Objective:A mineralized gelatin methacryloyl/hyaluronic acid methacryloyl (GelMA/HAMA) double network hydrogel was constructed, its performance was characterized, and its cell compatibility was studied.Methods:The hydrogel was constructed by photocrosslinking technology, and the biomimetic mineralized hydrogel was prepared by alternating mineralization with calcium and phosphorus solution. The hydrogel was mineralized for three times, for 24 h each time. The control groups were the non-mineralized groups, named the GelMA-0 group and GelMA/HAMA-0 group, respectively. The experimental groups were the mineralized treatment groups. The GelMA hydrogel and GelMA/HAMA double network hydrogel mineralized for 1, 2 and 3 times were named the GelMA-1, 2 and 3 group and GelMA/HAMA-1, 2 and 3 group, respectively. The microstructure of the mineralized hydrogel was characterized by scanning electron microscope and energy dispersive X-ray spectroscopy. The chemical composition of the mineralized hydrogel was characterized by Fourier transform infrared spectroscopy and X-ray diffraction. The mineralization rate of the mineralized hydrogel was evaluated by the ashing method. The compressive strength of the mineralized hydrogel was evaluated by a compression test. The cell compatibility of bone marrow mesenchymal stem cells (BMSCs) on the mineralized hydrogel surface was evaluated by a cell counting kit-8 assay and living/dead cell staining experiment. The datas were analyzed by one-way analysis of variance and Tukey test.Results:With the increase in the number of mineralization cycles, the mineralized layer gradually thickened, and the mineral layer on the cross section of the hydrogel uniformly penetrated into the interior of the hydrogel, with the amount of internal mineral deposition increased. The microstructure of the hydrogel and the distribution of inorganic particles also changed. The surface of the GelMA-3 group and the GelMA/HAMA-3 group contained Ca and P elements, and the distribution was consistent. The inorganic particles generated by mineralization may be immature hydroxyapatite (HAP) crystal precursors. To further verify the chemical composition of inorganic particles, the GelMA-0 and 3 groups as well as the GelMA/HAMA-0 and 3 groups of hydrogels showed GelMA amide Ⅰ, Ⅱ and Ⅲ bands. The GelMA-3 group and GelMA/HAMA-3 group hydrogels contained phosphate, and the diffraction peaks confirmed that the inorganic particles are mainly HAP crystal precursors with low crystallinity. The mineralization rates of the GelMA-1, 2 and 3 groups were (12.48±1.06)%, (21.12±0.62)% and (27.31±0.45)%, while those of the GelMA/HAMA-1, 2 and 3 groups were (15.54±1.03)%, (23.39±0.25)% and (32.26±0.62)%, and the compressive elastic modulus of the GelMA-0, 1, 2 and 3 groups were (69.01±1.04, 91.76±2.05, 105.16±2.95, 131.65±2.21) kPa, and those of the GelMA/HAMA-0, 1, 2 and 3 groups were (270.76±4.56, 347.47±4.60, 388.98±6.96, 430.6±15.47) kPa. Under the same cyclic mineralization times, the mineralization rates and compressive elastic modulus of the GelMA/HAMA double network hydrogel were significantly higher than those of the GelMA hydrogel (all P<0.01). The cell proliferation rates of BMSCs on the GelMA/HAMA-0, 1, 2 and 3 hydrogel surfaces were (49.80±3.38)%, (52.32±1.28)%, (58.00±4.64)% and (62.46±2.74)% on the first day of culture, (58.86±3.36)%, (58.26±3.45)%, (73.08±2.61)% and (76.40±3.45)% on the third day of culture, and (85.89±4.23)%, (90.75±3.21)%, (103.35±4.11)% and (113.42±3.40)% on the fifth day of culture. The cell proliferation rates of the GelMA/HAMA-3 group were significantly higher than those of GelMA/HAMA-0 and 1 groups (all P<0.01). Living/dead cell staining experiment showed that BMSCs on the surface of the GelMA/HAMA-0, 1, 2 and 3 groups were dominated by living cells on the third and fifth days of culture. Conclusions:The mineralized GelMA/HAMA double network hydrogel was constructed, which significantly improved the mineralization rate, mechanical performance and cell compatibility of the biomimetic mineralized hydrogel.

In vivo, cells exist within the mechanical microenvironment of the extracellular matrix (ECM), and their biological processes are regulated by various mechanical factors. However, existing technology limits the direct study of the interaction between cells and ECM in vivo. Using hydrogel to mimic the natural ECM and to construct a suitable mechanical microenvironment for isolated cells, which indirectly reflects the cell-ECM interactions in real organisms through in vitro studies. Thus, in this review, the effects of mechanical factors in hydrogel on different types of cells were summarized, and the development of cell mechanical detection technology using hydrogel as carriers was introduced. Moreover, the future work of using hydrogel to study cell mechanical behavior and related detection technology, as well as standardized preparation, was discussed.

Dynamic hydrogel is three-dimensional networks of hydrophilic polymer formed by dynamic reversible cross-linked bonds. It is widely used in the field of bone repair therapy. The dynamic reversible cross-linked bonds in the dynamic hydrogel endows it with shear-thinning rheological properties, resulting in excellent self-healing, injectability and tunable mechanical properties. In this review, the formation mechanism of dynamic hydrogel was summarized, including non-covalent interaction and dynamic covalent interaction. The advantages and disadvantages of the various cross-linked methods used to form dynamic hydrogel were discussed. The applications of dynamic hydrogel in bone repair therapy were summarized, including haemostasis for filling bone defects, drug delivery, modulation of stem cell behavior, and biomimetic mineralization, to provide a reference for the future development of dynamic hydrogel for bone repair therapy.

Objective Mitochondrial reactive oxygen species(mtROS)could cause damage to pancreatic β-cells,rendering them susceptible to oxidative damage.Hence,investigating the potential of the mitochondria-targeted antioxidant(Mito-TEMPO)to protect pancreatic β-cells from ferroptosis by mitigating lipid peroxidation becomes crucial. Methods MIN6 cells were cultured in vitro with 100 μmol/L sodium palmitate(SP)to simulate diabetes.FerroOrange was utilized for the detection of Fe2+fluorescence staining,BODIPY581/591C11 for lipid reactive oxygen species,and MitoSox-Red for mtROS.Alterations in mitophagy levels were assessed through the co-localization of lysosomal and mitochondrial fluorescence.Western blotting was employed to quantify protein levels of Acsl4,GPX4,FSP1,FE,PINK1,Parkin,TOMM20,P62,and LC3.Subsequently,interventions were implemented using Mito-TEMPO and Carbonyl cyanide 3-chlorophenylhydrazone(CCCP)to observe changes in ferroptosis and mitophagy within MIN6 cells. Results We found that SP induced a dose-dependent increase in Fe2+and lipid ROS in MIN6 cells while decreasing the expression levels of GPX4 and FSP1 proteins.Through bioinformatics analysis,it has been uncovered that mitophagy assumes a crucial role within the ferroptosis pathway associated with diabetes.Additionally,SP decreased the expression of mitophagy-related proteins PINK1 and Parkin,leading to mtROS overproduction.Conversely,Mito-TEMPO effectively eliminated mtROS while activating the mitophagy pathways involving PINK1 and Parkin,thereby reducing the occurrence of ferroptosis in MIN6 cells.CCCP also demonstrated efficacy in reducing ferroptosis in MIN6 cells. Conclusion In summary,Mito-TEMPO proved effective in attenuating mtROS production and initiating mitophagy pathways mediated by PINK1 and Parkin in MIN6 cells.Consequently,this decreased iron overload and lipid peroxidation,ultimately safeguarding the cells from ferroptosis.

OBJECTIVE: To investigate the effect of ABT-263,an anti-apoptotic protein inhibitor,on human cutaneous squamous cell carcinoma A431 cells,and to explore its molecular mechanisms. METHODS: i) Total protein was extracted from human immortalized epidermal cells( Ha Ca T cells) and A431 cells in logarithmic growth phase. The protein expression of B-cell lymphoma-2( BCL-2) and BCL2-like 1( BCL-XL) was detected by Western blotting. ii) The A431 cells were treated with ABT-263( inhibitor group) and dimethyl sulfoxide( control group) at a concentration of 50 μmol/L for 4 and 9 hours. The morphological changes of the cells were examined by transmission electron microscopy. iii) The A431 cells were treated with 0,10,25,40,and 50 μmol/L of ABT-263 for 24 hours,and the cell viability was determined by CCK-8 assay. iv) The A431 cells were treated with different doses of ABT-263,and the expression of cleaved Caspase-3, cleaved poly( ADP-ribose) polymerase-1( PARP-1), phosphorylated protein kinase B [p AKT(ser473)],phosphorylated glycogen synthase kinase-3β(p GSK3β) and phosphorylated histone H2 AX(γH2 AX) was detected by Western blot. RESULTS: The relative expression of BCL-2 and BCL-XL in A431 cells were higher than those in Ha Ca T cells( P < 0. 01). Transmission electron microscopy results showed that A431 cells in inhibitor group gradually changed from normal morphology to apoptotic morphology,showing loss of microvilli,increased nuclear chromatin density and aggregation around the nuclear membrane,and nuclear fragmentation. The cell viability of A431 cells in 10,25,40 and 50 μmol/L groups were lower than those in control group( P < 0. 05). The relative expression of cleaved Caspase-3 and cleaved PARP-1 in A431 cells in 10,30 and 50 μmol/L groups were higher than those in control group( P < 0. 05).The relative expression of p AKT( ser473) and p GSK3β in A431 cells in 10,25,40 and 50 μmol/L groups were lower than those of the control group( P < 0. 05) and γH2 AX protein expression was higher than that of the control group( P <0. 05). A431 cell viability and p GSK3β protein expression decreased with the increase of inhibitor dosage( P < 0. 01).The relative expression of cleaved Caspase-3 and γH2 AX protein increased with the increase of inhibitor dosage( P <0. 01),showing dose-effect relationship. CONCLUSION: ABT-263 can induce apoptosis of A431 cells through mitochondria pathway and induce the inactivation of AKT/GSK3β pathway,which can promote the apoptosis of A431 cells with a doseeffect relationship.