Remote triggering of TGF-β/Smad2/3 signaling in human adipose stem cells laden on magnetic scaffolds synergistically promotes tenogenic commitment

Abstract

Injuries affecting load bearing tendon tissues are a significant clinical burden and efficient treatments are still unmet. Tackling tendon regeneration, tissue engineering strategies aim to develop functional substitutes that recreate native tendon milieu. Tendon mimetic scaffolds capable of remote magnetic responsiveness and functionalized magnetic nanoparticles (MNPs) targeting cellular mechanosensitive receptors are potential instructive tools to mediate mechanotransduction in guiding tenogenic responses. In this work, we combine magnetically responsive scaffolds and targeted Activin A type II receptor in human adipose stem cells (hASCs), under alternating magnetic field (AMF), to synergistically facilitate external control over signal transduction. The combination of remote triggering TGF-β/Smad2/3 using MNPs tagged hASCs, through magnetically actuated scaffolds, stimulates overall expression of tendon related genes and the deposition of tendon related proteins, in comparison to non-stimulated conditions. Moreover, the phosphorylation of Smad2/3 proteins and their nuclear co-localization was also more evident. Overall, biophysical stimuli resulting from magnetic scaffolds and magnetically triggered cells under AMF stimulation modulate the mechanosensing response of hASCs towards tenogenesis, holding therapeutic promise.

Statement of Significance

The concept of magnetically-assisted tissue engineering may assist the development of innovative solutions to treat tendon disorders upon remote control of biological processes as cell migration or differentiation. Herein, we originally combine a fibrous aligned superparamagnetic scaffold, based on a biodegradable polymeric blend of starch and poly-ɛ-caprolactone incorporating magnetic nanoparticles (MNPs), and human adipose stem cells (hASCs) labelled with MNPs functionalized with anti-activin receptor type IIA (ActRIIA). Constructs were stimulated using alternating magnetic field (AMF), to activate the ActRIIA and subsequent induction of TGF-β signaling, through Smad2/3 phosphorylation cascade, enhancing the expression of tendon-related markers. Altogether, these findings contribute with powerful bio-magnetic approaches to activate key tenogenic pathways, envisioning future translation of magnetic biomaterials into regenerative platforms for tendon repair.

Introduction

Tendons are connective tissues which main function is the transmission of forces between muscles and bones, enabling body motion [1]. Many factors are likely to be involved in the onset of tendon injuries. Intrinsic factors include age, gender, anatomical variants, body weight, systemic disease, and genetic predisposition. Extrinsic factors include sporting activities, physical loading, occupation, and environmental conditions [2]. As a consequence of injury, tendon undergoes a repair process instead of complete regeneration which leads to the formation of a scar tissue limiting tendon gliding and motion amplitude, with a high risk of re-injury and/or associated pain [3]. Currently, classical treatments including oral administration of anti-inflammatory drugs and/or physical rehabilitation and surgical interventions fail in inducing a regenerative process and restoring the original functionality of the tissue [4]. In this context, tissue engineering (TE) emerges as a promising superior option for the development of competent bioartificial substitutes encouraging the regeneration of the damaged tissue. Moreover, magnetically-assisted strategies to remotely deliver stimuli directly to cells is a promising approach in tissue engineering. Magnetic actuation concomitantly combined with magnetic responsive materials represent tools with bioinstructive action in vitro but also upon implantation for remote cell mechanotransduction stimulation.

Previous studies suggest that the actuation of magnetic nanoparticles (MNPs) in response to an external magnetic force within a biomaterial substrate, causes its local deformation [5], which is able to activate/promote cells mechanotransduction mechanisms that will ultimately drive cellular responses [6,7]. Strategies applying magnetic stimuli combined with magnetic responsive scaffolds have shown positive outcomes in bone [8], cardiac [9], and vascular [10] TE. Our group has recently reported on magnetic responsive fibrous scaffolds for tendon TE, suggesting activation of YAP/TAZ signaling in response to magnetic stimulus [11]. These strategies may have an important role in tenogenic differentiation of stem cells [11], [12], [13], [14] and/or in the modulation of the inflammatory response [15,16], envisioning improved repair outcomes. Signaling cascades act as transducers of mechanical forces into downstream mechanosensory molecules. MNPs targeting cell membrane receptors or ion channels, by magnetic mechano activation technology, have been explored as a powerful instructive tool to actuate signaling pathways in human mesenchymal stem cells for TE approaches [13,[17], [18], [19]. Transforming growth factor (TGF)-β signaling, transduced by Smad2/3 cascade, has been most associated to tendon formation, differentiation, and homeostasis [20,21], highlighting its interest as a potential molecular target for magnetic actuated strategies. On the other hand, activin receptor type IIA (ActRIIA) has been suggested to be involved in the regulatory pathway of the mechanosensitive gene Tenomodulin [13,[22], [23], [24], known as a tendon lineage marker. Upon activation of this receptor, an intracellular signaling through phosphorylation of Smad2/3 is initiated [25].

In a previous work of our group, we reported that targeted ActRIIA in human adipose stem cells (hASCs), using MNPs and magnetic stimulation, induced tenogenic transcriptional responses, through TGF-β/Smad2/3 signaling pathway [13]. Moreover, 3D-printed polymeric composites incorporating MNPs within its aligned fiber structure were designed by us showing improved biological performance in comparison with non-magnetic counterparts [12].

Herein we hypothesized that combining the two approaches may potentiate the tenogenic differentiation of hASCs through magnetically assisted tissue engineering tools with magnetic biomaterials serving as mediators of mechanotransduction. Specifically, we propose to assess the synergistic effect of hASCs labelled with anti-ActRIIA functionalized MNPs, seeded onto magnetic scaffolds, and remotely actuated by external magnetic field. The remote actuation of an alternating magnetic field (AMF) was provided by a custom-designed solenoid device, to stimulate cells laden on the magnetic scaffolds and assist the regulation of tendon related markers to drive tenogenesis (Fig. 1).