Cellular and matrix contributions to tissue construct stiffness increase with cellular concentration

Abstract

The mechanics of bio-artificial tissue constructs result from active and passive contributions of cells and extracellular matrix (ECM). We delineated these for a fibroblast-populated matrix (FPM) consisting of chick embryo fibroblast cells in a type I collagen ECM through mechanical testing, mechanical modeling, and selective biochemical elimination of tissue components. From a series of relaxation tests, we found that contributions to overall tissue mechanics from both cells and ECM increase exponentially with the cell concentration. The force responses in these relaxation tests exhibited a logarithmic decay over the 3600 second test duration. The amplitudes of these responses were nearly linear with the amplitude of the applied stretch. The active component of cellular forces rose dramatically for FPMs containing higher cell concentrations.

Figure 1

(a) FPM width and (b) FPM thickness decreased after three days of incubation. The circles correspond to the experimental variation of FPM width as the initial cell concentration varied.

Figure 1

(a) FPM width and (b) FPM thickness decreased after three days of incubation. The circles correspond to the experimental variation of FPM width as the initial cell concentration varied.

Figure 2

(a) Cellular concentration after three days of incubation increased monotonically with starting cell concentration. The dashed line is the concentration at which cells are predicted to form a contiguous, steric network. (b) The dashed line in Fig. 2b represents no change in the cell number after incubation; values above this line indicate an increase in the number of cells, while values below this indicate a decreasing number of cells, as was the case for FPMs beginning with very high cell concentrations.

Figure 2

(a) Cellular concentration after three days of incubation increased monotonically with starting cell concentration. The dashed line is the concentration at which cells are predicted to form a contiguous, steric network. (b) The dashed line in Fig. 2b represents no change in the cell number after incubation; values above this line indicate an increase in the number of cells, while values below this indicate a decreasing number of cells, as was the case for FPMs beginning with very high cell concentrations.

Figure 3

Typical logarithmic relaxation response of a FPM subjected to a rapid stretch. The sample shown had a final cell concentration of 10.7 Mcell/ml and was tested in DMEM at strain levels ε=0.02, 0.08, 0.15, and 0.22, from (1) through (4), respectively. The standard deviation of the data shown did not exceed the size of the circles, and was 4, 8, 12 and 16 dynes, for (1) through (4), respectively.

Figure 4

Coefficient a as a function of strain for FPMs containing a final cell concentration of 11.8 Mcell/ml (“ECM” represents specimens tested in PBS with deoxycholate; “FPM” represents specimens tested in DMEM with serum).

Figure 5

Coefficient b as a function of tissue strain for FPMs containing a final cell concentration of 11.8 Mcell/ml (“ECM” represents specimens tested in PBS with deoxycholate; “FPM” represents specimens tested in DMEM with serum).

Figure 6

The overall FPM stiffness after three days of incubation increased monotonically with final cell concentration. Circles represent the experimental measurements of the FPM short-term secant modulus as a function of the final cell concentration, and the solid line is a power-law fit (Eq. 6).

Figure 7

The contribution of the ECM to overall FPM stiffness after three days of incubation increased monotonically with final cell concentration. Circles represent the experimental measurements of the FPM short-term secant modulus as a function of the final cell concentration, and the solid line is a power-law fit (Eq. 6).

Figure 8

The ECM stiffness increases with increasing cell concentration were far greater than those that would be expected from compaction of collagen in the absence of remodeling. The relationship between collagen concentration and ECM modulus followed a power law.

Figure 9

The contribution of the cell to overall FPM stiffness after three days of incubation increased with final cell concentration for all but the highest of cell concentrations. Circles represent the experimental measurements of the FPM short-term secant modulus as a function of the final cell concentration, and the solid line is the difference between the fits for FPM and ECM (Eq. 6).

Figure 10

Variation in the stress-free strain, ε_o, for the rate-independent component of the FPM mechanical response as a function of the cell concentration. This difference is related to the contribution of the active cellular contraction to the overall stress in the FPM.