Carboxymethyl kappa carrageenan-modified decellularized small-diameter vascular grafts improving thromboresistance properties
Authors/Creators
Description
Highlights of the Study
This study developed a thromboresistant small-diameter vascular graft by combining perfusion-based decellularization of sheep carotid arteries with luminal surface modification using carboxymethyl kappa carrageenan (CKC). The approach successfully preserved the extracellular matrix architecture while reducing platelet adhesion and maintaining endothelial cell viability, proliferation, and functionality. The findings introduce CKC as a promising anticoagulant surface modifier for improving the hemocompatibility of decellularized vascular grafts.
Key findings:
🟢 Perfusion-based decellularization: A custom-designed perfusion system successfully removed cellular and nuclear components from sheep carotid arteries while maintaining the original vessel dimensions and three-dimensional extracellular matrix architecture.
🟢 ECM preservation: Histological evaluation confirmed preservation of important extracellular matrix components following decellularization, including collagen and elastin, supporting the structural integrity of the resulting vascular scaffold.
🟢 CKC surface functionalization: Carboxymethyl kappa carrageenan was successfully immobilized onto the luminal surface of decellularized arteries through covalent interactions between amine groups of the vascular ECM and carboxyl groups of CKC.
🟢 Chemical confirmation: FTIR analysis demonstrated successful carboxymethylation of κ-carrageenan and subsequent CKC immobilization on the vascular scaffold, including characteristic changes associated with sulfate groups and amide bond formation.
🟢 Uniform CKC immobilization: Toluidine blue staining confirmed the presence of a continuous CKC-containing layer on the luminal surface, demonstrating successful surface functionalization of the decellularized graft.
🟢 Crosslinking optimization: The E3C1 formulation containing 8 mg EDC, 16 mg NHS, and 0.01 g CKC produced the highest degree of crosslinking among the investigated surface-modification conditions.
🟢 Preserved surface wettability: CKC modification did not significantly alter graft wettability, with contact angles of approximately 56.53° for unmodified grafts and 55.31° for the optimized E3C1 formulation.
🟢 Mechanical properties: Decellularization reduced Young’s modulus from 15.53 ± 0.73 MPa in native tissue to 7.82 ± 0.93 MPa, whereas CKC modification increased it to 9.85 ± 0.41 MPa, partially compensating for the mechanical changes caused by decellularization.
🟢 Reduced platelet adhesion: CKC modification produced a substantial improvement in thromboresistance, decreasing platelet adhesion from 44.53 ± 2.05% in unmodified decellularized grafts to 19.57 ± 1.37% in the optimized E3C1 group.
🟢 Antithrombotic surface behavior: SEM observations confirmed markedly fewer adhered platelets on CKC-modified grafts than on unmodified decellularized vessels, supporting the anticoagulant activity of the CKC-functionalized luminal surface.
🟢 Endothelial cell compatibility: CKC-modified grafts supported HUVEC viability and proliferation, with no significant adverse effect on endothelial cell numbers compared with unmodified scaffolds under static culture conditions.
🟢 Dynamic endothelial response: Under dynamic culture, an initial reduction in endothelial cell number occurred because of flow-induced shear stress; however, cell numbers subsequently increased as the cells adapted to the dynamic environment.
🟢 Flow-induced morphology: HUVECs exhibited a relatively round morphology under static conditions but developed a more spindle-shaped morphology under dynamic flow, demonstrating cellular adaptation to shear stress.
🟢 Endothelial phenotype: CD31 immunostaining demonstrated cell–cell contacts on the luminal surfaces of both unmodified and CKC-modified vascular grafts, supporting successful endothelialization.
🟢 Nitric oxide production: HUVECs cultured under flow shear stress produced detectable nitric oxide, whereas NO release under static conditions was substantially lower, demonstrating the importance of physiological mechanical stimulation for endothelial functionality.
🟢 Perfusion bioreactor: The custom-designed perfusion bioreactor enabled dynamic culture of recellularized vascular grafts and provided physiologically relevant flow conditions for evaluating endothelial cell behavior.
🟢 Heparin-alternative potential: CKC combines structural similarity to heparin with anticoagulant activity, technical simplicity, relatively low-cost preparation, and additional biological properties, supporting its potential as an alternative surface modifier for vascular grafts.
🟢 Small-diameter graft potential: The combination of preserved ECM architecture, markedly reduced platelet adhesion, suitable mechanical behavior, and maintenance of endothelial cell functionality supports CKC-modified decellularized vessels as promising small-diameter vascular graft candidates.
🟢 Future translation: Further in vivo studies are required to determine the long-term stability, hemocompatibility, patency, and remodeling behavior of CKC-modified vascular grafts before clinical translation.
Overall, this study demonstrates that covalent immobilization of carboxymethyl kappa carrageenan on perfusion-decellularized sheep carotid arteries can substantially improve graft thromboresistance while preserving a biologically supportive environment for endothelial cells. The marked reduction in platelet adhesion from 44.53% to 19.57%, together with maintained endothelial viability, proliferation, and nitric oxide production, identifies CKC surface functionalization as a promising strategy for the development of small-diameter vascular grafts and future vascular bypass applications.
ARTICLE LINK
https://doi.org/10.1002/jbm.a.36684
DOI
10.1002/jbm.a.36684
HOW TO CITE
Ilanlou, S.; Khakbiz, M.; Amoabediny, G.; Mohammadi, J.; Rabbani, H. Carboxymethyl kappa carrageenan-modified decellularized small-diameter vascular grafts improving thromboresistance properties. Journal of Biomedical Materials Research Part A 2019, 107A, 1690–1701. DOI: 10.1002/jbm.a.36684.
KEYWORDS
Small-Diameter Vascular Grafts • Decellularization • Thrombogenicity • Carboxymethyl Kappa Carrageenan • Sheep Carotid Arteries
Abstract
The study developed decellularized small-diameter vascular grafts from sheep carotid arteries using a custom-designed perfusion decellularization system. The process completely removed cell nuclei while preserving the three-dimensional structure and mechanical properties of the native tissue. To reduce thrombogenicity caused by the exposure of extracellular matrix components to blood, carboxymethyl kappa carrageenan (CKC) was introduced as an anticoagulant surface modifier and uniformly immobilized onto the decellularized arteries through interactions between the amine groups of the vascular matrix and the carboxyl groups of CKC.
CKC modification significantly reduced platelet adhesion from 44.53 ± 2.05% in the control grafts to 19.57 ± 1.37% in the modified grafts. In addition, the modified scaffolds supported endothelial cell viability and proliferation as well as nitric oxide production. These findings demonstrate that CKC modification can improve the thromboresistance of decellularized small-diameter vascular grafts while maintaining endothelial cell compatibility and function.
Overall, the CKC-modified scaffold represents a promising strategy for reducing thrombosis in small-diameter vascular grafts and has potential for future in vivo evaluation and vascular bypass applications.