RGD Immobilization on Carboxyl Surface-functionalized Electrospun Poly--caprolactone Scaffolds Promotes Endothelialization and Anti-thrombotic Activity in a Perfusion Bioreactor
RGD Immobilization on Carboxyl Surface-functionalized Electrospun Poly--caprolactone Scaffolds Promotes Endothelialization and Anti-thrombotic Activity in a Perfusion Bioreactor
Sonia Abbasi Ravasjani,1Hadi Seddiqi,2Ghassem Amoabediny,3Jianfeng Jin,4Behrouz Zandieh Doulabi,5Jenneke Klein Nulend,6,*
1. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA)-University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands 2. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA)-University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands 3. School of Chemical Engineering, College of Engineering, University of Tehran, Tehran, Iran 4. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA)-University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands 5. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA)-University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands 6. Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA)-University of Amsterdam and Vrije Universiteit Amsterdam, Amsterdam Movement Sciences, Amsterdam, The Netherlands
Introduction: Rapid endothelialization, endothelial cell stability, and prevention of thrombus formation by nitric oxide (NO) production, at the lumen of vascular electrospun poly--caprolactone (PCL) scaffolds under blood flow is still a challenge. Surface-functionalization of PCL scaffolds with negatively-charged carboxyl (COOH) or positively-charged amine (NH2) groups, followed by immobilization of arginine-glycine-aspartate (RGD) onto the scaffold surface affects endothelialization and cell stability. However, whether RGD immobilization on COOH surface-functionalized electrospun PCL scaffolds is more effective than on NH2 surface-functionalized scaffolds under blood flow is unknown. In this study we aimed to test whether RGD immobilization on COOH or NH2 surface-functionalized electrospun PCL scaffolds affects endothelialization, endothelial cell stability, and NO production in a perfusion bioreactor mimicking blood flow.
Methods: Electrospinning: Hollow tubular PCL scaffolds (ø: 4 mm, length: 30 mm, thickness: 0.26 mm) were electrospun (30 kV; feed rate: 1.5 ml/h; distance: 15 cm) using 13% (wt/wt) PCL in ¼ (v/v) acetic acid/formic acid.
Surface functionalization and RGD immobilization: PCL scaffolds were surface-functionalized by carboxyl (PCL/COOH) or amine (PCL-NH2) functional groups using wet-chemical treatment with 3 M NaOH or 10% (wt/v) 1,6-hexamethydiamine (HMDA). RGD was immobilized on carboxyl (PCL-COOH/RGD) or amine (PCL/NH2/RGD)-functionalized PCL scaffolds.
Scaffold characterization: The physicochemical properties of electrospun scaffolds, i.e. fiber diameter and distribution as well as pore size and distribution (ImageJ software), porosity (liquid-displacement assay), topography (scanning electron microscopy (SEM)), and hydrophilicity (water contact-angle) were determined.
Cell culture and scaffold bioactivity: Human umbilical vein endothelial cells (HUVECs) were seeded at 105 cells/cm2 at the lumen of the scaffolds, and cultured in a static or perfusion bioreactor for 8 days. HUVECs proliferation (AlamarBlue® assay), collagen production (picrosirius red staining), NO production (Griess assay), and stability (AlamarBlue® fluorescent-assay) were assessed.
Results: COOH and NH2 surface-functionalization followed by RGD immobilization (PCL-COOH/RGD and PCL-NH2/RGD) decreased fiber diameter (0.4-0.5-fold) and water contact angle (0.3-0.7-fold), but increased pore size (1.7-2.1-fold) and porosity (1.2-fold). PCL-COOH/RGD and PCL-NH2/RGD increased cell proliferation (2.2-5.6-fold) and collagen deposition (1.4-1.7-fold) in a perfusion bioreactor after 8 days. PCL-COOH/RGD and PCL-NH2/RGD increased cellular NO production (1.2-4.2-fold) after 30 min in a perfusion bioreactor. PCL-COOH/RGD and PCL-NH2/RGD increased cell stability by decreasing cell detachment (PCL-COOH/RGD: 0.07-fold; PCL-NH2/RGD: 0.2-fold) after 1 h in a perfusion bioreactor. Cells were more stable on COOH surface-functionalized RGD immobilized (PCL-COOH/RGD) scaffolds compared to NH2 surface-functionalized RGD immobilized (PCL-NH2/RGD) scaffolds after 1 h in a perfusion bioreactor.
Conclusion: Maximum endothelialization, cell stability, and NO production were observed on carboxyl surface-functionalized electrospun PCL scaffolds followed by RGD immobilization, which might be promosing for long-term application of endothelial cells to prevent thrombus formation at the lumen of vascular scaffolds under blood flow.