Cellularised collagen-based scaffolds for engineering the vascular wall: a tri-culture study

Abstract

Event Abstract Back to Event Cellularised collagen-based scaffolds for engineering the vascular wall: a tri-culture study Caroline Loy1, Daniele Pezzoli1, Jayachandran N. Kizhakkedathu2 and Diego Mantovani1 1 CHU de Quebec, Research Center, Laval University, Lab. for Biomaterials and Bioengineering, CRC-1, Dept. Min-Met-Materials Eng, Canada 2 The University of British Columbia, Centre for Blood Research, Dept of Pathology and Lab Med, Dept of Chemistry, Canada Introduction: Engineered vessel walls have the potential to be used instantly as an in vitro models of vascular tissue for the investigation of patho/physiological processes and as preclinical tests for drugs and devices[1]. Herein, we describe the development of a collagen-gel based tri-culture model closely mimicking the tri-layered cellular organization of native arteries. Materials and Methods: Cellularized collagen gels were prepared as reported elsewhere[2]-[4]. A tri-step process was developed to produce the tri-layered construct. The external (adventitia) layer was composed of fibroblasts (FBs) and smooth muscle cells (SMCs) at a density of 106 cells/ml within collagen; the middle (media) layer of only SMCs (106 cells/ml); the innermost (intima) layer was obtained by seeding a monolayer of endothelial cells (ECs, 80 000 cells/cm2) over the media layer. A rotating bioreactor was used to seed ECs in the lumen of tubular systems. The degree of endothelialisation of the construct was investigated by fluorescence microscopy, phenotypic protein expression of calponin and alpha-actin by SMCs by western blot, and the blood compatibility of constructs by clotting time assay. Also, histological staining of transversal sections was performed to observe the organization of the construct and of the produced extra-cellular matrix. Results and Discussion: A tri-culture model of vascular wall with an organisation in multi-layer and a complete monolayer of ECs within 24 h for the flat model and 48 h for the tubular model was developed. Figure 1 clearly shows the organisation in two cellularized collagen gel layers and the presence of a monolayer of ECs. After 7 days, the compaction of the collagen matrix by vascular cells was achieved, and the deposition of ECM components such as fibronectin and fibrillin-1 was observed. Figure 2 confirmed that the ECs formed a continuous monolayer over the construct and did not migrate inside the collagen. The blood compatible functionality of the EC monolayer was demonstrated by the clotting time assay (Fig 3) showing that after 7 days of maturation clotting was prevented, with a concentration of free hemoglobin higher than 80% after 60 min of incubation (p > 0.05 between the endothelialized constructs and the non-endothelialized constructs). Also western blot results suggest that SMCs lose their contractile phenotype in the mono-culture while the contractile phenotype was not significantly affected in the tri- and bi- culture with a heterogeneous population of synthetic and contractile SMCs after 7 days of culture. Conclusion: The design of an in vitro vascular wall model in disk and tubular shape using collagen as scaffold was reported. This tri-culture engineered tissue promotes cell-matrix remodeling and improves the anticoagulant surface properties. This model is expected to allow the investigation the mutual intimate relationships existing among vascular cells thus representing a step forward into the engineering of in vitro vascular tissue models. This work was partially funded by NSERC-Canada, CIHR-Canada, FRQ-NT-Quebec, CFI-Canada. CL was awarded of a doctoral scholarship from NSERC Create Program in Regenerative Medicine (www.ncprm.ulaval.ca)