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一个主要的衰弱但仍未解决的并发症。先前的研究表明，至少部分地，作为手术后初始炎症反应的一部分，粘连是通过向活的肌腱或自体移植的肌腱细胞传递转化生长因子-2信号来调节的。有趣的是，虽然各种病理中的纤维化与转化生长因子-21诱导纤溶酶原激活物抑制物-1(PAI-1)从而阻止基质金属蛋白酶的激活有关，但转化生长因子-23在皮肤创伤愈合中具有抗瘢痕作用。然而，转化生长因子-2亚型调节肌腱瘢痕形成的机制仍然知之甚少，也没有预防粘连的生物疗法可用。为了解决这些问题，建立了一种新的小鼠屈指肌腱成形术模型，该模型包括植入自体或冻干的自体或冻干同种异体肌腱于后爪远端屈指长腱(FDL)。在这个模型中，自体移植的跖趾(MTP)关节屈曲范围低于同种异体移植，这与术后14天和28天更多的纤维性瘢痕有关。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的MTP关节屈曲改善有关。此外，通过冷冻干燥的同种异体移植物局部和短暂的Gdf5基因转移显著减少了粘连，并恢复了屈肌腱成形术后的MTP关节屈曲。最近使用瘢痕组织的肌腱细胞种植的胶原凝胶模型的最新数据表明，GDF-5抑制了转化生长因子-21诱导的胶原凝胶的收缩，并挽救了基质金属蛋白酶的基因表达。因此，本研究试图验证以下假设：1)脱细胞同种异体肌腱不能响应内源性的转化生长因子-21信号，是屈肌腱成形术中有效的输送支架；2)抑制转化生长因子-21/PAI-1通过rAAV/同种异体移植物过表达Gdf5或TGFB3抑制MMPs，减少屈指肌腱成形术中的瘢痕形成和粘连。这项申请提出了一系列免费的研究来验证这些假设，并开发出一种用于屈肌腱粘连的组织工程解决方案。具体目的1试图通过确定活体移植物肌腱细胞和内源性转化生长因子-2信号在屈肌腱成形术粘连中的作用，正式挑战目前屈肌腱成形术中支持使用活体自体移植物的范式。为了开发一种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试图研究转化生长因子-2和GDF-5在体外瘢痕组织模型中对基质金属蛋白酶介导的重塑的影响。最后，特殊目标3将研究在小鼠屈肌腱成形术模型中，使用rAAV负载的同种异体肌腱移植过表达TGFB3和Gdf5如何影响粘连。已经组建了一个经验丰富的多学科团队来进行这些研究，这些研究将测试屈肌腱组织工程中具有重大临床意义的创新的机械驱动范例。

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.