Preparation and roles of sliver-loaded viscous hydrogel in healing of full-thickness skin defect wounds with bacterial colonization in mice
10.3760/cma.j.cn501120-20210906-00304
- VernacularTitle:含银黏性水凝胶的制备及其在小鼠细菌定植全层皮肤缺损创面愈合中的作用
- Author:
Yunqing DONG
1
;
Linlin LI
;
Xuanru ZHU
;
Longbao FENG
;
Keyao JIA
;
Rui GUO
;
Biao CHENG
Author Information
1. 南方医科大学第一临床医学院，广州 510515
- Keywords:
Wound healing;
Hydrogel;
Hyaluronic acid;
Nano silver;
Re-epithelialization;
Collagen deposition;
Angiogenesis
- From:
Chinese Journal of Burns
2021;37(11):1036-1047
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To prepare the modified hyaluronic acid viscous hydrogel loaded with sliver particles and to explore the roles and mechanism of the hydrogel in healing of full-thickness skin defect wounds with bacterial colonization in mice.Methods:The experimental research method was adopted. Dopamine modified hyaluronic acid (HA-DA) and phenylboric acid modified hyaluronic acid (HA-PBA) were prepared, and their characteristic peaks were detected by Fourier-transform infrared spectroscopy. Different mass of acrylamides was added to HA-DA and HA-PBA to prepare the viscous hydrogel with mass fraction of acrylamide in 10%, 15%, and 20%. The gelation of the viscous hydrogel with mass fraction of acrylamide in 20% was observed in the state of tilt and inversion at 37 ℃, and the storage modulus and loss modulus of the above 3 kinds of viscous hydrogels were detected by rotational rheometer. The sliver-loaded viscous hydrogel was prepared by adding nano silver ions to the viscous hydrogel with mass fraction of acrylamide in 20%. The concentration of silver ions released by sliver-loaded viscous hydrogel was measured by inductively coupled plasma mass spectrometer, and the cumulative release rate of silver ion was calculated ( n=5). The mouse fibroblasts L929 were divided into phosphate buffered saline (PBS) group, viscous hydrogel group, and sliver-loaded viscous hydrogel group, which were dealt correspondingly, and the cell survival was detected by cell counting kit 8 method after 1, 2, and 3 d of culture ( n=5). Twenty-four male C57BL/6 mice aged 6-8 weeks were selected, and forty-eight full-thickness skin defect wounds were inflicted and inoculated with the mixture of Escherichia coli and Staphylococcus aureus in the back of the mice, with two wounds in each mouse. The wounds were divided into normal saline group, viscous hydrogel group, and sliver-loaded viscous hydrogel group, which were dealt correspondingly, with 16 wounds in each group, and two wounds in each mouse were divided into different groups. On post injury day (PID) 3, 7, 10, and 14, the wound healing was observed and the wound healing rate was calculated. On PID 3, the colony forming units of Escherichia coli and Staphylococcus aureus in wounds were observed and counted. On PID 14, the epithelized epidermal thickness and the optical density of collagen fiber in wounds were observed and analyzed after hematoxylin eosin staining and Masson staining, respectively. On PID 3, 7, and 10, the expressions of tumor necrosis factor α (TNF-α), transforming growth factor β 1 (TGF-β 1), and vascular endothelial growth factor (VEGF) were detected by immunohistochemistry. The number of wounds in each index detecting at each time point was four. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and Bonferroni correction. Results:The characteristic peaks of HA-PBA were detected at the wave numbers of 1 369 and 1 425 cm -1, indicating that phenylboric acid had been successfully grafted on hyaluronic acid, and the characteristic peaks of HA-DA were detected at the wave numbers of 1 516 and 1 431 cm -1, indicating that dopamine had been successfully grafted on hyaluronic acid. The viscous hydrogel with mass fraction of acrylamide in 20% maintained the stable and no-flow condition of gelation in the state of tilt and inversion at 37 ℃. The storage modulus and loss modulus of the viscous hydrogel increased with the increase of acrylamide content, the storage modulus and loss modulus of the 3 kinds of viscous hydrogels had no obvious changes with the increase of the oscillation frequency or time, and the storage modulus of the 3 kinds of acrylamide hydrogels were greater than the loss modulus. The release of silver ion in the sliver-loaded viscous hydrogel lasted for 7 days, and the cumulative release rate of silver ion was up to 65%. After 1, 2, and 3 d of culture, the cell survival rates in sliver-loaded viscous hydrogel group were significantly lower than those in PBS group and viscous hydrogel group ( P<0.05 or P<0.01), while after 1 d of culture, the cell survival rate in viscous hydrogel group was significantly lower than that in PBS group ( P<0.01). With extension of time after injury, the wounds of mice in the 3 groups shrank gradually. On PID 3, 7, 10, and 14, the wound healing rates in sliver-loaded viscous hydrogel group were (53.0±3.6)%, (75.3±6.9)%, (93.3±1.2)%, and (96.7±0.8)%, which were significantly higher than (21.8±6.4)%, (53.9±8.2)%, (72.0±7.8)%, and (92.5±0.4)% in normal saline group ( P<0.01). On PID 3 and 14, the wound healing rates in sliver-loaded viscous hydrogel group were significantly higher than (43.5±2.4)% and (94.1±1.5)% in viscous hydrogel group ( P<0.05). On PID 3 and 10, the wound healing rates in viscous hydrogel group were significantly higher than those in normal saline group ( P<0.01). On PID 3, the colony forming units of two bacteria in wound of sliver-loaded viscous hydrogel group were significantly less than those in normal saline group and viscous hydrogel group ( P<0.01), while the colony forming units of two bacteria in wound of viscous hydrogel group were significantly less than those in normal saline group ( P<0.05). On PID 14, the wounds were basically epithelialized and the epidermis was thicker, with collagen protein content being increased significantly and more orderly arranged collagen in sliver-loaded viscous hydrogel group compared with those in the other 2 groups. On PID 14, the epidermal thickness in wounds of sliver-loaded viscous hydrogel group was significantly increased compared with that in the other two groups ( P<0.05), and the optical density of collagen fiber was significantly increased compared with those in normal saline group ( P<0.05). On PID 3, the expressions of TGF-β 1 and VEGF in wounds of sliver-loaded viscous hydrogel group were significantly higher than those in normal saline group ( P<0.05 or P<0.01), while the expression of VEGF in wounds of viscous hydrogel group was significantly higher than that in normal saline group ( P<0.01). On PID 7, the expression of TGF-β 1 in wounds of sliver-loaded viscous hydrogel group was significantly higher than that in the other 2 groups ( P<0.01), and the expression of VEGF was significantly higher than that in normal saline group ( P<0.01). On PID 10, the expression of TNF-α in wounds of sliver-loaded viscous hydrogel group was significantly lower than that in normal saline group ( P<0.05), the expressions of TGF-β 1 and VEGF in wounds of sliver-loaded viscous hydrogel group were significantly higher than those in normal saline group ( P<0.05 or P<0.01), and the expression of VEGF in wounds of sliver-loaded viscous hydrogel group was significantly higher than that in viscous hydrogel group ( P<0.05). Conclusions:The sliver-loaded viscous hydrogel prepared in this study has good stability and elasticity, which can continuously release silver ions and help to accelerate the healing of full-thickness defect wounds with bacterial colonization in mice. Besides, the sliver-loaded viscous hydrogel has low biological toxicity and can promote re-epithelialization, collagen deposition as well as angiogenesis of wounds, which may be related to the infiltration and regression of inflammatory cells.
