Session: Research Posters

Paper Number: 111690

111690 - Leveraging in Vitro Model Systems to Assess Uterine Mechanobiology During Pregnancy 

Introduction: Uterine tissue undergoes significant growth and remodeling throughout pregnancy to support fetal development and parturition. The smooth muscle laden myometrium is spatiotemporally regulated by unique biochemical and biomechanical signals that coordinate the transition from quiescence to highly contractile. Despite general knowledge of phenotypic modulation of other smooth muscle systems, there remains a limited understanding of the mechanisms regulating the contractile phenotype of myometrial cells. This hinders progress in new therapeutics address obstetric complications such as preterm labor and post-partum hemorrhaging- in which dysfunctional myometrial contractility underlies pathology.

Biomechanical signaling arises from progressive tissue stretch imposed by rapid fetal growth in the third trimester. Myometrial cells transition towards a contractile phenotype, indicated by higher expression of contraction-associated proteins, including oxytocin receptor and connexin-43, which coordinate synchronized and forceful muscle contractions during labor. Given that mechanical loading via stretch initiates the contractile phenotype of myometrial cells during pregnancy, our central hypothesis proposes that this mechanical signal is modulated by the cellular micro-environment. Consequently, aberrant transmission of mechanical cues contributes to dysfunctional contractility. In order to test this hypothesis, our group developed a mechanically tunable in vitro model with independent control over (1) matrix stiffness (2) matrix protein composition (3) externally applied mechanical load. This work explores the regulatory effects of cell-ECM interactions on this activation pathway through in mechanical activation of third trimester myometrial cells using our in vitro model.  

Methodology: PHM1-41 cells, immortalized myometrial cells derived from pregnant human myometrium​ during elective cesarean section (not in labor and without pregnancy complications​), are used in this work. Mechanical conditioning of 2D monolayers of PHM1-41 cells with uniaxial static strain is accomplished using the Flexcell Tension System. Bulk Contraction is a measurement % gel contraction of  mechanically conditioned cells resuspended in 3D collagen gels. Gene and protein expression levels of contraction-associated proteins (oxytocin receptor and connexin-43) are quantified using quantitative RT-PCR and Western blots respectively. Relative phosphorylation of MLC20 relies on antibody based immunofluorescence analysis. ImageJ was utilized to measure % gel contraction, cellular alignment, corrected total cell fluorescence.

Preliminary Results and Conclusion: Upregulation of fibronectin expression during late pregnancy in animal models indicates matrix-mediated strength reinforcement prior to labor commencement, which corresponds to an upregulation of fibronectin binding integrins (e.g. 𝛼5𝛽1). (Burkin et al. 2013) These matrix protein-mediated effects are seen in our centrifugal based cell adhesion assays in which PHM1-41 cells have higher adhesion strength on fibronectin-coated substrates relative to Type I collagen controls. Similarly, fibronectin substrates have higher basal phosphorylation levels of MLC20.

Our model enables new assessments of fibronectin-mediated activation of myometrial cells through mechanical conditioning with our Flexcell Instrument. Our initial studies have demonstrated in vitro mechanical activation of myometrial cells after 24 hours of 10% uniaxial static strain on fibronectin coated plates. Contractile responses were quantified by bulk contraction measurements in addition to expression levels of contraction-associated proteins. Additionally, statically strained cells show increased cellular alignment parallel to the applied strain direction. Interestingly, our studies have implicated the Rho/ROCK pathway as a mediator of mechanical activation in myometrial cells. Further studies will assess the role of specific fibronectin binding integrins in the transduction of activating mechanical signals.  

Overall, this preliminary work begins to expand our understanding of contraction regulation during pregnancy and has the potential to inform new directions in myometrial therapeutics.

Presenting Author: Isabella Claure Boston University

Presenting Author Biography: Isabella Claure a Biomedical Engineering PhD student at Boston University, co-advised by Dr. Joyce Wong and Dr. Catherine Klapperich. Our work employs science and engineering approaches to address current women's health issues. My project advances foundational understanding of uterine smooth muscle mechanobiology during pregnancy using in vitro model systems. Currently a fellow of the NIH Translational Research in Biomaterials training program.

University of Miami Alumni (Go Canes!), graduated in 2018 with a B.S. in Biomedical Engineering. Research experience in organ-on-a-chip systems, biomaterial characterization, and nanoparticle design.

Passionate about ensuring diversity in the sciences, and supporting the next generation of great minds.

Authors:

Isabella Claure Boston University
Anika Joglekar Boston University
Catherine Klapperich Boston University
Joyce Wong Boston University