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，其携带炎症、允许平滑关节运动的关节软骨表面的退化，并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，但迄今为止，还没有有效的治疗方法来治愈或预防这种疾病。近年来，研究人员采用组织工程的方法试图对退化的组织进行改革，但还没有成功地实现天然软骨的再生，这表明目前的策略错过了相关的步骤。大多数软骨组织工程方法集中在选择干细胞群和开发作为组织再生3D支架的生物材料，但较少关注给予细胞正确改革组织的诱导线索。这一事实在具有复杂结构的组织中特别重要，例如软骨，其中存在不对称的生长因子（GF）梯度，并且必须维持生长因子梯度以使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，因为蛋白质在滑液中很快被冲走，并且复发性关节炎在临床实践中是不切实际的。因此，将GF封装在生物可降解微球（MS）中并在生物材料支架内持续释放是一种更可行的方法，用于生成和维持GF梯度。Pouya Rezai的实验室在通过微流体生成MS方面拥有丰富的专业知识。微流体学在微观尺度上研究流体的行为，需要专业知识，专业知识和昂贵的设备，很少有实验室能够获得。利用微流体技术合成微粒允许完全控制微粒性质（即直径大小、层数、分子包封的高效率等）。Eileen Gentleman的实验室已经开发出一种可光交联的透明质酸生物材料，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，可以维持软骨细胞，假定它作为一个理想的系统，为临床原位组织工程的软骨。在这里，我们建议使用聚（乳酸-共-乙醇酸）（PLGA），FDA批准的可生物降解的聚合物，合成不同的MS配方GF缓释。MS可以与生物材料和干细胞一起用于OA背景下的软骨再生。因此，我们将克服目前软骨再生医学的局限性，并带来下一代的组织工程方法。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沿着使用。最后，我们将在相关的MS配方封装模型蛋白质，以研究其降解和释放动力学在生理条件下，随后，图什么配方是更适合复制软骨天然GF gradients. Results的结果，这个多学科的项目是在组织工程的当前限制的实现。这个机会将使我们更接近再生医学背景下的成功疗法。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）整合到一种新颖的，革命性的和有前途的方法的开发中，而且还为加拿大和英国实验室之间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.