Applications Of Porous Polyactic Acid In Tissue Engineering Dermis Substitutes And Immortalization Of Human Skin Fibroblasts

The field of tissue engineering exploits living cells in a variety of ways to restore, maintain, or enhance tissues and organs. To engineer living tissues in vitro, cultured cells are coaxed to grow on bioactive degradable scaffolds that provide the physical and chemical cues to guide their differentiation and assembly into three-dimensional tissues.In the first study we used porous PLA biodegradable polymers and developed artificial dermis. The investigation is based on these requirements and contains three main parts:1. To observe the biological behaviors of the dermal fibroblasts on the polyactid acid membrane. We planted the dermal fibroblasts on polyactid acid membrane and observe the attaching efficiency, proliferation ability. The morphological changes were investigated by HE staining and transmission electron microscopy. PLA showed very good bio-compatibility and can be used to construct dermal substitutes.2. The PLA sponge was prepared by a particulate-leaching technique usingsieved sodium chloride particulates. NaCl was leached out by washing the composite with deionized water until the weight of the dried sponge remained unchanged. Finally, the PLA sponge was formed after drying. 3. To evaluate the role of polyactic acid(PLA) in the formation of artificial dermis, skin fibroblasts were seeded into the scaffold and cultured in medium. The final product was analysed by histological and immunohistochemical methods as well as electron microscopy. It is concluded that porous PLA is an ideal scaffold for artificial dermis.Secondly, in order to obtain immortalized human skin fibroblasts, we performed a systematic study using plasmids that transduce either SV40 LT antigen or hTERT. The preliminary results were as followings:1. Establishment of primary cell culture fibroblasts were prepared from foreskin and maintained with DMEM, supplemented with 10% PCS and antibiotics.2. Either LT or hTERT was transduced singly in the fibroblasts, after selection with G418 containing medium, the G418-resistant clones were isolated and expanded. Immunocytochemical staining of LT antigen or hTERT and results of in situ hybridization showed that the clones had the positive gene expressions.3. In order to further the study, we constructed a new plasmid containing green fluorescen protein cDNA by inserting an expression cassette of hTERT into pIRES2-EGFP.4. LT and hTERT were transduced on combination in the fibroblasts. Selected the positive clones by fluorescent microscopy.5. To observe the biological alteration of the three transfected fibroblasts, we counted the population doublings and drawed a graph, perform karyotype assay. The results showed clearly that neither hTERT nor LT antigen alone wassufficient for immortalization of human skin fibroblasts. However sequential transduction of both genes results in immortal cultures that grew without either a senescent or crisis phase being observed.The study indicated that the dermis-forming model provides a system in which better artificial dermis formation can be readily followed. Such immortalized diploid fibroblast line may provide useful resources for dermis substitutes construction, studies on immortalization and in vitro transformation.