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    Tissue Engineering

    (L-446) Investigating cytotoxicity of the photoinitiator ruthenium and sodium persulfate on lymphatic endothelial cells seeded on methacrylated collagen

    Thursday, October 12, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row L - Poster # 446

      Presenting Author(s)

    • EX

      Eleni Xhupi

      Student
      Worcester Polytechnic Institute, United States

      • Co-Author(s)

    • BR

      Brian N.K Ruliffson

      PHD student
      Worcester Polytetic Institute, Massachusetts, United States

      • Last Author(s)

    • CW

      Catherine F. Whittington

      Assistant Professor
      Worcester Polytechnic Institute, United States

    Introduction:: Fibrosis relates to the formation of fibrous tissue to create a stiff environment that can be attributed to pathological illnesses such as lymphedema. Fibrotic tissue surrounding lymphatic vessels is seen in patients with lymphedema, which occurs when there is an insufficient number of functioning lymphatic vessels that decreases lymph fluid drainage and alters the body’s immune response. Currently, there are limited in vitro models that represent the formation of fibrous connective tissue at relevant pathological tissue stiffness levels and can investigate the response of lymphatic vessels to changing tissue stiffness. This gap prevents us from reproducing a lymphatic endothelial cell (LEC) response that occurs in a stiff tissue matrix (i.e., changes in growth, proliferation, and migration) and further investigating the progression and effects of fibrosis and tissue stiffening on diseases like lymphedema without using in vivo models.  Lymphatic endothelial cell response (viability, proliferation, cell-cell interaction) to a pre- stiffened collagen matrix and a progressively stiffened matrix will be explored in this work. To develop a model capable of progressive stiffening observed in fibrosis in vivo, photoinitiaors ruthenium and sodium persulfate will be used to stiffen methacrylated type I collagen, and this project investigates whether the photoinitiator is toxic to LECs, because there is limited information on photoinitiator to this cell population. Overall, this model contributes to work that aims to build an accurate in vitro fibrosis model with progressive stiffening to recreate the LEC response and better understand related pathological conditions.  

    Materials and Methods:: The cytotoxicity of photoinitiator ruthenium and sodium persulfate (RUT) was assessed through a live/dead viability study. Four experimental groups were used to examine the effects of different conditions on Human Dermal Lymphatic Endothelial cells (HDLEC; PromoCell). A summary of experimental groups and controls is shown in Table 1. All groups used methacrylated collagen (ColMA; 8 mg/mL; Advanced BioMatrix) coated with fibronectin and were seeded with HDLEC’s at 8,000 cells per well on Day 0. One sample was prepared with photoinitator (RUT) within ColMA at the start of culture (Group 1), and some were initially prepared without photoinitator then dosed with phonoinitator in cell culture medium on Day 6 (Groups 2 and 3) to allow fresh photoinitator to diffuse into ColMA overnight prior to photo -crosslinking. Any photo-crosslinked samples were exposed to 405 nm light for 90 secs. All samples were cultured for 7 days prior to staining with Live/Dead assay (calceinAM – live; ethidium homodimer - dead) and imaged immediately on Keyence fluorescence microscope. A total cell count was performed using ImageJ to calculate the percentage of living and dead cells. Two other experiments were run simultaneously under the same conditions to assess cell proliferation and formation of cell-cell junctions. After both plates were fixed, one was stained with VE-Cadherin to look at cell-cell junctions, and the other was stained with Ki- 67 proliferation antibody. Both samples were imaged on the Keyence fluorescent microscope, 4x magnification and analyzed using ImageJ.   

    Results, Conclusions, and Discussions::

    There was more growth and proliferation of HDLECs in Group 1 with 97% viability compared to 74% in Group 2 as shown in (Figures 1 and 2). There did not appear to be a stark difference in Group 3 and 4 cell viability compared to Group 1. Stiffening of the matrix prior to introduction of cells likely created a favorable environment for cell attachment and growth, while stiffening after seeding may have rendered cells less adaptable to change. To better understand how the cells that had attached to the progressively stiffened collagen, Group 2, a primary antibody to VE- Cadherin was applied. In Figure 3, Group 2 results showed that the HDLECs were not greatly impacted by the photoinitator, showing similar morphology observed to LEC’s cultured on 2D surfaces.  This positive staining suggests that although viability was lower in Group 2, cells still attached to ColMA and retained cell integrity when compared with controls. 




    Overall, the addition of RUT within and on top of ColMA shows promising results based on reasonable levels of cell death and normal cell phenotype. Further work must be done with additional replicate experiments to draw a definitive conclusion for RUT as a photoinitiator for progressive stiffening of ColMA. Future work will include introducing an experimental group to assess RUT retention and evaluating the effects of stiffening on cell-cell junction formation to gain more insight into the effects of fibrosis on lymphatic capillary function.   




    Acknowledgements (Optional): : We would like to acknowledge the National Cancer Institutes RO3 award (1 R03CA267449-01) and Genentech Research Award (PI: University of Michigan) for funding this work.

    References (Optional): :