Development Of Tissue Engineering Skin Kit And Application In Treatment Of M.canis Infection

Based on previous studies,it was found that 94.29%of the dogs with skin fungal diseases were infected by only one kind of fungal specie.Considering the high prevalence and zoonotic from animal to human,Microsporum Canis was selected as research subjects in sample dogs with skin fungal disease in present study.In order to reduce the number of experimental animals used in pre-clinical studies for new veterinary drugs and screen the better efficacy from a number of drugs,in the present study we developed a tissue engineering skin kit and selected antifungal drugs by tissue engineering skin kit for the individual treatment of sample dogs infected with M Canis.Meanwhile,with the aim to improve the structure stability of tissue engineering skin kit,in this study,epidermis structure and transport medium were standardized.1.Reconstruction of tissue-engineered skin and kit assembly Tissue-engineered skins were successfully reconstructed by cultured canine skin fibroblasts and keratinocytes.After slicing and HE staining,results showed that no living cells was observed in composites derived from fibroblasts and autocrine extracellular matrix after treatment.Keratinocytes attached closely to the outer surface of autocrine extracellular matrix composite,and proliferated well and differentiated to multi-cellular components,which is very similar with natural microstructure of canine skin epidermis,including the stratum comeum,the granular and base layer.2.Standardization of epidermis in tissue-engineered skin kit In order for standardization of epidermis in tissue-engineered skin kit,total scores of barrier function were calculated via quantitative methods.In the present study,total scores of experimental and control group were eight vs six,respectively,which meant that tissue engineering skin in the experimental group would rank as "excellent" vs just"good" in control group.It revealed clearly that barrier function in experimental group was better than that of control group.3.Standardization of transport conditions for tissue-engineered skin kit In order for standardization of transport conditions for tissue-engineered skin kit,especially nutrient supply during transport,total scores of barrier function were calculated via quantitative methods after transport.Results showed total scores of barrier function after transport were seven,ranking as "excellent",too.4.Application of tissue-engineered skin kit in treatment of M.Canis infection Fungi were isolated from affected area in sample dogs.After purification,culture and identification,M.Canis were inoculated in the prepared tissue-engineered skin kit and incubated for 6 hours.Then,histological staining and flow cytometer analysis were conducted for experimental and control group after treatment of different drugs.In the present study,average Anti-Infective Drug Efficacy(AIDE)was 87.45%in terbinafine hydrochloride group,72.50%in miconazole nitrate group and 69.14%in ciclopirox group,respectively.Animal treatment showed Symptom Score Reduction Index(SSRI)by terbinafine hydrochloride had already reached 100%after individual treatment for 3 weeks.But,SSRI in treatments by miconazole nitrate and ciclopirox for 4 weeks were just 50%and 100%.