(K-407) Functionalized Hydrogels to Investigate the Reciprocal Relationship Between Extracellular Matrix Stiffness and Cell Behavior

Introduction:: The development and pathology of different tissues in the body can be characterized by changes in composition and physical properties. Research has emphasized the mechanical conditions of extracellular environments and the role that tissue stiffness specifically plays in cell behavior. Literature suggests that matrix stiffness plays an essential role in dictating cell behavior during development, tissue homeostasis, and disease progression [1]. Researchers have relied on tunable hydrogels such as polyacrylamide (PA) to investigate tissue stiffness in this regard, as they can be adjusted to correlate with specific stiffnesses of various tissues and pathological states. However, cells do not adhere to the PA surface and do not allow researchers to account for fiber patterning found with different tissues and pathologies (e.g., lung tissue in pulmonary fibrosis). This research proposes a new method of functionalization that uses polymer fibers physically entangled onto the surface of the PA gel to provide a matrix for cell seeding. Polymer fibers mimic collagen, whereas the hydrogel component represents the proteoglycans found in the extracellular matrix. This work evaluates methods of adhering fibers to hydrogels of various stiffnesses and will serve as a springboard to study cell behavior in tunable microenvironments.

Materials and Methods:: Polymer fiber mats were fabricated via solution blow spinning technique with polycaprolactone (PCL) dissolved in dichloromethane at 6% mass/vol [2]. Samples for scanning electron microscopy (SEM) were deposited directly onto SEM specimen stubs, stored in a desiccator, and sputter coated with gold for imaging. A 16mm diameter hand punch was used to cut out scaffold disks from the PCL fiber mat. Scaffolds were prewetted by centrifuging for 3 minutes at 3000 rpm through a gradient series of ethanol, rinsed with deionized water, then stored in phosphate-buffered saline. Polyacrylamide (PA) hydrogels of various stiffnesses were prepared by combining different ratios of acrylamide monomer and bisacrylamide (bis) cross-linking agent with initiator and catalyst. Hydrogels were created within stiffness ranges from normal physiological to fibrotic pathological states [3].To prepare the PA hydrogel for PCL fiber scaffold interface, a 25mm cover slip was placed inside a 100mm petri dish, and then a Teflon O-ring was placed on the coverslip. The Teflon O-ring, 1.5mm wide with 17mm inner diameter, allows for molding the PA gel solution during crosslinking and the ability to adjust hydrogel thickness. The gel solution was pipetted into the O-ring, and the prewetted PCL scaffold was placed over the gel (Fig. 1). The petri dish was covered and left for 15-20 minutes for the hydrogel to solidify with the fiber scaffold.

Results, Conclusions, and Discussions:: ImageJ was used to analyze SEM images of PCL fiber scaffolds and the data were imported into OriginPro to generate a histogram showing the diameter distribution of fibers. Fibers ranged from 200nm to 1μm in diameter with an average of 512nm and standard deviation of 138nm. Solution blow spinning process parameters such as polymer concentration, nozzle to collector distance, and gas pressure can be adjusted to tailor fiber scaffold properties and the spot size of the mat generated. Fiber scaffolds were successfully adhered to hydrogels of various stiffnesses. Stiffnesses spanned physiological to pathological states by varying the monomer to cross-linking agent ratio. For normal physiological tissue valued at 500Pa, the gel formulation contained 3% acrylamide and 0.2% bis. For fibrotic tissue valued at 5,000Pa, the gel formulated contained 7.5% acrylamide and 0.2% bis. The next steps are to model advanced fibrosis with 50,000Pa gels and experimentally confirm hydrogel stiffnesses with and without adhered fibers. Future work will investigate cell attachment, morphology, and growth on fiber-hydrogel composite scaffolds. Development of this platform will enable research on cell function in a microenvironment that more closely resembles tissues to study the reciprocal relationship between extracellular matrix stiffness and cell behavior such as in fibrosis and tumor formation.

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References (Optional): : [1] Handorf AM, Zhou Y, Halanski MA, Li WJ. Tissue stiffness dictates development, homeostasis, and disease progression. Organogenesis. 2015;11(1):1-15. doi: 10.1080/15476278.2015.1019687. PMID: 25915734; PMCID: PMC4594591.

[2] Juliano E. Oliveira, Luiz H. C. Mattoso, William J. Orts, Eliton S. Medeiros, "Structural and Morphological Characterization of Micro and Nanofibers Produced by Electrospinning and Solution Blow Spinning: A Comparative Study", Advances in Materials Science and Engineering, vol. 2013, Article ID 409572, 14 pages, 2013. https://doi.org/10.1155/2013/409572

[3] Yeung, T., Georges, P. C., Flanagan, L. A., Marg, B., Ortiz, M., Funaki, M., Zahir, N., Ming, W., Weaver, V., & Janmey, P. A. (2005). Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell motility and the cytoskeleton, 60(1), 24–34. https://doi.org/10.1002/cm.20041