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

描述(申请人提供)：韧带断裂后的愈合和重建后的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，这些事件形成了新韧带，其特征比天然组织更像疤痕。修复过程从几个月到几年不等，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。这些缺陷很可能是关节过早退化和骨关节炎的原因。我们实验室的初步数据有力地表明，通过调节炎症过程，韧带愈合可以变得更具再生性。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，促进再生愈合。我们建议研究韧带愈合(大鼠MCL模型)和韧带移植愈合(大鼠前交叉韧带模型)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。我们将使用白介素4(IL-4)、白介素1受体拮抗剂(IL-1ra)和白介素17中和抗体(1-IL-17)来调节炎症。通过这些，我们期望在韧带和韧带移植物中诱导更多的再生愈合。临床翻译需要一个本地化的传递系统。因此，我们建议开发两种方法，一是从微球中“持续”释放炎症介质，在重建之前可以将其注射到断裂韧带的撕裂端或韧带移植物中；二是从微球中释放蛋白质，这种释放受炎症引起的局部pH变化的“控制”。治疗将进行优化(通过剂量和时间)，结果将在两个愈合时间进行比较。评估参数包括：伤口大小、细胞外基质成分、细胞外基质微观形态和组织、力学行为、细胞密度和细胞类型。本课程将探讨与修复/再生相关的信号因子的变化。以上参数越接近完整的韧带，越能被认为是可再生的。如果成功，这项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“疤痕”形成中的作用的更好的理解，2)高度发展的持续微球递送系统，以局部调节愈合3)复杂的pH开发的微球递送系统，以局部控制愈合，4)改进的诱导再生韧带愈合的方法，以及5)诱导再生的ALC移植物愈合的先进技术。其中的每一个都将具有重要的意义，并且每个都将具有比本文探讨的那些更广泛的应用。 公共卫生相关性：韧带损伤经常发生，但愈合存在问题。前交叉韧带的愈合潜力相对较小，必须重建才能恢复功能，但重建后的前交叉韧带移植物通常会延长，其强度可下降约50%。即使韧带愈合良好(例如MCL)，在受伤数年后，产生的“疤痕样”组织在机械上是劣质的，在成分上也是异常的。而且，这些“愈合”的韧带通常比天然组织长。因此，韧带移植愈合和韧带愈合经常导致关节松弛、关节不稳定和关节运动异常，这与骨关节炎的早期发病有关。因此，诱导韧带或韧带移植物再生愈合的新方法将是非常重要的。开发这些方法(通过可注射微球上的生物矿物涂层的炎症介质的受控输送)是这一应用的目标。

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.