Indentation Micromechanics of Fibroblast-Populated Fibrin Constructs

摘要

During normal wound healing, a fibrin clot is formed within the first few minutes and is replaced, over several days, by collagen and other extracellular matrix components which are populated with fibroblasts [Clark, 1996]. This process leads to the rebuilding of dermal tissue on which the epidermal tissue is slowly rebuilt. In severe acute and chronic wounds dermal replacement materials that mimic these processes are needed. Current trends are to use scaffolds as structural substitutes as well as carriers for growth factors and cells for wound treatment. Fibrin-based sealants have been used over the last 30 years in hemostasis and tissue sealing applications and in the last 5 years as a scaffold. Fibrin sealant consists of two primary components, fibrinogen and thrombin which form a fibrin clot when mixed. However, a basic understanding of the microstructure/property relationships of fibrin/collagen constructs is not yet fully established. Furthermore, while previous studies showed that cells proliferate and differentiate within 3D fibrin clots, their effect on structural mechanics of the fibrin clot has not been examined. Here, an indentation protocol was established to determine the effects of fibrin/collagen biochemistry and time-dependent cellular response on the elastic parameters.of the constructs. Specifically, 4 ml fibrin/collagen constructs were prepared with varying compositions of human-derived fibrinogen (5 - 33 mg/ml), thrombin (1 or 2 U/ml) and bovine collagen (0,25, 50,75, 100 weight % of 2.4 mg/ml collagen). Constructs were prepared with/without the presence of human foreskin fibroblasts (ATCC NIH3T3) seeded at a density of 100K cells/ml. Using a 3-mm diameter punch indenter, the indentation load-displacement response was measured after 1,5 and 10 days of incubation at 37℃ in a humidified air/5% CO_2 atmosphere. Four replicates per experimental condition were prepared, and three indentations per replicate were performed. For the unpopulated fibrin, there was a linear (R = 0.983) relationship between the indentation stiffness and fibrinogen concentration. Also, there was a nonlinear relationship between indentation stiffness and concentration of collagen in the fibrin/collagen constructs. Finally, although in most cases there was no measurable change in the stiffness of the cell-populated tissue constructs with incubation time, in one of the cell-populated formulations, however, the indentation stiffness decreased steadily with increasing incubation time. These results are analyzed within the context of existing analytical micromechanics models for the relationships between scaffold structure, e.g., porosity / fibril diameter, and stiffness.