ABSTRACT
Objective To investigate the in vitro effect of alginate (Alg)/glycol chitosan (GC) double-network hydrogel on immune function of splenic regulatory T cells (Treg) of mice. Methods Single network hydrogel of Alg or GC and double-network (DN) gel material (Alg/GC) were prepared. Splenic CD4+CD25+Treg of BABL/c mice were isolated and purified by Magnetic Activated Cell Sorting (MACS) kit and further stimulated with different hydrogels for 24 hours and 48 hours with or without polymyxin B (PMB) exposure. Cells treated with Alg, or GC, or DN were divided into Alg group, GC group, DN group, respectively. Cells without hydrogens were served as control group. Expressions of the forkhead/winged helix transcription factor p3 (Foxp3) and cytotoxic T cell-associated antigen 4 (CTLA-4) on CD4+CD25+Treg were measured by flow cytometry. Their expression levels would be used to determine the conditions for further experiments. The CFSE kit was applied for assessing the proliferative activity of effector T cells (Teffs) after co-cultured with CD4+CD25+Treg. Enzyme linked immunosorbent assay (ELISA) was used to determine the cytokine levels including interleukin (IL)-10 and transforming growth factor (TGF)-β, IL-2, IL-4, and interferon (IFN)-γ. Results With or without PMB exposure, the expression of CTLA-4 and Foxp3 showed no significant difference under the stimulation of various hydrogels (P>0.05), including Alg, GC, and DN. Therefore, for further studies, cells were stimulated by different hydrogens in the presence of PMB for 24 hours. Cells without hydrogen simulation were served as control. Compared with the control group [(47.73±5.35)%], CTLA-4 expression was obviously up-regulated in both GC [(95.99±2.79)%] and DN [(92.21±2.97)%] hydrogel groups (P<0.05). In addition, TGF-β levels were markedly elevated in CD4+CD25+Treg stimulated with GC [(957.06±138.70)pg/ml] or DN [(905.03±73.04)pg/ml], compared with the level in the cells in the control group [(558.75±48.58)pg/ml, P<0.05]. However, no difference was detected in the expression of Foxp3 among different groups. In comparison with control group [proliferation (42.04±1.35)%, IL-4/IFN-γ (1.45±0.27)], Treg cells that were stimulated with GC or DN showed enhanced inhibitory effect on Teffs, including suppressed proliferation of Teffs [DN group (37.22±1.39)%, GC group (35.20±2.03)%] and increased ratio of IL-4/IFN-γ of Teffs [DN group (2.30±0.22), GC group (2.57±0.23)] (P<0.05). Conclusion Alg/GC double-network hydrogel can greatly enhance the immunosuppressive response of Treg, which is dominantly seen with GC single network hydrogel.
ABSTRACT
Objective • To construct a natural polymers-based hydrogels with enhanced mechanical property through double network (DN) strategy under the premise of retaining the good biocompatibility of natural polymers, and then evaluate the beneficial effect of hydrogels on the osteogenic differentiation of rat bone mesenchymal stem cells (rBMSCs). Methods • Methacrylated hyaluronic acid (HAMA) and methacrylated natural gelatin (GelMA) were synthesized through reaction of HA and Gel with methacrylic anhydride (MA), respectively. Thereafter, HA-Gel DN hydrogels were fabricated through two-step photo-crosslinking. The basic physicochemical properties of hydrogels were evaluated by scanning electron microscopy, swelling, compression and degradation analysis. Hydrogels were applied as substrate materials for rBMSCs culture in vitro. Cell viability, attachment, spreading and proliferation were assessed by CCK-8 analysis and fluorescent staining analysis. The osteogenic differentiation of rBMSCs was determined by quantitative PCR and Western blotting analysis. Results • In comparison to HA hydrogels and Gel hydrogels, HA-Gel DN hydrogels showed more suitable physicochemical properties, such as more suitable water absorption and water retention [(12.6± 0.7) fold, (10.3± 0.4) fold], stronger mechanical property [(43.7± 5.6) kPa] and slower degradation rate [(82.3±3.9)% for 12 weeks] for osteogenic differentiation of rBMSCs. Experiment in vitro revealed that HA-Gel DN hydrogels had good biocompatibility, quantitative PCR revealed that it could promote the expression of osteogenic genes including Runx2, BSP, OPN, OCN, OSX and ALP. Western blotting revealed that the HA-Gel DN hydrogels also increased the levels of osteogenic proteins (OPN, OSX and BSP). Conclusion • HAGel DN hydrogels have good biocompatibility and promote the osteogenic differentiation of rBMSCs, which provide a new experimental basis for DN hydrogels becoming the potential material for bone defects repair.