Treatment of venous ulcers with autologous cultured bone marrow stem cells

描述（由申请人提供）：下肢静脉溃疡和其他严重慢性伤口影响着500万至700万美国人，每年至少造成200亿美元的直接损失。静脉溃疡患者生活质量差，感染频繁，失去工作和独立性。近年来，由于更好的常规护理和新的先进疗法，如使用活体生物工程皮肤，临床结果有所改善。然而，静脉高压的潜在问题会导致严重的继发性改变，如组织纤维化、感染倾向，以及越来越多的认识到的损伤细胞的表型改变，这些改变会损害皮肤修复和表皮表面重塑。静脉溃疡会变得非常难以愈合。事实上，即使是先进的治疗方法也只能治愈50%的静脉溃疡，这些溃疡已经存在了一年以上。我们最近有证据表明，局部应用自体骨髓来源的间充质干细胞（MSCs），从患者自己的骨髓中培养并在体外培养和扩增，可以显著加速慢性伤口的愈合，包括静脉溃疡。在我们的前期工作中，我们开发了一种新的纤维蛋白聚合物结构，用于在喷雾系统中将培养的MSCs输送到伤口。在此过程中，我们通过降低聚合所需的纤维蛋白原/凝血酶浓度和消除作为蛋白酶抑制剂成分的抑蛋白蛋白来修饰现有的纤维蛋白胶结构。一旦被喷洒到伤口中，MSCs就会以一种薄的纤维蛋白凝胶的形式存在，这种纤维蛋白凝胶不会影响它们的生存能力，并且会被内源性消除，从而将细胞释放到伤口中。我们现在准备进行一项临床试验，以验证患者自身的间充质干细胞可以加速伤口愈合的假设。我们已经开发了一个GMP设施来处理MSCs的生长，我们已经从FDA获得了临床试验的IND。我们计划以下两个具体目标：1)在三组随机对照盲法临床试验中确定MSCs对静脉溃疡愈合的影响。所有患者均采用常规腿部压迫绷带治疗。培养的自体间充质干细胞将以纤维蛋白喷雾剂的形式输送到伤口，这组患者将与单独接受腿部压迫治疗或仅接受纤维蛋白喷雾剂治疗的对照组患者进行比较。愈合将通过计算机平面测量来评估伤口边缘迁移和愈合率，伤口大小缩小和完全闭合；2)表征并密切关联伤口边缘分子标志物在治疗反应中愈合受损和上皮迁移的表达。在特定目的1治疗的静脉溃疡边缘进行基线和顺序活检将用于确定伤口边缘的c-myc、2-连环蛋白和角蛋白6/16和17的表皮表达。这些测量与伤口大小和边缘迁移密切相关，将帮助我们建立有希望的分子标记，参与受损愈合，以及MSCs是否可能通过影响这些特定分子标记的表达和定位而起作用。

DESCRIPTION (provided by applicant): Venous leg ulcers and other severe chronic wounds affect 5-7 million Americans, and are responsible for a direct cost of at least $20 billion annually. Patients with venous ulcers experience poor quality of life, frequent infections, and loss of work and independence. Clinical outcomes have improved in recent years because of better conventional care and new advanced therapies, such as the use of living bioengineered skin. However, the underlying problem of venous hypertension leads to severe secondary changes, such as tissue fibrosis, propensity for infection, and an increasingly recognized set of phenotypic changes in resident wound cells that impair dermal repair and epidermal resurfacing. Venous ulcers can become very difficult to heal. Indeed, even advanced therapies heal less than 50% of venous ulcers that have been present for more than a year. We have recently developed evidence that the topical application of autologous bone marrow-derived mesenchymal stem cells (MSCs), grown from the patients' own bone marrow aspirate and cultured and expanded in vitro, can lead to dramatic acceleration of healing in chronic wounds, including venous ulcers. During our preliminary work, we have developed a novel fibrin polymer construct for delivering the cultured MSCs to the wound in a spray system. In doing so, we modified an existing fibrin glue construct by decreasing the fibrinogen/thrombin concentrations required for polymerization and by eliminating aprotenin as the protease inhibitor component. Once sprayed into the wound, the MSCs are in a thin fibrin gel that does not affect their viability and is endogenously eliminated, thus releasing the cells to the wound. We are now ready to move forward with a clinical trial that will test the hypothesis that the patients' own MSCs can accelerate the healing of their wounds. We have developed a GMP facility for handling the the growth of MSCs, and we have received an IND from the FDA for our clinical trial. We plan the following two specific aims: 1) Determine the effect of MSCs on the healing of venous ulcers in a three-arm randomized controlled blinded clinical trial. All patients will be treated with conventional leg compression bandages. Cultured autologous MSCs will be delivered to the wound in a fibrin spray, and this group will be compared to control patients receiving leg compression treatment alone or the fibrin spray only. Healing will be assessed by computerized planimetry for wound edge migration and healing rate, wound size reduction, and complete closure; 2) Characterize and closely correlate the expression of wound edge molecular markers of impaired healing and epithelial migration in response to treatment. Baseline and sequential biopsies from the edges of venous ulcers treated in specific aim 1 will be used to determine the epidermal expression of c-myc, 2-catenin, and keratins 6/16 and 17 at the wounds' edges. These measurements, closely correlated with wound size and edge migration, will help us establish promising molecular markers involved in impaired healing and whether the MSCs may work by affecting the expression and localization of these specific molecular markers.