| Part1Experimental study of different quantity of autologous micro-skin graft for wound repair in a pig modelObjective:To investigate the survival and epithelization of the autologous micro-skin graft to the wound, observe the wound healing speed and quality after different quantity of micro-skin grafting, find out the best quantity of micro-skin graft for wound healing.Materials and methods:We made an animal model of full-thickness skin defect wound (2×2cm2) and used autologous micro-skin graft to repair the defect with area expansion ratio of10%,20%,30%,40%,50%,60%,70%,80%,90%,100%, totally10groups with6defects in each group. The rate of wound healing was measured, the macroscopy and histological samples by HE staining were examined on the2,3and4weeks after grafting; The rate of wound contracture was measured and HE staining were also examined on the4,8and12weeks after grafting.Results:1. The speed of wound healing was low and the rate of wound healing was about45%in10%-30%groups, there was no significant difference between the three groups (p>0.05); while in40%-100%groups the speed of wound healing was faster and the rate of wound healing was almost75%three weeks after grafting, there was also no significant difference between the seven groups (p>0.05); The wound healing rate of the latter seven groups was higher than it of the former three groups (P<0.05).2. The wounds were totally reepithelized4weeks after grafting, and the area of wound was stable after12weeks. By the time of12weeks, the rate of wound contracture was about70%in10%-30%groups, there was no significant difference between the three groups (p>0.05); while in40%-100%groups the wound contracture rate was about55%, there was also no significant difference between the seven groups (p>0.05); The wound contracture rate of the latter seven groups was lower than it of the former three groups (P<0.05).3. On the12th week post-operation, in groups of40%-100%, HE staining showed multiple layers of the epithelial cell with good polarity, dermal papilla was abundant and mature collagen fibers were ordered arrangement with fibrocyte diffused distribution in them; While in10%-30%groups, the layers of the epithelial cell and dermal papilla were less, a lot of fibrocytes could be seen in the subcutaneous layer, and collagen fibers were not mature enough.Conclusions:The best quantity of micro-skin graft for wound repair is40%of area expansion ratio. It means that such quantity of micro-skin could repair the wound faster with lower contracture rate. Part2long-term effects of autologous micro-skin and micro-mucosa combined with acellular dermal matrix graft for wound repair in a pig modelObjective:To investigate the long term effects of autologous micro-skin and micro-mucosa combined with acellular dermal matrix graft for wound repair, evaluate that if this method could achieve lower contracture rate.Materials and methods:We made an animal model of full-thickness skin defect wound (5×6cm2). The wounds were randomly divided into4groups depending on the different way of wound repair with6defects in each group. Micro-skin and micro-mucosa group (group A), micro-skin and micro-mucosa combined with acellular dermal matrix group (group B), acellular dermal matrix group (group C), and thick split thickness free skin graft group (group D). The area expansion ratio is40%in group A and B. The rate of wound healing and contracture was measured, the macroscopy and-histological samples by HE staining were examined on the2,4,8and12weeks after grafting; SEM examination was taken on the12week, as well as the biomechanical characteristics test of the four groups.Results:1. The wound was gradually healing over time postoperatively. The epithelization was observed in group A, B and C. Acellular dermal matrix was partially fallen off in group C, and the wound contracture was more significant than other groups. The wound in group D was completely healed. The wound healing rate of group D was much higher than those of other three groups (P<0.05), and the wound healing rate of group A and B was much higher than those of group C (P<0.05), there was no significant difference between group A and B (p>0.05).2. The wound contracture rate was measured4,8, and12weeks after the grafting. On the4week, there was no significant difference between group A, B and C (p>0.05), the wound contracture rate of D was much higher than those of other three groups (P<0.05). While on the8and12week, there was significant difference between all groups, D<B<A<C (P<0.05).3. Epithelial cell layers and polarity of all the groups increased gradually over time postoperatively, inflammatory cells gradually reduced, collagen fibers gradually increased and well ordered arrangement. The time of wound healing and inflammatory reaction in group C was longer, and most part of ADM was fallen off. When came to the12th week, the skin of group B and D were similar with normal skin, while the skin of group A and C were similar with scar tissue.4. The biomechanical characteristics of group D was much better than those of group A, B and C, and it was similar with normal skin. Although the elasticity of group B was improved, the tensile strength was reduced.Conclusions:Autologous micro-skin and micro-mucosa combined with acellular dermal matrix graft for wound repair is easy to perform, and such method can improve the long term quality of wound healing, but still not as good as thick split thickness free skin graft. Part3Experimental study of micro-skin combined adipose tissue derived stem cells for wound healing in nude mouseObjective:To evaluate the effect of adipose tissue derived stem cells (ADSCs) on wound healing when combined the micro-skin graft in a nude mouse model, to track and investigate the in vivo survival and differentiation of ADSCs on wound healing.Materials and methods:10BALB/c nude mice aged6weeks were divided into two groups. Two full-thickness circular excisional wound (diameter lcm) were created on the dorsum of each mouse. The area expansion ratio was40%, and the micro-skin was grafted to every wound. After that, ADSCs were injected intra-dermally around the wounds of treatment group. PBS was applied to control group. The wound area was measured at day7,9,12. And full thickness skin samples were taken from the wound sites for evaluation of volume density of vessels and-distribution of collagen fibers by HE stain. Immunofluorescence staining of sections was performed to investigate the in vivo survival and differentiation of ADSCs. Real-time PCR analysis was performed to compare the expression of these cytokines such as IL-1, IL-6, IL-10, VEGF, TGF-β,HGF, FGF2and Collagen I between the treatment group and control group.Results:1. The wound healing rate of treatment group was much higher than those of control group (P<0.05) at day7,9post-operation. The wound contracture rate of treatment group was much lower than those of control group (P<0.05) at day9,12post-operation.2. Histological evaluation of the wounds at7,12days after surgery showed enhanced re-epithelialization in the ADSCs group compared with control group. In addition, granulation tissue in the ADSCs injected group appeared to be thicker and larger. Poorly formed granulation tissue was significantly delayed in control group. Compared with the control group, blood vessel density was evidently increased in the ASDCs-treated group at postoperative day7,12(p<0.05).3. Immunofluorescence staining revealed that some GFP+ADSCs could differentiate into vascular endothelial cells, and the GFP and CD31double positive cells could been find at day7,12. No sign was observed that ADSCs differentiated into epithelial cells. But the rate of survival and differentiation of ADSCs was low.4. Real-time PCR:The expression of IL-1and IL-6in treatment group was significant lower than that in control groups (p<0.05), and the expression of IL-10, VEGF, FGF2, TGF-β, Collagen I in treatment group was significant higher than that in control group (p<0.05).Conclusions:When perform autologous micro-skin graft for wound repair, locally administered adipose tissue derived stem cells can accelerate wound healing through differentiation and vasculogenesis, which may be due to their regulation effect on inflammation and paracrine action during wound repair. |
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