Surface modification of bioresorbable electrospun matrices with heparin and lysozyme for vascular tissue engineering

P.C. CARACCIOLO; I. RIAL-HERMIDA; F. MONTINI BALLARIN; G.A. ABRAHAM; A. CONCHEIRO; C. ALVAREZ-LORENZO

Santiago de Compostela

Otro; IX Internacional Forum on Advances in Pharmaceutical Technology (CISDEM); 2015

Universidad de Santiago de Compostela - CISDEM

The development of tissue-engineered vascular prostheses is still a challenge, and many approaches are currently being investigated. Poly(L-lactic acid) (PLLA), a FDA approved bioresorbable polyester presenting mechanical properties close to collagen, such as high elastic modulus, and good electro-spinnability, and a bioresorbable segmented polyurethane (SPU) elastomer mimicking elastin mechanical properties, such as tensile strength and compliance, were processed by multilayer electrospinning to obtain a novel bilayered vascular graft. Thematerials were processed in two different compositions: PLLA/SPU 50/50 blend (inner layer) and PLLA/SPU 90/10 blend (outer layer) [1], each of them mimicking the collagen-elastin ratio presented in the media and adventitia layers of natural arteries, respectively. One of the main drawbacks to the use of implantable devices and matrices for tissue engineering is the high risk of infection and thrombosis. The immobilization of polymers or biomolecules on surfaces can avoid these difficulties. Heparin is a glycosaminoglycan, which has been widely used in vascular therapy due to its high anticoagulant capacity, preventing thrombosis events on synthetic surfaces [2]. Moreover, lysozyme is an antibacterial enzyme capable of hydrolyze the peptidoglycan layer of Gram-positive bacteria cell walls [3]. In this work, electrospun matrices of PLLA/SPU 50/50 blend (as the inner layer of vascular grafts) were surface modified through the immobilization of heparin and lysozyme, as anticoagulant and antibacterial, respectively. The modification through ester and urethane functional groups was explored. In the ester route, an alkaline treatment was employed to increase the density of PLLA reactive carboxylic groups. Then, diaminoPEG (DAPEG) was employed as spacer to avoid a reduction in heparin activity due to limited mobility. In the urethane route, a NaClO treatment was employed to activate SPU urethane functional groups. Further modification with allyl glycidyl ether generated epoxy-capped oligomers. This step was followed by DAPEG modification. From then on, both routes followed with heparin modification. Moreover, a matrix prepared by each route was exposed to a lysozyme solution to form a heparin-lysozyme complex, due to lysozyme has a relatively low stability in its free state. MSC proliferation on modified scaffolds resulted in the order of the control, being higher for the lysozyme modified scaffolds. Cell proliferation reached its maximum at day 7. After this, the values decreased for all scaffolds. It was previously reported that a highproliferation inhibits further cell growth. Moreover, live-dead staining showed cells were able to growth onto the scaffolds, displaying better cytocompatibility the matrices obtained through urethane route. Lysozyme activity was evaluated through m. lysodeikticus suspensions. The immobilized lysozyme remained active for both modification routes, being more active the matrix obtained through urethane route. Thus, from the obtained results, it can be concluded that the urethane route seems to be the most promising for covalent modification of PLLA/SPU 50/50 blends.