Fucoidan functionalization on poly(vinyl alcohol) hydrogels for improved endothelialization and hemocompatibility

Abstract

The performance of clinical synthetic small diameter vascular grafts remains disappointing due to the fast occlusion caused by thrombosis and intimal hyperplasia formation. Poly(vinyl alcohol) (PVA) hydrogels have tunable mechanical properties and a low thrombogenic surface, which suggests its potential value as a small diameter vascular graft material. However, PVA does not support cell adhesion and thus requires surface modification to encourage endothelialization. This study presents a modification of PVA with fucoidan. Fucoidan is a sulfated polysaccharide with anticoagulant and antithrombotic properties, which was shown to potentially increase endothelial cell adhesion and proliferation. By mixing fucoidan with PVA and co-crosslinked by sodium trimetaphosphate (STMP), the modification was achieved without sacrificing mechanical properties. Endothelial cell adhesion and monolayer function were significantly enhanced by the fucoidan modification. In vitro and ex-vivo studies showed low platelet adhesion and activation and decreased thrombin generation with fucoidan modified PVA. The modification proved to be compatible with gamma sterilization. In vivo evaluation of fucoidan modified PVA grafts in rabbits exhibited increased patency rate, endothelialization, and reduced intimal hyperplasia formation. The fucoidan modification presented here benefited the development of PVA vascular grafts and can be adapted to other blood contacting surfaces.

Keywords: End-to-side anastomosis; Endothelialization; Fucoidan; Hemocompatibility; Rabbit carotid artery; Small diameter vascular graft.

Figure 1.

(A) Schematic diagram of polyvinyl alcohol-fucoidan (PVA-F) preparation procedure. (B) Scanning electron microscopy (SEM) images showed the structures on PVA (left) and PVA-F (right) surfaces. (C) Static water contact angle of PVA and PVA-F surfaces. (n=6, p=0.0554). (D) Fucoidan density on PVA-F samples was determined by toluidine blue staining. (E) Stability of fucoidan modification was determined by toluidine blue staining at day 0, 15, 30 and 60. n=3, no significant decrease was observed.

Figure 2.

Mechanical properties of PVA and PVA-F samples. (A) Crosslinking density of PVA-F samples is significantly higher than PVA samples (n=5). (B) Water content of PVA and PVA-F hydrogels (n=3). (C) Compliance of PVA and PVA-F samples (n=3). (D) Burst pressure of PVA and PVA-F samples (n=5). (E) Longitudinal tensile Young’s modulus of PVA and PVA-F samples (n=3). (F) Circumferential tensile Young’s modulus of PVA and PVA-F samples (n=3). * denotes statistical significance using t-test with p < 0.05, n=3.

Figure 3.

Endothelial cell adhesion on PVA and PVA-F samples. (A) Representative fluorescence images of human umbilical vein endothelial cells (HUVECs) on PVA and PVA-F samples after 48 hr culture. Scale bar = 50 μm. (B) Average cell density on PVA and PVA-F samples. **** denotes statistical significance using one-way ANOVA with p < 0.0001, n=5. (C) CD31, VE-cadherin and eNOS expression of HUVEC monolayer on PVA-F samples (data normalized to glass). HUVECs on glass cover slip were included in analysis as a positive control. HUVECs on PVA samples were not taken into analysis. * denotes statistical significance using t-test with p < 0.05, n=5. (D) Representative fluorescence images of collagen type IV (COL IV) and laminin (LM) deposited by HUVECs on PVA and PVA-F samples after 24 hr culture samples (data normalized to glass). HUVECs on glass cover slip were included in analysis as a positive control. Scale bar = 50 μm. (E) Collagen type IV and laminin expression of HUVECs on PVA-F samples (data normalized to glass). HUVECs on PVA samples were not taken into analysis due to low cell number. No statistical significance was observed, n=3.

Figure 4.

In vitro hemocompatibility tests of PVA and PVA-F samples. (A) Platelet adhesion and morphology on collagen coated glass cover (positive control), ePTFE (clinical control, platelets are as indicated with arrows), PVA and PVA-F. (B) Quantification of platelet adhesion determined by lactate dehydrogenase (LDH) assay. (C) Real-time thrombin generation assay. Lag-time (D) represents the time taken to start forming thrombin. Peak (E) represents the maximum thrombin generation rate. (F) represents endogenous thrombin potential (ETP). n=5, *, **and *** indicate a significant difference using one-way ANOVA with p < 0.05, p < 0.01, and p < 0.001, respectively.

Figure 5.

Gamma sterilization effect on PVA-F samples. (A) Average cell density on PVA-F samples non-treated (PVA-F) and gamma sterilized (PVA-Fg). CD31 (B) and eNOS (C) expression of HUVECs on before and after gamma sterilized PVA-F sample (data normalized to glass). n=5, * and ** denotes statistical significance using one-way ANOVA with p < 0.05 and p < 0.01, respectively. Lagtime (D), Peak (E) and ETP (F) of real-time thrombin generation on non-treated and gamma sterilized samples (n=3).

Figure 6.

Ex-vivo shunt study. Platelet accumulation (A), fibrin accumulation (B) and average luminal patent area (C) data are displayed as mean ± SD for all samples. Collagen and ePTFE controls are shown for comparison but were not included in the statistical comparison due to low sample size. No significant differences were observed between the various PVA samples for the platelet (p=0.0523), fibrin (p=0.201 for the 1-way ANOVA), or patent area (p=0.109 for the 1-way ANOVA) data.

Figure 7.

Implantation of ePTFE, PVAg, and PVA-Fg grafts. (A) Dimensions of implanted grafts. (B) Patency rate of ePTFE (n=4), PVAg (N=5), and PVA-Fg (n=5). (C) Representative images of ePTFE graft, PVAg graft, and PVA-Fg graft after implantation. Scale bar=1cm. (D) Representative ultrasound images of ePTFE, PVAg, and PVA-Fg grafts and flow velocity (peak systolic velocity) inside the grafts after 1-month implantation. (N.D. not determined)

Figure 8.

In-vivo endothelialization of PVA-Fg grafts. (A) Hematoxylin and eosin (H&E) staining of middle section of explanted vascular grafts. PVA-Fg showed a layer of cells on the luminal surfaces. Scale bar = 50 μm. (B) Immunofluorescence staining of the middle section of explanted vascular grafts. Arrows indicate luminal surfaces. Scale bar = 100 μm. CD31 positive signals in PVA-Fg samples indicated the presence of endothelial cells. (C) Immunofluorescence staining of the middle section of explanted vascular grafts. Arrows indicate luminal surfaces. Scale bar = 100 μm. The eNOS positive signals indicated that the endothelial cells inside PVA-Fg samples expressed eNOS proteins.

Figure 9.

Proximal and distal anastomotic stenosis of ePTFE, PVAg, and PVA-Fg grafts. (A) Representative H&E staining images of patent ePTFE, occluded ePTFE, PVAg, and PVA-Fg grafts. IH indicates intimal hyperplasia. (B) Stenosis percentage of ePTFE, PVAg, and PVA-Fg grafts at proximal anastomoses. (C) Stenosis percentage of ePTFE, PVAg, and PVA-Fg grafts at distal anastomoses. (Graft sections with missing pieces were not taken into calculation.) PVA-Fg grafts showed lower percentage of stenosis at both proximal and distal anastomosis than unmodified PVAg grafts. (D) ⍺-SMA staining of patent ePTFE, occluded ePTFE, PVAg and PVA-Fg grafts confirmed the presence of smooth muscle cells in the intimal hyperplasia. Scale bar=200 μm. (E) Ki67 staining of patent ePTFE, occluded ePTFE, PVAg and PVA-Fg grafts showed the presence of proliferating cells, but in a low density. Scale bar=50 μm.