Fast stress relaxing gellan gum that enhances the microenvironment and secreting function of bone mesenchymal stem cells

https://doi.org/10.1016/j.ijbiomac.2022.10.011Get rights and content

Abstract

This study shows tunable stress relaxing gellan gum (GG) hydrogel for enhanced cell growth and regenerative medicine. The molecular weight and physical crosslinking density of GG were tuned and characterized with physicochemical analysis and mechanical tests. The result showed that a decrease in the molecular weight of the GG correlated with a decline in the mechanical properties but faster stress relaxing character. We also discovered that human-derived bone marrow stem cells (hBMSC) showed active viability, proliferation, and remodeling in the fast stress relaxing GG hydrogel. In particular, hBMSC showed an enhanced release profile of growth factors and exosomes (Exo) in the fast stress relaxing GG hydrogel. The secretome obtained from hBMSC embedded in hydrogel exhibited similar cytotoxicity and wound healing properties to that of secretome extracted from hBMSC cultured in a tissue culture plate (TCP) a standard culture condition. Thus, this work demonstrates the potential of fast stress relaxing GG hydrogels for medical application.

Introduction

Recently, mesenchymal stem cells (MSC) are suggested as promising candidates for regenerative medicine. Significant data and many clinical cases showed that besides the multipotency and self-renewal ability of MSC, the therapeutic effect of MSC derives from the paracrine property [1], [2]. The factors obtained from the paracrine mechanisms contain growth factors, cytokines, chemokines, and extracellular vesicles (EVs), collectively named secretomes. These secretomes contribute to cell migration, proliferation, anti-inflammatory, angiogenesis, and anti-apoptotic [1], [3], [4]. The efficacy of MSC-derived secretome may differ according to the culture conditions. Culture conditions can be modified by altering the genetic or protein expression, providing hypoxia/ischemia preconditioning or mechanical stimuli. Also, culturing cells in spheroids form or in a 3D microenvironment may improve therapeutic effects or increase the release of the secretome [5], [6], [7], [8].

Gellan gum (GG) hydrogel is widely applied for the 3-dimensional (3D) culture of MSC due to similar microenvironment and mechanical properties to nature-derived extracellular matrix (ECM). GG is a thermo-reversible polymer that exists as a coiled structure at a high temperature (~90 °C) and forms a double-helical structure when the temperature falls below a certain point [9], [10]. In addition, the gelation process is isotropic, meaning the presence of cations allows for the formation of a more stable hydrogel. Monovalent cations (e.g. Na+, K+) enhance the mechanical properties of the GG hydrogel by screening the electrostatic repulsion of the anionic GG backbone. Divalent cations (e.g. Ca2+, Mg2+) form more stable viscoelastic GG hydrogels by forming bonds between two carboxylate groups and divalent cations along with an above screening effect [11]. Nevertheless, it is difficult for cell growth to occur actively within pristine GG hydrogel. This is because, GG lacks cell adhesive sites for active cell-cell and cell-biomaterial interaction for cell migration, proliferation, and differentiation. Therefore, approaches to chemically modifying the GG backbone using adhesive ligands have been actively conducted [12], [13], [14], [15]. However, evidence from recent studies suggests that the time-dependent mechanical property of viscoelastic hydrogels also plays an important role in regulating the migration, proliferation, differentiation, and remodeling of the cellular matrix [16], [17], [18], [19], [20]. It was shown that the fast stress relaxing hydrogel induced more effective cell growth. Studies on cell growth and disease according to the stress relaxation property of hydrogels are actively underway using various biomaterials such as hyaluronic acid (HA), collagen, alginate (ALG), silk, etc. [20], [21], [22], [23], [24], [25], [26], [27]. Nonetheless, ECM/protein-derived biomaterials have poor physical properties and require chemical crosslinking to form a scaffold. ALG is widely used as it can be easily crosslinked with cations. However, a Luer lock syringe or two glass plates are needed to induce gelation. On the other hand, GG hydrogel can easily form a hydrogel by lowering the temperature or exposing it to cations. Also, GG hydrogel can form various shapes by injecting the GG solution into the desired shape of the mold.

In this paper, the fast stress relaxing GG hydrogel was designed by controlling molecular weight and physical crosslinking density by ionic crosslinking and guest-host interaction. The physical properties of the fabricated hydrogels were evaluated with physicochemical characterization and mechanical tests. Next, we examined the viability, proliferation, and matrix remodeling of the human-derived bone mesenchymal stem cells (hBMSC) that are embedded in the manufactured hydrogels to observe the applicability of the novel GG-based hydrogel in tissue engineering. The secretion profile of anti-apoptosis, pro-angiogenic, and anti-inflammatory growth factors and exosomes (Exo) of the encapsulated hBMSC were analyzed to characterize potential in regenerative medicine. In addition, the extracted secretome's cytotoxicity and wound healing effect on the fibroblasts were characterized.

To the best of our knowledge, this is the first study to design the GG hydrogel considering stress relaxation properties and analyzing the MSC cell growth and secretion property according to the stress relaxing property of GG-based hydrogel.

Section snippets

Preparation and chemical characterization of oxidized gellan gum (OGG) and reduced gellan gum (RGG)

Low-acyl gellan gum (GG, Gelzan™, Sigma-Aldrich, MO, USA) was dissolved in 90 °C distilled water (DW, 5 g/500 mL) until a homogeneous aqueous solution was obtained. The transparent GG solution was cooled to 40 °C and different dosages of 0.25 M sodium periodate (NaIO4, Sigma-Aldrich, USA) were added using a drop-wise method. The mixtures were gently stirred and reacted for 90 min to achieve various oxidization degrees. The oxidation reactions were quenched by adding an equimolar amount of

Preparation of fast stress relaxing hydrogels

Herein, the stress relaxation property of the GG was modulated through structural modification and physical crosslinking (Scheme 1, Fig. S2(A)). NaIO4 hydrolyzes the GG backbone's glycosidic linkages by cleaving the adjacent dihydroxyl groups [28]. The oxidation process was performed using three different amounts of 0.25 M NaIO4 (Table S1), while treatment time and temperature conditions were constant. The OGG showed oxidation degrees (%) of 5 % (HOGG), 15 % (MOGG), and 25 % (LOGG). A reduction

Conclusion

This work demonstrates an approach to the modulation of the stress relaxation properties of GG hydrogels and evaluates the effect of substrate stress relaxation on hBMSC biological activity (Scheme 2). A significant body of work has demonstrated the importance of the time-dependent mechanics of hydrogels to cell growth. Many research groups have developed viscoelastic hydrogels using various biomaterials. However, there remains a demand for biomaterials that are convenient for manufacturing.

The

Abbreviations

    ALG

    Alginate

    βCD

    Beta-cyclodextrin

    CaCl2

    Calcium chloride

    cPBS

    Calcium-containing phosphate buffered saline

    COL IV

    Collagen type (IV)

    CM

    Conditioned media

    D2O

    Deuterium oxide

    DMSO

    Dimethyl sulfoxide

    DW

    Distilled water

    DLS

    Dynamic light scattering

    Exo

    Exosomes

    ECM

    Extracellular matrix

    EVs

    Extracellular vesicles

    FBS

    Fetal bovine serum

    FN

    Fibronectin

    GG

    Gellan gum, Low-acyl gellan gum

    H&E

    Hematoxylin & Eosin

    HGF

    Hepatocyte growth factor

    hBMSC

    Human-derived bone marrow stem cells, StemPro® BM Mesenchymal Stem Cells

    HA

    Hyaluronic acid

CRediT authorship contribution statement

JH Choi and G Khang designed and conceived the study. JH Choi, SI Kim, JS Seo, and N Tmursukh synthesized the materials. JH Choi and SI Kim characterized the materials. JH Choi, SI Kim, SE Kim, SH Choe, and SJ Kim performed in vitro study and characterization. S Park supported in drawing of a scheme. JH Choi wrote the manuscript. JE Song and G Khang contributed reagents and materials for this study. G Khang supervised the overall research and manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was supported by a grant from the Basic Science Research Program administered through the National Research Foundation of Korea (NRF) and funded by the Ministry of Science, ICT & Future Planning (Grant No. NRF-2020R1A2C2103089).

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