Development of PLGA microsphere formulations for the sustained release of growth factors

骨关节炎(OA)是一种常见的关节疾病，它伴随着炎症，关节软骨表面的退化，使关节运动顺畅，最终导致慢性疼痛和运动障碍。目前的OA治疗旨在缓解症状，但到目前为止，还没有有效的治疗方法来治愈或预防这种情况。最近，研究人员利用组织工程学的方法试图重建降解的组织，但还没有成功地获得天然软骨的再生，这表明目前的策略缺乏相关的步骤。大多数软骨组织工程方法侧重于选择干细胞群体和开发生物材料作为组织再生的3D支架，但较少关注给细胞提供诱导线索来正确地改革组织。这一事实在软骨等结构复杂的组织中尤其重要，因为软骨中存在不对称的生长因子(GF)梯度，必须保持生长因子(GF)的不对称梯度，才能保证干细胞存活和正确的细胞分化。向关节腔外注射GF是不够的，因为蛋白质在滑液中很快就会被冲走，而且在临床实践中反复补充是不现实的。因此，将玻璃纤维包裹在生物可降解微球(MSS)中并在生物材料支架中进行缓释是生成和维护GF梯度材料的一种更可行的方法。Pouya Rezai的实验室在微流体生成MSS方面拥有丰富的专业知识。微流体学研究流体在微尺度上的行为，需要专门的知识、专业知识和昂贵的设备，而很少有实验室能够获得这些知识、专业知识和昂贵的设备。利用微流体合成微粒子，可以完全控制微粒子的性质(即直径大小、层数、分子包裹的高效率等)。否则，这将是无法实现的。艾琳·金特尔曼的实验室已经开发出一种基于透明质酸的可光交叉生物材料，用于修复受损的软骨。我们已经证明，这种生物材料是可注射的，可以支持软骨细胞，是临床原位软骨组织工程的理想系统。在这里，我们建议使用FDA批准的可生物降解聚合物聚(乳酸-乙醇酸)(PLGA)来合成不同的MS制剂，用于GF的缓释。在骨性关节炎的情况下，MSS可以与生物材料和干细胞一起用于软骨的再生。因此，我们将克服目前软骨再生医学的局限性，带来下一代组织工程方法。为了实现这一目标，我们将首先产生不同大小(从纳米到微米直径)和不同层(单层或双层)的MS。然后，我们将评估哪些配方适合与生物材料和细胞一起使用。最后，我们将模型蛋白包裹在相关的MS配方中，研究其在生理条件下的降解和释放动力学，并随后确定哪些配方更适合复制软骨自然生长因子梯度。这一多学科项目的结果是满足了目前组织工程的局限性。这一机会将使我们更接近再生医学背景下的成功治疗。该项目不仅将两个完全不同的学科(来自Gentleman实验室的组织工程学和来自Rezai实验室的微流体和微粒产生)结合在一起，开发出一种新颖、革命性和有前途的方法，而且还为加拿大和英国实验室之间的新的富有成效的合作铺平了道路。

Osteoarthritis (OA) is a common joint disorder that carries inflammation, degeneration of the cartilaginous surface of joints that allow smooth joint movement, and eventually, chronic pain and locomotor disability. Current OA treatments aim to alleviate the symptoms, but to date, there are no effective treatments to cure or prevent the condition. Recently, researchers have resorted to tissue engineering in an attempt to reform the degraded tissue, but no successful regeneration of native cartilage has been yet achieved, suggesting that current strategies miss a relevant step.Most cartilage tissue engineering approaches focus on selecting a stem cell population and on developing a biomaterial that serves as a 3D scaffold for tissue regeneration, but lesser attention is paid on giving inductive cues for cells to correctly reform the tissue. This fact is particularly important in tissues with complex structures, such as cartilage, where asymmetrical gradients of growth factors (GF) are present and must be maintained for stem cell survival and correct cell differentiation.External GF injection into joint cavities is not sufficient, as proteins quickly wash away in synovial fluid, and recurrent supplementations are impractical in clinical practice. Therefore, the encapsulation of GFs in biodegradable microspheres (MSs) and their sustained release within biomaterial scaffolds is a much more feasible approach for the generation and maintenance of GF gradients.Pouya Rezai's lab has vast expertise on the generation of MSs by microfluidics. Microfluidics studies the behaviour of fluids at the microscale and requires specialized knowledge, expertise and expensive equipment that very few laboratories have access to. Harnessing microfluidics for the synthesis of microparticles allows full control in microparticle properties (i.e diameter size, number of layers, high efficiency of molecule encapsulation, etc.) that would be unattainable otherwise.Eileen Gentleman's lab has developed a photocrossinkable hyaluronan-based biomaterial for the repair of damaged cartilage. We have demonstrated that this biomaterial is injectable and can sustain cartilage cells, postulating it as an ideal system for clinical in situ tissue engineering of cartilage.Here, we propose the use of poly(lactic-co-glycolic acid) (PLGA), a FDA-approved biodegradable polymer, to synthesise different MS formulations for GF sustained release. MSs can be used in concert with biomaterials and stem cells for the regeneration of cartilage in the context of OA. Thus, we will overcome current limitations on cartilage regenerative medicine and bring about the next generation of tissue engineering approaches.To achieve that, we will first generate MSs of different sizes (ranging from nanometric to micrometric diameters) and different layers (mono or bilayered MSs). Then, we will assess which formulations are suitable to be used along with biomaterials and with cells. Lastly, we will encapsulate model proteins in relevant MS formulations to investigate their degradation and release kinetics under physiological conditions, and subsequently, to figure what formulations are more adequate to replicate cartilage natural GF gradients.The result of this multidisciplinary project is the fulfilment of the current limitation in tissue engineering. This opportunity will bring us closer to a successful therapy in the context of regenerative medicine. The project not only brings in concert two completely different disciplines (tissue engineering, from Gentleman lab, and microfluidics and microparticle generation, from Rezai lab) into the development of a novel, revolutionizing and promising approach, but also sets the way for a new fruitful collaboration between Canadian and UK-based laboratories.