Discipline: Technology and Engineering
Subcategory: Biomedical Engineering
Session: 1
Room: Marriott Balcony B
Melissa Mendoza - Binghamton University
Co-Author(s): Bruce Murray, Binghamton University, Binghamton, NY; Peter Huang, Binghamton University, Binghamton, NY; Gretchen Mahler, Binghamton University, Binghamton, NY
Calcific aortic valve disease (CAVD) is a progressive disease ranging from mild valve thickening (aortic sclerosis) to severe valve calcification (aortic stenosis)1. Current treatments for CAVD include complete valve replacement and minimal drug interventions tailored to other cardiovascular diseases2,3. Early-CAVD has been characterized by the reorganization of the extracellular matrix, where glycosaminoglycans (GAGs) such as chondroitin sulfate (CS), normally found in the valve spongiosa layer, migrate to the collagen-rich fibrosa layer and are deposited near calcified nodules1, 4. Here we utilize three-dimensional (3D) microfluidic devices to study the effects of shear stress and CS on calcification in a model of the aortic valve fibrosa. We hypothesize that as the concentration of CS increases in cultures, there will be an increase in calcific nodules and in the presence of shear stress and endothelial cells, there will be a decrease in calcification. We developed a microfluidic bioreactor that is capable of 3D cell culture for up to 14 days. The multi-layer polydimethylsiloxane (PDMS) microfluidic devices are fabricated utilizing a wafer mold, soft lithography, and corona discharge. The PDMS device consists of a flow channel and a reservoir containing a 3D matrix5. The matrix hydrogels are collagen I-only healthy controls or collagen I with CS at 1 mg/mL and 20 mg/mL. Porcine aortic valve interstitial cells (PAVIC) are embedded within and endothelial cells (PAVEC) are seeded onto the matrix. Steady shear stress at 1 dyne/cm2 is applied using a peristaltic pump and placed at 37?C and 5% CO2. Alizarin Red S staining (ARS), an assay used to assess calcium deposition both qualitatively and quantitatively, is used to quantify calcific nodule formation after cultures. Healthy microfluidic models at 1 dyne/cm2 with only PAVIC embedded into the matrix have decreased ARS staining compared to static controls. The addition of 1 mg/mL CS to the collagen I hydrogels seeded with PAVEC and PAVIC cells resulted in increased ARS (0.050?0.027 mM) compared to static controls (0.023?0.002 mM). In the presence of 20 mg/mL CS, microfluidic PAVIC/PAVEC cultures result in less calcific nodules (0.023?0.0003 mM) compared to static controls (0.031?0.002 mM). Additionally, surface area analysis of calcified nodules in ImageJ, showed that increasing CS in dynamic models decreases the average size of the nodules. These results show that static culture conditions and an increase in CS results in increased calcification. In dynamic cultures, the presence of 1 dyne/cm2 results in lowered ARS levels and resulted in smaller nodules. Further research utilizing this fabricated microfluidic device involves studying additional biomechanical and biochemical factors that lead to valve disease progression in vitro. 1Mahler et al. ArterioThrombVascBio,2013.2Freeman & Otto. Circulation,2005.3Porras et al. PNAS,2018.4Dahal et al. JBiomedMaterResA,2017.5Mina et al. RSCAdvances,2016.
Funder Acknowledgement(s): This work was supported by NSF CMMI 1919438, the Clifford D. Clark Diversity Fellowship for Graduate Students, and LSAMP Bridge to Doctorate.
Faculty Advisor: Gretchen Mahler, gmahler@binghamton.edu
Role: I have conducted all aspects of this research under the supervision of Dr. Gretchen Mahler.