Alginate-magnetic short nanofibers 3D composite hydrogel enhances the encapsulated human olfactory mucosa stem cells bioactivity for potential nerve regeneration application

Highlights

• Alginate-magnetic short nanofibers 3D composite hydrogels were prepared.

• Prepared hydrogel could enhance the neuronal differentiation of encapsulated OE-MSCs.

• Increasing sonication power/time results in shorter nanofibers with better uniformity.

• Prepared composite hydrogel exhibited more bioactivity comparing with alginate hydrogel.

• The increment of storage modulus was recorded in proportion to M.SNFs content.

Abstract

The design of 3D hydrogel constructs to elicit highly controlled cell response is a major field of interest in developing tissue engineering. The bioactivity of encapsulated cells inside pure alginate hydrogel is limited by its relatively inertness. Combining short nanofibers within a hydrogel serves as a promising method to develop a cell friendly environment mimicking the extracellular matrix. In this paper, we fabricated alginate hydrogels incorporating different magnetic short nanofibers (M.SNFs) content for olfactory ecto-mesenchymal stem cells (OE-MSCs) encapsulation. Wet-electrospun gelatin and superparamagnetic iron oxide nanoparticles (SPIONs) nanocomposite nanofibers were chopped using sonication under optimized conditions and subsequently embedded in alginate hydrogels. The storage modulus of hydrogel without M.SNFs as well as with 1 and 5 mg/mL of M.SNFs were in the range of nerve tissue. For cell encapsulation, OE-MSCs were used as a new hope for neuronal regeneration due to their neural crest origin. Resazurin analyses and LIVE/DEAD staining confirmed that the composite hydrogels containing M.SNFs can preserve the cell viability after 7 days. Moreover, the proliferation rate was enhanced in M.SNF/hydrogels compared to alginate hydrogel. The presence of SPIONs in the short nanofibers can accelerate neural-like differentiation of OE-MSCs rather than the sample without SPIONs.

Introduction

In the body tissues, the extracellular matrix (ECM) surrounds the cells and directs the growth, differentiation, and phenotype of the cell by providing structural, mechanical, and biological support [1]. In the face of irreversible tissue damage, a synthetic matrix can be utilized for regeneration. The success of the synthetic matrix depends largely on their similarity to the ECM [2,3]. Among the various types of artificial matrices, the hydrogel and fibrous structure have received tremendous attention due to their potential to mimic the ECM as well as their remarkable properties and structure [4,5]. In this regard, hydrogels have unique features such as similar mechanical strengths to soft tissues, providing three-dimensional (3D) network, high biocompatibility, and simple synthesis [6,7]. Alginate has been studied in past as a 3D hydrogel for encapsulating neural stem cells because of its compatibility with nervous system tissue [8,9]. Banerjee et al. [10] reported that the decrease in the alginate hydrogel modulus increases the proliferation rate of neural stem cells. Their results showed that the optimum concentration of alginate hydrogel is 0.25% which have comparable modulus to that of nerve tissues (~100 pa). However, the alginate hydrogel can limit the cell adhesion and cell spreading due to its relatively inertness. Therefore, the encapsulated cells inside alginate hydrogel become immobile and have rounded morphology [11]. Yet this issue can be overcome by chemical modification or combination of an alginate hydrogel with cell-adhesive materials, which can promote neural development [12].

Due to structural similarity to the native ECM, electrospun nanofibers were extensively explored as substrates to cultivate, proliferate, and differentiate cells in biomimetic environments [[13], [14], [15], [16], [17]]. It has been reported that neural differentiation of mesenchymal stem cells was accomplished with gelatin nanofibers in presence of retinoic acid and sonic hedgehog [18]. However, most electrospun nanofibrous structures do not allow cells to penetrate and migrate within the fibers because nanofibrous meshes have small pore sizes and compact structure and performed as a two-dimensional membrane. Consequently, the in vitro evaluation of nanofibrous meshes still does not mimic the in vivo environment properly and requires further development [11].

Since native ECM is a combination of fibrous proteins within gelatinous ground substance, the separate production of hydrogels and nanofibrous structure does not lead to proper biomimicry [19]. In this regard, the combination of hydrogel and fibers causes the formation of a 3D composite hydrogel with enhanced functional properties, which combine the advantages of both structures as well as overcome their limitations [5,11]. Hsieh et al. [20] demonstrated that composites of poly(caprolactone-co-D,L-lactide) fibers in the hyaluronan/methylcellulose hydrogel maintained cell viability and proliferation and enhanced neuronal differentiation than pure hydrogel. In another study, fibroblasts were cultivated in the gelatin/alginate hydrogel incorporating polycaprolactone (PCL) nanofibers. Their results indicated that composite hydrogel showed superior cell adhesion and viability compared to hydrogel without short nanofibers [21]. The use of short nanofibers in the hydrogel can facilitate dissolution and dispersion of the fibers in the hydrogel matrix as well as showed higher water wettability in comparison with using nanofiber mats in the hydrogel [5,21]. Beyond different reported techniques, such as chemical treatment, cryogenic milling and patterned UV-crosslinking, the electrospinning/ultrasonication is an effective and easy method to produce short nanofibers. Moreover, the sonication could preserve the fiber surface morphology as well as their properties [22,23].

The physical properties of an artificial matrix greatly affect cell behavior such as cell survival and differentiation and tissue integration [24]. In the presence of magnetic nanoparticles, mainly superparamagnetic iron oxide nanoparticle (SPION), nerve regeneration can be boosted. Kim et al. [25] evaluated the effects of iron oxide nanoparticles on the neurite outgrowth during the differentiation of PC12 cells. They have demonstrated that the iron oxide nanoparticles directly interacted with proteins which activated the signaling pathways of neuronal differentiation. In another study, the nasal olfactory mucosa cells, which were encapsulated within the magnetic fibrin hydrogel, exhibited a 3D growth pattern and early neural differentiation [26].

Stem cells can be encapsulated inside 3D composite hydrogels, thereby improving their biological efficacy [27]. Beyond the different types of stem cells, olfactory ecto-mesenchymal stem cells (OE-MSCs) that extracted from human olfactory mucosa are promising candidates for nerve repair due to their neural crest origin [28,29]. It was demonstrated that OE-MSCs have highly similar gene expression profiles with bone marrow stem cells, but with some unique characteristics like the extensive overexpression of neuronal gene markers [7].

To the best of our knowledge, investigation of a construct made of the composite hydrogel, SPION, and OE-MSC on the nerve regeneration has not been studied previously. The main objective of this work is to improve alginate hydrogel bioactivity while maintaining its mechanical properties; moreover, we intend to evaluate viability and neural-like differentiation of OE-MSCs, which were encapsulated inside prepared 3D magnetic composite hydrogels in comparison with alginate hydrogel. The magnetic short nanofibers were used to provide contact points and differentiation cues to human OE-MSCs encapsulated inside 3D environment of novel alginate composite hydrogel. The prepared magnetic hydrogel allowed cells to recognize the magnetic fibrous structure as well as a water-rich environment that simulate cells in the ECM. We measured the rheological properties of the composite hydrogels to compare with those of nerve tissue. Differences in cytotoxicity were also compared to determine the effects of magnetic short nanofiber (M.SNF) on cell viability. Finally, the gene expression profiles of cells were evaluated to provide evidence of the magnetic effect of the composite hydrogel on the neural-like differentiation of OE-MSCs.