Objective:To explore the preparation and preliminary research on the characteristics of modified nano-bioglass hydrogel.Methods:(1) The nano-bioglass suspension was prepared by adding 67 mL nano-silica suspension into 400 mL saturated calcium hydroxide solution, and its suspension stability was observed. (2) The hydrogel with final mass fraction of 10% gelatin and 1% sodium alginate was prepared and set as control group. On the basis of the hydrogel in control group, the nano-bioglass suspension prepared in experiment (1) was added to prepare the hydrogel with the final mass fraction of 0.5% bioglass, 10% gelatin, and 1% sodium alginate, and the hydrogel was set as the experimental group. The gelling time at 4 and 25 ℃and the dissolution time at 37 ℃ of hydrogel in 2 groups were recorded, and the gelation at 4 and 25 ℃and dissolution condition at 37 ℃of the hydrogel in 2 groups were observed. The hydrogel in 2 groups were collected and cross-linked with 25 g/L calcium chloride solution after cold bath at 4 ℃, and the compression modulus was measured by Young′s modulus tester. In addition, the hydrogel in 2 groups were collected and cross-linked as before, and freeze-drying hydrogel was made at -20 ℃. The relative volumes were measured and the porosity of hydrogel in 2 groups was calculated. The number of sample in the experiment was 3. (3) Fibroblasts (Fbs) were isolated and cultured from 12 C57BL/6J mice of 24 hours old and the morphology was observed by inverted microscope, and the third passage of Fbs were cultured for the following experiment. Fbs were collected to make single cell suspension with the cell concentration of 1×10 5/mL. The single cell suspension was divided into experimental group and control group according the random number table (the same grouping method below), which were added with hydrogel in experimental group and control group prepared in experiment (2), respectively. At culture hour 12, 24, and 48, cells of 3 wells in each group were collected to detect the survival rate by cell counting kit 8 method. (4) The third passage Fbs were collected to prepare the single cell suspension with the cell concentration of (3.0~4.5)×10 7/mL, which was divided into experimental group and control group, with 1 tube in each group. The single cell suspension in 2 groups were added with green fluorescent probe DIO for staining and then added with 9 mL hydrogel in experimental group and control group prepared in experiment (2), respectively. The mixed solution of Fbs and hydrogel in 2 groups was cross-linked as before to make cell-loaded hydrogel. On culture day 3, the survival of cells in the hydrogel was observed by laser confocal microscope. The cell-loaded hydrogel was prepared as before and without added with green fluorescent probe DIO. On culture day 7, the adhesion and extension of cells in the hydrogel were observed by scanning electron microscope. (5) Twelve 6-week-old female BALB/c-nu nude mice were collected and divided into experimental group and control group, with 6 mice in each group. A round full-thickness skin defect wound with diameter of 1 cm was made on the back of each mouse. Immediately after injury, one cell-loaded hydrogel block in the experimental group and the control group prepared in experiment (4) was placed in the wound of each mouse in the experimental group and the control group, respectively. On post injury day (PID) 7 and 14, 3 nude mice in each group were sacrificed to collect the wound and wound margin tissue, which was stained with hematoxylin-eosin to observe the wound healing. Data were statistically analyzed with independent sample t test. Results:(1) The nano-bioglass particles could be uniformly dispersed in water and had good suspension stability. (2) The hydrogels of the 2 groups were molten at 37 ℃, and no precipitation of particle was observed. The dissolving time of the hydrogel in the experimental group and the control group at 37 ℃ was 5 and 10 min, respectively. The gelation time of the hydrogel in the experimental group and the control group at 25 ℃ was 30 and 180 min, respectively, and the gelation time of the 2 groups at 4 ℃ was 5 and 10 min, respectively. The compression modulus of hydrogel in the experimental group was (53±6) kPa, which was significantly higher than (23±6) kPa in the control group ( t=6.364, P<0.01). The porosity of the hydrogel in the experimental group was (86.1±2.1)%, which was similar to (88.2±4.4)% in the control group ( t=1.210, P>0.05). (3) The cells were in long fusiform, and the proportion of nuclei was high, which was accorded with the morphological characteristics of Fbs. At culture hour 12, 24, and 48, the survival rate of cells in the experimental group was (84±4)%, (89±4)%, and (130±10)%, which was similar to (89±5)%, (90±4)%, and (130±11)% in the control group, respectively ( t=1. 534, 0.611, 0.148, P>0.05). (4) On culture day 3, the cells in the two groups had complete morphology in the hydrogel, no nuclear lysis or disappearance were observed, the cytoplasm remained intact, and the fluorescence intensity of the cells in the experimental group was significantly stronger than that in the control group. On culture day 7, the cells in the experimental group and the control group adhered and stretched in the hydrogel, and the number of cells in the experimental group adhered to the hydrogel was significantly more than that in the control group. On PID 7, the wound area of the nude mice in the control group and the experimental group were reduced, the reduction area of mice in the experimental group was more obvious, and a large amount of inflammatory cells were seen in and around the wound in the 2 groups. On PID 14, the wound area of the nude mice in the control group was larger than that of the experimental group, and the number of inflammatory cells in and around the wound was significantly more than that in the experimental group. Conclusions:Nano-bioglass hydrogel possesses good physical, chemical, and biological properties, cell loading potential, and the ability to promote wound healing, which means it has a good potential in clinical application.

Objective:To observe the influence of the stiffness of three-dimensionally bioprinted extracellular matrix analogue on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into skin appendage cells.Methods:(1) Sodium alginate of 1 g and 4 g gelatin, 3 g sodium alginate and 8 g gelatin were mixed respectively, and the two mixtures were dissolved in 100 mL ultra-pure water respectively to prepare two sodium alginate-gelatin composite hydrogels, named 1A4G hydrogel and 3A8G hydrogel, which were used in the subsequent experiments. The morphology of the two hydrogels at room temperature, after condensation for 15-30 min at 4 ℃ (the same condensation condition below), after condensation and cross-linking with 25 g/L calcium chloride solution (the same cross-linking condition below), and after condensation and three-dimensional printing with a three-dimensional bioprinter (the same three-dimensional printer below) and cross-linking were observed respectively. Young′s modulus (stiffness) of the two kinds of hydrogels was measured by Young′s modulus tester after condensation and cross-linking ( n=3). Two kinds of hydrogels were cross-linked and freeze-dried, and their pore structure was observed by scanning electron microscope. Two hydrogels were cross-linked and freeze-dried, and the porosity was detected by anhydrous ethanol replacement method ( n=3). (2) BMSCs were isolated from femur and tibia of 20 C57BL/6 mice (no limitation with sex, born 7 days) and cultured, and the second passage of cells was used for further test. The BMSCs single cell suspension (1.0×10 7 /mL) was mixed with 1A4G hydrogel and 3A8G hydrogel respectively at 1∶9 volume ratio to prepare BMSCs-loaded 1A4G hydrogel and BMSCs-loaded 3A8G hydrogel for three-dimensional printing. One construct was printed with 1 mL cell-loaded hydrogel (the same dosage for printing below). Mesenchymal stem cells (MSCs) specific medium was added after cross-linking, and the printed constructs were divided into 1A4G group and 3A8G group according to the hydrogel. One construct of each group cultured for 7 days was tested with live/dead kit to count the live cells and dead cells in 50-fold field of view. Nine printed constructs from each of the two groups were taken, and BMSCs of nine wells (1.0×10 6 per well) cultured with 2 mL MSCs specific medium were set as two-dimensional culture group. After 1, 3, 5 day (s) of culture, three printed constructs from 1A4G group and 3A8G group respectively and three wells of cells from two-dimensional culture group were taken to detect the absorbance value in culture medium by cell counting kit 8, denoting the cell proliferation activity. (3) BMSCs-loaded 1A4G hydrogel and BMSCs-loaded 3A8G hydrogel of 10 mL respectively were prepared as in experiment (2), which were respectively mixed with 0.5 mL plantar dermis homogenate extracted from 10 C57BL/6 mice of 1 day postnatal with unknown sex, then three-dimensionally printed, cross-linked, cultured with MSCs specific medium for 3 days and then changed to sweat gland specific medium. The printed constructs were divided into 1A4G group and 3A8G group according to their hydrogel. After 7 days of culture with sweat gland specific medium, the expressions of epithelial cell surface markers cytokeratin-5 (CK5) and CK14, sweat gland cell surface markers CK18 and Na + /K + -ATPase (NKA), and hair follicle cell surface markers CK17 and alkaline phosphatase (ALP) at protein level in cells of printed constructs in the two groups were detected by immunofluorescence method. The expressions of CK5, CK14, CK18, NKA (detecting ATP1a1), CK17, and ALP at mRNA level in cells of printed constructs in the two groups were detected with real-time fluorescent quantitative reverse transcription polymerase chain reaction ( n=3). Data were statistically analyzed with independent sample t test, Fisher′s exact probability test, analysis of variance for factorial design, and Bonferroni method. Results:(1) Compared with that of 3A8G hydrogel, 1A4G hydrogel had lower viscosity and better fluidity at room temperature. Both kinds of hydrogels were gel-like after condensation, based on which, the shape of cross-linked hydrogels was uniform and regular, with three-dimensional printing and cross-linking made hrdrogels forming solid crisscross cylindrical constructs. The Young′s modulus of 1A4G hydrogel was (52±6) kPa, which was obviously lower than (218±5) kPa of 3A8G hydrogel ( t=40.470, P<0.01). The pore structure of the two hydrogels was similar, with all the cross-sections showing porous network structure. The porosity of the two hydrogels was similar ( t=0.930, P>0.05). (2) The distribution of live/dead cells between 1A4G group and 3A8G group was similar after 7 days of culture ( P>0.05), most of which were live cells. The absorbance value in culture medium of printed constructs among 1A4G group, 3A8G group, and two-dimensional culture group didn′t show statistically significant differences after 1, 3, 5 day (s) of culture ( P>0.05). Compared with that after 1 day of culture within each group, the absorbance value in culture medium of printed constructs in 1A4G group and 3A8G group was significantly increased after 3 and 5 days of culture ( P<0.05 or P<0.01), and the absorbance value in culture medium of cells in two-dimensional culture group was significantly increased after 5 days of culture ( P<0.01). Compared with that after 3 days of culture within each group, the absorbance value in culture medium of printed constructs in 1A4G group and 3A8G group and that of cells in two-dimensional culture group was significantly increased after 5 days of culture ( P<0.01). (3) After 7 days of culture with sweat gland specific medium, the CK5, CK14, CK18, NKA, CK17, and ALP were positively expressed at protein level in cells of printed constructs in the two groups. After 7 days of culture with sweat gland specific medium, the expressions of CK5, CK14, CK18, and NKA at mRNA level in cells of printed constructs were close between the two groups ( t=0.362, 0.807, 0.223, 1.356, P>0.05); the expressions of CK17 and ALP at mRNA level in cells of printed constructs in 3A8G group were 1.96±0.21 and 55.57±11.49, respectively, which were significantly higher than 1.05±0.42 and 2.01±0.27 in 1A4G group ( t=3.333, 8.074, P<0.05 or P<0.01). Conclusions:BMSCs cultured three-dimensionally in 1A4G and 3A8G hydrogels tend to differentiate into sweat gland cells, but the BMSCs cultured three-dimensionally in 3A8G hydrogel show a stronger tendency to differentiate into hair follicle cells than the cells cultured in 1A4G hydrogel. It suggests that relatively high stiffness of three-dimensionally bioprinted extracellular matrix analogue facilitates not only differentiation of BMSCs into sweat gland cells, but also their differentiation into hair follicle cells.

Background Natural pyrethrins have long been widely used in the fields of environmental and household hygiene. Studies have reported that natural pyrethrins have potential liver toxicity, but their specific mechanisms are still unclear yet. Objective To explore the effect of natural pyrethrins on DNA damage in human liver cells. Methods This study used human liver cell QSG7701 as an in vitro testing model. After exposure to DMSO and a series of concentrations of natural pyrethrins (5, 10, 20, and 40 μg·mL⁻¹) for 6 and 24 h, reactive oxygen species (ROS) was detected by fluorescence microscopy using a fluorescence probe, thiobarbituric acid reactive substance (TBARS) by colorimetric method using a microplate reader, DNA damage by comet assay through observing DNA fragment migration under microscope, and phospho H2AX (γH2AX) and 8-oxoguanine (8-oxoG) by immunofluorescence assay using a laser confocal microscope. Results As the exposure concentration of natural pyrethrins increased, the fluorescence intensity of ROS significantly increased in a concentration-dependent manner. The differences in ROS between the 10 μg·mL⁻¹ and above groups and the control group were statistically significant (P<0.01), and the ROS levels in the 20 μg·mL⁻¹ and 40 μg·mL⁻¹ treatment groups were 2.17 and 3.05 times higher than that in the control group respectively. The TBARS level increased in a concentration-dependent manner in natural pyrethrins treated cells (P<0.01), and the levels in the 20 μg·mL⁻¹ and 40 μg·mL⁻¹ treatment groups were 2.46 and 3.01 times higher than that in the control group respectively. The results of comet assay showed trailing formation of cellular DNA in each dose group; as the exposure concentration of natural pyrethrins increased, indicators such as tail DNA content (TDNA%), tail length (TL), tail moment (TM), and Olive tail moment (OTM) increased in a concentration-dependent manner. Compared with the control group, the differences in the indicators between the 20 μg·mL⁻¹ and above groups and the control group were statistically significant (P<0.01), especially in the 40 μg·mL⁻¹ treatment groups, where TDNA%, TL, TM, and OTM were (46.92 ± 3.52) %, (64.67± 4.16) μm, 30.96 ± 2.94, and 22.64 ± 3.89, respectively. The cellular immunofluorescence results showed that natural pyrethrins induced the formation of γH2AX and 8-oxoG, the fluorescence intensities of γH2AX and 8-oxoG increased in a concentration-dependent manner, and the differences between the 10 μg·mL⁻¹ and above groups and the control group were statistically significant (P<0.01). Conclusion Natural pyrethrins could induce DNA damage in human liver cells, and ROS-mediated oxidative stress may play an important role in its liver cell genotoxicity.

Objective To establish bovine corneal opacity and permeability （BCOP） test， and determine its predictive ability for the eye irritation evaluation of cosmetics. Methods A total of ten reference chemicals were selected to establish the BCOP test. Then eye irritation of 16 routinely collected cosmetics in our laboratory was predicted. In vitro scores were calculated by the change in the opacity and sodium fluorescein permeability after exposure to the testing cosmetics， and subsequently compared with the historical data by Draize test. Results Reference chemicals with known irritation classification were correctly classified by the BCOP test， which was consistent with the classification of UN globally harmonized system of classification and labeling of chemicals. Moreover， the specificity of the BCOP test for the classification of non-irritating cosmetics samples was 80.0% （8/10）， and the sensitivity for weak to mild irritating cosmetics samples was 83.3% （5/6）. The BCOP test demonstrated an overall classification consistency of 81.3% （13/16） with in vivo test. Conclusion BCOP test may be independently used to identify chemicals with potential eye irritation and serious eye damage， suggesting it is significant for in vitro integrated test strategy for predicting eye irritation due to cosmetics.