The promotion of in vitro vessel-like organization of endothelial cells in magnetically responsive alginate scaffolds

Abstract

One of the major challenges in engineering thick, complex tissues such as cardiac muscle, is the need to pre-vascularize the engineered tissue in vitro to enable its efficient integration with host tissue upon implantation. Herein, we explored new magnetic alginate composite scaffolds to provide means of physical stimulation to cells. Magnetite-impregnated alginate scaffolds seeded with aortic endothelial cells stimulated during the first 7 days out of a total 14 day experimental course showed significantly elevated metabolic activity during the stimulation period. Expression of proliferating cell nuclear antigen (PCNA) indicated that magnetically stimulated cells had a lower proliferation index as compared to the non-stimulated cells. This suggests that the elevated metabolic activity could instead be related to cell migration and re-organization. Immunostaining and confocal microscopy analyses supported this observation showing that on day 14 in magnetically stimulated scaffolds without supplementation of any growth factors, cellular vessel-like (loop) structures, known as indicators of vasculogenesis and angiogenesis were formed as compared to cell sheets or aggregates observed in the non-stimulated (control) scaffolds. This work is the first step in our understanding of how to accurately control cellular organization to form tissue engineered constructs, which together with additional molecular signals could lead to a creation of an efficient pre-vascularized tissue construct with potential applicability for transplantation.

Introduction

Tissue engineering and regenerative medicine can offer solutions to a number of compelling clinical problems that have not been adequately addressed through the use of permanent replacement devices. While encouraging results have been achieved with thin-walled tissues (e.g., engineered urinary bladder [1]), tissue engineering of dense tissues such as the heart, that depend on vascular supply, remains a challenge. One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro is important for maintaining cell viability during tissue growth, inducing structural organization and promoting vascularization upon implantation [2].

It has already been shown that apart from molecular signals (e.g. growth factors), additional physical cues, such as electrical signaling, mechanical stimulation, and medium perfusion are required in tissue engineering [3], [4], [5], [6]. Bioreactors developed to apply mechanical forces via piston/compression systems, substrate bending, hydrodynamic compression and fluid shear (relevant for vasculature homeostasis) [7], [8], [9], [10], require direct contact with the cell culture and have limited degrees of freedom in which these forces can be applied. In addition, it would be desirable to be able to apply the same physical cues in vivo as in vitro.

Hydrogel nanocomposites impregnated with magnetic particles are attractive materials for achieving remote actuation with adjustable direction and possibility of in vivo applicability. The magnetic field can be coupled to the particle to actuate a process within a target cell regardless of whether there are intervening structures, such as tissue. Stress parameters can also be varied dynamically simply by varying the properties (e.g. strength, frequency) of the applied field. Polymer and hydrogel nanocomposites laden with magnetic particles have already been demonstrated as potential candidates for pulsatile drug delivery and soft actuator applications [11], [12], [13], [14], [15].

The feasibility of mechanical stimulation induced by magnetic forces has been shown by binding of magnetic particles to membranes of mesenchymal stem cells (MSCs) to stimulate calcium stores, change membrane potential, upregulate genes related to bone and cartilage formation and promote bone matrix mineralization in vitro [16], [17], [18], [19], [20], [21]. However, to the best of our knowledge this approach has not been fully realized in 3D cell cultures within polymeric scaffolds.

In the present paper, we explore whether impregnation of alginate scaffolds with magnetically responsive nanoparticles (MNP) and further exposure to an alternating magnetic field would exert a direct effect on endothelial cell activity. We hypothesized that exposure of a magnetic scaffold to an alternating magnetic field would enable the mechanical stimulation of endothelial cells and influence their organization into early capillary-like structures in vitro. This work is the first step forward in our understanding of how to accurately control cellular organization into tissue engineered constructs, which together with additional molecular signals could lead to a creation of an efficient pre-vascularized tissue construct ready for transplantation.