猪胫骨移植术后血管生成的动物模型
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Xylazine | VetTek, Bluesprings, MO | N/A | 2mg/kg |
| Telazol | Pfizer Inc., NY, NY | 2103 | 5mg/kg |
| Buprenorphine | Zoo Pharm, Windsor, CO | N/A | 0.18mg/kg |
| Cefazoline | Hospira, Lake Forest, IL | RL-4539 | 1g |
| Ethilon sutures | Ethicon, Sommerville, NJ | BV 130-5 | 9-0 |
| Locking plate | DePuy Synthes Vet, West Chester, PA | VP4041.09 | 9-hole 3.5mm locking plate |
| Vicryl sutures | Ethicon, Sommerville, NJ | J808T | 2-0, 3-0 |
| Tegaderm | 3M Health Care, St. Paul, MN | 16006 | 15x10cm |
| Hickman catheter | Bard Access System Inc., Salt Lake City, UT | 600560 | 9.6 French |
| Carprofen | Zoetis Inc., Kalamazoo, MI | 1760R-60-06-759 | 4mg/kg |
| Tacrolimus | Sandoz Inc., Princeton, NJ | 973975 | (0.8-1.5mg/kg/day) |
| Mycophenolate Mofetil | Sandoz Inc., Princeton, NJ | 772212 | (50-70mg/kg/day) |
| Methylprednisolone sodium succinate | Pfizer Inc., NY, NY | 2375-03-0 | 500 mg |
| Gentamicin | Sparhawk Laboratories, Lenexa, KS | 1405-41-0 | 3mg/kg |
| Dermabond Prineo | Ethicon, San Lorenzo, Puerto Rico | 6510-01-6140050 | |
| Isoflurane 99.9% 250 ml | Abbott Animal Health | 05260-5 | |
| Lactated Ringer's 1L | Baxter Corporation | JB1064 | |
| Saline 0.9%, 1 L | Baxter Corporation | 60208 | |
| Ceftiofur | Pfizer Canada Inc. | 11103 | 5mg/kg |
| Microfil | Flow Tech Inc, Carver, MA | MV-122 | 125 ml |
| Decalcifying Solution | Thermo Fisher Scientific, Chesire, WA, UK | 8340-1 |
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