[Effects of porcine acellular dermal matrix combined with human epidermal stem cells on wound healing of full-thickness skin defect in nude mice]

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: The morphology of porcine ADM was analyzed by photograph of digital camera, the cell residues in porcine ADM were observed by hematoxylin-eosin (HE) staining, the surface structure of porcine ADM was observed by scanning electron microscope, the secondary structure of porcine ADM was analyzed by infrared spectrometer, the porcine ADM particle size was analyzed by dynamic light scattering particle size analyzer, and the porcine ADM potential was analyzed by nano-particle size potentiometer. The morphology of porcine ADM was observed by inverted fluorescence microscope when it was placed in culture medium for 30 min, 1 d, and 5 d (n=2). The porcine 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 below) in static state at normal temperature for the corresponding time to calculate the water absorption by weighing method (n=3). Swiss white mouse embryonic fibroblasts (Fbs) were divided into blank control group (culture medium only), and 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 which were added with the corresponding final concentrations of ADM extract respectively. At post culture hour (PCH) 24, 48, and 72, the cell survival rate was detected by cell counting kit 8 and the cytotoxicity was graded (n=5). The erythrocytes of a 6-week-old male Sprague-Dawley male rat were divided into normal saline group, ultra-pure water group, and 5 mg/mL ADM extract group, 10 mg/mL ADM extract group, and 15 mg/mL ADM extract group which were treated with the corresponding final concentrations of porcine ADM extract respectively. After reaction for 3 h, the absorbance value of hemoglobin was detected by microplate reader to represent the blood compatibility of porcine ADM (n=3). ESCs were isolated and cultured from the discarded prepuce of a 6-year-old healthy 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, and then identified by flow cytometry. The porcine 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 scanning 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) 1, 7, 11, and 15, the wound healing was observed and the wound healing rate was counted (n=3). On PID 7, the epithelialization of wounds was observed by HE staining and the length of un-epithelialized wound was measured (with this and the following sample numbers of 4). On PID 11, the dermal area and collagen deposition of wounds were observed by Masson staining and the dermal area of wound section was 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) in ESC after wound transplantation was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with independent sample t test, one-way analysis of variance, analysis of variance for repeated measurement, and least significant difference t test. Results: The porcine ADM was white particles and composed of reticular structure, with no cells inside, disordered structure, and rough surface. The absorption peak of porcine ADM appeared at the wave numbers of 1 659, 1 549, and 1 239 cm^-1, respectively. The main particle size distribution of porcine ADM in solution was 500 to 700 nm, with negative charge on the surface. The morphology of porcine ADM in static state at 30 min and on 1 and 5 d was relatively stable. The water absorption of porcine ADM remained relatively high level in static state from 30 min to 120 min. The cytotoxicity of mouse embryonic 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 of erythrocytes in ultra-pure water group was significantly higher than the values in normal saline group and 15 mg/mL ADM extract group (with t values of 8.14 and 7.96, respectively, P<0.01). After 3 days of culture, the cells of the fourth passage showed pebble-like morphology, with low expression of CD71 and high expression of CD49f, which were identified as ESCs. There was ESC attachment and growth on porcine 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, 11, 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 (with t values of 2.83 and 4.72 respectively, 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 (t=4.86, 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 (with t values of 2.71, 2.90, and 3.23 respectively, 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), and (163±32) μm, respectively, which were significantly shorter than (1 199±43) μm in PBS alone group (with t values of 5.69, 10.19, and 27.54 respectively, P<0.01). On PID 11, the dermal areas of wound section of nude mice in ADM alone group, ESC alone group, and ESC/ADM group were significantly larger than the area in PBS alone group (with t values of 27.14, 5.29, and 15.90 respectively, 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 the collagen production of nude mice in ESC alone group and PBS alone group was similar. On PID 11, in the wounds of nude mice in ESC alone group and ESC/ADM group, the cells with positive expression of CD49f were respectively 135±7 and 185±15, and the mRNA expressions of GAPDH were positive; while there were no expressions of CD49f nor mRNA of GAPDH in the wounds of nude mice in PBS alone group and ADM alone group. Conclusions: ESC/ADM particles can promote the wound healing of full-thickness skin defects in nude mice, which may be related to the improved survival rate of ESCs after transplantation and the promotion of dermal structure rearrangement and angiogenesis by ADM.

图 1

猪脱细胞真皮基质（ADM）物理特性分析结果。1A.数码相机下猪ADM呈白色；1B.猪ADM中无细胞成分存在苏木精-伊红×200, 图中标尺为200 μm；1C.猪ADM表面呈细丝状扫描电子显微镜×1 000, 图中标尺为2 μm；1D.红外光谱仪分析显示猪ADM在1 659、1 549、1 239 cm^-1波数处出现吸收峰；1E.动态光散射粒度分析仪分析显示, 猪ADM粒径主要为500~700 nm；1F.纳米粒度电位仪分析显示, 猪ADM表面带负电荷

图 2

猪脱细胞真皮基质（ADM）在角质形成细胞无血清培养基中放置各时间点形态倒置荧光显微镜×40, 图中标尺为500 μm。2A.放置30 min, 猪ADM形态不规则, 呈网状；2B、2C.分别为放置1、5 d, 猪ADM大小和形态与图 2A相近

图 3

复合人表皮干细胞（ESC）的猪脱细胞真皮基质（ADM）颗粒构建结果。3A.第4代细胞呈多角形、细胞核大, 鹅卵石样排列倒置荧光显微镜×200, 图中标尺为200 μm；3B.表达CD71^-CD49f⁺的细胞比例约97%；3C.培养3 d, ADM中有ESC生长苏木精-伊红×200, 图中标尺为200 μm；3D.培养3 d, ADM上有ESC生长激光扫描共聚焦显微镜×200, 图中标尺为200 μm

图 4

4组裸鼠伤后各时间点全层皮肤缺损创面愈合情况。4A、4B、4C、4D.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组伤后1 d创面情况, 大小均与参照卡片相近；4E、4F、4G、4H.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组伤后7 d创面情况, 图 4F、4G、4H创面较图 4E明显缩小；4I、4J、4K、4L.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组伤后15 d创面情况, 图 4J、4K、4L创面较图 4I明显缩小

图 5

4组裸鼠伤后7 d全层皮肤缺损创面上皮化情况和伤后11 d创面胶原沉积和切面真皮化情况。5A、5B、5C、5D.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组创面上皮化情况, 其中图 5B、5C、5D上皮化较图 5A明显更快, 苏木精-伊红×200, 图中标尺为200 μm。5E、5F、5G、5H.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组伤后11 d创面胶原沉积和切面真皮化情况, 图 5F和5H创面胶原沉积较图 5E多, 图 5F、5G、5H创面切面真皮面积较图 5E大, Masson×200, 图中标尺为200 μm

图 6

免疫荧光法检测4组裸鼠伤后11 d全层皮肤缺损创面处ESC特异性标志物CD49f表达情况。6A、6B、6C、6D.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组, 红框为各组创面上皮化位置花青素3-4', 6-二脒基-2-苯基吲哚×40, 图中标尺为200 μm；6E、6F、6G、6H.分别为单纯PBS组、单纯ADM组、单纯ESC组、ESC/ADM组, 图 6E、6F中无CD49f表达, 图 6G、6H中CD49f表达阳性花青素3-4', 6-二脒基-2-苯基吲哚×200, 图中标尺为50 μm