Basic Study Of The Repair Of Epithelial Defect In Surgical Cavity Of Ear With Tissue Engineering Technique

Surgical resection is the treatment of choice for ear diseases such as tumor,congenital ear malformation, and chronic suppurative atticoantral disease in the middle ear cleft, which results in concha skin defect and mastoid cavity open or exposure of the temporal bone surface. Clinical studies have found that, in 3%-40% patients,epithelialization does not occur at all or quickly enough before the onset of infection.Leakage of tissue cause bacterial infection and granulation formation when allowed from bone wall into the mastoid cavity. When infection sets in, will easy to form scar tissue and postoperative stenosis. Attempts have been made to repair with skin,periosteal flap, bone pate, temporal muscle fascia–periosteum flap and fat, also with part of allograft or xenograft. Although the application of these repair materials have a certain effect, part of re-operative patients have injury of the donor site or insufficient number. We have not yet found an ideal and stable repair materials and related technology for facilitating cell colonization and supporting temporal skin. The method of reconstruction for structure and function recover have not been conducted.The aim of this study is combining cells and materials based on tissue engineering technology to construct skin tissue for repairing epithelial defect in otological cavity after surgery or temporal bone wound.Objective:(1) To establish the isolation and cultivation methods of ADSCs in vitro and investigate the basic biological characteristics of ADSCs as seeding cells in ear cavity epithelium tissue engineering.(2) To construct ADSC aggregates–ECM and to observe the growth status of ADSCs seeded in the ECM scaffold, meanwhile, to investigate the feasibility of ECM as a scaffold for tissue engineering.(3) Two terms of ADSC aggregates–ECM and simple ECM scaffold shall we adopt to treat the New Zealand rabbit auricle and auditory meatus wound model which would be made in order to compare wound closure and inflammation, whereby to explore the healing-promoting effect and mechanism of complex patch.Methods:(1) Optimizated the methods of Zuk’ADSCs isolation, purification and culture.Induced differentiation, surface molecules, growth curve of ADSCs were made to verify for stem cell biological characterization.(2) ADSCs were co-cultured with ECM. The aggregates formation were observed when ADSCs cultured in the ECM by the scanning electron microscopy, the laser scanning confocal microscope and flow Cytometer. The autocrione activity factors of ADSCs were determined by RT-PCR.(3) Two models of New Zealand rabbit auricle and auditory meatus were randomized into three groups for each of terms after cutting off and punching. The composite patch and simple ECM scaffold were transplanted into the wound, and the blank control group was established. Morphology data of wound area were collected on week 2, 4, 7.Regeneration tissue samples were subjected to HE and to compare healing degree in terms of discharge, grannulation and infection.The expression profile of new cytokines for promoting tissue healing were examined through western blot on the protein extracted from wound healing tissue on week 1, 2.Results:(1) ADSCs obtained were similar in size, spindle-shaped fibroblasts-liked cells and had Adipocytes and osteoblast differentiation potential. Flow cytometry showed that cells were highly positive for CD29, CD90 and weakly positive for CD14 and CD45.ADSCs had obvious exponential growth phase.(2) ADSCs tightly adhered to the ECM and overlapped. The cells closely connected with less apoptosis. HGF, TGF-β, b FGF, and EGF gene expression levels were increased in ADSCs cultured on ECM compared with control group.(3) In auditory meatus models, skin formed, as was almost consistent with the surrounding tissue in the ADSC aggregates–ECM group when compared to ECM group and control group at 4 week. HE staining also showed that ADSC aggregates–ECM group had a larger range of normal skin-like structure when compared with the other groups.(4) At 1 and 2 weeks, levels of b FGF, EGF, HGF, VEGF, and TGF-β1 in the ADSC aggregates–ECM and ECM groups were significantly higher than those in the control group.(5) In ear auricle model, At 2 weeks, Wound healing rate in ADSC aggregates-ECM group was higher than that the ECM group and the control group. HE staining showed that the infiltration of inflammatory cells was found to be more evident in the wound region of the control group. At 4 weeks, the wounds in all groups were completely healed, inflammatory cells reduced. At 7 weeks, a significant decrease in skin thickness of ADSC aggregates–ECM and ECM groups when compared to the control group(p<0.05).Conclusions:(1) Isolated stem cells show the typical characterization of ADSCs, and can be used as seeding cells in ear cavity epithelium tissue engineering.(2) ECM scaffold provides a good environment for cell growth and promotes the expression of multiple cytokines. It is a kind of potential application value of tissue engineering scaffold material.(3) ADSC aggregates–ECM and ECM are beneficial to ear wound healing. ADSC aggregates–ECM hold the capability of reducing the intensity of inflammatory reaction after trauma, the capability of promoting better wound healing and the capability of reducing the thickness of scar than both simple ECM and control Group. Our study will provide meaningful experimental basis for temporal bone epithelium repairing.