三维胶原基质支架植入作为肝脏再生策略
1Liver, Pancreas and Motility Laboratory; Unit of Experimental Medicine; School of Medicine,Universidad Nacional Autónoma de Mexico (UNAM), 2Department of Surgery, School of Medicine,Universidad Nacional Autónoma de México (UNAM), 3Department of Cells and Tissue Biology, School of Medicine,Universidad Nacional Autónoma de México (UNAM), 4Department of Pathology,Hospital General de Mexico, 5Materials Research Institute,Universidad Nacional Autónoma de México (UNAM)
Summary
肝脏疾病是由许多促进纤维化或肝硬化的原因引起的。移植是恢复健康的唯一选择。然而，鉴于可移植器官的稀缺性，必须探索替代方案。我们的研究提出了从动物模型中将胶原支架植入肝脏组织。
Abstract
肝病是全球死亡的主要原因。过量饮酒、高脂肪饮食和丙型肝炎病毒感染会促进纤维化、肝硬化和/或肝细胞癌。肝移植是临床推荐的手术，以改善和延长晚期疾病患者的寿命。然而，只有10%的移植是成功的，器官可用性，术前和术后手术以及与该结果直接相关的成本升高。细胞外基质（ECM）支架已成为组织修复的替代方案。生物相容性和接枝接受度是这些生物材料的主要有益特征。尽管在肝脏切除术模型中已经评估了恢复肝脏大小和正确功能的能力，但尚未评估使用支架或某种支持来替换已消失的肝脏肿块的体积。
在大鼠肝脏中进行部分肝切除术，从牛髁异种植入胶原基质支架（CMS）。切除左肝叶组织（约40%），手术植入相同比例的CMS。在手术前后评估肝功能检查。第3天，第14天和第21天后，对动物实施安乐死，并进行宏观和组织学评估。在第3天和第14天，在CMS周围观察到脂肪组织，没有排斥或感染的临床证据，第21天的血管新形成和CMS重吸收也是如此。有组织学证据表明，炎症过程和邻近细胞向CMS的迁移是微不足道的，用苏木精和曙红（H&E）以及Masson的三色染色观察到。CMS被证明在肝组织中表现良好，可能是研究慢性肝病组织再生和修复的有用替代方案。
Introduction
肝脏是参与维持体内平衡和蛋白质产生的最重要器官之一1。不幸的是，肝病是全球死亡的主要原因。在肝损伤的晚期阶段，包括肝硬化和肝细胞癌，肝移植是临床推荐的程序。然而，由于供体的稀缺和成功移植率低，组织工程（TE）和再生医学（RM）的新技术已经开发出来2，3。
TE涉及使用干细胞，支架和生长因子4来促进发炎，纤维化和水肿器官和组织的恢复1，5，6。支架中使用的生物材料模仿天然ECM，为引导细胞重塑提供物理，化学和生物线索7。胶原蛋白是从真皮，肌腱，肠和心包8，9中获得的最丰富的蛋白质之一。此外，胶原蛋白可以作为生物聚合物获得，通过生物打印或静电纺丝产生二维和三维支架10，11。该小组是第一个报告使用骨源胶原蛋白进行肝组织再生的组织。另一项研究报告了使用从牛胶原蛋白合成的支架，该支架是从皮肤获得的，具有均匀且紧密排列的毛孔，它们之间没有任何通讯12。
去细胞化保留了天然的ECM，允许随后掺入具有干细胞电位的细胞13，14。然而，该程序仍处于实验阶段，在肝脏，心脏，肾脏，小肠和膀胱中来自小鼠，大鼠，兔子，猪，羊，牛和马3，14。目前，切除的肝脏质量体积在任何动物肝切除术模型中都没有被替换。然而，使用额外的支持或网络（生物材料）使细胞增殖和血管生成对于迅速恢复肝实质功能可能是必不可少的。因此，支架可以用作再生或修复慢性肝病组织的替代方法，从而消除由于捐赠和肝移植的临床并发症而产生的局限性。
Protocol
本研究已获得墨西哥国立自治大学医学院伦理委员会（DI/115/2015）和墨西哥总医院伦理委员会（CI/314/15）的批准。该机构符合实验动物生产，护理和使用的所有技术规范，并通过了国家法律（NOM-062-ZOO-1999）的法律认证。本研究从墨西哥国立自治大学医学院实验动物设施获得体重150-250克（6-8周龄）的雄性Wistar大鼠。 1. 从牛股骨获得胶原基质支架 从墨西哥卫生和农业当?…
Representative Results
骨脱矿质会影响CMS的机械性能，而不会改变其毛孔的原始形状或相互连接。CMS可以具有任何形状，因此，可以调整到所选器官或组织的大小和形状19。在目前的协议中，我们使用三角形CMS（图1A-D）。使用大鼠模型评估CMS异种激素在肝脏中的再生能力。尽管肝脏是一个易碎和柔软的器官，但在该协议中进行的外科手术确?…
Discussion
器官移植是肝纤维化或肝硬化患者的主要治疗方法。少数患者从这一程序中受益，因此有必要为等待名单上的患者提供治疗替代方案。组织工程是一种有前途的策略，采用支架和具有再生潜力的细胞2，4，13。切除肝脏的一部分是该过程的关键步骤，因为该血管化器官大量出血。因此，必须进行手术床止血以防止这种并发症。?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者要感谢实验医学部门实验动物设施的工作人员，护士Carolina Baños G.的技术和外科支持，Marco E. Gudiño Z.对显微照片的支持，以及Erick Apo对肝脏组织学的支持。国家委员会支持这项科学技术研究（CONACyT），批准号SALUD-2016-272579和PAPIIT-UNAM TA200515。
Materials
| Anionic detergent | Alconox | Z273228 | |
| Biopsy cassettes | Leica | 3802453 | |
| Camera DMX | Nikon | DXM1200F | |
| Centrifuge | Eppendorf | 5424 | |
| Chlorhexidine gluconate 4% | BD | 372412 | |
| Cover glasses 25 mm x 40 mm | Corning | 2980-224 | |
| Eosin | Sigma-Aldrich | 200-M | CAS 17372-87-1 |
| Ethyl alcohol, pure | Sigma-Aldrich | 459836 | CAS 64-17-5 |
| Flunixine meglumide | MSD | Q-0273-035 | |
| Glass slides 75 mm x 25 mm | Corning | 101081022 | |
| Hematoxylin | Merck | H9627 | CAS 571-28-2 |
| Hydrochloric acid 37% | Merck | 339253 | CAS 7647-01-0 |
| Ketamine | Pisa agropecuaria | Q-7833-028 | |
| Light microscopy | Nikon | Microphoto-FXA | |
| Microtainer yellow cape | Beckton Dickinson | 365967 | |
| Microtome | Leica | RM2125 | |
| Model animal: Wistar rats | Universidad Nacional Autónoma de México | ||
| Nylon 3-0 (Dermalon) | Covidien | 1750-41 | |
| Polypropylene 7-0 | Atramat | SE867/2-60 | |
| Povidone-iodine10% cutaneous solution | Diafra SA de CV | 1.37E+86 | |
| Scaning electronic microscopy | Zeiss | DSM-950 | |
| Sodium hydroxide, pellets | J. T. Baker | 3722-01 | CAS 1310-73-2 |
| Software ACT-1 | Nikon | Ver 2.70 | |
| Stereoscopy macroscopy | Leica | EZ4Stereo 8X-35X | |
| Sterrad 100S | Johnson and Johnson | 99970 | |
| Surgipath paraplast | Leica | 39601006 | |
| Synringe of 1 mL with needle (27G x 13 mm) | SensiMedical | LAN-078-077 | |
| Tissue Processor (Histokinette) | Leica | TP1020 | |
| Tissue-Tek TEC 5 (Tissue embedder) | Sakura Finetek USA | 5229 | |
| Trichrome stain kit | Sigma-Aldrich | HT15 | |
| Unicell DxC600 Analyzer | Beckman Coulter | BC 200-10 | |
| Xylazine | Pisa agropecuaria | Q-7833-099 | |
| Xylene | Sigma-Aldrich | 534056 | CAS 1330-20-7 |
References
- Li, N., Hua, J. Immune cells in liver regeneration. Oncotarget. 8 (2), 3628-3639 (2017).
- Langer, R., Vacanti, J. Tissue Engineering. Science. 260 (5110), 920-926 (1993).
- Lee, H., et al. Development of liver decellularized extracellular matrix bioink for three-dimensional cell printing-based liver tissue engineering. Biomacromolecules. 18 (4), 1229-1237 (2017).
- Shafiee, A., Atla, A. Tissue engineering: Toward a new era of medicine. Annual Review of Medicine. 68, 29-40 (2017).
- Hu, C., Zhao, L., Wu, Z., Li, L. Transplantation of mesenchymal stem cells and their derivatives effectively promotes liver regeneration to attenuate acetaminophen-induced liver injury. Stem Cell Research & Therapy. 11 (1), 88 (2020).
- Sancho-Bru, P. Therapeutic possibilities of stem cells in the treatment of liver diseases. Gastroenterologia y Hepatologia. 34 (10), 701-710 (2011).
- Kobolak, J., Dinnyes, A., Memic, A., Khademhosseini, A., Mobasheri, A. Mesenchymal stem cells: Identification, phenotypic characterization, biological properties and potential for regenerative medicine through biomaterial micro-engineering of their niche. Methods. 99, 62-68 (2016).
- Freedman, B. R., Mooney, D. J. Biomaterials to mimic and heal connective tissues. Advanced Materials. 31 (19), 1806695 (2019).
- Meyer, M. Processing of collagen based biomaterials and the resulting materials properties. Biomedical Engineering Online. 18 (1), 24 (2019).
- El Baz, H., et al. Transplant of hepatocytes, undifferentiated mesenchymal stem cells, and in vitro hepatocyte-differentiated mesenchymal stem cells in a chronic lver failure experimental model: a comparative study. Experimental and Clinical Transplantation. 16 (1), 81-89 (2018).
- Nedjari, S., Awaja, F., Guarino, R., Gugutkov, D., Altankov, G. Establishing multiple osteogenic differentiation pathways of mesenchymal stem cells through different scaffold configurations. Journal of Tissue Engineering and Regenerative Medicine. 14 (10), 1428-1437 (2020).
- Chan, E. C., et al. Three dimensional collagen scaffold promotes intrinsic vascularisation for tissue engineering applications. PLoS One. 11 (2), 0149799 (2016).
- Arenas-Herrera, J. E., Ko, I. K., Atala, A., Yoo, J. J. Decellularization for whole organ bioengineering. Biomedical Materials. 8 (1), 014106 (2013).
- Parmaksiz, M., Dogan, A., Odabas, S., Elçin, A. E., Elçin, Y. M. Clinical applications of decellularized extracellular matrices for tissue engineering and regenerative medicine. Biomedical Materials. 11 (2), 022003 (2016).
- Gacek, G. Stereo microscope, neglected tool. Postepy Biochemii. 63 (1), 68-73 (2017).
- Oldham, S., Rivera, C., Boland, M. L., Trevaskis, J. L. Incorporation of a survivable liver biopsy procedure in mice to assess non-alcoholic steatohepatitis (NASH) resolution. Journal of Visualized Experiments: JoVE. (146), e59130 (2019).
- The University of Texas at Austin Institutional Animal Care and Use Committee. . Guidelines for the Use of Chemical Depilatory Agents on Laboratory Animals. , (2021).
- Sivridis, L., Kotini, A., Anninos, P. The process of learning in neural net models with Poisson and Gauss connectivities. Neural Networks. 21 (1), 28-35 (2008).
- León-Mancilla, B. H., Araiza-Téllez, M. A., Flores-Flores, J. O., Piña-Barba, M. C. Physico-chemical characterization of collagen scaffolds for tissue engineering. Journal of Applied Research and Technology. 14 (1), 77-85 (2016).
- León, A., et al. Hematological and biochemical parameters in Sprague Dawley laboratory rats breed in CENPALAB, Cenp:SPRD. Revista Electronica de Veterinaria. 12, 1-10 (2011).
- Tsuchiya, A., et al. Mesenchymal stem cell therapies for liver cirrhosis: MSCs as “conducting cells” for improvement of liver fibrosis and regeneration. Inflammation and Regeneration. 39, 18 (2019).
- Badylak, S. F. The extracellular matrix as a biologic scaffold material. Biomaterials. 28, 3587-3593 (2007).
- Acevedo, G. C. Xenoimplante de colágena en uretra de perro. Universidad Nacional Autónoma de México. , (2011).
- Montalvo-Jave, E. E., et al. Absorbable bioprosthesis for the treatment of bile duct injury in an experimental model. International Journal of Surgery. 20, 163-169 (2015).
Cite This Article
León-Mancilla, B., Martínez-Castillo, M., Medina-Avila, Z., Pérez-Torres, A., Garcia-Loya, J., Alfaro-Cruz, A., Piña-Barba, C., Gutierrez-Reyes, G. Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy. J. Vis. Exp. (172), e62697, doi:10.3791/62697 (2021).