Mammoto Tadanori, Mammoto Akiko
Vascular Biology Program, Department of Surgery, Boston Children's Hospital and Harvard Medical School;
J Vis Exp. 2014 Dec 21(94):52012. doi: 10.3791/52012.
Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.
干细胞研究和生物工程技术最近取得的重大进展，在利用生物材料再生和修复骨科及牙周领域的简单组织损伤方面取得了巨大进步。然而，尝试再生更复杂的三维（3D）器官（如肺）的结构和功能却不太成功，因为器官再生的生物学过程尚未得到充分探索。越来越清楚的是，血管生成，即新血管的形成，在器官再生中起着关键作用。新形成的脉管系统不仅输送器官再生所需的氧气、营养物质和各种细胞成分，还为再生的局部组织提供指导性信号。因此，要在成体中成功再生肺，有必要重现肺特异性微环境，在这种微环境中血管生成驱动局部肺组织的再生。尽管传统的体内血管生成检测方法，如皮下植入富含细胞外基质（ECM）的水凝胶（如纤维蛋白或胶原蛋白凝胶或Engelbreth-Holm-Swarm小鼠肉瘤细胞分泌的基质胶 - ECM蛋白混合物），被广泛用于探索血管生成的一般机制，但肺特异性血管生成尚未得到很好的表征，因为将生物材料原位植入肺的方法尚未完全建立。本方案的目标是介绍一种独特的方法，将纤维蛋白凝胶植入成年活体小鼠的肺表面，从而在凝胶内部成功重现源自宿主肺的血管生成。这种方法使研究人员能够探索在正常和病理条件下肺特异性微环境控制血管生成和肺泡再生的机制。由于植入的生物材料向相邻肺组织释放并提供物理和化学信号，将这些生物材料植入患病肺可能会使相邻的患病组织恢复正常，从而使研究人员能够开发针对各种类型肺病的新治疗方法。
