Ectopic Implantation Research Articles

O-030 Disturbed homeostasis of regulatory T-cells and imbalanced signal transducer and activator of transcription 5 (STAT5) in women with ovarian endometriosis

Abstract Study question Do women with endometriosis have altered cytokine signalling and regulatory T-cell (Treg) homeostasis compared to controls? Summary answer Altered homeostasis of circulating Treg cells seems to be associated with STAT5 phosphorylation, which is significantly increased in women with ovarian endometriosis compared to controls. What is known already Cytokine signaling influences homeostasis of regulatory T-cells (Treg), which play a crucial role in cell-mediated immunity and are responsible for certain immune disorders. Endometriosis is a condition associated with altered immune response to endometrial cells. While some animal studies have shown that Treg deficiency facilitates ectopic implantation of endometriosis, a recent study performed in a small number of women with endometriosis has shown the decreased proportion of a specific-activated subset of Treg cells in the endometrioma compared to controls. There is no data characterising cytokine signalling in circulating Treg subsets, which could underlie their altered homeostasis in women with endometriosis. Study design, size, duration A prospective cohort study including 14 women with endometriosis and 10 controls at a university-based tertiary care centre for endometriosis. The data were collected from August 2020 to September 2021. Serum blood samples were collected in the early follicular phase of the menstrual cycle, in women not using any hormonal treatment. Early follicular steroid hormone levels were measured. A detailed expert ultrasound examination was performed to diagnose and accurately map endometriotic lesions. Participants/materials, setting, methods We investigated signal transducer and activator of transcription (STAT) signalling in peripheral blood Treg subsets, including CD45RA+FOXP3low+ naive Tregs (nTregs). Expression of proliferation marker Ki-67 and STAT activation also in FOXP3- conventional (Tcon) CD4+T-cells was monitored, hypothesising that defects in this pathway could be associated with altered Treg/Tcon homeostasis in endometriosis. Immunophenotyping and flow cytometry methods with phospho-specific antibodies that allow analysis of basal STAT activation/phosphorylation also in rare subsets of Treg cells were used. Main results and the role of chance Among women with endometriosis, 5 had evidence of ovarian and deep infiltrating endometriosis (DIE), 5 had only ovarian endometriosis and 4 had only evidence of DIE. Groups were comparable in their clinical characteristics, there was no difference in the median age of women with endometriosis and controls; 31 (range 23-45) vs. 34 (range 27-39). We demonstrated significantly higher levels of phosphorylated STAT5 (pSTAT5) in nTreg from women with ovarian endometriosis compared to controls (p = 0.026). To assess the homeostatic balance between the nTreg and Tcon, we compared pSTAT5 levels between these cell subsets in each patient. STAT5 phosphorylation was significantly higher in Tcon than in nTreg subset (p < 0.001). Considering STAT5 activation in nTreg subset, this was positively correlated with the proportion of nTreg among CD4+T-cells (rs=0.54, p = 0.027) in all women with endometriosis. We also found a significant positive correlation between pSTAT5 levels in Tcon from women with endometriosis and the percentage of Ki-67+ cells in the Tcon subset (rs=0.66, p = 0.005) as well a significant correlation between luteinizing hormone (LH) levels and pSTAT5 in Tcon subset of CD4 cells (rs=0.71, p = 0.02). This implies LH-dependent imbalanced STAT5 signalling that could be responsible for increased Tcon homeostatic proliferation. Limitations, reasons for caution Our sample size is relatively small and the findings should be validated in future prospective studies. Due to the sample size, there is a risk for Type I error, although the results are coherent in indicating disturbance of Treg homeostasis. Wider implications of the findings Our study suggests that altered STAT5 signalisation may be associated with disturbed nTreg/Tcon homeostasis, which seems to be mostly prominent in women with ovarian endometriosis. If confirmed by further studies in the future, targeted treatment options could be developed to supress progress of endometriosis in this group of women. Trial registration number not applicable

In Vitro and Ectopic In Vivo Studies toward the Utilization of Rapidly Isolated Human Nasal Chondrocytes for Single-Stage Arthroscopic Cartilage Regeneration Therapy.

Nasal chondrocytes (NCs) have a higher and more reproducible chondrogenic capacity than articular chondrocytes, and the engineered cartilage tissue they generate in vitro has been demonstrated to be safe in clinical applications. Here, we aimed at determining the feasibility for a single-stage application of NCs for cartilage regeneration under minimally invasive settings. In particular, we assessed whether NCs isolated using a short collagenase digestion protocol retain their potential to proliferate and chondro-differentiate within an injectable, swiftly cross-linked and matrix-metalloproteinase (MMP)-degradable polyethylene glycol (PEG) gel enriched with human platelet lysate (hPL). NC-hPL-PEG gels were additionally tested for their capacity to generate cartilage tissue in vivo and to integrate into cartilage/bone compartments of human osteochondral plugs upon ectopic subcutaneous implantation into nude mice. NCs isolated with a rapid protocol and embedded in PEG gels with hPL at low cell density were capable of efficiently proliferating and of generating tissue rich in glycosaminoglycans and collagen II. NC-hPL-PEG gels developed into hyaline-like cartilage tissues upon ectopic in vivo implantation and integrated with surrounding native cartilage and bone tissues. The delivery of NCs in PEG gels containing hPL is a feasible strategy for cartilage repair and now requires further validation in orthotopic in vivo models.

Unusual Case of Tubal Stump Pregnancy after Salpingectomy

Ectopic pregnancy implantation on the tubal stump after salpingectomy is a rare location for extrauterine pregnancy, whose pathogenesis is still unknown. We report in this work the case of a 37 years old patient who had been operated 10 years ago for a right ectopic pregnancy and who was presented to the emergency department for a ruptured ectopic pregnancy and in the surgical exploration showed a ruptured pregnancy on a salpingectomy stump.

Tubal Endometriosis: From Bench to Bedside, A Scoping Review.

Tubal endometriosis (EM) refers to the detection of ectopic endometrial implants on tubes. It may cause a significant defect of the tubes, translating into dysmenorrhea, pelvic pain, and infertility. We aimed to evaluate the disease characteristics, prevalence, histopathological findings and genetic profile of patients with tubal EM. A thorough search of three electronic databases was performed for studies that presented outcomes of patients with tubal EM. Thirteen studies (four observational, seven case reports, two genetic) were considered eligible for inclusion. The prevalence of tubal EM ranged from 6.9% to 69%. The predominant symptoms for referral of patients were infertility and abdominal pain. Women of reproductive age underwent salpingectomy for the management of the disease. Only one case of malignant transformation was recorded in a 60-year-old patient. The prevalence of tubal EM ranges depending on the indication for surgery, the presence of concomitant pelvic EM and the type of diagnosis and treatment. Further, more extensive, larger studies are warranted to evaluate the impact of tubal EM in the progression and prognosis of EM, the effect of salpingectomy in the improvement of disease-related symptoms and to designate the group of patients that could benefit from risk-reducing salpingectomy based on the risk of developing ovarian malignancy.

The role of ZBTB16 in odontogenic differentiation of dental pulp stem cells

ObjectiveOdontogenic differentiation of dental pulp stem cells (DPSCs) is highly controlled by the activation of several transcription factors. The zinc finger and BTB domain-containing 16 (ZBTB16) gene encodes a BTB/POZ domain and zinc finger containing transcription factors and is involved in several biological processes, but little is known about its role in odontogenic differentiation. The main goal of the current study was to determine the role of ZBTB16 in odontogenic differentiation of DPSCs. DesignZBTB16, runt-related transcription factor 2 (RUNX2), and osterix (OSX) were silenced via small-hairpin RNA (shRNA) lentivirus. The odontoblastic differentiation of DPSCs was detected by alkaline phosphatase (ALP) staining, activity measurement, and alizarin red S staining in vitro. The gene and protein expression levels were assessed by RT-qPCR and western blotting. Further, an ectopic implantation experiment was performed to explore the role of ZBTB16 in mineralization regulation in vivo followed by histological examination. ResultsThe silencing of ZBTB16 attenuated ALP activity and mineralized nodules formation by DPSCs. In addition, knockdown of ZBTB16 impaired the expression of markers involved in odontogenic differentiation, including dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, and collagen 1 in vitro and vivo. Silencing the OSX gene suppressed ZBTB16 expression and, in turn, OSX expression decreased after ZBTB16 knockdown. However, shRUNX2 did not suppress ZBTB16 expression and shZBTB16 did not affect RUNX2 expression. ConclusionsZBTB16 may play an important role in modulating the odontoblastic differentiation of DPSCs and act as a regulator of OSX in a possible feed-back cycle independent of RUNX2.

Ultrasound-guided percutaneous implantation of rabbit VX2 carcinoma, using a coaxial technique and gelfoam pellet injection combination to establish a rabbit liver tumor model.

PURPOSE We aimed to investigate the safety and tumor seeding rate of a coaxial implantation technique combined with injection of a gelfoam pellet in establishing a VX2 liver tumor model in rabbits. METHODS A VX2 liver tumor model was established in 60 male New Zealand white rabbits, which were randomly divided into 3 groups (20 in each group) based on implantation technique (all performed under ultrasound guidance): group A, single needle only; group B, single needle with injection of a gelfoam pellet; or group C, coaxial technique with injection of a gelfoam pellet. The rates of liver tumor formation and tumor seeding to extrahepatic tissues were compared 2 weeks after implantation. Data were also collected regarding procedure time, number of punctures, occurrence of complications, and mortality rate. RESULTS A VX2 liver tumor model was established in all 60 rabbits (100%, 60/60). Ectopic implantation rate was 70% (14/20) in group A, 35% (7/20) in group B, and 5% (1/20) in group C, with significant difference among the groups (p < 0.001). Post hoc analysis showed significant difference between group A and group C (p < 0.001). However, there were no significant differences between group B and group A or group C (p = 0.027, p = 0.048, respectively). There were no significant differences among the groups in terms of procedure time (p = 0.405) or number of punctures (p = 0.612). No complications or deaths occurred. CONCLUSION A coaxial technique with injection of a gelfoam pellet is an effective and safe method for VX2 liver tumor implantation in rabbits, and this technique can reduce the risk of tumor seeding to the abdominal wall and omentum.

Graphene-Oxide Porous Biopolymer Hybrids Enhance In Vitro Osteogenic Differentiation and Promote Ectopic Osteogenesis In Vivo.

Over the years, natural-based scaffolds have presented impressive results for bone tissue engineering (BTE) application. Further, outstanding interactions have been observed during the interaction of graphene oxide (GO)-reinforced biomaterials with both specific cell cultures and injured bone during in vivo experimental conditions. This research hereby addresses the potential of fish gelatin/chitosan (GCs) hybrids reinforced with GO to support in vitro osteogenic differentiation and, further, to investigate its behavior when implanted ectopically. Standard GCs formulation was referenced against genipin (Gp) crosslinked blend and 0.5 wt.% additivated GO composite (GCsGp/GO 0.5 wt.%). Pre-osteoblasts were put in contact with these composites and induced to differentiate in vitro towards mature osteoblasts for 28 days. Specific bone makers were investigated by qPCR and immunolabeling. Next, CD1 mice models were used to assess de novo osteogenic potential by ectopic implantation in the subcutaneous dorsum pocket of the animals. After 4 weeks, alkaline phosphate (ALP) and calcium deposits together with collagen synthesis were investigated by biochemical analysis and histology, respectively. Further, ex vivo materials were studied after surgery regarding biomineralization and morphological changes by means of qualitative and quantitative methods. Furthermore, X-ray diffraction and Fourier-transform infrared spectroscopy underlined the newly fashioned material structuration by virtue of mineralized extracellular matrix. Specific bone markers determination stressed the osteogenic phenotype of the cells populating the material in vitro and successfully differentiated towards mature bone cells. In vivo results of specific histological staining assays highlighted collagen formation and calcium deposits, which were further validated by micro-CT. It was observed that the addition of 0.5 wt.% GO had an overall significant positive effect on both in vitro differentiation and in vivo bone cell recruitment in the subcutaneous region. These data support the GO bioactivity in osteogenesis mechanisms as being self-sufficient to elevate osteoblast differentiation and bone formation in ectopic sites while lacking the most common osteoinductive agents.

Development and characterization of poultry collagen-based hybrid hydrogels for bone regeneration.

ABSTRACTPurpose:Poultry by-products can contribute as an innovative natural source for the development of composites based on polymers and minerals aiming at bone regeneration. The objective of this study was the physicochemical and biological characterization of collagen-based hydrogels crosslinked with ultraviolet (UV)-riboflavin.Methods:Pure hydrogels of 100% collagen (G1) or hybrid hydrogels, 90% collagen:10% apatite (G2), 90% collagen:10% nanokeratin (G3), and 90% collagen:5% apatite:5% nanokeratin (G4) were characterized by scanning electron microscope, Fourier-transform infrared spectroscopy, differential scanning calorimetry, swelling degree and quali-quantitative histological analysis. Ectopic implantation in subcutaneous tissue in mice at one, three and nine weeks allowed to assess the inflammation (neutrophils, lymphocytes, macrophages, and giant cells) and repair (neovascularization, and connective tissue) to determine biocompatibility and the integrity of biomaterials to score their biodegradability. Histomorphometry on critical size defects in rat calvaria at one and three months evaluated the percentage of bone, connective tissue, and biomaterials in all groups.Results:The hydrogels presented porous microstructure, water absorption and physicochemical characteristics compatible with their polymeric and/or mineral composition. All materials exhibited biocompatibility, biodegradability, and low osteoconductivity. G2 showed greater density of new bone and biomaterial than the G1, G3 and G4.Conclusions:The collagen-apatite group formulation suggests potential for development as osteopromoting membrane.

Endometriosis an Ayurvedic View

Endometriosis is a chronic estrogen dependent condition characterized by the ectopic implantation of functional tissue lining of uterus. The exact cause of endometriosis is not detected yet even though there are some theories which explain the pathogenesisof endometriosis. Retrograde menstruation and Coelomic metaplasia theories are included under this. Based on the site of appearance classification of endometriosis can be done as pelvic, extra pelvic and remote. Appearance of endometrial tissue in myometrium is termed as adenomyosis and endometriotic deep lesions in ovary is Endometrioma. The risk factor for aggravating endometriosis is cyclical hormonal changes especially estrogen. Ayurveda does not name endometriosis as a separate disease title. In Ayurveda the diseases related with genital tract is discussed under yoniroga. Brihatrayee and laghutrayee describe 20 yoni rogaunder various dosha vitiation. Due to the peculiar characteristic of pain, generally endometriosis is taken under vatika yoni vyapath. Even though vatais predominant, pithaand kaphadoshaare also involved in various stages. Retrograde flow of menstrual blood, genetic factors, lymphatic and vascular spread are considered as the causative factors. Nidanaof endometriosis include mithayahara,-vihara, arthava dusti, bija dushti,use of apadravyas (iatrogenic causes) and daiva(genetic or chromosomal factors). Various type pain and infertility are the main clinical features of endometriosis. Pain is due to increased vata in kadee desa. Through the study the Nidana of endometriosis is analyzed and compared with latest theories. Endometriosis is not considered as a life threatening disease it is a life altering disease that require timely diagnosis and treatment. Currently there is no universally acceptable treatment protocol for endometriosis is available

Uterine factors essential in embryo implantation are also associated with ectopic implantations

Implantation is fundamental to successful establishment of both the trophoblast (placenta) and embryo. Free-floating cells originating from the oviduct and uterus leads to benign ectopic lesions (endosalpingiosis, ES and endometriosis, EM respectively) and may also play a role in the pathogenesis of other ectopic implantations. We hypothesized that mechanisms driving ES will give crucial insight into early pregnancy implantations and failures, including recurrent pregnancy loss, placenta accrete spectrum, and ectopic pregnancies. To fully test this hypothesis, we developed a novel mouse model to interrogate the role of implantation signaling factors in promoting non-cancerous ectopic lesions. We developed an animal model of ES using auto-fluorescing (inherent in the donor mouse cells) oviductal tissue. To test how implantation factors affect ES and EM development donor mice were treated daily with progesterone from postnatal day 2-10 to induce infertility by blocking implantation (progesterone-induced uterine gland knock out, PUGKO). Oviductal and endometrial tissue from PUGKO and vehicle control (VC) mice was harvested. Recipient mice received either oviductal or endometrial tissue alone or co-injected with endometrial tissue (non-auto-fluorescing) to reintroduce implantation factors. After a month, recipient mice were euthanized to count auto-fluorescing legions. PUGKO endometrium had significantly reduced implantation (2.14 lesions/mouse (L/M), p=0.042) compared to VC (4.71 L/M, figure 1). PUGKO endometrium with WT endometrium restored implantation. Additionally, oviduct implantation (0.92 L/M, p=0.028) was significantly increased when injected with WT endometrium (2.75 L/M, figure 2). Implantation factors are crucial to initiate and promote ectopic tissue growth. Our novel mouse model demonstrates that the removal of endometrial implantation factors reduces ectopic growth. Adding implantation factors increases oviductal tissue implantation. This model allows us to study the of implantation of these lesions, yielding the development of novel therapies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Enzymatically functionalized RGD-gelatin scaffolds that recruit host mesenchymal stem cells in vivo and promote bone regeneration

Critical-size bone defects are imposing a substantial biomedical burden. Despite being long regarded as a potential approach to mitigate this burden or an alternative to bone grafts, bone tissue engineering (BTE) has virtually not proceeded to widespread clinical practices. In the BTE field, it is highly required to find a facile method to prepare active scaffolds with tailored biological functions. Here, we immobilized cell adhesive RGD motifs onto gelatin sponge (GS) scaffolds through enzymatic linking. On the basis of the resulting RGD-functionalized GS (RGD/GS) scaffolds, we developed a new and convenient strategy for bone defect repair, in which the scaffolds were first used to recruit mesenchymal stem cells (MSCs) from skeletal muscle, immediately followed by their engraftment into bone defect. We demonstrated significantly enhanced host cells homing into RGD/GS scaffolds as a result of specific RGD-integrin interactions, and the recruited host cells showed a strong osteogenic differentiation potential. After ectopic implantation of cell-laden RGD/GS scaffolds into critical-size mouse bone defects, marked bone tissue regeneration occurred. The presented strategy not only provides an agile route for the preparation of bioactive scaffolds and the construction of osteoinductive bone-graft substitutes, but also avoids or minimizes the complicated and laborious cell isolation, in vitro expansion and cell seeding procedures used in the conventional BTE.

Hyaluronan size alters chondrogenesis of mesenchymal stem cells cultured on tricalcium phosphate-collagen-hyaluronan scaffolds.

Hyaluronan (HA) provides a favorable environment for chondrogenesis of bone marrow mesenchymal stem cells (BMSCs). A previous report from our group indicated that addition of HA increases the chondro-inductive capacity of scaffolds. Therefore, this study aimed to investigate whether the Mw of the HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. Human BMSCs (hBMSCs) and rabbit BMSCs (rBMSCs) were isolated and expanded. TCP-COL scaffolds and TCP-COL-HA scaffolds with two different HA Mws were assessed for their capacity to induce cartilage regeneration from hBMSCs in vitro and in vivo. The results showed that about 96.96% of hBMSCs expressed CD44. Moreover, Hyal-1 and chondrogenic marker genes expressions were increased in hMSCs seeded on TCP-COL-HA scaffolds, and blocking the HA-CD44 interaction with an anti-CD44 antibody reduced the expression levels of Hyal-1 and chondrogenic marker genes. Additionally, TCP-COL-HA scaffolds with 2000 kDa Mw showed greater induction of BMSC chondrogenesis induction compared with those with 80 kDa Mw. Similar results were observed in an ectopic implantation nude mouse model. In a rabbit osteochondral defect repair model, rBMSCs seeded on TCP-COL-HA scaffolds with 2000 kDa Mw showed greater cartilage regeneration than those seeded with 80 kDa Mw. In addition, hBMSC-seeded TCP-COL-HA scaffolds with 2000 kDa Mw showed a significantly higher mechanical strength than those with 80 kDa Mw. Collectively, these results indicate that the Mw of HA could affect chondrogenesis of BMSCs seeded on TCP-COL-HA scaffolds. The TCP-COL-HA scaffolds might be used as allogenic off the shelf products in cartilage tissue engineering in future.

Prefabricated 3D-Printed Tissue-Engineered Bone for Mandibular Reconstruction: A Preclinical Translational Study in Primate.

The advent of three dimensionally (3D) printed customized bone grafts using different biomaterials has enabled repairs of complex bone defects in various in vivo models. However, studies related to their clinical translations are truly limited. Herein, 3D printed poly(lactic-co-glycolic acid)/β-tricalcium phosphate (PLGA/TCP) and TCP scaffolds with or without recombinant bone morphogenetic protein −2 (rhBMP-2) coating were utilized to repair primate’s large-volume mandibular defects and compared efficacy of prefabricated tissue-engineered bone (PTEB) over direct implantation (without prefabrication). 18F-FDG PET/CT was explored for real-time monitoring of bone regeneration and vascularization. After 3-month’s prefabrication, the original 3D-architecture of the PLGA/TCP-BMP scaffold was found to be completely lost, while it was properly maintained in TCP-BMP scaffolds. Besides, there was a remarkable decrease in the PLGA/TCP-BMP scaffold density and increase in TCP-BMP scaffolds density during ectopic (within latissimus dorsi muscle) and orthotopic (within mandibular defect) implantation, indicating regular bone formation with TCP-BMP scaffolds. Notably, PTEB based on TCP-BMP scaffold was successfully fabricated with pronounced effects on bone regeneration and vascularization based on radiographic, 18F-FDG PET/CT, and histological evaluation, suggesting a promising approach toward clinical translation.

The role of adropin, salusin-α, netrin-1, and nesfatin-1 in endometriosis and their association with insulin resistance

Objective:The pathogenesis of endometriosis has not been clearly explained. Inflammatory factors of ectopic implantation and the growth of ectopic endometrial cells have been subjects of major interest. The number of studies evaluating salusin-α and nesfatin-1 markers in patients with endometriosis is limited. No studies have evaluated the levels of anti-inflammatory markers for adropin and netrin-1 in patients with endometriosis. This study investigates how some important inflammatory regulatory markers in the inflammatory process affect the pathogenesis of endometriosis and determines whether any relationship exists between serum levels of these parameters and endometriosis and insulin resistance.Materials and Methods:This prospective study included 73 patients with endometriosis diagnosed histopathologically after laparoscopic surgery and 75 healthy controls. Serum adropin, salusin-α, netrin-1, and nesfatin-1 levels and homeostatic model assessment of insulin resistance (HOMA-IR) values of the participants were measured.Results:The endometriosis group had significantly lower nesfatin-1 levels than the control group (3.0±0.53 vs 9.5±0.94, p=0.005). Between the patient and control groups, there was no difference regarding serum adropin, salusin-α, and netrin-1 levels (p=0.36, p=0.34, p=0.75, respectively). Nesfatin-1 had a significant positive correlation with adropin, salusin-α, and netrin-1 (r=0.563, p<0.01; r=0.738, p<0.01; r=0.700, p<0.01, respectively), but had a negative correlation with fasting blood glucose (r=−0.343, p<0.05). HOMA-IR values were comparable between both groups.Conclusion:The lower nesfatin-1 levels leading to increased inflammatory pathway activity in patients with endometriosis might play a role in endometriosis pathogenesis. Without causing systemic insulin resistance, decreased nesfatin-1 might contribute to endometriosis pathogenesis locally by leading to the reduced insulin susceptibility of endometriosis cells.

The Challenge of the Non-Compliant Patient: A Case of Caesarean Section Scar Ectopic Pregnancy

Ectopic pregnancy is a quite common and life-threatening pregnancy. The most common site of ectopic implantation of a fertilized embryo is the fallopian tube. In extremely rare situations, the embryo can implant in other locations, which makes the diagnosis and management even more complex. Although close observation of a new pregnancy is key in the diagnosis and treatment of an ectopic pregnancy, there is still a major risk of life-threatening outcomes. This is a case report of a 31-year-old patient with a history of multiple pregnancies who presented to a community hospital in the spring of 2021 with an ectopic caesarean scar pregnancy. A diagnosis of ectopic pregnancy was on a timely basis, and surgical management was advised. Upon refusal of treatment and admission, pharmacological management was initiated, but patient compliance challenged the success of the therapy. Patient non-compliance to close follow-up resulted in a ruptured uterus. Emergency laparotomy with supracervical hysterectomy was performed as a life-saving procedure.

PD37-09 INTERMITTENT WST-11 VASCULAR TARGET PHOTODYNAMIC THERAPY IN A MOUSE ORTHOTOPIC BLADDER MODEL

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-09 INTERMITTENT WST-11 VASCULAR TARGET PHOTODYNAMIC THERAPY IN A MOUSE ORTHOTOPIC BLADDER MODEL Jie Chen, Jasmine Thomas, Karan Nagar, Lucas Nogueira, Peter Reisz, Kwanghee Kim, and Jonathan Coleman Jie ChenJie Chen More articles by this author , Jasmine ThomasJasmine Thomas More articles by this author , Karan NagarKaran Nagar More articles by this author , Lucas NogueiraLucas Nogueira More articles by this author , Peter ReiszPeter Reisz More articles by this author , Kwanghee KimKwanghee Kim More articles by this author , and Jonathan ColemanJonathan Coleman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Vascular targeted photodynamic therapy (VTP) is a non-surgical tumor ablation approach used to treat early-stage prostate cancer and may also be effective for urothelial cancer based on preclinical data in heterotopic models. While orthotopic models are valuable as they reproduce factors such as local tissue environment and cancer progression pathogenesis compared to ectopic implantation, its implementation is technically difficult. Herein we describe our work in developing an orthotopic murine model for bladder urothelial cancer using intermittent laser pulses to ensure mouse survival and local tumor control. METHODS: The MB-49 murine urothelial cancer line was used for all experiments after establishing stable luciferase expression. Tumors were implanted in 16 1C57BL-6 mice by surgical approach by direct injection of 1x105 MB49-Luc cells into the bladder. Successful tumor implantation was assessed by IVIS images 72 hours after implantation. Mice were divided in two groups: control and VTP treated. VTP was performed 96 hours after cell injection at a power of 100mW in 30 seconds pulses, with 30 seconds interval. Mouse bladders were harvested 13 days post implantation. Safety, tumor control, and histology parameters were assessed. RESULTS: Adequate tumor growth was observed in all mice, and no major toxicity was observed in both groups. Final tumor size was 71% smaller, and histological viability was decreased in VTP treated tumors when compared to controls (p <0.001, figure 1). CD31, Ki67 expression were decreased 2.75 and 1.87-fold respectively, and tunnel staining was increased 3.2-fold in control tumors when compared to VTP treated. (p <0.05, figure 2). CONCLUSIONS: A mouse orthotopic bladder urothelial model via injection of MB-49 cell line is feasible and displays a very high tumor take rates. Intermittent VTP treatment has an effect on mouse bladder tumors as shown by the decrease in tumor size, weight and change in histology. Additional refinement to the model may provide a means to establish an appropriate timeline and utility for future studies. Source of Funding: NIH/NCI (P30 CA008748) and the Thompson Family Foundation © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e658-e659 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jie Chen More articles by this author Jasmine Thomas More articles by this author Karan Nagar More articles by this author Lucas Nogueira More articles by this author Peter Reisz More articles by this author Kwanghee Kim More articles by this author Jonathan Coleman More articles by this author Expand All Advertisement Loading ...

Free Transplantation of a Tissue Engineered Bone Graft into an Irradiated, Critical-Size Femoral Defect in Rats.

Healing of large bone defects remains a challenge in reconstructive surgery, especially with impaired healing potential due to severe trauma, infection or irradiation. In vivo studies are often performed in healthy animals, which might not accurately reflect the situation in clinical cases. In the present study, we successfully combined a critical-sized femoral defect model with an ionizing radiation protocol in rats. To support bone healing, tissue-engineered constructs were transferred into the defect after ectopic preossification and prevascularization. The combination of SiHA, MSCs and BMP-2 resulted in the significant ectopic formation of bone tissue, which can easily be transferred by means of our custom-made titanium chamber. Implanted osteogenic MSCs survived in vivo for a total of 18 weeks. The use of SiHA alone did not lead to bone formation after ectopic implantation. Analysis of gene expression showed early osteoblast differentiation and a hypoxic and inflammatory environment in implanted constructs. Irradiation led to impaired bone healing, decreased vascularization and lower short-term survival of implanted cells. We conclude that our model is highly valuable for the investigation of bone healing and tissue engineering in pre-damaged tissue and that healing of bone defects can be substantially supported by combining SiHA, MSCs and BMP-2.

The Inflammatory Feed-Forward Loop Triggered by the Complement Component C3 as a Potential Target in Endometriosis.

The complement system is a major component of humoral innate immunity, acting as a first line of defense against microbes via opsonization and lysis of pathogens. However, novel roles of the complement system in inflammatory and immunological processes, including in cancer, are emerging. Endometriosis (EM), a benign disease characterized by ectopic endometrial implants, shows certain unique features of cancer, such as the capacity to invade surrounding tissues, and in severe cases, metastatic properties. A defective immune surveillance against autologous tissue deposited in the peritoneal cavity allows immune escape for endometriotic lesions. There is evidence that the glandular epithelial cells found in endometriotic implants produce and secrete the complement component C3. Here, we show, using immunofluorescence and RT-qPCR, the presence of locally synthesized C3 in the ectopic endometriotic tissue, but not in the eutopic tissue. We generated a murine model of EM via injection of minced uterine tissue from a donor mouse into the peritoneum of recipient mice. The wild type mice showed greater amount of cyst formation in the peritoneum compared to C3 knock-out mice. Peritoneal washings from the wild type mice with EM showed more degranulated mast cells compared to C3 knock-out mice, consistent with higher C3a levels in the peritoneal fluid of EM patients. We provide evidence that C3a participates in an auto-amplifying loop leading to mast cell infiltration and activation, which is pathogenic in EM. Thus, C3 can be considered a marker of EM and its local synthesis can promote the engraftment of the endometriotic cysts.

P–293 Ovarian endometriomas are heterogenous for the steroidogenic function and the expression of estrogen and progesterone receptors

Abstract Study question Do all ovarian endometriomas have steroidogenic function and express estrogen and progesterone receptors? Summary answer No, they are heterogenous for the steroidogenic function and the expression of the estrogen and progesterone receptors. What is known already Excessive ectopic estrogen production and up-regulation of estrogen receptor- β, which drives inflammation together with aberrant progesterone signaling leading to impaired decidualization and establishment of ectopic endometrial implants together with down-regulated progesterone receptor (PR) expression are the cardinal molecular features of the disease. However, several fundamental questions still remain to be answered as to whether all ovarian endometriomas carry these molecular aberrations and are steroidogenically active; and if so, the amount of sex steroids they produce correlate with the level of expression of steroidogenic enzymes. We aimed to address these questions in the current study. Study design, size, duration A molecular research study on the surgical specimens collected between April 2020 and December 2020. Seven histopathologically confirmed benign endometriotic cyst capsules obtained from the patients undergoing laparoscopic excision of unilateral ovarian endometriomas without deep infiltrating endometriosis during early follicular phase were used in the study. Participants/materials, setting, methods The mean age±SD (range) of the patients were 32.8±4.9 (30–39). The mean endometrioma size was 5±1.2cm (5–7.5 cm). The samples were cut into equal size pieces of 0.5x0.5cm size and cultured for one day to measure their E2and P4production; and analyzed for the expression of steroidogenic enzymes with quantitative immunoblotting and for the expression of FSH-R, ER and PR with real-time qRT-PCR methods. Luteinized granulosa cells and ovarian cortex were set as references. Main results and the role of chance StAR expression was consistently observed in all samples. However, we noticed significant discrepancies among the samples regarding their steroidogenic function and the expression of aromatase and 3 β-HSD enzymes. E2 production exhibited significant variation (from 5 to 1177pg/mL) from sample to sample despite comparable levels of aromatase expression. ER- βup-regulation as a cardinal molecular feature of endometriosis, was observed in all but one samples (1.46 to 5.48 folds, p &amp;lt; 0.0001). However, its expression level did not correlate with either aromatase expression or the amount of E2 the samples produced. A similar phenomenon was observed in P4 arm of steroidogenesis. Even though 3 β-HSD was expressed by all but one samples detectable amount of P4 was produced only by two samples (up to 15ng/mL). PR expression was down-regulated only in two samples (0.3 to 0.07 folds, p &amp;lt; 0.0001), and significantly up-regulated in the other samples (1.2 to 4.7 folds, p &amp;lt; 0.001). No correlation was found among the samples regarding the expression of PR, 3 β-HSD and P4 output. FSH-R was detected in all samples at the levels comparable to ovarian cortex but its expression level did not show any correlation with ER, aromatase expression and E2 production. Limitations, reasons for caution These results need to be confirmed in studies with larger sample size and different types of endometriotic lesions. Wider implications of the findings: The regulation of steroidogenic activity of endometriomas cannot simply be explained by the expression level of the steroidogenic enzymes, underscoring the importance of other mechanisms that post-translationally regulate their enzymatic activity and metabolism of estrogen and progesterone. PR is not always down-regulated and FSH-R is commonly up-regulated in ovarian endometriomas. Trial registration number Not applicable

Serial cellular events in bone formation initiated by calcium phosphate ceramics

To better understand the biological mechanisms triggered by osteoinductive materials in vivo, we evaluated the timeline of cellular responses to osteoinductive materials subcutaneously implanted in FVB mice. More F4/80-positive macrophages were present in osteoinductive tri-CaP ceramic (TCP) with submicron surface topography (TCPs) than non-osteoinductive TCP with micron surface topography (TCPb) at week 1. Moreover, TCPs (but not TCPb) significantly enhanced osteoclastogenesis, and induced macrophages to polarize from M1 to M2 in the first week. The time sequence and relevance of macrophages and osteoclasts responses involved in bone formation was then evaluated through peri-implant injection of specific chemicals in mice implanted with osteoinductive TCPs. Day-1 injection of clodronate liposomes (LipClod) depleted macrophages, inhibited macrophage polarization to M2, blocked osteoclastogenesis and bone formation, while the day-6 injection was less effective. Anti-RANKL antibody (aRANKL) did not affect macrophage colonization but inhibited osteoclastogenesis. Injection of aRANKL before week 2 aborted bone formation in TCPs, while injection at week 4 partially inhibited bone formation. The overall data show that following ectopic implantation, osteoinductive materials allow macrophage colonization in hours to days, macrophage polarization to M2 in days (within 7 days), osteoclastogenesis in weeks (e.g. in 2 weeks) and bone formation thereafter (after 4 weeks). The serial cellular events verified herein bring a new insight on material-induced bone formation and pave the way to further explore the mechanisms triggered by osteoinductive materials. Statement of significanceA series of key cellular events triggered by osteoinductive calcium phosphate ceramic was revealed: macrophages colonized within hours to days, polarization of M2 macrophages occurred within 7 days, osteoclastogenesis mainly occurred in weeks (e.g. in 2 weeks) and bone formation finally arose thereafter (after 4 weeks). Moreover, such time sequence of cellular events was confirmed with specific chemicals (clodronate liposomes and anti-RANKL antibody). The findings verified herein bring a new insight on material-induced bone formation and pave the way to further explore the mechanisms triggered by osteoinductive materials.