Modulation of the Immune System to Improve Ligament/Ligament Graft Healing

描述（由申请人提供）：断裂后韧带愈合和重建后韧带移植仍然是一个巨大的挑战。愈合涉及一系列复杂、协调的事件，这些事件形成新韧带，其特征比天然组织更像疤痕。修复过程从数月到数年，受伤的韧带永远不会完全恢复其原始的机械性能。重建的膝关节通常不太稳定，无法恢复正常的关节运动学。这些缺陷可能是过早关节退化和骨关节炎的原因。 我们实验室的初步数据强烈表明，韧带愈合可以通过调节炎症过程变得更加再生。初步数据还表明，微球可以被设计成释放蛋白质，这些蛋白质将在空间和时间上调节炎症以实现再生愈合。我们建议调查韧带愈合（大鼠MCL模型）和韧带移植愈合（大鼠ACL模型）过程中的炎症过程，以研究它们在瘢痕形成和/或组织再生中的作用。我们将用白细胞介素-4（IL-4）、白细胞介素-1受体拮抗剂（IL-1 Ra）和白细胞介素-17中和抗体（1-IL-17）调节炎症。通过这些，我们希望在韧带和韧带移植物中诱导更多的再生愈合。临床翻译需要本地化的交付系统。因此，我们建议开发两种方法，第一，从微球中“持续”释放炎症介质，可以在重建前注射到断裂韧带的撕裂端或韧带移植物中，第二，从微球中释放蛋白质，由炎症引起的局部pH值变化“控制”。将优化治疗（通过剂量和时间），并在两个愈合时间比较结果。评价参数为：伤口尺寸、ECM成分、ECM微观结构形态和组织、机械性能、细胞结构和细胞类型。将探讨与愈合/再生相关的信号传导因子的变化。上述参数越接近完整的韧带，则认为再生愈合越多。 如果成功，本研究将产生五个非常重要的结果：1）对炎症过程及其在韧带“瘢痕”形成中的作用的更好理解，2）高度发展的局部调节愈合的持续微球递送系统，3）复杂的pH发展的局部控制愈合的微球递送系统，4）诱导再生韧带愈合的改进方法，和5）诱导再生ALC移植物愈合的先进技术。其中每一个都将具有重大意义，每一个都将具有比本文所探讨的更广泛的应用。 公共卫生相关性：韧带损伤经常发生，但愈合是有问题的。ACL愈合潜力相对较小，必须重建以恢复功能，但ACL移植物通常会延长，重建后其强度可能下降约50%。即使韧带愈合良好（例如MCL），受伤后数年，产生的“疤痕样”组织在机械上也是较差的，并且在成分上是异常的。而且，这些“愈合”的韧带通常比天然组织长。因此，韧带移植物愈合和韧带愈合经常导致松弛、关节不稳定和异常关节运动，这与骨关节炎的早期发作相关。新的方法来诱导韧带或韧带移植物的再生愈合将是非常重要的。开发这些方法（通过可注射微球上生物矿物涂层的炎症介质的受控递送）是本申请的目标。

DESCRIPTION (provided by applicant): Healing of ligaments after rupture and ligament grafts after reconstruction remains a great challenge. Healing involves a complex, coordinated series of events that form a neo-ligament which is more scar-like in character than the native tissue. The repair process extends from months to years and the injured ligament never fully recovers its original mechanical properties. Reconstructed knees are often less stable and fail to restore normal joint kinematics. These deficiencies are likely the cause of premature joint degeneration and osteoarthritis. Preliminary data from our lab strongly suggest that ligament healing can become much more regenerative by modulation of the inflammatory processes. Preliminary data also suggest that microspheres can be designed to release proteins that will spatially and temporally modulate inflammation for regenerative healing. We propose to investigate inflammatory processes during ligament healing (rat MCL model) and during ligament graft healing (rat ACL model) to study their role in scar formation and/or tissue regeneration. We will modulate inflammation with interleukin-4 (IL-4), interleukin-1 receptor antagonist (IL-1Ra) and neutralizing antibody for interleukin-17 (1-IL-17). Through these, we expect to induce more regenerative healing in ligaments and ligament grafts. Clinical translation requires a localized delivery system. We therefore propose to develop two methods, first, a "sustained" release of inflammatory mediators from microspheres that can be injected into the torn ends of a ruptured ligament or into a ligament graft prior to reconstruction, and second, protein release from microspheres that is "controlled" by local pH changes due to inflammation. Treatments will be optimized (via dosage and time) and outcomes will be compared at two healing times. Parameters of evaluation will be: size of wound, ECM composition, ECM microstructural morphology and organization, mechanical behavior, cellularity, and cell types. Changes in signaling factors relevant to healing/regeneration will be explored. The closer to an intact ligament the above parameters are, the more healing will be considered regenerative. If successful, five very significant outcomes will arise from this study: 1) an improved understanding of inflammatory processes and their role in ligament "scar" formation, 2) a highly developed sustained microsphere delivery system to locally modulate healing 3) a sophisticated pH-developed microsphere delivery system to locally control healing, 4) an improved method to induce regenerative ligament healing, and 5) an advanced technique to induce regenerative ALC graft healing. Each one of these would be of great significance and each would have much broader applications than those explored herein. PUBLIC HEALTH RELEVANCE: Ligament injuries frequently occur, but healing is problematic. The ACL, with relatively little healing potential, must be reconstructed to recover function, but ACL grafts usually lengthen and their strength can drop by ~50% after remodeling. Even with ligaments that heal well (e.g. the MCL) the resulting "scar-like" tissue is mechanically inferior and compositionally abnormal years after injury. And, these "healed" ligaments are usually longer than the native tissue. Therefore, ligament graft healing and ligament healing frequently result in laxity, joint instability, and abnormal joint motion, which correlate with an early onset of osteoarthritis. New methods to induce regenerative healing of ligaments or ligament grafts would then be very significant. Developing these methods (via controlled delivery of inflammatory mediators from biomineral coatings on injectable microspheres) is the goal of this application.