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Equine Bone Marrow Mesenchymal Stem Cell-Derived Extracellular Vesicle Anti-Inflammatory Properties on Tenocyte-Macrophage In-Vitro Co-Cultures

Cooper, Hannah Erin
http://orcid.org/0000-0002-3350-5641
http://rave.ohiolink.edu/etdc/view?acc_num=osu1703852505234168

Abstract Details

2024, Master of Science, Ohio State University, Veterinary Clinical Sciences.
Equine superficial digital flexor tendon (SDFT) injuries heal primarily through fibrosis, in part due to persistent macrophage-induced inflammation throughout the healing process. Despite the low retention and short life of bone marrow mesenchymal stem cells (BM-MSC) following intratendinous injections, benefits including reducing local inflammation, improving histological structure, and reducing tendon re-injury rates, in experimental and naturally occurring equine tendonitis, are well-known. Extracellular vesicle (EV) release at the healing milieu is one of the speculated mechanisms for the immunomodulatory and anti-fibrotic benefits of BM-MSC. EVs are secreted membrane-bound cytokine, growth factor and mRNA/miRNA cargo reflective of the parent cells and facilitate intercellular communication. From a clinical standpoint, EVs circumvent donor site morbidity, time lag for culture expansion and allogenic cell-related immunogenicity limitations of BM-MSC and comprise an ‘off-the-shelf’ cell-free therapy for equine tendon injuries.    This research investigates the mitogenic, anti-inflammatory, and extracellular matrix (ECM) remodeling effects of equine BM-MSC-derived EVs (BM-EV) on tenocytes cultured on acellular SDFT matrix. We hypothesized that (1) BM-EV increases the adherence and proliferation of tenocytes during culture on acellular SDFT matrix, and (2) BM-EV modulates inflammation and stimulates extracellular matrix (ECM) remodeling during direct (cell-to-cell contact) tenocyte-macrophage co-culture on acellular SDFT matrix.   Undifferentiated (7-day basal media), pro-inflammatory (6-day GM-CSF and 24-hour LPS+IFN-γ priming) and regulatory (6-day basal media and 24-hour IL-4+IL-10 priming) macrophages were generated from peripheral blood CD14+ monocytes. Passage 3 tenocytes (CD90+105+27-45-) were isolated from freshly euthanized cadaveric forelimb mid-metacarpal SDFT via collagenase I digestion and monolayer culture.   BM-EV were isolated via ultracentrifugation method (2,000g for 20 minutes and 100,000g for 2 hours at 2-8C) from pooled culture media of passage 2 to 5 equine BM-MSC (CD44+ 29+45-). BM-EV size and concentration were determined through electron microscopy and BCA assay, respectively.     SDFT explants (1cm3) were decellularized through 3 freeze-thaw cycles. Tenocytes (300,000/explant) were seeded on explants and acclimatized for 24 hours. Undifferentiated, pro-inflammatory or regulatory macrophages (1:1 ratio) were introduced to tenocytes seeded on matrices and treated with or without 100ug/mL BM-EV. All co-culture groups were established in triplicates and maintained for 72 hours.     Tenocyte adherence (histology) and proliferation (MTT assay) were assessed in tenocyte-matrix cultures treated with media alone or media supplemented with 100ug/mL BM-EV for 72 hours. Data were analyzed via t-test (p<0.05).     Cytokine and ECM remodeling enzymes (ELISA) were quantified in tenocyte-macrophage matrix co-culture media (aliquoted and stored at -80C). Co-culture matrices were snap frozen for analyzing ECM synthesis (ELISA). Differences between tenocyte-macrophage co-culture and respective BM-EV treatment groups were evaluated via one-way ANOVA or Kruskal Wallis test and subsequent pair wise comparisons (p<0.05).   BM-EV treatment enhanced tenocyte adherence to acellular SDFT matrix and significantly increased the tenocyte proliferation when cultured by themselves (p=0.029) and on matrices (p<0.0001).     BM-EV treatment increased (p=0.0430) IL-6 levels in pro-inflammatory macrophage-tenocyte co-cultures compared to respective regulatory macrophage co-cultures. Media interleukin-1 receptor antagonist (IL-1Ra) concentration of BM-EV-treated tenocyte-pro-inflammatory macrophage co-culture was significantly increased compared to the respective untreated co-culture (p=0.016). IL-1Ra concentrations of BM-EV treated tenocyte-regulatory macrophage co-culture were increased (p=0.011) compared to the remainder experimental groups. Media IL-1β of BM-EV treated pro-inflammatory macrophage-tenocyte co-cultures were 1000-fold increased (p=0.02) as compared to BM-EV treated regulatory groups. Tumor necrosis factor-a (TNFa) BM-EV treated media significantly (p=0.021) reduced TNFa concentrations as compared to media alone. Matrix metallopeptidase -3 (MMP-3) concentrations in BM-EV treated regulatory macrophage-tenocyte co-cultures was 3-fold lower (p=0.008) compared to respective pro-inflammatory co-cultures. BM-EV treatment did not impact co-culture media TIMP-1 and TIMP-2 concentrations.   BM-EV treatment demonstrated mitogenic effect on equine tenocytes and modulated pro-inflammatory macrophages by increasing the secretion of anti-inflammatory molecule, IL-1Ra during co-culture. Interleukin-6 is a multifactorial cytokine with pro-/anti-inflammatory and cell proliferative properties, and consistent with these results are also enriched in rat BM-EV. The lack of concurrent significant differences in IL-6 or TNFa concentrations among BM-EV treated and untreated tenocyte-macrophage co-cultures on SDFT matrix reflect the inter-horse variability noted in our results analyzed thus far and supports further optimization of the co-culture model used. Tenocytes are central to the quality of the ‘healed’ tendon tissue as they are responsible for ECM synthesis during healing. Our ongoing analyses of ECM gene expression and synthesis (collagen type I, III and sulfated GAGs) in matrix co-cultures, in conjunction with ECM remodeling enzyme levels, are vital to determine how polarized macrophages and their secreted cytokines with and without BM-EV treatment impact tenocyte phenotype and ECM turnover.     This is the first report of BM-EV mitogenic effects and immunomodulation properties on equine tenocytes. EVs mitigates the limitations linked to cell-based tendon therapeutics. Given that BM-MSC therapy has demonstrated benefits for enhancing equine tendon healing, our results provide a foundation for evaluating the in-vivo healing attributes of BM-EV in experimental equine SDFT injury.
Sushmitha Durgam (Advisor)
Alison Gardner (Committee Member)
Laura Hostnik (Committee Member)
Amit Sharma (Committee Member)
70 p.
Comparative; Medicine
Equine; Superficial Digital Flexor Tendon; SDFT; Bone Marrow Derived Extracellular Vesicles; Extracellular Vesicles; EV; BM-EV; Tendon Injury; Tendon Healing

Recommended Citations

Citations

  • Cooper, H. E. (2024). Equine Bone Marrow Mesenchymal Stem Cell-Derived Extracellular Vesicle Anti-Inflammatory Properties on Tenocyte-Macrophage In-Vitro Co-Cultures [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1703852505234168

    APA Style (7th edition)

  • Cooper, Hannah. Equine Bone Marrow Mesenchymal Stem Cell-Derived Extracellular Vesicle Anti-Inflammatory Properties on Tenocyte-Macrophage In-Vitro Co-Cultures. 2024. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1703852505234168.

    MLA Style (8th edition)

  • Cooper, Hannah. "Equine Bone Marrow Mesenchymal Stem Cell-Derived Extracellular Vesicle Anti-Inflammatory Properties on Tenocyte-Macrophage In-Vitro Co-Cultures." Master's thesis, Ohio State University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=osu1703852505234168

    Chicago Manual of Style (17th edition)

osu1703852505234168
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