International Journal of Biological Macromolecules
Fast stress relaxing gellan gum that enhances the microenvironment and secreting function of bone mesenchymal stem cells
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).
References (57)
- et al.
Stem cell secretome and its effect on cellular mechanisms relevant to wound healing
Mol. Ther.
(2018) - et al.
Mesenchymal stem cells: clinical applications and biological characterization
Int. J. Biochem. Cell Biol.
(2004) - et al.
Secretome released from hydrogel-embedded adipose mesenchymal stem cells protects against the Parkinson’s disease related toxin 6-hydroxydopamine
Eur. J. Pharm. Biopharm.
(2017) - et al.
Characterizing the impact of 2D and 3D culture conditions on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing in vitro and ex vivo
Acta Biomater.
(2019) - et al.
Gel-sol transition in gellan gum solutions. I. Rheological studies on the effects of salts
Top. Catal
(1994) - et al.
Recent trends on gellan gum blends with natural and synthetic polymers: a review
Int. J. Biol. Macromol.
(2018) - et al.
Micropatterned gellan gum-based hydrogels tailored with laminin-derived peptides for skeletal muscle tissue engineering
Biomaterials
(2021) - et al.
Dual functional modification of gellan gum hydrogel by introduction of methyl methacrylate and RGD contained polypeptide
Mater. Lett.
(2020) - et al.
Hydrogel substrate stress-relaxation regulates the spreading and proliferation of mouse myoblasts
Acta Biomater.
(2017) - et al.
Varying PEG density to control stress relaxation in alginate-PEG hydrogels for 3D cell culture studies
Biomaterials
(2019)
Tunable fast relaxation in imine-based nanofibrillar hydrogels stimulates cell response through TRPV4 activation
Biomacromolecules
An improved complex gel of modified gellan gum and carboxymethyl chitosan for chondrocytes encapsulation
Carbohydr. Polym.
Preparation and characterization of an injectable dexamethasone-cyclodextrin complexes-loaded gellan gum hydrogel for cartilage tissue engineering
J. Control. Release
Evaluation of double network hydrogel of poloxamer-heparin/gellan gum for bone marrow stem cells delivery carrier
Colloids Surf. B
Glutaraldehyde cross-linked chitosan microspheres for controlled release of centchroman
Carbohydr. Res.
Stress relaxing hyaluronic acid-collagen hydrogels promote cell spreading, fiber remodeling, and focal adhesion formation in 3D cell culture
Biomaterials
Influence of polymer molecular weight on the in vitro cytotoxicity of poly (N-isopropylacrylamide)
Mater. Sci. Eng. C.
Biomaterials that promote cell-cell interactions enhance the paracrine function of MSCs
Biomaterials
Using exosomes, naturally-equipped nanocarriers, for drug delivery
J. Control. Release
Manufacturing exosomes: a promising therapeutic platform
Trends Mol. Med.
Stress-relaxing double-network hydrogel for chondrogenic differentiation of stem cells
Mater. Sci. Eng. C.
The mesenchymal stem cell secretome: a new paradigm towards cell-free therapeutic mode in regenerative medicine
Cytokine and Growth Factor Rev.
Mesenchymal stem cells: a new trend for cell therapy
Acta Pharmacol. Sin.
3D cell culture stimulates the secretion of in vivo like extracellular vesicles
Sci. Rep.
Biomaterials functionalized with MSC secreted extracellular vesicles and soluble factors for tissue regeneration
Adv. Funct. Mater.
Recent progress in gellan gum hydrogels provided by functionalization strategies
J. Mater. Chem. B
Peptide modification of purified gellan gum
J. Mater. Chem. B
Laminin-modified gellan gum hydrogels loaded with the nerve growth factor to enhance the proliferation and differentiation of neuronal stem cells
RSC Adv.
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