Fabrication And Properties Of Gene- Activated Skin-regenerative Materials For Appendages Regeneration

The regeneration of skin appendages is an important symbol of skin functionalization during skin tissue repair and regeneration. Even though many commercialized artificial skin substitutes can repair the structure of epidermis and dermis, the skin appendages are still lack. To achieve the regeneration of skin appendages during wound healing, the targeted gene was encorded into the plasmid by gene-engineering to get the HGF-pDNA that could continually express the targeted protein Hepatocyte Growth Factor (HGF). After incorporated the seed cells - bone marrow mesenchymal stem cells (BMSCs), the gene-activated scaffold (GAS) with BMSCs was fabricated.The nanocomplexes of liposome and plasmid were fabricated by electrostatic interaction to protect the plasmid. After incorporated into the collagen-chitosan scaffold, the nanocomplexes was found to be distributed evenly and adhere to the wall of hole through confocal laser scanning microscopy (CLSM) and scanning electron microscope (SEM). After BMSCs were incorporated into GAS, the result of Western Blotting showed that GAS could promote the express of target protein HGF compared with blank scaffold. Through RT-qPCR and Western Blotting, the characteristic factors - Versican, Alkaline phosphatase (ALP), type IV collagen (COL IV) showed a significant increase in GAS compared with blank scaffold which meant BMSCs were induced to differentiate into hair follicle cells.GAS with BMSCs (GASM) as experiment group was implanted into the SD-Rat full-thickness skin defect compared with the blank scaffold with BMSCs (BSM) and the GAS without BMSCs as control groups. From gross views, the experiment group showed some hair structures across the surface of regenerative epidermis. The H&E staining showed some incompact hair follicle-like structures 10 d post-surgery and more mature hair follicle-like structures 21 and 35 d post-surgery. To verify the structures of hair follicles, immunohistochemical staining (IHC) was carried out. The result showed the hair follicle-like structures 10,21 and 35 d post-surgery was positive for Versican, ALP and Alpha smooth muscle actin (a-SMA) which demonstrated the hair follicle specificity of these structures. RT-qPCR and Western Blotting showed the expression of Versican, ALP and a-SMA in gene and protein level was higher compared with the control groups. After BMSCs from male rats as seed cells were implanted into the female rats to verify the source of cells in regenerative hair follicles by Y chromosome in situ hybridization, the result showed some of cells in regenerative hair follicle-like structures 10 d post-surgery were positive, but the positive percentage was decreased significantly 21 d post-surgery. It demonstrated that both BMSCs and the autologous cells contributed to the regeneration of hair follicle structures.To achieve in situ induced regeneration, the GAS with SDF-1 was expected to induce autologous BMSCs to participate in the regeneration of hair follicle. The OCS was used to form the nanoparticles with polylysine (PLL) and stromal cell derived factor-1 (SDF-1) and enzyme-linked immunosorbent assay (ELISA) demonstrated the nanaparticles could achieve encapsulation rate of 63.01%. After nanoparticles immersed in PBS, the release rate of SDF-1 was 40% at 168 h. The experiment in vivo demonstrated that GAS with SDF-1 could regenerate the hair follicle-like structures 35 d post-surgery but GAS without SDF-1 couldn’t regenerate hair follicle-like structure 35 d post-surgery.