| BackgroundPelvic organ prolapse(POP)is a common condition in adult women all over the world,which greatly reduces patients,quality of life due to urinary,lower genital,and gastrointestinal tract symptoms.The vagina is a supporting hammock for the pelvic viscera and well supported vagina is essential for normal pelvic organ support.The vaginal wall is comprised of four layers.The lamina propria layer composed primarily of ?/? collagen and the muscularis layer confer the greatest tensile strength to the vaginal wall.The biochemical changes of these constituents can compromise the biomechanical properties of the vagina,which can further induce the development of POP.In this study,we focused on the change in collagen subtypes in lamina propria layer and the smooth muscle in muscularis layer of vaginal wall in the women with POP.Until now,the pathophysiology of prolapse remains poorly understood.The exploration of animal model of POP is of great importance not only to make clear the etiology and underlying pathophysiology of POP,but also to develop new treatments for POP.Firstly,we study the effects of menopause on collagen subtypes,smooth muscle content and the related biomechanical property changes in the rat vagina.In addition,we assess whether the ovariectomized rat model can serve as a suitable animal model to develop new treatments for POP.Secondly,we used the more suitable animal model-non-human primates and studied the prevalence rate of spontaneous POP in older multiparous rhesus macaque.Mesenchymal stem cell(MSCs)-based tissue engineering technique can restore the normal function in injured tissues or organs and have been successfully employed in the field of stress urinary incontinence(SUI).However,little effort has been made to treat POP using the technique.In this study,we aimed to study if the human umbilical cord mesenchymal stem cells(HUMSCs)can restore the weakened vagina tissue in ovariectomized rats through stimulating the regeneration of vaginal fascia and musculature.Materials and Methods1.POP vagina sample collection and histomorphological analysis:Participants were recruited between Jun 2018 and Dec 2018 at the department of obstetrics and gynecology of Peking Union Medical College Hospital(PUMCH).The case group of patients underwent hysterectomy and reconstructive pelvic surgery because of advanced anterior vagina prolpase,while control group underwent hysterectomy for benign gynaecological diseases with no sign of POP.For the case group,full thickness tissue biopsies were collected from most distal prolapse points(Aa or Ba),while full thickness tissue biopsies were taken from the anterior vaginal cult in the control group.Tissues was used for histology and a-SMA immunohistochemistry for the analysis of collagen ?/? and smooth muscle content.Vimentin immunofluorescence was used to test the changes in fibroblast.2.Exploration of animal model of POP:The anterior vagina of Sprague-Dawley(SD)rats was assessed 2,4 and 16 weeks after ovariectomy.Rats without ovariectomy were used as the normal control group.Tissues were used for the expression analysis of collagen ?/? and a-SMA performed by histology,immunohistochemistry and western blot.Biomechanical testing was conducted for control rats and experimental rats 16 weeks post ovariectomy.In addition,we performed POP-Q exams in 34 rhesus macaque to learn the prevalence rate of spontaneous POP in older multiparous rhesus macaque.3.Tissue engineering:In this study,we used ovariectomized rats.All of the rats were divided into four groups:saline group,collagen group,saline+HUMSCs group and collagen+HUMSCs group.0.3ml saline or 2mg/ml collagen solution alone,and 3xlO6 cells in 0.3 ml of saline or collagen solution was injected into the anterior vaginal wall of the ovariectomized rats.Tissue samples were collected at 2?4 and 12 weeks after injection.Bioluminescence imaging(BLI),immunofluorescence were performed to detect the behavior of cells in vivo.In addition,histology,immunohistochemistry and Real time PCR were performed to analyze collagen ?/?,a-SMA and the related cytokines.Biomechanical testing was conducted for the rats 12 weeks post injection.Results1.Histomorphological change in the anterior vagina of POP patients:In the control group,the collagen exhibited thicker and more ordered,while the collagen arranged at random and loose in the case group.Collagen I(16.65±6.63%versus 25.27±5.54%,P<0.001)and smooth muscle content(26.04±4.36%versus 44.74±5.65%,P<0.001)were significantly decreased in the case group.The fibroblast changed to be round shaped and became less in the case group(37.0±11.7vs47.5±8.3,P<0.001).2.Ovariectomised SD rat model and the prevalence rate of POP in rhesus macaque:The expression of collagen I was increased significantly from 2 weeks after ovariectomy compared with the control group,while collagen ? showed a declining trend.Two weeks after ovariectomy,the smooth muscle bundles began to become disorganized,and the fraction of smooth muscle in the nonvascular muscularis of the proximal vagina was significantly decreased(P<0.001).Uniaxial biomechanical tests showed that the vagina exhibited a significantly higher tangent modulus 16 weeks after ovariectomy(0.74±0.2 7 MPa versus 0.25.0.11 MPa,P=0.016).In addition,the prevalence rate of spontaneous POP in older multiparous rhesus macaque was 11.8%.3.HUMSCs can restore the vaginal wall of the ovariectomised SD rats:BLI signal was disappeared within one week after MSCs injection.Scant DIR-labeled cells were detected in the lamina propria and muscularis layers in the collagen+HUMSCs group at 2 weeks.We did not find MSCs differentiation in vivo.Compared with the control groups,the content of collagen I decreased significantly at 4 and 12 weeks in the MSCs groups,while collagen ?increased significantly(P<0.001).The fraction of smooth muscle in the nonvascular muscularis increased significantly in the MSCs groups at 12 weeks(P<0.001).mRNA level of ACTA2 in the collagen+HUMSCs group was significantly higher than the saline group at 2 and 4 weeks(P=0.042 and P=0.040).In addition,the mRNA levels of angiogenic factors(bFGF or VEGF)in the MSCs groups were significantly higher than the control groups at different time point.Conclusions1.In the anterior vaginal wall of the POP group,the collagen arranged at random and loose and collagen I content in the lamina propria layer was significantly decreased.The fraction of smooth muscle in the muscularis was significantly decreased.In addition,the fibroblast changed to be round shaped and became less in the POP group.2.The histological structore of rats vagina was similar to human's.In this study,the rat vagina showed continuous biochemical and biomechanical changes from 2 weeks after ovariectomy.Ovariectomized rats might be served as an animal model for developing new treatments for POP.However,it is not the most appropriate animal model because collagen ?is the dominating collagen type in the rat vagina,which is different from human.3.Non-human primates-rhesus macaque is the most appropriate animal model of POP.Further studies of the changes in collagen,elastin and smooth muscle in the vagina of rhesus macaque with spontaneous POP are needed.4.HUMSCs can restore and normalize the weakened vagina tissue of the ovariectomized rats.HUMSCs exert a therapeutic effect via a paracrine manner,rather than differentiating into target tissue types. |
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