Designing Microfabricated Basal Lamina Analogs to Enhance Skin Regeneration

描述（由申请人提供）：在美国，每年大约有12000人被严重烧伤，需要植皮。在美国，糖尿病和静脉溃疡以及压疮每年影响着另外300万到400万人。虽然工程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，但再生皮肤的愈合时间延长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化成分介导角质细胞粘附、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微观结构和细胞外基质（ECM）线索，这些线索指导角质形成细胞的功能，促进精确设计的基层类似物表面上健壮的表皮层的快速再生。我们假设，具有三维特征和ECM线索的微制造基底层类似物模拟真皮-表皮交界处的细胞微环境，将增加表皮干细胞在生物工程皮肤替代品表面的聚集，并促进强健表皮层的快速再生。为了系统地验证这一假设，我们将在含有成纤维细胞和基底层类似物的皮肤替代品表面培养角质形成细胞，这些替代品具有微制造的地形特征和离散的ECM成分，我们将使用定量形态计量学分析细胞对地形线索和成纤维细胞旁分泌信号的反应。因此，我们提出以下具体目标：具体目标1：建立角化细胞功能与基底层类似物表面ECM生物化学之间的定量关系。特异性目的2：确定支架微结构和成纤维细胞信号传导对角化细胞功能以及微合成基板类似物表面表皮再生的定量作用。预期的结果是确定一系列对改进生物工程皮肤替代品设计至关重要的参数，以及促进具有更高结构和机械稳定性的高功能皮肤组织的快速再生。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成分介导角质细胞粘附、增殖和分化的机制，并促进强健上皮组织的快速再生。该项目的总体目标是定量分析微观结构和细胞外基质（ECM）线索，这些线索指导角质形成细胞的功能，促进微制造组织类似物表面强健表皮层的快速再生。最终，我们预计这些研究的发现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.