Allografts and Gene Therapy in Flexor Tendon Tissue Engineering

描述（由申请人提供）：术后粘连是自体移植屈肌肌腱成形术的一种主要的衰弱性并发症，但仍未解决。先前的研究表明，粘连至少部分地通过TGF-2信号传导至活肌腱或自体移植肌腱细胞来调节，作为对手术的初始炎症反应的一部分。有趣的是，虽然多种病理中的纤维化与TGF-21诱导纤溶酶原激活物抑制剂-1（派-1）（其防止MMP的激活）相关，但TGF-23在皮肤伤口愈合中具有抗瘢痕形成作用。然而，TGF-2亚型调节肌腱瘢痕形成的机制仍然知之甚少，预防粘连的生物疗法也不可用。为了解决这些问题，开发了一种新的屈肌腱成形术小鼠模型，该模型涉及将中间活自体移植物或冻干同种异体移植物植入后爪的远侧趾长屈肌腱（FDL）。在该模型中，自体移植物的跖趾（MTP）关节屈曲范围低于同种异体移植物，这与术后14天和28天时更多的纤维化瘢痕形成相关。Tgfb 1的早期表达与Mmp 9和瘢痕形成的抑制相关，而Gdf 5的晚期表达与Mmp 2和Mmp 14的延迟上调相关，随后改善MTP关节屈曲。此外，通过冻干同种异体移植物的局部和瞬时Gdf 5基因递送显著减少了粘连，并在屈肌腱成形术后恢复了MTP关节屈曲。最近的数据使用体外肌腱细胞接种的胶原蛋白凝胶模型的瘢痕组织表明，GDF-5抑制TGF-21诱导的凝胶收缩和挽救MMP基因表达。因此，该提议试图测试以下假设：1）无细胞同种异体移植物，其不能响应内源性TGF-21信号，是屈肌腱成形术中治疗剂的有效递送支架;和2）使用rAAV/同种异体移植物介导的Gdf 5或Tgfb 3的过表达抑制TGF-21/派-1诱导的MMP抑制，减少屈肌腱成形术中的瘢痕形成和粘连。本申请提出了一系列的补充研究，以测试这些假设，并开发一个组织工程解决方案的屈肌腱粘连。具体目标1旨在通过确定活移植肌腱细胞和内源性TGF-2信号传导在屈肌腱成形术粘连中的作用，正式挑战屈肌腱成形术中有利于使用活自体移植物的当前范例。为了开发用于屈肌腱粘连的机械驱动的组织工程解决方案，特定目标2试图研究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，特定目标3将研究使用rAAV负载的FDL肌腱同种异体移植物的Tgfb 3和Gdf 5的过表达如何影响小鼠屈肌腱成形术模型中的粘连。一个经验丰富的多学科团队已经组装进行这些研究，将测试一个创新的机械驱动的范例，在屈肌腱组织工程具有重大的临床意义。

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.