ECM biomaterials for diabetic foot ulcers

A.具体目标： 糖尿病在美国很普遍，其并发症对健康和生活质量有毁灭性的影响。创面愈合受损是一种严重的并发症，会导致下肢慢性创面的发展。这种伤口的患者行动不便，总体健康状况不佳，是最常接受截肢手术的患者。大截肢术后五年死亡率超过80%。伤口护理做法可能是有效的，但很大一部分糖尿病溃疡仍然存在或导致截肢。新的治疗策略必须集中在损害愈合的病理机制上，以改善患者的预后。由于糖尿病高血糖，伤口变得慢性，包括血流减少，缺氧，以及对愈合至关重要的免疫细胞、成纤维细胞和角质形成细胞的异常行为。最近的无偏见分析，如单RNA序列，揭示了成纤维细胞和细胞外基质(ECM)产生的关键参与。令人惊讶的是，几乎没有人注意到ECM蛋白作为负向调节因子对愈合的作用。我们实验室的工作表明基质细胞蛋白血栓反应蛋白-2(TSP-2)在糖尿病伤口中的作用。正常情况下，TSP-2在创面表达，并通过增加和降低基质金属蛋白酶(MMP2/9)、赖氨酸氧化酶(LOX)和ECM交联物的水平，促进ECM重塑和血管退行性变。我们最近的工作表明，TSP-2在糖尿病患者和小鼠的伤口中升高，这表明伤口愈合受损与TSP-2的高表达之间存在相关性。这些发现使我们能够提出一个重要的前提，即异常的ECM产生和组装有助于防止伤口闭合。我们通过产生缺乏TSP2的糖尿病小鼠(DBDB-DKO小鼠)并观察到愈合的正常化，部分证实了这一假设。在这一应用中，我们提出了探索和开发糖尿病ECM表型的研究。具体地说，我们计划在体外和体内表征细胞外基质的异常及其对细胞功能障碍的贡献。此外，新产生的DBDB-DKO小鼠将被用于探索一种新的ECM表型。我们还致力于将我们的发现转化为生物工程方法，为使用新型ECM作为组合治疗策略的一部分提供原则证明。这些研究将包括从我们为提供临床级材料而创建的TSP2 KO猪身上获得的细胞外基质。因此，本申请的具体目标是： 具体目的1.验证糖尿病患者成纤维细胞功能障碍和ECM合成和组装改变导致伤口延迟闭合的假说。 我们将在这些小鼠身上进行伤口愈合，并监测生物力学完整性的恢复，这是愈合的关键方面。我们还将进行体外研究，通过研究来自WT或DBDB小鼠的真皮成纤维细胞与ECM的相互作用，来研究糖尿病对细胞功能的间接影响。具体地说，我们将评估移民、牵引力的生产和收缩。包括细胞来源的ECM和来自DBDB-DKO小鼠的细胞将使我们能够更详细地探讨TSP2的作用。为了更广泛地了解糖尿病引起的成纤维细胞功能障碍所涉及的信号通路，我们将对所有的基因类型进行批量RNA测序和通路分析。 具体目的2.验证新型工程天然结构可以恢复糖尿病动物的愈合的假说。 我们之前已经证明，来自TSP2KO小鼠的脱细胞皮肤结构和皮肤衍生水凝胶可以促进糖尿病小鼠的细胞整合、血管成熟和愈合。在这个目标中，我们将把这种结构与药物输送方法结合起来，以开发组合策略。这些研究将得到来自我们新创造的TSP2 KO猪的皮肤来源的ECM的补充。具体地说，我们将准备携带药物的TSP2零结构与藻酸盐微球一起嵌入，以对抗感染，减少慢性炎症和恢复愈合。将制作全层切除创面，并通过每日测量创面面积来量化愈合率。此外，伤口将接受组织学和免疫组织化学染色，以评估伤口闭合、上皮化、炎症、血管生成和细胞外基质重塑。 该项目的完成将使我们能够在损伤的伤口愈合中建立一个主要的分子机制，这将为开发非愈合伤口的工程解决方案提供信息。在令人兴奋的平行研究中，我们培育了一种TSP2 KO猪，我们预计它将用于生产临床使用的基于ECM的产品。我们相信，在这一应用中拟议的严格研究将为再生性ECM结构走向临床提供重要支持。

A. SPECIFIC AIMS: Diabetes is widespread in the United States, and its complications have devastating effects on health and quality of life. Impaired wound healing is a serious complication that leads to the development of chronic wounds in the lower extremities. Patients with such wounds display decreased mobility, and overall health, and are the most frequently subjected to limb amputation. Five year mortality following major amputation is over 80%. Wound care practices can be effective, but a large fraction of diabetic ulcers still persist or lead to amputation. Novel therapeutic strategies must focus on the pathological mechanisms underlying impaired healing in order to improve patient outcomes. Wounds become chronic due to diabetic hyperglycemia, including reduced blood flow, hypoxia, and aberrant behavior of immune cells, fibroblasts, and keratinocytes that are critical to healing. Recent unbiased analyses, such as single RNA seq, have revealed the critical involvement of fibroblasts and extracellular matrix (ECM) production. Surprisingly, little attention has been paid to the role of ECM proteins as negative modulators of healing. Work in our laboratory suggests the contribution of the matricellular protein thrombospondin-2 (TSP-2) to diabetic wounds. Normally, TSP-2 is expressed in wounds and contributes to ECM remodeling and vascular regression, in part, by increasing and decreasing the levels of matrix metalloproteinase (MMP)-2/9 and lysyl oxidase (LOX) and ECM crosslinking, respectively. Our recent work has shown that TSP-2 is elevated in the wounds of diabetic patients and mice suggesting a correlation between compromised wound healing and high TSP-2 expression. These findings allowed us to put forward the overarching premise that abnormal ECM production and assembly contribute to prevention of wound closure. We obtained partial confirmation for this hypothesis by generating diabetic mice lacking TSP2 (dbdb-DKO mice) and observed normalization of healing. In this application, we propose studies to explore and exploit ECM phenotypes in diabetes. Specifically, we plan to characterize ECM abnormalities and their contribution to cell dysfunction in vitro and in vivo. In addition, the newly generated dbdb-DKO mouse will be used to probe a novel ECM phenotype. We also aim to translate our findings into a bioengineering approach that will provide proof-of-principle for the utilization of novel ECMs as part of a combinatorial therapeutic strategy. These studies will include ECM obtained from a TSP2 KO pig that we have created for the provision of clinical grade materials. Accordingly, the specific aims of this application are: Specific Aim 1. To test the hypothesis that fibroblast dysfunction and altered ECM production and assembly in diabetes contribute to delayed wound closure. We will perform wound healing in these mice and monitor recovery of biomechanical integrity, a critical aspect of healing. We will also perform in vitro studies to investigate the indirect impact of diabetes on cell functions by studying the interactions of dermal fibroblasts with ECM derived from WT or dbdb mice. Specifically, we will evaluate migration, production of traction forces, and contraction. Inclusion of cell-derived ECM and cells from dbdb-DKO mice will allow us to probe the role of TSP2 in greater detail. In order to obtain a broader understanding of the signaling pathways involved in the diabetes-induced fibroblast dysfunction we will perform bulk RNA-sequencing and pathway analysis of all genotypes. Specific Aim 2. To test the hypothesis that novel engineered native constructs can restore healing in diabetic animals. We have previously shown that decellularized skin constructs and skin-derived hydrogels from TSP2 KO mice can increase cellular integration, vessel maturation, and healing in diabetic mice. In this aim, we will combine such constructs with drug delivery approaches to develop combinatorial strategies. These studies will be complemented by skin-derived ECM derived from our newly created TSP2 KO pigs. Specifically, we will prepare drug-loaded TSP2-null constructs embedded with alginate microspheres to combat infections, reduce chronic inflammation and restore healing. Full thickness excisional wounds will be made and the rate of healing will be quantified through daily measurements of wound area. Additionally, wounds will be subjected to histological and immunohistochemical stains in order to assess wound closure, epithelialization, inflammation, angiogenesis, and ECM remodeling. Completion of this project should allow us to establish a major molecular mechanism in impaired wound healing, which could inform the development of engineering solutions to non-healing wounds. In exciting parallel studies, we have generated a TSP2 KO pig, which we anticipate will be used to produce ECM-based products for clinical use. We believe the proposed rigorous studies in this application will provide significant support for the advancement of regenerative ECM constructs towards the clinic.