Fabrication Of Gene-activated Bilayer Dermal Equivalent For Skin Regeneration

Gene therapy and tissue-engineered scaffolds were combined to fabricate bioactive scaffolds. A cationic gene delivery vector, N,N,N-trimethyl chitosan chloride (TMC), was synthesized and used to condense DNA. Then, the TMC/DNA complexes were incorporated into collagen-chitosan scaffold. The in vitro releasing test showed that TMC/DNA complexes had a faster releasing rate in the initial stage, and then slowly release until 28 days. The released plasmid DNA with supercoiled structure was detected; the released TMC/DNA complexes were still capable of transfecting a high percentage of cells. TMC/pDNA-VEGF complexes were incorporated into the collagen-chitosan scaffold to build a gene-activated scaffold. The in vitro results demonstrated that cells kept their phenotype and had a higher viability on the scaffold. The loaded TMC/pDNA-VEGF complexes possessed higher transfection efficiency too.In the previous studies, collage-chitosan/silicone membrane bilayer dermal equivalents (BDEs) were prepared. To develop the next generation BDE with higher performance, a faster rate of angiogenesis is highly demanded. Therefore, the TMC/pDNA-VEGF complexes were incorporated into BDEs to obtain the gene-activated BDEs. To evaluate the angiogenesis property in vivo, the gene-activated BDEs were transplanted into the full thickness incisional wounds. At day 7,10 and 14 after surgery, immunohistochemistry and immunofluorescence results showed that the gene-activated BDE group had the highest number of newly-formed and mature blood vessels. RT-qPCR results showed that the TMC/pDNA-VEGF complexes could effectively transfect cells in vivo and express VEGF. The ultra-thin skin graft was further transplanted onto the dermis regenerated by the gene-activated BDEs at day 10 and well survived. At 112 days after grafting, the healing skin had a similar structure and～80% tensile strength of the normal skin. The epidermis had formed papillary structure, conveying its well differentiation.Meanwhile, the effects and the possible dermal repair mechanism of the gene-activated BDEs were evulated in the full-thickness incisional wound repair process. The in vivo transfection of the TMC/pDNA-VEGF complexes was sustained as long as 70 days. The wound tissue treated by gene-activated BDEs had higher VEGF and TGF-β3 mRNA expression, but the TGF-β1 mRNA expression was lower. The healed skin had a tighter connection between epidermis and dermis, and the papillary structure was also observed. The use of the gene-activated BDE could reduce the formation of scar tissue. Moreover, the healing effects of the blank BDE were better than petrolatum gauze.Burn wounds are dynamic wounds, which can deepen with time, thereby the repair of burn wounds are more difficult than the incisional wounds. The gene-activated BDEs were also used to repair the full-thickness burn wounds. The in vivo results showed that, at day 7,14 and 21 after surgery, the gene-activated BDE group had the highest number of newly-formed and mature blood vessels, and the enhanced angiogenesis was realized. The ultra-thin skin graft was further transplanted onto the dermis regenerated by the gene-activated BDEs at day 14 and well survived. At 105 days after grafting, the healing skin had a similar structure and～70% tensile strength of the normal skin.Finally, the blank BDE and the gene-activated BDE were compared with a clinical dermal equivalent J-1 ADM in the treatment of incisional and burn wounds. The results showed that as for incisional wounds treatment, the gene-activated BDE was superior to blank BDE and J-1 ADM, and the blank BDE was nearly equal to J-1 ADM. As far as burn wounds treatment was concerned, the gene-activated BDE was superior to blank BDE and J-1 ADM, and J-1 ADM was slightly better than the blank BDE.