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. Author manuscript; available in PMC: 2022 Aug 1.
Published in final edited form as: Colloids Surf B Biointerfaces. 2021 Apr 29;204:111805. doi: 10.1016/j.colsurfb.2021.111805

Synthesis of Sonicated Fibrin Nanoparticles that Modulate Fibrin Clot Polymerization and Enhance Angiogenic Responses

Colleen A Roosa 1,2, Ismaeel Muhamed 1,2, Ashlyn T Young 1,2, Kimberly Nellenbach 1,2, Michael A Daniele 1,2,3, Frances S Ligler 1,2, Ashley C Brown 1,2,*
PMCID: PMC8217261  NIHMSID: NIHMS1701129  PMID: 33964527

Abstract

Chronic wounds can occur when the healing process is disrupted and the wound remains in a prolonged inflammatory stage that leads to severe tissue damage and poor healing outcomes. Clinically used treatments, such as high density, FDA-approved fibrin sealants, do not provide an optimal environment for native cell proliferation and subsequent tissue regeneration. Therefore, new treatments outside the confines of these conventional fibrin bulk gel therapies are required. We have previously developed flowable, low-density fibrin nanoparticles that, when coupled to keratinocyte growth factor, promote cell migration and epithelial wound closure in vivo. Here, we report a new high throughput method for generating the fibrin nanoparticles using probe sonication, which is less time intensive than the previously reported microfluidic method, and investigate the ability of the sonicated fibrin nanoparticles (SFBN) to promote clot formation and cell migration in vitro. The SFBNs can form a fibrin gel when combined with fibrinogen in the absence of exogenous thrombin, and the polymerization rate and fiber density in these fibrin clots is tunable based on SFBN concentration. Furthermore, fibrin gels made with SFBNs support cell migration in an in vitro angiogenic sprouting assay, which is relevant for wound healing. In this report, we show that SFBNs may be a promising wound healing therapy that can be easily produced and delivered in a flowable formulation.

Keywords: Fibrin, nanoparticles, wound healing, regenerative medicine, fibrin sealant

Graphical Abstract

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1. Introduction:

Chronic wounds are caused by a disruption in the normal wound healing process. Over 6.5 million patients in the United States are affected by chronic wounds with an estimated cost of treatment of $28 to $96 billion per year1. Untreated chronic wounds can lead to significant loss of function, especially for patients who require limb amputations due to repeated wound infection. In normal wound healing physiology, the first stage of healing begins with the formation of a platelet plug (hemostasis) and subsequent infiltration of immune cells to eliminate pathogenic organisms and foreign debris (inflammation). Following inflammation and immune cell clearance, the wound enters the proliferative phase marked by extracellular matrix deposition, angiogenesis, and reepithelization. Wound remodeling starts several weeks after injury and marks the transition from granulation tissue to a scar. Chronic wounds can form as a result of chronic venous insufficiency, arterial disease, prolonged pressure, or peripheral neuropathy2 and fail to shift from the inflammatory to the proliferative stage of wound healing, resulting in progressive tissue damage3,4.

The fibrin clot matrix is a key structure in the proliferative stage of wound healing. During hemostasis, the plasma protein fibrinogen is cleaved by the enzyme thrombin to form insoluble fibrin strands. The fibrin fibers form a crosslinked network that is stabilized by thrombin-activated FXIIIa and activated platelets5, which is followed by an inflammatory stage in which leukocytes infiltrate the clot and eliminate dead cells and pathogenic material. The fibrin matrix facilitates cell migration and stimulates fibroblast invasion to form new tissue. As the wound begins to remodel, endothelial cells invade the fibrin matrix and align to form new vascular structures which is vital to tissue regeneration. Chronic wounds contain a plethora of proteases released by immune cells that digest components of the fibrin clot, which results in a deficient provisional matrix that cannot adequately support cell infiltration and reepithelization6. Therefore, the delivery of an exogenous fibrin scaffold to a fibrin-deficient chronic wound may promote cell migration and tissue regeneration that will accelerate the rate of wound healing.

Fibrin-based biomaterials in the forms of patches, sponges, and sealants have been clinically used for wound management7,8. Biological agents such as cells, drugs, and growth factors can also be incorporated into fibrin biomaterials to enhance the therapeutic response9,10. Fibrin sealants are widely used to promote hemostasis and wound closure11, but common formulations do not provide an optimal environment for healing. To ensure rapid and effective clotting, commercial fibrin sealants mix fibrinogen and thrombin components at very high concentrations, between 80-120 mg/mL and 300-600 NIH U/mL, respectively12. In contrast, the physiological concentrations of fibrinogen and thrombin in the blood are between 2-4 mg/mL and 0.25-1 U/mL13,14, respectively. The high fibrinogen concentrations in commercial fibrin sealants form a very dense fibrin network, which can inhibit cell infiltration and proper tissue regeneration. Fibrin structure plays an important role in the wound healing process, since variables such as fiber thickness, network porosity, and permeability affect its biological function15. Recently, it has been shown that fibrin network porosity plays an important role in cell behavior and that fibrin gels with low fibrinogen concentration contain larger pores that enable quicker cell migration.16 Additionally, mesenchymal stromal cells cultured in fibrin gels were found to proliferate better in gels of low fibrinogen concentration (5 mg/mL) than higher concentrations (17 and 50 mg/mL)17. Increased cell proliferation also correlates with improved blood vessel growth, or angiogenesis18. Therefore, the ideal fibrin biomaterial would contain physiological concentrations of fibrinogen and maintain high porosity that is conducive to cell infiltration.

To that end, we have previously created pre-polymerized fibrin nanoparticles (FBN) comprised of physiologically relevant concentrations of fibrinogen and thrombin using a microfluidic droplet generator19. These FBNs can be delivered as a flowable solution directly to a wound to fill defects of various sizes and shapes. Unlike traditional fibrin sealants, FBNs are pre-polymerized prior to administration to wound sites, and can be comprised of physiologically relevant fibrinogen and thrombin concentrations. Therefore, we hypothesized that FBNs would promote enhanced migration into wound beds compared to traditional fibrin sealants. Our prior studies reported that when coupled to keratinocyte growth factor, FBNs enhanced fibroblast cell migration out of a 3D collagen gel and supported wound closure in a murine full-thickness wound model19. However, the microfluidic method for synthesizing the FBNs was time intensive (>48 hours) and yielded a relatively small volume of particles. Here, we report a new method to produce fibrin nanoparticles via a high throughput sonication procedure. In addition, we evaluated the ability of the sonicated fibrin nanoparticles (SFBN) to form clots and promote angiogenesis. Overall, the results described herein demonstrate that SFBNs promote clot formation in fibrinogen solutions, even in the absence of exogenous thrombin, and promote angiogenesis in vitro.

2. Materials and preparatory procedures applied

2.1. Preparatory procedures:

2.1.1. SFBN synthesis

Prepare a 5mL fibrin clot by polymerizing 2 mg/mL human fibrinogen (Enzyme Research Laboratories) with 1 U/mL thrombin (Enzyme Research Laboratories) in 1X HEPES buffer (25 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH 7.4) in a 15 mL tube. Allow the clot to polymerize for at least 1 hour at room temperature.

2.1.2. Sprouting Assay

At least one week prior to the sprouting assay experiment, thaw 1 vial of human umbilical vein endothelial cells (HUVEC) and maintain culture according to ATCC protocols. Passage HUVEC cells when culture has reached approximately 80% confluence and ensure cells have high viability prior to starting sprouting assay.

2.2. Equipment set-up.

The fibrin particles were suspended in a 50 mL tube containing 10 mL ultrapure water and placed in a beaker with ice to prevent overheating. A microtip probe (Branson Ultrasonics) was carefully inserted into the fibrin mixture and sonicated at 28 Watts (maximum power setting for the microtip probe) in pulse mode (60% duty cycle; to avoid overheating of the probe and surrounding solution) for six minutes, which was the least amount of time needed to produce SFBNs. After sonication, the solution was filtered through a 70 μm tissue strainer to remove large fibrin particles and debris.

3. Methods according to protocol:

3.1. SFBN Synthesis:

  1. Create a 5mL fibrin clot by polymerizing 2 mg/mL human fibrinogen (Enzyme Research Laboratories) with 1 U/mL thrombin (Enzyme Research Laboratories) in 1X HEPES buffer (25 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH 7.4) in a 15 mL conical tube.

  2. Allow enough time for complete fibrin gel polymerization (~1 hour), then remove the gel from the tube using a spatula, slice it finely with a scalpel (wear gloves and take adequate precautions working with sharps), and extrude through a 70 μm tissue strainer with a pestle.

  3. Suspend fibrin particles in a 50 mL conical tube containing 10 mL ultrapure water and place tube in a beaker filled with ice.

  4. Carefully insert sonicator microtip probe (take care not to touch sides or bottom of tube with the probe) and sonicate at 28 Watts in pulse mode (60% duty cycle) for six minutes.

  5. After sonication, filter solution through a 70 μm tissue strainer to remove large fibrin particles and debris.

  6. After purification, SFBNs can be lyophilized and stored at −20 °C. This allows the SFBNs to be re-suspended at desired concentration in the buffer of choice. If SFBNs will be used for cell culture or in vivo applications, these steps should be performed in a sterile environment using sterile buffers.

3.2. Atomic Force Microscopy (AFM) Characterization:

  1. AFM samples can be prepared by first cleaning glass coverslips in a series of solutions including 5% Alconox detergent solution, deionized water, acetone, absolute ethanol, and isopropanol and allowed to dry overnight in a fume hood.

  2. Incubate glass coverslips with 3-aminopropyltrimethoxysilane (1:100 dilution in absolute ethanol; Sigma-Aldrich, Missouri USA; safety glasses required when handling silanes) for 2 hours with shaking.

  3. Aspirate the 3-aminopropyltrimethoxysilane and wash the coverslips with deionized water.

  4. Add 3 mL of SFBN solution at 0.01 mg/mL to each coverslip in a 12 well plate and centrifuge the plate for 10 minutes at 3,700 RPM. The centrifugation step deposits the particles on the coverslips faster and more uniformly than if air drying.

  5. Aspirate SFBN solution and let coverslips air dry overnight in a fume hood or dry quickly with nitrogen. Drying at this stage is not required, but drying with nitrogen helps to ensure that no impurities are deposited on the surface prior to imaging.

  6. The AFM is operated in air in tapping mode with aluminum reflex coated silicon tips. Tips should have a force constant of 42 N/m (NanoAndMore). A 20 x 20 μm area is imaged and individual particles (n = 50) are analyzed in Asylum Research Software. Alternatively, measurements can be performed in liquid.

3.3. Clot Polymerization Assay

  1. Prepare 50 μL SFBN clots (1 mg/mL, 5 mg/mL, or 10 mg/mL SFBN concentrations) in HEPES buffer (25 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH 7.4) with 1 mg/mL fibrinogen in a 96 well plate. Positive control fibrin clots were prepared with 1 mg/mL fibrinogen and 1 U/mL thrombin in HEPES buffer and negative control is fibrin clot mixture without thrombin treatment.

  2. Use a plate reader to measure absorbance values of the wells at 350 nm every thirty seconds for two hours.

  3. The absorbance curves can be analyzed to determine final turbidity, rate of polymerization, and half-maximum polymerization time as previously described20. Subtract baseline absorbance values from each time point.

3.4. Confocal microscopy and fiber density of SFBN clots

  1. Prepare 50 μL SFBN clot solutions with 1 mg/mL fibrinogen spiked with 10 μg/mL Alexa-Flour 488 labeled fibrinogen (for visualization) and SFBN concentrations at 1 mg/mL, 5 mg/mL, or 10 mg/mL in 1X HEPES buffer (no thrombin).

  2. Positive control fibrin clot solutions should be prepared with 1 mg/mL fibrinogen and 1 U/mL thrombin in HEPES buffer and negative control is fibrin clot mixture without thrombin treatment.

  3. Place 100 μL of fibrin clot solution directly on a clean glass slide, cover solution with a glass coverslip, and allow it to polymerize for at least one hour at room temperature prior to imaging. Avoid air bubble formation in the preparation of fibrin clot.

  4. The fiber density of clots from each group (n=3) can be analyzed in ImageJ by converting images to binary and dividing cumulative black pixels (fibrin fibers) by cumulative white pixels (unoccupied free space) normalized to same confocal area, image scale, magnification, and laser intensity under different SBFN dosages.

3.5. Sprouting Assay

  1. Hydrate 0.5 grams of Cytodex 3 microcarrier beads in 1X PBS and suspend at 60,000 beads/mL. Cytodex beads are dextran beads coated with denature porcine-skin collagen, which is a good substrate for binding of anchorage-dependent cells, such as HUVECs.

  2. Remove 100μL of bead solution (6,000 beads), spin down solution, then remove PBS supernatant and wash with EGM-2 Media (Catalogue CC3162, Lonza, Switzerland). Combine bead solution with 1 million human umbilical vein endothelial cells (HUVECs) in 1.5 mL of EGM-2 and place in a 5 mL round bottom tube.

  3. Place the bead-HUVEC mixture vertically in an incubator (37°C, 5% CO2, 100% humidity), and shake gently every 20 minutes for 4 hours. Transfer coated beads to a T25 culture flask in a final volume of 5mL of media and incubate overnight.

  4. Gently wash the beads off the flask surface with media using a pipette (beads are nonadherent). Transfer beads into a 15 mL conical tube, wait 1-2 minutes for beads to settle, aspirate supernatant, and resuspend beads at desired concentration of 500 beads/mL in EGM-2 media. Aliquot into 1.5 mL Eppendorf tubes.

  5. Wait 1-2 minutes for beads to settle to the bottom of the tubes, then aspirate the media.

  6. Prepare 500 μL of fibrinogen solution at 1 mg/mL in sterile 1X PBS buffer (0.137M NaCl, 0.0027M KCl, and 0.0119M phosphates) in separate 1.5 mL Eppendorf tubes. Add 0.03 mg/mL of aprotinin to the fibrinogen solution to prevent clot degradation over time.

  7. Add SFBNs to the fibrinogen solution at final concentrations of 1 mg/mL, 5 mg/mL, or 10 mg/mL, then immediately transfer this solution to the tubes containing beads. Gently mix solutions by pipetting up and down, then transfer 500 μL of solution from each tube/condition into one well of a 24 well plate.

  8. Fibrin clot controls should be prepared in the absence of SFBNs by adding 1 U/mL thrombin to the fibrinogen-bead solution immediately prior to pipetting into 24 well plate.

  9. Allow clots to polymerize for 1 hour in an incubator at 37°C. After full polymerization, add 1 mL of EGM-2 media dropwise on top of each clot. Incubate clots for 48 hours to allow sprouting to occur.

  10. After 2 days, remove media and fix samples in 4% paraformaldehyde for 15 minutes at room temperature.

  11. After fixation, permeabilize the cells with 0.5% Triton X-100 for 10 minutes at room temperature, wash with PBS, and block with 5% BSA in 1X PBS for 2 hours.

  12. After blocking, stain cells with 1:4000 dilution of FITC-phalloidin (Catalogue U0281, Abnova, Taiwan) in 0.1% BSA at 4° C overnight to visualize actin fibers.

  13. After incubation with phalloidin, stain nuclei with 1:1000 dilution of DAPI solution (Catalogue D1306, Invitrogen, USA) in 1X PBS for 5 minutes at room temperature to visualize cell nuclei.

  14. Image samples with a fluorescent microscope at 10X magnification. The average sprout length can be calculated in ImageJ by drawing a line from the edge of the bead to the end of the sprout and measuring the total length.

4. Expected results:

4.1. Characterization of SFBNs

Air dried SFBNs were characterized in air topography mode using AFM. For the particle trace analysis, a horizontal line was drawn across the center of 50 different particles and the height and diameter of each particle was calculated using Asylum Research Software. SFBNs exhibited an average diameter of 377 ± 103 nm and an average height of 30 ± 26 nm (n=50, Fig. 1A-C). The hydrodynamic diameter of SFBNs was characterized using Nanosight Nanoparticle Tracking Analysis and average diameter was found to be 245 ± 69 nm (Fig. 1D). NanoSight measures the diffusion coefficient of particles by tracking the Brownian motion of the particles. By equating the diffusion coefficient to a homogeneous sphere in a solution of identical viscosity and temperature, the hydrodynamic diameter is indirectly quantified using the Einstein-Stokes equation. Analyzing the AFM results, it suggests that SFBN displays a flattened pancake structure when dehydrated during AFM prep. The AFM imaging technique used here analyzes air-dried SFBN, while NanoSight measures particles in solution. Materials can have different intramolecular forces when hydrated in solvent or dried. These intramolecular forces affect the surface area-to-volume ratio of particles. In this case, it appears that the surface interactions between the SFBNs and the glass causes the SFBNs to spread on the glass surface to diameters that are larger than the hydrodynamic diameter. However, it should be noted that the heights of the particles observed in AFM are quite small (~30 nm).

Figure 1:

Figure 1:

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(A) Representative AFM image of SFBN particles (air topography mode). (B) Zoomed in image of individual SFBN particle (scale bar = 300 nm) (C) Particle height and width trace of two individual particles. Average diameter = 377 ± 103 nm and average height = 30 ± 26 nm (n=50). (D) Hydrodynamic diameter measurement of SFBNs using Nanosight Nanoparticle Tracking Analysis indicating average diameter of hydrated SFBNs = 245 ± 69 nm.

4.2. Clot polymerization assay

The polymerization kinetics of fibrin clots ±SFBNs and ±thrombin were monitored using an absorbance-based assay. Representative absorbance curves are shown in Fig. 2A. Final clot turbidity increased with increasing concentration of SFBNs, and the fibrinogen + thrombin (Th) control clot had the highest turbidity, which correlates with confocal images presented in Figure 3. The 1 mg/mL SFBN clot was significantly less turbid (p<0.0001, one-way ANOVA with Tukey’s post-hoc analysis) than the other three conditions. The time to half maximum polymerization was also dependent on SFBN concentration, and it was found that the 1mg/mL SFBN clot was the slowest to polymerize. The polymerization rate (calculated from the slope of the linear portion of the curves) of SFBN clots increased with increasing concentration of SFBNs, but the polymerization rate of the positive control clot was significantly faster (p<0.0001, one-way ANOVA with Tukey’s post-hoc analysis) than the other conditions, due to the specific activity of the enzyme thrombin. For traditional fibrin bulk gel polymerization, thrombin is necessary for fibrin polymerization. Here we show that SFBNs can promote fibrin polymerization without the addition of exogenous thrombin. These results demonstrate that SFBNs can substitute thrombin and modulate the rate of fibrinogen gel polymerization.

Figure 2:

Figure 2:

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(A) Averaged absorbance polymerization curves from nine clots per group. (B) Final clot turbidity at 2 hours. (C) Time to half maximum polymerization rate, which correspond to the time to reach half maximum turbidity value. (D) Polymerization rate of clots, which is calculated by dividing half maximum turbidity value by the time to reach this value. *p<0.05; ***p<0.001; ****p<0.0001, one-way ANOVA with Tukey’s post-hoc analysis (GraphPad Prism).

Figure 3:

Figure 3:

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(A) Confocal microscopy images of clots polymerized with 1 mg/mL fibrinogen and 1, 5, or 10 mg/mL SFBN, and 1 mg/mL fibrinogen + 1 U/mL thrombin control. Scale bar = 20 μm. (B) Mean clot fiber density (n=3 per condition) +/− SD calculated in ImageJ for each clot condition. *p<0.05; ***p<0.001; ****p<0.0001, one-way ANOVA with Tukey’s post-hoc analysis (GraphPad Prism).

4.3. Confocal microscopy

Fibrin clots were generated by polymerizing 1 mg/mL fibrinogen with 1, 5, or 10 mg/mL SFBNs in the absence of exogeneous thrombin. The positive control clot was polymerized in the absence of SFBNs and addition of 1 U/mL thrombin. All clots were examined with a Zeiss laser scanning confocal microscope, and fiber density was analyzed in ImageJ. The fiber density increased with increasing concentration of SFBNs (Fig. 3). The 1 mg/mL SFBN clots were significantly less dense than all other conditions. There was no significant difference in fiber density between the 10 mg/mL SFBN clots and control fibrinogen + thrombin (Th) clots. These results demonstrate that clot structure is tunable based on SFBN concentration.

4.4. Sprouting Assay

The in vitro sprouting assay demonstrated that HUVEC cells can sprout in SFBN gels within 48 hours (Fig 4). The beads from the sprouting assay were imaged with a fluorescent microscope. Average sprout length was calculated using a blinded analysis in ImageJ by drawing a line from the edge of the bead to the end of the sprout and measuring the total length. It was found that cells had significantly longer sprouts (p<0.0001, one-way ANOVA with Tukey’s post-hoc analysis) in the clots containing SFBNs compared to the fibrinogen + thrombin controls. Cells also sprouted significantly better in 1 mg/mL SFBN clots compared to the other conditions. Confocal microscopy showed that these clots are the most loosely crosslinked, which may better support cell migration and longer sprouts. There were no significant differences between sprout length in 5 mg/mL and 10 mg/mL clots. This is likely due to their similar clot structure, as shown in the fiber density results. The HUVECs formed significantly longer sprouts in the fibrin clots made with the lowest concentration of SFBNs (Fig. 4). The confocal images of 1 mg/mL SFBN clots also showed that this formulation had the lowest fiber density, which can be attributed to less crosslinking between fibrin fibers (Fig. 3). Therefore, we draw the conclusion that the HUVECs can recapitulate native angiogenesis much better in fibrin clots with lower fiber density.

Figure 4:

Figure 4:

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(A) Representative images of HUVEC-coated beads embedded in fibrin gels containing 1 mg/mL fibrinogen and 1, 5, or 10 mg/mL SFBN (no thrombin) or 1 mg/mL fibrinogen + 1 U/mL thrombin, after 48 hours of culture. Cell actin stained with FITC-Phalloidin (green) and nuclei with DAPI (blue). Scale bar = 100 um. (B) Average sprout length calculated from beads in each gel condition (n=16). *p<0.05; ***p<0.001; ****p<0.0001, one-way ANOVA with Tukey’s post-hoc analysis (GraphPad Prism).

5. Future Prospects:

In this protocol, we demonstrate that sonicated fibrin nanoparticles (SFBN) can be produced in large quantities using a probe sonication method. SFBNs induce fibrin clot formation without addition of exogenous thrombin and clot polymerization kinetics are tunable based on SFBN concentration. Confocal microscopy showed that the clot fiber density increased with increasing concentrations of SFBNs. In the sprouting assay, HUVEC cells sprout significantly better in SFBN clots compared to the controls, which correlates with improved angiogenesis outcomes. Since SFBN clots are less dense than the fibrin + thrombin control, it is likely that cells had sufficient 3D space to move into the more porous matrix and thus exhibited increased migratory distance. Previous results demonstrate that mesenchymal stromal cells cultured in fibrin gels proliferate better in gels of low fibrinogen concentrations than higher concentrations and that increased cell proliferation correlates with improved blood vessel growth, which is likely due to the increased porosity in these lower concentration gels17-18. Our results are in line with these previous findings in that angiogenesis was found to correlate with porosity.

SFBNs can also be functionalized to attach wound healing growth factors like vascular endothelial growth factor and keratinocyte grown factor to further improve healing outcomes. The ability to control fibrin clot density, fiber thickness and porosity using SFBNs will aid wound healing by supporting cell migration, inflammation clearance, neovascularization and tissue regeneration. The method for producing SFBNs presented here facilitates facile production of fibrin nanoparticles and is easily scalable. This method could be used to create flowable fibrin-based scaffolds without the need for exogenous thrombin and could be applied to a number of tissue engineering and regenerative medicine applications.

Table 1.

Reagents used.

Name of reagent Manufacturer City and country
Human Fibrinogen Enzyme Research Laboratories South Bend, IN, USA
Thrombin Enzyme Research Laboratories South Bend, IN, USA
1X HEPES Buffer (25 mM HEPES, 150 mM NaCl, 5 mM CaCl2, pH 7.4) Prepared in lab Raleigh, NC, USA
Alconox detergent MilliPore Sigma Darmstadt, Germany
Acetone MilliPore Sigma Darmstadt, Germany
Ethanol (absolute) MilliPore Sigma Darmstadt, Germany
Isopropanol (>99.5%) MilliPore Sigma Darmstadt, Germany
3-aminopropyltrimethoxysilane MilliPore Sigma Darmstadt, Germany
Fibrinogen from human plasma, Alexa Fluor 488 conjugate Thermo Fischer Scientific Waltham, MA, USA
Cytodex 3 microcarriers Cytiva Marlborough, MA, USA
1X PBS (0.137M NaCl, 0.0027M KCl, and 0.0119M phosphates) Thermo Fischer Scientific Waltham, MA, USA
EGM-2 Endothelial Cell Growth Medium Lonza Basel, Switzerland
Human Umbilical Vein Endothelial Cells (HUVEC) ATCC Manassas, VA, USA
Aprotinin MilliPore Sigma Darmstadt, Germany
Paraformaldehyde solution (4% in PBS) Thermo Fischer Scientific Waltham, MA, USA
Triton X-100 MilliPore Sigma Darmstadt, Germany
Bovine serum albumin MilliPore Sigma Darmstadt, Germany
Fluorescent dye 488-I Phalloidin (FITC) Abnova Taipei, Taiwan
DAPI nuclear counterstain Thermo Fischer Scientific Waltham, MA, USA
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Table 2.

Equipment used.

Name of equipment Manufacturer City and country
Branson Sonifier Cell Disrupter 200 Branson, Model W-200P Danbury, CT, USA
Branson Ultrasonics Microtip Probe (diameter = 3/16”) Branson Danbury, CT, USA
70 μm cell strainer Corning Inc. Corning, NY, USA
Atomic Force Microscope (Asylum MFP3D-Bio) Asylum Research Santa Barbara, CA, USA
Zeiss LSM 880 Airyscan confocal microscope Zeiss Oberkochen, Germany
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Highlights.

  • A high throughput method for creating fibrin nanoparticles using probe sonication is described.

  • These particles can form a fibrin gel when combined with fibrinogen without exogenous thrombin.

  • Polymerization rate and fiber density of fibrin clots is tunable based on particle concentration.

  • Particle-containing fibrin gels promote angiogenesis.

Acknowledgments

Funding: This work was funded in part by DMR-1847488 from the National Science Foundation (Alexandria, Virginia) and NIH NHLBI 1R01HL146701-01A1 from the National Institutes of Health (Bethesda, Maryland).

Footnotes

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