Microphysiological systems of the placental barrier.

Methods to evaluate maternal-fetal transport across the placental barrier have generally involved clinical observations after-the-fact, ex vivo perfused placenta studies, or in vitro Transwell assays. Given the ethical and technical limitations in these approaches, and the drive to understand fetal development through the lens of transport-induced injury, such as with the examples of thalidomide and Zika Virus, efforts to develop novel approaches to study these phenomena have expanded in recent years. Notably, within the past 10 years, placental barrier models have been developed using hydrogel, bioreactor, organ-on-a-chip, and bioprinting approaches. In this review, we discuss the biology of the placental barrier and endeavors to recapitulate this barrier in vitro using these approaches. We also provide analysis of current limitations to drug discovery in this context, and end with a future outlook.

Characterizing placental stiffness using ultrasound shear-wave elastography in healthy and preeclamptic pregnancies.

PURPOSE: To measure the stiffness of the placenta in healthy and preeclamptic patients in the second and third trimesters of pregnancy using ultrasound shear-wave elastography (SWE). We also aimed to evaluate the effect of age, gestational age, gravidity, parity and body mass index (BMI) on placental stiffness and a possible correlation of stiffness with perinatal outcomes. METHODS: In a case-control study, we recruited a total of 47 singleton pregnancies in the second and third trimesters of which 24 were healthy and 23 were diagnosed with preeclampsia. In vivo placental stiffness was measured once at the time of recruitment for each patient. Pregnancies with posterior placentas, multiple gestation, gestational hypertension, chronic hypertension, diabetes, autoimmune disease, fetal growth restriction and congenital anomalies were excluded. RESULTS: The mean placental stiffness was significantly higher in preeclamptic pregnancies compared to controls in the third trimester (difference of means = 16.8; 95% CI (9.0, 24.5); P < 0.001). There were no significant differences in placental stiffness between the two groups in the second trimester or between the severe preeclampsia and preeclampsia without severe features groups (difference of means = 9.86; 95% CI (-5.95, 25.7); P ≥ 0.05). Peripheral regions of the placenta were significantly stiffer than central regions in the preeclamptic group (difference of means = 10.67; 95% CI (0.07, 21.27); P < 0.05), which was not observed in the control group (difference of means = 0.55; 95% CI (- 5.25, 6.35); P > 0.05). We did not identify a correlation of placental stiffness with gestational age, maternal age, gravidity or parity. However, there was a statistically significant correlation with BMI (P < 0.05). In addition, pregnancies with higher placental stiffness during the 2nd and 3rd trimesters had significantly reduced birth weight (2890 ± 176 vs. 2420 ± 219 g) and earlier GA (37.8 ± 0.84 vs. 34.3 ± 0.98 weeks) at delivery (P < 0.05). CONCLUSION: Compared to healthy pregnancies, placentas of preeclamptic pregnancies are stiffer and more heterogeneous. Placental stiffness is not affected by gestational age or the severity of preeclampsia but there is a correlation with higher BMI and poor perinatal outcomes.

ZEB2, a master regulator of the epithelial-mesenchymal transition, mediates trophoblast differentiation.

STUDY QUESTION: Does the upregulation of the zinc finger E-box binding homeobox 2 (ZEB2) transcription factor in human trophoblast cells lead to alterations in gene expression consistent with an epithelial-mesenchymal transition (EMT) and a consequent increase in invasiveness? SUMMARY ANSWER: Overexpression of ZEB2 results in an epithelial-mesenchymal shift in gene expression accompanied by a substantial increase in the invasive capacity of human trophoblast cells. WHAT IS KNOWN ALREADY: In-vivo results have shown that cytotrophoblast differentiation into extravillous trophoblast involves an epithelial-mesenchymal transition. The only EMT master regulatory factor which shows changes consistent with extravillous trophoblast EMT status and invasive capacity is the ZEB2 transcription factor. STUDY DESIGN, SIZE, DURATION: This study is a mechanistic investigation of the role of ZEB2 in trophoblast differentiation. We generated stable ZEB2 overexpression clones using the epithelial BeWo and JEG3 choriocarcinoma lines. Using these clones, we investigated the effects of ZEB2 overexpression on the expression of EMT-associated genes and proteins, cell morphology and invasive capability. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used lentiviral transduction to overexpress ZEB2 in BeWo and JEG3 cells. Stable clones were selected based on ZEB2 expression and morphology. A PCR array of EMT-associated genes was used to probe gene expression. Protein measurements were performed by western blotting. Gain-of-function was assessed by quantitatively measuring cell invasion rates using a Transwell assay, a 3D bioprinted placenta model and the xCelligenceTM platform. MAIN RESULTS AND THE ROLE OF CHANCE: The four selected clones (2 × BeWo, 2 × JEG3, based on ZEB2 expression and morphology) all showed gene expression changes indicative of an EMT. The two clones (1 × BeWo, 1 × JEG3) showing >40-fold increase in ZEB2 expression also displayed increased ZEB2 protein; the others, with increases in ZEB2 expression <14-fold did not. The two high ZEB2-expressing clones demonstrated robust increases in invasive capacity, as assessed by three types of invasion assay. These data identify ZEB2-mediated transcription as a key mechanism transforming the epithelial-like trophoblast into cells with a mesenchymal, invasive phenotype. LARGE SCALE DATA: PCR array data have been deposited in the GEO database under accession number GSE116532. LIMITATIONS, REASONS FOR CAUTION: These are in-vitro studies using choriocarcinoma cells and so the results should be interpreted in view of these limitations. Nevertheless, the data are consistent with in-vivo findings and are replicated in two different cell lines. WIDER IMPLICATIONS OF THE FINDINGS: The combination of these data with the in-vivo findings clearly identify ZEB2-mediated EMT as the mechanism for cytotrophoblast differentiation into extravillous trophoblast. Having characterized these cellular mechanisms, it will now be possible to identify the intracellular and extracellular regulatory components which control ZEB2 and trophoblast differentiation. It will also be possible to identify the aberrant factors which alter differentiation in invasive pathologies such as preeclampsia and abnormally invasive placenta (AKA accreta, increta, percreta). STUDY FUNDING AND COMPETING INTEREST(s): Funding was provided by the Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine and Surgery at Hackensack Meridian Health, Hackensack, NJ. The 3D bioprinted placental model work done in Drs Kim and Fisher's labs was supported by the Children's National Medical Center. The xCELLigence work done in Dr Birge's lab was supported by NIH CA165077. The authors declare no competing interests.

Trophoblast-endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model.

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium-trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast-endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast-endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.

Biomimetic Placenta-Fetus Model Demonstrating Maternal-Fetal Transmission and Fetal Neural Toxicity of Zika Virus.

Recent global epidemics of viral infection such as Zika virus (ZIKV) and associated birth defects from maternal-fetal viral transmission highlights the critical unmet need for experimental models that adequately recapitulates the biology of the human maternal-fetal interface and downstream fetal development. Herein, we report an in vitro biomimetic placenta-fetus model of the maternal-fetal interface and downstream fetal cells. Using a tissue engineering approach, we built a 3D model incorporating placental trophoblast and endothelial cells into an extracellular matrix environment and validated formation of the maternal-fetal interface. We utilized this model to study ZIKV exposure to the placenta and neural progenitor cells. Our results indicated ZIKV infects both trophoblast and endothelial cells, leading to a higher viral load exposed to fetal cells downstream of the barrier. Viral inhibition by chloroquine reduced the amount of virus both in the placenta and transmitted to fetal cells. A sustained downstream neural cell viability in contrast to significantly reduced viability in an acellular model indicates that the placenta sequesters ZIKV consistent with clinical observations. These findings suggest that the placenta can modulate ZIKV exposure-induced fetal damage. Moreover, such tissue models can enable rigorous assessment of potential therapeutics for maternal-fetal medicine.

Placental basement membrane proteins are required for effective cytotrophoblast invasion in a three-dimensional bioprinted placenta model.

Fetal cytotrophoblast invasion of maternal decidual vasculature is necessary to normal pregnancy. In preeclampsia, there is shallow invasion and abnormal remodeling of the uterine vasculature that lead to significant maternal and perinatal morbidity and mortality. The placental basement membrane (BM) proteins (e.g., laminin and collagen) has been implicated in the development of placenta while the level of laminin is significantly lower in preeclampsia. However, there are very limited studies, if any, on the effect of extracellular matrix (ECM) microenvironment on the invasion of cytotrophoblast. In this study, we hypothesized that placental BM proteins are required for effective cytotrophoblast invasion. Using proteomics, we found that more than 80% of ECM proteins in placental basal plate (pECM) were BM proteins. In addition to upregulating expressions of MMP2 (1.5-fold) and MMP9 (6.3-fold), pECM significantly increased the motility rates of cytotrophoblasts by 13-fold (from 5.60 ± 0.95 to 75.5 ± 21.8 µm/day) to achieve an effective invasion rate that was comparable to in vivo results. Treatments with PI3K inhibitors completely removed the pECM-enhanced invasive phenotypes and genotypes of cytotrophoblasts, suggesting its dominant role in cytotrophoblast-ECM interactions. Our results described, for the first time, the substantial effects of the ECM microenvironment on regulating cytotrophoblast invasion, an area that is less investigated but appear to be critical in the pathogenesis of preeclampsia. Moreover, the approach presented in this work that fabricates organ models with organ-specific ECM can be an attractive option to screen and develop novel therapeutics and biomarkers not only in preeclampsia but also other diseases such as cancer metastasis. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1476-1487, 2018.

Repair of Tympanic Membrane Perforations with Customized Bioprinted Ear Grafts Using Chinchilla Models.

The goal of this work is to develop an innovative method that combines bioprinting and endoscopic imaging to repair tympanic membrane perforations (TMPs). TMPs are a serious health issue because they can lead to both conductive hearing loss and repeated otitis media. TMPs occur in 3-5% of cases after ear tube placement, as well as in cases of acute otitis media (the second most common infection in pediatrics), chronic otitis media with or without cholesteatoma, or as a result of barotrauma to the ear. About 55,000 tympanoplasties, the surgery performed to reconstruct TMPs, are performed every year, and the commonly used cartilage grafting technique has a success rate between 43% and 100%. This wide variability in successful tympanoplasty indicates that the current approach relies heavily on the skill of the surgeon to carve the shield graft into the shape of the TMP, which can be extremely difficult because of the perforation's irregular shape. To this end, we hypothesized that patient specific acellular grafts can be bioprinted to repair TMPs. In vitro data demonstrated that our approach resulted in excellent wound healing responses (e.g., cell invasion and proliferations) using our bioprinted gelatin methacrylate constructs. Based on these results, we then bioprinted customized acellular grafts to treat TMP based on endoscopic imaging of the perforation and demonstrated improved TMP healing in a chinchilla study. These ear graft techniques could transform clinical practice by eliminating the need for hand-carved grafts. To our knowledge, this is the first proof of concept of using bioprinting and endoscopic imaging to fabricate customized grafts to treat tissue perforations. This technology could be transferred to other medical pathologies and be used to rapidly scan internal organs such as intestines for microperforations, brain covering (Dura mater) for determination of sites of potential cerebrospinal fluid leaks, and vascular systems to determine arterial wall damage before aneurysm rupture in strokes.

3D Printed Pericardium Hydrogels To Promote Wound Healing in Vascular Applications.

Vascular grafts that can support total replacement and maintenance by the body of the injured vessel would improve outcomes of major surgical reconstructions. Building scaffolds using components of the native vessel can encourage biological recognition by native cells as well as mimic mechanical characteristics of the native vessel. Evidence is emerging that incorporating predetermined building-blocks into a tissue engineering scaffold may oversimplify the environment and ignore critical structures and binding sites essential to development at the implant. We propose the development of a 3D-printable and degradable hybrid scaffold by combining polyethylene glycol (PEG)acrylate and homogenized pericardium matrix (HPM) to achieve appropriate biological environment as well as structural support. It was hypothesized that incorporation of HPM into PEG hydrogels would affect modulus of the scaffold and that the modulus and biological component would reduce the inflammatory signals produced from arriving macrophages and nearby endothelial cells. HPM was found to provide a number of tissue specific structural proteins including collagen, fibronectin, and glycosaminoglycans. HPM and PEGacrylate formed a hybrid hydrogel with significantly distinct modulus depending on concentration of either component, which resulted in scaffolds with stiffness between 0.5 and 20 kPa. The formed hybrid hydrogel was confirmed through a reduction in primary amines post-cross-linking. Using these hybrid scaffolds, rat bone marrow derived macrophages developed an M2 phenotype in response to low amounts (0.03%, w/v) of HPM in culture but responded with inflammatory phenotypes to high concentrations (0.3%, w/v). When cultured together with endothelial cells, both M1 and M2 macrophages were detected, along with a combination of both inflammatory and healing cytokines. However, the expression of inflammatory cytokines TNFα and IL1ß was significantly (p < 0.05) lower with hybrid hydrogels compared to single component PEG or HPM hydrogels. This reduction in inflammatory cytokines could impact the healing environment that persists at the implantation site. Finally, using this developed hybrid hydrogel, models of neonatal vasculature were manufactured using digital light projection (DLP) 3D printing. The structural control achieved with this novel biomaterial suggests a promising new tool in vascular graft development and research, with potential for complex structures for use in congenital heart defect reconstruction.

Bioengineering Strategies to Treat Female Infertility.

Bioengineering strategies have demonstrated enormous potential to treat female infertility as a result of chemotherapy, uterine injuries, fallopian tube occlusion, massive intrauterine adhesions, congenital uterine malformations, and hysterectomy. These strategies can be classified into two broad categories as follows: (i) Transplantation of fresh or cryopreserved organs into the host and (ii) tissue engineering approaches that utilize a combination of cells, growth factors, and biomaterials that leverages the body's inherent ability to regenerate/repair reproductive organs. While whole organ transplant has demonstrated success, the source of the organ and the immunogenic effects of allografts remain challenging. Even though tissue engineering strategies can avoid these issues, their feasibilities of creating whole organ constructs are yet to be demonstrated. In this article we summarize the recent advancements in the applications of bioengineering to treat female infertility.

Development of a 3D Printed, Bioengineered Placenta Model to Evaluate the Role of Trophoblast Migration in Preeclampsia.

Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity and mortality. Current research suggests that the impaired trophoblastic invasion of maternal spiral arteries contributes significantly to the development of PE. However, the pathobiology of PE remains poorly understood, and there is a lack of treatment options largely due to ineffective experimental models. Utilizing the capability of bioprinting and shear wave elastography, we developed a 3D, bioengineered placenta model (BPM) to study and quantify cell migration. Through BPM, we evaluated the effect of epidermal growth factor (EGF) on the migratory behavior of trophoblast and human mesenchymal stem cells. Our results demonstrate a positive correlation between cell migration rates and EGF concentration. These results indicate that a feasible ex vivo placental model can be bioprinted to examine cellular, molecular, and pharmacologic interactions. In addition, EGF clearly affects the celluar migration, a potential therapeutic agent to treat preeclampsia. We envision that our ex vivo tissue modeling approach can be readily transferred to study other normal biologic and abnormal pathologic processes such as fibroblast migration in wound healing and stem cell homing.