Modeling Biomechanical Transformation of Keratinocyte/ or Fibroblast/ Fibrin Gels

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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.