Increasing the Lifetime of Artificial Lung Device by Improving Biocompatibility of PDMS Antonio Bunce and Johnson Huynh

Abstract The artificial lung developed by Joseph A. Potkay of Case Western Reserve University is one of the first artificial lung devices that can use air as the ventilating gas. The increase in oxygen exchange efficiency of the device marks a huge step forward in terms of portability for artificial lung devices. The enhancements were made through use of new mathematical modeling and the use of a bio-inspired design in the device microfabrication. While this device is on the cutting edge in terms of portability and efficiency, there are still problems that need to be overcome such as thrombus formation which can limit device lifetime. Currently the device lifetime can be measured in hours, which is not ideal for patients suffering from lung disease. The current material that is used in the microfabrication of the device is PDMS. In order for the artificial lung device to prolong its lifetime, improvements must be made in terms of PDMS biocompatibility. The designers of the device have postulated that these improvements can be achieved through surface functionalization of the PDMS with hemocompatible bio-molecules or through microchannel endothelialization. We intend to look at methods of modifying the surface characteristics of PDMS such as ion irradiation which can improve cell adhesion, thus allowing the device to have a longer lifetime. 1. Introduction Millions of individuals all over the world are influenced by lung disease. In the United States, in particular, it is one of the leading causes of death (1). Different approaches have been utilized to tackle this terrible phenomenon. For patients in clinical settings, doctors have recommended using positive pressure ventilation to somewhat make up for the lungs inability to function. In other situations, temporary respiratory supports provide injured lungs some time to heal. For more chronic issues, lung transplantation and artificial devices are options. Unfortunately, patients must wait approximately 1.2 years to actually acquire another lung and about 11% die before ever getting one (1). Therefore, artificial lungs have arisen as the better alternative. However, significant improvements must be made first to these devices, and Joseph Potkays artificial lung will lead the scientific community in the right direction. Potkay, a research assistant professor at Case Western Reserve University, has developed an artificial lung that is 3 to 5 times more efficient than current devices (1). Gas exchange, biocompatibility, and portability have been limiting conventional devices from their full clinical potential. Potkays smallscale, microfabricated artificial lung utilizes new mathematical modeling and a bio-inspired design to allow air (as opposed to pure oxygen) to be its ventilating gas (1). Because of the devices similar characteristics and dimensions to the natural lung, there is hope that this advancement will represent a momentous step towards the first truly portable and implantable artificial lung. Figure 1 showcases two different views of the blood and air channels within the artificial lung device. The microchannels flow at perpendicular directions with respect to one another. The two channels are separated by a PDMS gas diffusion membrane to allow

Figure 1: Schematic of Artificial Lung Device

the exchange of oxygen and carbon dioxide. Despite the promise that this device has shown, improvements are still necessary for Potkays invention. The construction of the device itself needs to be further analyzed, but more importantly, the biocompatibility must be enhanced. It has a current lifetime measured only in hours, due to the formation of clots within the device (1). Experimental results indicated that the blood-side pressures increased with more exposure to blood, and microscopic inspection showed thrombus formation at larger channels and at size changes. Before any changes are made to increase the biocompatibility of the channels inside the device, the tubing that connects the blood supply to the device should first be heparinized in order to avoid clot formation outside the device (1). This paper will analyze a variety of possible ways to advance the biocompatibility of the devices internal channels. First, the effects of the combination of dermatan sulfate and heparin on a PDMS surface will be examined. Second, fibronectin will be evaluated as an approach to endothelialize microfluidic channels. Lastly, the third method involves the use of single ion irradiation and how it modifies PDMS surface properties. Each technique has its own advantages and disadvantages, and this work will investigate what exactly is the best course of action for Potkays artificial lung. 2. Methods of Increasing Biocompatibility Currently, many PDMS devices intended to interact with blood are primed with heparin in order to reduce the instance of blood coagulation. Heparin inhibits thrombus formation and helps in natural degradation of existing thrombi. The effect of the use of heparin on PDMS microfluidic channels can be clearly seen in Figure 2 (2).

Figure 2: (a) Heparinized PDMS channel. (b) Regular PDMS In the case of the artificial lung device previously discussed, a mixture of phosphate buffered saline and heparin was used to prime the device before the introduction of blood (1). Even though the device was primed with heparin, clotting was still evident in the device after exposure to blood. A proposed method of increasing the effectiveness of heparin in preventing clot formation is the addition of dermatan sulfate to the system through priming or controlled dosages to the patient. Dermatan sulfate is a glycoaminoglycan that can be found deep in the walls of human vasculature (3). Typically, heparin cofactor II found in the blood stream acts as an inhibitor of thrombin by forming a covalent 1:1 complex. However, in the present of dermatan sulfate, the rate of formation of this complex increases 1000-fold resulting in more successful protection against coagulation (3). The activation of heparin cofactor II by dermatan sulfate is usually accomplished after an injury to the endothelium of a blood vessel exposes dermatan sulfate to heparin cofactor II. This activation of heparin cofactor II is typically observed in the presence of higher

concentrations of heparin, which explains the necessity for dermatan sulfate introduction in combination with that of heparin (3). A second method to increase the biocompatibility of blood microchannels is through fibronectinassisted endothelialization. In the paper MEMS-assisted spatially homogeneous endothelialization of a high length-to-depth aspect ratio microvascular network, endothelial cells are seeded onto the interior walls of a PDMS microchannel under dynamic conditions. First, the PDMS channel is fabricated utilizing standard microfabrication techniques (4). Nickel electroplating, photoresist removal, and PDMS micromolding are used to construct the deformable scaffold. It has a length to depth (l/d) aspect ratio of greater than 200 (length of 2 cm and depth of 80 m), and it has a width of about 80 m. After assembly, the channel is treated with oxygen plasma (for about 30 seconds) to prepare for endothelial cell seeding (4). This results in a more hydrophilic structure, as it is then sterilized in ethanol for 2 hours. Following vacuum drying, the microchannel is incubated in 8 mL of 50 g/mL fibronectin for 24 hours at 4 C. This important glycoprotein will assist in endothelial cell adhesion as it binds to extracellular matrix components. Subsequently, 5 mL of PBS (phosphate buffered saline) is then used to remove the excess fibronectin (4). After that is finished, the channel is placed in media at 37 C for 2 hours prior to cell seeding. As the channel is stored in media, cytopreserved human umbilical vein endothelial cells (HUVEC) can be thawed and cultured in a cell culture flask with endothelial cell growth medium. They are spun down to a pellet and resuspended in media at 10 6 cells/mL. Once everything is correctly prepared, endothelialization of the channel can take place. The PDMS channel is placed on a microplate shaker to create a dynamic environment for incoming HUVECs (4). This type of dynamic seeding allows the cells to accumulate into the trenches and stick to the interior walls of the channel. The channel is only on the microplate shaker for 20 minutes as the cell media is introduced, and the HUVECs culture in the relaxed channel for 5 days in a 37 C incubator. The microchannel is now biocompatible without mass transport limitations (which is typical of high aspect ratio channels). Another method of increasing the biocompatibility of PDMS is single ion irradiation. For this method, an ion acceleration voltage of 40 kV and a beam current of 0.1 mA were used to implant iron, magnesium, and tantalum atoms into separate PDMS films approximately 4 m in thickness (5). The PDMS samples were irradiated for five seconds to twenty minutes. The implantation of the ions resulted in topographical changes that can be seen in Figure 3 (5). Contact angle measurements were then taken 48 hours after the samples had been irradiated in order to determine changes in surface energy from normal PDMS. It was found that the ion irradiation had rendered the surfaces more hydrophilic and increased the total Figure 3: (a) Untreated PDMS (b) Ion Irradiated PDMS surface energy (5). Focused ion beam milling was also used to cut cross-section samples in order to verify the hypothesis that the topographical changes only occurred at the surface of the PDMS film. It was found that the ions did not penetrate the film and the topographical changes were only surface features. The samples of PDMS that had been irradiated for twenty minutes were then used in an in vivo biocompatibility test to see if cell adhesion to the PDMS had increased due to the irradiation. The samples for each different ion (Fe, Mg, and Ta) were first sterilized and then placed into a 24-well plate. The

samples were then seeded with L929 fibroblast cells that had been cultured in Dulbeccos Modified Eagle Medium supplemented with 20% fetal bovine serum (5). Samples of regular PDMS were also subjected to the same processes to serve as a control. All of the samples were cultured for 48 hours at 37C and 5% CO2 before removal from the well plates and subsequently immersed in fresh medium to remove any unattached cells. The samples were then placed in a new 24-well plate with 0.5 mL of DMEM:F12 containing 1 mM calcein-AM for thirty minutes at 37C. The irradiated and regular PDMS samples were then examined in order to determine the number of viable cells attached. Optic microscopy images of the irradiated samples and normal PDMS after cell

Figure 4: Examples of Cell Seeding on Pure PDMS and Ion Implanted PDMS Surfaces seeding can be seen in Figure 4 (5). It was determined upon examination that there was a 450% increase in cell viability for the Mg and Ta implanted PDMS and a 650% increase in cell viability for the Fe-implanted sample over normal PDMS (5). This conclusion represents a very significant increase in the biocompatibility of PDMS. 3. Evaluation of Methods for Artificial Lung Device The use of dermatan sulfate in the artificial lung device in conjunction with continued heparin priming has the potential to significantly reduce the formation of thrombi in the device. For use in the device, dermatan sulfate could be pumped through the device along with the phosphate buffered saline and heparin to prime it, or the patient could be given controlled dosages of dermatan sulfate while using the artificial lung device. The disadvantages to the use of dermatan sulfate are that it is not readily understood and has been linked to several medical complications (6). Due to its high ability to decrease thrombus formation in the body, an excess of dermatan sulfate could be detrimental as it may inhibit the normal clotting response in the host. Dermatan sulfate is a glycoaminoglycan and requires multiple specific lysosomal enzymes in order to be degraded in the body (7). This complex degradation process can be severely disrupted if a single one of the enzymes is not functioning properly and result in lysosomal storage of the glycoaminoglycan. The storage of glycoaminoglycans such as dermatan sulfate is the cause for a range of diseases known as mucopolysaccharidoses (7). This set of diseases can lead to several different complications in the human body such as bone lesions and blindness (6). One class of mucopolysaccharidoses, Sanfilippo diseases, has been correlated with accumulations of dermatan sulfate, and results in severe mental handicaps for those afflicted due to dysfunction in the central nervous system. Accumulation of dermatan sulfate in the mitral valve has been found to lead to mitral valve prolapse as well (6). As seen before, it is beneficial to utilize fibronectin-assisted endothelialization on the interior walls of a PDMS microchannel. Oxygen plasma renders a microchannel more hydrophilic, which allows the fibronectin to attach to the walls. Fibronectin plays a key role towards endothelial cell adhesion, as the cells attach to it under dynamic conditions. It is a fairly simple process, and can be applied to the artificial lung device. A point of interest that must be addressed when discussing this relationship is that the dimensions of the artificial lung device are different than the dimensional characteristics of the channel in the fibronectin paper. While both of the widths are relatively the same (around 80 m), the two

length-to-depth aspect ratios are comparably not very close. The fibronectin papers aspect ratio is around 250 (2 cm / 80 m), while the artificial devices aspect ratio is around 31 (310 m / 10 m) (1, 4). Even though the fibronectin microchannel has proven to be effective in allowing normal flow for high aspect ratio channels, the artificial devices lower aspect ratio should not be a detriment towards endothelialization of its blood channels. Single ion irradiation is a novel and straightforward method for increasing the biocompatibility of PDMS. It has shown significant ability to increase the cell viability of fibroblasts on PDMS (5). The adhesion of fibroblasts to the implanted PDMS could serve as a precursor to eventual endothelial cell adhesion on the PDMS surface. Fibroblasts are an integral part of the endothelial layer in blood vessels and secrete many of the components of the extracellular matrix that could provide areas of adhesion for endothelial cells. The applicability of single ion irradiation of Mg, Ta, and Fe ions on microfluidic channels has yet to be addressed as it has only been tested on thin polymer films (5). As such, irradiation could only reliably be used on the PDMS gas diffusion membrane in the artificial lung device, which is a flat 15 m thick PDMS layer. It is not known whether the implanted ions may have an effect on the diffusion of oxygen or carbon dioxide. Also, it has not been determined how the implanted PDMS may respond to other common treatments such as oxygen plasma. 4. Discussion The use of dermatan sulfate in combination with heparin has too many possible complications and is not well enough understood to be an ideal method of increasing the biocompatibility of PDMS. Similarly, single ion radiation, while able to dramatically increase cell viability, is also not well understood in terms of its interactions with other common micro fabrication techniques. Currently, the gas diffusion membrane in the artificial lung device is attached to the air and blood channels by means of using oxygen plasma treatment on the surfaces to be bonded, then bringing them into contact to form a liquid-tight seal (1). Since it is currently not known how ion irradiated PDMS responds to oxygen plasma treatment and vice versa, the bonding of the gas diffusion membrane to the blood channel may prove difficult. It is possible that since both processes increase the hydrophilicity of the PDMS, an ion irradiated layer may be able to bond to an oxygen plasma treated layer, but it is unknown what effect the implanted ions may have on this bondage. Lastly, the effect of the ions implanted in the PDMS surface on the diffusion of oxygen and carbon dioxide through the PDMS is not known and could potentially decrease the diffusion of the gases leading to a less effective device, which is not a desired outcome. Based on examining the advantages and disadvantages to each method outlined previously, it was determined that fibronectin-assisted endothelialization of the blood microchannels is the best method to improve the biocompatibility of PDMS for the artificial lung device. In this method, oxygen plasma is used to make the surface of the PDMS channels more hydrophilic before the addition of the fibronectin (4). Therefore, in order to use this method in the artificial lung device, the channels can be oxygen plasma treated as normal and bonded to the gas diffusion membrane, and then the blood channel can be endothelialized using a similar process to that described previously for the fibronectinassisted endothelialization. The major difference between the processes is that the fibronectin and cell culture solutions will have to be pumped through the device for incubation. This method Figure 5: Final Solution

will allow the sides of the blood channel to be endothelialized as well as the gas diffusion membrane on top of the channel, which will lead to less thrombus formation in the device. This solution can be seen in the illustration in Figure 5. While according to the designers of the artificial lung device a channel that is 10 m in height has the best gas diffusion, the other geometry tested, 20 m high channels will be used (1). This conclusion is due to the fact that an endothelial cell is typically about 12-15 m in diameter, so a channel height of 20 m will allow more successful endothelialization than a channel height of 10m. 5. Conclusion The improvements stated in this paper should be applicable to the artificial lung device developed by Joseph Potkay. The use of fibronectin in previous research has shown promise in the endothelialization of microfluidic channels (4). Endothelialization of the microfluidic channels will allow the reduction of thrombus formation in the device thereby increasing its lifetime. The other methods discussed, the use of dermatan sulfate in combination with heparin and single ion irradiation, while promising, need to be further researched before their applicability can be fully understood. Single ion irradiation appears to offer the most dramatic increase in biocompatibility of PDMS by increasing the cell viability by up to 650% (5). However, the interaction of ion irradiated surfaces with other common PDMS treatments has presently not been investigated, and should be further assessed as the applications of this treatment appear to be a very encouraging alternative for increasing the biocompatibility of PDMS for future iterations of the artificial lung device and other devices as well. References 1. J. A. Potkay, M. Magnetta, A. Vinson and B. Cmolik, "Bio-inspired, efficient, artificial lung employing air as the ventilating gas," Lab on a Chip, pp. 2901-2909, 2011. Online. Available. <http://pubs.rsc.org/en/content/articlepdf/2011/lc/c1lc20020h>.

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