To compare the effects of two different cryoprotectants on human desmoglein 1 (Dsg 1), and to provide experimental basis for the optimization of cryoprotectant. Methods Five donated thin spl it-thickness skin grafts were used, and the experiment was conducted within 4 hours after skin grafts harvest. The skin grafts were divided into 3 groups: group A (n=2) in which skin grafts were immersed in 0.5 mol/L trehalose/DMSO; group B (n=2) in which skin grafts were immersed in DMSO/propanediol; group C (n=1) in which fresh skin graft received no further treatment. Groups A and Bwere stored in — 196℃ l iquid nitrogen for 7 and 21 days, respectively, and then underwent experiment. mmunohistochemistry staining observation was performed on each group, RT-PCR method was used to detect the expression of Dsg 1 in skin. Results The immunohistochemistry staining showed that the protein in groups A and B was stained brown-yellow and distributed evenly 7 days after cryopreservation; the expression signal of epidermal basal cell was similar to that of group C; absorbance (A) value of groups A, B and C was 0.285 ± 0.006, 0.284 ± 0.004 and 0.287 ± 0.008, respectively, suggesting there was no significant difference between groups A and B and group C (P gt; 0.05). At 21 days after cryopreservation, the expression of positive cells in group B decreased; no obvious decrease was observed in group A, A value of groups A and B was 0.282 ± 0.004 and 0.275 ± 0.005, respectively, indicating there was a significant difference between group B and groups A and C (P lt; 0.05). RT-PCR detection showed that A value of groups A and B at 7 days after cryopreservation was 0.810 ± 0.012 and 0.803 ± 0.008, respectively; A value of groups A and B at 21 days after cryopreservation was 0.806 ± 0.008 and 0.782 ± 0.013, respectively; and the A value of group C was 0.814 ± 0.012, indicating there was significant difference between group B and groups A and C at 21 days after ryopreservation (P lt; 0.05), and no significant differences among groups were noted at other time points (P gt; 0.05).Conclusion Trehalose/DMSO is better than traditional cryoprotectant DMSO/propanediol in protecting Dsg 1 of human skin.
To compare the effect of trehalose with that of different traditional cryoprotectants on human skin and to detect the new protection mechanism of trehalose in hypothermia. Methods The skins to be cryopreserved were first treated with DMSO/Propyleneglycol (D/P group), trehalose/DMSO (T/D group), DMSO/ serumfree keratinocyte medium(D/K group), DMEM (DMEM group), respectively, so as to be compared with fresh skin (control grouop). Then the histological structure of skin of different groups was observed and analyzed by pathological technology (SP immunohistochemistry, DAB staining). Furthermore, the influence of trehalose on α-actinin at gene level with RT-PCR was investigated. The viabil ity of skin in 5 respective groups was evaluated by using succinate dehydrogenase (SDH). The experiments were carried out 14 days after cryopreservation. Results The results of immunohistochemistry showed that A values of control group, T/D group, D/P group, D/K group and DMEM group were 27.50 ± 7.92, 18.40 ± 5.81, 13.10 ± 5.11, 11.50 ± 4.54 and 5.30 ± 2.14, respectively. There was no significant difference between control group and T/D group (P gt; 0.05), but control group was significantly different from the other groups (P lt; 0.05). The results of PCR studies showed that A values of control group, T/D group, D/P group, D/K group and DMEM group were 0.816 ± 0.134, 0.723 ± 0.245, 0.564 ± 0.265, 0.245 ± 0.071 and 0.148 ± 0.048, respectively. Control group was not significantly different from T/D group and D/P group (P gt; 0.05), but was significantly different from D/K group and DMEM group (P lt; 0.05). The results of SDH showed that A valuse of control group, T/D group, D/P group, D/K group and DMEM group were 18.2 ± 3.7, 12.3 ± 3.6, 10.2 ± 2.4, 7.3 ± 2.1 and 5.7 ± 1.5, respectively. There was no significant difference between control group and T/D group (P gt; 0.05), while control group was significantly different from the other groups (P lt; 0.05). Conclusion The results suggest that cryopreservation protocol-trehalose/DMSO is better than the traditional cryoprotectant for ryopreservation on α-actinin of human skin.
The freeze-drying is a technology that preserves biological samples in a dry state, which is beneficial for storage, transportation, and cost saving. In this study, the bovine pericardium was treated with a freeze-drying protectant composed of polyethylene glycol (PEG) and trehalose (Tre), and then freeze-dried. The results demonstrated that the mechanical properties of the pericardium treated with PEG + 10% w/v Tre were superior to those of the pericardium fixed with glutaraldehyde (GA). The wet state water content of the rehydrated pericardium, determined using the Karl Fischer method, was (74.81 ± 1.44)%, which was comparable to that of the GA-fixed pericardium. The dry state water content was significantly reduced to (8.64 ± 1.52)%, indicating effective dehydration during the freeze-drying process. Differential scanning calorimetry (DSC) testing revealed that the thermal shrinkage temperature of the pericardium was (84.96 ± 0.49) ℃, higher than that of the GA-fixed pericardium (83.14 ± 0.11) ℃, indicating greater thermal stability. Fourier transform infrared spectroscopy (FTIR) results showed no damage to the protein structure during freeze-drying. Hematoxylin and eosin (HE) staining demonstrated that the freeze-drying process reduced pore formation, prevented ice crystal growth, and resulted in a tighter arrangement of tissue fibers. The frozen-dried bovine pericardium was subjected to tests for cell viability and hemolysis rate. The results revealed a cell proliferation rate of (77.87 ± 0.49)%, corresponding to a toxicity grade of 1. Additionally, the hemolysis rate was (0.17 ± 0.02)%, which is below the standard of 5%. These findings indicated that the frozen-dried bovine pericardium exhibited satisfactory performance in terms of cytotoxicity and hemolysis, thus meeting the relevant standards. In summary, the performance of the bovine pericardium treated with PEG + 10% w/v Tre and subjected to freeze-drying could meet the required standards.