2021 Virtual Undergraduate Research Symposium
2021 Virtual Undergraduate Research Symposium
In vitro analysis of hMSCs on Polyelectrolyte Complexes in the Context of Physeal Injuries
In vitro analysis of hMSCs on Polyelectrolyte Complexes in the Context of Physeal Injuries
HONORABLE MENTION
PROJECT NUMBER: 10 | AUTHOR: Sydney Marchando, Chemical and Biological Engineering
MENTOR: Melissa Krebs, Chemical and Biological Engineering
ABSTRACT
The growth plate, or epiphyseal plate, is a cartilage region at the end of long bones which is responsible for longitudinal bone growth in children. Growth plate fractures can result in the formation of a “bony bar”, which can cause limb shortening and growth defects. Current treatment involves surgical resection of the bony bar and filling of the injury site with an intermediate, such as adipose tissue or other inert biomaterials. These treatments often fail because of insufficient mechanical support and lack of mimicry to the native extracellular matrix (ECM) structure which is necessary for proper tissue regeneration. In this project, hydrogel polyelectrolyte complexes (PECs) composed of various concentrations of the biopolymers alginate, chitosan, hyaluronic acid, and calcium were investigated. This project involved the development of scaffolds for tissue culture in vitro and the analysis of these polymers on differentiation and growth of human mesenchymal stem cells (hMSCs). In particular, PECs fabricated with RGD and HA, as well as with lower molecular weight alginate, significantly decreased alkaline phosphatase (ALP) expression. The PECs were also tested as a drug delivery system for sustained release of small interfering RNA (siRNA). The effect of siRNA on gene knockdown for the p38 MAPK-14 pathway, as well as the effect of this siRNA on the downstream Runx2 and Col1A1 pathways was analyzed utilizing quantitative polymerase chain reaction (qPCR). A decrease in the expression of both Runx2 and Col1A1 was observed after treatment with p38 MAPK14 siRNA. The adhesion and proliferation of the cells grown on the scaffolds was visualized utilizing confocal microscopy of cells stained with DAPI.
PRESENTATION
AUTHOR BIOGRAPHY
Sydney Marchando is a senior studying Chemical Engineering with minors in Biomedical Engineering and Public Affairs. She began working in Dr. Melissa Krebs’ laboratory in Spring 2020 to develop a novel treatment method for growth plate injuries. Her project involves the development of biopolymers and tissue culture scaffolds to determine their effect on stem cell growth and differentiation. She has enjoyed conducting research related to an injury she suffered as a young child and is planning to continue research in the orthopedic field with a Master’s degree in Biomedical Engineering following her graduation in May.
Congratulations, Sydney! Enjoyed your presentation and reading about your research.
Very cool research Sydney! I enjoyed your presentation and look forward to reading/hearing about those next steps you talked about. Those confocal images are also super awesome!
Thank you Drake! I’m excited for what the next steps might mean in terms of an actual treatment for patients down the road!
Great work and congratulations! You’re research is fascinating and I enjoyed your presentation!
Thank you so much!
Thank you for your support and for watching my presentation!
Woohoo Sydney rocks! Nice job on the presentation and great job this semester!
Thank you Michael, and thanks for all your support this past year!
Hooray Sydney, well done, such great work! Thank you for contributing so much to our group!
Thank you so much for all your support and guidance, Dr. Krebs!! You are the best!
I liked your talk. It seems like your research topic has some really nice medicinal utility moving forward! Are higher molecular weight PECs more effective as a treatment since they are associated with greater alkaline phosphatase expression? Good job on your talk!
Thanks Griffin! Alkaline phosphatase expression is associated with osteogenesis, which is the formation of bone. In our case, we are hoping to prevent the formation of a bony bar in the growth plate fracture, so the lower expression of ALP with lower molecular weight alginate is what we are investigating more. Similar to the decline seen with HA and RGD.
Nice work, Sydney! You demonstrated that the period of release is at least 6 months. What are the relevant time scales for bony bar formation in the clinical setting?
Thank you Dr. Diniz Behn! Bony bar formation usually occurs 24-28 days after the injury to the growth plate, so the sustained release is exciting. Something of note that we will definitely have to consider in future studies is the fact that this release occurred without degradation factors like shear force or enzyme induced degradation, which would likely have an affect on the duration the scaffolds could release in the body. We think this sustained release is due to charge interactions between the scaffolds and the siRNA, so we will need to further look into these findings, particularly for application in a patient down the road.
I really enjoyed the use of figures in your poster. You mention the need for adequate mechanical support in your abstract, do you have plans to do more material analysis in the future in terms of mechanical properties? Also, after all of the enzyme has been released, how does the scaffold interact with the body. Will it bio-degrade or is it integrated into the new tissue?
Thanks Haidyn! On these hydrogels, we’ve conducted rheology to look at their mechanical properties, as well as swelling and degradation analysis to observe how the hydrogels respond to fluid flow to try to mimic what they would experience in the body. The goal with this treatment is prevent bone formation, so we are also very interested in the result of our scaffolds on the stem cells we grow on them, which is why we are looking at osteogenic (bone) markers, such as ALP. Typical treatments that fail occur after the bony bar has formed, requiring removal of the bony bar, and then filling this location with something to try to prevent the bar from forming again. The goal of this project would be to prevent bony bar formation. In the body, the gels would degrade, ideally after they had sufficient time to release within the injury site. Thanks for watching and for your questions!