Preparation Of Laser Micropore Porcine Acellular Dermal Matrix And Its Application

Objective: To investigate the feasibility of punching porcine acellular dermal matrix (PADM) with frequency- tripled Nd:YAG laser, Ytterbium fiber laser and CO₂ laser, and affirm the optimal laser and its work parameter. To prepare PADM with laser-punched micropores of different interpore distance, aiming at seeking an optimal inter-pore distance of PADM to be covered with split-thickness autograft to improve the quality of wound repair. To investigate the feasibility of preparing living dermal matrix by inoculating fibroblasts on the surface of laser micropore PADM.Methods: 1. PADM was prepared by the trypsin/Triton X-100 method and divided randomly into two groups, freeze-dry group and wet group. Micropores were produced on the PADM in two groups with frequency-tripled Nd:YAG laser, fiber laser and CO₂ laser, by duplication method and circuitous method, respectively. The formation of micropores was observed grossly. The shrinkage rate and thermal damage rate of PADM were calculated. With output power and frequency constant, scanning speed of frequency-tripled Nd:YAG laser varied from 10 mm/s to 600 mm/s. The diameter of ingate and outgate of micropores was measured, and thermal damage rate of PADM was calculated. 2. Micropores were produced on PADM with CO₂ laser. The distance between micropores varied from 0.8 mm, 1.0 mm, 1.2 mm and 1.5 mm. Full thickness defect wounds were created on the back of 144 SD rats. The rats were divided into 6 groups randomly, with 24 rats in each group. Micropore groups I -IV: the wounds were grafted with PADM with micropores in four different intervals respectively, and covered with split-thickness autograft. Mesh group: PADM was meshed and covered with split-thickness autograft. Control group: simple split-thickness autograftingwithout PADM. The gross observation of wound healing and histological observation were performed at 2, 4, 6 weeks after surgery. The wound healing rate and contraction rate were calculated. Laser micropore PADM, meshed PADM and split-thickness autograft were cografted to cover the secondary wound after scar excision in 9 plastic patients. 3. Human fibroblasts were inoculated on the surface of laser micropore PADM in two groups, freeze-dry group and wet group. The concentration of TGF-β1 and bFGF in the supernatant fluid of the culture medium were measured by ELISA method on 2, 4, 6, 8, 10, 12 days after inoculation.Results: 1. The micropores of PADM processed by duplication method were in irregular sharp and differed in size, while the micropores of PADM processed by circuitous method were in regular sharp and uniform in size. PADM in freeze-dry group did not shrink when processed by laser, while that in wet group shrank in different degrees. The shrinkage rate of PADM processed with fiber laser by duplication method was significantly higher than that with CO₂ laser (P < 0.05). The shrinkage rate of PADM processed with frequency-tripled Nd:YAG laser by circuitous method was significantly higher than those with fiber laser and CO₂ laser (P < 0.05). The thermal damage rate of PADM in wet group, which was processed with fiber laser by duplication method, was significantly higher that with CO₂ laser (P < 0.05), however, there was no statistical difference in freeze-dry group when processed with these two lasers by duplication method (P > 0.05). By circuitous method, the thermal damage rate of PADM in wet group processed with frequency-tripled Nd:YAG laser was the highest, fiber laser took the second place, and CO₂ laser the lowest. On the contrary, the thermal damage rate of PADM in freeze-dry group processed with frequency-tripled Nd:YAG laser was the lower, and CO₂ laser the highest. When scanning speed of frequency-tripled Nd:YAG laser was no more than 80 mm/s, the thermal damage rate was lower, and the aperture size of micropores varied unobvious. The thermal damage rate and aperture size increased as scanning speed varied from 100 mm/s to 400 mm/s. However, the aperture size decreased as scanning speed up to 500mm/s. Laser could not penetrated PADM when scanning speed reached 600 mm/s. 2. Two and four weeks after surgery, the wound healing rate in micropore groups I and II was lower than that in control group (P < 0.05). There was no statistically significant difference between the two groups and mesh group (P > 0.05). Six weeks after surgery, there was no statistically significant difference in wound healing rate between micropore groups I and II and control group (P > 0.05), but it was significantly higher than that of mesh group (P < 0.05). The wound contraction rate in micropore groups I and II was remarkably lower than that in control group 4 and 6 weeks after surgery (P < 0.05), and it was significantly lower than that in mesh group 6 weeks after surgery (P < 0.05). Histological examination revealed good epithelization, regularly arranged collagenous fibers, and integral structure of basement membrane in composite skin grafting groups. The compound grafts consisting of laser micropore PADM and split-thickness autograft took well in 8 patients. 3. The concentration of TGF-β1 and bFGF in freeze-dry group was higher than that in wet group on each time point (P < 0.05).Conclusions: 1. It is the optimal choice to process freeze-dried PADM with frequency-tripled Nd:YAG laser by circuitous method. In this way, the better efficiency, milder thermal damage and micropores in regular sharp and size can be attained, and it is convenient to adjust the aperture size of micropores. The optimal scan speed is 80 mm/s. 2. Laser micropore PADM (0.8 mm or 1.0 mm in distance) grafting in combination with split-thickness autografting can improve the quality of wound healing. The PADM with laser micropores in 1.0 mm distance is the better choice. 3. Living dermal matrix can be obtained by inoculating fibroblasts on the surface of laser micropore PADM, and the freeze-dried PADM is more suitable for adhesion and proliferation of fibroblasts.