MECHANISMS OF WOUND HEALING WITH CULTURED HUMAN SKIN

培养人类皮肤的伤口愈合机制

• 批准号: 2188387
• 负责人: Steven T Boyce
• 金额: $ 20.23万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2188387
• 关键词: angiogenesis; angiogenesis factor; artificial skin; athymic mouse; autoradiography; blood flow measurement; burn therapy; cardiovascular agents; cell adhesion molecules; collagen; cytokine; enzyme linked immunosorbent assay; fibroblasts; human tissue; immunochemistry; keratinocyte; melanocyte; mixed tissue /cell culture; monophenol monooxygenase; oxygen transport; polymers; skin transplantation; ultrasound blood flow measurement; western blottings; wound healing

Rapid and effective healing of burn wounds with cultured analogues of human skin is the central objective of this proposal. Medical benefits from improved healing may include, but not be limited to; reduced requirements for split-thickness skin autograft, shorter hospitalization time, and reduced long-term debilitation after recovery. However, anatomic and physiologic deficiencies of all current models of cultured skin have restricted realization of these benefits. Major deficiencies result from culture conditions that do not produce an epidermal analogue with true barrier properties, and from absence of a vascular plexus in dermal analogues. The current model of cultured skin in this laboratory is a collagen-based sponge populated with cultured human keratinocytes and fibroblasts. Proposed studies will address and correct some or all of the deficiencies of cultured skin by integrated studies in vitro, in animals, and in burn patients. In vitro studies will address: a) media (e.g.; nutrient, mitogen) and biophysical conditions that affect cellular metabolism of keratinocytes, fibroblasts, and melanocytes; b) composition (structure and biochemistry) of implantable biopolymer substrates for cell delivery; and c) expression of cytokines and adhesion proteins by skin analogues before grafting. Evaluation of these factors will be performed by: a) measurements of cell growth (DNA synthesis) and epidermal differentiation (barrier ultrastructure, lipids, trans epidermal water loss, surface hydrophobicity); b) electron microscopy and biochemical assay of biopolymer substrates; and c) western blot analyses of expressed cytokines and adhesion proteins. Skin analogues with optimal composition will be compared to controls (human xenograft, autograft, no graft) for efficacy (wound contraction, HLA-ABC expression), skin pigmentation (melanin content, tyrosinase) and expression of cytokines and adhesion proteins by grafting to full-thickness skin wounds on athymic mice. Experimental wounds will also be treated to stimulate vascularization during initial engraftment by topical delivery of angiogenic agents (bFGF, TGF-beta, and nutrients to improve cell survival. Angiogenesis will be studied versus time after grafting by: a) acrylic casts of the neovascular plexus; b) transcutaneous oxygen; and c) laser Doppler of capillary blood flow. Clinical studies will focus on stimulation of angiogenesis, and control of microbial contamination of the skin substitute during engraftment. Excised burns treated with composite cultured skin will be studied in the clinic by paired-site comparison to treatment with meshed split-thickness skin autograft. Comparative parameters of outcome will include: a) engraftment of cultured skin (incidence of failure, degree of infection, time to wound closure, need for reconstruction); and b) long- term results (function, contraction and cosmesis). The investigators possess all of required expertise in cell biology, skin biochemistry and biophysics, wound physiology, and clinical burn care to perform these studies successfully. Accomplishment of these objectives may contribute to reduced mortality and morbidity from burns, improved materials for plastic and reconstructive surgery, validated models as alternatives to animal testing of consumer products, and development of other tissue and organ substitutes.

用培养的类似物快速有效地治愈烧伤伤口 人皮是该提议的核心目标。医疗益处 从改善的康复中可能包括但不限于；减少 厚度厚度皮肤自体移植的要求，住院较短 时间，减少了恢复后的长期衰弱。但是，解剖学 以及所有当前培养皮肤模型的生理缺陷都有 这些好处的限制。主要缺陷来自 不产生表皮类似物的培养条件 屏障特性，以及缺乏皮肤上的血管丛 类似物。该实验室中培养的皮肤的当前模型是 基于胶原蛋白的海绵，含有培养的人角质形成细胞和 成纤维细胞。拟议的研究将解决并纠正一些或全部 通过体外研究，动物中的整合研究，培养皮肤的缺乏， 和烧伤的患者。 体外研究将解决：a）培养基（例如； 营养，有丝分裂原）和影响细胞的生物物理条件 角质形成细胞，成纤维细胞和黑素细胞的代谢； b）组成 （结构和生物化学）细胞的可植入生物聚合物底物的（结构和生物化学） 送货; c）皮肤的细胞因子和粘附蛋白表达 嫁接前的类似物。将对这些因素进行评估 作者：a）测量细胞生长（DNA合成）和表皮 分化（屏障超微结构，脂质，反式表皮水 损失，表面疏水性）； b）电子显微镜和生化 生物聚合物底物的测定； c）表达的蛋白质印迹分析 细胞因子和粘附蛋白。具有最佳成分的皮肤类似物 将与对照组（人异种移植，自体移植，无移植）进行比较 功效（伤口收缩，HLA-ABC表达），皮肤色素沉着 （黑色素含量，酪氨酸酶）和细胞因子的表达和粘附 蛋白质通过嫁接到无胸腺小鼠的全厚性皮肤伤口。 实验伤口也将被治疗以刺激血管化 在最初的植入期间，通过局部递送血管生成剂（BFGF， TGF-β和营养素可改善细胞存活。血管生成将是 研究与嫁接后的时间：a）新生血管的丙烯酸铸件 丛b）经皮氧； c）毛细血管血的激光多普勒 流动。临床研究将集中于刺激血管生成，并 控制皮肤替代的微生物污染 植入。用复合培养的皮肤处理的切除的烧伤将是 在诊所中通过配对的位点进行了研究与网状治疗 厚度厚的皮肤自体移植。结果的比较参数将 包括：a）植入培养的皮肤（失败的发生率，程度 感染，伤口闭合时间，需要重建）； b）长期 术语结果（功能，收缩和化合物）。调查人员 拥有细胞生物学，皮肤生物化学和 生物物理学，伤口生理和临床烧伤护理以执行这些 研究成功。这些目标的实现可能有助于 燃烧的死亡率和发病率降低，改进的塑料材料 和重建手术，经过验证的模型作为动物的替代品 测试消费产品以及其他组织和器官的开发 替代品。