Primary Study On Simulating Skin Development In Tissue-engineered Skin Construction With Skin Appendages

Objective：Application blade scraping method and enzyme digestion method toobtain keratinocyte（KC）and fiber cells of fetal rat and. Culture and identification ofthese cells,and applications the KC as seed cells, PGA as a stand, to constructtissue-engineered skin with skin appendages, to provide a good experimental modelfor fetal rat KC pluripotent, and seeks to obtain a more complete biological propertiesof tissue-engineered skin provide a better biological repair materials for clinicalapplication.Methods:The fetal rat and mouse KC obtained by blade scraping method werecultured by keratinocyte serum-free medium (K-SFM), observation of cell growthand colony formation, and identify cells with the Pan-CK monoclonal antibodies. Thefetal rat and mouse fiber cells obtained by enzymatic digestion were cultured inDMEM, observation of cell growth and colony formation identify cells with vimentinmonoclonal antibodies; application of KC, fiber cells and PGA stand to constructtissue-engineered skin with skin appendages initially to observe the ability of the KCformation skin appendages.Results:1.The observation revealed that the fetal rat and mouse skin growth anddevelopment of skin appendages in different development stages. At E14.5, skinappendage development has begun although only1-2layers of KC was seen, atE16.5,it was the most productive period, at E18.5, it was basically completed, atP7,skin appendages were mature.2.The E14.5day fetal rat KC can be obtained successfully and purely byscraping method, and a stable culture system can be established.3.The E16.5, E18.5, P7days fetal rat and mouse KC and fiber cells can beobtained successfully and purely by enzymatic digestion, and a stable culture systemcan be established.4.The tissue engineering skin construct by E14.5day fetal rat KC, fiber cellsand PGA have complete structure. The epidermis and dermis were linked closely. Conclusion:1.Observation of the different developmental stages of fetal rat andmouse skin structure, we mastered the growth and development of skin appendagesinitially.2.Blade scraping method is a better way to get E14.5fetal rat KC, we can get alarge number of cell viability better.3.Enzyme digestion is a stable cell culture technology to get E16.5, E18.5andP7days fetal rat and mouse KC and fiber cells.4.The tissue engineering skin construct by E14.5day embryo rat KC, fiber cellsand PGA was closed to normal skin.