Compression-induced structural and mechanical changes of fibrin-collagen composites

Abstract

Fibrin and collagen as well as their combinations play an important biological role in tissue regeneration and are widely employed in surgery as fleeces or sealants and in bioengineering as tissue scaffolds. Earlier studies demonstrated that fibrin-collagen composite networks displayed improved tensile mechanical properties compared to the isolated protein matrices. Unlike previous studies, here unconfined compression was applied to a fibrin-collagen filamentous polymer composite matrix to study its structural and mechanical responses to compressive deformation. Combining collagen with fibrin resulted in formation of a composite hydrogel exhibiting synergistic mechanical properties compared to the isolated fibrin and collagen matrices. Specifically, the composite matrix revealed a one order of magnitude increase in the shear storage modulus at compressive strains>0.8 in response to compression compared to the mechanical features of individual components. These material enhancements were attributed to the observed structural alterations, such as network density changes, an increase in connectivity along with criss-crossing, and bundling of fibers. In addition, the compressed composite collagen/fibrin networks revealed a non-linear transformation of their viscoelastic properties with softening and stiffening regimes. These transitions were shown to depend on protein concentrations. Namely, a decrease in protein content drastically affected the mechanical response of the networks to compression by shifting the onset of stiffening to higher degrees of compression. Since both natural and artificially composed extracellular matrices experience compression in various (patho)physiological conditions, our results provide new insights into the structural biomechanics of the polymeric composite matrix that can help to create fibrin-collagen sealants, sponges, and tissue scaffolds with tunable and predictable mechanical properties.

Keywords: Collagen; Compression; Fibrin; Fibrin-collagen composites; Structural properties; Viscoelasticity.

Figure 1

Schematic diagram of the experimental procedure to measure shear viscoelastic response of compressed fibrin, collagen, or fibrin-collagen matrices.

Figure 2

A, B, C The elastic or storage modulus G′ (A), the loss or viscous modulus G″ (B), and the viscosity/elasticity ratio [tan(δ)= G″/ G′] as a function of compressive strain for the fibrin-collagen, fibrin, and collagen networks. Fibrin matrix was formed from two-fold diluted human platelet-poor plasma (fibrinogen concentration 2–3.5 mg/mL) and the final collagen concentration was 1.5 mg/mL. Each curve is an average over three samples prepared and measured under the same conditions (M±SD).

Figure 3

A, B, C The elastic or storage modulus G′ (A), the loss or viscous modulus G″ (B), and the viscosity/elasticity ratio [tan (δ)=G″/G′], (C) as a function of compressive strain for the fibrin clots formed from undiluted (fibrinogen concentration 2–3.5 mg/mL) and diluted (fibrinogen concentration 1–1.75 mg/mL) platelet-poor plasma (PPP). D, E, F: The elastic or storage modulus G′, (D), the loss or viscous modulus G″, (E), and viscosity/elasticity ratio (F) as a function of compressive strain for the collagen network at two different protein concentrations, 1.5 mg/ml and 2.5 mg/ml. Each curve is an average over three collagen samples prepared and measured under the same conditions (M±SD).

Figure 4

Normal stress as a function of compressive strain for fibrin-collagen, fibrin, and collagen networks. Fibrin matrix was formed from two-fold diluted human platelet-poor plasma (fibrinogen concentration 2–3.5 mg/mL) and the final collagen concentration was 1.5 mg/mL. Each curve is an average over three constructs prepared and measured under the same conditions (M±SD).

Figure 5

Scanning electron microscopy images of fibrin (A) collagen (B) and fibrin-collagen (C) matrices compressed to more than 1/10th of their initial thickness. D, E: Quantitative measurements of collagen, fibrin and mixed scaffolds: (D) the mean diameter of individual fibers (M±SD) and (E) the mean thickness of fiber bundles (M±SD). An asterisk (*) is used to indicate the statistical difference when compared with collagen gel (two-tailed Mann-Whitney test, P<0.05, n = 220). The final fibrinogen and collagen concentrations were 2–3.5 mg/mL and 1.5 mg/mL, respectively (see also Supplementary Figure S2 and Figure S3).

Figure 6

Confocal microscopy images of uncompressed (top) and compressed (bottom) fibrin (A, D), collagen (B, E), and composite (C, F) matrices shown as confocal z-stack slices of 3D networks. Images of compressed networks were taken at a compressive strain γ=0.5. Collagen was imaged in reflection mode (red) and fluorescently labeled fibrin was visualized using fluorescence mode (green). The final fibrinogen and collagen concentrations were 2–3.5 mg/mL and 1.5 mg/mL, respectively (see also Supplementary Figure S4 and Figure S5).

Figure 7

A: Absolute node density for uncompressed (γ=0) and two-fold compressed (γ=0.5) networks of individually prepared fibrin and collagen matrices and fibrin-collagen fibers in the composite (M±SD); *P<0.05, 1000 nodes in 3 samples, two-tailed Mann-Whitney test. B: Connectivity distribution in terms of true and apparent branching points resulted from oblique impact of fibers. Here, ρ_T is the absolute node density of the network and n is the number of fibers irradiating from true or apparent branching points: true branching points (n=3), criss-crossing points (n=4), and their superposition (n=5 and n=6). Each symbol is an average over three different regions of a network (M±SD). Fibrin matrix was formed from two-fold diluted human platelet-poor plasma (fibrinogen concentration 2–3.5 mg/mL) and the final collagen concentration was 1.5 mg/mL.

Figure 8

A confocal microscopy image of the fibrin-collagen composite network exhibiting bundling (A) and criss-crossing (B) of fibers in the compressed matrix (γ=0.5). Here, ‘c’ stands for collagen and ‘f’ indicates fibrin fibers. The final fibrinogen and collagen concentrations were 2–3.5 mg/mL and 1.5 mg/mL, respectively.

Figure 9

Bottom: The absolute node density as a function of the node connection degree, n, in uncompressed and two-fold compressed fibrin networks formed from diluted plasma and in the presence of collagen (fibrin-collagen composite). Here, n is the number of fibers irradiating from true or apparent branching points: true branching points (n=3), criss-crossing points (n=4), and their superposition (n=5, 6). Each bar is an average over three different regions of a network (M±SD); *P<0.05, 1000 nodes in 3 samples, two-tailed Mann-Whitney test. Top: Confocal microscopy images of the uncompressed fibrin network in the absence (fibrin) and presence of collagen (fibrin in composite). Fibrin matrix was formed from two-fold diluted human platelet-poor plasma (fibrinogen concentration 2–3.5 mg/mL) and the final collagen concentration was 1.5 mg/mL.

Figure 10

Fiber segment length variations in uncompressed (γ=0) and compressed (γ=0.5) fibrin, collagen and fibrin-collagen networks. The box shows the interquartile range, a line in the box marks the median, whiskers indicate minimum and maximum values and the circle in the box shows the mean value. Fibrin matrix was formed from two-fold diluted human platelet-poor plasma (fibrinogen concentration 2–3.5 mg/mL) and the final collagen concentration in collagen and fibrin-collagen constructs was 1.5 mg/mL. Differences are statistically significant when compared to uncompressed matrices and collagen samples (P<0.01, 1200 segments in 3 samples, two-tailed Mann-Whitney test).