Over the past 20 years, the midurethral sling has become the most common operation for stress incontinence in women, due to its proven safety, reproducibility, and long-term efficacy. Although generally considered a permanent implant, on rare occasions surgeons may need to remove the synthetic mesh. While the suburethral portion can be excised transvaginally, removal of the retropubic portion requires an abdominal approach. In this video, we demonstrate the laparoscopic approach in two patients who requested removal of their slings. The first patient is a 42-year-old G4P3 with a history of Ehlers-Danlos Syndrome, who underwent a TLH and retropubic sling for menorrhagia, pelvic pain, and SUI. At her two-week postoperative visit, she was noted to have a vaginal mesh exposure and pain. Her surgeon removed the mesh exposure, but the pain persisted. She requested removal of the remaining mesh and was referred for laparoscopic treatment. After the bladder was backfilled with sterile saline through a three-way Foley, the peritoneum superior to the bladder reflection was incised between the medial umbilical folds with monopolar electrocautery. Sharp and blunt dissection was then carried out and the space of Retzius was entered. Once Cooper's ligaments were visualized, the bladder was drained. While this space is largely avascular, the tissue around the mesh itself can be somewhat vascular due to neovascularization. Once the areolar tissue has been dissected away, the location of the mesh becomes obvious. One can appreciate that the blue color of the sling in this case facilitates identification and dissection of the sling from the surrounding tissues. As can be seen here, the surrounding tissue may be quite adherent to the sling. Once the mesh has been isolated, we transect the mesh as close as possible to the rectus muscle anteriorly. Once the anterior portion has been freed, the surgeon has additional mobility that facilitates further dissection of the sling. Again while largely avascular, the tissue ingrowth around the sling can be more vascular and one should be prepared to address any bleeding with monopolar or bipolar electrocautery. Again one can see that the blue color of the mesh helps with the dissection since the tissue planes can be somewhat obscured by scar tissue. Once the sling segment is dissected out inferiorly as far down as possible to the level of the endopelvic fascia, the mesh is transected and removed. The mesh segments can easily fit through a 5mm laparoscopic port. The blue color of the polypropylene mesh helps identify some remnants of the sling that remained attached to the backside of the pubic bone and these were dissected away. We then turned our attention to the contralateral side and repeat the same procedure. When transecting the mesh at the anterior abdominal wall with hook scissors helps increase mobility and facilitates dissection. We find that a locking needle driver actually provides the best traction on the mesh to aid with dissection. When once the segment of mesh has been isolated, hook scissors are used to transect the mesh at the level of the endopelvic fascia. The area is checked for residual mesh and hemostasis is assured. We close the space of retzius with a running barbed suture. The next patient is a 55 year old G1P1 with a history of anxiety, depression and fibromyalgia who had a retropubic sling 6 years earlier and presented with stage 2 anterior wall prolapse, suprapubic pain and overactive bladder. She underwent a sling release and anterior repair. Over the next several years, she had worsening of her pelvic pain and overactive bladder. She was treated conservatively with minimal improvement in her symptoms and requested removal of the sling. This case was started in much the same manner as the previous case. Here you can see the realer tissue in the space of retzius as we dissect back towards Cooper's ligament. This patient had a clear midurethral sling making it significantly more difficult to locate and identify the two arms of the sling. As you can see, the arms of the sling were slightly more medial than in the previous case. Here you can see the mesh anteriorly against the pubic bone. We were able to isolate the sling down to the endopelvic fascia before transecting the mesh arms. A finger placed vaginally in the distal lateral sulcus can be helpful when transecting the mesh at the level of the endopelvic fascia. Here the mesh is being transected at the level of the rectus muscles anteriorly. Once the segment of mesh was removed on the right side, we were able to access additional fragments of mesh within the endopelvic fascia and these were resected as well. We then turn to the contralateral side and similarly use traction and counter traction to isolate the mesh. Notice how much less the clear mesh stands out from the surrounding tissues. Use of tactile and visual clues can be helpful in locating the mesh arms when a clear mesh was used for the sling procedure rather than a blue mesh. As can be seen in this as well as the previous case, the sling arm needs to be peeled away from the back of the pubic bone which can lead to some bleeding that may need to be addressed with electrocautery. Once we reach the most distal portion of the mesh at the insertion of the endopelvic fascia, the mesh segment is transected and removed. Retropubic removal of mesh arms is a reasonable surgical approach with minimal morbidity compared to laparotomy. Understanding the type of mesh used in the prior retropubic sling can change the surgical approach slightly as the dyed mesh is significantly easier to identify than clear mesh. I'd like to thank the scientific committee for allowing us to present our work. And we're grateful to the NIH for funding this research. Bacterial biofilms are ubiquitous in nature as well as institutional settings including hospitals. In fact, all permanent implanted devices are vulnerable to colonization with bacteria and therefore are susceptible to biofilm formation. Biofilms cause chronic inflammation and tissue degradation similar to what is observed in mesh complications specifically or particularly mesh exposure. Biofilms are problematic because they are relatively resistant to antibiotics in the immune system. And interestingly, biofilm forming capability has been reported for a number of pathogens commonly found in the urogenital tract. Biofilm formation occurs when planktonic or floating bacteria adhere to a hard surface. They then proliferate and form microcolonies which then synthesize an extracellular polymeric matrix which shields it from the surrounding environment. As the biofilm matures, it periodically releases bacteria into the environment which stimulates an immune response and inflammation. And as this process repeats itself, it induces a chronic inflammatory response and increases the risk of device complication. We hypothesize that bacterial biofilm formation is one of the mechanisms contributing to mesh complications and that biofilms are more common in meshes removed for the indication of mesh exposure than for pain. Our objectives were to determine whether polypropylene mesh exposed to bacteria is susceptible to biofilm formation and determine whether polypropylene meshes removed from women with mesh complications contain biofilms. We had two arms to our study, an in vitro arm in which three polypropylene meshes were cold cultured with E. coli at 37 degrees, guiding mesh PS, UltraPro, and Restorel. We then examined them for biofilms at 24 and 48 hours. We also had an in vivo arm in which mesh tissue complex removed from women with mesh complications, 10 from women with pain, and 21 from women with exposure, where we examined only the portion of mesh in exposure patients that had not been in contact with the vaginal contents. We then used fluorescent in situ hybridization with a panbacterial probe that binds bacterial 16S ribosomal RNA and was tagged to a fluorescent tag. For biofilm to be counted as a biofilm, the bacteria had to be present in clusters or microcolonies and encased in an extracellular polymeric matrix. There was no differences in clinical and demographic variables between our exposure and pain groups. The average age was roughly, women were in their 50s, with an average BMI of roughly 30 and a median parity of 3. The implantation time averaged 50 to 60 months, or 5 to 6 years. And interestingly, half of the women in each cohort reported smoking, which is significantly greater than the CDC-reported prevalence of 15% in the U.S. population. There were otherwise no differences in comorbidities, the use of vaginal estrogen, the presence of vaginal discharge, or the distribution of sling versus prolapse mesh across the two groups. The in vitro results are shown here, and as you can see, multiple biofilms or bacterial microcolonies embedded in a matrix were observed on the surface of mesh fibers. We observed no difference in the number of biofilms at 24 and 48 hours, and no difference according to mesh type. The in vivo results are shown here, with the positive controls on the bottom for our bacterial probe with an E. coli culture in the bottom left and a vaginal swab on the right. And what we found was biofilms were present in mesh tissue complexes from women with both pain and exposure, with a total prevalence of 45%, 40% in the pain group, and 48% in the exposure group, with no difference in pain versus exposure. We found no difference in biofilm formation when comparing sling versus prolapse mesh, the presence of vaginal discharge, smoking status, the use of vaginal estrogen, or comorbidities. So in conclusion, biofilms form easily on polypropylene mesh surfaces. Biofilms are common in meshes removed from women with complications, with no difference observed in pain versus exposure, and in this way, biofilms may comprise one of the mechanisms leading to mesh complications. Further research into specific organisms associated with biofilms on urogynecologic meshes and the patient populations most susceptible to biofilm formation is needed. Thank you. I have the title of the next presentation, is Mesh-Induced Fibrosis the Role of T-Regulatory Cells? Oh, I'm sorry. Do I have the wrong one? Sorry. Evaluation of Host, Immune Cellular, and Extracellular Matrix Responses to Sacral-Copropexy Mesh with and without Tension. And Dr. Bighouse will present. Thank you for the opportunity to present our research. We have no relevant disclosures. Augmentation with biomaterials is common and is used to reduce the risk of recurrent prolapse. Polypropylene is a synthetic mesh currently in use for prolapse repair, and over time, multiple studies have driven modifications in the material properties of polypropylene mesh, including lower weight mesh with a larger pore size in an effort to decrease the complications associated with mesh use. Despite these modifications to mesh properties, there are still complications associated with mesh, some of which are listed here. Our goal is to decrease complications from mesh use. In order to do that, we need to learn more about how the body responds to mesh on tension, because tension is required for prolapse repair. When tissues experience mechanical forces like tension, a biochemical response occurs, and that process is called mechanotransduction. This leads to an activation of inflammatory signaling and matrix remodeling. Therefore, we hypothesize that mesh implanted on tension will cause an increase in the vaginal pro-inflammatory host response and impaired matrix remodeling compared to mesh implanted without tension. In order to test our hypothesis, we created a study to determine whether vaginal host immune and matrix responses are altered in a rat sacrocopopexy model when lightweight polypropylene is attached without tension versus with tension. To achieve this objective, we performed hysterectomy and overectomy on 32 rats. We then divided them into four groups. The first group was the control group, and no mesh was attached. The second group, mesh was attached to the vagina only. In the third group, sacrocopopexy on no tension, the mesh was attached to the vagina and then to an overlying vertebrae with no tension applied. And then in the fourth group, mesh was attached to the vagina, tension was applied, and then attached to the overlying vertebrae. The tension was measured in vivo with a tensiometer. The vaginal mesh complexes, or vagina for controls, were excised 90 days after surgery. A pathologist blinded to the treatment groups evaluated H&E and mason-trichrome same slides to determine his morphologic response. He used the system with these eight categories listed here. We evaluated the immune cellular response by examining macrophages present, M1s being the pro-inflammatory macrophages and M2 the remodeling type. And we also measured pro-inflammatory and anti-inflammatory cytokines. In addition to the cellular response, we evaluated the matrix response by measuring MMP concentrations as well as type 1 and 3 collagen. We analyzed our data with the following tests. Now for the results. Here are representative H&E and mason-trichrome images from each group. The black arrows are pointing to the mesh fibers. Overall there was a minimal inflammatory response around the mesh fibers. This table shows the comparison of overall his morphologic score among groups. All groups with mesh had higher scores than the control group. Both sacrocopopexy groups had higher scores than the vaginal mesh only group. However, there was no difference between the sacrocopopexy groups with and without tension. In general, there were low levels of inflammation as evidenced by the following series of box plots. These box plots show the number of macrophages on the Y axis and the treatment groups along the X axis. There was no difference in the number of M1 or M2 macrophages among groups. Here are the box plots showing the cytokine concentrations by group. There was no difference in pro-inflammatory or anti-inflammatory cytokines between groups. These box plots show MMP concentration by group. There was no difference in MMPs between groups. And lastly, there was a predominance of type 1 collagen in all groups. And this box plot shows there was no difference in the type 1 to 3 collagen ratio among groups. In conclusion, attachment of mesh in a sacrocopopexy technique resulted in an increased histological inflammatory response unrelated to whether tension was applied to the mesh. Other markers of cellular inflammation and matrix remodeling showed no differences among experimental groups 90 days after implantation. Thank you. Thank you. Thank you for this opportunity to present our work. We're grateful to the NIH for their support of our research. Over 350,000 urogynecologic meshes are placed annually for repair of prolapse and incontinence with an approximately 5% complication rate. Inflammation of any foreign body, as you've heard, generates a foreign body response, seen here with macrophages surrounding a mesh fiber. In addition, all mesh undergoes some level of deformation, which can impact the magnitude and direction of this immune response. In normal wound healing, the initial macrophage response is primarily an M1 pro-inflammatory macrophage response, which helps promote clearance of bacteria and debris. This is followed by an M2 response, which promotes wound healing. An increased M1 response can result in tissue degradation and exposure. M2 response promotes tissue integration, but if the proliferative response to each fiber overlaps, it can cause fibrosis, encapsulation, and pain. A critical question in this pathogenous paradigm is what promotes an appropriate M2 response. T-regulatory cells are induced from T helper cells at the site of an injury. They play a key role in regulating the adaptive immune system by producing inflammatory cytokines like IL-10 and TGF-beta and performing direct cytolysis of effector CD8 cells. They also modify antigen presentation by dendritic cells. The overall response is a decreased magnitude of the immune response. It is the balance between T-regulatory cells and CD8 effector cells that determine whether immune suppression or activation dominates. T-regulatory cells promote the switch from an M1 to an M2 response. Cytotoxic T cells contribute to an over-robust M1 response. T-regulatory cells can help moderate the fibrosis response where an absence or low T-regulatory cells can contribute to fibrosis. Because T-regulatory cells inhibit a fibrotic response, we hypothesize that Tregs will be inversely related to the amount of fibrosis and that pain samples will have fewer T-regulatory cells than women with exposure. Our aims for this study were to quantify the number of T-regulatory cells relative to the amount of fibrosis in mesh tissue complexes removed from women with complications and to compare the T-regulatory response in women with pain versus exposure. Meshes were explanted from patients with pain or exposure. They were labeled with CD4, a marker of T-helper cells, and FOXC3, a marker of T-regulatory cells. They were imaged under a digital microscope and then the same samples were washed and stained with a standardized trichrome staining protocol which stains collagen blue. These samples were cropped to the exact dimensions and 0.25 millimeter squared grids were overlaid. T-regulatory cells and T-helper cells were counted in each grid and collagen content was quantified using an intensity profile. High levels of collagen content was significantly different than vaginal control tissues and was considered pathologic compared to mild or moderate collagen content. Of pain samples, 4 were prolapse and 4 were sling. Of exposure samples, 3 were prolapse and 5 were sling. Average age was 58, BMI was 29, and median parity was 3. There were no differences in comorbidities such as rate of diabetes, smoking, menopausal status, hormone use. Median duration of implementation was 38 months with a range of 3 to 67 months. On the left side here, you can see that there are a few T-regulatory cells. Those are the red and yellow dots are the T-helper cells and T-regulatory cells. You can see that this corresponds to areas with thickest collagen. On the right side of the sample, there are areas of high T-regulatory cells and low fibrosis. On average, there were 11.3 T-regulatory cells per millimeter squared in areas of high fibrosis compared to 13 in areas of low fibrosis. Overall, there were 100 fewer T-regs per centimeter squared in areas of high fibrosis. There was no difference between pain and exposure. In conclusion, T-helper cells, primarily T-regs, were present years after mesh implantation, challenging the common notion that this immune response is transient. The inverse relationship between fibrosis and T-regulatory cells suggests a protective role. Pain and exposure complications may exist along the same spectrum, and a future area of research would be a potential therapeutic role of T-regulatory cells. Thank you for your time, and I look forward to your questions. Thank you. Dr. Function and Dr. Knight will present this work. Good morning, everyone, and thank you to the Society for allowing me to present our research. We are also grateful to the DoD and to the NIH for funding this work. Animal models have played a critical role in identifying mechanisms of mesh complications. Currently, the non-human primate, the rhesus macaque, is the gold standard model for evaluating prolapsed mesh. This model is advantageous, as it is a large animal model that has similar pelvic anatomy and physiology to humans. Additionally, the vagina is large enough to allow for the implantation of mesh using surgical techniques that are used in humans. However, the non-human primate is limited in that it is a limited resource itself, and studies utilizing the primate can be quite expensive. Thus, there is a need for a less costly large animal model for evaluating prolapsed mesh. The New Zealand white rabbit may serve as an alternative model, as it is relatively inexpensive and is a large animal model. Like the non-human primate, the vagina of the rabbit is large enough to allow for the implantation of mesh using surgical techniques that are used in humans. It is important to note that the rabbit vagina consists of two portions, an external portion and an internal portion. The internal vagina lacks vaginal support similar to the prolapsed vagina in women. Thus, for the purposes of this study, we utilized the internal vagina. Specifically, the objective of this study was to determine the impact of prolapsed mesh on rabbit vaginal smooth muscle morphology and function as compared to the non-human primate. We hypothesized that the thickness and contractile function of the vaginal smooth muscle will decrease as a result of mesh implantation. To assess the impact of prolapsed mesh on the rabbit vagina, restorol was implanted onto the vagina of 10 rabbits for 12 weeks via a lumbar copalpexy. 10 rabbits served as a sham control. After 12 weeks, the mesh vagina complexes were explanted, and the morphology of the smooth muscle was assessed using myosin trichrome staining, while the contractile function of the smooth muscle was evaluated by stimulating the mesh vagina complexes with 120 millimolar KCL to assess smooth muscle function. We found that the rabbit vagina consists almost entirely of smooth muscle. In this myosin trichrome staining with full thickness cross-section of the rabbit vagina, muscle is stained red and collagen is stained blue. As you can see, the majority of the staining is red, signifying a dominance of smooth muscle. Implanting restorol resulted in significant thinning of the vaginal smooth muscle by 23%. Despite implanting mesh on the rabbit vagina in flat configuration, we observed mesh buckling in three animals. Here's an image of a cross-sectional view of an explanted mesh vagina complex with the mesh buckle highlighted. Looking at the mesh buckle histologically, we observed significant thinning of the underlying vagina in the area where the mesh eroded towards the vaginal lumen, and this result is similar to what is seen in women with mesh exposure. The contractile force of the rabbit vagina significantly decreased by 43% with the implantation of restorol, and this result is similar to the 46% decrease observed in the contractile force of the non-human primate vagina with the implantation of restorol via an abdominal sacral copal plexus. If you take a closer look at the data, you will also notice a difference in the scales between the rabbit and non-human primate. The scale for the rabbit data is an order of magnitude higher than that of the non-human primate. Changing the scale so that they match, you can clearly see that the rabbit sham vagina was 558% more contractile than the non-human primate vagina. Similarly, the rabbit mesh implanted vagina was 593% more contractile than the non-human primate mesh implanted vagina. The increased contractile function of the rabbit vagina is largely a result of the rabbit vagina consisting primarily of smooth muscle as compared to the non-human primate in which the vagina, the smooth muscle within the vagina of the non-human primate is not as high. In summary, polypropylene mesh negatively impacts vaginal smooth muscle morphology and function similar to the non-human primate which suggests that the rabbit may serve as an alternative model. Given the abundance of smooth muscle, the rabbit may also serve as a relevant model for studies on vaginal smooth muscle morphology. The associated thinning within the area of a mesh buckle suggests that the rabbit may also serve as a model for understanding mesh exposures. Future studies will investigate the impact of mesh on collagen structure, macrophage characterization and the extracellular matrix of the rabbit vagina. I would like to thank my mentors, Dr. Wiley and Abramowitz for their guidance with this work. I would also like to thank lab members Amanda, Gabby, Stacy, Megan and Chana for their contributions to this work. Again, I would like to acknowledge the DOD and the NIH for supporting this work. Thank you all for your time. Thank you. We'd like to invite our last three authors to come and accept questions. Dr. Bighouse, when you did your surgery for the implantation, it was really quite impressive there how beautiful all of the mesh incorporated so beautifully at 90 days. Now, your rats, were they all over-erectomized at the same time as the surgery? And do you think the remodeling that's induced by the over-erectomy could encourage engraftment of the mesh into the vagina? So the over-erectomy was performed at the same time that the mesh was implanted and certainly can definitely play a role, I think. And, you know, it would be something we could look at having a group that was over-erectomized versus not to tease out those differences. Yes. Hi. Kristen Jacobs from Chicago. I have a question regarding the last presentation. And thank you for your work and I appreciate the proposal or the presentation in the first meeting. Question about the smooth muscle functionality and its relationship and importance with pelvic organ prolapse and subsequent repair. Is it otherwise a marker for overall disorganization of connective tissue or where is the link between why smooth muscle contractility is important for prolapse repair? We know that with women with prolapse, they already have impaired smooth muscle, at least in terms of the bundles and the organization of the actual smooth muscle within the vagina. And we've seen that in our priming studies. The smooth muscle is always, feels like it's negatively impacted a lot more than the collagen, for example. So it's just that right now we're really, that's why in this particular study we focus more so on the smooth muscle because it's always so negatively impacted. And we want to maintain the function and the tone of the vagina. So that's smooth muscle's function. So we want to make sure that we're not really negatively impacting or impairing that function. Could I ask a question about your quantification of smooth muscle force? So you expressed it for a cross-sectional area? With the smooth, we do about the volume. So this is, I do, so when I, when we do the vaginal contractility assay, I clamp the mesh vagina complex together. I don't isolate the smooth muscle. So then I, that will, that particular mesh tissue complex will respond to the KCL and we'll have a force that results. And we divide that force by the actual volume of the tissue because we found that the length really could impact our force. So when you do it that way, of course it's going to be less because there's less smooth muscle cells. But if you normalize it to the number of smooth muscle cells in the tissue, does it mean that the smooth muscle, the terrible organization of that smooth muscle, is it still contracting normally or is it also impaired? Not only is it reduced, but it doesn't contract well at all. You see what I'm saying? Yeah. So you're saying your first part, you said that if we normalize it by the number of smooth muscle cells, it's hard to do that with these tissues because we would have to, this particular assay, right when we sacked the animals, it's tested right away. And after that, I don't know if during the whole testing process, we might disrupt some of the smooth muscle. So I can't really give an exact number in terms of how many cells are present. That would be great if I could do that to normalize it, to make that normalization better. But unfortunately I can't. For some people, they just fix it, fix the tissue while it's hung in the bath and then look at it histologically to see how much smooth muscle was in that tissue at the time of contraction. Yeah, that's going to be something we'll definitely look into in the future. It's just an interesting question about the actual cellular nature of the problem. Yes, likewise, that was my question. I work with a group where we actually would always fix the tissue and section it in a cross-sectional area and count the cells in the cross-sectional area of smooth muscle. And in fact, in post-menopausal women that were age-matched, we actually found higher contractile forces in prolapse, advanced prolapse, than in normal controls. And so you sometimes find surprising results. So you may want to consider correlating what you do have with the cross-sectional area as well. Thank you. Thank you. Thank you to our presenters. Thank you. Good morning. Polypropylene mesh is used in the treatment of gynecologic conditions, including pelvic organ prolapse. Mesh use is beneficial for many women, but it's not without complications. These complications can include exposure through the vagina, erosion and chambering structures, and pain, among others. Polypropylene mesh is commonly considered inert, but has been increasingly associated with a persistent inflammatory response. The macrophage is considered to be the primary mediator of this foreign body reaction to synthetic biomaterials. In the absence of a foreign body, when looking at the physiologic wound healing response, we see that macrophage response escalates at around two to three days post-injury. This response initially consists of macrophages of the M1 phenotype, with a transition to the M2 phenotype later on. As we have heard in some of the previous presentations today, M1 macrophages are pro-inflammatory in nature, whereas M2 macrophages are more anti-inflammatory and pro-wound remodeling. Recent studies have indicated that a specific macrophage subtype driving the early host response is really critical in determining the long-term response to the implant. And so this is really an interesting idea, because potentially the early response could help to predict if a mesh-related complication might develop. If we combine the ideas of physiologic wound healing with the foreign body reaction, in an ideal situation, after an implant is introduced into the body, we would first have this nice, necessary M1-type response, which transitions in a timely manner to an M2-like profile. And what we do know is that in the biomaterials community, materials that are associated with this early host response that mimics this timely phenotypic switch that you see above, are associated with better downstream outcomes. But polypropylene has been associated with both an early and prolonged response that is predominantly pro-inflammatory. And so we do have interleukin-4, which is a cytokine that can induce or force this M1 to M2 phenotypic transformation artificially. In our lab, we have previously developed a coating that releases IL-4 from mesh, and can release more or less IL-4, depending on how much coating we place. Currently though, we are limited by in vitro release kinetics data, which we know can oftentimes be very different from the in vivo reality. In vitro profiles show an immediate release with early exhaustion of our cytokine, which may not be completely physiologically relevant. So if our goal is to release IL-4 in a way that mimics normal wound healing, we need to see what is happening in vivo. To do this, our strategy is to fluorescently tag our IL-4 protein, coat it onto implantable mesh, and then monitor with in vivo daily imaging and longitudinal cohorts of animals. First, to fluorescently tag IL-4, we subjected to a fluorescent labeling reaction with Alexa Fluor 594. To verify this, we used high performance liquid chromatography. Then we assessed if the bioactivity of IL-4 had been maintained in the presence of the fluorophore tag. When IL-4 is added to naive macrophages, the production of the M2-centric enzyme Arginase 1 is upregulated. So we supplemented both untagged and tagged IL-4 into the culture media of naive macrophages, fixed cells after 48 hours, and immunofluorescently labeled them for Arginase 1, which you will see as a green color on the next slide, co-localized with cellular nuclei in blue. Qualitatively, compared to our appropriate control groups, macrophages subjected to untagged and tagged IL-4 showed similarly increased Arginase staining. Quantitatively, this was confirmed with cell profiler image analysis software. Next, we wanted to show that tagged IL-4 could be incorporated into our previously developed coating, so we first modified polypropylene in a way that gave it a net negative charge. Then this was dipped into a solution of positively charged chitosan polymer, followed by a dip into negatively charged dermatin sulfate polymer complex to our tagged IL-4. This process can then be repeated as many times as you want in order to build up a coating that contains IL-4. The presence of tagged IL-4 in the coating was verified by using confocal fluorescence microscopy, and then we wanted to verify that it could also be seen in an in vivo animal imaging device, and for this we chose an IVAS-200 imaging platform. Compared to the appropriate control meshes, mesh that had tagged IL-4 even after terminal sterilization with ethylene oxide gas showed a nice, strong signal in the red channel, so we were able to verify that the IVAS could be used moving forward for our animal studies. Future directions include implanting this tagged mesh subcutaneously onto the abdomens of mice, and then monitoring loss of fluorescence signal with daily in vivo imaging in longitudinal cohorts of animals with the ultimate goal of correlating downstream tissue responses to the in vivo release pattern seen upstream. In conclusion, IL-4 was successfully tagged with maintained bioactivity and was able to be incorporated into our previously developed layer-by-layer electrostatic coating. It is the hope that with in vivo release profiles elucidated, we can find a physiologic pattern of release that optimizes downstream outcomes of mesh incorporation with the ultimate goal of finding some sort of a pattern of release that reduces mesh-associated complications. Thank you very much, and I look forward to your questions. Thank you. Thank you. Thank you. Okay, thank you so much for the kind introduction, and thank you all for being here today. This work was supported in part by NSF. In the U.S., one in 10 women receive surgical intervention for pelvic organ prolapse, with 30% undergoing reoperation, or 10 to 25% having complications. Efficacy of treatment may be impacted by an incomplete understanding of the underlying mechanisms for POP. Elements such as injury to the levator ante muscles during childbirth and connective tissue dysfunction have been implicated. In the setting of prolapse, studies have shown vaginal tissue to display a decrease in smooth muscle cell composition, organization, as well as contractile function. Contractile function has been primarily investigated independently in the circumferential or axial direction. However, the vaginas under multiaxial loading, which may affect the contractile response. The objective of the study was to assess the contractile response of a marine vagina under various circumferential and axial loads, where it was hypothesized that loading, that loads above a specific range can cause a diminished in the contractile response. Animal models have been widely utilized to study vaginal contractility. This study utilized vaginal tissue from control mice. In order to preserve the cylindrical tissue geometry, as well as maintain the native cell matrix interaction, an extension inflation device was used. The length of the tissue was manually adjusted with a linear stage and a constant pressure was applied through the lumen. Axial force was measured with a force transducer and the outer diameter was optically tracked real time. The tissue was explanted and the vagina was secured within the testing device with sutures. Explaining the vagina, removing the natural connections resulted in tethering or a reduction in length of the tissue. Therefore, a physiologic length was estimated and restored via axial extension, followed by preconditioning of the two muscle cells. At the estimated physiologic length, the tissue was contracted with 40 millimolars of KCL inducing maximum contraction while being subjected to various constant pressures. The experimental protocol was repeated assessing the contraction as a function of the length or axial extension of the tissue below and above the estimated physiologic length while being subjected to a constant pressure of seven millimeters of mercury as determined by a pilot study with intravaginal pressure catheterization. Histological analysis of the two muscle cells was performed on circumferential and axial sections with Mason's Trichrome staining. Statistical analysis was conducted with a one-way ANOVA as well as T-TEF. In figure A, pressure is on the X-axis and on figure A, along the X-axis pressure is located and the change in diameter with contraction is along the Y-axis. From zero millimeters of mercury to 15 millimeters of mercury, there was a significant decrease in the magnitude of circumferential contraction. In figure B, we see length along the X-axis and the magnitude or change in diameter along the Y where no statistical significance was detected with respect to loading. In figures A and B, it shows the change in the axial force readings with respect to loading where no statistical significance was detected. Histological analysis resulted in a significant composition within the circumferential direction. This study investigated contraction of the mouse vagina as a function of circumferential as well as axial loading. Findings suggest that within the mouse vagina that the two muscle cells are more aligned within the circumferential direction and are sensitive to circumferential loads such as pressure used within the study. Methods within the study may be leveraged to further investigate surgical outcomes as well as pharmacological treatment and agents used in assessing contractile function of tissue. I would like to thank my lab collaborators as well as funding sources and Dr. Murtada in training in the bioxial contractility protocol. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you. Thank you for the opportunity to present our research. We have no relevant disclosures. Polypropylene is a permanent mesh material that's in current use to augment prolapse repair to reduce the risk of occurrence. In the past, heavier weight, smaller pore polypropylene mesh was used. Heavyweight mesh elicited a robust pro-inflammatory chronic response. To avoid this altered host response, lightweight large pore mesh is currently in use. Despite these changes made to the material properties of polypropylene, we still have complications with its use that you can see listed here. One problem with polypropylene is that it's 100 times stiffer than that of vaginal tissue. The stiffness mismatch can lead to stress shielding, maladaptive remodeling, and long-term complications. Ultimately, there's altered biomechanics due to a mismatch in mechanical properties between polypropylene and the vagina. We need a material that better matches the properties of the vagina. One such material is polycarbonate urethane, or PCU. It is an elastomer and is currently being used in cardiac research and orthopedic applications in humans. PCU is compliant yet strong and has toughness. It can be made to have the same properties of that of the vagina with an elastic modulus around 10 megapascals. Since PCU has similar mechanical properties to that of the vagina, we hypothesize that PCU mesh implanted on the rat vagina will elicit a similar host response with normal remodeling to that of lightweight polypropylene implanted on the rat vagina. In order to test this new material, we created this study to compare the vaginal host immune and ECM responses to PCU mesh to those of lightweight polypropylene mesh. We used the PCU mesh that was 3D printed and had one millimeter pore sizes, and then we used the lightweight polypropylene that we're currently using in our sacral copepexy surgeries that had a pore size of 1.5 millimeters. Next, we performed hysterectomy and overectomy on 24 rats. We divided them into three groups. The first group was the control with no material attached to the vagina. The second group had PCU attached to the vagina, and the third group had polypropylene attached to the vagina. The same histomorphologic scoring system was used as in my other project. The pathologist was blinded to the treatment groups and evaluated the H&E and mason-trichrome-sane slides with the eight categories listed here. The immune cellular response was evaluated by measuring the types of macrophages present, and an Illuminex assay was performed to measure pro-inflammatory and anti-inflammatory cytokines. Finally, we evaluated the extracellular matrix by measuring MMPs in type one and three collagen. We performed the following statistical tests, and here are our results. On the left are the H&E representative images for each group and mason-trichrome on the right. Overall, there was a minimal inflammatory response. Both mesh groups had higher inflammatory histologic scores than the control group. When looking at PCU compared to polypropylene, there was no difference in score between those two groups. Here are the box plot results for the type of macrophages. The number of macrophages is on the Y-axis, and the treatment groups are on the X-axis. There was no difference in the number of macrophages between groups. These box plots show the cytokine concentrations. Cytokines are on the Y-axis, and again, treatment groups are on the X-axis. There was no difference in pro-inflammatory or anti-inflammatory cytokines between groups. Here are the box plots for MMP concentrations. Here are the box plots for MMP concentrations, and again, there were no differences between groups. Finally, there was a predominance of type 1 collagen in all groups and no difference in 1 to 3 collagen ratio. In summary, there was a minimal inflammatory response around the lightweight polypropylene, which is in contrast to the response to heavyweight mesh that I showed you at the beginning of this presentation. As you can see here with the heavyweight mesh on the left and the lightweight polypropylene in the middle. The host response to PCU was similar to that of lightweight polypropylene, as you can see in the PCU image on the right. Furthermore, PCU did not have a more robust response than currently used lightweight polypropylene and was similar to other in vivo studies using PCU. In conclusion, PCU offers strength with less stiffness than polypropylene and does not elicit more inflammation in the rat vagina. Therefore, PCU is a promising material for prolapse mesh. Thank you. Thank you. Thank you. I'd like to invite our last three presenters back to the podium. Dr. Bickel and these presentations are open for questions. Yes. I take it you don't want to sit down over here? No. So I'm wondering about, what was the time point again on the last presentation? What time point? Yeah. 90 days. So do you think there could be changes early on? You know, the immediate acute reaction could modify the long-term outcome. Of course, the long-term outcome is the most important, but you think that an early time point may have shown differences? It possibly could, but then, you know, that's in thinking of designing the study, we thought of an early time point, but then the time point that really matters is going to be the chronic end point. And so that's why we decided to go with the 90 days only. Okay. Yes. Thank you. My name is Austin Hill with Troy Health in Cincinnati. My question's for the third presenter. Thank you for that presentation. It's very interesting thinking of new meshes that we could use. My question's regarding, we talked about the stiffness of the material and the stiffness of the vagina, and you said we could keep the strength of the vagina with that material, but we didn't talk about it in the presentation, at least how that material compares with strength of the vagina. So with us using these meshes for prolapse repair, if we were going to change something that was less stiff and more stretchy, how could that impact the possibility for recurrence of prolapse or the strength of the material over time? And so the hope is that since it is a strong material, just like polypropylene is strong, we would have good outcomes in terms of decreased recurrence. But since we are matching more of the properties, we would decrease complications like the mesh erosion and mesh exposure. So I guess the strength of the material is the same and its ability to maintain that strength is the same, but it's just more stretchy. Correct. Okay, great, thanks. Ali. Ali Lugg, Henry Ford, Detroit, Michigan. My question is in regards to your last presentation. So thank you for your work. Just, I'm curious, what would be your next step in terms of translating the PCU, the information that you received, to the next stage, more clinical? Thank you for your question. So the next step that I anticipate is using a larger animal model and then also using it in a surgical technique, so a sacrocopalpexy surgery. I have a question for Ms. Clark. In the studies that you all performed looking at this biaxial contractile response, did you see any changes based on the region of the vagina that you were taking specimens from? Certainly we have a lot of evidence that there are regional differences in the contractile smooth muscle function. Great, yes, that's an awesome question. And particularly with the testing protocol we used, we were able to keep the full length of the vagina intact. So one thing that we were looking at initially was respect to regional differences, looking at the changes in contractions between the two regions. However, with utilizing KCL, we particularly didn't find any differences, but I'm pretty sure utilizing other pharmacological agents, that could be detected. Thank you. Are there any further questions? I have one more, one last one about the IL-4. IL-4. Okay. Why do you want to put the IL-4 on a coated mesh? Have you thought about just using a tagged slow-release IL-4 biomaterial? Could you use that as a control at least to see if it's just as good as or better than mesh? Yeah, I mean, we could use a different biomaterial that had properties similar to IL-4. Is that the premise of your question? Oh, you know, like a nanoparticle, a gel, or something like that. Just inject it in there, because when you really want this macrophage regulatory process, it would be good just to have it right there during the early healing process. And why do you need the mesh? Yes, that's an excellent question. And so you could definitely inject something. What we're trying to do is mimic a more natural physiologic wound healing response. And so it's my ideal goal to wait until about day three to start the release. And so I feel like maybe, right, if I was to inject something upon implantation, it could be compromised, maybe be destroyed by day three. But I really want to get at, I think we kind of get into a rut thinking that, you know, M1 is bad and M2 is good, but you really do need that M1 profile initially. And it's my belief that you need that till about day three, just as if we didn't have a foreign body there. And so by coating it onto the mesh, I can actually try to protect it and delay the release, and then let it come out naturally at day three without the need for an additional injection or a breach of the epithelium again. Yeah, thank you for your question. Very interesting, thank you. Thank you. And thank you for everyone joining us. Short, please join the Basic Science Big at your earliest convenience for those of you who do this work. Thank you. Thank you. Thank you. Thank you.

interleukin-4

mesh-related complications

implantable mesh

vaginal tissue

circumferential loads

polypropylene mesh

polycarbonate urethane mesh

prolapse repair

host immune response