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，会导致炎症、关节软骨表面的退化（使关节能够顺利运动），并最终导致慢性疼痛和运动障碍。目前的骨关节炎治疗旨在减轻症状，但迄今为止，还没有有效的治疗方法来治愈或预防这种情况。最近，研究人员诉诸组织工程试图改造退化的组织，但尚未成功实现天然软骨的再生，这表明当前的策略错过了相关步骤。大多数软骨组织工程方法侧重于选择干细胞群和开发用作组织再生3D支架的生物材料，但较少关注为细胞提供诱导线索以正确改造组织。这一事实对于具有复杂结构的组织尤其重要，例如软骨，其中存在不对称的生长因子 (GF) 梯度，必须维持生长因子 (GF) 才能保证干细胞存活和正确的细胞分化。将外部 GF 注射到关节腔中是不够的，因为蛋白质会很快在滑液中被冲走，并且反复补充在临床实践中是不切实际的。因此，将 GF 封装在可生物降解的微球 (MS) 中并在生物材料支架内持续释放，是生成和维持 GF 梯度的更可行的方法。Pouya Rezai 的实验室在微流体生成 MS 方面拥有丰富的专业知识。微流体学研究微尺度流体的行为，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。利用微流体合成微粒可以完全控制微粒的特性（即直径大小、层数、分子封装的高效率等），这是其他方法无法实现的。Eileen Gentleman 的实验室开发了一种可光交联的透明质酸生物材料，用于修复受损软骨。我们已经证明这种生物材料是可注射的，并且可以维持软骨细胞，将其视为临床软骨原位组织工程的理想系统。在这里，我们建议使用聚乳酸-乙醇酸共聚物（PLGA）（一种 FDA 批准的生物可降解聚合物）来合成不同的 MS 制剂，用于 GF 缓释。 MS 可以与生物材料和干细胞配合使用，用于 OA 中的软骨再生。因此，我们将克服目前软骨再生医学的局限性，并带来下一代组织工程方法。为了实现这一目标，我们将首先生成不同尺寸（从纳米到微米直径）和不同层（单层或双层MS）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，我们将把模型蛋白封装在相关的 MS 配方中，以研究它们在生理条件下的降解和释放动力学，随后找出哪些配方更适合复制软骨天然 GF 梯度。这个多学科项目的结果是满足了当前组织工程的局限性。这个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 实验室的组织工程和来自 Rezai 实验室的微流体和微粒生成）结合起来，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新的富有成效的合作奠定了基础。

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.