Designing Microfabricated Basal Lamina Analogs to Enhance Skin Regeneration

描述（由申请人提供）：每年，美国约有12，000人严重烧伤，需要植皮。糖尿病和静脉溃疡以及压疮每年影响美国另外300万至400万人。虽然工程化组织类似物作为受损皮肤的替代品已经取得了一些临床成功，但再生皮肤的延长愈合时间和机械诱导的移植失败仍然是持续存在的问题。为了解决生物工程皮肤替代品的局限性，需要了解其三维微结构和生物化学组成介导角质形成细胞粘附，增殖和分化以及高功能上皮组织再生的机制。该项目的总体目标是定量分析指导角质形成细胞功能的微观结构和细胞外基质（ECM）线索，这些角质形成细胞功能促进精确工程化基底层类似物表面上稳健表皮层的快速再生。我们假设，具有三维特征的微制造基底层类似物和模仿真皮-表皮交界处的细胞微环境的ECM线索将增加生物工程皮肤替代品表面上的表皮干细胞聚集，并促进稳健表皮层的快速再生。为了系统地测试这一假设，我们将培养角质形成细胞的皮肤替代品的表面含有成纤维细胞和基底层类似物与微制造的地形特征和离散ECM组合物，我们将使用定量形态测定分析细胞的地形线索和成纤维细胞旁分泌信号。因此，我们提出了以下具体目标：具体目标1：建立角质形成细胞功能和基底层类似物表面上ECM生物化学之间的定量关系。具体目标二：确定支架微结构和成纤维细胞信号传导对角质形成细胞功能以及对微制造基底层类似物表面上的表皮再生的定量作用。预期的结果是确定一系列参数，这些参数对于改进生物工程皮肤替代品的设计以及促进具有增加的结构和机械稳定性的高功能皮肤组织的快速再生至关重要。为了解决生物工程皮肤替代品的局限性，需要了解其三维微结构和生化组成介导角质形成细胞粘附，增殖和分化，并促进快速再生的强大的上皮组织的机制。该项目的总体目标是定量分析指导角质形成细胞功能的微观结构和细胞外基质（ECM）线索，这些角质形成细胞功能促进微制造组织类似物表面上稳健表皮层的快速再生。最终，我们预计这些研究的结果将为我们提供改进生物工程皮肤替代品设计的参数，这些替代品将促进烧伤和糖尿病溃疡等具有挑战性的伤口的愈合。

DESCRIPTION (provided by applicant): Every year, approximately 12,000 people in the United States are burned severely enough to require skin grafting. Diabetic and venous ulcers, as well as pressure sores affect an additional 3 to 4 million people in the United States each year. While engineered tissue analogs have achieved some clinical success as substitutes for damaged skin, prolonged healing times for regenerated skin and mechanically-induced graft failure remain persistent problems. To address the limitations of bioengineered skin substitutes requires an understanding of the mechanisms by which their three-dimensional microarchitecture and biochemical composition mediates keratinocyte adhesion, proliferation and differentiation, and the regeneration of highly functional epithelial tissue. The overall goal of this project is to quantitatively analyze the microstructure and extracellular matrix (ECM) cues that direct keratinocyte functions which promote the rapid regeneration of a robust epidermal layer on the surfaces of precisely engineered basal lamina analogs. We hypothesize that microfabricated basal lamina analogs with three-dimensional features and ECM cues that mimic the cellular microenvironments of the dermal-epidermal junction will increase epidermal stem cell clustering on the surface of bioengineered skin substitutes and promote the rapid regeneration of a robust epidermal layer. To systematically test this hypothesis, we will culture keratinocytes on the surfaces of skin substitutes containing fibroblasts and basal lamina analogs with microfabricated topographic features and discrete ECM compositions, and we will use quantitative morphometric assays to analyze cellular responses to topographic cues and fibroblast paracrine signaling. As such, we propose the following Specific Aims: Specific Aim 1: Establish quantitative relationships between keratinocyte functions and the ECM biochemistry on the surfaces of basal lamina analogs. Specific Aim 2: To determine quantitative roles of scaffold microarchitecture and fibroblast cell signaling on keratinocyte functions as well as on epidermal regeneration on the surfaces of microfabricated basal lamina analogs. The expected outcome is the identification of a series of parameters critical for improving the design of bioengineered skin substitutes, as well as for promoting the rapid regeneration of highly functional skin tissue with increased structural and mechanical stability. To address the limitations of bioengineered skin substitutes requires an understanding of the mechanisms by which their three-dimensional microarchitecture and biochemical composition mediates keratinocyte adhesion, proliferation and differentiation, and promotes the rapid regeneration of robust epithelial tissue. The overall goal of this project is to quantitatively analyze the microstructure and extracellular matrix (ECM) cues that direct keratinocyte functions which promote the rapid regeneration of a robust epidermal layer on the surfaces of microfabricated tissue analogs. Ultimately, we anticipate that the findings from these studies will provide us with parameters for improving the design of bioengineered skin substitutes that will facilitate healing of challenging wounds such as burns and diabetic ulcers.