Optimisation of Plasma Coatings for Bone-Replacement Scaffolds Using Mesenchymal Stem Cells.

肌肉骨骼疾病是世界各地发病率的主要原因，与疼痛、行动不便、畸形以及在某些情况下死亡有关。疾病患病率随着年龄的增长而增加。在未来几年，影响骨骼疾病的发病率将上升，造成巨大的医疗保健和社会经济负担。目前的治疗通常局限于疼痛管理，随后进行终末期全关节置换术。基于细胞的治疗方法在骨科中是一种很有吸引力的生物选择，因为它们可以通过利用间充质干细胞（MSCs）分化成骨和软骨的内在能力来提供骨骼组织功能的持久恢复，通常与仿生支持材料相结合，以实现3D重建。虽然已经研究了许多材料来增强骨修复，但没有一种材料像磷酸钙那样被广泛应用于临床，最突出的是羟基磷灰石（HA）。据报道，这种材料具有骨导电性（支持成骨细胞和新骨沉积）和潜在的骨诱导性（刺激成骨细胞向成骨细胞分化）。这种对HA支架或HA包被植入物的细胞反应已经在体外和体内用MSCs和终分化成骨细胞证明了。间充质干细胞是一种多能祖细胞群，存在于包括骨髓在内的人体许多组织中；它们可以被分离并诱导分化成许多不同的细胞类型，包括成骨细胞。透明质酸支架/涂层生产中固有的可变性导致最终材料特性的巨大变化。这一点尤其重要，因为细胞对这些材料表面的反应已被证明依赖于多种因素。这些包括表面形貌、（微观）结构和成分，它们在某种程度上是由初始生产过程变量（如时间、温度、进料速度）决定的。虽然计算地形和固体自由形状制造的最新发展使生成控制总体结构的组件成为可能，允许针对患者的设计，但细胞反应（例如附着、增殖、分化）的最佳条件的确定仍然依赖于低效、耗时和昂贵的分析，通常使用“一次一个因素”（OFAT）方法。这个过程没有考虑在所选过程中重要的因素之间的相互作用，也没有考虑这些因素变量对细胞反应的影响。本研究的目的是实施实验统计设计（DOE）技术，使用多变量分析来评估多个输入参数对间充质干细胞对HA的成骨反应的影响；因此，避免了由于传统OFAT方法的因素间相互作用而导致的混淆结果。在这种情况下，实验方法将围绕矫形植入装置的HA等离子喷涂实施，这是一个复杂的生产过程，已知可以增强细胞反应和骨骼生长，但与影响临床效果的输入参数有关，存在很大程度的差异。具体来说，我们提出了一个四因素DOE筛选HA等离子涂层过程输入参数，并结合成骨能力的测定。这将允许快速筛选多个HA材料输出参数及其在细胞水平上的影响。主要的透明质酸制剂将被用于进一步分析成骨诱导和作用机制。该提案将同样依赖于材料科学（利兹大学，DePuy），工艺工程，DOE筛选（DePuy）， MSC生物学，细胞分化和细胞-生物材料相互作用（约克大学）的跨机构专业知识，使其成为一种真正的跨学科方法，以加速优化临床应用的仿生支架开发。

Musculoskeletal disease is a leading cause of morbidity across the world, associated with pain, immobility, deformities and in some cases, death. Disease prevalence increases with age. In the coming years, the incidence of disorders affecting the skeleton will rise, causing huge healthcare and socioeconomic burden. Current treatments are typically restricted to pain management followed by end-stage total joint replacement. Cell-based therapies are an appealing biological option in orthopaedics, as they may provide long-lasting restoration of skeletal tissue function by exploiting the intrinsic capacity of mesenchymal stem cells (MSCs) to differentiate into bone and cartilage, often in association with a biomimetic support material to enable 3D reconstruction.Although many materials have been investigated to enhance bone repair, none have seen as much clinical use as the calcium phosphates, the most prominent being hydroxyapatite (HA). This material has been reported as both osteoconductive (support osteoprogenitors and new bone deposition) and potentially osteoinductive (stimulate differentiation of osteoprogenitors towards bone-forming osteoblasts). This cellular response to HA scaffolds or HA-coated implants has been demonstrated in vitro and in vivo using MSCs and terminally differentiated osteoblasts. MSCs are a multipotent progenitor cell population found in many tissues throughout the body including bone marrow; they can be isolated and induced to differentiate into many different cell types, including osteoblasts.The inherent variability in HA scaffold/coating production leads to large variation in the final material characteristics. This is particularly important as cell responses to these material surfaces have been demonstrated to rely on multiple factors. These include surface topography, (micro)structure and composition, which are in some part determined by initial production process variables (such as time, temperature, feed rate). Whilst recent developments in computational topography and solid free-form fabrication make it possible to generate components with control over gross architecture, allowing patient-specific design, the determination of the optimal conditions for cellular responses (e.g. attachment, proliferation, differentiation) still relies on inefficient, time-consuming and expensive assays commonly using a 'One Factor At a Time' (OFAT) approach. This process does not account for the significant between-factor interactions within the chosen process, nor the influence of these factor variables on the cellular response.The purpose of this study is to implement statistical Design of Experiments (DOE) techniques using multivariate analysis to assess multiple input parameter effects on osteogenic responses of MSCs to HA in parallel experimental runs; therefore avoiding confounding results due to between-factor interactions of traditional OFAT approaches. In this instance the experimental methodology will be implemented around the HA plasma spraying of orthopaedic implant devices, a complex production process known to enhance cellular responses and bone growth, but have a large degree of variation in relation to input parameters that will influence clinical effect.Specifically, we propose a 4-factor DOE screening of HA plasma-coating process input parameters in conjunction with assays of osteogenic capacity. This will allow rapid screening of multiple HA material output parameters and their effects at a cellular level. Leading HA formulations will be taken forward for further analysis of osteogenic induction and mechanism of action. This proposal will rely equally on cross-institutional expertise in materials science (Leeds, DePuy), process engineering, DOE screening (DePuy), MSC biology, cell differentiation and cell-biomaterial interaction (York), making it a truly interdisciplinary approach to expedite optimised biomimetic scaffold development for clinical applications.