BIOE 410 Term Paper

7/30/2019 BIOE 410 Term Paper

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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 throughmicrochannel 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 actuallyacquire 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 small-

scale, 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


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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 atsize 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 arti ficial lung.

2. Methods of Increasing Biocompatibility

Currently, many PDMS devices intended to interact with blood are primed with heparin in order toreduce 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


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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 fibronectin-

assisted endothelialization. In the paperMEMS-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 fabricatedutilizing 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 106

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 intopographical 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

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

Figure 3: (a) Untreated PDMS (b) Ion Irradiated PDMS


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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

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

Figure 4: Examples of Cell Seeding on Pure PDMS and Ion Implanted PDMS Surfaces


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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 lungemploying air as the ventilating gas,"Lab on a Chip, pp. 2901-2909, 2011. Online. Available.

.

2. S. Thorslund, J. Sanchez, R. Larsson, F. Nikolajeff and J. Bergquist, "Bioactive heparinimmobilized onto microfluidic channels in poly(dimethylsiloxane) results in hydrophilic surface

properties," Colloids and Surfaces, pp. 106-113, 2005. Online. Available..

3. D. M. Tollefsen, "Vascular Dermatan Sulfate and Heparin Cofactor II," Progress in MolecularBiology and Translational Science, vol. 93, pp. 351-367, 2010. Online. Available.

.

4. N. Naik, V. Kumar, E. L. Chaikof and M. G. Allen, "MEMS-assisted spatially homogeneousendothelialiization of a high length-to-depth aspect ratio microvascular network," 33rd Annual

International Conference of the IEEE EMBS, pp. 290-293, 2011. Online. Available.

.

5. M. Ionescu, B. Winton, D. Wexler, R. Siegele, A. Deslantes, E. Stelcer, A. Atanacio and D.Cohen, "Enhanced biocompatibility of PDMS (polydimethylsiloxane) polymer films by ion

irradiation,"Nuclear Instruments and Methods in Physics Research B, pp. 161-163, 2011. Online.

Available. .

6. "What is Dermatan Sulfate?," Conjecture Corporation, 2003-2012. [Online]. Available:http://www.wisegeek.com/what-is-dermatan-sulfate.htm. [Accessed 8 December 2012].


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7. S. Thorslund, J. Sanchez, R. Larsson, F. Nikolajeff and J. Bergquist, "Bioactive heparinimmobilized onto microfluidic channels in poly(dimethylsiloxane) results in hydrophilic surface

properties," Colloids and Surfaces, pp. 106-113, 2005. Online. Available.

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