Blood Compatibility of Degradable Polar Hydrophobic Ionic Polyurethane (D-PHI) Designed for Blood Contact Applications

Brockman, Kathryne S (Department of Chemical Engineering and Applied Chemistry, University of Toronto)
Kizhakkedathu, Jayachandran N (Department of Pathology and Laboratory Medicine and Centre for Blood Research, UBC)
Santerre, J. Paul (Institute of Biomaterials and Biomedical Engineering and Faculty of Dentistry, University of Toronto)

Introduction

Cardiovascular disease is a major cause of death in Canada, often requiring bypass graft surgery. Ideally, the bypass graft would consist of autologous vessels; however, due to a lack of suitable natural vessels, artificial vascular grafts are required. While materials such as Dacron and polytetrafluoroethylene currently exist for large diameter (>6 mm) vascular grafts, they are unsuitable for small diameter applications[1]. Degradable polar hydrophobic ionic polyurethane (D-PHI) has been developed for small diameter vascular graft applications[2], and it shows mechanical properties similar to that of natural vessels[2] and a reduced pro-inflammatory cell response[3]. However, the blood compatibility of this material has yet to be studied, which is important since D-PHI may contact blood during this application. The current work will use in vitro methods to assess the blood compatibility of D-PHI with a focus on coagulation cascade initiation, complement activation, and platelet activation.

Materials and Methods

D-PHI films and particles generated from scaffolds were fabricated as previously described[2]. Films were cast on glass slides, delaminated, and cut to size. Particles were ground from scaffolds and sieved to an average particle size of 80.5 ± 28.6 μm. Films were used as is and particles were suspended in HEPES buffered saline solution by sonication to a concentration of 50 g/L. Coagulation in whole blood was assessed by thromboelastography (TEG) analysis wherein 40 μL of D-PHI particle suspension in HEPES was mixed with 360 μL of fresh whole human blood (protocol was approved by UBC's clinical ethics board) or platelet poor plasma (PPP), then 340 μL of the resulting solution was recalcified by the addition of 20 μL of 0.2 M CaCl2 and added to a TEG cup warmed to 37°C. Whole blood or PPP incubated with HEPES buffer was used as a control. The TEG measures the speed and kinetics of clot formation to determine overall change in blood coagulation. The intrinsic and extrinsic pathways of the coagulation cascade were evaluated by activated partial thromboplastin time (APTT) and dilute prothrombin time (PT) assays respectively. For these assays, PPP and particle suspension were mixed in a ratio of 9:1 and added to a coagulometer with the appropriate reagent(s) (APTT: Siemen's Actin FSL and 0.2 M CaCl2; PT: thromboplastin), and the time to clot formation was measured. The total complement consumption (or activation) was measured by a hemolytic assay using sheep erythrocytes (CH50 sheep erythrocyte lysis assay). Briefly, D-PHI suspension was mixed with dilute human serum (1:5 serum:GVB++) in a 1:9 ratio and incubated for 1 h at 37°C, then diluted 1:2 with GVB++ and mixed in a 1:1 ratio with antibody-sensitized sheep erythrocytes (CompTech Complement Technology, Inc.). After a second 1 h incubation at 37°C, cold GVB is used to stop the reaction and the lysis of erythrocytes measured colourimetrically. The amount of lysis was correlated to the level of complement activation. Platelet activation and adhesion were assessed by the expression of CD62P on the surface of platelets by flow cytometry using anti-CD62P antibodies and SEM analysis of surface adhered platelets, respectively. Hemolysis in the presence of D-PHI particle suspension was also measured.

Results

TEG studies demonstrate a statistically significant decrease in coagulation time (CT) for D-PHI in whole blood compared to the control sample; however, no significant change in CT is seen between D-PHI and control in PPP (Fig. 1). The CT for D-PHI is significantly longer for APTT (intrinsic pathway) and significantly shorter for PT (extrinsic pathway) when compared to control (Fig. 2). There is no significant change in hemolysis, or activation of complement or platelets compared to buffer control (Fig. 3).

Discussion and Conclusion

The results suggest increased potential of thrombus generation in whole blood exposed to D-PHI when compared to whole blood under normal conditions; however, there is little change in thrombus formation for PPP exposed to D-PHI. Furthermore, the intrinsic pathway of the coagulation cascade does not appear to initiate clotting in the presence of D-PHI. Since complement activation, platelet activation and adhesion, and hemolysis are also negligible, it is argued that the plasma proteins (which includes the coagulation cascade and complement activation proteins), platelets and red blood cells are not predominantly responsible for the decreased CT in whole blood upon exposure to D-PHI. This suggests that white blood cells (WBCs) may be playing a significant role in the CT changes observed with whole blood and D-PHI. However, hemolysis and platelet activation can change under flow. Therefore, future studies will further investigate platelet and RBCs response under flow, as well as WBC activation.


Figure 1: Clotting time of whole blood and platelet poor plasma (PPP) when exposed to D-PHI measured with TEG


Figure 2: Clotting time of PPP exposed to D-PHI and specific solutions to initiate (A) the intrinsic pathway of the coagulation cascade and (B) the extrinsic pathway of the coagulation cascade


Figure 3: (A) Complement activation and (B) CD62 platelet activation of D-PHI particle suspension compared to HEPES (negative) and positive controls

Acknowledgements

I would like to acknowledge Interface Biologics, Inc. and the following individuals: Kyle Battiston and Meilin Yang for PU synthesis; Jian Wang and Kai Yu for SEM help; and Benjamin Lai for blood compatibility methodology. Thank you to the following funding agencies: NSERC Grant %23SYN 430828; Queen Elizabeth II, NSERC CGSM, and NCPRM scholarships.

References

1. Xue, L.; Greisler, H. P. Biomaterials in the development and future of vascular grafts, Journal of vascular surgery. 2003, 37, 472-480. 2. Sharifpoor, S.; Labow, R. S.; Santerre, J. P. Synthesis and Characterization of Degradable Polar Hydrophobic Ionic Polyurethane Scaffolds for Vascular Tissue Engineering Applications, Biomacromolecules. 2009, 10, 2729-2739. 3. Battiston, K. G.; Labow, R. S.; Santerre, J. P. Protein binding mediation of biomaterial-dependent monocyte activation on a degradable polar hydrophobic ionic polyurethane. Biomaterials. 2012, 33, 8316-8328.

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