In vitro and Ex vivo Assessments of the Compatibility of Fibrin Sealant with Antimicrobial Compounds

Abstract

Background:

Fibrin sealants are used as antimicrobial-releasing carriers for preventing surgical site infections; however, it is important to determine the release kinetics and antimicrobial effects of drugs added to fibrin sealants and the effects of drugs on clot/clotting properties.

Materials and Methods:

The antimicrobial and antibiofilm activity of cefazolin, colistin, gentamicin, oxacillin, tobramycin, and silver nitrate released from fibrin sealant were characterized using in vitro and ex vivo assays against bacteria commonly found on the skin. The effects of antimicrobial agents on the physical structure of the fibrin sealant were assessed with scanning electron microscopy (SEM) and on the clotting rate and strength of fibrin clots using run-off tests and rheology.

Results:

Generally, antibiotic agents were released gradually from fibrin sealant and were stable after release, with antimicrobial effects evident up to three days. Cefazolin, gentamicin, and oxacillin prevented biofilm formation of Staphylococcus aureus in porcine skin explants; gentamicin and colistin prevented biofilm formation of Pseudomonas aeruginosa. Gentamicin, cefazolin, colistin, and tobramycin did not affect the structural integrity or viscoelastic properties of fibrin sealant; changes were observed with oxacillin (SEM) and particularly silver nitrate (SEM and rheology). No antimicrobial agents caused deterioration of clotting time (run-off tests).

Conclusions:

From the antimicrobial agents tested, gentamicin and cefazolin showed prolonged release from fibrin sealant, sustained antimicrobial activity, and biofilm prevention properties against Staphylococcus aureus; similar results were observed for gentamicin and colistin against Pseudomonas aeruginosa. For each of these findings, the physical structure of the fibrin sealant, clotting rate, and strength of fibrin clots were unaffected.

Surgical site infections can lead to significant morbidity and may result in extended hospital stay, re-admission, and the need for repeated surgical procedures.¹ An approach to reduce infection rate after surgical procedures is the local application of antimicrobial agents, with the aim of achieving and maintaining effective concentrations in the wound space. Antimicrobial agents with activity against bacteria commonly involved in infections of skin and skin structures are likely to be of particular interest in this context.^2,3 The ability of bacteria to produce biofilms, which can be highly tolerant to antimicrobial agents, can further impede wound healing.^4,5 The ability to prevent biofilm formation is, therefore, an important consideration for antimicrobial agents.

Fibrin sealants have hemostatic and adhesive properties and have been used increasingly in general surgical procedures to reduce bleeding and improve wound healing. The components of the fibrin sealant are mixed to form a fibrin clot that is biocompatible and biodegradable. The potential of fibrin sealants as an antimicrobial-releasing carrier for prevention of infection during surgery has also been explored.^6–8 Surgical specialisms such as otorhinolaryngologists generally work in semi-sterile settings and fibrin sealants are used frequently^9,10; the addition of effective antimicrobial agents could be an attractive option to prevent complications from infection.

Although fibrin sealants could provide a slow-release system for antimicrobial agents, certain factors must be considered when assessing the suitability of specific combinations. The fibrin sealant might be affected by the antimicrobial, which could lead to a rapid release of the drug, or the sealant properties might affect antimicrobial efficacy. A potential rapid decline in antimicrobial concentration or changes in the effectiveness of treatment could promote antimicrobial resistance.⁷ Equally, antimicrobial agents might change the biochemical properties of the fibrin sealant mixture and affect the rate of clot formation or the strength of the resultant fibrin clot.^6,7,11

TISSEEL^® (Baxter Healthcare Corporation, Deerfield, IL) and ARTISS^® (Baxter Healthcare Corporation, Deerfield, IL) are fibrin sealants that comprise a two-component system containing stable formulations of thrombin and fibrinogen.^12,13 Indications include hemostasis during surgical repair (TISSEEL), use as a sealant to prevent leakage from colonic anastomoses (TISSEEL), or adherence of autologous skin grafts to prepared wound beds resulting from burns (ARTISS). Here, we characterized the antimicrobial and antibiofilm activity of compounds released from fibrin sealant using in vitro and ex vivo assays and assessed effects of the antimicrobials on the physical structure of the fibrin sealant, the rate of fibrin clotting, and clot viscoelastic properties.

Materials and Methods

Complete descriptions for all methods used are given in the Supplementary Materials.

Assessing the impact of antimicrobial agents on fibrin clot formation

The impact of antimicrobial agents on fibrin clot formation was assessed with a turbidity test and pipette test. To identify antimicrobial compounds that allow clot formation in a similar time as controls (fibrin sealant formulated without antimicrobial), a broad spectrum of antimicrobial agents (antibiotic agents; antifungals; antimicrobial agents based on iodine, biguanides, metal ions, and quaternary ammonium compounds) was initially selected. When mixed with sealer protein and/or thrombin, iodine-based antimicrobials, Silvasorb gel, and Protosan had high absorbance that interfered with the testing and vancomycin, teicoplanin, colistin, and chlorohexidine digluconate formed a precipitate.

Compounds chosen for further testing were cefazolin, colistin, gentamicin, oxacillin, tobramycin, and silver nitrate. As noted in specific sections below, not all antimicrobial agents were assessed for some evaluations.

Ex vivo porcine dermal biofilm prevention model

As a suitable model of biofilm formation in human skin, a porcine skin model was used in this study given the morphologic (e.g., dermal–epidermal thickness, hair follicles) and biochemical similarities (e.g., collagen and lipid composition) between swine and human skin.^14,15 This study specifically focused on prevention of biofilm formation within the skin using an ex vivo model of mature biofilm. Left untreated, mature biofilms result within 24 hours and are genotypically and phenotypically reminiscent of infected wounds observed clinically. It should be noted, however, that the tissue is non-viable and sterilized, so it serves to model only the biofilm component of chronic wounds.

For biofilm prevention studies, porcine dermal tissue acquired from a U.S. Department of Agriculture-certified vendor was cut into 12.7 mm punches and simulated wounds were created in the center of the punch by removing the epidermis using a rotary power tool (approximately 3 mm in diameter and 1–2 mm deep). The simulated wound area with exposed dermis was then inoculated with 10⁵–10⁶ colony forming units (CFU) of early log phase microbial growth and allowed to incubate for 20 minutes. Inoculated explants were treated with a given fibrin clot formulation and formation of mature biofilm was monitored over time and compared to untreated controls. Ex vivo experiments were completed in three sets.

Subcultures of Staphylococcus aureus or Pseudomonas aeruginosa were prepared by adding 0.15 mL overnight culture to baffled flasks with 30 mL tryptic soy broth (TSB). They were incubated at 125 revolutions per minute (rpm) and 37°C until reaching 10⁸ CFU/mL (optical density [OD]₆₄₀ 0.2–0.4), and 20 μL of the subculture was used for inoculation at log₁₀ 6 CFU per explant.

Cefazolin, colistin, gentamicin, oxacillin, tobramycin, and silver nitrate were tested for biofilm prevention against Staphylococcus aureus. Colistin, gentamicin, tobramycin, and silver nitrate were tested for biofilm prevention against Pseudomonas aeruginosa. Test compounds were prepared for use in the Baxter Duploject system. Antimicrobial agents were prepared at their instructions for use concentration, mixed with two parts thrombin (4 IU/mL), and 2 mL were loaded into a syringe. A second syringe was loaded with 2 mL sealer protein according to instructions. After incubation, biofilm bacteria were recovered from explants, diluted in phosphate buffered saline (PBS) and spot plated on tryptic soy agar (TSA). Colonies on plates were counted, and recoveries were reported as CFU per explant.

Zone of inhibition assays

Initial screening

The in vitro effects of antimicrobial agents contained in fibrin sealant were examined by zone of inhibition (ZOI) assays. Zone of inhibition plates were prepared using agar overlay. Initial ZOI screening was done to determine dose-response effects of antimicrobial agents. Three antibiotic agents (gentamicin, oxacillin, tobramycin) and silver nitrate were tested against Staphylococcus aureus and two antibiotic agents (gentamicin, tobramycin) and silver nitrate were tested against Pseudomonas aeruginosa.

Each set of experiments tested fibrin clots with water (negative control) or four concentrations of each antimicrobial in water, as well as a positive control (9/32-inch paper disk with an equivalent mass as the highest concentration of antimicrobial found in the clots). After 24 hours at 37°C, the diameter of each zone clear of bacterial growth was measured with calipers.

Duration of efficacy

Clots were prepared with water (negative control) or antimicrobial compound as for the initial ZOI testing. Positive controls were made on paper discs using equivalent mass of antimicrobial contained in the clot with the highest antimicrobial concentration. To determine the duration of efficacy, clots and discs were transferred every 24 hours to fresh ZOI plates until a zone of inhibition was no longer observed.

Up to six different species of bacteria (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis, Acinetobacter baumannii, and Enterococcus faecium) were tested with five antimicrobial compounds (cefazolin, colistin, gentamicin, oxacillin, and tobramycin) predicted to show a zone of inhibition. Silver nitrate was excluded from further testing because of low efficacy at 1 mM in initial screening.

Antimicrobial efficacy of fibrin clots with antimicrobial agents

For high-throughput antimicrobial screening assays, challenge plates were set up in 96-well microtiter plates similar to standard minimum bactericidal concentration (MBC) assays, and testing was done in simulated wound fluid (SWF). Staphylococcus aureus and Pseudomonas aeruginosa were tested with cefazolin, gentamicin, tobramycin, oxacillin, and silver nitrate. The starting concentration of the antimicrobial agents were cefazolin, 4 mg/mL; gentamicin, 8 mg/mL; oxacillin, 8 mg/mL; tobramycin, 0.16 mg/mL; and silver nitrate, 0.2 mM. For each 96-well plate assay, the starting concentration was then serial diluted (twofold) across the columns of the plate.

Scanning electron microscopy

The effects of all six antimicrobial agents on the structure of fibrin clots were examined. Clots were prepared with antimicrobial concentrations: cefazolin, 4 mg/mL; gentamicin, 8 mg/mL; oxacillin, 8 mg/mL; tobramycin, 0.16 mg/mL; colistin, 10 mg/mL; and silver nitrate, 0.2 mM. Clots with fibrin sealant, without antimicrobial agents and human plasma, were prepared for comparison. Test groups were imaged at 40 × , 200 × , 500 × , 2,000 × , and 10,000 × magnification in replicates of two using a JSM-6510 Series Scanning Electron Microscope (JEOL Ltd., Freising, Germany).

Run-off test

Run-off tests on an incline glass plate at 30-degree angle were used to examine the effects of antimicrobial agents on clot formation time after mixing the components of the fibrin sealant. Antimicrobial agents were added to the thrombin component. The components were drawn into Duploject System syringes, with 1 mL total sealant applied via the associated application cannula. Time from application start to curing was recorded.

Rheology

Evaluation of viscoelastic properties of fibrin sealant was performed with a modular Anton Paar MCR-300 rheometer (Anton Paar GmbH, Graz, Austria). Thrombin solutions were supplemented with antimicrobial agents prior to measurements.

Storage modulus (G′) describing the elastic portion and loss modulus (G′′) describing the viscous portion of the sample behavior were recorded as functions of time. Mean and standard deviation of G′ and G′′ were calculated at 300, 600, and 900 seconds of the gelation process as well as the loss factor (tanδ, ratio G′′/G′) as a measure of viscoelastic behavior.

Results

Impact of antimicrobials on fibrin clot formation

The results for all antimicrobial compounds evaluated are shown in Supplementary Table S1. Based on the clotting time test and turbidity test results, iodine-based antimicrobial agents, quaternary ammonium antimicrobial agents, and biguanides were excluded from further evaluation; antifungals also were not studied further because of the rarity of fungal infection after surgical procedures, and their limited solubility in aqueous buffers.^16–19 Although all metal ions tested showed little impact on clotting time, silver nitrate was used in subsequent antimicrobial testing because it is the most used metal-based antimicrobial.

Table 1 shows results of the turbidity and clotting time tests for compounds subsequently evaluated for antimicrobial testing (cefazolin, colistin, gentamicin, oxacillin, tobramycin, and silver nitrate); clotting time was similar to that of controls (fibrin sealant without antimicrobial).

Table 1.

Impact of Antimicrobial Agents on Fibrin Clot Formation

Antimicrobial	MIC^a (mg/mL)	IFU^b (mg/mL)	Concentration in clot (mg/mL)^e	Highest test concentration	Turbidity test result	Pipette test clotting time (s)
Control^c (water)	—	—	—	—	—	61
Cefazolin sodium salt	0.002–0.008	20	4	N/A	No impact	82
Colistin sulfate	0.004	50	10^d	N/A	No impact	105
Gentamicin sulfate	0.00025–0.016	40	8	N/A	No impact	90
Oxacillin sodium salt	0.00025–0.001	40	8	N/A	Delays turbidity by 3 ×	85
Tobramycin	0.0002–0.002	0.8	0.16	N/A	No impact	68
Silver nitrate	N/A	N/A	N/A	2 mM	No impact	52.5

MIC = minimum inhibitory concentration; IFU = instructions for use; N/A = not applicable.

Denotes the range of MICs for each drug reported in the literature.

The recommended concentration per the drug IFU.

Pure water used in place of antimicrobial as a control. Note: fibrin clot has the same dilution as the treatment groups (e.g., fibrin clots containing antimicrobials).

Colistin sulfate added to the thrombin solution as it could cause precipitation of the sealer protein

Concentration of material added to the fibrin clot. Final concentration in clot is 1/5 of starting material. All dilutions were done in pure water.

Biofilm prevention

Figure 1 shows biofilm prevention data for the antimicrobial treatments and their respective controls. Positive controls in aqueous antimicrobial solutions were recovered at 24 and 72 hours. Antimicrobial agents in fibrin clots were at least as effective as positive controls, indicating that fibrin clot does not impact antimicrobial efficacy.

FIG. 1.

Viable biofilm-associated Staphylococcus aureus or Pseudomonas aeruginosa isolated from pig skin explants over time after being inoculated with 10⁶ colony forming units (CFU) and treated with antimicrobial-loaded fibrin formulations or negative controls (water/fibrin without antibiotic and saline [‘Control’]).

Cefazolin, gentamicin, and oxacillin were all effective at preventing mature biofilm of Staphylococcus aureus (Fig. 1A). Similarly, colistin and gentamicin were at least as effective in fibrin clots as in aqueous solution at preventing biofilm formation of Pseudomonas aeruginosa (Fig. 1C). Negative controls with fibrin clots were made with water containing no antibiotic, as well as untreated explants gave similar results experiment-to-experiment and showed high levels of biofilm associated bacteria growing in the explants (Figs. 1B and 1D).

Zone of inhibition assays

A dose-response was observed for the antimicrobial agents studied within fibrin clots in the initial screening as shown in Figure 2. Some clots without sufficient levels of antimicrobial for a given microbe appeared to shrink in size within 24 hours. Zone of inhibition data from the time course study are summarized in Table 2.

FIG. 2.

Initial zone of inhibition (ZOI) screening results for various antimicrobial-loaded fibrin clots against Staphylococcus aureus or Pseudomonas aeruginosa

Table 2.

Summary Data for the Dose and Time Relation of Various Antibiotic Agents Against a Broad Spectrum of Bacteria

	Cefazolin sodium salt	Colistin sulfate	Gentamicin sulfate	Oxacillin sodium salt	Tobramycin
IFU (mg/mL)^a	20	50	40	40	0.8
Concentration in fibrin clot (mg/mL)	4	10	8	8	0.16
Species tested	Duration of antimicrobial release and visible zone of inhibition (hr)
Staphylococcus aureus	<72	<48	<96	<96	<72
Pseudomonas aeruginosa	Not tested	<72	<72	Not tested	<48
Escherichia coli	<72	<96	<72	Not tested	<48
Staphylococcus epidermidis	Not tested	<48	<72	<72	Not tested
Acinetobacter baumannii	Not tested	<96	<48	Not tested	<48
Enterococcus faecium	Not tested	Not tested	<72	Not tested	<48

If the duration of release was <72 hr clear zones were present at 48 hr, but not at 72 hr.

IFU = instructions for use.

The recommended concentration per the drug IFU.

For all combinations of antimicrobial agents and species tested, there was a clear burst release at 24 hours, followed by a decrease in ZOI time, where many of the zones were comparable to negative controls at 48 hours. Some clots were reduced in size in samples showing no ZOI at longer time points (e.g., 72 and 96 hours), indicating that bacteria were degrading the clot in the absence of antibiotic; complete clot degradation was only seen with Pseudomonas aeruginosa.

In vitro assessment of efficacy for antimicrobial fibrin sealant formulations

Results shown in Supplementary Figure S1 indicate a dose-dependent effect for gentamicin, oxacillin, tobramycin, and silver nitrate against both Staphylococcus aureus and Pseudomonas aeruginosa when these antimicrobial agents were formulated with the fibrin sealant.

Scanning electron microscopy

Clots containing tobramycin, cefazolin, colistin, and gentamicin showed structure comparable to standard fibrin sealant (Supplementary Figure S2). The presence of open pores and large fibers similar to a normal plasma clot indicated that at the used concentrations these antimicrobial agents did not notably impact the clot structure of the fibrin sealant. Oxacillin caused a condensed structure without pores, which affected the macroscopic clot, causing it to be more transparent (Supplementary Figure S2). This is in-line with the three times delayed increase in turbidity seen during initial screening (Table 1). Silver nitrate had a marked impact on the structure of the fibrin sealant that was visible with the naked eye, with samples appearing white rather than opaque (Supplementary Figure S2).

Run-off test

Results are shown in Supplementary Figure S3. Run-off times of fibrin sealant with antimicrobials were comparable (colistin, 3.88 seconds; tobramycin, 3.55 seconds; silver nitrate, 4.30 seconds) or tendentially lower (cefazolin, 3.12 seconds; gentamicin, 2.60 seconds; oxacillin, 2.08 seconds) than that of standard fibrin sealant (4.51 seconds). Polymerization of the fibrin sealant was rapid in all cases. No antimicrobial caused a severe deterioration of the run-off time, and average time from application start to curing was less than 4.51 seconds for all antimicrobial agents.

Rheology

The obtained G′, G′′, and tanδ values for the investigated clotting processes at 300, 600, and 900 seconds after gelation initiation by component mixing are shown in Figure 3. The time-dependent evolution of G′ with silver nitrate suggested clotting was slowed when compared with the other test groups. Further analysis revealed that formulation with silver nitrate differed the most from all other investigated groups: G′ after 900 seconds (2.61 ± 2.09 kPa) and G′′ after 900 seconds (0.93 ± 0.66 kPa) were lower than for all other groups, including the control group with G′ after 900 seconds 25.03 ± 6.27 kPa and G′′ after 900 seconds (3.10 ± 1.23 kPa). Notably, clots with silver nitrate had a non-homogeneous appearance, low adhesion to the measuring system, and were white in color in contrast to the opaque materials obtained for all other groups. Aside from samples with silver nitrate, largest differences in final mean G′ values (i.e., after 900 seconds of clotting time) were recorded between groups control and tobramycin with 25.03 ± 6.27 kPa and 42.47 ± 16.59 kPa, respectively.

FIG. 3.

Visualization of (A) storage modulus data, (B) loss modulus data, (C) loss factor data at 300, 600, and 900 seconds after clotting initiation of investigated sealant formulations obtained from time-sweep oscillation rheology measurement. Bars and whiskers indicate mean ± standard deviation of the data.

At initial stages of gelation (i.e., at 300 seconds and 600 seconds) slightly lower G′ values for control, tobramycin, and colistin were recorded compared to the other test groups; however, final G′ at 900 seconds was similar to the other groups. Calculated loss factor values for investigated fibrin clots were in the typical viscoelastic range (100 > tanδ > 0.01). For all investigated groups except silver nitrate, the loss factor was between 0.1 and 0.2 at 300, 600, and 900 seconds, respectively. Thus, after 300 seconds onwards, a gel-like state was achieved for all test groups except silver nitrate due to clotting of the sealant protein formulation. A summary of all results is shown in Supplementary Table S2.

Discussion

There is a lack of consensus and evidence from prospective clinical trials concerning the role of topical antimicrobial agents to prevent infection after specific surgical procedures.^20–22 Although fibrin sealants have been investigated as an antimicrobial releasing carrier for prevention of surgical site infections,^6–8 clinical use of fibrin sealants is variable across different types of surgical procedures^23–27 and they are often used in off-label indications. For example, it has been reported that autologous platelet-rich fibrin co-delivered with antibiotic agents including colistin or tobramycin is a feasible method for topical antibiotic treatment supplementary to sinus surgery, with the antibiotics released for more than seven days.²⁸

Given that many surgeries use peri-operative and post-operative prophylaxis with antibiotic agents, combining fibrin sealant with antimicrobial agents that can target microbes commonly involved in skin and soft tissue or wound infections^2,3 may provide a rational option for preventing surgical site infections. Among the antimicrobial agents included in this study, gentamicin is used topically to prevent surgical site infections^21,22 because of its broad spectrum of activity and cefazolin is used for perioperative prophylaxis.^29,30 Fibrin glue combined with antibiotic agents including gentamicin or colistin can be used successfully in combination with standard anti-infection therapy for treatment of acute and chronic bone infections with soft tissue involvement.³¹ In previous studies, addition of gentamicin to fibrin glue gave durable antimicrobial activity against the most common pathogens involved in post-operative and traumatic ocular infections.³²

When gentamicin was combined with fibrin glue to examine antibacterial effects in vitro, two-thirds of the drug diffused out within two to three days.³³ This finding has been supported by other studies reporting that gentamicin is released from fibrin sealant clots during the first three days.^11,34 Equally, studies with cefazolin have shown that fibrin clots containing this antibiotic maintained antimicrobial activity against Staphylococcus aureus for up to seven days.³⁵ The suggestion that fibrin sealants containing cefazolin may offer an effective approach to postoperative antibiotic delivery is further supported by evidence that the antibiotic was stable in clots and released within 48 hours with a strong burst over the first six to eight hours.³⁶

These previous findings were generally supported by our current study. We found that cefazolin, colistin, gentamicin, oxacillin, and tobramycin appeared compatible with normal clot formation even at relatively high concentrations. Moreover, our results showed that gentamicin and cefazolin have prolonged release kinetics from fibrin sealant and are stable after release with an antimicrobial effect evident for at least two days (cefazolin) or three days (gentamicin). Importantly, these antibiotic agents, along with colistin and tobramycin did not seem to affect the structural integrity or viscoelastic properties of fibrin sealant, whereas some changes were noted with oxacillin (with SEM) and particularly silver nitrate (with both SEM and rheology).

We found that cefazolin and gentamicin along with oxacillin in fibrin clots were all effective at preventing mature biofilms of Staphylococcus aureus, whereas gentamicin and colistin were at least as effective in fibrin clots as in aqueous solution at preventing biofilm formation of Pseudomonas aeruginosa. Although the ex vivo model used lacks critical components typical of wound infections, (e.g., presence of an active host immune system and a polymicrobial infection), the biofilms strongly resemble those found in a clinical setting and are relevant to the real world, as biofilm formed in a natural tissue matrix is more robust and is more difficult to prevent than on a plastic surface (e.g., petri dish, etc.).¹⁵

In ZOI assays the tested antimicrobial agents in fibrin clots were at least as effective as their positive controls (aqueous antimicrobial agents) and exhibited concentration-dependent responses, indicating that the fibrin sealant does not impact antimicrobial efficacy. Release of the antimicrobial from the clot also occurs both on a semisolid matrix (TSA) and within simulated wound fluid (SWF). Although the ZOI typically disappeared by two to three days for most antimicrobial agents, gentamicin and oxacillin were still effective at three days against Staphylococcus aureus, and colistin and gentamycin were effective for three days against Pseudomonas aeruginosa; these data suggest that there may be preferential in procedures in which there is a risk of these infections. Furthermore, fibrin formulations of gentamicin exhibited full kill at concentrations higher than the minimum bactericidal concentration (MBC) reported in the literature.³⁷

Scanning electron microscopy revealed that fibrin sealant containing cefazolin, colistin, gentamicin, and tobramycin showed a structure comparable to standard TISSEEL fibrin sealant and physiologic plasma clot whereas oxacillin and silver nitrate caused changes to the fibrin sealant structure. Because TISSEEL and ARTISS have the identical composition except for the thrombin content and the TISSEEL thrombin was diluted to 4 IU/mL in most tests (except plate run-off) it can be expected that both fibrin sealants are not impacted by cefazolin, gentamicin, colistin, and tobramycin. To test clotting of fibrin sealants we used a run-off test on an 30-degree inclined glass plate. This setup allows testing of clotting speed of full-strength fibrin sealants with high thrombin concentrations. None of the antimicrobial agents tested caused a severe deterioration of the run-off time, and average time from application start to curing was less than 4.51 seconds for all antimicrobial agents.

Silver nitrate considerably altered the viscoelastic properties of the clot formation process in comparison to the control group, which is most likely due to precipitation and non-homogeneous clots being formed upon mixing with the sealant solutions. Other studies have investigated the influence of antimicrobial substances on clotting time.^11,34 Gentamicin (35 mg/mL) drastically increased the clotting time and decreased the rigidity of a fibrin sealant that was counter-acted by addition of factor XIII.¹¹ In the current study, a lower concentration of gentamicin (i.e. 8 mg/mL) was used and final clot stiffness was comparable to the control group. Furthermore, a previous study reported that concentrations of gentamicin and tobramycin approximately 1 mg/mL did not alter clotting behavior whereas increasing concentrations 10-fold resulted in increased clotting times.³⁴

Conclusions

The data presented show the potential utility in combining antimicrobial agents with fibrin sealants for antimicrobial and antibiofilm properties, which may provide in vivo protection against microbial infection. Although not all antimicrobial agents were suitable for mixing with fibrin sealants, among those that were, gentamicin and cefazolin showed prolonged release from fibrin sealant, sustained antimicrobial activity and biofilm prevention properties against Staphylococcus aureus, without affecting the physical structure of the fibrin sealant or the rate and strength of fibrin clotting. Similar properties were observed for colistin and gentamicin against Pseudomonas aeruginosa, also without affecting the physical structure of the fibrin sealant or the rate/strength of fibrin clotting. The evidence suggests further clinical investigations into the role of fibrin sealants in preventing surgical site infections are merited.

Footnotes

Acknowledgments

Editorial support was provided by Meridian HealthComms, sponsored by Baxter Healthcare Corporation. The study and final manuscript were kindly discussed with Heinz Redl, PhD, Vienna.

Authors' Contributions

HG and PS contributed to the design of the studies and the interpretation of the data. SM was involved in conducting the experiments. RR contributed to data evaluation and interpretation. CL provided input to the manuscript and contributed to data interpretation. EV and AS contributed to the design of experiments and interpretation of the data. EV was responsible for conducting the experiments and data validation. BS was involved in the conduction of the experiments and in data interpretation. All authors reviewed and approved the manuscript for submission.

Author Disclosure Statement

HG is an employee of Baxter Medical Products GmbH. PS is Chief Executive Officer of Trauma Care Consult GmbH, and AS is Vice President of Research and Development at iFyber, which are the two companies that received funding for conducting this study. EV is an employee of iFyber. RR is an employee of Trauma Care Consult GmbH. SM received funding through Trauma Care Consult GmbH. CL and BS have no conflicts of interest to declare.

Funding Information

This study was sponsored by Baxter Healthcare Corporation, One Baxter Parkway, Deerfield, Illinois.

Supplementary Material

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