Effects of mechanical properties of gelatin methacryloyl hydrogels on encapsulated stem cell spheroids for 3D tissue engineering
Cell spheroids are three-dimensional cell aggregates that have been widely employed in tissue engineering. Spheroid encapsulation has been explored as a method to enhance cell-cell interactions. However, the effect of hydrogel mechanical properties on spheroids, specifically soft hydrogels (<1 kPa), has not yet been studied. In this study, they determined the effect of encapsulation of stem cell spheroids by hydrogels crosslinked with different concentrations of gelatin methacryloyl (GelMA) on the functions of the stem cells. To this end, human adipose-derived stem cell (ADSC) spheroids with a defined size were prepared, and spheroid-laden hydrogels with various concentrations (5, 10, 15%) were fabricated. The apoptotic index of cells from spheroids encapsulated in the 15% hydrogel was high. The migration distance was five-fold higher in cells encapsulated in the 5% hydrogel than the 10% hydrogel. After 14 days of culture, cells from spheroids in the 5% hydrogel were observed to have spread and proliferated. Osteogenic factor and pro-angiogenic factor production in the 15% hydrogel was high. Collectively, our results indicate that the functionality of spheroids can be regulated by the mechanical properties of hydrogel, even under 1 kPa. These results indicate that spheroid-laden hydrogels are suitable for use in 3D tissue construction.
Schematic diagram of the overall process used to encapsulate spheroids in gelatin methacryloyl (GelMA) hydrogel of various concentrations. Adipose-derived stem cell (ADSC) spheroids were encapsulated within hydrogels crosslinked with 5, 10, and 15 w/v% GelMA.
Hydrogels are crosslinked networks of hydrophilic polymers, and have been widely used in tissue engineering applications because of their extracellular matrix (ECM)-like elastic properties and high water content. Hydrogels can be formed from a number of synthetic and natural water-soluble polymers. Gelatin methacryloyl (GelMA) is particularly attractive for cell encapsulation due to the presence of cell adhesion moieties in the polymer chain, high biocompatibility, rapid network formation by photo-induced reaction, and the mildness of the cross-linking conditions required (room temperature (RT) and neutral pH).
(a) Illustration of the harvesting of ADSC spheroids from micro-patterned and temperature-responsive hydrogels. (b) Optical image of the hydrogels (scale bar = 5 mm) (c) phase contrast image of the polydopamine pattern on the hydrogel surface, (d) micro-sized ADSC layers at 37 °C, and (e) spheroidal assembly of micro-sized ADSC layers at 4 °C (scale bar = 100 μm). (f) Phase contrast image of ADSC spheroids harvested from the hydrogel (scale bar = 100 μm). (g) Confocal microscopy images of F-actin-stained ADSC spheroids (scale bar = 50 μm) (h) Live/Dead staining images of ADSC spheroids (scale bar = 200 μm). (i) Size distribution of ADSC spheroids.
Various cell types (e.g. human mesenchymal stem cells (MSCs), endothelial cells (ECs), fibroblasts) have been encapsulated in GelMA hydrogels and encapsulation has been shown to support in vitro proliferation, elongation, migration, and differentiation of these cells. In addition, tissue regeneration using GelMA hydrogels as carriers of transplanted cells has been investigated. For instance, human MSCs encapsulated by GelMA hydrogels enhanced in vitro mineralization and osteogenesis and had a strong therapeutic effect in a bone defect model.GelMA-encapsulated MSCs/ECs supported vascular network formation and were easily transferred in an in vivo subcutaneous model by transdermal photo-polymerization to improve neovascularization. In addition, human adipose-derived stem cells (ADSCs) encapsulated with GelMA hydrogels blended with hyaluronic acid (HA) were developed for 3D bio-printing of bone tissue.
Hydrogels for cell encapsulation should model the diverse mechanical properties (0.5–20 kPa) of natural tissue, and thus the effects of the mechanical properties of hydrogels on cell functionality (viability, spreading, migration, proliferation, and/or differentiation) require investigation. For example, the viability of fibroblasts encapsulated within a 20 kPa GelMA hydrogel was lower than that of fibroblasts encapsulated within hydrogels with a lower storage modulus due to a limited supply of nutrients in the 20 kPa GelMA hydrogel [8]. Migration of human MSCs encapsulated within fibrin hydrogels decreased when the modulus of the hydrogel was increased from 50 Pa to 1 kPa by controlling the concentration of fibrinogen.
(a) Optical images of GelMA and spheroids-laden hydrogels (scale bar = 5 mm) and phase contrast images of ADSC spheroids encapsulated within 5% GelMA hydrogel (scale bar = 500 μm). (b) Live/Dead staining images of ADSC spheroids encapsulated in 5, 10, and 15% hydrogels (scale bar = 200 μm). (c) Encapsulation rate of spheroids according to GelMA concentration. (d) SEM images of 5, 10, and 15% GelMA spheroid-laden hydrogels (scale bar = 200 μm). (e) Pore area, (f) Young's modulus, and (g) swelling ratio of spheroid-laden hydrogels prepared with 5, 10, and 15% GelMA. * and $ indicate a significant difference relative to the 5% and 10% cells, respectively (n = 4, p < 0.05, ANOVA for comparison of GelMA concentration). (h) SEM images of spheroids after encapsulation in 5, 10, and 15% GelMA hydrogels.
In addition, the spreading of MSCs encapsulated within alginate hydrogels was enhanced by fast degradation of hydrogels with a lower Young's modulus. Spreading and migration of mouse MSCs were controlled by pore area changes caused by HA hydrogel network degradation through matrix metalloproteinase (MMP) activity. The proliferation of human MSCs within poly (ethylene glycol)-fibrinogen hydrogel increased in softer hydrogels due to the influence of von Willebrand factor (vWF), which regulates proliferation. In addition, the morphology of human MSCs encapsulated within HA hydrogels has been shown to vary depending on the mechanical properties of the hydrogels; cells became rounder and showed up-regulation of adipogenesis when encapsulated in softer hydrogels, while cells grown in stiffer hydrogels had a spindle morphology and showed enhanced osteogenesis . These previous studies all highlight the importance of controlling the mechanical properties of the hydrogels used for cell encapsulation.
Cell spheroids are three-dimensional spherical cell aggregates formed by self-assembly of cells and have been widely employed in tissue engineering, regenerative medicine, and cell therapy.
(a) Phase contrast images of ADSC spheroids encapsulated at 6000 spheroids/ml (high density (HD)) or 1500 spheroids/ml (low density (LD)) spheroids/ml in 5% GelMA hydrogel after 14 days of culture (scale bar = 500 μm) and Live/Dead staining images of spheroids after 14 days of culture (scale bar = 200 μm). (b) Average sprout length of spheroids encapsulated in hydrogels according to spheroid density. (c) Relative proliferation of spheroids in hydrogels according to spheroid density for 14 days. * indicates a significant difference relative to 1 day (n = 4, p < 0.05, t-test for comparison of 1 day). (d) Dry weight of spheroid-laden hydrogels after 14 days of culture. (e) Young's modulus of spheroid-laden hydrogels after 14 days of culture.
Stem cell spheroids exhibit high viability, cytokine release (pro-angiogenic, anti-inflammatory), and differentiation efficiency due to intensive cell-cell interactions compared to those that occur among cells cultured in two dimensional (2D) environments. Recent studies have encapsulated spheroids within hydrogels to determine if this encapsulation enhances cell-cell interactions within spheroids. For example, ADSC spheroids encapsulated in enzyme-crosslinked gelatin hydrogels demonstrated enhanced expression of adipogenesis/chondrogenesis-related genes (PPAR-γ, Col II, Sox-9) and MSC spheroids encapsulated within an alginate hydrogel modified with RGD (Arg-Gly-Asp) peptide secreted more vascular endothelial growth factor (VEGF) under hypoxic conditions than control spheroids.
In addition, the effect of hydrogel mechanical properties on the functionality of stem cell spheroids has been investigated. Human MSC spheroids encapsulated in RGD peptide-conjugated alginate hydrogels with a storage modulus ranging from 1 to 15 kPa showed a difference in differentiation according to storage modulus; osteogenesis was observed in 5 kPa hydrogel while chondrogenesis was observed in 1 kPa hydrogel [24]. MSC spheroids encapsulated in fibrin hydrogels with a compressive modulus ranging from 20 to 80 kPa secreted more VEGF the higher the compressive modulus (i.e. the stiffer the gel), while prostaglandin E2 (PGE2) secretion was increased in more compliant gels [25]. However, information about the effects of hydrogel mechanical properties on spheroids is still limited. In particular, how stem cell spheroids respond to changes in the mechanical properties of relatively soft GelMA-based hydrogels (< 1 kPa) has not yet been studied.
Given that, our purpose in this study was to determine the effects of GelMA hydrogel mechanical properties on the functions of stem cell spheroids following encapsulation. To this end, stem cell spheroids with a defined size were prepared, and spheroid-laden hydrogels with various mechanical properties (0.3–2.0 kPa) were fabricated. Then, the effect of mechanical properties on cell morphology and functionality (migration, apoptosis, proliferation) of cells within spheroids was investigated. Finally, the osteogenic differentiation potential and expression of pro-angiogenic factors by stem cells encapsulated within spheroids in hydrogels were studied to evaluate the potential of spheroid-laden hydrogels in 3D tissue construction.
Eun Mi Kim, Gyeong Min Lee, Sangmin Lee, Se-jeong Kim, Dongtak Lee, Dae Sung Yoon, Jinmyoung Joo, Hyunjoon Kong, Hee Ho Park, Heungsoo Shin,
Effects of mechanical properties of gelatin methacryloyl hydrogels on encapsulated stem cell spheroids for 3D tissue engineering,
International Journal of Biological Macromolecules,
2021,
,
ISSN 0141-8130,
https://doi.org/10.1016/j.ijbiomac.2021.11.145.
(https://www.sciencedirect.com/science/article/pii/S0141813021025502)