Research Article
Influence of viscosity on chondrogenic differentiation of mesenchymal stem cells during 3D culture in viscous gelatin solution-embedded hydrogels

https://doi.org/10.1016/j.jmst.2020.05.018Get rights and content

Abstract

Differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) is regulated by a variety of cues of their surrounding microenvironments. In particular, mechanical properties of cell culture matrices have been recently disclosed to play a pivotal role in stem cell differentiation. However, it remains elusive how viscosity affects the chondrogenic differentiation of hMSCs during three-dimensional (3D) culture. In this study, a 3D culture system that was established by embedding viscous gelatin solution in chemically cross-linked gelatin hydrogels was used for 3D culture of hMSCs in gelatin solutions with different viscosities. The influence of solution viscosity on chondrogenic differentiation of hMSCs was investigated. Viscous gelatin solutions promoted cell proliferation in the order of low, middle and high viscosity while elastic hydrogels restricted cell proliferation. High viscosity gelatin solution led to increased production of the cartilaginous matrix. Under the synergistic stimulation of chondrogenic induction factors, high viscosity was beneficial for the chondrogenic differentiation of hMSCs. The results suggested the role of viscosity should be considered as one of the dominant mechanical cues affecting stem cell differentiation.

Introduction

Stem cells are one of the dominant cell sources for tissue engineering and regenerative medicine. They have been widely used for repairing cartilage injuries and defects. Controlling the chondrogenic differentiation of stem cells is critical for cartilage tissue engineering. Mimicking the extracellular matrix (ECM) microenvironment surrounding cell in vivo is an attractive strategy to control stem cell functions [1]. In vivo ECM microenvironments dynamically change not only their composition but also their physical properties such as elasticity and topography during stem cell differentiation process and tissue development [[2], [3], [4]]. A variety of approaches such as immobilization of cell growth factors and cytokines, use of decellularized matrices and regulation of physicochemical properties have been proposed and established to create biomimetic ECM microenvironments for controlling stem cell functions and guiding functional tissue regeneration [5,6]. In particular, mechanical properties such as stiffness have been broadly investigated for their impact on stem cell differentiation and fate decision [[7], [8], [9], [10], [11], [12]]. Matrix stiffness has been reported to specify lineage of human mesenchymal stem cells from neurons to myoblasts and osteoblasts when the matrix stiffness changes from the brain-mimicking soft matrix to the muscle-mimicking stiffer matrix and collagenous bone-mimicking rigid matrix, respectively [8]. Since this report, many researches have reported the correlation of stiffness with cell functions such as cell adhesion, morphology, mobility, self-renewal, proliferation and differentiation [[9], [10], [11], [12]].

In addition to elastic property, ECM microenvironments have viscous property because of their viscoelastic characters. To elucidate the impact of viscous property on stem cell differentiation, the separate influence of viscosity and stiffness has been investigated by using viscoelastic hydrogels with dissipative viscous matrix or viscous interfaces [13]. Viscoelastic hydrogels with dissipative viscous matrix have been prepared by sterically entrapping high molecular weight linear polyacrylamide molecules without covalent bonding in covalently cross-linked networks of polyacrylamide. Primary rat hepatic stellate cells have been cultured on the hydrogels to disclose the influence of viscous dissipation on cell differentiation. The dissipative component, linear polyacrylamide molecules, can reduce cell differentiation and help the cells to maintain their phenotype [13]. Murine myoblasts cultured on lipid bilayers of different viscosities exhibit higher spreading area but lower circularity on higher viscosity lipid bilayers [14]. These studies have used viscoelastic surfaces or viscous interfaces to discriminate the viscosity impact on cell functions [[13], [14], [15]]. However, it is unclear how viscosity of viscous fluid affects chondrogenic differentiation of stem cells because there are no good models to culture stem cells in three-dimensional (3D) viscous liquid for such investigation. Therefore, the aim of this study was to investigate the influence of viscosity on chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) by using a 3D culture system that could provide viscous microenvironments for 3D cell culture of hMSCs. The cells were cultured in gelatin solutions of different viscosities that were embedded in chemically crosslinked gelatin hydorgels. The viscosity of gelatin solution showed a significant influence on the chondrogenic differentiation of hMSCs.

Section snippets

Synthesis of gelatin methacryloyl macromer

Gelatin methacryloyl (GelMA) macromer was synthesized by introducing methacrylate groups in gelatin molecules [16]. 5 g of gelatin powder from porcine skin (type A, 300 bloom, Sigma-Aldrich, St Louis, Missouri, USA) was dissolved at 10 (w/v)% in phosphate buffered saline (PBS, pH 7.4, Nacalai Tesque, USA) at 50 °C under stirring until gelatin was dissolved. 5 mL of methacrylic anhydride (Sigma-Aldrich, St Louis, Missouri, USA) was added dropwise to the gelatin solution at a rate of 0.5 mL min−1

Viscosity of gelatin solution of different concentration and stiffness of GelMA hydrogel

To obtain gelatin solutions of different concentrations and viscosities, different amount of gelatin powder was dissolved in serum-free H-DMEM. The three gelatin solutions showed a clear difference in viscosity. Their viscosities increased with an increase in gelatin concentration (Fig. 1). Therefore, the 5 (w/v) %, 10 (w/v) % and 15 (w/v) % gelatin solutions were used as cell culture matrix with low, medium and high viscosity, respectively.

Preparation of biphasic hydrogels loaded with cell-laden gelatin solutions

To investigate the influence of gelatin solution

Discussion

A variety of materials such as mucin, dextran and glycol (PEG) have been used to create viscous microenvironments [18,19]. Unlike these materials, gelatin derived from thermal denaturation of the collagen which is a major component of ECMs can provide more closely mimicking ECM microenvironments [20]. In addition, hMSCs can be encapsulated in gelatin hydrogels because gelatin solution can form hydrogels by physical crosslinking at a low temperature such as 4 °C. Gelatin hydrogels can change to

Conclusion

This study disclosed the influence of gelatin solution viscosity on proliferation and chondrogenic differentiation of hMSCs in a 3D biphasic hydrogel culture system. The cells showed high viability during the 3D culture. High viscosity promoted cartilaginous sGAG production while inhibited cell proliferation. On the other hand, low viscosity promoted cell proliferation while suppressed cartilaginous sGAG production. Under the presence of chondrogenic induction factors, high viscosity gelatin

Acknowledgement

This work was financially supported by JSPS KAKENHI (Nos. 18K19947, 18K19945 and 19H04475).

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