Matriz de Riesgos

From Experiment 2, fluorescent images of platelet adhesion each coating after blood exposure are

in Fig. A. 1.15, taken at 40X magnification to visualize a wide view of platelet distribution on the

surfaces. On the unmodified PDMS surface (Fig. A. 1.15a), platelets can be seen distributed across the

surface at a consistent, low density. Some platelets appear stained with only FITC, indicating adhesion

but no activation, while some are also cross-stained with Cy-3, pointing to activation. Of the coatings, HA

(Fig. A. 1.15b) shows the least platelet adhesion/activation, with only a few platelets present on the

146 activation compared to the unmodified PDMS surface, and the PAA-His surface (Fig. A. 1.15d) shows a

modest increase. In particular, the Hemin surface is covered by a high density of cross-stained platelets,

some of which have started to form larger aggregates. IHC imaging was not conducted on samples from

Experiment 1.

Fig. A. 1.15: Experiment 2 – 40X magnification IHC cross-stain of platelet adhesion (CD41, FITC green)

and platelet activation (CD62p, Cy-3 red) on membranes removed from the in vivo devices after nearly 4 hours of blood contact. Coatings are: (a) unmodified PDMS, (b) HA, (c) Hemin, and (d) PAA-His.

DISCUSSION

Considering the data sets from all experiments, the biomolecule coatings strongly altered the

hemocompatibility of the Si-PDMS membrane surfaces. Whether the benefit was positive or negative, the

coatings generally showed consistent trends between in vitro wettability and in vivo hemocompatibility,

and mostly differed from the unmodified PDMS surfaces. Between the two experiments, it is unclear if

the coatings have a detrimental impact on the inherent gas permeability of the Si-PDMS membranes.

147 evidence. Although the coatings in Experiment 2 consistently decreased the in vitro O2 flux through the

membranes, it is unclear if this trend was also observed in vivo due to the high measurement error. As

such, the biomolecule coatings demonstrated a clear hemocompatibility benefit compared to unmodified

PDMS, but were unable to show any blood-gas exchange improvements on gas exchange membranes.

The presence of the biomolecule coatings on the surface was confirmed by contact angle changes.

Characterization of the coatings was largely consistent with prior research on these coatings. To validate

the presence of the coatings on the surface, two additional characterization techinques were employed in

this study: colorimetric staining for amine and carboxyl groups and x-ray photoelectron spectroscopy.

However, these datasets were not sufficiently complete to include in this analysis. As such, coating

characterization in this study is currently limited to contact angle analysis. Still, other characterization

techniques may have been useful to further define the functionality of the coatings. Data on thickness and

conformity via ellipsometry could have identified possible links between coating thickness and

thrombogenicity. Atomic force microscopy could have been used to image the topography of the coated

surfaces, since surface roughness is also linked to thrombogenicity. Additionally, a technique to quantify

the surface charge of the biomolecule coatings could have verified the charge state of the coatings under

physiologic conditions.

Comparison of the characterization and hemocompatibility data revealed some trends consistent

with all data sets. Namely, the HA coating was the most hydrophilic, and also the most resistant to cell

and platelet adhesion. This finding was consistent between Experiments 1 and 2. Conversely, the most

hydrophobic coatings – PAA-Lys and Hemin – were more thrombogenic than HA, but to different

extents. PAA-His was intermediate in both regards. These comparisons indicated a strong connection

between hydrophilicity and hemocompatibility with the coatings. However, if hydrophobicity alone

determined hemocompatibility, the unmodified PDMS and PAA-Lys surfaces should have been as

148 unmodified PDMS material control despite being equally hydrophobic. Also, in Experiment 2, the

unmodified PDMS surface attracted relatively few cells and platelets, comparable to the PAA-His surface

and far less than Hemin. As such, factors beyond wettability likely contributed towards

hemocompatibility, although hydrophilicity was a clear factor in preventing thrombosis.

Between all data sets, the influence of the coatings on gas permeability was unclear. The two in

vitro data sets indicated opposite conclusions. Whereas Experiment 1 showed no significant difference

with coating application, Experiment 2 showed a clear reduction in permeability with the application of

any coating to the surface. Normalizing the flux measured through unmodified SµM-PDMS membranes

from Chapter 2 (Fig. 2.7) to surface area, the O2 flux measured in prior studies at 10 mL/min flow rate

was 306±35.2 mL/min/m2_{. Both datasets from Experiment 1 and 2 (Fig. A. 1.6 and Fig. A. 1.7) showed}

far less flux on the unmodified membranes than this study at the same flow rate, although the membranes

used in these studies had different pore designs that may have influenced the normalization. Still, the flux

measured in Experiment 2 was much closer to this previous measurement than Experiment 1, reaching

close to the same flux at a higher water flow rate. Given the increased number of data points in

Experiment 2, as well as the use of higher flow rates, the in vitro dataset in Experiment 2 is likely more

reflective of the coatings’ effect on the inherent gas permeability of the membranes. As such, the coatings appear to strongly decrease gas permeability of SµM-PDMS membranes.

The reduction seen with DETA application alone may be the most significant contributor to the

overall lower permeability of the coatings, since most of the coatings did not decrease flux further than

DETA alone. One possibility for this observation may be related to the effects of the plasma treatment

required to apply the DETA coating. Plasma treatment of PDMS causes the formation of a stiff, glassy

SiO2 layer on the PDMS surfaces, which has been shown to strongly reduce the gas permeability of

PDMS 115,117_{. Usually, this SiO}

2 layer dissipates over time due to surface rearrangement of the PDMS

149 microns thick, unlike the ultrathin spin-coated layer on the SµM-PDMS. It is possible that an SiO2 layer

on SµM-PDMS membranes may not experience much surface rearrangement over time due to its

thinness, and lack of sufficient PDMS required for rearrangement. Therefore, it is possible that the gas

permeability reduction associated with plasma treatment may linger on SµM-PDMS membranes for an

indefinite amount of time. Plasma-treated PDMS without DETA application was not independently

evaluated in this experiment, so further studies would need to confirm this observation.

The in vivo datasets from Experiments 1 and 2 were also unclear as to the impact of the coatings

on gas exchange. Neither experiment was able to demonstrate oxygenation, unlike in Chapter 3. Lack of

demonstrable oxygenation in both experiments is likely due to the instability of the ventilation regimen of

the pigs. Unstable control over the incoming venous blood, and a limited collection of data points per

device, resulted in high variance in O2 flux measurements. Similarly, CO2 flux measurements were

affected by this instability in ventilation, particularly in Experiment 2 with the wide variation in blood pH

(7.3-7.7 over the course of the experiment). Still, the extremely high CO2 removal seen in Experiment 1

was striking, especially compared to past results from Chapter 5 under similar hypercarbic conditions.

This CO2 removal vastly exceeds data from Arazawa, et al. (411 mL/min/m2), although – given the lack

of a control uncoated device in this experiment – it is unclear if the coatings were the main driver of the

high CO2 removal. When a control device was added in Experiment 2, no significant difference in CO2

removal was seen between uncoated and coated devices, possibly attributable to the inconsistency in the

pig’s ventilation. Considering all of the in vivo datasets, the impact of the coatings on in vivo gas exchange cannot be determined without additional experiments.

Future in vivo experiments would need to significantly modify the animal handling procedures in

order to maintain consistent O2 and CO2 control over the incoming venous blood from the pig. In

Experiments 1 and 2, the pig was ventilated with only O2 because equipment to mix multiple gases for the

150 venous blood (SO2 ~100%), and the subsequent hand bag ventilation regimen to create a slightly hypoxic

state. Titration of the inhaled ventilator gas with room air or N2 would prevent this venous hyperoxia, and

allow for controllable hypoxia within the venous blood. If such a ventilation regimen is used, better

quality in vivo data could be collected to demonstrate O2 and CO2 transport.

CONCLUSION

Ultimately, the biomolecule coatings on SµM-PDMS membranes did significantly improve the

hemocompatibility of the surfaces, but were unable to demonstrate consistent impacts on gas exchange

through the membranes. Of the coatings, the HA coating proved to be the most hydrophilic and

hemocompatible when exposed to flowing blood. Oxygen flux in vitro may have been significantly

lessened by the application of the coatings, possibly due to the plasma treatment required for surface

modification. However, in vivo no difference was observed in oxygenation with or without coatings.

Strikingly high CO2 removal was seen in one of the experiments, signaling significant promise for the

coatings in a CO2 removal device. However, this result was not validated by the second experiment. Both

of the experiments were impacted by the instability of the in vivo setup, and so neither experiment can

conclusively demonstrate the impact of the coatings on in vivo gas exchange. Nonetheless, this work

raises significant interest in the use of these coatings for improving PDMS-based oxygenator

hemocompatibility, and possibly the gas exchange efficiency through the membranes. Future work will

focus on increasing sample numbers to improve overall data quality for characterization and membrane

151