Covalent linkage of heparin provides a stable anti‐coagulation surface of decellularized porcine arteries

Abstract

Establishing thrombosis‐resistant surface is crucial to develop tissue‐engineered small diameter vascular grafts for arterial reconstructive procedures. The objective of this study was to evaluate the stability and anti‐coagulation properties of heparin covalently linked to decellularized porcine carotid arteries. Cellular components of porcine carotid arteries were completely removed with chemical and physical means. Heparin was covalently linked to the decellularized vessels by a chemical reaction of the carboxyl end of amino acids with hydroxylamine sulphate salt and heparin‐EDC. Bound heparin contents were measured by quantitative colorimetric assay of toluidine blue staining. The average content of heparin in treated vessels was 35.6 ± 11.6 mg/cm2 tissue, which represented 6.21 ± 2.03 UPS heparin/cm2 tissue. The stability of heparin linkage was tested by incubating the heparin‐linked vessels either in PBS at 37°C or in 70% alcohol at room temperature up to 21 days, showing no significant reduction of heparin content. Anti‐coagulation property of bound heparin was determined with a clotting time assay using fresh dog blood. Standardized small pieces of non‐heparin‐bound vessels were clotted in fresh dog blood within 10 min., whereas all heparin‐bound vessels did not form clot during 1‐hr observation. In vivo platelet deposition of the vessel was determined with a baboon model of the femoral arteriovenous external shunt and 111Indium labelling of platelets. There were 1.38 ± 0.07 × 109 and 0.64 ± 0.11×109 baboon platelets deposited on the control and heparin‐linked vessels, respectively, at 60 min. These data demonstrate that covalent linkage of heparin provides an effective and stable anti‐coagulation surface of decellularized porcine carotid arteries. This study may suggest a new strategy to develop tissue‐engineered biological vascular grafts, which could be used for human coronary or low extremity artery bypasses.

Heparin nanomodification improves biocompatibility and biomechanical stability of decellularized vascular scaffolds

“…14,25 Briefly, the formaldehyde-fixed BJV scaffold samples modified or not modified by seven cycles heparin/DHI were embedded in paraffin and cut into slices. The histological sections were deparaffinized and hydrated routinely, then stained with 0.1% toluidine blue for 10 minutes.…”

“…Establishing a thromboresistant surface is crucial to prevent formation of blood clots in the initial phase of graft implantation. 14 Poor biomechanical stability can induce aneurysms. Formation of an aneurysm is another reason for transplant failure because of poor biomechanical stability of xenografts.…”

“…15 This approach can reduce thrombus formation without bleeding complications and also provide a substrate to bind growth factors, including basic fibroblast growth factor, epithelial growth factor, and vascular endothelial growth factor, which accelerate endothelialization and regeneration. 14,[16][17][18] Heparin can be linked to an extracellular matrix using a crosslinker such as glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or chitosan, but the amount of heparin linked is small, and long-term results are not ideal. 14,[19][20][21] Much effort has been devoted to improving the antithrombogenicity of xenogeneic skin grafts by surface modification using anticoagulant molecules.…”

“…14,[16][17][18] Heparin can be linked to an extracellular matrix using a crosslinker such as glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), or chitosan, but the amount of heparin linked is small, and long-term results are not ideal. 14,[19][20][21] Much effort has been devoted to improving the antithrombogenicity of xenogeneic skin grafts by surface modification using anticoagulant molecules. Dai et al reported that complex multilayers of iron polysaccharide, which are used to modify a nitinol surface, provide a stable thromboresistant surface with good biocompatibility.…”

Click-coated, heparinized, decellularized vascular grafts

“…Typically, heparin is linked to the extracellular matrix (ECM) using a “one-to-one” (one active group of ECM-to-one heparin chain) crosslinker such as glutaraldehyde [9] and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) [10–12]. As there are few surface accessible amino groups on the ECM, the “one-to-one” approach results in a low surface coverage of heparin.…”