DTAF dye concentrations commonly used to measure microscale deformations in biological tissues alter tissue mechanics

Abstract

Identification of the deformation mechanisms and specific components underlying the mechanical function of biological tissues requires mechanical testing at multiple levels within the tissue hierarchical structure. Dichlorotriazinylaminofluorescein (DTAF) is a fluorescent dye that is used to visualize microscale deformations of the extracellular matrix in soft collagenous tissues. However, the DTAF concentrations commonly employed in previous multiscale experiments (≥2000 µg/ml) may alter tissue mechanics. The objective of this study was to determine whether DTAF affects tendon fascicle mechanics and if a concentration threshold exists below which any observed effects are negligible. This information is valuable for guiding the continued use of this fluorescent dye in future experiments and for interpreting the results of previous work. Incremental strain testing demonstrated that high DTAF concentrations (≥100 µg/ml) increase the quasi-static modulus and yield strength of rat tail tendon fascicles while reducing their viscoelastic behavior. Subsequent multiscale testing and modeling suggests that these effects are due to a stiffening of the collagen fibrils and strengthening of the interfibrillar matrix. Despite these changes in tissue behavior, the fundamental deformation mechanisms underlying fascicle mechanics appear to remain intact, which suggests that conclusions from previous multiscale investigations of strain transfer are still valid. The effects of lower DTAF concentrations (≤10 µg/ml) on tendon mechanics were substantially smaller and potentially negligible; nevertheless, no concentration was found that did not at least slightly alter the tissue behavior. Therefore, future studies should either reduce DTAF concentrations as much as possible or use other dyes/techniques for measuring microscale deformations.

Figure 1. DTAF synthesis and structure.

DTAF is synthesized by conjugating aminofluorescein with cyanuric chloride. Extracellular matrix proteins can then be fluorescently labeled through the reaction between the remaining chloro groups (highlighted in red) attached to the triazine ring and free amine groups on the protein.

Figure 2. Experimental setup and quantification of tissue macroscale response.

(A) Uniaxial tension device mounted on a confocal microscope. (B) Representative plot of tissue mechanical response highlighting portions used to quantify the macroscale tensile properties (i.e. quasi-static tensile modulus and incremental percent stress relaxation).

Figure 3. Fascicle macroscale response.

Plots of the average stress-strain response to incremental loading for samples stained at (A) 2.5, (B) 10, (C) 100, and (D) 2000 µg/ml along with their paired non-stained controls.

Figure 4. Macroscale mechanical properties as a function of DTAF concentration and applied grip-to-grip strain level.

(A) Samples stained at high concentrations (>10 µg/ml) maintain large positive quasi-static moduli at greater applied grip-to-grip strains, suggesting that DTAF increases the tissue yield strain. (B) Staining also reduced the amount of stress relaxation throughout testing, with greater effects observed with increasing DTAF concentration. Note: The Non-Stained group contains all the paired non-stained control samples (n = 16). (C,D) Paired differences between stained samples and non-stained controls confirm that high DTAF concentrations produce (C) large increases in quasi-static modulus and (D) decreases in stress relaxation at all applied grip-to-grip strain levels. Lower DTAF concentrations (≤10 µg/ml) exhibited relatively small effects at 6-8% grip-to-grip stains. *p<0.05, ^#p<0.10.

Figure 5. Results of multiscale testing and modeling.

Multiscale testing demonstrates that higher concentrations of DTAF (A) decrease interfibrillar sliding and (B) increase fibril strains. However, the relationship between both of these microscale deformations and the macroscale tissue strains are similar between the two DTAF concentrations. Additionally, a shear lag model incorporating a perfectly plastic interfibrillar shear stress was successful in (C) fitting the macroscale fascicle mechanics (R² = 0.997) and (D) predicting the microscale fibril strains (R² = 0.68) of tendon fascicles stained at 10 µg/ml. These results are similar to the model performance for samples stained at 2000 µg/ml . Therefore, these data suggest that while DTAF alters fascicle multiscale mechanics it doesn' change the physical mechanisms underlying fascicle behavior.

Figure 6. Results of constant strain rate testing.

(A) Plots of the stress-strain response to constant strain rate testing for 2000 µg/ml samples and non-stained controls. (B) While the linear modulus was unaffected by DTAF staining, (C) an increase was observed for the ultimate tensile strength (UTS). *p<0.05.

Figure 7. Explanation for how effects of DTAF may be masked during constant strain rate testing.

Although the non-stained fascicles have a lower quasi-static modulus, they have a greater viscous response than the stained samples. Therefore, the non-stained samples stiffen more in response to the higher strain rate during the constant strain rate testing, possibly causing the stress-strain curves to overlap.