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.具体目的： 糖尿病在美国很普遍，其并发症对健康和生活质量产生了破坏性的影响。伤口愈合受损是一种严重的并发症，导致下肢慢性伤口的发展。这种伤口的患者表现出降低的活动性和整体健康状况，并且最常受到肢体截肢的影响。重大截肢后的五年死亡率超过80％。伤口护理练习可能是有效的，但是很大一部分糖尿病性溃疡仍然持续或导致截肢。新颖的治疗策略必须集中于愈合受损的病理机制，以改善患者的结局。伤口由于糖尿病高血糖而慢性病，包括血液流量减少，缺氧和免疫细胞的异常行为，成纤维细胞和角质形成细胞，对愈合至关重要。最近的无偏分析（例如单RNA SEQ）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，很少关注ECM蛋白作为治疗的负调节剂的作用。在我们的实验室中的工作表明，基质蛋白血小板蛋白-2（TSP-2）对糖尿病伤的贡献。通常，TSP-2在伤口中表达，并通过分别增加和降低基质金属蛋白酶（MMP）-2/9和赖氨酸氧化酶（LOX）和ECM交联的水平，从而有助于ECM重塑和血管回归。我们最近的工作表明，在糖尿病患者的伤口和小鼠的伤口中，TSP-2升高，表明伤口愈合和高TSP-2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，即ECM异常产量和组装有助于预防伤口闭合。我们通过产生缺乏TSP2（DBDB-DKO小鼠）的糖尿病小鼠并观察到愈合的归一化来获得该假设的部分证实。在此应用中，我们提出了研究，以探索和利用糖尿病中的ECM表型。具体而言，我们计划表征ECM异常及其对细胞功能障碍的体外和体内的贡献。此外，新生成的DBDB-DKO小鼠将用于探测一种新型的ECM表型。我们还旨在将我们的发现转化为一种生物工程方法，该方法将为新型ECMS作为组合治疗策略的一部分提供原则证明。这些研究将包括我们为提供临床级材料而创建的TSP2 KO Pig获得的ECM。因此，本申请的具体目的是： 具体目的1。为了测试糖尿病中成纤维细胞功能障碍以及ECM产生和组装的假设有助于延迟伤口闭合。 我们将在这些小鼠中进行伤口愈合，并监测生物力学完整性的恢复，这是愈合的关键方面。我们还将通过研究皮肤成纤维细胞与源自WT或DBDB小鼠的ECM的相互作用来研究糖尿病对细胞功能的间接影响。具体而言，我们将评估迁移，牵引力的产生和收缩。包括细胞来源的ECM和来自DBDB-DKO小鼠的细胞将使我们更详细地探究TSP2的作用。为了更广泛地了解糖尿病诱导的成纤维细胞功能障碍所涉及的信号传导途径，我们将对所有基因型进行大量的RNA序列和途径分析。 具体目的2。为了测试以下假设：新型工程的天然构建体可以恢复糖尿病动物的愈合。 我们先前已经表明，来自TSP2 KO小鼠的脱细胞构建体和皮肤衍生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。在此目标中，我们将将此类结构与药物输送方法相结合，以制定组合策略。这些研究将由我们新创建的TSP2 KO猪衍生而来的皮肤衍生的ECM补充。具体而言，我们将准备装有藻酸盐微球嵌入药物的TSP2-NULL构建体，以对抗感染，减少慢性炎症并恢复愈合。将通过每日的伤口区域测量来量化全厚度的切除伤口，并量化愈合率。此外，为了评估伤口闭合，上皮化，炎症，血管生成和ECM重塑，伤口将受到组织学和免疫组织化学染色。 该项目的完成应使我们能够在伤口愈合受损中建立主要的分子机制，这可以为非愈合伤口的工程解决方案提供开发。在令人兴奋的平行研究中，我们产生了一个TSP2 KO Pig，我们预计将用于生产基于ECM的产品供临床使用。我们认为，本应用程序中提出的严格研究将为再生ECM构造向诊所的发展提供重大支持。