3D Printed Biomimetic Bioglass-Gradient Matrices for ACL Reconstruction

项目总结： 前十字韧带(ACL)损伤非常常见，特别是在有积极生活方式的人中。 根据美国整形外科学会运动医学会最近的一份报告，超过15万美国人 每年遭受前交叉韧带损伤，估计造成5亿美元的损失。当前的治疗策略 例如自体移植物和同种异体移植物由于供体部位发病率和免疫排斥风险而受到高度限制， 分别进行了分析。合成移植物是一种很有前途的替代品，但存在包括移植物质量差在内的主要缺点。 移植物-组织界面的整合和机械失配导致次优愈合，重复 移植物失败，临床结果不佳。移植物-组织界面的组织工程具有巨大的潜力 目的：提高人工前交叉韧带重建手术的临床疗效。的中心假说 拟议研究是结合仿生生物玻璃梯度，该梯度在成分、机械上 在生物上模拟天然的前交叉韧带连接将通过促进细胞来改善合成移植物的整合 迁移、成分导向的细胞分化和从头形成基质。目标1中的研究 提案将使用基于3D拉曼光谱映射的方法来识别胶原蛋白：矿物 兔前交叉韧带天然骨-纤维软骨-韧带界面的成分梯度。拉曼 光谱数据将被转换为STL文件，并用于3D打印基于胶原的仿生生物玻璃梯度 包含复制原生前交叉韧带成分梯度的基质(BioGIM)。AIM 2中的研究 该提案的重点将是通过模仿 生物凝胶的成分和力学性能与天然前交叉韧带吻合的成分和力学性能相当。第一，生物玻璃颗粒 尺寸将被调制(35纳米和10微米)，并产生最优的生物玻璃颗粒尺寸，以产生组成- 将确定定向差异化。其次，将采用和调制京尼平交联剂 具有最优颗粒大小的BioGIM可聚集在天然凹槽的机械性能上，并 从而进一步增强物质导向的差异化。细胞分化研究将在 不同的培养基条件(正常生长介质和成骨介质)评价其生物活性 在没有添加外部因素的情况下，生物GIM的性能。提案目标3中的研究将调查矩阵 重组BioGIM，产生符合目的的材料定向分化2.BioGIM功能将 通过细胞合成的组织特异性基质的确认和定型进行评估，评估 Bio GIMs培养后的生物力学特性及细胞分布和新生基质的评价 通过拉曼光谱和常规的生物检测方法检测成分。总体而言，预期结果 建议的研究是提供具有机械能力的具有材料导向的MSC分化的Bio-Gim 和基质重组，然后可以整合到合成移植物上，以改善前交叉韧带的结果 在未来的小动物模型研究中的手术。最后，将拉曼光谱技术与3-甲基-3-甲基-4-甲基-4-甲基-4-甲基-3-甲基-3-甲基-3-吡咯烷酮的方法相结合 用于开发仿生梯度的3D打印不限于ACL，并且很容易应用于 其他重要关节的再生，如肩袖肌腱和关节软骨。

Project Summary: Anterior cruciate ligament (ACL) injuries are highly common especially in individuals with active lifestyles. Based on a recent report from American Orthopedic Society for Sports Medicine, more than 150,000 Americans suffer from ACL injuries each year costing an estimated 500 million US dollars. Current treatment strategies such as autografts and allografts are highly limited due to donor site morbidity and risk of immune rejection, respectively. Synthetic grafts are a promising alternative but suffer from major drawbacks that include poor graft integration and mechanical mismatch at the graft-tissue interface resulting in suboptimal healing, repetitive graft failure and poor clinical outcome. Tissue engineering of the graft-tissue interface has significant potential to improve the clinical outcome of ACL reconstruction surgeries using synthetic grafts. The central hypothesis of the proposed study is that incorporation of a biomimetic bioglass gradient that compositionally, mechanically and biologically mimics the native ACL enthesis will improve synthetic graft integration by promoting cell migration, composition-directed cell differentiation and de novo matrix formation. Studies in Aim 1 of the proposal will employ a 3D Raman spectral mapping based approach to discern the collagen:mineral compositional gradient at the native bone-fibrocartilage-ligament interface of the rabbit ACL. The Raman spectral data will be converted into STL files and used to 3D print collagen-based biomimetic bioglass gradient incorporated matrices (BioGIMs) that replicate the compositional gradient of the native ACL. Studies in Aim 2 of the proposal will focus upon achieving material-directed differentiation of MSCs by mimicking the compositional and mechanical properties of the BioGIMs to that of native ACL enthesis. First, bioglass particle size will be modulated (35 nm and 10 µm) and the most optimal bioglass particle size that yields composition- directed differentiation will be identified. Second, genipin crosslinking will be employed and modulated for BioGIMs with the most optimal particle size to converge upon the mechanical properties of native enthesis and thus further enhance material-directed differentiation. Cellular differentiation studies will be performed in different culture medium conditions (normal growth medium, and osteogenic medium) to assess the bioactivity of the BioGIMs without the addition of external factors. Studies in Aim 3 of the proposal will investigate matrix reorganization on BioGIMs that yield material-directed differentiation as per Aim 2. BioGIM functionality will be assessed via confirmation and typification of cell-synthesized tissue-specific matrix, evaluation of biomechanical properties of BioGIMs after culture and assessment of cellular distribution and de novo matrix components by Raman spectroscopy and conventional biological assay methods. Overall, the expected outcomes of the proposed study is to deliver a mechanically competent Bio-GIM with material-directed MSC differentiation and matrix reorganization which can then be integrated onto synthetic grafts to improve the outcome of ACL surgeries in future small-animal models studies. Finally, this combined approach of Raman spectroscopy and 3- D printing for the development of biomimetic gradients is not limited to ACL and is easily applicable for the regeneration of other vital joints such as the rotator cuff tendon and articular cartilage.