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

Abstract

Background Cardiovascular disease is the main cause of mortality in the USA and Western countries, leading to a demand for new materials that can be used as substitutes for blood vessels. Bacterial cellulose (BC) is a material with excellent biocompatibility and mechanical properties, thus holding great potential for arterial replacement. To improve the adhesion of human microvascular endothelial cells (HMECs) to BC, chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to cover the cellulose fibers. The effects of these proteins were studied. Materials & methods The attachment/viability and apoptosis were measured by the MTS and TUNEL methods, respectively. Confocal microscopy was used to analyze the morphology of microvascular cells grown on BC membrane. The visualization of cellulose fibers was also possible once the recombinant protein RGD-CBM was chemically linked to the fluorescein isothiocyanate. The formation of capillary-like structures by the endothelial cells was observed with optical microscopy. To evaluate the RGD effect on the migration of the cells through the BC, a migration chamber and an attractant were used to stimulate the cells to grow into the BC. Results The results of the MTS test demonstrated that the recombinant proteins containing adhesion sequences were able to increase significantly the attachment of HMEC to BC surfaces, and TUNEL assay showed that after 24 h of adhesion, independently of the treatment, cells remained viable on the BC pellicle, even when less cells adhered to cellulose treated with the CBM or buffer comparing to RGD. The images by confocal microscopy showed that the cells on the BC treated with the RGD presented a more elongated morphology and proliferated on the pellicle, forming a confluent cell layer on the membrane after 7 days. The results showed that the RGD decreased the ingrowth of the HMEC cells through the BC and also stimulated the early assembly of endothelial cells into capillary-like structures. Conclusions In this work, a new approach was successfully developed to functionalize the BC. The use of recombinant proteins containing a CBM domain, exhibiting high affinity and specificity for cellulose surfaces, allows the control on the interaction of this material with cells. The CBM may virtually be combined to any biologically active protein for the modification of cellulose-based materials, for in vitro or in vivo applications.