P020 - Amniotic Suspension Allograft Modulates Inflammation in an in vitro Human Synoviocyte Model
Abstract
Purpose
Osteoarthritis (OA) affects 30.8 million Americans; IL-1 and TNF-α are two potent pro-inflammatory cytokines that affect the joint1. Amniotic membranes contain cytokines and growth factors2, including IL-1Ra and TIMPs, that may help modulate the inflammation associated with OA. A previous clinical pilot study using amniotic suspension allograft (ASA), which contains amniotic membrane and cells from the amniotic fluid, was shown to improve pain and function scores out to a year3. In the current in vitrostudy, a synoviocyte inflammation model was used to determine the effect of ASA treatment on inflammation; we hypothesized that ASA treatment would reduce inflammation.
Methods and Materials
Human fibroblast-like synoviocytes (HFLS, Cell Applications) were cultured under standard conditions. Cells were primed with 1ng/mL IL-1β, 10ng/mL TNF-α, or a combination for 72 hours prior to treatment with or without ASA conditioned media (CM). A dosing study ranging from 1-100% was done to determine the optimal CM concentration (25% CM) used for this study. Supernatant was collected 96 hours later for measuring protein production using commercially-available ELISA kits (R&D Systems), while cells were collected in RNAzol for gene expression analysis. Targets examined included IL-1β, TNF-α, MMP-1, TIMP-3, IL-1Ra, and IL-6.
Results
ASA treatment resulted in a significant downregulation in gene expression of IL-1β and MMP-1, and a significant decrease in protein production of IL-1β, TNF-a (Figure 1A), and MMP-1. IL-6 is a dual pro-/anti-inflammatory cytokine; we saw transient changes in gene expression. ASA treatment resulted in a significant downregulation in IL-1Ra gene expression. Treatment with ASA resulted in an increase in protein levels of IL-1Ra (Figure 1B), IL-6, and TIMP-3 (Figure 1C).
Conclusion
In this synoviocyte inflammation model, treatment with ASA resulted in decreased pro-inflammatory cytokines and proteases along with increased anti-inflammatory cytokines and inhibitors. This study provides evidence of the potential for ASA to modulate inflammation.
