Effects of porcine acellular dermal matrix combined with human epidermal stem cells on wound healing of full-thickness skin defect in nude mice / 中华烧伤杂志

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.

Clinical application and long-term follow-up study of acellular dermal matrix combined with auto-skin grafting / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the clinical effect of acellular dermal matrix (ADM) combined with auto-skin grafting on deep burn wound ,and the result of long-term follow-up and histological examination.</p><p><b>METHODS</b>One hundred and fifty-two patients with deep burn hospitalized from February 2000 to July 2003 were repaired with porcine ADM and auto split-thickness graft. Wound healing rate was assessed 1 week after operation. Degree of cicatricial hyperplasia was examined 1, 3, 6, 12 months after operation. Wound samples from 5 patients were harvested for histological examination 72 months after operation, for which transmission electron microscopy were employed in 2 cases.</p><p><b>RESULTS</b>Grafts completely survived was seen in 116 patients (accounting for 76.3% of cases), survival rate over 95% were observed in 23.7% of cases. One hundred and twenty-seven patients were followed up 1 month after operation, in whom mild local contraction, cord like scar was seen along its junction with skin, its texture was soft ,and there was no pruritus or blister formation. One hundred and one patients were followed up 3 months after operation, and the graft showed mild contraction less marked when compared with that of the site where auto split-thickness skin grafting was used. Articular function was good. Eighty-two patients were followed up 6 months after operation,color and texture of grafts were similar to normal skin with no obvious cicatricial hyperplasia. Fifty-eight patients were followed up 12 months after operation, the texture of grafts was similar to normal skin without obvious reject reaction. Sixteen patients were followed up over 72 months after operation, the grafts appeared dry compared with normal skin. Histological examination showed: tissue structure of grafts was similar to normal skin, intact small sweat gland and sweat gland cells were not found in dermal layer.</p><p><b>CONCLUSION</b>Heterologous ADM combined with auto split-thickness graft can survive in human body without obvious immune rejection reaction for a long time. No intact small sweat gland or sweat gland cells in dermis is a problem worth of study in regeneration of skin function.</p>

Treatment of deep partial thickness burns by a single dressing of porcine acellular dermal matrix / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the effect of one dressing of porcine acellular dermal matrix on deep partial thickness burns.</p><p><b>METHODS</b>From January 1997 to January 2004, sixty-seven cases of deep partial thickness total burned surface area (TBSA) from 50% to 90% burn wound were treated by a single dressing of porcine acellular dermal matrix (the porcine acellular dermal matrix group). Ten cases of deep partial thickness burned patients with the same TBSA treated by exposure method served as the exposure method group. The healing time of the wound was observed. The patients were followed up for 3 months to 2 years, and the scar proliferation was observed.</p><p><b>RESULTS</b>The deep partial-thickness wound would be healed without dressing change in the porcine acellular dermal matrix group, and the average healing time was (12.2 +/- 2.6) days. The average healing time of the exposure method group was (27.4 +/- 3.5) days. Follow up of the patients within 3 months to 2 years showed that scar proliferation in the porcine acellular dermal matrix group was much less than that in the exposure method group, even no scar proliferation was observed in some patients.</p><p><b>CONCLUSION</b>Without tangential excision, autografting and dressing change, a single dressing of porcine acellular dermal matrix on deep partial thickness burn wound could shorten the healing time and inhibit scar proliferation.</p>

The volume change of micronized acellular dermal matrix after injection / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore a new injectable material.</p><p><b>METHODS</b>Micronized swine acellular dermal matrix was injected into the ear of rabbits. The augmentation was observed and measured with pathological and transmission electron microscopic technique.</p><p><b>RESULTS</b>There were fibroblasts and capillaries in the injected material. The absorption rate of injected acellular dermal matrix was 26.93% in the experimental group at 48 weeks after injection.</p><p><b>CONCLUSION</b>The acellular dermal matrix is an ideal injectable material for lower absorption and easy usage.</p>