Allografts and Gene Therapy in Flexor Tendon Tissue Engineering

描述（由申请人提供）：术后粘连是自体屈肌腱成形术的主要并发症，但仍未解决。先前的研究表明，作为手术初始炎症反应的一部分，粘连至少在一定程度上是由TGF-2向活肌腱或自体移植物肌腱细胞发出的信号调节的。有趣的是，虽然多种病理纤维化与TGF-21诱导纤溶酶原激活物抑制剂-1 （PAI-1）相关，可阻止MMPs的激活，但TGF-23在皮肤伤口愈合中具有抗疤痕作用。然而，TGF-2同种异构体调节肌腱瘢痕形成的机制仍然知之甚少，防止粘连的生物治疗方法也不可用。为了解决这些问题，我们建立了一种新的小鼠屈肌腱成形术模型，该模型包括在后爪远端指长屈肌腱（FDL）中植入骨间活体自体移植物或冻干同种异体移植物。在这个模型中，自体移植物的跖趾关节屈曲范围比同种异体移植物低，这与术后14天和28天更多的纤维化瘢痕有关。Tgfb1的早期表达与Mmp9的抑制和瘢痕形成有关，而Gdf5的晚期表达与Mmp2和Mmp14的延迟上调以及随后MTP关节屈曲的改善有关。此外，通过冻干同种异体移植物局部和瞬时传递Gdf5基因可显著减少粘连并恢复屈肌腱成形术后的MTP关节屈曲。最近使用体外瘢痕组织细胞种子胶原凝胶模型的数据表明，GDF-5抑制TGF-21诱导的凝胶收缩并挽救MMP基因表达。因此，本研究旨在验证以下假设：1)对内源性TGF-21信号无反应的脱细胞异体移植物是屈肌腱成形术治疗中有效的递送支架；2)利用rAAV/同种异体移植物介导的Gdf5或Tgfb3的过表达抑制TGF-21/PAI-1诱导的MMPs抑制，可减少屈肌腱成形术中的瘢痕和粘连。本应用程序提出了一系列的补充研究来测试这些假设，并开发了屈肌腱粘连的组织工程解决方案。特异性目标1旨在通过确定活体移植物肌腱细胞和内源性TGF-2信号在屈肌腱成形术粘连中的作用，正式挑战当前屈肌腱成形术中倾向于使用活体自体移植物的范式。为了开发一种机械驱动的屈肌腱粘连组织工程解决方案，Specific Aim 2旨在研究TGF-2和GDF-5对mmp介导的体外瘢痕组织模型重塑的影响。最后，Specific Aim 3将研究负载raav的FDL肌腱异体移植物过度表达Tgfb3和Gdf5如何影响小鼠屈肌腱成形术模型中的粘连。我们组建了一个经验丰富的多学科团队来进行这些研究，以测试屈肌腱组织工程中创新的机械驱动范式，具有重要的临床意义。

DESCRIPTION (provided by applicant): Postoperative adhesion is a major debilitating yet still unsolved complication of autograft flexor tendoplasty. Previous studies have shown that adhesions are modulated, at least in part, by TGF-2 signaling to the live tendon or autograft tenocytes as part of the initial inflammatory response to the surgery. Interestingly, while fibrosis in a variety of pathologies is associated with TGF-21 induction of plasminogen activator inhibitor-1 (PAI-1) that prevents activation of MMPs, TGF-23 has anti-scarring effects in cutaneous wound healing. However, the mechanisms by which TGF-2 isoforms modulate tendon scarring remain poorly understood, and biologic therapeutics to prevent adhesions are not available. To address these questions, a novel mouse model of flexor tendoplasty that involves the implantation of intercalary live autograft or freeze-dried allograft is implanted in the distal flexor digitorum longus (FDL) tendon of the hind paws was developed. In this model, the range of metatarsophalangeal (MTP) joint flexion was lower with autografts than allografts and this was associated with more fibrotic scarring at 14 and 28 days post-surgery. Early expression of Tgfb1 was associated with suppression of Mmp9 and scar formation, while late expression of Gdf5 was associated with delayed upregulation of Mmp2 and Mmp14, and subsequent improvement in the MTP joint flexion. Furthermore, local and transient Gdf5 gene delivery via freeze-dried allografts significantly reduced adhesions and restored the MTP joint flexion following flexor tendoplasty. Recent data using an in vitro tenocyte-seeded collagen gel model of scar tissue suggest that GDF-5 suppresses TGF-21 induced contraction of the gel and rescues MMP gene expression. Therefore, this proposal seeks to test the following hypotheses: 1) Acellular allografts, which cannot respond to endogenous TGF-21 signals, are effective delivery scaffolds for therapeutics in flexor tendoplasty; and 2) Inhibition of TGF-21/PAI-1 induced suppression of MMPs using rAAV/allograft-mediated over expression of Gdf5 or Tgfb3 reduces scarring and adhesions in flexor tendoplasty. This application proposes a series of complimentary studies to test these hypotheses and develop a tissue engineering solution for flexor tendon adhesions. Specific Aim 1 seeks to formally challenge the current paradigm in flexor tendoplasty that favors the use of live autografts by determining the role of the live graft tenocytes and endogenous TGF-2 signaling in flexor tendoplasty adhesions. Towards the development of a mechanistically- driven tissue engineering solution for flexor tendon adhesions, Specific Aim 2 seeks to investigate the effects of TGF-2 and GDF-5 on MMP-mediated remodeling of an in vitro scar tissue model. Finally, Specific Aim 3 will investigate how over-expression of Tgfb3 and Gdf5 using rAAV-loaded FDL tendon allografts influences adhesion in the mouse flexor tendoplasty model. An experienced multi-disciplinary team has been assembled to conduct these studies that will test an innovative mechanistically-driven paradigm in flexor tendon tissue engineering with significant clinical implications.