Zhonghua Shao Shang Za Zhi. 2021 Nov 25;37:1-12. doi: 10.3760/cma.j.cn501120-20200920-00418. Online ahead of print.
ABSTRACT
Objective: To explore the effects of porcine acellular dermal matrix (ADM) combined with human epidermal stem cells (ESCs) on wound healing of full-thickness skin defect in nude mice. Methods: Experimental research methods were applied. The physical properties of ADM were analyzed by photograph of digital camera, hematoxylin eosin (HE) staining, scanning electron microscope, infrared spectrometer, particle size analyzer and nano-particle size potentiometer. The biological properties of ADM were analyzed by fluorescence inverted microscope, weighing method (ADM was divided into 5 min group, 10 min group, 20 min group, 30 min group, 60 min group, and 120 min group according to the random number table (the same grouping method as below) and in static state at normal temperature for the corresponding time to calculate the water absorption, with sample number of 3), cytotoxicity test and rating (mouse embryonic fibroblasts (Fbs) were divided into blank control group (culture medium only), 50.0 g/L ADM extract group, 37.5 g/L ADM extract group, 25.0 g/L ADM extract group, 12.5 g/L ADM extract group, and 6.5 g/L ADM extract group (the latter 5 groups were added with the corresponding final concentrations of AMD extract respectively). At post culture hour (PCH) 24, 48, and 72, the cell survival rate was detected by cell counting kit 8, with sample number of 5), and blood compatibility test (the erythrocytes of a 6-week-old male Sprague-Dayley rat were divided into normal saline group, ultra-pure water group, 5 mg/mL ADM extract group, 10 mg/mL ADM extract group, and 15 mg/mL ADM extract group and treated with the corresponding reagents of corresponding concentration. After reaction for 3 h, the absorbance value was detected by microplate reader, with sample number of 3). ESCs were isolated, cultured, and identified from the discarded prepuce of a 6-year-old boy who was treated in the Department of Urology of the First Affiliated Hospital of Army Medical University (the Third Military Medical University) in July 2020. ADM particles of composite ESC (hereinafter referred to as ESC/ADM) were constructed by mixed culture. After 3 days of culture, the composite effect of ESC/ADM was observed by HE staining and laser confocal microscope. Thirty-six 7-8-week-old male non-thymic nude mice were divided into phosphate buffer solution (PBS) alone group, ADM alone group, ESC alone group, and ESC/ADM group, with 9 mice in each group, and the wound model of full-thickness skin defect was established. Immediately after injury, the wounds were treated with the corresponding reagents at one time. On post injury day (PID) 7, 11, and 15, the wound healing was observed and the wound healing rate was counted (with sample number of 3). On PID 7, the epithelialization of wounds was observed by HE staining and the epithelialization length was measured (with following sample numbers of 4). On PID 11, the dermis area and collagen deposition of each group were observed by Masson staining and calculated, the number of cells expressing CD49f, a specific marker of ESC, was calculated with immunofluorescence staining, the mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in ESC after wound transplantation with detected by real-time fluorescence quantitative PCR. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, and least signigicant difference t test. Results: ADM was white particles and composed of reticular structure, with disordered structure, rough surface, and no cells inside. The absorption peak of ADM appears at the wave numbers of 1 659, 1 549 and 1 239 cm-1, respectively. The main particle size distribution of ADM in solution was 500 to 700 nm, with negative charge on the surface. The morphology of ADM in static state at 30 min and on 1 and 5 d were relatively stable. The water absorption of ADM reached its peak in static state at 30 min and remained relatively stable after that. The cytotoxicity of mouse Fbs in 6.5 g/L ADM extract group, 12.5 g/L ADM extract group, and 25.0 g/L ADM extract group was grade 1 at PCH 24, and the cytotoxicity of the other groups was 0 grade at each time point. After reaction for 3 h, the absorbance value of hemoglobin in ultrapure water group was significantly higher than the values in normal saline group and 15 mg/mL ADM extract group (t=8.14, 7.96, P<0.01). The results showed that hemolysis did not occur in 5 mg/mL ADM extract, 10 mg/mL ADM extract, nor 15 mg/mL ADM extract. The cells of the fourth passage showed pebble-like morphology, low expression of CD71 and high expression of CD49f, which were identified as ESCs. There was ESC attachment and growth on ADM particles. On PID 1, the wound sizes of nude mice were almost the same in PBS alone group, ADM alone group, ESC alone group, and ESC/ADM group. On PID 7 and 15, the wound contraction of nude mice in each group was observed, especially in ADM alone group, ESC alone group, and ESC/ADM group. On PID 7, the wound healing rates of nude mice in ESC alone group and ESC/ADM group were significantly higher than the rate in PBS alone group (P<0.05 or P<0.01). On PID 11, the wound healing rate of nude mice in ESC/ADM group was significantly higher than that in PBS alone group (P<0.01). On PID 15, the wound healing rates of nude mice in ADM alone group, ESC alone group, and ESC/ADM group were significantly higher than the rate in PBS alone group (P<0.05). On PID 7, the length of un-epithelialized wound of nude mice in ADM alone group, ESC alone group, and ESC/ADM group was (816±85), (635±66), (163±32) μm, respectively, which were significantly lower than (1 199±43) μm in PBS alone group (t=5.69, 10.19, 27.54, P<0.01). On PID 11, the dermal areas of wounds of nude mice in ADM alone group, ESC alone group, and ESC/ADM group were significantly larger than those in PBS alone group (t=27.14, 5.29, 15.90, P<0.01); the collagen production of nude mice in ADM alone group and ESC/ADM group was more obvious than that in PBS alone group, and that in ESC alone group and PBS alone group was similar. On PID 11, the expression of CD49f and mRNA of GAPDH gene were positive in the wounds of nude mice in ESC alone group and ESC/ADM group. Conclusions: ESC/ADM particles can promote the healing of full-thickness skin defects in nude mice, which may be related to the fact that particles can improve the survial rate of ESC after transplant and ADM can promote rearrangement and vascularization of dermis.
PMID:34839596 | DOI:10.3760/cma.j.cn501120-20200920-00418
