Improving bacterial cellulose for blood vessel replacement: Functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide

Abstract

Chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to improve the adhesion of human microvascular endothelial cells (HMEC) to bacterial cellulose (BC). The effect of these proteins on the HMEC–BC interaction was studied. The results obtained demonstrated that recombinant proteins containing adhesion sequences were able to significantly increase the attachment of HMEC to BC surfaces, especially the RGD sequence. The images obtained by scanning electron microscopy showed that the cells on the RGD-treated BC present a more elongated morphology 48 h after cell seeding. The results also showed that RGD decreased the in-growth of HMEC cells through the BC and stimulated the early formation of cord-like structures by these endothelial cells. Thus, the use of recombinant proteins containing a CBM domain, with high affinity and specificity for cellulose surfaces allows control of the interaction of this material with cells. CBM may be combined with virtually any biologically active protein for the modification of cellulose-based materials, for in vitro or in vivo applications.

Introduction

Cardiovascular disease is the leading cause of mortality in Western countries. Surgical bypass with autologous grafts remains the most used treatment, saphenous veins and mammary arteries being preferably used. However, many patients do not have suitable vessels, due to pre-existing vascular disease, amputation or previous harvest for prior vascular procedures. Moreover, a second surgical procedure is needed to obtain the vessel [1], [2]. For the reconstruction of arteries of large caliber currently available synthetic grafts (e.g. Dacron, ePTFE and polyurethane) offer a reasonable solution and proven clinical efficacy. However, for small sized (<6 mm) grafts these materials generally give poor performance, due to anastomotic intimal hyperplasia and surface thrombogenicity [3], [4], [5]. This scenario prompts the search for new materials suitable for the effective replacement of small blood vessels.

Bacterial cellulose (BC) produced by Acetobacter spp. is a biomaterial that has gained interest in the field of tissue engineering due to its unique properties. BC has been studied by several research groups as a scaffold for cartilage [6], [7], [8], wound dressing [9], [10], dental implants [11], [12], [13], [14], [15], [16], [17], nerve regeneration [18], [19] and vascular grafts [18], [20], [21], [22], [23]. The in vivo biocompatibility of BC was also evaluated in a study conducted by Helenius and colleagues [24].

Many strategies have been pursued to improve the compatibility and effectiveness of vascular grafts, through the production of unreactive surfaces, the surface modification of existing synthetic grafts (e.g. modifying surface properties and the incorporation of biologically active substances) and coating with autologous cells [4]. Seeding the graft surface with endothelial cells [25] is a promising approach; this mimicks the native vessel, thereby decreasing thrombosis. However, the high loss of endothelial cells on the restoration of blood flow after implantation presents a major challenge [4], [26], [27]. The rate and quality of endothelialization of a synthetic vascular graft depends on the interaction of endothelial cells with these cardiovascular materials. Several approaches have been attempted to increase endothelial cell adhesion to typically non-adhesive polymeric biomaterials used for synthetic vascular grafts [28]. One such approach involves pre-coating with endothelial cell-specific adhesives. The tripeptide Arg–Gly–Asp (RGD), an amino acid sequence found in many adhesive plasma and extracellular matrix proteins, has been used to enhance cell adherence. Binding of cells to the RGD sequence occurs via integrin receptors on the cell membrane. An improvement in the biocompatibility and performance of BC – envisaging its use as small diameter vascular grafts – by enhancing adhesion to human microvascular endothelial cells (HMEC-1) was attempted in this work by coating BC with adhesion peptides.

Many strategies have been developed to modify the materials used as synthetic grafts (e.g. Dacron, ePTFE and polyurethane). The adsorption of active substances like heparin, RGD, albumin–heparin conjugates, dipyridamole have little or no effect, due to the coatings being washed away [27]. In a previous work we described the production of recombinant proteins containing adhesion sequences fused to a CBM (cellulose-binding module) [29]. For artificial grafts based on cellulose the use of a CBM (exhibiting high affinity and specificity for cellulose surfaces) that can be combined with virtually any biologically active protein is an important strategy to avoid loss of the biological agents coating the graft.