Local Control of Pyoderma Gangrenosum Using Human Amniotic Membrane and Transcriptome Analysis.

Pyoderma gangrenosum (PG) is a rare, chronic, ulcerative disease characterized by non-healing wounds that worsen with debridement, a phenomenon called pathergy. No consensus regarding pathogenesis, diagnosis, or treatment exists for PG. A previous pilot study using dehydrated human amniotic/chorionic membrane (dHACM), following excisional debridement, augmented PG wound healing and allowed for subsequent wound closure through split-thickness skin grafting (STSG). In this clinical trial (NCT05120726), four patients with an established PG diagnosis were enrolled to undergo treatment with dHACM and characterize the pre- and post-treatment transcriptome profiles. RNA sequencing was used to isolate the total RNA from specimens. Genes of particular interest were quantified through real-time quantitative reverse transcription polymerase chain reaction. We observed varied changes to the local expression of inflammatory response, positive regulators of cellular proliferation, and extracellular matrix disassembly cytokines. All PG wounds produced granulation tissue following treatment and were closed using split-thickness skin grafts.

Decellularized amniotic membrane hydrogel promotes mesenchymal stem cell differentiation into smooth muscle cells.

Previous studies showed that the bladder extracellular matrix (B-ECM) could increase the differentiation efficiency of mesenchymal cells into smooth muscle cells (SMC). This study investigates the potential of human amniotic membrane-derived hydrogel (HAM-hydrogel) as an alternative to xenogeneic B-ECM for the myogenic differentiation of the rabbit adipose tissue-derived MSC (AD-MSC). Decellularized human amniotic membrane (HAM) and sheep urinary bladder (SUB) were utilized to create pre-gel solutions for hydrogel formation. Rabbit AD-MSCs were cultured on SUB-hydrogel or HAM-hydrogel-coated plates supplemented with differentiation media containing myogenic growth factors (PDGF-BB and TGF-ß1). An uncoated plate served as the control. After 2 weeks, real-time qPCR, immunocytochemistry, flow cytometry, and western blot were employed to assess the expression of SMC-specific markers (MHC and α-SMA) at both protein and mRNA levels. Our decellularization protocol efficiently removed cell nuclei from the bladder and amniotic tissues, preserving key ECM components (collagen, mucopolysaccharides, and elastin) within the hydrogels. Compared to the control, the hydrogel-coated groups exhibited significantly upregulated expression of SMC markers (p ≤ .05). These findings suggest HAM-hydrogel as a promising xenogeneic-free alternative for bladder tissue engineering, potentially overcoming limitations associated with ethical concerns and contamination risks of xenogeneic materials.

Intra-uterine injection of amnion-derived acellular bioscaffold product in mares, a description of systemic and intra-uterine effects over 21 days.

Amnion-derived acellular bioscaffold (ADABP) products demonstrate interesting anti-inflammatory and healing properties which could be beneficial for intrauterine use. The objective of this study was to evaluate the safety of intrauterine injection of ADABP on systemic and uterine health. The study design randomly assigned subjects to one of two groups, control mares (n = 3) which received 3 mL injection of sterile saline in the base of each uterine horn, and treatment mares (n = 9) which received 3 mL of ADABP in the base of one uterine horn and 3 mL injection of sterile saline in the base of the other uterine horn. The leukogram had no significant effect of group and no group by day interaction. The serum biochemistry panel had no effect of group on any of the parameters examined. There were no significant differences in uterine culture or uterine biopsy results. The data suggests intrauterine injection of ADABP has no negative systemic or uterine effects.

Temporally resolved early bone morphogenetic protein-driven transcriptional cascade during human amnion specification.

Amniogenesis, a process critical for continuation of healthy pregnancy, is triggered in a collection of pluripotent epiblast cells as the human embryo implants. Previous studies have established that bone morphogenetic protein (BMP) signaling is a major driver of this lineage specifying process, but the downstream BMP-dependent transcriptional networks that lead to successful amniogenesis remain to be identified. This is, in part, due to the current lack of a robust and reproducible model system that enables mechanistic investigations exclusively into amniogenesis. Here, we developed an improved model of early amnion specification, using a human pluripotent stem cell-based platform in which the activation of BMP signaling is controlled and synchronous. Uniform amniogenesis is seen within 48 hr after BMP activation, and the resulting cells share transcriptomic characteristics with amnion cells of a gastrulating human embryo. Using detailed time-course transcriptomic analyses, we established a previously uncharacterized BMP-dependent amniotic transcriptional cascade, and identified markers that represent five distinct stages of amnion fate specification; the expression of selected markers was validated in early post-implantation macaque embryos. Moreover, a cohort of factors that could potentially control specific stages of amniogenesis was identified, including the transcription factor TFAP2A. Functionally, we determined that, once amniogenesis is triggered by the BMP pathway, TFAP2A controls the progression of amniogenesis. This work presents a temporally resolved transcriptomic resource for several previously uncharacterized amniogenesis states and demonstrates a critical intermediate role for TFAP2A during amnion fate specification.

A Systematic Review of Human Amnion Enhanced Cartilage Regeneration in Full-Thickness Cartilage Defects.

Cartilage defects present a significant challenge in orthopedic medicine, often leading to pain and functional impairment. To address this, human amnion, a naturally derived biomaterial, has gained attention for its potential in enhancing cartilage regeneration. This systematic review aims to evaluate the efficacy of human amnion in enhancing cartilage regeneration for full-thickness cartilage defects. An electronic search was conducted on MEDLINE-PubMed, Web of Science (WoS), and the Scopus database up to 27 December 2023 from 2007. A total of 401 articles were identified. After removing 125 duplicates and excluding 271 articles based on predetermined criteria, only 5 articles remained eligible for inclusion in this systematic review. All five eligible articles conducted in vivo studies utilizing rabbits as subjects. Furthermore, analysis of the literature reveals an increasing trend in the frequency of utilizing human amnion for the treatment of cartilage defects. Various forms of human amnion were utilized either alone or seeded with cells prior to implantation. Histological assessments and macroscopic observations indicated usage of human amnion improved cartilage repair outcomes. All studies highlighted the positive results despite using different forms of amnion tissues. This systematic review underscores the promising role of human amnion as a viable option for enhancing cartilage regeneration in full-thickness cartilage defects, thus offering valuable insights for future research and clinical applications in orthopedic tissue engineering.

Injectable multifunctional hydrogel containing Sphingosine 1-phosphate and human acellular amniotic membrane for skin wound healing.

Objectives: The skin serves as the main defense barrier, protecting against injuries, and preventing infection and water loss. Consequently, wound healing and skin regeneration are crucial aspects of wound management. A novel hydrogel scaffold was developed by incorporating carboxymethyl cellulose (CMC) and gelatin (Gel) hydrogels cross-linked with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) containing Sphingosine 1-phosphate (S1P). This hydrogel is applied topically to treat acute wounds and is covered with a human acellular amniotic membrane (hAAM) as a secondary dressing. Materials and Methods: The scaffold was subjected to in vitro cell viability, red blood cell hemolysis, blood clotting index, and in vivo assays. Real-time PCR was implemented to verify the expression of genes involved in skin wounds. The physical and chemical properties of the scaffolds were also tested using weight loss, swelling ratio, scanning electron microscopy (SEM), Fourier transform infrared (FTIR), and mechanical tensile analysis. Results: The synthetic scaffold is biocompatible as evidenced by the high percentage of 3T3 cell viability (127%) after 72 hr. Additionally, excellent hemocompatibility with a low hemolytic effect (2.26%) was observed. Our in vivo wound healing assay demonstrated that CMC/Gel/S1P/hAAM wound dressing led to faster wound healing in treated rats compared to the control group over 14.Also, the mechanical tests showed that the amniotic membrane and the hAAM had very different Young's modulus and elongation at break values. Conclusion: This study demonstrates the effectiveness of the CMC/Gel/EDC hydrogel with S1P as a wound dressing. Additionally, hAAM exhibits excellent characteristics as a protective layer for the treatment of acute wounds.

Dehydrated Amnion Chorion Membrane versus standard of care for diabetic foot ulcers: a randomised controlled trial.

OBJECTIVE: Diabetic foot ulcers (DFUs) continue to challenge wound care practitioners. This prospective, multicentre, randomised controlled trial (RCT) evaluated the effectiveness of a dehydrated Amnion Chorion Membrane (dACM) (Organogenesis Inc., US) versus standard of care (SoC) alone in complex DFUs in a challenging patient population. METHOD: Subjects with a DFU extending into dermis, subcutaneous tissue, tendon, capsule, bone or joint were enrolled in a 12-week trial. They were allocated equally to two treatment groups: dACM (plus SoC); or SoC alone. The primary endpoint was frequency of wound closure determined by a Cox analysis that adjusted for duration and wound area. Kaplan-Meier analysis was used to determine median time to complete wound closure (CWC). RESULTS: The cohort comprised 218 patients, and these were split equally between the two treatment groups with 109 patients in each. A Cox analysis showed that the estimated frequency of wound closure for the dACM plus SoC group was statistically superior to the SoC alone group at week 4 (12% versus 8%), week 6 (22% versus 11%), week 8 (31% versus 21%), week 10 (42% versus 27%) and week 12 (50% versus 35%), respectively (p=0.04). The computed hazard ratio (1.48 (confidence interval: 0.95, 2.29) showed a 48% greater probability of wound closure in favour of the dACM group. Median time to wound closure for dACM-treated ulcers was 84 days compared to 'not achieved' in the SoC-treated group (i.e., ≥50% of SoC-treated DFUs failed to heal by week 12; p=0.04). CONCLUSION: In an adequately powered DFU RCT, dACM increased the frequency, decreased the median time, and improved the probability of CWC when compared with SoC alone. dACM demonstrated beneficial effects in DFUs in a complex patient population. DECLARATION OF INTEREST: This study was funded by Organogenesis Inc., US. JC serves as a consultant and speaker for Organogenesis. RDD serves as a speaker for Organogenesis. OMA and MLS serve as consultants for Organogenesis. The authors have no other conflicts of interest to declare.

Decellularized Amnion Membrane Triggers Macrophage Polarization for Desired Host Immune Response.

Appropriate regulation of immunomodulatory responses, particularly acute inflammation involving macrophages, is crucial for the desired functionality of implants. Decellularized amnion membrane (DAM) is produced by removing cellular components and antigenicity, expected to reduce immunogenicity and the risk of inflammation. Despite the potential of DAM as biomaterial implants, few studies have investigated its specific effects on immunomodulation. Here, it is demonstrated that DAM can regulate macrophage-driven inflammatory response and potential mechanisms are investigated. In vitro results show that DAM significantly inhibits M1 polarization in LPS-induced macrophages by inhibiting Toll-like receptors (TLR) signaling pathway and TNF signaling pathway and promotes macrophage M2 polarization. Physical signals from the 3D micro-structure and the active protein, DCN, binding to key targets may play roles in the process. In the subcutaneous implant model in rats, DAM inhibits the persistence of inflammation and fibrous capsule formation, while promoting M2 macrophage polarization, thereby facilitating tissue regeneration. This study provides insights into DAM's effect and potential mechanisms on the balance of M1/M2 macrophage polarization in vitro and vivo, emphasizing the immunomodulation of ECM-based materials as promising implants.

Application of a practical amniotic membrane ring made on-site for restoration of ocular surface health in dry eye disease.

PURPOSE: Dry eye disease (DED) is one of the most common ocular surface disorders worldwide. Despite different underlying pathogenic processes, DED is characterized by ocular surface inflammation, which in turn induces further damage to the corneal epithelium and its underlying structures. Amniotic membrane transplants are known to have potent anti-inflammatory effects and also have the ability to enhance epithelial healing. In this study, we aimed to evaluate the clinical efficacy of amniotic membrane ring (AMR) application in treating refractory dry eye disease. METHODS: A retrospective analysis of 22 patients treated with contact lens-like amniotic membrane rings was performed. This amniotic membrane ring was formed using an aspiration catheter covered by a large piece of amniotic membrane graft. The amniotic membrane was sutured to the catheter using eight sutures. In this way, a customized amniotic membrane ring was prepared for each patient. Patients' demographics, symptoms, use of medications, conjunctival inflammation, corneal staining, and visual acuity were compared before and after treatment. In addition, the amniotic membrane retention duration, the amniotic membrane's effect on ocular surface healing, follow-up time, and complications were evaluated. RESULTS: Twenty-eight eyes of 22 patients (18 females and 4 males) aged 53.32±13.36 (6-73) years were included. The AMR retention duration ranged from 5 to 16 days, with a mean of 11±3.09 days, at which time the amniotic membrane had dissolved or been removed inadvertently by the patient. Discomfort with the ring was seen in 1 of 28 eyes (3.6%). The patients reported symptomatic relief for a period of 3.64±1.25 months. Symptomatic relief was accompanied by a reduction of OSDI scores (from 63.39±17.24 to 33.19±12.45) (P<0.001), use of topical medications (from 4.21±1.03 to 2.42±0.50) (P<0.001), conjunctival hyperemia (from 1.57±1.19 to 0.35±0.48) (P<0.001), corneal staining (from 2.89±1.16 to 0.57±0.74) (P<0.001), and improvement in visual acuity (from 0.23±0.16 to 0.16±0.25 logMAR) (P=0.001). CONCLUSIONS: Amniotic membrane ring treatment might be used to treat refractory dry eye disease. This technique has an economic advantage over other commercially available amniotic bandage tissues and can be easily removed and replaced during a follow-up examination.

IL-1 and TNF mediates IL-6 signaling at the maternal-fetal interface during intrauterine inflammation.

Introduction: IL6 signaling plays an important role in triggering labor and IL6 is an established biomarker of intrauterine infection/inflammation (IUI) driven preterm labor (PTL). The biology of IL6 during IUI at the maternal-fetal interface was investigated in samples from human subjects and non-human primates (NHP). Methods: Pregnant women with histologic chorioamnionitis diagnosed by placenta histology were recruited (n=28 term, n=43 for preterm pregnancies from 26-36 completed weeks of gestation). IUI was induced in Rhesus macaque by intraamniotic injection of lipopolysachharide (LPS, n=23). IL1 signaling was blocked using Anakinra (human IL-1 receptor antagonist, n=13), and Tumor necrosis factor (TNF) signaling was blocked by anti TNF-antibody (Adalimumab n=14). The blockers were given before LPS. All animals including controls (intraamniotic injection of saline n=27), were delivered 16h after LPS/saline exposure at about 80% gestation. Results: IUI induced a robust expression of IL6 mRNAs in the fetal membranes (chorion-amnion-decidua tissue) both in humans (term and preterm) and NHP. The major sources of IL6 mRNA expression were the amnion mesenchymal cells (AMC) and decidua stroma cells. Additionally, during IUI in the NHP, ADAM17 (a protease that cleaves membrane bound IL6 receptor (IL6R) to release a soluble form) and IL6R mRNA increased in the fetal membranes, and the ratio of IL6 and soluble forms of IL6R, gp130 increased in the amniotic fluid signifying upregulation of IL6 trans-signaling. Both IL1 and TNF blockade suppressed LPS-induced IL6 mRNAs in the AMC and variably decreased elements of IL6 trans-signaling. Discussion: These data suggest that IL1 and TNF blockers may be useful anti-inflammatory agents via suppression of IL6 signaling at the maternal-fetal interface.

Efficacy of Amniotic and Chorionic Membrane in Facial Wound Healing: A Comparative Study.

Background Advancements in regenerative techniques have been utilized in placental amnion and chorion for a variety of purposes. Their ability to regenerate tissues has led to their usage in tissue engineering, wound healing, and other therapeutic applications. This study aims to evaluate and compare the efficacy of amnion and chorion in facial tissue wound healing. Methodology  The study was an observational comparative study conducted in the Department of Oral and Maxillofacial Surgery, involving 20 participants divided into two groups (Group I and Group II). Study groups were selected according to the inclusion and exclusion criteria. A dehydrated human amnion/ chorion membrane was applied to the affected site of each group respectively. Its efficacy in wound healing was analyzed in the first, third, seventh day, and second week. Statistical analysis was done using SPSS software (IBM Corp., Armonk, NY). Results Patients treated with amnion membrane showed a decrease in wound size and the wound was completely healed by second week with mean scores of wound sizes of 0.00 whereas the wound remained unhealed by second week with mean of 1.70 to those treated with chorion membrane. Conclusion Amnion showed superior efficacy in wound healing at two-week intervals when compared to the chorion. Hence, this could be used in regenerative medicine as a graft to induce healing in facial wounds.

Stem-Cell-Driven Chondrogenesis: Perspectives on Amnion-Derived Cells.

Regenerative medicine harnesses stem cells' capacity to restore damaged tissues and organs. In vitro methods employing specific bioactive molecules, such as growth factors, bio-inductive scaffolds, 3D cultures, co-cultures, and mechanical stimuli, steer stem cells toward the desired differentiation pathways, mimicking their natural development. Chondrogenesis presents a challenge for regenerative medicine. This intricate process involves precise modulation of chondro-related transcription factors and pathways, critical for generating cartilage. Cartilage damage disrupts this process, impeding proper tissue healing due to its unique mechanical and anatomical characteristics. Consequently, the resultant tissue often forms fibrocartilage, which lacks adequate mechanical properties, posing a significant hurdle for effective regeneration. This review comprehensively explores studies showcasing the potential of amniotic mesenchymal stem cells (AMSCs) and amniotic epithelial cells (AECs) in chondrogenic differentiation. These cells exhibit innate characteristics that position them as promising candidates for regenerative medicine. Their capacity to differentiate toward chondrocytes offers a pathway for developing effective regenerative protocols. Understanding and leveraging the innate properties of AMSCs and AECs hold promise in addressing the challenges associated with cartilage repair, potentially offering superior outcomes in tissue regeneration.

Clinico-radiological outcome of Arthroscopic Anterior Cruciate Ligament Reconstruction with Augmentation of Dehydrated Human Amnion Chorion Allograft Membrane using Peroneus Longus Autograft.

Introduction: For many sportsmen, anterior cruciate ligament (ACL) tears are unfortunate but common injuries. Several growth factors, cytokine, chemokine, and protease inhibitors functions in stimulation of paracrine reactions in fibroblast, endothelial, and stem cells thereby promoting the tissue restorative processes. Augmented with dehydrated Human Amnion Chorion Membrane (dHACM) allograft reinforces the reconstructed ligament and aids in effective restoration. Materials and methods: In this case control study 15 patients undertaking ACL reconstruction with tripled peroneus augmented dHACM (G1) were prospectively monitored up for a period of 8 months along with 15 control patients (G2) without dHACM augmentation. Clinical and radiological outcomes were analysed and assessed about effect of augmenting the peroneus longus graft using dHACM. Clinical analysis included pre-operative two, four, six, and eight months post-operative Tegnor-Lysholm score, and radiological analysis included the 6th month postoperative MRI signal-to-noise ratio (SNR) measurements by mean signal-value at femoral insertion, midsubstance and tibial insertion of ACL graft. Results: Clinically, as a mean Lysholm score of all patients, they were revealed to be consecutively high in G1 than in Group 2 at four, six, and eight months. The signal-to-noise ratio from the MRI results showed majority having good healing in G1 group. Conclusions: Based on 6-month MRI, an effective ligamentization (SNR<75) was noticed in 53.33% of patients in the dHACM allograft enhanced group on comparison with 33% in the controls. The overall results show that the augmentation of dHACM allograft to ACL reconstruction yields in good patient outcomes at post-operative follow-up.

Introduction to Amniotic Membranes in Maxillofacial Surgery-A Scoping Review.

Background: Amniotic membrane (AM) holds significant promise in various medical fields due to its unique properties and minimal ethical concerns. This study aims to explore the diverse applications of the human amniotic membrane (HAM) in maxillofacial surgery. Methodology: A comprehensive search was conducted on databases, namely Google Scholar, PubMed, and Scopus, from January 1985 to March 2024. Articles in English, Polish, and Spanish were included, focusing on keywords related to amniotic membrane and oral surgery. Results: Various preservation methods for HAM were identified, namely fresh, decellularized, cryopreserved, lyophilized, and air-dried formats. Clinical studies demonstrated the efficacy of HAM in repairing oral mucosal defects, vestibuloplasty, oronasal fistula closure, cleft palate treatment, bone defect repair, and medication-related osteonecrosis of the jaw (MRONJ). Surgeon evaluations highlighted the ease of handling but noted challenges in suturing and stability during application. Conclusions: Amniotic membranes offer a versatile and effective option in maxillofacial surgery, promoting wound healing, reducing inflammation, and providing a scaffold for tissue regeneration. Further research, including randomized trials and comparative studies, is warranted to validate the efficacy and optimize the utilization of HAM in clinical practice.

Transcriptomic analysis delineates preterm prelabor rupture of membranes from preterm labor in preterm fetal membranes.

BACKGROUND: Globally, preterm birth remains the leading cause of death in children younger than 5 years old. Spontaneous preterm birth is comprised of two events that may or may not occur simultaneously: preterm labor and preterm prelabor rupture of membranes (PPROM). To further explore the concept that spontaneous preterm birth can result from the initializing of two separate but overlapping pathological events, we compared fetal membrane tissue from preterm labor deliveries to fetal tissue from preterm labor with PPROM deliveries. We hypothesized that the fetal membrane tissue from preterm labor with PPROM cases will have an RNA-seq profile divergent from the fetal membrane tissue from preterm labor controls. METHODS: Chorioamnion, separated into amnion and chorion, was collected from eight gestationally age-matched cases and controls within 15 min of birth, and analyzed using RNA sequencing. Pathway enrichment analyses and functional annotations of differentially expressed genes were performed using KEGG and Gene Ontogeny Pathway enrichment analyses. RESULTS: A total of 1466 genes were differentially expressed in the amnion, and 484 genes were differentially expressed in the chorion (log2 fold change > 1, FDR < 0.05) in cases (preterm labor with PPROM), versus controls (preterm labor only). In the amnion, the most significantly enriched (FDR < 0.01) KEGG pathway among down-regulated genes was the extracellular matrix receptor interaction pathway. Seven of the most significantly enriched pathways were comprised of multiple genes from the COL family, including COL1A, COL3A1, COL4A4, and COL4A6. In the chorion, the most significantly enriched KEGG pathways in up-regulated genes were chemokine, NOD receptor, Toll-like receptor, and cytokine-cytokine receptor signaling pathways. Similarly, KEGG pathway enrichment analysis for up-regulated genes in the amnion included three inflammatory pathways: cytokine-cytokine interaction, TNF signaling and the CXCL family. Six genes were significantly up regulated in chorionic tissue discriminated between cases (preterm labor with PPROM) and controls (preterm labor only) including GBP5, CXCL9, ALPL, S100A8, CASP5 and MMP25. CONCLUSIONS: In our study, transcriptome analysis of preterm fetal membranes revealed distinct differentially expressed genes for PPROM, separate from preterm labor. This study is the first to report transcriptome data that reflects the individual pathophysiology of amnion and chorion tissue from PPROM deliveries.

Comparative, Controlled, Retrospective Study of Patient-Reported Outcomes After Meniscectomy With Adjunctive Use of Platelet-Rich Plasma or Amniotic Umbilical Cord Tissue.

Background: Meniscal tears are one of the most frequent injuries to the knee, with an estimated incidence of 222 per 100,000 individuals aged 18 to 55 years based on magnetic resonance imaging. Poor outcomes following meniscal surgical interventions are common and have led many surgeons to use biologic augmentation strategies to enhance the healing. Methods: We conducted a single-center, retrospective, observational study of patients who underwent arthroscopic meniscectomy with and without adjunctive platelet-rich plasma (PRP) or the particulate form of amniotic umbilical cord (AMUC) tissue. We evaluated patient-reported outcomes on the visual analog scale for pain, International Knee Documentation Committee (IKDC) Subjective Knee Evaluation Form, Lysholm Knee Scoring Scale, 12-Item Short Form Survey, and Knee Injury and Osteoarthritis Outcome Score (KOOS) during a 1-year postoperative period. Complications and follow-up procedures were also evaluated. Results: We evaluated 113 patients who underwent meniscectomies from November 2010 to March 2017. Pain severity was significantly decreased only in the AMUC group at 6 months (P=0.0143). Patients in the AMUC group demonstrated significant improvement in functional recovery based on the IKDC and the KOOS subscales of pain, symptoms, activities of daily living, and sport and recreation function at 6 months. Patients in the PRP group had a significant benefit in the KOOS subscales of pain, symptoms, sport and recreation function, and knee-related quality of life at 3 months. Improvement in the control group was less substantial. Patients in the PRP group had more complications and follow-up procedures (30.0%) than patients in the AMUC group (8.3%). Conclusion: In our study population, arthroscopic meniscectomy with adjunctive use of AMUC tissue improved patient-reported outcomes and reduced the reoperation rate compared to conventional technique or adjunctive use of PRP.

Evaluation of Leukocyte Chemotaxis Induced by Human Fetal Membranes in an In Vitro Model.

Leukocyte infiltration into the maternal-fetal interface is a consequence of the robust inflammation in the gestational tissues during term labor and preterm labor with or without infection. During pregnancy, the fetal membranes act as a physical barrier that isolates the fetus into the amniotic cavity, keeping it in an optimal environment for its development. In addition, the fetal membranes possess immunological competencies such as the secretion of cytokines and chemokines in response to different stimuli. Clinical and experimental evidence indicates that these tissues are involved in the extensive chemotaxis of immune cells in normal or pathological conditions.Few studies have evaluated the chemotactic capacities of the fetal membranes considering that this tissue is composed of two adjacent tissues, the amnion and the chorion, which have different characteristics. Although these tissues function as a unit, their response is complex since there is an interaction between them, where each tissue contributes differently. The protocol described here allows us to evaluate the in vitro chemotactic capacities of fetal membranes in response to various applied stimuli, considering the contribution of each of their components (amnion and choriodecidua) using a Boyden chamber assay and phenotyping the chemo-attracted leukocytes by flow cytometry.

Culture of Human Fetal Membranes in a Two Independent Compartment Model: An Ex Vivo Approach.

During pregnancy, the fetal membranes composed of the amnion and chorodecidua constitute a selective barrier separating two distinct environments, maternal and fetal. These tissues have the function of delimiting the amniotic cavity. Their histological complexity gives them physical, mechanical, and immunological properties to protect the fetus. Although the study of the amnion, chorion, and decidua separately provides knowledge about the functions of the fetal membranes, the protocol we describe in this chapter has the advantage of maintaining the biological and functional complexity of these tissues. In addition, this experimental model allows the researcher to recreate various pathological scenarios because this model allows for differential stimulation of the amnion or choriodecidua.

Obtaining Tissues of Human Amniotic Membrane and Identification of Pluripotent Markers.

The immunofluorescence technique has been used to identify pluripotent markers in the human amniotic epithelial cells (hAEC). hAEC belonging to human fetal membranes, specificamently to amnion layer, and are arising by epiblast, this sugest that the hAEC have characteristics of epiblast cells, in other words, characteristcs of pluripotent stem cells. Here we describe obtaining human amnion tissue and identifying pluripotent markers by immunofluorescence.

Feto-Maternal Interface Organ-on-Chip: A New Technology to Study Ascending Infection.

Modeling human pregnancy is challenging as two subjects, the mother and fetus, must be evaluated in tandem. To understand pregnancy, parturition, and adverse pregnancy outcomes, the two feto-maternal interfaces (FMi) that form during gestation (i.e., the placenta and fetal membrane) need to be investigated to understand their biological roles, and organ dysfunction can lead to adverse outcomes. Adverse pregnancy outcomes such as preterm rupture of the membranes, spontaneous preterm birth, preeclampsia, intra-uterine growth restriction, and gestational diabetes rates are on the rise worldwide, highlighting the need for future studies and a better understanding of molecular and cellular pathways that contribute to disease onset. Current in vivo animal models nor in vitro cell culture systems can answer these questions as they do not model the function or structure of human FMis. Utilizing microfabrication and soft-lithography techniques, microfluidic organ-on-chip (OOC) devices have been adapted by many fields to model the anatomy and biological function of complex organs and organ systems within small in vitro platforms.These techniques have been adapted to recreate the fetal membrane FMi (FMi-OOC) using immortalized cells and collagen derived from patient samples. The FMi-OOC is a four-cell culture chamber, concentric circle system, that contains both fetal (amniochorion) and maternal (decidua) cellular layers and has been validated to model physiological and pathological states of pregnancy (i.e., ascending infection, systemic oxidative stress, and maternal toxicant exposure). This platform is fully compatible with various analytical methods such as microscopy and biochemical analysis. This protocol will outline this device's fabrication, cell loading, and utility to model ascending infection-related adverse pregnancy outcomes.