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Logo of teaMary Ann Liebert, Inc.Mary Ann Liebert, Inc.JournalsSearchAlerts
Tissue Engineering. Part A
 
Tissue Eng Part A. 2009 July; 15(7): 1865–1876.
Published online 2009 March 20. doi:  10.1089/ten.tea.2008.0319
PMCID: PMC2749875
NIHMSID: NIHMS131096

Modulation of 3D Fibrin Matrix Stiffness by Intrinsic Fibrinogen–Thrombin Compositions and by Extrinsic Cellular Activity

Haison Duong, M.Sc., Benjamin Wu, D.D.S., Ph.D., and Bill Tawil, Ph.D.corresponding author

Abstract

Fibrin is a substance formed through catalytic conversion of coagulation constituents: fibrinogen and thrombin. The kinetics of the two constituents determines the structural properties of the fibrin architecture. We have shown previously that changing the fibrinogen and thrombin concentrations in the final three-dimensional (3D) fibrin matrix influenced cell proliferation and differentiation. In this study, we further examined the effect of changing fibrinogen and thrombin concentrations in the absence or presence of fibroblasts on the structural modulus or stiffness of 3D fibrin matrices. We have prepared fibroblast-free and fibroblast-embedded 3D fibrin matrices of different fibrinogen and thrombin formulations, and tested the stiffness of these constructs using standard mechanical testing assays. Results showed that there was a corresponding increase in stiffness with increasing thrombin and fibrinogen concentrations; the increase was more notable with fibrinogen and to a lesser degree with thrombin. The effect of fibroblasts on the stiffness of the fibrin construct was also examined. We have observed a small increase in the stiffness of the fibroblast-incorporated fibrin construct as they proliferated and exhibited spreading morphology. To our knowledge, this is the first comprehensive report detailing the relationship between fibrinogen and thrombin concentrations, cell proliferation, and stiffness in 3D fibrin matrices. The data obtained may lead to optimally design suitable bioscaffolds where we can control both cell proliferation and structural integrity for a variety of tissue engineering applications.

Introduction

Biomaterials are essential for the advancement in tissue engineering because they can function as delivery vehicles for bioactive substances or cells. The preferred materials should have mechanical and biochemical properties that are biocompatible, bioabsorbable, and biodegradable; furthermore, the materials should easily be manipulated and easily reproducible. Fibrin has long been recognized as a biopolymer possessing all of the above-mentioned properties.14 Physiologic fibrin, as in a fibrin clot, is formed subsequent to tissue injury, and its formation is critical to the initiation of wound healing and to the remodeling process of the wound site. The fibrin clot is formed by enzymatic cleavage and reorganization of two key plasma-derived coagulation components, fibrinogen and thrombin. In the presence of transglutaminase factor XIIIa and calcium ions, a meshwork of cross-linked polyfibrils is formed producing a provisional matrix. Similar to physiologic fibrin, biologic fibrin can be polymerized through fibrinogen and thrombin purified from human or animal source. The polymerized fibrin can be reproduced externally and can be modified to create three-dimensional (3D) porous structures of different size, shape, viscosity, porosity, and tensile strength for a variety of tissue engineering applications: from tissue sealants5 to delivery vehicles for drugs,6 growth factors,7 and cells.811

The relationships between fibrin microstructure, rate of polymerization, and structural stiffness have been studied extensively1214 in a cell-free fibrin clot system; however, the relationship between fibrin microstructure, structural stiffness, and cell biocompatibility in a cell-incorporated system is still unclear. The stiffness of the surface to which cells adhere can affect cell morphology, protein expression, and migration. Yeung et al. have shown that fibroblasts and endothelial cells develop a spread morphology and actin stress fiber expression when grown on matrix surface with a stiffness of at least 2 kPa; whereas, stiffness has little effect on neutrophils embedded within the matrix.15 Similarly, we have shown that changing the concentration of fibrinogen and thrombin has significant effect on monocyte adhesion and proliferation.16 It has been established that the modulation of the intrinsic factors such as fibrin constituents, fibrinogen, and thrombin can have profound effect on the mechanical properties of the fibrin microstructure. Modifying the thrombin concentration may affect the rate of polymerization, fiber size, and porosity.1719 Similarly, modifying the fibrinogen concentration has also been shown to affect fiber size and porosity.20 It is still unclear whether extrinsic factors such as presence of cells can affect the stiffness of the fibrin microstructure. Most studies have focused on how the different fibrinogen to thrombin formulations may have an effect on cell morphology and survival.2123 However, the relationship between fibrinogen and thrombin with respect to structural stiffness and cellular response still needs to be addressed. In this study, we have taken a comprehensive approach to construct 3D fibrin matrices using a full range of fibrinogen to thrombin formulations to compare final structural stiffness and to determine how stiffness may correlate to cell proliferation.

Materials and Methods

Materials

Fibrin Sealant (Tisseel® Sealant Kit) containing human fibrinogen, thrombin, tris-glycine buffer, calcium chloride, and aprotinin (fibrinolysis inhibitor) was generously provided by Baxter Bioscience (Westlake, CA). Normal human dermal fibroblasts (lot number 5F1351) were purchased from Lonza (Walkersville, MD). Live/Dead® Viability Assay Kit and alamarBlue® Cell Proliferation Assay Kit were purchased from Invitrogen (Carlsbad, CA). Mechanical compressive indentation measurements of 3D fibrin matrices were performed on an Instron 5564 Universal Material Testing machine equipped with a load cell capacity of 1 kN and a Bluehill2®-integrated material testing software (Instron, Norwood, MA).

3D fibrin matrices of varying fibrinogen to thrombin concentrations

Fibrinogen (100 mg/mL) was reconstituted in 40 mM tris-glycine containing 3000 KIU/mL of aprotinin at 37°C. Thrombin (500 IU) was reconstituted in 40 mmol/mL of CaCl2; whereas, 1 IU of thrombin is defined as the activity contained in 0.0853 mg of the First International Standard of Human Thrombin. The reconstituted fibrinogen was prediluted to various concentrations ranging from 2 to 50 mg/mL. Thrombin was prediluted to various concentrations ranging from 2 to 100 IU. The resulting formulations between the two components were indicated in Table 1. Three-dimensional fibrin matrices were formed in individual wells of a 24-well tissue culture plate. Two milliliters of 3D fibrin matrices was prepared by quickly mixing equal volume of the appropriately diluted fibrinogen and thrombin solutions. A dual-barrel syringe (Discus Dental, Culver City, CA) was used to mix each matrix (Fig. 1A). The components were allowed to polymerize undisturbed at room temperature for 1 h. The resulting polymerized 3D fibrin matrices were covered with 1 mL of 40 mmol CaCl2 and incubated at 37°C. The 3D fibrin matrices measured 18 mm in diameter and 5 mm in height with average volume of 1271 mm3.

FIG. 1.
Illustration of experimental methodology. (A) Construction of acellular and fibroblast-embedded 3D fibrin matrices. A resulting 2 mL cylindrical 3D fibrin matrix was formed in each well of 24-well tissue culture plates. (B) Illustration of the ...
Table 1.
3D Fibrin Matrix Formulations Derived from Fibrinogen and Thrombin Components

Fibroblast-embedded 3D fibrin matrices of different fibrinogen to thrombin concentrations

Normal human dermal fibroblasts of early passage (<10) were counted and combined with the fibrinogen and thrombin components to yield desired fibrin formulations and cell densities (ranging from 50,000 (50k) to 5,000,000 (500k) cells). Fibroblast-embedded 3D fibrin matrices and corresponding control acellular 3D fibrin were allowed to polymerize for 1 h followed by submersion in DMEM (Mediatech, Manassas, VA) supplemented with aprotinin (3000 KIU/mL), 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (Fisher Scientific, Pittsburg, PA). The 3D fibrin matrices were maintained at 37°C and 5% CO2 in a humidified incubator until processed.

Mechanical testing

Compressive indentation measurements on acellular 3D fibrin matrices and fibroblast-embedded 3D fibrin matrices were taken using an Instron 5564 Universal Material Testing Machine. Briefly, excess liquid was removed from individual 3D fibrin matrix at the time of testing leaving the fibrin matrix attached to the walls of the well. A cylindrical flat-ended stainless steel indenter with a diameter of 3.0 mm was attached to the testing machine and was programmed to press into the 3D fibrin matrix surface at a controlled displacement rate of 5 mm/min until a final displacement of 2 mm was achieved by the indenter (Fig. 1B). It should be noted that during testing, the area and the volume of the fibrin matrix remained unchanged by matrix adhesion to the well surface; in addition, the 3.0 mm indenter diameter was specifically chosen to rule out total matrix volume displacement during testing. The indenter only displaces the matrix at a focal indentation point, whereas a larger diameter indenter would displace a greater surface area and volume of the matrix of which may affect our measurements. We have tested displacement rate from 1 to 10 mm/min and have determined that a 5 mm/min displacement rate was a reliable strain rate for these 3D fibrin matrix formulations of varying concentrations. The force displacement data were then analyzed for the mechanical stiffness of both the acellular and fibroblast-embedded fibrin matrix groups. Specifically, a nominal Young's modulus, E, was estimated based upon linearizing portions of the load displacement data and then analyzing the slope of the line in accordance with the Boussinesq elastic solution for the flat punch indentation,24 where

equation M1

where a is the punch radius (1.5 mm), υ is the Poisson's ratio of the fibrin matrix, F is the applied load force, and U is the displacement. A Poisson's ratio of 0.25 was assumed for the fibrin matrix. The Young's modulus, E, was calculated by fitting the equation to the experimental data obtained from displacements between 0.0 and 1.0 mm.

Fibroblast viability and proliferation in 3D fibrin matrices

The Live/Dead Viability/Cytotoxicity Kit (Invitrogen) employs two color fluorescent dyes: Calcein-AM that produces an intense green fluorescent (ex/em ~495/~515 nm) for live cells and EthD-1 that produces an intense red fluorescent (ex/em ~495/~635 nm) for dead cells. Direct visual assessment of fibroblast viability inside the 3D fibrin matrices was conducted using this assay kit. Briefly, at various time points of interest, fibroblast-embedded 3D fibrin matrices were washed once with 1 mL of Dulbecco's phosphate buffer saline (D-PBS). The wash buffer was removed and replaced with 1 mL of fresh serum-free DMEM containing 5 μM of Calcein-AM and 5 μM of EthD-1. A 3 h incubation period was required to ensure that the reagents had diffused through the cell-embedded fibrin matrix. After incubation, the fibroblast-embedded 3D fibrin matrices were washed three times with 2 mL of D-PBS at 10 min intervals to remove unbounded reaction products. The bicolor labeling was viewed through a Leica IRB inverted microscope (McBain Instruments, Los Angeles, CA) equipped for fluorescent detection and fitted with an Optronics® digital CCD camera for image capturing (Optronics, Goleta, CA). A Bioquant® Image Analysis System (Bioquant, Nashville, TN) was used for image analysis.

Fibroblast proliferation inside 3D fibrin matrices of varying formulations was also determined using alamarBlue Cell Proliferation/Cytotoxicity Kit (Invitrogen, Irvine, CA). alamarBlue is designed to provide a rapid and sensitive measurement of cell proliferation and cytotoxicity in various human and animal cell lines. alamarBlue is a redox indicator that yields a colorimetric change and a fluorescent signal in a response to cellular metabolic activity. The compound is nontoxic, soluble, and stable in tissue culture medium. Fibroblast-embedded 3D fibrin matrices were incubated with 1 mL of fresh DMEM and 10% FBS containing alamarBlue diluted 1:10. At various time intervals, the redox reaction, in which alamarBlue is reduced by the cells, was measured by fluorescent reading at excitation wavelength of 535 nm and emission wavelength of 595 nm. The fluorescent measurements were translated into cell number against a standard curve using different cell densities. The fluorescent measurements were performed on an Infinite® 200 series microplate reader (Tecan, San Jose, CA).

Inhibition of matrix contraction of fibroblast by staurosporine

To assess whether fibroblast contraction may exert an effect of matrix stiffness, fibroblast-embedded fibrin matrices were treated with or without 0.5 μM of staurosporine (Sigma, St. Louis, MO). The fibroblast-embedded matrices were cultured up to 14 days followed by Live/Dead assessment and mechanical indentation testing.

Statistical analysis

Data were presented as mean ± standard deviation from three or more experiments with each experimental parameter done in triplicates. The statistical significance was assessed by paired Student's t-test, and a p-value of less than 0.05 was considered statistically significant, while a p-value of 0.01 was considered highly significant. Some data were analyzed by linear regression analysis, and statistical significance was assessed by two-way ANOVA with a Bonferroni posttesting. Statistical analysis was performed using GraphPad Prism® 4 analysis software (GraphPad Software, San Diego, CA).

Results

Structural modulus or stiffness of acellular 3D fibrin matrices comprised of different fibrinogen or thrombin concentrations

To determine how changing the fibrinogen or thrombin concentrations may affect the structural modulus of the 3D fibrin construct, we have prepared broad range of matrices comprising of different fibrinogen and thrombin formulations (Table 1). The structural modulus of the 3D fibrin constructs herein shall be loosely defined as an index of stiffness.

Modulating the fibrinogen and thrombin concentrations represent two separate variables that we have evaluated for the stiffness correlation of the 3D fibrin matrices: (i) by comparing different concentrations of fibrinogen (ΔF) with respect to a constant thrombin concentration (~T) to obtain a stiffness versus changing fibrinogen concentrations correlation; (ii) by comparing different thrombin concentrations (ΔT) with respect to a constant fibrinogen concentration (~F) to obtain a stiffness versus changing thrombin concentrations correlation. For ΔF test, we have prepared six groups of 3D fibrin matrices with each group comprising 2, 5, 10, 20, 50, or 100 IU/mL of thrombin. For each group, the fibrinogen concentrations varied from 2, 5, 17, 34, and 50 mg/mL. For the ΔT test, we have constructed five groups of 3D fibrin matrices with each group comprising 2, 5, 17, 34, or 50 mg/mL of fibrinogen, and the thrombin concentrations were varied within each group (2–100 IU/mL).

Mechanical indentation for the ΔF tests (Fig. 2A) showed that the structural modulus of the 3D fibrin matrices increased with increasing fibrinogen concentration. This effect was measured in all groups of thrombin concentrations from 2 to 100 IU/mL (Fig. 2A). Paired Student's t-test analysis showed statistical significance (*p < 0.001) of increasing stiffness in response to ΔF with matrices having lower kPa values at 2 mg/mL of fibrinogen and higher kPa values as the fibrinogen concentration increased up to 50 mg/mL. This was observed in all groups of ~T. In addition, there were significant differences (**p < 0.001) in the change in stiffness between ΔF at 2 and 5 mg/mL between 5 and 17 mg/mL, between 17 and 34 mg/mL, and between 34 and 50 mg/mL (Fig. 2A). The percent difference in stiffness corresponding to ΔF was also evaluated (Fig. 3A). At ~T of 2 IU/mL, changing the fibrinogen concentration from 2 to 5 mg/mL resulted in a fivefold increase in the stiffness of the matrices. At 17 mg/mL, the increase was 12-fold; at 34 mg/mL, the increase was 24-fold; and at 50 mg/mL, the increase was 46-fold. Similar increases in stiffness were measured in all groups of thrombin (2, 5, 10, 20, 50, and 100 IU/mL) (Fig. 3A). To evaluate whether the corresponding increases were linear, we have performed a linear regression analysis against the data obtained (data not shown). Results showed that the increase in the structural modulus of the matrices linearly corresponds to increasing fibrinogen concentrations at ~T. The R2 values were greater than 0.97, and two-way ANOVA analysis showed that fibrinogen had the same significant effect (p < 0.001) at all values of thrombin.

FIG. 2.
Structural modulus values of stress over strain indentation test as expressed in kPa to indicate the stiffness of 3D fibrin matrices. (A) Change in matrix stiffness correlates to increasing fibrinogen concentrations (2–50 mg/mL) in six ...
FIG. 3.
Percent difference in the stiffness of the 3D fibrin matrices calculated from the mean of the measured stiffness. (A) Evaluation of data at constant thrombin, ~T, relative to changing fibrinogen concentrations (ΔF). Inset numbers in bars ...

Evaluating the data with respect to ΔT and at ~F showed that the structural modulus of the 3D fibrin matrices increased when the thrombin concentration was increased (Fig. 2B), but in the lowest fibrinogen formulation of 2 mg/mL, the stiffness varied with increasing thrombin concentration. At ~F of 5 mg/mL, the stiffness steadily increased as the thrombin concentration increased (from 2 to 20 IU/mL); however, at thrombin concentrations of 50 and 100 IU/mL, the stiffness appeared to have declined compared to the 20 units/mL of thrombin formulation. Similarly, at ~F of 17, 34, and 50 mg/mL, changing the thrombin concentrations showed significant increases in the stiffness, with decreasing values measured in matrices comprising 100 IU/mL of thrombin. Paired Student's t-test showed statistical significance where p < 0.05 for all ΔT values compared to lower 2 IU/mL concentrations. In addition, statistical significance was observed when compared 2 to 5 IU/mL, 5 to 10 IU/mL, 10 to 20 IU/mL, 20 to 50 IU/mL, and 50 to 100 IU/mL. Evaluating the percent differences of the stiffness corresponding to ΔT showed that at ~F of 2 mg/mL, the percent differences varied with at various thrombin values (Fig. 3B). Increasing the thrombin concentrations, from 2 to 5 IU/mL, resulted in a 3-fold difference; at 10 IU/mL, a 4.6-fold difference; at 20 IU/mL, 2.3-fold; at 50 IU/mL, 4-fold; and at 100 IU/mL, 3-fold. This variability in values was diminished as the fibrinogen concentration increased. At ~F of 5 mg/mL, a 1.3-fold difference was observed when the thrombin concentration was increased from 2 to 5 IU/mL; 10 IU/mL resulted in a 1.6-fold difference; 20 IU/mL resulted in a 1.8-fold difference; 50 IU/mL resulted in a 1.2-fold difference; and 100 IU/mL resulted in a 1.5-fold difference. Similar result was observed at ~F of 17, 34, and 50 mg/mL. Linear regression showed that increasing the thrombin concentration at lower ~F of 2 mg/mL does not affect the gel stiffness linearly, where R2 < 0.5 for most values and that rate of change or differences varied greatest at lower 2 mg/mL of fibrinogen; however, in matrices of higher fibrinogen concentrations, >5 mg/mL, the effect of thrombin on the matrices' stiffness appeared to follow a more linear trend. With the exception at the 100 IU/mL of thrombin formulation, the stiffness decreased compared to the 20 IU/mL formulation. Two-way ANOVA showed that thrombin does not have the same effect at all values of fibrinogen, and that the effect is only significant in matrices consisted of fibrinogen concentrations >5 mg/mL. In summary, fibrinogen accounted for 84% of the total variance, while thrombin accounted only 5.8% of the total variance. The data analysis showed that changing the fibrinogen concentrations had a greater effect on the structural modulus of the 3D matrices than changing the thrombin concentrations.

Fibroblast proliferation in 3D fibrin matrices of different fibrinogen and thrombin concentrations

To evaluate how fibrin composition may affect cell proliferation, we seeded normal human fibroblasts (200,000 (200k) cells) in 3D fibrin matrices of different fibrinogen formulations: 5, 17, and 34 mg/mL at ~T of 2 IU/mL. To exclude the degradation effect, 3000 KIU/mL of aprotinin was added to the matrices, and the cells were allowed to grow in the 3D fibrin matrices for 1 to 10 days in normal culture media supplemented with aprotinin. At various time points, Live/Dead assessment of fibroblasts viability and mortality was visualized by Calcein-AM/EthD-1 labeling. For proliferation studies, cellular metabolic activity of fibroblast embedded in 3D fibrin matrices was determined by alamarBlue. Absorbance reading from the assay was interpreted as the relative number of cellular activity or cell number. Results showed that in the 3D fibrin matrices consisted of 5 mg/mL of fibrinogen, fibroblast continued to proliferate from day 1 through day 10 in culture (Fig. 4A). In the 17 mg/mL formulation, the proliferation values increased at day 7; by day 10, the values had remained the same as that of day 7; conversely, in the 34 mg/mL formulation, fibroblast proliferation did not increase with respect to day 1; rather, there was a slight decrease at both days 7 and 10 (Fig. 4A). Paired Student's t-tests showed that there was significant fibroblast growth inhibition in 3D fibrin constructs consisted of fibrinogen concentration >5 mg/mL; further, the effect was also significant as time progressed. Fibroblast proliferation in the 5 mg/mL formulation increased linearly by as much as 80% after 7 days in culture, and at day 10, the proliferation was greater than 100%. In the 17 mg/mL formulation, the proliferation was less linear and only increased by 24% by 7 days and 26% by 10 days. Proliferation was not observed in formulation of 34 mg/mL; in fact, fibroblast growth inhibition was observed (Fig. 4B). The lack of EthD-1 labeling for dead cells in all fibroblast-embedded 3D fibrin matrices suggested that cell death was not the cause of growth inhibition as observed in higher fibrin formulation, where they displayed abnormal morphology. Morphological assessment showed that fibroblasts formed densely packed cell network and extensions (cell spreading) in lower 5 mg/mL fibrin formulation; whereas, in the 17 and 34 mg/mL formulations, the cells appeared spherical and cell spreading was minimal (Fig. 4C).

FIG. 4.
Fibroblast proliferation in 3D fibrin matrices of different fibrinogen concentrations (5–34 mg/mL) and at ~T of 2 units/mL. (A) alamarBlue determination of fibroblast-embedded 3D fibrin matrices from 1 to 10 days showed ...

Effect of initial cell seeding density on the structural stiffness of 3D fibrin matrices

To examine whether mixing cells with the fibrin formulation components would affect the kinetic rate of fibrin polymerization and, ultimately, the microstructure and structural modulus of the 3D matrices, fibroblasts, from low to high cell densities (0–5000k cells), were incorporated into 3D fibrin matrices comprising ~F of 5 mg/mL and ~T of 5 IU/mL. Mechanical indentation tests showed that there were insignificant differences (<5%) between the structural modulus of the acellular 3D fibrin matrices (no cells) and that of those matrices with fibroblasts embedded in them (Fig. 5). There were little differences in the stiffness of 3D fibrin matrices containing 200k fibroblasts and that of the constructs with high cell density (5000k cells).

FIG. 5.
Cell seeding density and matrix stiffness test. Fibroblasts seeded from 0 to very high density, 5000k cells, in 3D fibrin matrices were evaluated 1 h after polymerization of matrices. Values are expressed as mean ± SD ( ...

Effect of cell density on proliferation in 3D fibrin matrices

To find the optimum cell seeding density for the 3D fibrin matrices, we incorporated fibroblasts at various densities into matrices of ~F = 5 mg/mL and ~T = 2 IU/mL. AlamarBlue assessment showed that there was a linear increase in fibroblast proliferation from day 0 up to day 21. This increase was observed in all cultures of different seeding densities, and the paired Student's t-test showed significance, p < 0.05, when compared day 1 culture to all other time points (Fig. 6A). The percent change in cell number was also evaluated (Fig. 6B). After 7 days, in the 50k cell density group, fibroblast number increased significantly by 91%, by 23% in the 100k cell density group, by 105% in the 200k cell density group, and by 15% in the 400k cell density group. After 14 days, the 50k cell density group had a 650% increase in cell number, 319% in the 100k cell density group, 183% in the 200k cell density group, and 16% in the 400k cell density group. By 21 days, the 50k cell density group had increased by 1000%, the 100k cell density group by 660%, the 200k cell density group by 274%, and the 400k cell density group by only 61%. From the evaluation, it was clear that there was a correlation between cell seeding density and cell proliferation over time. In this case, seeding fibroblasts at 50,000 cells appeared to be the optimal density for incorporation into 3D fibrin matrices, where cells showed continued growth for up to 21 days and beyond. In comparison, in matrices where the initial seeding density was at 400,000 cells, fibroblast proliferation was minimal.

FIG. 6.
Effect of cell seeding density on fibroblast proliferation in 3D fibrin matrices at ~F = 5 mg/mL and ~T = 5units/mL. Fibroblasts were embedded in the matrices at different cell densities ranging ...

Effect of seeding density and changes in cell proliferation on structural modulus of 3D fibrin matrices

The relationship between initial seeding density, proliferation over time, and effect on stiffness was examined. After 24 h in culture, the structural modulus of the matrices was measured. Compared to the acellular matrices, the 50k cell density and the 100k cell density group had lower kPa values than those of the acellular matrices, whereas the 200k cell density and the 400k cell density group had high kPa values. Throughout the course of 21 days in culture, all cell groups showed increases in stiffness. This increase was most significant (p < 0.05) at day 21. Comparing stiffness values to the acellular matrices, seeding cells within the fibrin matrices does affect the stiffness over time. The effect by cells was further investigated by comparing the effect on stiffness by cell densities. Results showed that there were definite differences between the 50k cell density group and the other higher cell density groups. Most notably, at 21 days, the stiffness of the 400k cell density group was greater than the stiffness of the 50k cell density group (Fig. 7A).

FIG. 7.
Relationships between fibroblast initial seeding density and matrix stiffness. (A) Average values ± SD (n = 3) for the structural modulus of matrices of different cell densities measured at days 1, 7, 14, and 21. ...

In comparison to time in the fibrin matrices, the 50k cell density group and the 100k cell density group started with lower stiffness values; however, as the culture time increased, the percent change in stiffness of the matrices correspondingly increased with significance at day 21. The percent change was calculated by dividing the stiffness values of the day 7-21 groups to that of the day 1 groups. This would indicate the degree of change in the stiffness every 7 days of culture time up to 21 days for various cell seeding densities. The 200k and 400k groups had small increases in stiffness from day 7 and reaching significant increases after 21 days (Fig. 7B).

Correlation between cell proliferation and change in structural modulus

The percent change in cell proliferation was compared to the percent change in the stiffness of the matrices (Fig. 8A). After 7 days of culture, the 50k cell density group had an average increase of 91% in cell number; correspondingly, the average change in stiffness for the matrices was recorded at 45%, thereby yielding a change in proliferation to change in stiffness index ratio of 2:1. At 100k cell density, the ratio was 0.8:1; at 200k cell density, the ratio was 5.6:1; and at 400k cell density, the ratio was 6.8:1 (Fig. 8B). This index ratio would vary among all groups, with the 50k cell density group recording a continued increase in the change in proliferation to change in stiffness index ratio at days 14 and 21. The 100k cell density and the 200k cell density groups recorded fluctuations in the change in proliferation to change in stiffness index ratio at those time points. Whereas in the 400k cell density group, the change in proliferation to change in stiffness index ratio decreased to 1.3:1 at day 14 and 0.8:1 at day 21. The larger index ratio value suggested that the disparity between the change in cell proliferation and the change in stiffness was smaller. As time progressed, the 50k cell density groups had increasing ratio values, suggesting that the large increase in fibroblast proliferation within the 3D fibrin matrices affected the structural modulus of the matrices minimally. In the 100, 200, and 400k cell density groups the change in proliferation to change in stiffness ratios appeared to be decreasing as culture time progressed to 21 days. Most notably, the 400k cell density had the smallest ratio after 21 days in culture. This would suggest that by day 21, the 400k density groups had decreased proliferation, while the corresponding structural modulus continued to increase, thereby suggesting that some other factor besides cellular proliferation is contributing to the increase in the structural stiffness. We have observed that by day 14, the fibroblast-embedded fibrin matrices appeared compacted with decreasing size and volume and that the compaction was more significant in the 400k density groups.

FIG. 8.
Correlation between cell proliferation and 3D fibrin matrix stiffness. (A) Plot obtained from data shown in Figures 6 and and7.7. At various time periods (7–21 days), the effect of cell seeding density on the change in cell number was ...

Effect of cell contraction of the 3D fibrin matrix and corresponding structural modulus

As the culture time increased, the 3D fibrin matrices decreased in volume (Fig. 9A). Morphological assessment of the fibroblasts within the matrices revealed that over time, the cells proliferated and formed densely packed networks by cell spreading. We next tested whether fibroblast proliferation had an effect on matrix contraction, thereby affecting the structural modulus. Fibroblast cell spreading was inhibited by adding staurosporine to the culture media. After 14 days, Live/Dead labeling showed that the staurosporine had no toxic effect on the cells, and that cell spreading was inhibited (Fig. 9C). We have shown that the 3D fibrin matrices of the 50k cell density group did not contract. The effect of staurosporine in this cell density group was not clear; however, in the other higher cell density groups, the effect of staurosporine clearly showed that cell spreading does contribute to the stiffness of the matrices. The 100k group recorded stiffness differential of 39% in presence of staurosporine; similarly, the 200k group recorded 16.7%, and the 400k cell density group recorded 34.4% (Fig. 9B).

FIG. 9.
Effect of fibroblast contraction on matrices and stiffness. (A) Day 14 fibroblast 3D fibrin matrices showing contraction or loss of volume and matrix size as indicated by double-headed arrows. (B) Plot showing day 14 differences in matrix stiffness between ...

Discussion

The unique feature of fibrin is that it can be modified to create microstructures of different porosities and stiffness to simulate an environment that is optimized for cell growth and survival. This would include matching the mechanical properties of the specific tissue type. For cell-incorporated fibrin matrices, porosity and structural stiffness are important mechanical properties to consider. Porosity allows for diffusion of macromolecules and nutrients for cell signaling and proliferation, as well as facilitating cell retention. For example, fibrin matrices with large, opened network of fibrils may result in increased cell loss and faster degradation of the fibrin material, whereas matrices with dense network may retain cells and resist fibrinolysis.25 The porosity of the fibrin matrices can be controlled by modifying the thrombin and fibrinogen concentration.26 Modifying the thrombin and fibrinogen concentrations has been shown to affect the rigidity of the fibrin microstructure.14,15,21

The relationship between structural stiffness and cell processes has been investigated. Lo et al.27 have shown that fibroblasts exhibit spreading morphology and preferentially migrate from a soft to hard surface. Similarly, Yeung et al.15 have reported that fibroblasts developed spreading morphology when grown on surfaces with a stiffness modulus of greater than 2 kPa, and that different cell types may prefer different stiffness for growth and survival. Most cells rely on a stiffness-sensing feedback mechanism to which they can interact with the substrate surface.28

In this study, we have explored a wide range of formulations involving different concentrations of fibrinogen and thrombin to create 3D fibrin matrices of varying stiffness. The range in stiffness was measured between 0.058 kPa (for lower concentration of fibrinogen/thrombin formulation) and 4 kPa (for higher concentration of fibrinogen/thrombin formulation). Much of the increase in the stiffness was directly related to increasing fibrinogen concentration, and to a lesser degree to thrombin concentration. Physiologic fibrinogen and thrombin concentration for clot formation has been measured around 2 mg/mL and 0.01 to 0.1 units/mL of thrombin.17,29 Our formulations include the low 2 mg/mL of fibrinogen and the low 2 units/mL of thrombin, and we have extended range of the formulation to a high 50 mg/mL of fibrinogen and 100 units/mL of thrombin. From this study, we now have a correlation between stiffness and fibrinogen/thrombin formulation; in general, a low stiffness value of 0.05 kPa to a high stiffness value of 4 kPa could be achieved using this range of formulations. This range falls within the required substrate stiffness of 0.001 to 10 kPa for cell attachment as reported in other studies.30,31 Interestingly, in our fibroblast proliferation study within 3D fibrin matrices comprising 5, 17, and 34 mg/mL of fibrinogen at constant thrombin of 5 units/mL, the respective stiffness was 0.4, 0.7, and 1.5 kPa. These values fall short of the 2 to 3 kPa value as indicated in Yeung et al.15 for fibroblast mechanosensing and cell spreading. However, our morphological assessment showed that the fibroblast favored lower 5 mg/mL concentration, at 0.4 kPa, as opposed to the 34 mg/mL concentration of 0.7 kPa. Clearly, at least in our system, the fibroblast preferred formulations that yielded lower kPa values. Fibroblasts in lower formulation continued to proliferate and expressed spreading morphology, whereas the proliferation was decreased and cell spreading inhibited in higher formulations.

We next asked whether proliferation of fibroblasts within the fibrin matrix could contribute to changes in the structural stiffness. Seeding fibroblasts at various cell densities (50,000 to 400,000 cells per matrix) and culturing them up to 21 days showed that cell proliferation and culture time do not significantly alter the structural stiffness of the matrix. Interestingly, we have observed that an initial seeding density of 50,000 cells was optimal for ensuring continued proliferation within the matrices, whereas with higher initial cell seeding density of 400,000 cells, the rate of proliferation had diminished over time.

Degradation and shrinkage are major factors that can affect the 3D fibrin architecture and, ultimately, its stiffness. Fibrinolysis was inhibited by the addition of aprotinin into the matrices at the time of polymerization. When fibroblasts were incorporated, initial cell seeding density does not alter the stiffness of the matrices; however, as fibroblasts proliferated, matrix compaction or contraction was observed, and correspondingly, there was a significant increase in the structural stiffness of those matrices that have compacted. Inhibition of fibroblast spreading and expression of actin stress fibers through exposure to staurosporine showed that the compaction of the 3D fibrin matrices by fibroblasts may increase the stiffness by as much as 39%.

In conclusion, we have presented a detailed analysis of how changing the intrinsic factors such as fibrinogen and thrombin correlates to change in the structural stiffness of the 3D fibrin matrices; in addition, we have explored various extrinsic factors, such as cell proliferation, cell seeding density, and cell contraction of matrix, and how these can affect the structural stiffness.

Acknowledgment

This study was supported by the NIH/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) (AR05325001A).

Disclosure Statement

No competing financial interests exist.

References

1. Tawil B. Fibrinits applications. Review article. In: Guelcher S.A., editor; Hollinger J.O., editor. An Introduction to Biomaterials. Chapter 7. London: CRC Taylor & Francis; 2005. pp. 105–120.
2. Ye Q. Zund G. Benedikt P. Jockenhoevel S. Hoestrup S.P. Sakyama S. Hubbell J.A. Turina M. Fibrin gel as a three dimensional matrix in cardiovascular tissue engineering. Eur J Cardiothorac Surg. 2000;17:587. [PubMed]
3. Joekenhoevel S. Zund G. Hoerstrup S.P. Chalabi J.S. Demircan L. Messmer B.J. Turina M. Fibrin gel—advantages of a new scaffold in cardiovascular tissue engineering. Eur J Cardiothorac Surg. 2001;19:424. [PubMed]
4. Grassl E.D. Oegema T.R. Tranquillo R.T. Fibrin as an alternative biopolymer to type I collagen for fabrication of a media-equivalent. J Biomed Matter Res. 2002;60:607. [PubMed]
5. Sierra D.H. Fibrin sealant adhesive systems: a review of their chemistry, material properties, and clinical applications. J Biomater Appl. 1993;7:309. [PubMed]
6. MacPhee M.J. Singh M.P. Brady R., Jr. Akhyani N. Liau G. Lasa C., Jr. Hue C. Best A. Drohan W. Fibrin sealant: a versatile delivery vehicle for drugs and biologics. In: Sierra D.H., editor; Saltz R., editor. Surgical Adhesives and Sealants Current Technology and Applications. Lancaster, PA: Techomic Publishing Co.; 1996. pp. 109–120.
7. Wong C. Inman E. Spaethe R. Helgerson S. Fibrin-based biomaterials to deliver human growth factors. Thromb Haemost. 2003;89:573. [PubMed]
8. Silverman R.P. Passaretti D. Huang W. Randolph M.A. Yaremchuk M.J. Injectable tissue-engineered cartilage using a fibrin glue polymer. Plast Reconstr Surg. 1999;103:1809. [PubMed]
9. Wechselberger G. Schoeller T. Stenzl A. Ninkovic M. Lille S. Russell R.C. Fibrin glue as a delivery vehicle for autologous urothelial cell transplantation onto a prefabricated pouch. J Urol. 1998;160:583. [PubMed]
10. Bensad W. Triffitt J.T. Blanchat C. Oudina K. Sedel L. Petite H. A biodegradable fibrin scaffold for mesenchymal stem cell transplantation. Biomaterials. 2003;24:2497. [PubMed]
11. Christmann K.L. Vardanian A.J. Fang Q. Sievers R.E. Fok H.H. Lee R.J. Injectable fibrin scaffold improves cell transplant survival, reduces infarct expansion, and induces neovasculature formation in ischemic myocardium. J Am Coll Cardiol. 2004;44:654. [PubMed]
12. Roberts W.W. Lorand L.L. Mockros L.F. Viscoelastic properties of fibrin clots. Biorheology. 1973;10:29. [PubMed]
13. Fukada E. Kaibara M. The dynamic rigidity of fibrin gels. Biorheology. 1973;10:129. [PubMed]
14. Ferry J.D. Morrison P.R. Preparation and properties of serum and plasma proteins, VIII, the conversion of human fibrinogen to fibrin under various conditions. J Am Chem Soc. 1947;69:388. [PubMed]
15. Yeung T. Georges P.C. Flanagan L.A. Marg B. Ortiz M. Funaki M. Zahir N. Ming W. Weaver V. Janmey P.A. Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil Cytoskelet. 2005;60:24. [PubMed]
16. Mana M. Cole M. Cox S. Tawil B. Human U937 monocyte behavior and protein expression on various formulations of three-dimensional fibrin clots. Wound Repair Regen. 2006;14:72. [PubMed]
17. Bateman R.M. Leong H. Podor T. Hodgson K.C. Walley K.R. The effect of thrombin concentration on fibrin clot structure imaged by multiphoton microscopy and quantified by fractal analysis. Microsc Microanal. 2005;11:1018.
18. Wolberg A.S. Thrombin generation and fibrin clot structure. Blood Rev. 2007;21:131. [PubMed]
19. Ryan E.A. Mockros L.F. Weisel J.W. Laszlo L. Structural origins of fibrin clot rheology. Biophys J. 1999;77:2813. [PubMed]
20. Zhao H. Ma L. Zhou J. Mao Z. Gao C. Shen J. Fabrication and physical and biological properties of fibrin gel derived from human plasma. Biomed Mater. 2008;3:1. [PubMed]
21. Rowe S.L. Lee S.Y. Stegemann J.P. Influence of thrombin concentration on the mechanical and morphological properties of cell-seeded fibrin hydrogels. Acta Biomater. 2007;3:59. [PMC free article] [PubMed]
22. Cox S. Cole M. Tawil B. Behavior of human dermal fibroblasts in three-dimensional fibrin clots: dependence on fibrinogen and thrombin concentration. Tissue Eng. 2004;10:942. [PubMed]
23. Catelas I. Sese N. Wu B. Dunn J. Helgerson S. Tawil B. Human mesenchymal stem cell proliferation and osteogenic differentiation in fibrin gels in vitro. Tissue Eng. 2006;12:2385. [PubMed]
24. Sneddon I.N. The relation between load and penetration in the axisymmetric Boussinesq problems for a punch of arbitrary profile. Int J Eng Sci. 1965;3:47.
25. Collet J.P. Park D. Lesty C. Soria J. Soria G. Montalescot J. Weisel J.W. Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches confocal microscopy. Arterioscler Thromb Vasc Biol. 2000;20:1353. [PubMed]
26. Linnes M.P. Ratner B.D. Giachelli C.M. A fibrinogen-based precision micrporous scaffold for tissue engineering. Biomaterials. 2007;28:5298. [PMC free article] [PubMed]
27. Lo C.M. Wang H.B. Dembo M. Wang Y.L. Cell movement is guided by the rigidity of the substrate. Biophys J. 2000;79:144. [PubMed]
28. Pedham R.J. Wang Y. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci USA. 1997;94:13661. [PubMed]
29. Wolberg A.S. Thrombin generation and fibrin clot structure. Blood Rev. 2007;21:131. [PubMed]
30. Bao G. Suresh S. Cell and molecular mechanics of biological materials. Nat Mater. 2003;2:715. [PubMed]
31. Wakatsuki T. Kolodney M.S. Zahalak G.I. Elson E.L. Cell mechanics studied by a reconstituted model tissue. Biophys J. 2000;79:2353. [PubMed]

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