Name: surgical angiogenesis in porcine tibial allotransplantation: a new large animal bone vascularized composite allotransplantation model
Uploaded: 2017-08-13T13:00:00.000+00:00
Duration: 10 min 31 s
Description: Watch this Scientific Journal Video about Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model at JoVE.com

作者感谢媒体支持服务部, 梅奥诊所罗切斯特, 视频制作的锰, 以及伊亚 Kotsougianis 的视频编辑。优秀的艺术品是由吉姆 Postier, 罗切斯特, 锰。此外, 作者希望感谢德国研究基金会 (德意志 Forschungsgemeinschaft) 为 Dr. Dimitra Kotsougiani (DFG 补助金: KO 4903/1-1) 提供薪资支持。这项工作得到了来自苏拉亚·奥贝德的慷慨馈赠的支持。这项工作是在微血管研究实验室, 骨科手术部梅奥诊所罗切斯特, 锰。

这项研究由机构动物保育和使用委员会 (IACUC) 批准, 在梅奥诊所罗切斯特。尤卡坦半岛小型猪在这一手术 VCA 过程中作为捐赠者和受赠人。供者和受体配对是基于 DNA 序列猪白细胞抗原 (sla) haplotyping, 以确保 sla 2223中的主要不匹配。动物是年龄和体重匹配和相同的血型。两个外科手术组同时从捐献者的营养血管中采集猪胫骨骨段, 并准备接受 orthopically 的同种异体胫骨骨段。同时, 在骨营养血管的微血管修复, 一个受体衍生的动静脉束被放置在胫骨段的自体血管生成。
1. 前的准备工作
<ol><li>快速尤卡坦半岛小型猪在手术前的前一天, 并称他们为受控药物管理。</li>
	<li>与嗪 (2 毫克/千克) 和 tiletamine 盐酸和唑盐酸 (5 毫克/千克) 的组合, 皮下注射的镇静动物。</li>
	<li>在耳静脉内放置一个外周导管, 用于静脉注射药物和生理盐水, 并管理丁丙诺啡 (0.18 毫克/千克) 和预防性抗生素 (1 克 cefazoline 静脉注射和5毫克/千克呋肌)。</li>
	<li>剃须的右后肢和左颈部, 将作为收获地点的带血管的胫骨骨段和现场放置的中心静脉导管, 分别。</li>
	<li>通过测试口腔肌肉的松弛来检查生命体征和镇静程度。</li>
	<li>插管的动物与适当大小的气管导管在胸骨卧床24。</li>
	<li>将微型猪转移到手术台上, 并通过管理异氟醚 (1-3%) 连接到呼吸机以维持麻醉。</li>
	<li>通过测试眼睑、瞳孔光和角膜反射来确认麻醉深度。</li>
	<li>监测氧气饱和度与脉冲血传输探头连接到耳朵。使用血压袖带和体温探头进行术中生命标志的监测。</li>
	<li>将尤卡坦微型猪放在暖垫上的仰卧位。此外, 在手术中使用强制空气加温毯来防止体温过低。</li>
	<li>用眼睛上的兽医药膏来预防麻醉时的干燥。</li>
</ol>2. 带血管的胫骨骨段的收获
<ol><li>用聚维酮碘溶液清洗每个小型猪的右腿。用无菌毛巾擦干皮肤, 用无菌的方式将四肢披上。用碘浸渍的粘合剂切口包裹和隔离肢体, 以尽量减少污染的风险。</li>
	<li>用手术刀 anterolaterally 在后肢进行切口, 从膝关节开始, 沿胫骨前脊延伸至胫关节。</li>
	<li>用剪刀解剖皮肤和皮下组织, 并从胫骨侧面缩回前室肌。 注: 胫骨前肌源的释放有助于暴露。骨间膜现在暴露了。</li>
	<li>确定颅骨胫动脉和静脉 (以后用作血管新生的动静脉束)。 注意: 胫骨干动脉和血管位于骨间膜的前表面。</li>
	<li>为了改善手术的视野, 从胫骨前肌的插入部分释放一部分, 用振荡锯去除胫骨脊的一部分。</li>
	<li>保护颅内胫血管, 切开骨间膜, 从胫骨结节的水平开始, 用剪刀切割。</li>
	<li>可视化胫骨尾部的血管, 在膜下远端运行。 注意: 他们从胫腓骨血管分支, 并引起胫骨骨干的营养蒂, 只是远端的结节。现在可以可视化的营养孔和血管进入胫骨的后侧面只是胫骨结节的远端。</li>
	<li>用 microclamp 标记营养蒂。不要分离血管蒂。</li>
	<li>在营养孔附近发现胫骨前室的肌支;可用于吻合血管的骨移植营养血管。用 microclamp 标记肌肉分支。</li>
	<li>
3.5 厘米胫骨骨段包括血管蒂的收获。
	<ol>
<li>使用切割夹具, 以确保精确和可再生的骨切除。在胫骨内侧表面放置和固定切割夹具, 包括营养孔和血管。<ol>
<li>在跳汰机指导下, 用振荡锯进行平行骨切割, 以去除3.5 厘米胫骨段。使用相同的定位和夹具的捐助者和受体动物, 以最大限度的大小和形状匹配。</li>
		</ol>
</li>
	</ol>
</li>
	<li>一旦两个切口已经用振荡锯, 旋转胫骨骨段, 以可视化的营养椎弓根在后表面。用剪刀将其来源的营养蒂从颅骨胫动脉中分离出来。用剪刀解剖和释放胫骨段, 在其表面留下一层骨膜和肌肉的薄袖。</li>
	<li>将胫骨骨段缩回, 并用锋利的钳夹将胫骨骨段抬高, 使胫骨干动脉就位。 注: 带血管的骨段现已准备好微血管转移, 并在每一个尤卡坦小型猪中建立了3.5 厘米胫骨骨缺损。</li>
	<li>结扎用可吸收的 polyglactin 3-0 缝合线在踝关节上的胫腓骨血管, 释放他们与血管组织的袖口, 创造一个动静脉 (AV) 捆绑。保持缝线至少5厘米长, 以减轻植入胫骨骨段。</li>
</ol>3. 胫骨原位骨 VCA 重建联合外科血管生成
<ol><li>
交换收获的胫骨骨段与它的营养蒂之间的两个动物使用它们作为骨 VCAs。
	<ol>
<li>为了允许将颅内胫骨动静脉 (AV) 束进入胫骨骨段, 用振荡锯从近端交界部位取出 V 形段。</li>
		<li>在胫骨骨缺损部位的远端钻一个直径0.5 厘米的孔, 并进入胫骨骨段的髓质管, 并引入已结扎远端的受体 AV 束, 进入髓内管, 促进随后的自生新的血液供应</li>
	</ol>
</li>
	<li>
将带血管的胫骨骨段原位入受体缺损。
	<ol>
<li>使用简单间断缝合技术和9-0 缝合25, 将胫骨骨段的营养蒂吻合到胫骨前室的预备肌肉支。</li>
	</ol>
</li>
	<li>用挤奶试验确认微血管吻合通畅26。</li>
	<li>
使用9孔 3.5 mm 锁定板实现接骨。
	<ol>
<li>将9孔板放在胫骨 anteromedially 上。用三双螺钉固定在胫骨骨段上方和下方的钢板上。此外, 放置皮质螺钉在胫骨骨段内固定。为了确认正确定位的骨 VCA 和板, 使用前后和横向射线照相。</li>
	</ol>
</li>
	<li>使用间断的3-0 和2-0 可吸收缝合线进行筋膜和层状皮肤闭合。最后用密闭的透明敷料封住伤口。</li>
</ol>4. 中心静脉导管置入颈静脉外静脉
<ol><li>
术后药物管理和免疫抑制 (是) 药物水平监测, 使用开放的技术, 放置静脉导管到颈外静脉。在移植手术结束时进行麻醉下的放置 (见1节)。
	<ol>
<li>用手术刀在颈部进行前外侧切口。用剪刀解剖皮下组织, 并暴露左颈静脉。</li>
		<li>通过颈外静脉小孔将希克曼导管插入颈静脉, 并用吸收缝合固定。取出导管在背部通过隧道皮下注射。</li>
		<li>使用间断的3-0 和2-0 可吸收缝合线, 将导管固定在皮肤上, 并在层上缝合颈部。</li>
		<li>在切口处放置咬合绷带。用渔网绷带将绷带和导管固定到位。</li>
	</ol>
</li>
</ol>5. 术后治疗及随访
<ol><li>手术后立即治疗尤卡坦半岛小型猪, 肌肉注射卡洛芬 (4 毫克/千克) 用于术后镇痛。管理丁丙诺啡 (0.18 毫克/千克), 根据需要治疗高强度疼痛。</li>
	<li>允许猪恢复60分钟, 然后将猪返回到一个特别的重症监护病房, 并密切监视直到完全恢复。</li>
	<li>将尤卡坦半岛的小型猪移到正常的笼子里, 并提供随意的水和食物。</li>
	<li>执行他克莫司 (0.8-1.5 毫克/千克/天) 和霉酚酸酯 (MMF) (50-70 毫克/千克/天) 口服和甲基泼尼松龙琥珀酸钠 (开始500毫克/天) 两周。</li>
	<li>根据槽血水平调整每日剂量的免疫抑制药物, 目的为克莫司和 1.0-3.5 µg/毫升的 MMF, 分别为 5.0-30.0 ng/毫升.逐步减少甲基泼尼松龙的剂量, 直至达到每天50毫克的维持剂量。</li>
	<li>用庆大霉素 (3 毫克/千克静脉注射) 和呋 (5 毫克/千克注) 进行两周的预防性抗生素的管理。</li>
</ol>

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作者声明他们没有竞争的金融利益。

移植同种异体骨 (骨 VCA) 可能代表未来的重建选择大节段性骨缺损。然而, 需要长期免疫抑制 (是) 和它的重要副作用的骨骼 VCA 生存是很难证明这些寿险关键应用程序6。
虽然实验鼠的自交系在同种异体移植研究中被广泛应用, 以测试避免长期免疫抑制的各种方法, 但猪模型可能会提供显著的优势8,9.尤卡坦半岛迷你猪是研究骨 VCA 排斥反应复杂过程的理想选择。从生理学上来说, 新的骨形成率与人 (猪 1.2-1.5 µm 每天; 人 1.0-1.5 µm 每天分别)32。解剖学上的相似性使使用基本相同的外科植入物和技术的原位骨重建。也许最重要的是, 定义明确的猪 alloresponse--在猪细胞因子检测和 anti-porcine 簇分化抗体的发展中取得了可能--使得这种和其他 VCA 研究更加严格33。
与任何类似的临床应用一样, 用骨 VCA 修复猪胫骨骨缺损的方法在技术上要求很高, 需要两个团队的方法, 在微血管手术和骨重建方面有足够的外科专业知识, 以实现可重现的结果。严格维护无菌术中的条件和围手术期的抗生素预防, 是减少感染并发症的风险。
在以前的研究中使用大鼠和兔短期是维持生存的血管化骨 allotransplants 在前2周通过灌注的骨 VCA 通过其异体营养血管。免疫抑制剂后, 在髓管内的受体衍生血管提供了新生血管, 允许长期骨 VCA 愈合和生存能力10,11,12。在研究端点, 可以检测到大量的移植嵌34,35,36。我们已经向前迈进, 并应用了我们的鼠和兔模型的行之有效的方法。这种模型是可行的, 以测试一种新的方法, 以维持组织的生存能力, 而不是长期在骨 VCA 研究, 使用自体血管的血管生成与短期结合, 有效地切换骨循环异基因的自生血管。
这个模型的主要优势, 在其他现有的猪骨含有 VCA 模型是它的原位设计, 使功能评估的重量轴承和评估的机械性能, 特别是稀疏的数据14,37. 通过对 allotransplanted 胫骨骨段的影像学和组织学评价以及外周血的分子生物学分析, 可以很容易地监测局部和全身性骨 VCA 排斥反应的复杂机制。最终, 低发病率的手术骨 VCA 程序, 使长期骨 VCA 生存和分析。
稳定的内固定, 正确的同种异体胫骨骨段并列和肢体对齐是至关重要的, 允许在第一天后, 猪的移动和需要仔细前规划。我们所选择的方法, 使用专门设计的切割夹具, 精确和可再生的骨切除结合钢板内固定是足够稳定的允许僵硬的 allotransplants, 即使在那些最小的大小不匹配。
所提出的技术的一个限制是, 它不允许评估不同的组织成分的皮肤和肌肉除了血管化的骨成分。虽然包括不同组织成分的复合皮瓣是可能的, 该模型的目的是研究独特的同种异体骨移植作为各种 VCA 组织 compontents 的免疫原性不同的38。
最后, 本文提供的信息, 建立一个可再生的大动物模型与定义遗传学的骨 VCA 研究。该模型可作为未来研究的基础, 调查血管生成对骨血流和骨重塑的影响, 并可能消除长期免疫抑制的需要。此外, 它可用于描述骨 VCA 排斥的复杂过程, 并测试其他创新免疫调节策略。定义的 SLA-型和定量的 SRY 基因在性别不匹配的猪可以允许确定的程度嵌的移植和外周血。

大节段性骨缺损是由外伤、感染或恶性肿瘤后保肢手术引起的。目前的重建选择, 如血管化自体骨移植、骨转运、人工关节置换和冷冻保存的坏死同种异体移植, 单独或联合使用, 都有显著的发病率, 并有很高的率并发症1,2,3。
微血管网络的存在对于骨骼的形成和稳态至关重要, 支持骨修复所需的成骨、软骨和间充质干细胞4。
活体同种异体骨移植是一种带血管的复合组织异体 (骨 VCA), 以其营养蒂的显微外科吻合术, 可作为未来重建的替代品。与冷冻异体骨一样, 通过紧密匹配的骨缺损形态提供即刻稳定性。像自体血管移植, 它提供了加强愈合和重塑活骨组织。任何移植程序的障碍仍然是长期免疫抑制的需要。这个问题在肌肉骨骼组织中更为尖锐, 这需要药物剂量比器官移植的2-3 倍5。伴随的风险, 包括器官毒性, 恶性肿瘤, 感染或发展移植物抗宿主病是很难证明在这些寿险关键-应用程序6。然而, 急性和慢性排斥的事件仍然是当前长期存在的一个主要问题, 即7。持续的努力, 以密切配合组织相容性抗原, 诱导捐赠特定的耐受性和/或改善药物免疫治疗尚未成功地在允许临床无药物组存活8,9。
我们先前已经证明了通过促进移植骨内的新的自生循环来维持骨 VCA 活力和增强小动物模型骨重塑的方法。这是由植入自体组织的外科血管生成的额外使用10,11,12。异体骨段移植 microsurgically 与营养骨段蒂吻合。此外, 宿主衍生血管被植入同种异体血管化骨段的髓质管。在这2周的过程中, 同种异体营养血管的通畅保持了药物免疫抑制。在取出后, 营养蒂将最终 thrombose13。新的毛细床, 基于宿主衍生的血管提供足够的循环, 以维持组织的生存能力。骨愈合和重塑是加强, 因为成骨和血管生成耦合10,11,12。不需要进一步的免疫治疗, 尽管有免疫的合格宿主和缺乏供体特定的耐受性, 但长期维持骨骼的生存能力。
将这种新的同种异体骨移植到临床实践中, 最好先对大动物模型的愈合、力学性能和免疫学进行进一步的研究。猪模型是理想的这样 VCA 研究14,15,16。微型猪在大小和解剖学上是可比较的人, 允许骨骼重建使用本质上完全相同的外科植入物和技术。猪免疫的定义很明确, 包括猪白细胞抗原 (SLA) 型和血型, 是移植手术所必需的。细胞谱系研究是可能的与性别不匹配的移植, 如详细分析免疫应答17,18,19,20,21。
在这里, 我们描述了一个骨 VCA 移植模型在尤卡坦半岛小型猪, 适合研究节段性骨丢失和重建。该模型可用于研究外科血管生成和短期的相互作用, 包括骨谱系、骨血流、愈合和重塑能力、alloresponsiveness 和生物力学的 VCA 存活和功能, 以及测试其他创新的免疫调节策略。

<table><tbody><tr><td>Xylazine</td><td>VetTek, Bluesprings, MO</td><td>N/A</td><td>2mg/kg</td></tr><tr><td>Telazol</td><td>Pfizer Inc., NY, NY</td><td>2103</td><td>5mg/kg</td></tr><tr><td>Buprenorphine</td><td>Zoo Pharm, Windsor, CO</td><td>N/A</td><td>0.18mg/kg</td></tr><tr><td>Cefazoline</td><td>Hospira, Lake Forest, IL</td><td>RL-4539</td><td>1g</td></tr><tr><td>Ethilon sutures</td><td>Ethicon, Sommerville, NJ</td><td>BV 130-5</td><td>9-0</td></tr><tr><td>Locking plate</td><td>DePuy Synthes Vet, West Chester, PA</td><td>VP4041.09</td><td>9-hole 3.5mm locking plate</td></tr><tr><td>Vicryl sutures</td><td>Ethicon, Sommerville, NJ</td><td>J808T</td><td>2-0, 3-0</td></tr><tr><td>Tegaderm</td><td>3M Health Care, St. Paul, MN&amp;nbsp;</td><td>16006</td><td>15x10cm</td></tr><tr><td>Hickman catheter</td><td>Bard Access System Inc., Salt Lake City, UT</td><td>600560</td><td>9.6 French</td></tr><tr><td>Carprofen</td><td>Zoetis Inc., Kalamazoo, MI</td><td>1760R-60-06-759</td><td>4mg/kg</td></tr><tr><td>Tacrolimus</td><td>Sandoz Inc., Princeton, NJ&amp;nbsp;</td><td>973975</td><td>(0.8-1.5mg/kg/day)</td></tr><tr><td>Mycophenolate Mofetil&amp;nbsp;</td><td>Sandoz Inc., Princeton, NJ&amp;nbsp;</td><td>772212</td><td>(50-70mg/kg/day)&amp;nbsp;</td></tr><tr><td>Methylprednisolone sodium succinate</td><td>Pfizer Inc., NY, NY</td><td>2375-03-0</td><td>500 mg</td></tr><tr><td>Gentamicin</td><td>Sparhawk Laboratories, Lenexa, KS</td><td>1405-41-0</td><td>3mg/kg&amp;nbsp;</td></tr><tr><td>Dermabond Prineo</td><td>Ethicon, San Lorenzo, Puerto Rico</td><td>6510-01-6140050</td><td></td></tr><tr><td>Isoflurane 99.9% 250 ml</td><td>Abbott Animal&amp;nbsp; Health&amp;nbsp;</td><td>05260-5</td><td></td></tr><tr><td>Lactated Ringer&#039;s 1L</td><td>Baxter Corporation</td><td>JB1064</td><td></td></tr><tr><td>Saline 0.9%, 1 L</td><td>Baxter Corporation</td><td>60208</td><td></td></tr><tr><td>Ceftiofur</td><td>Pfizer Canada Inc.</td><td>11103</td><td>5mg/kg</td></tr><tr><td>Microfil</td><td>Flow Tech Inc, Carver, MA</td><td>MV-122</td><td>125 ml</td></tr><tr><td>Decalcifying Solution</td><td>Thermo Fisher Scientific, Chesire, WA, UK</td><td>8340-1</td><td></td></tr></tbody></table>

surgical angiogenesis in porcine tibial allotransplantation: a new large animal bone vascularized composite allotransplantation model

创伤、感染性恶性肿瘤和先天性畸形导致的节段骨丢失仍然是一项重大的重建挑战。目前的治疗方案有重大的失败风险和大量的发病率。
使用骨带血管的复合同种异体移植 (VCA) 将提供一个密切吻合的骨大小和形状, 愈合和重塑潜力的活骨。目前, life-long 药物免疫抑制 (是) 是必需的。器官毒性、机会感染和肿瘤风险是治疗这种非致死性适应症的关注。
我们先前已经证明, 在大鼠和家兔中可以维持骨骼和关节 VCA 的生存能力, 而不需要通过植入 VCA 内的受体血管进行长期免疫抑制。它产生一个自生, neoangiogenic 循环与可测量的流量和主动骨重塑, 只需要2周的是。由于小动物在解剖学、骨骼生理学和免疫学方面与人类有很大的不同, 我们在进行临床应用之前, 已经开发出了一种猪骨 VCA 模型来评价这一技术。小型猪目前被广泛用于同种异体移植的研究, 其免疫学, 解剖, 生理和大小相似的人。在这里, 我们描述了一个新的猪原位胫骨骨 VCA 模型, 以测试的作用, 自体外科血管生成保持 VCA 生存能力。
该模型利用大小和形状匹配的同种异体胫骨骨段, 移植在尤卡坦半岛小型猪的主要猪白细胞抗原 (SLA) 错配中, 重建胫骨节段骨缺损。将接受者自体血管移植到同种异体胫骨骨段髓管中的营养血管的修复和植入是与同期短期相结合进行的。这允许 neoangiogenic 自体循环从植入的组织中发展, 在短时间内维持通过同种异体营养血管的流动。一旦建立, 新的自体循环维持骨活力后, 停止药物治疗和随后的营养血管血栓形成。

该技术成功地在四 SLA 主要不匹配的尤卡坦半岛小型猪和胫骨缺损重建使用大小匹配的胫骨 VCA。同时营养血管修复的骨移植和植入的 AV 捆绑从接受动物在移植髓质管允许立即骨循环和发展新的自体血液供应超过时间 (图 1)。在16周内, neoangiogenic 循环已建立在所有胫骨 VCAs, 可视化的微计算机断层扫描 (微 CT) 造影后注射显影血管造影聚合物 (125 毫升) 到股骨血管和脱胫骨 VCA (图 2)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55238/55238fig1.jpg"> 图 1: 胫骨原位骨 VCA 术.显示手术过程的图示。(a) 供体程序: 以其营养蒂为胫骨骨段的收获。(B) 在主要的 SLA 不匹配的猪之间交换胫骨骨段。(C) 接受程序: 动静脉束植入: 在髓质管中仔细插入颅骨胫血管。(D) 营养蒂的显微血管吻合术, 用于胫骨前室肌支和胫骨骨干的接骨板。在梅奥医学教育和研究基金会的许可下使用。保留所有权利。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig1large.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/55238/55238fig2.jpg"> 图 2: 代表性3D 脱胫骨 VCA 段的微 CT 血管造影图像.neongiogenic 循环 (黄色箭头) 是在灌注显影硅溶液后描述的。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
微型猪没有表现出任何窘迫或自残的迹象。所有的伤口愈合, 没有感染和动物行走正常, 最终能够承担全部重量的操作右下肢从第一个术后一天。在研究端点在 16th星期所有尤卡坦微型猪获得了超过150% 他们的原始的体重 (前: 56.0 ±6.1 与16星期后: 84.5 ± 6.0)。
两周的免疫抑制, 包括克莫司, 霉酚酸酯 (MMF) 和甲基泼尼松龙琥珀酸是用来维持血液流经营养蒂, 直到一个新的自体血液供应已经建立在异基因骨头移植在2周的免疫抑制周期性血液样本取自颈静脉导管, 以评估药物血水平。剂量进行了调整, 以保持槽血水平的 5-30 ng/毫升的克莫司和 1-3. 5 µg/毫升 MMF (表 1)。没有药物相关的并发症发生, 并瞄准槽水平的克莫司和霉酚酸酯可以实现 (图 3和图 4)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>免疫</td>
			<td>初始剂量</td>
			<td>槽水位</td>
			<td>维护剂量</td>
		</tr>
<tr>
<td>莫</td>
			<td>0.8-1.5 毫克/公斤/天</td>
			<td>5-30 ng/毫升</td>
			<td></td>
		</tr>
<tr>
<td>霉酚酸酯</td>
			<td>50-70 毫克/千克/天</td>
			<td>1-3 µg/毫升</td>
			<td></td>
		</tr>
<tr>
<td>甲基泼尼松龙琥珀酸钠</td>
			<td>500毫克</td>
			<td></td>
			<td>50毫克</td>
		</tr>
</tbody></table>表 1: 短期免疫抑制协议描述的是免疫抑制协议的第一2周后移植与开始剂量他克莫司, 霉酚酸酯和泼尼松龙。此外, 还针对他克莫司和霉酚酸酯的水平和维持剂量的泼尼松龙显示。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55238/55238fig3.jpg"> 图 3: 他克莫司的血液水平.在前2周的移植后, 他克莫司达到的槽位水平和分范围被描述。误差线表示分范围。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55238/55238fig4.jpg"> 图 4: 为霉酚酸酯的血液水平.对2周短免疫期的霉酚酸酯的槽血水平和分范围进行了分析。误差线表示分范围。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
尽管两周后停止免疫抑制, 在不同时间点 (2、4、6、10和16周) 的放射治疗右后肢体的 x 射线显示, 渐进性骨愈合的研究期间, 16 周时, 评分两个独立的和盲的观察器 (图 5)27,28。16th周的微 CT 分析用于量化骨痂的体积和密度, 以及在宿主/骨 VCA 接合处和骨 VCA 移植出现的骨骼形成,27。由新的自体血液供应促进的内部固定的维护, 不用减少损失或丢失, 可以证明28。骨愈合是在所有胫骨 (图 6) 中实现的。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55238/55238fig5.jpg"> 图 5:骨愈合进展超过16周.为确定骨愈合进展, 采用了非线性回归模型。值 R2用于定义模型与数据的匹配。使用评分系统, 基于前后和横向射线照相骨骨 VCA 入节段性骨缺损在研究期间 (2、4、6、10和16周) 的不同时间点的最高值为25分, 由两独立和盲观察器30,31。非线性回归模型描述了研究期间骨愈合值的中值和分范围 (R2 = 0.931), 显示了连续的骨愈合进展, 其价值接近于16周的25。误差线表示分范围。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig5large.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/55238/55238fig6.jpg"> 图 6: 三维重建猪胫骨骨干在微计算机断层扫描后的评价.有代表性的三维重建胫骨内板固定在2x 实际大小的图像。在16周的完整愈合后胫骨骨 VCAs 与外科血管生成显示。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
苏木精-曙红脱染色切片的组织学分析使用先前描述的比例分级排斥 (无, 轻度, 中度和严重) 没有发现严重排斥的迹象, 由此轻微和适度的拒绝迹象可以演示了三只猪 (图 7)29。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55238/55238fig7.jpg"> 图 7:来自胫骨 VCA 的水平苏木素-曙红染色切片的代表性图像显影硅溶液填充的容器显示为褐色 (星号)。轻度内浸润和反应 (粗箭头) 是看到与超过三分之二的缺陷填充骨 (小箭头) 按照可行的骨骼。10X 放大倍数。缩放栏 = 300 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55238/55238fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model at JoVE.com

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

目前, 任何一种带血管的复合异体移植都依赖于长期免疫抑制, 难以支持非致命的适应症。我们提出了一个新的猪胫骨 VCA 模型, 可用于研究骨 VCA 和示范使用的外科血管生成, 以维持骨骼的生存能力, 而无需长期免疫调节。

猪胫骨移植术后血管生成的动物模型

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

surgical-angiogenesis-porcine-tibial-allotransplantation-new-large

Nanyang Technological University

Research

JoVE Journal

Medicine

7.6K 次观看.  Mayo Clinic. 目前, 任何一种带血管的复合异体移植都依赖于长期免疫抑制, 难以支持非致命的适应症。我们提出了一个新的猪胫骨 VCA 模型, 可用于研究骨 VCA 和示范使用的外科血管生成, 以维持骨骼的生存能力, 而无需长期免疫调节。

视频: Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

自体内皮祖细胞播种技术和生物相容性在大动物模型的心血管设备的测试

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

科研

生物工程

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

原位主动脉瓣移植在小鼠血管疾病的研究

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

医学

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

平移血管复合同种异体移植（VCA）研究一种改进的异位猪后肢移植模型

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

计算机断层扫描和成骨血管生成耦合光学成像评估头盖骨自体移植和移植物的整合

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

下腔静脉间置移植物在小鼠模型中植入

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

在小型动物模型的动静脉（AV）循环研究血管生成和血管组织工程

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

异位自体肾移植的猪模型:有步骤，分步协议

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

A Reliable Porcine Fascio-Cutaneous Flap Model for Vascularized Composite Allografts Bioengineering Studies

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, failed by the first steps of the reconstructive ladder. Despite promising aesthetic and functional outcomes, the main limiting factor remains the need for a drastically applied lifelong immunosuppression and its well-known medical risks, preventing broader indications. Therefore, lifting the immune barrier in VCA is essential to tip the ethical scale and improve patients&#39; quality of life using the most advanced surgical techniques. De novo creation of a patient-specific graft is the upcoming breakthrough in reconstructive transplantation. Using tissue engineering techniques, VCAs can be freed of donor cells and customized for the recipient through perfusion-decellularization-recellularization. To develop these new technologies, a large-scale animal VCA model is necessary. Hence, swine fascio-cutaneous flaps, composed of skin, fat, fascia, and vessels, represent an ideal model for preliminary studies in VCA. Nevertheless, most VCA models described in the literature include muscle and bone. This work reports&#160;a reliable and reproducible technique for saphenous fascio-cutaneous flap harvest in swine, a practical tool for various research fields, especially vascularized composite tissue engineering.

The present protocol describes the porcine fascio-cutaneous flap model and its potential use in vascularized composite tissue research.

一种用于血管化复合同种异体移植物生物工程研究的可靠猪筋膜-皮肤皮瓣模型

Vascularized Composite Allografts (VCA) such as hand, face, or penile transplant represents the cutting-edge treatment for devastating skin defects, ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

改良异位异体下肢骨皮瓣模型在大鼠转化血管化复合同种异体移植研究中的应用

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

一种猪异位心脏移植方案，用于向心脏同种异体移植物提供治疗药物

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...