3D Printed Biomimetic Bioglass-Gradient Matrices for ACL Reconstruction

项目摘要 前交叉韧带（ACL）是连接股骨和胫骨的结缔组织带， 在稳定膝关节方面起着至关重要的作用。ACL断裂是非常严重的肌肉骨骼损伤， 自愈能力，并且通常需要手术干预来恢复正常的关节功能。超过175，000人 ACL重建（ACLR）手术在美国每年进行超过170亿美元的寿命 医疗费用。虽然有可行的移植物选择，但10%的病例报告移植物失败。此外，1 在5名青少年运动员中，ACLR的长期后果包括受影响者的流动性有限 高达80%的患者出现关节炎和早期骨关节炎。此外，治疗结果的性别差异 据报道，与男性相比，女性更容易发生ACL再撕裂。集成性差 骨-韧带界面处的ACL移植物（即，据报道，是次优移植物的主要原因 性能和高移植失败率。移植物-组织界面的组织工程是一种很有前途的选择 解决方案，以克服现有移植物选择的局限性，并改善ACLR后的临床结局。在这 reproducing the heterogeneity in composition，architecture and cell phenotype of the native enthesis post ACLR被认为对于改善移植物-骨结合、促进可靠的载荷转移， 并恢复ACL的长期正常功能。为了实现这一目标，我们建议开发一种新的连续 基于梯度的构建，其在单个平台上组合了多种生物物理和生物化学线索，以引导 ACL附着点的功能性再生。我们假设，组织特异性线索的空间呈现将 促进细胞迁移，刺激多系分化和基质重塑，改善移植物整合， 并使ACLR后的附着点功能性愈合。利用R15项目第一阶段的支持，我们 提供了一种连续的仿生生物玻璃梯度整合胶原蛋白基质（BioGIM）， 天然附着点矿化梯度的组成。这次更新申请有三个具体目标。 在Aim 1研究中，3D打印将与4D打印方案中的磁性对齐方法相结合， 在BioGIMs中实现胶原纤维定向和> 90%的胶原排列指数。排列的胶原蛋白可以提供 引导BioGIM纯胶原侧韧带分化的地形线索。在Aim 2研究中， 将优化TGF-β1剂量和递送策略，以在血管内皮细胞上实现可靠的纤维软骨基质形成。 BioGIM接口。此外，多谱系细胞分化和基质重塑沿着BioGIM将 进行评估。在Aim 3研究中，4D打印的BioGIM皮瓣将被整合到肌腱自体移植物的末端， 植入兔子体内不含BioGIM的自体移植物将作为临床对照，未手术的健康家兔将 作为阳性对照。植入后3周，测量的结局将包括 骨-韧带界面、从头基质重塑的典型化和炎症反应。成功 完成所提出的研究对于将新型BioGIM皮瓣用于ACL重建的临床转化至关重要。

Project Summary Anterior cruciate ligament (ACL) is a band of connective tissue that connects the femur and the tibia and plays a crucial role in stabilizing the knee joint. ACL ruptures are very serious musculoskeletal injuries with poor self-healing capability and often mandate surgical intervention to restore normal joint function. Over 175,000 ACL reconstruction (ACLR) surgeries are performed in the US each year with over 17 billion dollars in lifetime healthcare costs. While viable graft choices are available, graft failure is reported in 10% of cases. In addition, 1 in 5 teenage athletes reinjure their ACL. Long-term consequences of ACLR include limited mobility of the affected joint and early onset of osteoarthritis in up to 80% of patients. Further, sex-based differences in healing outcomes have been reported with females more susceptible to ACL re-tear compared to males. Poor integration of the ACL graft at the bone-ligament interface (i.e., enthesis) is reported to be the main reason for suboptimal graft performance and high graft failure rates. Tissue engineering of the graft-tissue interface is a promising alternative solution to overcome the limitations of existing graft choices and improve the clinical outcome post ACLR. In this realm, recreating the heterogeneity in composition, architecture and cell phenotype of the native enthesis post ACLR is deemed to be critically important for improving graft-bone integration, facilitating reliable load transfer, and restoring long-term normal ACL function. Towards this goal, we propose to develop a novel continuous gradient-based construct that combines multiple biophysical and biochemical cues on a single platform to guide functional regeneration of the ACL enthesis. We hypothesize that spatial presentation of tissue-specific cues will promote cell migration, stimulate multilineage differentiation and matrix remodeling, improve graft integration, and enable functional healing of enthesis post ACLR. Using support from the first period of this R15 project, we delivered a continuous biomimetic Bioglass-gradient integrated collagen matrix (BioGIM) that emulates the composition of the mineralized gradient of the native enthesis. This renewal application has three specific aims. In Aim 1 studies, 3D printing will be combined with a magnetic alignment approach in a 4D printing scheme to orient collagen fibers and achieve > 90% collagen alignment index in BioGIMs. Aligned collagen can provide topographical cues to guide ligamentous differentiation on pure collagen side of the BioGIM. In Aim 2 studies, TGF-β1 dosage and delivery strategy will be optimized to attain reliable fibrocartilaginous matrix formation on the BioGIM interface. In addition, multi-lineage cell differentiation and matrix remodeling along the BioGIM will be assessed. In Aim 3 studies, 4D printed BioGIM flaps will be integrated onto ends of a tendon autograft and implanted in rabbits. Autograft without BioGIM will serve as clinical control and unoperated healthy rabbits will be the positive control. Three weeks post implantation, outcomes measured will include mechanical strength of the bone-ligament interface, typification of de novo matrix remodeling, and inflammatory response. Successful completion of proposed studies is critical for clinical translation of the novel BioGIM flap for ACL reconstruction.