Engineering A Hydrogel Based Biomaterial Encapsulating FTY720 and RAW Macrophages to Stimulate Accelerated Wound Healing in an Oro-Nasal Fistula
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Author(s)
Behara, Monica
Advisor(s)
Goudy, Steven
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Abstract
Aberrant wound healing can sometimes be characterized as a prolonged exposure of pro-inflammatory immune cells and cytokines to the site of injury. This prolonged immune response can further damage tissue, often resulting in irreparable damage. Current studies in the field of wound healing attempt to modulate the immune system response to injury to reduce further complications from aberrant wound healing. FTY720 is an FDA approved drug for the treatment of multiple sclerosis to control immune cell circulation into the blood, reducing relapsing symptoms of poor voluntary muscle control and spasms. This work assesses the engineering and development of a biomaterial fabrication that encapsulates both FTY720 and RAW264.7 macrophages to serve as a supplement to wound healing in an oro-nasal fistula. An oro-nasal fistula arises as a complication to cleft palate repair, that can often lead a child to have developmental issues with speaking and eating. The oro-nasal fistula describes the abnormal opening between the oral and nasal cavities that is difficult to close once the wound forms. This leaves a pressing need to find a minimally invasive technique to heal this wound organically, leading to the development of an immunoregenerative strategy.
In this work, a hydrogel fabrication with PEG-4MAL, RGD and GPQ was developed to fully encapsulate RAW264.7 macrophages and FTY720 into one construct that will deliver these components to the area of the oronasal fistula wound to accelerate wound healing. This innovation was hypothesized to accelerate wound healing and generate cytokines to stimulate the pro-regenerative environment and effect of this biomaterial. This delivery of macrophages was introduced after studies involving nanofiber scaffold release of FTY720-P showed promise to a greater pro-regenerative effect through optimal M2 polarization and decreased migration. The hydrogel biomaterial was optimized to find the number of macrophages required for proliferation within the hydrogel matrix, as well as cytokine release and migratory capacity of these encapsulated macrophages. It was found that the macrophages optimally proliferate at 200,000 cells per 20 μL hydrogel and show decreases in pro-inflammatory cytokines and increases in pro-regenerative cytokines at the 10 μM concentration of FTY720-P. Additionally, the migratory capacity of these macrophages was assessed in normal growth media conditions as well as swelling with artificial saliva, and it was found that the migratory capacity in both cases was optimal at 5 μM of FTY720-P stimulation. Once the biomaterial was characterized in vitro, an in vivo experimental model was carried out to further test the wound healing capacity of this biomaterial in an oro-nasal fistula. It was found that the implantation of an FTY720-P and RAW264.7 macrophage hydrogel does significantly enhance wound healing of an oro- nasal fistula and can be further corroborated by the proven recruitment of significantly greater M2 macrophages and significantly fewer M1 macrophages over 7 days.
Overall, the development of this new biomaterial was found to have pro-regenerative effects on the oro-nasal fistula injury environment, showing that the implantation of both macrophages and FTY720 in one biomaterial does have potential to accelerate wound healing in humans. The next steps for this study would be to incorporate human macrophages and optimize based on size and behavior of the human macrophages, harvested from human tissue, like tonsil tissue. This therapeutic can potentially help a variety of pediatric patients suffering from oro-nasal fistula and can aid in accelerating wound healing to limit developmental problems that may ail the patient later in life.
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2023-04-25
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