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-功能关系的基本了解，设计具有可预测的机械、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-纤维蛋白结构中的蛋白酶和胶原表达的程度可能与结构结构中随时间变化的定量相关：(I)纤维直径平均和(Ii)纤维网络孔大小超过15天。我们进一步假设，这些结构的变化可能与机械功能的变化有关：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、基于物理的模型，并结合启发式分析，用于定制纤维蛋白生物材料的潜在结构-功能关系。我们将这些假说建立在以下观察基础上：首先，体外培养10天后，成纤维细胞种植的纤维蛋白结构表现出不变或降低的机械硬度[Mooney等人，2004年]。第二，纤维蛋白结构表现出与纤维蛋白原浓度成正比的机械硬度[Mooney等人，2004]。第三，纤维蛋白原和凝血酶在三维纤维蛋白/细胞结构中的浓度影响：(I)成纤维细胞和角质形成细胞的增殖、形态和定性的结构完整性[Cox等，2004]和(Ii)IL-8、PDGF受体和特定整合素的差异表达。第四，初步分析表明，机械刚度的潜在结构-功能关系可以建立基于物理的模型。具体目标是：1.实验表征体外时间对纤维蛋白凝块结构中纤维结构、机械硬度和细胞生物学的影响。对于11种纤维蛋白/凝血酶比例的构建物，每种都种植两种密度的成纤维细胞或角质形成细胞，间隔1、5、10和15天，我们将：1)测量机械硬度参数；2)测量平均纤维原纤维直径和纤维网络孔径；3)确定各种蛋白酶和胶原类型的蛋白表达水平；4)确定细胞增殖。Ii.基于SA I的数据，建立并严格验证基于物理和启发式的模型，以确定i)机械硬度和ii)纤维直径和平均纤维网络孔径之间的结构-功能关系。关键自变量：i)纤维蛋白/凝血酶比值，ii)有无细胞，iii)细胞密度(50000个/ml或100000个/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)。主要因变量：i)机械硬度，ii)纤维直径和纤维网络孔径，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.