3D Printed Biomimetic Bioglass-Gradient Matrices for ACL Reconstruction

项目摘要： 前交叉韧带（ACL）损伤非常常见，尤其是在生活方式活跃的人群中。 根据美国运动医学骨科学会最近的一份报告， 每年遭受ACL损伤的损失估计为5亿美元。当前治疗策略 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的风险而受到高度限制， 分别合成移植物是一种有前途的替代品，但存在主要缺点，包括移植物质量差， 移植物-组织界面的整合和机械不匹配导致愈合不佳，重复 移植失败和临床结果差。移植物-组织界面的组织工程具有巨大的潜力 改善使用合成移植物的ACL重建手术的临床结果。的中心假设 拟议的研究是结合仿生生物玻璃梯度，在成分、机械方面 并且生物学上模拟天然ACL附着点将通过促进细胞增殖来改善合成移植物整合， 迁移、组成导向的细胞分化和从头基质形成。目标1的研究 一项提案将采用基于3D拉曼光谱映射的方法来辨别胶原：矿物质 在兔ACL的天然骨-纤维软骨-韧带界面处的组成梯度。该拉曼 光谱数据将被转换成STL文件，并用于3D打印基于胶原蛋白的仿生玻璃梯度 在一个实施方案中，所述方法包括使用复制天然ACL的组成梯度的掺入基质（BioGIM）。研究目的2 该提案的重点是通过模拟细胞的分化来实现MSC的材料定向分化。 BioGIM的成分和机械性能与天然ACL附着点的成分和机械性能相比。第一，玻璃粒子 尺寸将被调制（35 nm和10 µm），最佳的玻璃颗粒尺寸产生的组合物- 将识别定向分化。第二，将采用京尼平交联，并调节其用于 具有最佳粒度的BioGIM，以满足天然附着点的机械性能， 从而进一步增强材料定向分化。细胞分化研究将在 不同的培养基条件（正常生长培养基和成骨培养基），以评估生物活性 在没有外部因素的情况下。提案目标3中的研究将调查矩阵 在BioGIM上进行重组，根据目标2产生材料定向分化。BioGIM功能将 通过细胞合成的组织特异性基质的确认和典型化进行评估， 培养后BioGIM的生物力学特性以及细胞分布和从头基质评估 通过拉曼光谱法和常规生物测定方法测定组分。总体而言，预期成果 提出的研究的目的是提供一个机械能力的生物GIM与材料定向MSC分化 和基质重组，然后可以整合到合成移植物上，以改善ACL的结果 在未来的小动物模型研究中进行手术。最后，这种结合拉曼光谱和3- 用于仿生梯度开发的D打印不限于ACL，并且容易适用于 其他重要关节如肩袖肌腱和关节软骨的再生。

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.