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.
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