Equine bone marrow MSC-derived extracellular vesicles mitigate the inflammatory effects of interleukin-1ß on navicular tissues in vitro.

BACKGROUND: Safe, efficacious therapy for treating degenerate deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) in navicular horses is critically necessary. While archetypal orthobiologic therapies for navicular disease are used empirically, their safety and efficacy are unknown. Mesenchymal stem cell-derived extracellular vesicles (EV) may overcome several limitations of current orthobiologic therapies. OBJECTIVES: To (1) characterise cytokine and growth factor profiles of equine bone marrow mesenchymal stem cell (BM-MSC)-derived extracellular vesicles (BM-EV) and (2) evaluate the in vitro anti-inflammatory and extracellular matrix (ECM) protective potentials of BM-EV on DDFT and NBF explant co-cultures in an IL-1ß inflammatory environment. STUDY DESIGN: In vitro experimental study. METHODS: Cytokines (IL-1ß, IL-6, IL-10, IL-1ra and TNF-α) and growth factors (TGFß1, VEGF, IGF1 and PDGF) in equine BM-EV isolated via ultracentrifugation and precipitation methods were profiled. Forelimb DDFT and NBF explant co-cultures from seven horses were exposed to media alone, or media containing 2 × 109 ± 0.1 × 109 particles/mL or 10 µg/mL BM-EV (BM-EV), 10 ng/mL interleukin-1ß (IL-1ß), or IL-1ß + BM-EV for 48 h. Co-culture media IL-6, TNF-α, MMP-3, MMP-13 concentrations and explant sulphated glycosaminoglycan (sGAG) content were quantified. RESULTS: IL-6, IGF1 and VEGF concentrations were 102.1 (37.61-256.2) and 182.3 (163.1-226.3), 72.3 (8-175.6) and 2.4 (0.1-2.6), 108.3 (38.3-709.1) and 211.4 (189.1-318.2) pg/mL per 2 × 109 ± 0.1 × 109 particles/mL or 10 µg/mL 10 µg of BM-EV isolated via ultracentrifugation and precipitation methods, respectively. Co-culture media MMP-3 in BM-EV- (p = 0.03) and BM-EV + IL-1ß-treated (p = 0.01) groups were significantly lower than the respective media and IL-1ß groups. DDFT explant sGAG content of BM-EV (p = 0.003) and BM-EV + IL-1ß groups were significantly higher compared with IL-1ß group. MAIN LIMITATIONS: Specimen numbers are limited, in vitro model may not replicate clinical case conditions, lack of non-MSC-derived EV control group. CONCLUSIONS: Equine BM-EV contains IL-6 and growth factors, IGF1 and VEGF. The anti-inflammatory and ECM protective potentials of BM-EV were evident as increased IL-6 and decreased MMP-3 concentrations in the DDFT-NBF explant co-culture media. These results support further evaluation of BM-EV as an acellular and 'off-the-shelf' intra-bursal/intrasynovial therapy for navicular pathologies.

Interleukin-1ß and methylprednisolone acetate demonstrate differential effects on equine deep digital flexor tendon and navicular bone fibrocartilage cells in vitro.

OBJECTIVE: To investigate the effects of interleukin-1ß (IL-1ß) and methylprednisolone acetate (MPA) on equine intrabursal deep digital flexor tendon (DDFT) and navicular bone fibrocartilage (NBF) cells in vitro. SAMPLE: Third passage DDFT and NBF cells from 5 healthy donor horses ages 11-17 years euthanized for reasons unrelated to musculoskeletal conditions. PROCEDURES: Aggregate cultures were incubated with culture medium alone (control), 10 ng/mL IL-1ß, 10 ng/mL IL-1ß + 0.05 mg/mL MPA, or 10 ng/mL IL-1ß + 0.5 mg/mL MPA for 24 hours. Extracellular matrix (ECM) gene expressions were assessed via real-time polymerase chain reaction (rtPCR). Culture media matrix metalloproteinase (MMP) -3 and -13 concentrations were quantified via ELISA. Total glycosaminoglycan (GAG) content in the cell pellets and culture media was also assessed. RESULTS: IL-1ß and IL-1ß combined with MPA significantly downregulated ECM gene expression to a greater extent in NBF cells compared with DDFT cells. IL-1ß and IL-1ß combined with MPA significantly upregulated MMP-3 culture media concentrations in DDFT cells only, and MMP-13 culture media concentrations to a greater extent in NBF cells compared with DDFT cells. CLINICAL RELEVANCE: NBF cells were more susceptible to IL-1ß and MPA-mediated ECM gene expression downregulation in vitro. These results serve as a first step for future work to determine intrabursal corticosteroid regimens that limits or resolve the inflammation as well as take into consideration NBF cell biosynthesis in horses with navicular disease, for which currently no information exists.

Evaluation of a Novel Bone Substitute Injection Technique for Potential Treatment of Impact Injury to the Equine Palmar Metacarpal Condyle.

OBJECTIVE: The aim of this study was to evaluate the injection of a bone substitute material (BSM) into an impact lesion in the palmar condyle of the third metacarpal bone. STUDY DESIGN: This was an in vivo controlled study performed on six horses. MATERIALS AND METHODS: Medial metacarpal condyles were exposed via arthrotomy and a compressive lesion created in anaesthetized horses using 80 psi (27.6 MPa) onto the articular surface (n = 12). Paired limbs were randomly selected as a control or for extra-articular injection of BSM towards the subchondral bone near the compressive lesion. Parameters of the surgical techniques and BSM distribution outcomes were evaluated using magnetic resonance imaging analysis, histology and histomorphometry. RESULTS: Injection of the BSM required significant pressure, as well as the use of a pilot hole. The BSM was visible in all magnetic resonance imagings in treatment limbs. Post-impact treatment limbs had greater average grey scale values than controls (p = 0.041), and greater average grey scale values than pre-impact treatment limbs (p = 0.004). Histology demonstrated haemorrhage and microfractures at the site of compression with no evidence of bone disruption from BSM injection. CONCLUSION: Injection of BSM into the dense subchondral bone of the equine palmar condyle could be targeted to a site of injury, distributed subchondrally and without further injury to bone or cartilage. CLINICAL SIGNIFICANCE: This procedure has potential for the treatment of clinical impact injury or osteoarthritis in horses, and long-term studies are warranted.