Experimental Study And Clinical Investigation On Vaginal Reconstruction With A Tissue-engineering Biological Material

Objective To investigate the effect on using a tissue-engineering biological material in vaginal reconstruction in the animal model and patients.Materials and Methods In 12 Chinese experimental miniature pigs, total vaginal excision were performed and vaginal reconstruction with homemade tissue-engineering biological material (Renov) followed. This tissue-engineering biological material is a kind of acellular dermal matrix (ADM). The control group was two of normal vagina specimens resected. After 1,2,4,6,8,12 weeks, the animals were sacrificed, the vaginal length was reevaluated and the neovaginas were prepared for histologic evaluation. The formalin-fixed vaginal specimens were dehydrated in graded ethanol solutions and embedded in paraffin; 4-μm sections were cut mounted on precoated slides, and stained for H&E, VG. Immunohistochemical analyses were performed with pancytokeratins AE1/AE3 to identify for epithelial cell layers, and with smooth muscle-specialα-actin antibodies for smooth muscle fibers. Western blot analyses with monoclonal antibodies to AE1/AE3 andα-actin were performed to confirm the reconstituted tissue components further. Organ bath studies were performed for strips of neovaginas compared with strips of the native vagina. The strips were attached by 4-0 silk sutures to a tissue support hook at one end and isometric force transducer at other end. The specimens were mounted in isolated baths containing 50 mL of Kreb's solution equilibrated with 95%O₂ and 5%CO₂ and maintained at 37℃. Peak contractions were recorded for each individual strips of tissue exposed to a variety of electrical and chemical stimuli. The chemical medicines such as KCL and phenylephrine were added in and electrical field simulation were included to further characterize the tissue-engineered constructs. At the same time, clinical investigation was carried out. 6 cases were performed for vaginoplasty with the tissue-engineering biological material. The clinical outcome were evaluated and PISQ questionnaire scales were noted.Results1. The Chinese experimental miniature pig model of vaginal reconstruction surgery was performed successfully. The anatomy of genital tract of female miniature pig is explored. The neovaginas were regenerated well. Vaginal apex stenosis and shrinkage were not observed.2. Within 1 weeks, we observed epithelialization of the graft at the site of 2/3 neovagina. Moderate adhesions from surrounding tissue was noted around the neovaginal apex. The neovagina was 8cm in length. Light microscopy inspection showed that the epithelium was flat, 2～3 layers, which was mildly infiltrated by leucocytes and erythrocytes. Within 4～6weeks, epithelialization was continued in the neovaginal canal. The neovagina was longer than before and more like native. The epithelium was thicker, and multi-layered. There were few lymphocytes below the epithelium. After 12 weeks, the neovagina was hardly differentiated from the native one at either macro or micro appearance. Epithelial cell layers were identified with broadly reacting monoclonal anti-pancytokeratins AE1/AE3 since 1 week after surgery, which was accordant to the result in H&E stain. Western blot analyses with monoclonal antibodies to cytokeratins (AE1/AE3) were performed to compare protein expression between native vaginal tissue, which served as a control, and reconstructed vaginal structure in order to confirm the maintenance of epithelial cell phenotypes.3. After 4 weeks, a few smooth muscle cells were observed with VG stain and immunohistochemical analyses positive monoclonalα-smooth muscle actin antibodies. Smooth muscle fascicle was appeared after 12 weeks. Western blot analyses with monoclonal antibodies toα- actin were performed to confirm the maintenance of smooth muscle cell phenotypes. There were first traces at 2 weeks and increasing expression at 4weeks.4. In organ bath studies, contractile responses were observed in the neovaginal constructs when chemical medicines and electrical stimulated. Similar amplitudes of response were observed between the neovaginal constructs and normal vaginal tissue at KCL and electrical stimulus duration, whereas no response was observed at phenylephrine, carbachol duration in the neovagina.5. With clinical investigation, there were 6 patients with Mayer-Rokitansky-Kuster-Hauser syndrome use acellular dermal matrix for vaginoplasty. At each visit after surgery, vaginal depth and epithelium were noted to assess the functional length of the neovagina. Total epithelialization of the vaginal epithelium was confirmed by examination at 4～6 weeks after the operation, and no vaginal apex stenosis was observed. The neovagina was 9cm in length and smooth. 2 cases who had sexual intercourse had not need mould any more. The PISQ scores of these two patients were high which suggested their sex life qualities were satisfied.Conclusions1. This is the first report of the creation of a neovagina with acellular matrix in Chinese experimental miniature pig model. Histological and functional analyses are evaluated to the neovagina. Good results confirm the animal model made successful. In the course of operation, the anatomy of gestational tract of female miniature pigs is explored, which may be served as the basis for animal model of pelvic reconstruction in future.2. The neovagina length is enough. Neither vaginal apex stenosis nor shrinkage is observed. Native cell infiltrate the acellular matrix and epithelium and smooth muscle are regenerated, which are confirmed with histological inspection and Western blot analyses.3. The contractile properties of the neovaginal constructs in response to electrical field stimulation were similar to those of normal vaginal tissue, which seem to imply an intact membrane system and similar smooth muscle structure.4. With clinical investigation, the neovaginal with tissue-engineering biological material appeared the same length and elasticity as normal. The use of tissue-engineering biological material to create a neovagina seems to appear as an easy, less invasive and useful method. Nevertheless, long-term outcome should be evaluated further with more cases.