3.1. Biocompatibility of GO and UCMSC
To observe the cytotoxicity of GO to UCMSC, the specific method is: take P4 generation UCMSC cells and GO solid particles, and conduct group culture observation. The control group is a pure UCMSC culture group, and the UCMSC concentration is 5.0×104 MSC/ml; the experimental group is In the UCMSC + GO co-culture group, the cell concentration was also 5.0×104 MSC/ml; the GO pellet mass was 5 mg, after relatively mixing, inoculated into a 24-well plate, inoculated 500ul per well. Incubate overnight in a 37°C, 5% CO2 incubator, continuously observe, and take photos for recording. Among them, Day-1, Day-3, Day-5 and Day-7 are for CCK8 detection. Day-1, Day-3 and Day-7 for life and death staining.
The cck8 test results in Fig. 1 and Fig. 2 showed that the cells had no proliferation within 7 days with the 5mg/ml drug. As the GO concentration is too high, the cell survival space is compressed, and the overall culture density is too high to cause cell death. High concentrations of GO can produce toxicity to cells and mediate cell apoptosis.
3. 2. In vitro culture of UCMSC and GO at different concentrations
UCMSCs with concentrations of 8.0×104/ml, 4.0×104/ml, and 2.0×104/ml were cultured in vitro with GO nanoparticles at a concentration of 30 µg/ml, and P4 generation umbilical cord UCMSCs were mixed with GO Inoculate into a 24-well plate, inoculate 500 ul per well, inoculate 2 wells in parallel for each concentration. Incubate overnight in a 37°C, 5% CO2 incubator, continuously observe and take photos for recording. Among them, Day-1, Day-3, Day-5 and Day-7 are for CCK8 detection. Day-1, Day-3 and Day-7 for life and death staining.
The cck8 test results from Fig. 3 to Fig. 6 showed that the proliferation degree of 8.0×104/m, 4.0×104/ml, 2.0×104/ml plus 30 µg/ml GO was lower than that of the control group. With the increase of stem cell concentration, 30 µg/ml GO was the effect of MSC proliferation is reduced. Combining the images under the magnification lens and the stained images of life and death, the low concentration of UCMSC cells cannot well reflect the migration and proliferation effects in the low concentration of GO environment. It is considered that the effect of GO particles on the cells is limited to a certain distance If the cell density is too low, the distance between GO and the cells is too far, so GO and UCMSC can be cultured separately, which cannot form a co-cultivation environment. However, it does not mean that the higher the density of UCMSC, the better, and the higher the seeding density will cause cell death. Therefore, in general, GO particles need a low-concentration environment to ensure their safety, while UCMSC must at least ensure a certain concentration to form a co-cultivation system.
3. 3. In vitro co-culture of UCMSC and GO granular lubricant
To observe the in vitro culture of UCMSC and GO granular lubricant. The inoculation density of UCMSC is 2.0×104/ml. The mixed concentration of GO granular lubricant is 15 µg/ml GO + 0.5% HA and 30 µg/mlGO + 0.25% HA; Inoculate 0.5ml into each well, inoculate 2 wells in parallel at each concentration; incubate overnight in a 37℃, 5% CO2 incubator, observe continuously and take pictures for recording.
The cck8 test results in Fig. 7 and Fig. 8 showed that within 72 hours, the proliferation of 15 µg/ml GO + 0.5% HA and 30 µg/ml GO + 0.25% HA was lower than that of the control group, and with the increase of GO concentration, the degree of MSC proliferation decreased. On the 5th day, the stem cell proliferation of the 15µg/ml GO + 0.5% HA group was higher than that of the control group. The CCK8 value of the 15 µg/ml GO + 0.5% HA group was 1.160 and that of the control group was 1.121; the proliferation degree of 30 µg/ml GO + 0.25% HA was higher than that of the control Group is low, as the GO concentration decreases, the degree of MSC proliferation increases. On the 7th day, the stem cell proliferation of the 15 µg/ml GO + 0.5% HA group was higher than that of the control group. The cck8 value of the 15 µg/ml GO + 0.5% HA group was 1.169 and that of the control group was 1.111; the proliferation degree of 30 µg/mlGO + 0.25% HA was higher than that of the control group, as the GO concentration decreases, the degree of MSC proliferation increases. The proliferation of stem cells in the 15 µg/ml GO + 0.5% HA group in group C was higher than that in the control group. It may be that the stem cells in the control group have proliferated to the maximum value and have been lost. Therefore, the cck8 value decreased from 1.121 to 1.111 at 5th day. The adsorption effect of GO on stem cells, the growth surface area in the well plate is larger than that of the control group, so the degree of stem cell proliferation is higher than that of the control group. The ratio of the mixed lubricant of GO and HA is also related to the cultivation of UCMSC. First of all, because sodium hyaluronate injection is a kind of macromolecular structure, it has high viscosity, theoretically, it will have a certain impact on the migration of MSC and hinder its aggregation to GO particles, so the concentration of HA should not be too high. However, HA will also hinder the aggregation of GO particles, which is conducive to the uniform distribution of GO particles. The results also show this. Small particles of GO will be safer and less toxic than large particles of GO. Therefore, the mixing of HA is conducive to the long-term uniform distribution of UCMSC + GO, and from the results, the low concentration of HA is more suitable for co-cultivation of the two.