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"Tendon injuries are the top reported musculoskeletal injuries with Achilles, patellar, and rotator cuff tendons as the most common types of tendon injuryy (Ackermann & Institutet, 2013). Tendon injuries are a common cause of disability and account for decreased productivity. Both chronic and acute injuries can result in long-term pain and disability(Pabón & Naqvi, 2021).Extracellular vesicles, specifically exosomes, treatment has proven to enhance regenerative strategy by improving ; biomechanical properties, accelerating tenocyte proliferation and migration, reducing inflammation, and facilitating the healing at tendon–bone interface(Ren et al., 2021).
A THP-1 cell line activation model used to quantify the effects of tenocyte conditioned media (CM) on the proliferation and morphology of the activated THP-1 cells. When THP-1 cells are activated, they polarize into macrophages and monocytes depending on environmental signals. Flow cytometry was used to accurately characterize specific macrophage phenotypes in the presence of tenocyte CM by using CD-markers (CD14,CD25,CD197,CD204 CD206 and HLADR) to understand if the modulation triggered inflammation(M1) or supressed inflamation (M2) phenotype Exosomes isolated from tenocyte CM cultured at 21% and 2% oxygen concentrations then filtered (0.22um) before differential centrifugation (100,000g 90mins), pellets were analyzed for protein concentration and particle size then added to a density gradient prepared with Optiprep solution and spun at 100,000g for 16 hours. Protein concentration was obtained for 12 fractions, iodixanol content(via UV vis 230nm) and particle size (zetasizer). Fractions 4-7 (1.08g-1.22g) were spun again for 2hrs at 120,000g to remove idoxoanol ,finally the presence of exosomes was confirmed via Scanning electron microscopy and zeta sizer particle size data.
Tenocyte CM obtained at 21% and 2% oxygen concentrations, have higher levels of inflammatory cytokines when compared with serum free DMEM. Cytokine levels obtained via quantitative cytokine array (eve technologies Canada). Significant differences observed in cytokine levels (IL-2, IL-6 and IL-8) with 21% CM displaying higher levels (28ug,22,20ug/ml) than 2% oxygen.(19,16,15ug/ml) .The most abundant cytokines were; IL-2 IL-6, IL8, TNFa in CM in both oxygen concentrations. Flow cytometry utilizing; CD197, HLADR and CD86 we were able to demonstrate that activated THP-1 cells cultured in tenocyte CM showed a phenotypic shift towards higher levels of M1 macrophages (inflammatory state), at 21%. In 2% tenocyte conditioned media a lower level of expression of all but CD25 when compared to 21%, pointing towards hypoxia inducing a suppression of M1 and M2 macrophages. However, 2% CM THP-1 cells demonstrated higher levels of dendritic cells when compared to 21% (established by CD25 expression. Exosome preparations confirmed a protein concentration of 120ug/ml after density gradient ultracentrifugation, we further characterized via zetasizer which confirmed particles from 30nm-150nm and SEM imaging to confirm the presence of exosomes (via morphology and size).
Futhur characterisation using ; tetraspanins and CD markers. Exosome preparations will be added to the THP-1 activation model in order to observe how exosomes influence the macrophage phenotype. Tenocyte conditioned media is able to modulate the immune system and stimulate healing."
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