Three-Dimensional Spheroid Culture Increases Exosome Secretion from Mesenchymal Stem Cells

BACKGROUND: Mass production of exosomes is a prerequisite for their commercial utilization. This study investigated whether three-dimensional (3D) spheroid culture of mesenchymal stem cells (MSCs) could improve the production efficiency of exosomes and if so, what was the mechanism involved. METHODS: We adopted two models of 3D spheroid culture using the hanging-drop (3D-HD) and poly(2-hydroxyethyl methacrylate) (poly-HEMA) coating methods (3D-PH). The efficiency of exosome production from MSCs in the 3D spheroids was compared with that of monolayer culture in various conditions. We then investigated the mechanism of the 3D spheroid culture-induced increase in exosome production. RESULTS: The 3D-HD formed a single larger spheroid, while the 3D-PH formed multiple smaller ones. However, MSCs cultured on both types of spheroids produced significantly more exosomes than those cultured in conventional monolayer culture (2D). We then investigated the cause of the increased exosome production in terms of hypoxia within the 3D spheroids, high cell density, and non-adherent cell morphology. With increasing spheroid size, the efficiency of exosome production was the largest with the least amount of cells in both 3D-HD and 3D-PH. An increase in cell density in 2D culture (2D-H) was less efficient in exosome production than the conventional, lower cell density, 2D culture. Finally, when cells were plated at normal density on the poly-HEMA coated spheroids (3D-N-PH); they formed small aggregates of less than 10 cells and still produced more exosomes than those in the 2D culture when plated at the same density. We also found that the expression of F-actin was markedly reduced in the 3D-N-PH culture. CONCLUSION: These results suggested that 3D spheroid culture produces more exosomes than 2D culture and the non-adherent round cell morphology itself might be a causative factor. The result of the present study could provide useful information to develop an optimal process for the mass production of exosomes.

The effects of 2-hydroxyethyl methacrylate on the proliferation and migration of human dental pulp cells / 实用口腔医学杂志

Objective:To assess the effects of HEMA on the proliferation and migration of human dental pulp cells(hDPCs).Meth-ods:hDPCs were obtained using tissue explant culture technique in vitro,and then cells of the 3rd -5th passages were treated by differ-ent concentrations of HEMA for 24,48 and 72 h respectively.Cell proliferation was examined by MTT assay.Cell migration was ob-served by Transwell method.Results:The proliferation ability of hDPCs decreased when exposed to HEMA in both time and concentra-tion dependent manner(vs control,P <0.05).Cell proliferation at 24 h expossure was statistically higher than that at 48 h and 72 h(P<0.05).The migration of hDPCs was significantly reduced in HEMA groups at different concentrations(vs control,P <0.05).Con-clusion:HEMA inhibits the proliferation and migration of human dental pulp cells in vitro.

Effect of moisture and drying time on the bond strength of the one-step self-etching adhesive system

OBJECTIVES: To investigate the effect of dentin moisture degree and air-drying time on dentin-bond strength of two different one-step self-etching adhesive systems. MATERIALS AND METHODS: Twenty-four human third molars were used for microtensile bond strength testing of G-Bond and Clearfil S3 Bond. The dentin surface was either blot-dried or air-dried before applying these adhesive agents. After application of the adhesive agent, three different air drying times were evaluated: 1, 5, and 10 sec. Composite resin was build up to 4 mm thickness and light cured for 40 sec with 2 separate layers. Then the tooth was sectioned and trimmed to measure the microtensile bond strength using a universal testing machine. The measured bond strengths were analyzed with three-way ANOVA and regression analysis was done (p = 0.05). RESULTS: All three factors, materials, dentin wetness and air drying time, showed significant effect on the microtensile bond strength. Clearfil S3 Bond, dry dentin surface and 10 sec air drying time showed higher bond strength. CONCLUSIONS: Within the limitation of this experiment, air drying time after the application of the one-step self-etching adhesive agent was the most significant factor affecting the bond strength, followed by the material difference and dentin moisture before applying the adhesive agent.

Influence of HEMA content on the mechanical and bonding properties of experimental HEMA-added glass ionomer cements

The purpose of this study was to determine the influence of incrementally added uncured HEMA in experimental HEMA-added glass ionomer cement (HAGICs) on the mechanical and shear bond strength (SBS) of these materials. Increasing contents of uncured HEMA (10-50 wt. percent) were added to a commercial glass ionomer cement liquid (Fuji II, GC, Japan), and the compressive and diametral tensile strengths of the resulting HAGICs were measured. The SBS to non-precious alloy, precious alloy, enamel and dentin was also determined after these surfaces were subjected to either airborne-particle abrasion (Aa) or SiC abrasive paper grinding (Sp). Both strength properties of the HAGICs first increased and then decreased as the HEMA content increased, with a maximum value obtained when the HEMA content was 20 percent for the compressive strength and 40 percent for the tensile strength. The SBS was influenced by the HEMA content, the surface treatment, and the type of bonding surface (p<0.05). These results suggest that addition of an appropriate amount of HEMA to glass ionomer cement would increase diametral tensile strength as well as bond strength to alloys and teeth. These results also confirm that the optimal HEMA content ranged from 20 to 40 percent within the limitations of this experimental condition.

Effect of hema and tegdma on the properties of experimental composite resins / 대한치과보철학회지

PURPOSE: The purpose of this study was to compare HEMA and TEGDMA as diluents for the composite resin. MATERIAL AND METHODS: Eight kinds of experimental light curable composite resins were prepared and used. Concentrations of monomer and filler were same for all experimental composites, except, the diluent's ratios to the monomer. The ratio of diluents to Bis-GMA were 15%, 20%, 25%, and 30%, and two kinds of diluents were used, so total experimental groups were eight including one control group of 25% TEGDMA. RESULTS: Depth of cure, flexural strength, shear bond strength to bovine enamel, shear bond strength to bovine dentin, water absorption and solubility of composites in water were measured. Sample size for each groups were 10. Arithmetic means were used as each groups representative values, and regression test for two diluents and four concentrations, Duncan's multiple range test, and Two-way ANOVA test were done for kinds of diluents and its concentrations at level of 0.05. CONCLUSION: Following results were obtained; 1. There were not significant differences in effects of HEMA and TEGDMA to depth of cure, flexural strength of composites and shear bond strength to bovine enamel (p>0.05). 2. Increase of the concentrations of the diluents made the depth of cure (p<0.001) and flexural strength (p<0.05) a little higher. 3. Shear bond strength to dentin was higher on HEMA containing composites than TEGDMA containing composites (p<0.001). 4. Water absorption was higher on HEMA containing composites than TEGDMA containing composites (p<0.01).