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.

Concurrent multi-lineage differentiation of mesenchymal stem cells through spatial presentation of growth factors.

Severe tendon and ligament injuries are estimated to affect between 300 000 and 400 000 people annually. Surgical repairs of these injuries often have poor long-term clinical outcomes because of resection of the interfacial tissue-the enthesis-and subsequent stress concentration at the attachment site. A healthy enthesis consists of distinct regions of bone, fibrocartilage, and tendon, each with distinct cell types, extracellular matrix components, and architecture, which are important for tissue function. Tissue engineering, which has been proposed as a potential strategy for replacing this tissue, is currently limited by its inability to differentiate multiple lineages of cells from a single stem cell population within a single engineered construct. In this study, we develop a multi-phasic gelatin methacrylate hydrogel construct system for spatial presentation of proteins, which is then validated for multi-lineage differentiation towards the cell types of the bone-tendon enthesis. This study determines growth factor concentrations for differentiation of mesenchymal stem cells towards osteoblasts, chondrocytes/fibrochondrocytes, and tenocytes, which maintain similar differentiation profiles in 3D hydrogel culture as assessed by qPCR and immunofluorescence staining. Finally, it is shown that this method is able to guide heterogeneous and spatially confined changes in mesenchymal stem cell genes and protein expressions with the tendency to result in osteoblast-, fibrochondrocyte-, and tenocyte-like expression profiles. Overall, we demonstrate the utility of the culture technique for engineering other musculoskeletal tissue interfaces and provide a biochemical approach for recapitulating the bone-tendon enthesis in vitro.

Model Placental Barrier Phenotypic Response to Fluoxetine and Sertraline: A Comparative Study.

Medications taken during pregnancy may significantly impact fetal development, yet there are few studies that rigorously assess medication safety due to ethical concerns. Selective serotonin reuptake inhibitors (SSRIs) are a class of drug increasingly being prescribed for depression, yet multiple studies have shown that taking SSRIs during pregnancy can lead to preterm birth and potential health concerns for the baby. Therefore, a biomimetic placental barrier model is utilized herein to assess transport profiles and phenotypic effects resulting from SSRI exposure, comparing fluoxetine and sertraline. Results show that the placental barrier quickly uptakes drug from the maternal side, but slowly releases on the fetal side. Phenotypically, there is a dose-dependent change in cell adhesion molecule (CAM) and transforming growth factor beta (TGFß) secretions, markers of cell adhesion and angiogenesis. Both drugs impact CAM secretions, whereas sertraline alone impacts TGFß secretions. When evaluating cell type, it becomes clear that endothelial cells, not trophoblast, are the main cell type involved in these phenotypic changes. Overall, these findings further the understanding of SSRI transplacental transport and drug-induced effects on the placental barrier.

Assessing SSRIs' effects on fetal cardiomyocytes utilizing placenta-fetus model.

Selective serotonin reuptake inhibitors (SSRIs) have been shown to hinder cardiomyocyte signaling, raising concerns about their safety during pregnancy. Approaches to assess SSRI-induced effects on fetal cardiovascular cells following passage of drugs through the placental barrier in vitro have only recently become available. Herein, we report that the SSRIs, fluoxetine and sertraline, lead to slowed cardiomyocyte calcium oscillations and induce increased secretion of troponin T and creatine kinase-MB with reduced secretion of NT-proBNP, three key cardiac injury biomarkers. We show the cardiomyocyte calcium handling effects are further amplified following indirect exposure through a placental barrier model. These studies are the first to investigate the effects of placental barrier co-culture with cardiomyocytes in vitro and to show cardiotoxicity of SSRIs following passage through the placental barrier. STATEMENT OF SIGNIFICANCE: Use of selective serotonin reuptake inhibitors (SSRIs), a class of antidepressants, during pregnancy continues to rise despite multiple studies showing potential for detrimental effects on the developing fetus. SSRIs are particularly thought to slow cardiovascular electrical activity, such as ion signaling, yet few, if any, methods exist to rigorously study these drug-induced effects on human pregnancy and the developing fetus. Within this study, we utilized a placenta-fetus model to evaluate these drug-induced effects on cardiomyocytes, looking the drugs' effects on calcium handling and secretion of multiple cardiac injury biomarkers. Together, with existing literature, this study provides a platform for assessing pharmacologic effects of drugs on cells mimicking the fetus and the role the placenta plays in this process.

In Vitro Models for Studying Transport Across Epithelial Tissue Barriers.

Epithelial barriers are the body's natural defense system to regulating passage from one domain to another. In our efforts to understand what can and cannot cross these barriers, models have emerged as a reductionist approach to rigorously study and investigate this question. In particular, in vitro tissue models have become prominent as there is an increased exploration of understanding biological molecular transport. Herein, we introduce the pertinent physiology, then discuss recent studies and approaches for building models of five epithelial tissues: skin, the gastrointestinal tract, the lungs, the blood-brain barrier, and the placenta. In particular, we evaluated literature from the past 5 years utilizing a tissue model to evaluate molecular transport. We then compare physiology of these tissues and discuss similarities in approaches, across tissues, to validate these models. We conclude with a summary of the approaches of growing interest across multiple tissues and an outlook on future steps to improve these models.

Development of surface functionalization strategies for 3D-printed polystyrene constructs.

There is a growing interest in 3D printing to fabricate culture substrates; however, the surface properties of the scaffold remain pertinent to elicit targeted and expected cell responses. Traditional 2D polystyrene (PS) culture systems typically require surface functionalization (oxidation) to facilitate and encourage cell adhesion. Determining the surface properties which enhance protein adhesion from media and cellular extracellular matrix (ECM) production remains the first step to translating 2D PS systems to a 3D culture surface. Here we show that the presence of carbonyl groups to PS surfaces correlated well with successful adhesion of ECM proteins and sustaining ECM production of deposited human mesenchymal stem cells, if the surface has a water contact angle between 50° and 55°. Translation of these findings to custom-fabricated 3D PS scaffolds reveals carbonyl groups continued to enhance spreading and growth in 3D culture. Cumulatively, these data present a method for 3D printing PS and the design considerations required for understanding cell-material interactions. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2566-2578, 2019.

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.

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.

3D printing for the design and fabrication of polymer-based gradient scaffolds.

To accurately mimic the native tissue environment, tissue engineered scaffolds often need to have a highly controlled and varied display of three-dimensional (3D) architecture and geometrical cues. Additive manufacturing in tissue engineering has made possible the development of complex scaffolds that mimic the native tissue architectures. As such, architectural details that were previously unattainable or irreproducible can now be incorporated in an ordered and organized approach, further advancing the structural and chemical cues delivered to cells interacting with the scaffold. This control over the environment has given engineers the ability to unlock cellular machinery that is highly dependent upon the intricate heterogeneous environment of native tissue. Recent research into the incorporation of physical and chemical gradients within scaffolds indicates that integrating these features improves the function of a tissue engineered construct. This review covers recent advances on techniques to incorporate gradients into polymer scaffolds through additive manufacturing and evaluate the success of these techniques. As covered here, to best replicate different tissue types, one must be cognizant of the vastly different types of manufacturing techniques available to create these gradient scaffolds. We review the various types of additive manufacturing techniques that can be leveraged to fabricate scaffolds with heterogeneous properties and discuss methods to successfully characterize them. STATEMENT OF SIGNIFICANCE: Additive manufacturing techniques have given tissue engineers the ability to precisely recapitulate the native architecture present within tissue. In addition, these techniques can be leveraged to create scaffolds with both physical and chemical gradients. This work offers insight into several techniques that can be used to generate graded scaffolds, depending on the desired gradient. Furthermore, it outlines methods to determine if the designed gradient was achieved. This review will help to condense the abundance of information that has been published on the creation and characterization of gradient scaffolds and to provide a single review discussing both methods for manufacturing gradient scaffolds and evaluating the establishment of a gradient.

Oncogene Knockdown via Active Loading of Small RNAs into Extracellular Vesicles by Sonication.

Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as promising drug delivery vehicles for small RNAs (siRNA and miRNA) due to their natural role in intercellular RNA transport. However, the application of EVs for therapeutic RNA delivery may be limited by loading approaches that can induce cargo aggregation or degradation. Here, we report the use of sonication as a means to actively load functional small RNAs into EVs. Conditions under which EVs could be loaded with small RNAs with minimal detectable aggregation were identified, and EVs loaded with therapeutic siRNA via sonication were observed to be taken up by recipient cells and capable of target mRNA knockdown leading to reduced protein expression. This system was ultimately applied to reduce expression of HER2, an oncogenic receptor tyrosine kinase that critically mediates breast cancer development and progression, and could be extended to other therapeutic targets. These results define important parameters informing the application of sonication as a small RNA loading method for EVs and demonstrate the potential utility of this approach for versatile cancer therapy.