Correlation Between Innervations In Sigmoid Neovagina And Sexual Activity Quality And Reconstruction Of Tissue Engineering Vagina In Rat

Vaginoplasty is indicated in female with congenital absence or hypoplasia of the vagina, example for Mayer-Rokitansky-Kuster-Hauser syndrome (MRKHS). Many vaginal reconstruction methods have been described. The fundamental method is that a potential neovaginal space is created by blunt dissection of the space between the rectum and the bladder, and then the successive lining with autos or xenoma tissues were placed. Sigmoid colon vaginoplasty is particularly useful because it is anatomically close to the perineum, it is sufficiently long and the mobility of its vascular pedicle which allows it to be brought into the perineum. To date many studies have addressed that sigmoid vaginoplasty, which have shown the surgical procedure provides excellent results including adequate vaginal length, natural lubrication, a low incidence of shrinkage and early intercourse. Furthermore, over 80% studies had reported patients were satisfied with the psychosexual outcomes. After transplantation of sigmoid colon with vascular pedicle, its local mucosa have occurred some adaptative changes in morphology, secretion, and defense function with stimulation of intercourse and prolonged time from surgery. However, what change do the innervations and nerves density occur in the sigmoid vagina, and whether the changs correlate with sexual function of patients, which have not been examined to our knowledge. The contents will be explored in clinical part of our study.Patients with MRKHS implement sexual intercourse using neovagina constructed by these traditional procedures. However, shortcomings of surgeries have been found, for example supernumerary trauma following autos tissues transplantation, rejection and propagation of diseases induced by xenoma tissues. Furthermore, the neovagina constructed by these ways show significantly difference in morphology and histology compared with the natural vagina. Therefore, it is necessary and emergent to explore and development the ideal vaginoplasty. With the development of tissue engineering, the ideal neovagina might be constructed with the technique. In fundament study part of the paper, we will attempt to reconstruction of tissue engineering vagina in rat, and expect to establish a new way for clinical vaginoplasty with more excellent outcomes.Part one:Innervations, Expression of Estrogen Receptor in the Neovagina Mucosa and Correlation Analyses with Sexual Activity Quality after Sigmoid VaginoplastyObjective:To investigate the innervation and expression of estrogen receptor (ER) in the neovaginal wall of the patients undergoing sigmoid vaginoplasty for Mayer-Rokitansky-Kistner-Hauser Syndrome (MRKHS), and to assess whether the changs of nerve density and ER expression correlate with sexual function of patientsMethods:Twenty-four patients with MRKHS undergone sigmoid colon vaginoplasty were selected as research subjects from January 2006 to December 2010 in the Department. of OB & GYN of second hospital of HeBei Medical University. Surgical outcomes were observed during follow-up, and sexual activity quality were surveyed using female sexual function index (FSFI) to patients with intercourse. Biopsies of the intact sigmoid colon were obtained from these subjects during sigmoid vaginoplasty in the same period (sigmoid colon group, n=24). Tissue biopsies were taken out transvaginally from the posterior neovagina at the upper third position (neovagina group, n=24). Biopsies of natural vagina were obtained at the same position with neovagina from age-matched female without vaginal diseases (natural vagina group, n=10). The protein and mRNA expressions of protein gene product 9.5 (PGP9.5), and ER were detected by immunohistochemical method and real-time fluorescence quantitative PCR (FQ-PCR) in order to observed the distribution and density of nerve fibers containing particular neuropeptide. The expression of ER was studied with the same methods, and furtherly the correlation above parameters with interval time to surgery were analyzed. The relationship was also studied among nerves density, ER expression, and FSFI in neovagina.Results:1 The follow-up was 3 to 39 months. The appearances of neovagina were similar to that of the natural vagina, the mean length of the neovagina was 10.6±2.4cm, the mean width was 2.9±0.6cm, and no postoperative mortality and minimal morbidity occurred. Seventeen patients (70.8%) had regular vaginal intercourse. The range of sexual history was 2 to 30 months. The mean full FSFI score was 24.11±1.33, fifteen of these patients had a normal score whose range was 23 to 29, and two of them had lower score than 23. After creation of a sigmoid neovagina, patients with MRKHS can be considered normal in terms of desire (4.18±0.51), arousability (4.04±0.55), lubrification (3.57±0.49), orgasm (4.00±0.48), satisfaction (5.06±0.65), and comfort (3.25±0.90).2 A larger number of PGP 9.5 nerve fibers were observed in lamina propria, muscle of the mucosa, submucosa, and muscle layer of the neovagina. The nerve fibers with VIP-and NPY-immunoreactivity (IR) mainly distributed around blood vessels and in the smooth muscles. In the neovagina, the expression intensity of nerve fibers was PGP 9.5> VIP and NPY (38.22±8.93 vs 7.45±2.01,6.13±2.38, P<0.05). These characters in neovagina were similar to the intact sigmoid colon in the study. In the natural vagina, the immunoreactivity nerve fibers distributed in the lamina propria and muscle layer.3 An integral optical density (IOD) of PGP9.5, VIP, and NPY-IR was measured to evaluate total innervations in neovagina. PGP9.5, VIP, and NPY-positive expression were significantly lower in the neovagina than that in the intact sigmoid colon (38.22±8.93 vs 46.12±6.63, P1<0.05; 7.45±2.01 vs 9.74±2.19, P2<0.05; 6.13±2.38 vs 8.38±2.51, P3<0.05), but higher than that in the natural vagina (38.22±8.93 vs 22.76±6.61, P;<0.05; 7.45±2.01 vs 4.59±1.76,P2<0.05; 6.13±2.38 vs 3.58±1.23, P3<0.05) These parameters mRNA expression displayed a similar tendency, which were the values in sigmoid colon> neovagina> vagina (PGP9.5:10.17±2.24 vs 7.94±1.92 vs 5.68±0.47, P1<0.05; VIP:4.14±1.32 vs 2.37±0.87 vs 1.04±0.36, P2<0.05; NPY:3.79±0.98 vs 2.68±0.75 vs 1.15±0.33, P3<0.05)4 In the neovagina, PGP9.5 and VIP-IR nerve fibers and mRNA expression had a positive linear correlation with time interval to surgery r1=0.73, P1=0.01; r2=0.16, P2=0.04), which indicated the nerve density tended to decrease firstly and gradually upturn by time after surgery. Whereas, correlation between NPY expression and time interval to surgery wasn't found(r=0.30, P=0.38).5 The expression ER protein and mRNA were significantly increase compared with the sigmoid colon (19.56±5.29 vs 10.49±2.84, P,<0.05; 4.36±1.41 vs 2.31±0.75, P2<0.05), but didn't reach the levels of that in the vagina (19.56±5.29 vs 27.83±6.78, P,<0.05; 4.36±1.41 vs 9.59±2.64, P2<0.05). ER expression had a positive linear correlation with time interval to surgery (r=0.92, P=0.00), which it gradually increased by time after surgery, but lower than that of in the natural vagina in our follow-up.6 In the neovagina, the expression of PGP9.5 and VIP mRNA had a positive linear correlation with that of ER (n=0.66, P1=0.03; r2=0.65, P2=0.03), NPY mRNA expression hadn't correlation with ER (r=0.46, P=0.15). FSFI hadn't correlation with PGP9.5, NPY, VIP and ER mRNA expression (r1=0.12, P1=0.72; r2=0.25, P2=0.43; r3=-0.47, P3=0.13; r4=0.50, P4=0.12).Conclusions:1 Sigmoid neovagina allowed a normal sexual life and provided a good quality of sexual activity in patients with MRKHS who had sexual intercourse.2 The distribution of sensory PGP9.5-, VIP-, and NPY- positive nerve fibers in the neovagina wall was similar to the pattern observed in the intact sigmoid colon wall. The density of nerve fibers in the neovagina was lower than that in the intact sigmoid but higher than that in the natural vagina, and gradually increased with time during our follow up. The adaptative change may be helpful in enhancing the sensitivity of the neovaginal mucosa and improving long-term quality of sexual activity.3 The expression of ER protein and mRNA were increase by time, which was probably physiological foundation on the adaptative changes of histomorphology and decrease of pH value in neovagina.4 The expression of PGP9.5 and VIP mRNA had a positive linear correlation with ER mRNA expression, which supported the protection of estrogen to nerves and maybe promoted incomplete reinnervation in neovagina by this way.5 No correlation between FSFI of patients and innervations and ER expression in the neovagina suggested the factors of effecting sexual function were complicated and needed further study.Part two:Primary Culture of Rat Vaginal Epithelial Cells in vitroObjective:Vaginal epithelial cells from Sprague-Dawley rats were primary cultured by tissue explants and enzyme digestion methods in vitro respectively in order to explore the better culture technique and provide seed cells for vaginal tissue engineering.Methods:1) Tissue explants method:Vaginal tissues of rats were pruned to 0.5-1mm2, and then planted on the culture capsule. After 3 hours in incubator with 37℃and 5%CO2, the Keratinocyte Serum-Free Medium (K-SFM) were added and continuously cultured.2) Enzyme digestion method:The rat vaginal tissues were digested by dispase enzyme to isolate the epithelial layer, and furtherly digested by trypsin. The cell suspension was cultured continuously in K-SFM.3) Time of primary culture, the cell morphology, proliferation characteristics and ultrastructure were oberserved under light and electron microscope, and the growth curve was drawn. The cells were identified with Anti-pan-cytokeratin (P-CK) by immunohistochemistry stain.Results:1) The cells started to growth within 3-5 days by tissue explants, and confluent growth was noted for subculturing after 3-4 weeks. The culture cells with enzyme digestion method began to adhere within 24-36 hours, and reached 80% confluency after 7-10 days.2) Vaginal epithelial cells were polygon-like or irregular globular and show typical appearances of "paving stone" after confluention under inversion microscope. Microvilli ridge were observed on cells surface with scanning electron microscope. The culture cells morphology and growth characteristics were essentially consistent by the two methods.3) The success ratio of culture cells by tissue explants was significantly low compared with enzyme digestion method (37.5% vs 50%, P<0.05). The cell productivity by the two methods didn't show significant differences (2.67±0.23×106/L vs 2.89±0.94×106/L, P>0.05).4) The expression of P-CK staining of the cultured cells was positive. Purity of cells using explants and enzyme digestion method were respectively 92% and 98%. The fourth generation cells pocess normal diploid karyotype.Conclutions:Vaginal epithelial cells were obtained by the two methods. Compared with explants method, the culture cells using enzyme digestion method display faster adhesion, shorter growth time, and higher success rate. The larger numbers of epithelial cells were rapidly obtained to construct engineering of vaginal tissue by enzyme digestion method.Part three:Biocompatibility Between PLGA/ⅠCollagen Scaffold and the Rat Vaginal Epithelial CellsObjective:To explore the biocompatibility of the PLGA/Ⅰcollagen with rat vaginal epithelial cells, and verify the feasibility of using PLGA/Ⅰcollagen as scaffold to reconstruct vagina by the tissue engineering.Methods:The rat vaginal epithelial cells were cultured by enzyme digestion method.Ⅰcollagen was combined with poly lactic-co-glycolic acid (PLGA) to form PLGA/Ⅰcollagen biodegradable polymer scaffold. The epithelial cells were culture in the leaching liquor of scaffold to detect its toxicity to cells by MTT. The epithelial cells were inoculated on the scaffold to calculate cell adhesion rate, and cells growth were observed in the epithelial cells-scaffold complexes with scanning electron microscope. Epithelial cells-scaffold complexes were implanted under cutis of rat back. At 2,4, and 8 weeks after implantation, the cells-scaffold were assessed by histological HE stain and immunohistochemical analysisResults:The Epithelial cells grew and proliferated well in the leaching liquor of scaffold. On the PLGA/Ⅰcollagen scaffold, the cell adhesion rate was 71.8%±9.2%, which significantly higher than that on the PLGA scaffold. The epithelial cells were well distributed and adhered to the PLGA/Ⅰcollagen scaffolds under scanning electron microscope. At 2 weeks after implanted under cutis of rat back, the epithelial cells attached and proliferated in the space, and the fibroblasts were seen. At 4 weeks, the epithelium were seen about 1-3 layer. At 8 weeks, the epithelial cells increased and arranged regularitility, which formed the membrane-like layer on the scaffold. The expression of P-CK of the epithelium was positive.Conclusions:The PLGA/Ⅰcollagen scaffolds are well-cytocompatible to the epithelial cells, and can be used as the biodegradable polymer scaffold of the vaginal tissue engineering.Part four:Reconstruction of Tissue Engineering Vagina in RatObjective:To explore feasibility of reconstructing completely rat vagina by tissue engineering, and provide technical function on clinical application.Methods:The epithelial cells were cultured from rat vaginal tissues, and innoculated on the medial surface of fistular PLGA/Ⅰcollagen biological material. The epithelial cells-scaffold complexes were transplanted to rat vagina in situ after culture in vitro to reconstruct vagina. After 1,3, and 6 months, we measured the vaginal length and observed the appearance at maero. The neovagina tissues were prepared for histological evaluation with HE, Masson's, and immunohistochemistry stain.Results:1) The experimental rat model of vaginal reconstruction surgery was performed sueeessfully.1 rat died to overdose of anesthesia after surgery, and during observation,3 rats subjected serious infection in abdominal cavity,2 of these died and 1 of these had ankylocolpos. and coleostenosis weren't be seen in terms of survival 6 rats.2) At 1 month after operation, we observed thin and red epithelialization on the biological material in the vaginal canal, which distributed like islands. The little of biological material took place degradation, and the length of vagina was 1.4cm. Within 1-3 months, the vaginal mucosa get thick gradually and pink. The biological material had degradated more than a half, and the vagina was 1.2cm in length. After 6 months, the neovagina was hardly differentiated from the native one at either maero or miero appearance, and the vagina was 1.2cm in length and no shrink.3) Light microscopy inspection showed the epithelium was 1-3 layers after 1 month, no smooth muscle, rare blood vessel and collagen fibers. At 3 months after surgery, the epithelium was thicker and multi-layere and the minor smooth muscle were seen occasionally. At 6 months, histology of neovagina was similar to the native one except less rete pegs in the epithelium basilar part, more smooth muscle and compact collagen fibers were observed by Masson's stain. Epithelial cells layer were identified by immunohistoch-emistry stain with P-CK since 1 month after surgery which was accordant to the result in HE stain.Conclusins:The neovagina which was reconstructed using rat vaginal epithelial cells and PLGA/I collagen scaffold was hardly differentiated from the native one at either maero or miero appearance. A tissue engineering approach to clinical vaginal reconstruction in women is now a realistic possibility.