Elucidating and modulating macrophage and stem cell responses to bioactive nanoclays for bone regeneration

医学进步带来了可喜的预期寿命延长。然而，这一进展带来了新的挑战：与年龄有关的疾病增加，生活质量相应下降。全世界每年有超过900万例骨折，其中约5%-10%与延迟愈合或骨不连有关。再生医学领域正在寻找新的疗法，利用干细胞再生受损或患病组织的潜力。这些疗法可以包括干细胞移植，或者使用药物和材料来招募和刺激已经存在于成人组织中的干细胞。粘土为再生医学提供了令人惊讶的丰富可能性。这在很大程度上是因为它们能够吸附和结合生物分子；这一特性长期以来一直被用于设计控制某些药物的释放和作用的片剂。英国研究小组已经开发出合成纳米大小(百万分之一毫米)的粘土颗粒，这种颗粒可以形成可注射的凝胶，在体内凝固。当粘土颗粒与从血液中吸附的蛋白质结合并相互作用时，凝胶化就会自发发生。这些富含蛋白质的“纳米粘土凝胶”为干细胞的生长创造了有利的环境。通过混合被称为生长因子的蛋白质，我们可以招募和刺激干细胞，将纳米粘土凝胶重塑为新的骨组织。由于纳米粘土凝胶可以结合并不释放生长因子，这些强大的干细胞刺激分子未来可以用于融合骨骼和修复不可愈合的骨折，具有更高的精确度、安全性和效率。虽然非常有希望，但关于这种修复过程有很多我们还不了解的地方。例如，我们知道，免疫系统细胞的反应将在决定人体在注射纳米粘土后的最初几小时和几天内如何反应方面发挥至关重要的作用。日本研究小组在了解和控制这种对植入材料的免疫反应方面处于世界领先地位。例如，该小组已经证明，有可能在修复部位以高度受控的方式释放药物，以吸引和刺激免疫细胞，以限制炎症和促进再生。因此，这项英国和日本的合作将研究免疫细胞如何发挥作用，清除纳米粘土颗粒，同时吸引干细胞形成骨。通过合作，我们将开发新的凝胶技术，将纳米粘土凝胶与明胶相结合，以促进一种影响免疫细胞再生的药物的受控释放。我们将测试的药物吡格列酮已经用于治疗糖尿病，因此，如果成功，很有可能在未来被用于再生医学。我们将通过将凝胶注射到小鼠的皮肤下，在体外和体内测试我们的方法，包括在培养皿中生长的细胞和在体内的细胞。体外实验将使用高倍显微镜来探索粘土纳米颗粒被免疫细胞吞噬后的命运。我们还将测量免疫细胞对纳米粘土的反应释放的生化信号，并测试是否可以修改细胞信号以促进吡格列酮的再生。在体内，我们将依靠最先进的活体动物成像技术来跟踪免疫细胞对纳米粘土凝胶的反应，并通过同步微型CT(3D X-Ray)将这些反应与新骨形成关联起来。这种令人兴奋的方法使我们能够跟踪单个动物随时间的反应，这大大减少了实验所需的小鼠数量。最后，在开发和优化了我们的新方法后，我们将测试该技术在大鼠颅骨手术准备的骨缺损中加速骨修复的能力。这些实验得到了很好的证实，对于测试我们的方法并证明后续对临床翻译的研究投资对患者的利益是必要的。

Medical advances have led to a welcome increase in life expectancy. However, this progress presents new challenges: increases in age-related diseases, and associated reductions in quality of life. There are over 9 million bone fractures per year, worldwide, of which approximately 5%-10% are associated with delayed healing or non-union. The field of regenerative medicine seeks new therapies that harness the potential of stem cells to regenerate damaged or diseased tissue. These therapies can involve stem cell transplantation, or using drugs and materials to recruit and stimulate stem cells already present in adult tissues.Clays offer surprisingly rich possibilities for regenerative medicine. This is largely due to their ability to adsorb and bind biological molecules; a property that has for a long time been used in the design of tablets to control the release and action of certain drugs. The UK research group have developed synthetic nano-sized (1 millionth of a millimetre) clay particles that form injectable gels that set in the body. Gelation occurs spontaneously as the clay particles bind and interlock with proteins adsorbed from blood. These protein-rich 'nanoclay gels' create environments favourable for stem cells to colonise. By mixing-in proteins called growth factors, we can recruit and stimulate stem cells to remodel the nanoclay gel into new bone tissue. Given nanoclay gels bind and do not release growth factors, these potent stem cell stimulating molecules can be used in the future to fuse bones and repair non-healing fractures with greater precision, safety and efficiency.While very promising, there is a lot about this repair process that we do not yet understand. For example, we know that the response of cells of the immune-system will play a vital role in determining how the body responds to nanoclay in the first hours and days after injection. The Japanese research group are world-leaders in understanding and controlling this immune response to implanted materials. For example the group have shown that it is possible to release drugs in a highly controlled manner at the site of repair to attract and stimulate immune cells to limit inflammation and promote regeneration. This UK-Japan collaboration will therefore study how immune cells function to clear away nanoclay particles and simultaneously attract stem cells for bone formation. In partnership, we will develop new gel technologies that combine nanoclay gels with gelatin to facilitate controlled release of a drug that influences immune cells towards regeneration. The drug we will test, pioglitazone, is already used in the treatment of diabetes and so, if successful, has a good chance of being adopted for regenerative medicine in the future. We will test our approach both 'in vitro', on cells grown in a petri dish and 'in vivo', within the body by injecting the gels under the skin of mice. In vitro experiments will employ high powered microscopes to explore the fate of clay nanoparticles after they are engulfed by the immune cells. We will also measure the biochemical signals released by immune cells in response to nanoclay and test whether cell signalling can be modified to promote regeneration by pioglitazone. 'In vivo', we will rely on state of the art live animal imaging techniques to track immune cell responses to nanoclay gels over time and correlate these responses to new bone formation via simultaneous micro CT (3D X-Ray). This exciting approach allows us to track responses in a single animal over time which substantially reduces the number of mice needed for experiments. Finally, having developed and optimised our new approach we will test the ability of the technology to accelerate bone repair in a surgically prepared bone defect in a rat cranium. These experiments are well established and essential to test our approach and justify subsequent research investment towards clinical translation for patient benefit.