| TM perforations are often caused by infection or trauma. A variety of autografts and allografts have long been used in the surgical closure of a TM perforation. A variety of autografts and xenografts have been used so far in the surgical closure of a tympanic membrane perforation. Autografts commonly applied are temporalis fascia, auricular cartilage, fat, etc, and xenografts are often allografts and synthesized biological material. Compared with xenografts, autografts are much more popular and enjoy a higher success rates of perforation closure, however it needs an additional surgical incision or extended dissection with its associated surgical morbidity so that it is more difficult to obtain the autografts for patients with second tympanoplasty.Being an emerging multidisciplinary field involving the material science, engineering and biomedicine, tissue engineering comes into being with the development of tissue culture, material, and transplantation. Up till now, tissue engineering has made great progress in the researches on the tissue engineered products of the skin, the muscle, the mucosal, the bone, the cartilage, the blood vessel, the nerve, and the cornea, and some of them have been used in patients in the clinical practice. However, tissue engineered TM has not beenresearched or used in practice yet.In present study, we explored the feasibility of constructing the tissue engineered TM and the tympanoplasty with the tissue engineered TM obtained in this study.1 ObjectiveTo develop a rapid method for the isolation of pure epithelial cells and fibroblasts from tympanic membrane. To reconstruct tympanic membrane in vitro by tissue engineering technique. To construct the optimal animal model of persistent tympanic membrane perforation. To evaluate the effect of tympanoplasty with tissue engineered tympanic membrane.2 Methods2.1 The epithelial cells and fibroblasts were isolated and purified from tympanic membrane by a new two-step method. The cells were identified by cell morphorlogy and immunostaining.2.2 The skin and dura of pig and human acellular amniotic membrane (HAMM) were processed with high satuated saline and enzymes to makeextracellular matrix (ECM) and then these 3 kinds of scaffold were grafted under the skin of guinea pigs. The reaction of guinea were examined by HE stain for 4 weeks. Meanwhile, these scaffold were cultured with the fibroblasts of tympanic membrane. Light microscopy and scanning electron microscopy were also used to evaluate the proliferation of fibroblasts on the surface of biological scaffolds after 4days.2.3 Cultured fibroblast and epithelial cells were seeded on the surface of ECM which had been prepared by removing of the original cells .The composite tympanic membrane were cultured in vitro for 1 week. the reconstructed tissue were examined in histological structure, and mechanicalproperties.2.4 Followed by medial infolding of TM microflaps, myringectomy was performed on 10 guniea pigs. Each of guinea pig was microscopically examined the healing of tympanic membrane perforation. The tympanic membrane were taken out randomly at 8 weeks for histological examination.2.5 Thirty guinea pigs underwent creation of bilateral chronic tympanic membrane perforations over a 8-weeks period. After stable perforations were created successfully. In present study each animal served as its own control by assigning right ear treatment and left ear control to each. The grafts were surgically placed through a postauricular incision and laid under the TM remnant.. The tympanic membrane were examined every week and then harvested for histopathological analysis. The changes in the auditory threshold of the guinea pigs before and after the treatment with the tissue engineered grafts were observed by auditory brainstem response audiometry (ABR) with tone bursts and click sounds at 8 weeks.3 Results3.1 Tympanic membrane epithelial cells cultured were of characteristic cobblestone morphology and showed a positive reaction keratin. Fibroblasts cultured were spindle -shaped and showed a positive reaction to antibodies against vimentin. Tympanic membrane epithelial cells and fibroblasts cultures of this processing were free of any contaminants.3.2 The fibroblast of tympanic membrane could be attached to and extended on these scaffold. Guinea pigs showed no evidence of rejection or extrution. These allografts appeared to integrate well and had good bio-compatibility.3.3 The tissue engineered tympanic membrane were of good biological andmechanic properties. The methods used by our study were effective and optimal.3.4 Of the 10 guinea (20TMS) pigs perforated ,persistent subtotal perforations were successfully created in 16 ears, 8weeks after initial procedure( 80%success). Suppurative Otitis media or TM healing spontaneously despite reperforation was found in 4 ears(20%failure).3.5 Sixty-nine ear repaired with two kind of grafts were completely healed after 8 weeks and had a better result than control ear. The same effect found in two kind of bio-scaffolds in term of tympanoplasty. The tympanic membrane repaired with grafts were the same as normal tympanic membrane in histological examination. The auditory threshold of ear repaired with grafts reached to 15dB at 8 week.4 Conclusions4.1 The pure tympanicmembrane epithelial cells and fibroblasts can be easily isolated by this method.4.2 After processed with enzymes, the derma and dural and human acellular amniotic membrane showed excellent bio-compatibility and then could be used as tissue engineered scaffold.4.3 The tissue engineered tympanic membrane could be an optimal biological dressing with good histocompatibility and inert immunogenicity.4.4 The ideal animal model of persistent tympanic membrane perforation can be successfully obtained with myringectomy combined with TM infolded.4.5 The application of the tissue engineered TM in the surgical enclosure of TM perforation will show tremendous promise with its availability, readiness to survive, predilection for host tissue integration, and lack of immunogenicity. Its most significant advantage probably lies in its ability to facilitate the...
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