Effects Of Transplantation Of PADM Containing Hyaluronic Acid With Autologous Split-thickness Skin On Wound Healing And Its Mechanisms In Rabbit

Objective The purpose of this study was to investigate the effect of composite skin grafts with hyaluronic acid on wound healing quality via detecting the grafts contracture rate and the biomechanics performance, and to explore its mechanism by microvessel density,typeⅠandⅢcollagen expression,changes of HA and its receptor.Methods 1 Full-thickness skin defects were performed on back of 20 Japanese white rabbits as a wound model, each wound was 5cm×3cm size. These wounds were randomly divided into 5 groups according to the different methods of transplantation:HA1+PADM+thin skin autograft group (HA1 group), HA2+PADM +thin skin autograft group (HA2 group), PADM+thin skin autograft group (PADM group), thin skin autograft group (TS group), normal skin group (NS group). The gross was observed and recorded to determine contracture rates of the grafts at 28 days and 56 days postoperatively. Transplanted skin was haverested for pathological examination, observing collagen fiber structure and fusion with surrounding tissue. The ZWICK material testing machine was used to test the relaxation, creep and stress-strain properties of the implanted skin. Then the statistical analysis was performed.2 Full-thickness skin defects were performed on back of 50 Japanese white rabbits as a wound model, each wound was 3cm×3cm size. These wounds were randomly divided into 5 groups according to the different methods of transplantation: HA1+PADM+thin skin autograft group (SHA1 group), HA2+PADM+thin skin autograft group (SHA2 group), PADM+thin skin autograft group (SPADM group), thin skin autograft group (STS group), normal skin group (SNS group). Wound healing condition was observed and recorded. Transplanted skin was harvested at 3 days,7 days,14 days,28 days and 56 days postoperatively. HE staining of specimen was performed and the structure change was observed. The expression of CD31 were examined by immunohistochemical staining to determine the microvessel density (MVD). TypeⅠandⅢcollagens were determined by Western blotting and IHC. The expression of CD44 and the concentration of HA were assayed by ELISA and RIA respectively. Statistical analysis was performed correspondingly.Results 1 The grafts contracture rate:On postoperation day 28, the graft contracture rate in TS group was higher (p<0,05) than that in other groups. However, there was no significant difference among the PADM group, HAl group and HA2 group (p>0.05). On day 56 post-operation, the contracture rates in PADM group and TS group were higher than that in the other groups (p<0.05), and that in HA2 group was the lowest among the 4 grafting groups (p<0.05).2 Biomechanics properties:(1) The relaxation curve showed that, on day 28 post-operation, the HA2 group's curve was closed mostly to NS's, so did HA1 group and TS group. On day 56 post-operation, HA2 group's and HA1 group's curves were closed mostly to NS's, PADM group's curve was most deviated from NS's.(2) Creeping curve showed that PADM group curve was approximate mostly to NS group, followed by TS group curve, and the farthest were HA2 group and HAl group curves on day 28 post-operation. All the experimental groups curves was approximate to NS group curve on day 56 post-operation.(3) Stress-strain curve showed that HA2 group curve was mostly near to NS group curve on day 28 post-operation and day 56. The skin flexibility in HA2 group was preferable for the stress being smallest than other grafts groups for inducing the same strain, but the strength was bad. While the PADM group curves mostly deviated from NS's, and the PADM group can endured much stress, but they were less flexibility than that in other groups.3 Microvessel density (MVD):There was no capillary formation in SHA1 group and SHA2 group, but some capillaries were appeared in STS group and SPADM group on day 3 post-operation (p<0.01). On day 7 post-operation, capillaries in SHA1 group and SHA2 group were observed, there was no significant difference in capillary density between the SHA1 group and SPADM group (p>0.05), and the value of MVD in SHA2 group was higher than that in SPADM group and SHA1 group (p<0.05), but lower than that in STS group. On day 14 post-operation, there was no significant difference in the value of MVD between the SPADM group and STS group (p>0.05), both of which were lower than that in SHA1 group, and the value of MVD in SHA2 group was higher than that in SHA1 group (p<0.05). On day 28 post-operation and day 56, the value of MVD in SHA2 group was the highest, followed by the value in SHA1 group, STS group, SPADM group, SNS group ordinally (p<0.01). From 3 days to 56 days postoperatively, the value of MVD in all experimental groups increased firstly, followed by a decline (p<0.01).4 Expression of collagensⅠandⅢ:On day 28 post-operation and day 56, the expressions of collagensⅠandⅢin SHA1 group and SHA2 group were higher than that in SPADM group (p<0.05), and expression of collagensⅠandⅢin SHA2 group were higher than that in SHA1 group (p<0.05).5 Contents of CD44:On day 3 post-operation and day 7, there were notable differences in contents of CD44 among the SHA1 group, SHA2 group and SPADM group (p<0.05), and the contents of CD44 in SHA1 group was higher than that in SPADM group, but lower than that in SHA2 group. On day 14 post-operation, there was no significant difference in CD44 levels between SHA2 group and SHA1 group (p>0.05), and the content of CD44 in SPADM group were higher than that both in SHA1 group and SHA2 group (p<0.05). On day 28 post-operation, there was no significant difference in CD44 levels between the SHA1 group and SPADM group (p>0.05), but the contents of CD44 in SHA1 group and SPADM group were higher than that in SHA2 group (p<0.05). On day 56 post-operation, there was no significant difference in CD44 levels between the SHA1 group and SPADM group (p>0.05), and the contents of CD44 in SHA1 group and SPADM group were lower than that in SHA2 group (p<0.05).6 HA Concentration:There were no significant differences in HA concentrations between SHA1 group and SHA2 group (p>0.05), both of which were higher than that in SPADM group on day 3 post-operation (p<0.05). No significant difference was detected among SHA1 group, SHA2 group and SPADM group (p>0.05) on day 7 post-operation. On day 14 post-operation, there was no significant difference between SHA1 group and SHA2 group (p>0.05), both of which was lower than that in SPADM group (p<0.05). On day 28 post-operation, there was no significant difference between the SHA1 group and SPADM group (p>0.05), but concentration of HA in SHA2 group was lower than that in SHA1 group and SPADM group (p<0.05). On day 56 post-operation, there was no significant difference between the SHA1 group and SPADM group (p>0.05), concentration of HA in SHA2 group was higher than that both in SHA1 group and SPADM group (p<0.05).Conclusion Topical hyaluronic acid can be used to alleivate contracture of composite grafts in rabbit wound, and to enhance the biomechanics performances of the transplanted skin, such as the relaxation, creeping and stress-strain properties. The the aforementioned effects may be attributed to HA helping to induce expression of its receptor (CD44), and strengthen the enzymolysis metabolism, etc. These metabolic products can promote grafts vascularity, increase the expressions of typeⅠandⅢcollagen, decrease the ratio of collagen typeⅠ/Ⅲ, inhibit contracture of the transplanted skin, and improve the quality of composite grafts, finally benefiting the wound healing.