Modeling Biomechanical Transformation of Keratinocyte/ or Fibroblast/ Fibrin Gels

描述（由申请人提供）：总体目标是基于对其结构-功能关系的基本理解，设计具有可预测机械、生物和生化特性的定制纤维蛋白生物材料，用于治疗皮肤损伤。我们假设成纤维细胞-纤维蛋白或角质形成细胞-纤维蛋白构建体中蛋白酶和胶原蛋白表达的程度可能与构建体结构的时间依赖性变化定量相关：15天内的平均（i）原纤维直径和（ii）原纤维网络孔径。我们进一步假设，这些结构的变化可能与机械功能的变化相关：即机械刚度。因此，这项工作的主要成果将是开发和验证严格的，基于物理学的模型，加上启发式分析，定制的纤维蛋白生物材料的潜在结构-功能关系。我们将这些假设基于以下观察：首先，成纤维细胞接种的纤维蛋白构建体在体外10天后表现出不变或降低的机械刚度[Mooney等人，2004年]。其次，纤维蛋白构建体表现出与纤维蛋白原浓度成比例的机械刚度[Mooney等人，2004年]。第三，3-D纤维蛋白/细胞构建体中的纤维蛋白原和凝血酶浓度影响：（i）成纤维细胞和角质形成细胞增殖、形态，以及定性地影响它们的结构完整性[考克斯等人，2004]和（ii）IL-8、PDGF受体和特异性整联蛋白的差异表达。第四，初步分析表明，基于物理的模型可以开发的基本结构功能关系的机械刚度。具体目标是：一。实验表征体外时间对基于纤维蛋白的凝块结构中的原纤维结构、机械刚度和细胞生物学的影响。对于11种纤维蛋白/凝血酶比率的构建体，每种构建体以1、5、10和15天的间隔接种有/没有两种密度的成纤维细胞或角质形成细胞的细胞，我们将：1）测量机械刚度参数; 2）测量平均纤维蛋白原纤维直径和原纤维网络孔径; 3）测定各种蛋白酶和胶原类型的蛋白表达水平和4）测定细胞增殖。二.基于来自SA I的数据，开发并严格验证i）机械刚度与ii）原纤维直径和平均原纤维网络孔径之间的结构-功能关系的基于物理学的启发式模型。关键独立变量：i）纤维蛋白/凝血酶比率，ii）有/无细胞，iii）细胞密度（50，000个细胞/ml或100，000个细胞/ml）; iv）细胞类型（成纤维细胞或角质形成细胞）和v）时间（1、5、10和15天）。关键因变量：i）机械刚度，ii）原纤维直径和原纤维网络孔径，iii）蛋白质水平，iv）细胞数量。

DESCRIPTION (provided by applicant): The overarching goal is to enable the design of tailored fibrin biomaterials with predictable mechanical, biological and biochemical properties for treatment of dermal injuries based on a fundamental understanding of their structure-function relationships. We hypothesize that the extent of protease and collagen expression in fibroblast - fibrin or keratinocyte - fibrin constructs may be quantitatively correlated with time-dependent changes in construct structure: the average (i) fibril diameter and (ii) fibril network pore size over 15 days. We further hypothesize that these changes in structure may be correlated with changes in mechanical function: namely, mechanical stiffness. Therefore, a major outcome of this work will be the development and validation of rigorous, physics-based models, coupled with heuristic analyses, for the underlying structure- function relationships of tailored fibrin biomaterials. We base these hypotheses on the following observations: First, fibroblast-seeded fibrin constructs exhibit an invariant or decreasing mechanical stiffness after 10 days in vitro [Mooney et al., 2004]. Second, fibrin constructs exhibit a mechanical stiffness that is proportional to fibrinogen concentration [Mooney et al., 2004]. Third, the fibrinogen and thrombin concentrations in 3-D fibrin/cell constructs affect: (i) fibroblast and keratinocyte proliferation, morphology, and, qualitatively, their structural integrity [Cox et al., 2004] and (ii) differential expression of IL-8, PDGF receptor and specific integrins. Fourth, preliminary analysis reveals that physics-based models may be developed for the underlying structure-function relationships for mechanical stiffness. The specific aims are: I. Experimentally characterize the effects of time in vitro on fibril structure, mechanical stiffness and cell biology in fibrin-based clot constructs. For constructs of eleven fibrin/thrombin ratios, each seeded with/without cells of two densities of either fibroblasts or keratinocytes, at intervals of 1, 5, 10 and 15 days, we will: 1) Measure the mechanical stiffness parameters; 2) Measure the average fibrin fibril diameter and fibril network pore size; 3) Determine protein expression levels for various proteases and collagen types and 4) Determine cell proliferation. II. Based on the data from SA I, develop and critically validate physics-based and heuristic models for the structure-function relationships between i) the mechanical stiffness and ii) fibril diameter and average fibril network pore size. Key independent variables: i) fibrin/thrombin ratio, ii) with/without cells, iii) cell density (50,000 cells/ml or 100,000 cells/ml); iv) cell type (fibroblasts or keratinocytes) and v) time (1, 5, 10 and 15 days). Key dependent variables: i) mechanical stiffness, ii) fibril diameter and fibril network pore size, iii) protein level, iv) cell number.