Effectiveness of Glycerol Mono-oleate as a Biosealant

Vascular access site complications are common following cardiac catheterization.¹ These complications, as high as 14% of patients undergoing percutaneous coronary intervention (PCI), increase hospital length of stay and cost.² Accordingly, vascular closure devices (VCD) were developed to decrease these complications and the time-to-hemostasis and ambulation. Since their introduction in 1995, many improvements such as utilizing biologics (collagen, thrombin and others) have helped to address these needs. However, little has been done to address the infectious complication rates which range from 0.0% to 5.1%.^3–6
Glycerol mono-oleate (GMO) (C₁₂H₄₀O₄) is a synthetic, biodegradable amphiphilic, lipid compound with many applications.^7,8 It is used as a nonionic surfactant and emulsant such as antifoam in juice processing and as a lipophilic emulsifier for water-in-oil applications. It also serves as a moisturizer in cosmetics, flavoring agent and as an excipient in antibiotics and other drugs.⁹ This oil-soluble compound undergoes a biophysical phase transition at a melting point below the physiological temperature of the human body, solid phase to a bioadhesive semisolid hydrogel phase. It also exhibits an expansion action thereby swelling in physical size when exposed to aqueous solutions.¹⁰ Furthermore, this family of compounds displays bacterial deterrent properties^11–15 which, in combination with its biophysical properties, may be optimal for use as a physiological sealant.
The aim of this study was to evaluate whether GMO would: 1) achieve hemostasis more effectively than control when injected into a swine liver biopsy tract; and 2) inhibit common percutaneous procedure pathogens.

Methods
The study was conducted through collaboration with the Departments of Vascular Surgery, Pathology, and Biomedical Engineering at Duke University Medical Center, Durham, North Carolina.
Bleeding model. All activities were preapproved by the Duke University Institutional Animal Care and Use Committee. An acute porcine model was used to observe the attainment of hemostasis after liver biopsy. Data were collected on seven Yorkshire crossbred swine weighing at least 50 kg. The primary outcome of the study was the time to achieve hemostasis following treatment after a core needle biopsy. Hemostasis was defined by a bleeding score equal to 0.
Coagulation status. Immediately prior to the creation of the first biopsy, blood was drawn and the baseline activatedclotting time (ACT) was measured and recorded. Unfractionated heparin was administered achieving a goal ACT > 250 throughout the study. Swine were anticoagulated to promote bleeding for accurate assessment of intervention versus control and mimic the anticoagulation status of patients undergoing common percutaneous and surgical procedures.

Surgical procedure (Figure 1). Currently acceptable practices for experimental surgery were observed for this study. At all times throughout the period of manipulation, the animal was maintained at a surgical plane of anesthesia, such that the animal did not experience pain or distress. Blood pressure was monitored throughout the experiment and maintained within a normal range.
A midline laparotomy was performed on 7 pigs. The liver was exposed and isolated by packing the perimeter, being careful not to compromise circulation. Ten open-liver biopsies were performed in each swine with a 14 gauge biopsy needle. Each biopsy was treated with an 8 Fr plastic sheath loaded with GMO (treatment) or nothing (control). The treatment and control biopsies were equally divided between the medial and lateral lobes of the liver. Bleeding was scored (0 = no bleeding or 1 = bleeding) immediately, 2 minutes, 5 minutes and 10 minutes after treatment.
At the end of the evaluation, animals were euthanized in a manner consistent with the recommendations of the American Veterinary Medical Association Panel on Euthanasia.
Bacteria (Day 1). Bacteria culture plates were made for Enterococcis faecalis (trypticase soy agar + 5% defibrinated sheep’s blood), Staphylococcus aureus (trypticase soy agar), Escherichia coli (nutrient agar), and Klebsiella pneumonia (nutrient agar). Freeze-dried cultures were obtained from ATCC and reconstituted in 1 ml of appropriate broth. This was added to 4 ml of broth and mixed by pipetting. Three drops of bacteria were streaked on their respective agar plates, and both tubes and plates were incubated overnight at 37 degrees Celsius. All plates and cultures displayed growth.
(Day 2). New plates were made as above and 2 ml of GMO were placed in the agar (Two 1 ml strips pipetted into hot agar). One colony of each bacterium was then selected from day 1 and added to 1 ml of broth and vortexed. One hundred ul of this mixture was then plated on both the control and GMO plates and left overnight at 37 degrees Celsius. The following day, bacterial growth was evaluated.
Biophysical properties. To characterize GMO’s biophysical properties, we performed melting point, size expansion and adherence experiments.
Melting point. Three microcentrifuge tubes containing 0.75 g of GMO were placed in a water bath. At 5-minute intervals the temperature was increased by 0.2 degrees Celsius. When the GMO began to change from a solid to a semisolid phase, the temperature was noted. This experiment was repeated 3 times with a total of 9 vials of GMO tested.
Size expansion. 38.5 cm of molecular porous membrane tubing (Spectra/POR^®, Heidelberg, Germany) was attached to a metal sinker clip and weighed. Approximately 7.5 g of GMO was melted and 7 ml of this GMO was placed inside the tubing. The weight of the tubing, clip and GMO was recorded. Fifty milliliters of water was placed in a beaker and the metal clipped tubing filled with GMO was submerged in water and the amount of water displaced was recorded. This experiment was repeated four times. Next, 4 different GMOfilled tubing sections, each clipped with a metal sinker, were weighed and submerged in a water-filled glass container for 3 minutes. The tubing was then removed and patted dry and placed in a beaker filled with 50 ml of water. The amount of water displaced was recorded for each of the 4 tubing sections. The results of the 4 measurements with and without initial water submersion were compared.

Adherence (Figure 2). Chicken breasts were chosen to mimic human tissue. Waterproof athletic adhesive tape (2.5 cm wide) was chosen to test the adhesion in this study. The nonadhesive side was applied to the chicken. A battery of tests were designed to include a control study, in which no material was applied to the tape; a water study, where the tape was soaked in water prior to testing; and lastly, the GMO study, in which the tape was soaked in the GMO before testing. Each test was performed 10 times and compared.
Each adherence test was performed on a servo-controlled hydraulic testing machine by Minneapolis Testing System (MTS). The chicken breasts were warmed to 37 degrees Celsius to mimic human tissue. While the chicken was warming, the GMO was melted. Each piece of tape was cut to 45 cm in length. This allowed for the pull-angle to remain small throughout the test. Seven and a half centimeters of the 45 cm of tape was soaked in either nothing, water or GMO for 2 minutes. The chicken breast was placed on the ram platform and raised until only 7.5 cm was touching the chicken. The tape was kept at a 90 degree angle and lightly pressed against the chicken to ensure full contact. After the tape was in position, the chicken was left to bond with the tape for 2 minutes. Each test was performed at a rate of 5 mm/second to 76.2 mm (7.5 cm) as specified in the American Society forTesting and Materials (ASTM) Standard D 6252/D 6252M- 98.¹⁶ In between each test the chicken was placed back in the water bath to ensure the temperature remained at 37 degrees Celsius.
Histology. To determine if GMO promotes inflammation, histologic sections from swine were evaluated. Ten swine, 4 acute and 6 survivals (7 and 28 days), underwent open laparotomy and liver biopsy as described in the bleeding study. Six open-liver biopsies were performed in the acute swine (3 control and 3 treatment); 10 open-liver biopsies were performed in the 7-day survival swine (5 control and 5 treatment); and 6 open-liver biopsies were performed in the 28-day survival swine (3 control and 3 treatment). Each biopsy in the survival pigs was marked with methylene blue for future identification. After the initial surgery, the survival pigs underwent closure of their laparotomy and were monitored daily until sacrifice. Post euthanasia, each liver biopsy was excised en bloc (~2.5 cm x 2.5 cm). Each liver specimen was placed in formalin and delivered to the histology technician who sectioned the block into 5 perpendicular slices. Each slice was processed and embedded in paraffin. Slices were then sectioned to create histologic slides which were stained with Masson trichrome. A representative slide from each block was evaluated microscopically for the number of macrophage cell layers and the radius of the fibroblast cell layer surrounding the biopsy tract.
To evaluate inflammation by GMO in subcutaneous tissue, each survival pig underwent a subcutaneous puncture on both sides of the neck, with right being treatment (GMO), and left being control (nothing). Post euthanasia, the neck biopsies were excised and sectioned to make slides which were stained with Masson trichrome. Each slide was evaluated microscopically for inflammation.
Statistical analysis. Comparisons of continuous variables for 2 groups were made with the Wilcoxon rank sum test. The GMO adherence group was compared with the water and control adherence groups. Difference in adherence was also compared for the water versus control groups.
Multiple logistic regression¹⁷ was used to evaluate the treatment effect of bleeding at each time point post treatment. A dichotomous model for bleeding (yes/no) was evaluated. In addition to the treatment effect, covariates in the models included pig and lobe of the liver (medial or lateral). The treatment differences were adjusted for the differences in covariates, if any, in the multiple logistic regression models. For each model, the odds ratio for the effect of intervention relative to control and confidence intervals were calculated at each time period.

Results

Figure 3 shows the rapid biosealant effect of GMO biopsy sites compared to control. At 30 seconds, 1/35 control biopsy sites had stopped bleeding compared to 26/35 treatment biopsy sites. Also, the difference in number of biopsy sites achieving hemostasis decreased over time.
Table 1 displays the significant hemostatic effect of GMO as compared to control at 30 seconds, 2 minutes and 5 minutes. At 10 minutes, the bleeding results were not significant, likely explained by a pig’s innate ability to clot at this time period.
Bacteria culture plates coated with Enterococcis faecalis, Staphylococcus aureus, E. coli, and Klebsiella pneumonia showed no growth on the GMO-filled sections. Growth of Enterococcis faecalis, E. coli, and Klebsiella pneumonia was not inhibited by the presence of GMO in the culture media. However, the plate coated with Staphylococcus aureus displayed a 200 micron halo surrounding the GMO.
The melting point of GMO was approximately 29.7 degrees Celsius, below that of body temperature. The average water displacement for tubing/clip/GMO without being soaked in water was 8 ml of water, whereas the average water displacement when soaked in water was 11.75 ml, a 46% increase. Table 2 depicts the amount of force required to pull a piece of athletic tape coated with GMO from a chicken breast is significantly more than athletic tape coated in water (p = 0.0027) and athletic tape coated with nothing (p = 0.0013). There was no significant difference in the amount of force generated between athletic tape coated with water and athletic tape coated with nothing (p = 0.3287).

Histologically, the GMO was apparent out to 28 days. For the acute swine, GMO formed an effective hemostaticseal around the biopsy tract (Figure 4) and there were no macrophages or fibroblasts seen. The GMO-treated liver biopsies displayed minimal to no erythrocytes in the biopsy lumen or within adjacent hepatic sinusoids, whereas the control liver biopsies displayed extravasation of blood into the biopsy lumen and into adjacent hepatic sinusoids, with early evidence of hepatocyte injury including cellular discohesion and shrinkage.
Figures 5 and 6 display the number of macrophage cell layers and fibroblast radius by histology for the acute and survival swine. The change in macrophage cell layers and fibroblast radius are typical of normal scar formation. A “foreign body giant cell”-type macrophage response was not identified in any of the intervention livers. Additionally, GMO did not polarize when analyzed under polarized light for the acute and survival histology. There were only 3 control biopsy sites found at day 28 because the methylene blue used to mark the control biopsy disappeared and the sites were not apparent.
Also, some of the subcutaneous neck biopsies in the survival swine displayed a “foreign body giant cell”-type macrophage reaction.

Discussion
In this study evaluating GMO as a percutaneous vascular biosealant, a shortened time to achieve hemostasis for swine treated with GMO was seen compared to those treated with nothing. Treatment with GMO resulted in the majority of biopsy sites attaining hemostasis at 30 seconds, a time faster than 4 commonly used vascular closure devices.¹⁸ This is the first study of its kind to evaluate the properties of GMO as a novel vascular closure device.
This study quantifies the inherent properties of GMO as a biosealant. The melting point of GMO is below that of normal body temperature, allowing a phase transition to occur from a solid to a semisolid phase when placed in the body. During phase transition, the GMO expands and displays adhesive properties allowing occlusion of the needle tract and positional stability, respectively. These biosealant properties are unique to GMO, which promote hemostasis compared to the hemostatic closure properties of the collagen and/or thrombin vascular closure devices that activate the coagulation cascade or the direct vessel closure of suture devices.
Histologically, GMO formed a circumferential seal surrounding the injured liver biopsy tissue compared to control, supporting its biosealant effect. GMO displayed a normal inflammatory response, promoting scar formation in the liver. However, when GMO was placed subcutaneously in the neck, a foreign body giant cell response was witnessed. This histologic difference between the liver and neck may be attributed to the different environments of these two areas within the body. The liver is a solid organ laced by sinusoidal and vascular spaces that prevent hemorrhage by rapid activation of the coagulation cascade, resulting in scar formation. On the other hand, the subcutaneous environment in the neck is composed of soft tissues. It is less vascular, resulting in a longer exposure time for GMO to scavenger immune cell populations, resulting in a foreign-body cell response.
Infection is an unusual but serious complication of vascular closure devices. GMO is the only biologic used by a vascular closure device that displays bacterial deterrent properties. Several reports have indicated that free fatty acids and their 1-monoglycerides have microbicidal activity against various bacteria in vitro.^11–15 The proposed antimicrobial mechanism is disruption of the bacterial cell membrane, leading to its demise.¹² This modest but beneficial characteristic of GMO may apply to a broad spectrum of bacteria and yeasts, which may decrease the infectious complication rate of these procedures. Future trials should focus on the mechanism of action of these deterrent properties and test a broader spectrum of pathogens.
Study limitations. Several limitations are inherent in this study. First, there is no “gold standard” bleeding model in the surgical literature. This study used a liver bleeding model described by Paulson and colleagues,²⁰ which tested the hemostatic properties of a fibrin sealant. This was an appropriate model for testing GMO’s biosealant properties, considering the liver is highly vascular and commonly biopsied. Histologically, differences were noted for the inflammatory response to GMO between the swine liver and subcutaneous tissue. This study did not investigate the mechanism for these histologic differences, whereas future studies should focus on this issue before implementation in humans.

Conclusions
This study identified several questions regarding the use of GMO as a vascular closure device. The mechanism of action for the rapid attainment of hemostasis by GMO can be explained through its biosealant properties. Accordingly, other hemostatic mechanisms such as activation of the coagulation cascade should be evaluated through additional studies. The bacterial deterrent properties of GMO are unique to vascular closure devices and should be further defined (bacteriostatic or bacteriocidal) in future studies. The survival study concluded at 28 days, and GMO was still present. Future studies should measure the time it takes for GMO to completely biodegrade. Furthermore, risks associated with GMO displacement during restick and GMO entry into the vasculature should be assessed.
The future application of GMO as a vascular closure device is encouraging. However, before human application, a femoral artery animal model with adequate subcutaneous tissue should be investigated since the inflammatory response characterized in the liver likely does not provide the optimum model for human femoral artery sites. This is evidenced by the different inflammatory response seen in our preliminary experiments with the swine neck.
In conclusion, the melting point, expansion, adherence, biosealant and bacterial deterrent properties of GMO are promising for a vascular closure device. Based on these results, phase 1 trials are being developed.

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