)-derived exosomes was developed, which can be surgically applied to the epicardial surface distal to LIMA anastomosis. The material conforms to the epicardium, is absorbable, and provides the scaffold for the sustained release of signaling factors. This regenerative therapy can stimulate myocardial recovery that does not respond to revascularization alone. This model translates to the clinical arena by providing means of physiological and mechanistic explorations regarding recovery in HIB.

This study presents a surgical porcine model of chronic myocardial ischemia due to progressive coronary artery stenosis, resulting in impaired cardiac function without infarction. Following ischemia, animals undergo off-pump coronary artery bypass graft with epicardial placement of stem cells-derived exosomes-laden collagen patch. This adjunctive therapy improves myocardial function and recovery.