Construction Of Tissue Engineered Skin Using Fetal Fibroblasts

Construction of tissue engineered skin using fetal fibroblastsSkin defects are main problems in plastic and burns surgery.Usually, autograft of skin or allograft of skin is used to repair skin defects. However,these therapies have many disadvantages,such as insufficient autografts,a severe morbidity of donor site,immune elimination towards allografts,etc.So it is necessary to invent a new method for constructing tissue-engineered skin which will be useful in clinical practice.Since Rheinwald and Green(1975) established the culture method of the keratinocytes,tissue-engineered skin became approached.In the present time,a lot of biological dressing,like Apligraft,Dermograft,Intergra, and Allodone etc,are widely used in clinical for wound healing treatments. But most of these tissue-engineered skin products have their disadvantages,such as poor physical performances,scarce of autologous seed cells,immune exclusive reaction towards xenogenous seed cells,long culturing time,expensive cost,etc.In this study,based on the previous researches,we amplify allogenic fetal fibroblasts in vitro and construct tissue engineering skin to repair mice skin deficiency and simultaneously observe clinical effects on patients.So it provides a new method to find ideal seed cells of tissue engineering skin and to treat skin deficiency.ObjectiveTo establish a strategy for amplify human embryonic fibroblasts in vitro,then to analyse if cultured fetal fibroblasts haves ome advantages to be the seed cells and to reveal how immunogenicity of cultured fetal fibroblasts changed,those were mixed with allogenic lymphocytes. To establish a method for construction of tissue-engineered skin using human fetal fibroblasts and rat tail collagen,then to observe structures and secretion characteristics of this TE skin.To repair experimental animal skin defects using TE skin,then to observe therapeutic effectiveness and its structure after wound healing.The final aim is to testify the possibility,effectiveness of clinical repairment of skin defects with the tissue engineered skin substitute.MethodsHuman embryonic and common fibroblasts were cultured with the method of tissue clump and enzyme digestion.The cell shape,cell activity, growth cycle were observed.The different generations of the two types of fibroblasts were individually mixed with allogenic lymphocytes in vitro to observe how the allogenic lymphocytes proliferate.Rat tail collagen was prepared and mixedwith the cultured human fetal or common fibroblasts suspension in calf serum,concentrated culture medium and sodium hydroxide to form dermis equivalent.The cell collagen gel was stained with HE staining to observe.The concentration of TGF-β1 and IL-6 in the supernatant fluid of the culture medium were measured by ELISA method.HEFs were stained with fluorescent dye Hoechst33342,these HEFs were seeded into rat tail collagen to form dermis equivalent and implanted into nude mice with dorsal skin defects.Effectiveness was observed postoperation and equivalent skin was excised and observed under light microscope.PAS staining was also performed on the basement membrane of living TE skin.Fibroblasts were isolated and cultured from the dorsum of SD rat embryo.Tissue-engineered skin was prepared using the cultured SD rat embryonic fibroblasts.We transplanted the TE skin to adult SD rats and compared them with simple rat tail collagen graft to observe their developments and their histological changes.The expressions of CD31 and vimentin of wound healing were also detected with immunohistochemisty staining.Twelve cases of full-thickness skin defects after soft tissue expansion were repaired with tissue-engineered fetal skin.The other near wound were selected as control group.The simple rat tail collagen and the petrolatum gauze cover the control region respectively.The inflammatory reaction,bleeding were observed during the treatment period. The effectiveness and the safety of TE skin had been assessed in patients. The healing condition and the bad reaction of the wound were observed, and the time to complete closure between application and control were calculated.ResultsWe obtained 7×10~7 cells by method of enzyme digestion,and needed very short time,but the cell shape and activity were not good.Using the tissue clump,the shape and activity were both good.Compared with common fibroblasts,human embryonic fibroblast had the characters such as fast growing rate,high mitosis index and rapidly and strong metabolism.In explore of the cells' immunogenicity,we found both the common and the fetal fibroblasts,with the passages of fibroblasts increasing,the cpm gradually decreased.The cpm of initial passage and first passage descended sharply,especially common fibroblasts.In the fetal fibroblasts,the cpm of the second passage,the third passage,the fourth passage and the fifth passage are approximation to the blank control.The rat tail collagen gel made fibroblasts have a suitable condition and the fibroblasts can be stretched slightly without being torn or deformed.The concentration of TGF-β1 and IL-6 in HEF group were lower and higher individually than in common fibroblasts group on each time point.(P＜0.05) 20days postopration,the wounds on the back of nude mice covered by TEF skin had been healed with a thin layer of epithelium.But the wound covered with simple rat tail collagen was still crust.There was a continuous blue stained basement membrane fluctuating between the epithelium and the dermis.These marked HEFs were visible in the implant by fluorescent microscopy ten days after implantation and then gradually disappeared.In the experiment on SD rats,histological examination revealed good epithelization,regularly arranged collagenous fibers and fibroblast,and satisfactory vascularization in the TEF skin grafting group.During the clinical application,the patients' condition after operation were stable without significant inflammatory reaction and bleeding on the wound.The healing time were significantly shortened 2-4 days compared with that of control group using rat tail collagen and 8-10 days compared with that of control group using petrolatum gauze.Safety assessment showed no clinical or laboratory evidence of rejection during the trial.There was not any ill effect after healing.ConclusionsHuman embryonic fibroblast is a promising source for tissue engineering for their characteristics such as easy acquirability, extensive proliferation,etc.The method of tissue clump is more suitable to our investigation.Human embryonic fibroblasts nearly have no antigenicity after being cultrued.Using the rat tail collagen can form dermis equivalent with the favorable biological traits of the fibroblasts interacting with extracellular matrix,which was the key to provide the artificial skin. The TEF skin we constructed can induce wounds healing with satisfactory effectiveness when grafted into experimental animals.In view of the therapeutic effects of this technique along with the simplicity in application,fetal skin cells could have great potential in tissue engineering.