Blood barrier medical devices essential for critical care often face thrombus formation, compromising function, and increasing systemic thrombosis risks. Clinical anticoagulants mitigate thrombosis, but heightened bleeding risks. Protein absorption on biomaterial surfaces triggers coagulation and inflammation.
We investigated the impact of zoura polymer wash through coating on ECMO circuit protein absorption. Currently, there is no commercial anticoagulant that selectively and effectively inhibits thrombosis in medical devices without affecting the patient. Additionally, it is essential to optimize and demonstrate the effectiveness of surface coatings and engineering over durations, ranging from days to weeks and even months for various medical devices.
Our new scientific objectives include evaluating the influence of different pre-processing approaches on coating application, assessing the impact of handling methods for the wash through coating solution on coating uniformity, investigating the effects of alternative antifouling polymer grafts on nonspecific protein fouling, and determining the ability of optimized coatings to limit thrombosis in vivo. To clean the lung circuit, recirculate 30%methanol in deionized water for 20 minutes. Follow this by recirculating 10%methanol in deionized water and then deionized water alone for another 20 minutes.
Dry the lung circuit with filtered house air set to low flow for two hours. Place the cleaned and dried lung circuit into the ultraviolet ozonolysis plasma generator. Close the generator and turn on the instrument to initiate plasma exposure for 20 minutes.
After completing plasma exposure, proceed directly to the coating step to prevent surface chain rearrangement, and loss of reactive sites generated by plasma interaction. While the device undergoes surface modification, dissolve 1.2 grams of dopamine hydrochloride in 600 milliliters of tris buffer in a glass beaker. Subsequently, dissolve the sulfobetaine methacrylate monomer at a ratio of one to 15 dopamine hydrochloride to sulfobetaine methacrylate.
Then add five millimolars of sodium periodate as droplets into the coating solution. Use a magnetic stir bar and stir plate set to 150 RPM to mix the solution thoroughly. Next, prime the lung device with the coating solution using a large syringe to draw and fill the circuit.
Clamp one end of the circuit and prime it from the other end while guiding air bubbles out through a circuit access connector. Once the circuit is fully primed, place the device under an ultraviolet light source for two hours. Agitate the solution by reorienting the ends of the prime device up and down every 10 minutes.
After the light treatment, drain the circuit completely. Gently rinse all samples with deionized water by priming and draining repeatedly using a 60 cubic centimeter syringe until the rinse effluent is clear. Finally, store the device filled with deionized water in a refrigerator set at four degrees Celsius for autopsy and surface analyses.
To perform the fibrinogen absorption test, transfer fiber mat samples in one milliliter of three milligrams per milliliter fibrinogen solution in well plates and incubate the plate at 37 degrees Celsius for 90 minutes at 60 RPM. Wash the samples with PBS. Transfer them into new wells and add one milliliter of one milligram per milliliter BSA solution to each well.
Incubate for another 90 minutes. Then wash the samples three more times with PBS solution and transfer them into new wells. Next, add one milliliter of a one to 1000 dilution of horse radish peroxidase conjugated fibrinogen antibody in PBS solution to each well.
After incubating the samples for 30 minutes and washing them, transfer them into new wells. In the new wells, add 500 microliters of ophenylenediamine in citrate phosphate buffer with 0.03%hydrogen peroxide, adjusted to a pH of 5.0 at 30 second intervals. Incubate the samples away from light for 30 minutes.
To stop the peroxidase and ophenylenediamine reaction, add 500 microliters of one normal hydrochloric acid to each well. Transfer the supernatant from each well into a cuvette. Measure the absorbance of the supernatant at 492 nanometers using a UV visible spectrophotometer.
Thaw the lactate dehydrogenase assay kit for 20 minutes. While the assay kit is thawing, prepare pooled adult human plasma to obtain platelet rich plasma. To prepare platelet rich plasma, centrifuge thawed human plasma sample tubes at 483 G to separate the plasma into two regions.
Identify the lower one third containing platelet rich plasma and the upper two thirds containing platelet poor plasma. Carefully remove the platelet pellets at the bottom of the tubes and the upper two thirds containing platelet poor plasma. Gently disperse the platelet pellet into the upper plasma by shaking the tubes.
Before testing, add calcium chloride to the platelet rich plasma to reverse the effects of citrates. Next, incubate the fiber mat samples in 500 microliters of platelet rich plasma for 90 minutes at 37 degrees Celsius. After incubation, transfer the samples into new wells and rinse three times with PBS solution.
Again, transfer the samples into new wells. Add 300 microliters of PBS solution and 10 microliters of 10 X lysis buffer to each well. Incubate the samples for 45 minutes, then add 50 microliters of the reaction mix to each well and incubate for 30 minutes away from light.
Add 50 microliters of hydrochloric acid to each well to stop the reactions. To detect lactate dehydrogenase activity from lysates of absorbed platelets, measure the light absorbance of the developed well solutions at 490 nanometers and 680 nanometers. Fibrinogen fouling was significantly reduced in coded PP-PDMS fibers compared to uncoated controls.
There was no significant difference in fibrinogen fouling between outer and inner bundles in PP-PDMS fibers under no flow conditions. Under 24 hour PBS flow, fibrinogen fouling in the outer and inner bundles remained low and did not show significant differences. Platelet fouling was higher in the outer bundle of PP-PDMS fibers than in the inner bundle, and uncoated control.
Platelet fouling was significantly higher in the outer bundle compared to the inner bundle encoded PP-PDMS fibers without flow exposure. Under 24 hour PBS flow, no significant difference in platelet fouling was observed between outer and inner bundles with low fouling levels.
