ECM biomaterials for diabetic foot ulcers

用于糖尿病足溃疡的 ECM 生物材料

• 批准号: 10432878
• 负责人: THEMIS R KYRIAKIDES
• 金额: $ 45.2万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10432878
• 关键词: Alginates; American; Amputation; Animals; Area; Attention; Behavior; Biocompatible Materials; Biomechanics; Biomedical Engineering; Blood flow; Cell physiology; Cells; Chronic; Clinic; Clinical; Complement; Complication; Complications of Diabetes Mellitus; Dangerousness; Dermal; Development; Diabetes Mellitus; Diabetic Foot Ulcer; Diabetic mouse; Drug Delivery Systems; Engineering; Epithelial; Excision; Extracellular Matrix; Extracellular Matrix Proteins; Family suidae; Fibroblasts; Functional disorder; Gelatinase A; Genotype; Health; Histologic; Hydrogels; Hyperglycemia; Hypoxia; Immune; Impaired healing; Impaired wound healing; In Vitro; Infection; Inflammation; Knockout Mice; Laboratories; Lead; Leg; Lower Extremity; Measurement; Microspheres; Molecular; Monitor; Mus; Pathologic; Pathway Analysis; Patient-Focused Outcomes; Patients; Persons; Pharmaceutical Preparations; Phenotype; Population; Prevention; Production; Protein-Lysine 6-Oxidase; Proteins; Quality of life; Recovery; Role; Signal Pathway; Skin; Stains; Testing; Therapeutic; Thick; Traction; Translating; United States; Work; angiogenesis; base; chronic wound; combat; combinatorial; crosslink; diabetic; diabetic patient; diabetic ulcer; healing; improved; in vivo; keratinocyte; limb amputation; migration; mortality; mouse model; non-healing wounds; novel; novel therapeutic intervention; regenerative; thrombospondin 2; transcriptome sequencing; vessel regression; wound; wound care; wound closure; wound healing

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.

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具有毁灭性影响。伤口愈合受损是一种严重的并发症，会导致下肢形成慢性伤口。患有此类伤口的患者表现出活动能力和整体健康状况下降，并且最常遭受截肢。大截肢后五年死亡率超过 80%。伤口护理措施可能有效，但很大一部分糖尿病溃疡仍然持续存在或导致截肢。新的治疗策略必须关注愈合受损的病理机制，以改善患者的治疗结果。由于糖尿病高血糖，伤口会变成慢性伤口，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。最近的公正分析，例如单 RNA 测序，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，很少有人关注 ECM 蛋白作为愈合负调节剂的作用。我们实验室的工作表明基质细胞蛋白血小板反应蛋白-2 (TSP-2) 对糖尿病伤口的影响。通常，TSP-2 在伤口中表达，并部分通过分别增加和减少基质金属蛋白酶 (MMP)-2/9 和赖氨酰氧化酶 (LOX) 和 ECM 交联的水平来促进 ECM 重塑和血管消退。我们最近的工作表明，糖尿病患者和小鼠的伤口中 TSP-2 升高，表明伤口愈合受损与 TSP-2 高表达之间存在相关性。这些发现使我们能够提出一个总体前提：异常的 ECM 生产和组装有助于防止伤口闭合。我们通过生成缺乏 TSP2 的糖尿病小鼠（dbdb-DKO 小鼠）并观察到愈合正常化，部分证实了这一假设。在此应用中，我们提出了探索和利用糖尿病 ECM 表型的研究。具体来说，我们计划描述 ECM 异常及其对体外和体内细胞功能障碍的贡献。此外，新生成的 dbdb-DKO 小鼠将用于探测新的 ECM 表型。我们还旨在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理验证。这些研究将包括从我们为提供临床级材料而创建的 TSP2 KO 猪中获得的 ECM。因此，该应用程序的具体目标是： 具体目标 1. 检验糖尿病中成纤维细胞功能障碍和 ECM 产生和组装改变导致伤口闭合延迟的假设。 我们将对这些小鼠进行伤口愈合，并监测生物力学完整性的恢复，这是愈合的一个关键方面。我们还将进行体外研究，通过研究真皮成纤维细胞与来自 WT 或 dbdb 小鼠的 ECM 的相互作用，研究糖尿病对细胞功能的间接影响。具体来说，我们将评估迁移、牵引力的产生和收缩。包含细胞源性 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，我们将对所有基因型进行大量 RNA 测序和通路分析。 具体目标 2. 检验新的工程天然构建体可以恢复糖尿病动物的愈合的假设。 我们之前已经证明，来自 TSP2 KO 小鼠的脱细胞皮肤结构和皮肤来源的水凝胶可以增加糖尿病小鼠的细胞整合、血管成熟和愈合。为此，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建的 TSP2 KO 猪的皮肤源性 ECM 的补充。具体来说，我们将制备嵌入藻酸盐微球的载药 TSP2-null 构建体，以对抗感染、减少慢性炎症并恢复愈合。将制作全层切除伤口，并通过每日测量伤口面积来量化愈合率。此外，将对伤口进行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 ECM 重塑。 该项目的完成将使我们能够建立伤口愈合受损的主要分子机制，这可以为不愈合伤口的工程解决方案的开发提供信息。在令人兴奋的平行研究中，我们培育出了一头 TSP2 KO 猪，我们预计它将用于生产基于 ECM 的临床产品。我们相信，本申请中提出的严格研究将为再生 ECM 结构走向临床提供重要支持。