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. 2017 Aug;25(8):1353-1361.
doi: 10.1016/j.joca.2017.03.015. Epub 2017 Mar 30.

Toward understanding the role of cartilage particulates in synovial inflammation

Affiliations

Toward understanding the role of cartilage particulates in synovial inflammation

A M Silverstein et al. Osteoarthritis Cartilage. 2017 Aug.

Abstract

Objective: Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with osteoarthritis (OA). Here, we developed an in vitro model to study the interaction of cartilage wear particles with fibroblast-like synoviocytes (FLS) to better understand the interplay of cartilage particulates with cytokines on cells of the synovium.

Method: In this study sub-10 μm cartilage particles or 1 μm latex particles were co-cultured with FLS ±10 ng/mL interleukin-1α (IL-1α) or tumor necrosis factor-α (TNF-α). Samples were analyzed for DNA, glycosaminoglycan (GAG), and collagen, and media samples were analyzed for media GAG, nitric oxide (NO) and prostaglandin-E2 (PGE2). The nature of the physical interaction between the particles and FLS was determined by microscopy.

Results: Both latex and cartilage particles could be phagocytosed by FLS. Cartilage particles were internalized and attached to the surface of both dense monolayers and individual cells. Co-culture of FLS with cartilage particulates resulted in a significant increase in cell sheet DNA and collagen content as well as NO and PGE2 synthesis compared to control and latex treated groups.

Conclusion: The proliferative response of FLS to cartilage wear particles resulted in an overall increase in extracellular matrix (ECM) content, analogous to the thickening of the synovial lining observed in OA patients. Understanding how cartilage particles interface with the synovium may provide insight into how this interaction contributes to OA progression and may guide the role of lavage and synovectomy for degenerative disease.

Keywords: Cartilage; Inflammation; Synovium; Wear particles.

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Conflict of interest statement

Conflict of Interest Statement

None of authors had a financial or personal relationship with organizations that could influence the work presented in this manuscript.

Figures

Figure 1
Figure 1
Schematic of the experimental study design. In study 1, FLS were cultured for 5 days with increasing concentrations of latex or cartilage particulates (up to 1000:1 particles:cells). In study 2, FLS were cultured for 5 days in the presence or absence of 250 latex or cartilage particles per cell and 10 ng/mL IL-1α or TNF-α. In study 3, FLS were cultured in the presence (FLS + CART) or absence (FLS) of 250 cartilage wear particles per cell for 5 days. Concurrently, media alone and particles alone were also cultured. Conditioned media from each donor group was combined with fresh media and added to recipient cultures for 5 days.
Figure 2
Figure 2
Collagen I (green, A) and lubricin (green, B) vs. negative (C) staining of a FLS cells sheet model of synovium. DAPI staining appears blue. Scalebar = 100um.
Figure 3
Figure 3
MTT assay for assessing the effect of increasing concentrations of cartilage (A) and latex (B) particles on cellular metabolic activity. Data presented as mean and 95% confidence interval. * p<0.05 vs. control (0:1 particles: cell), **p<0.05 vs. 100:1 cartilage particles: cell, *** p<0.05 vs. 250:1 cartilage particles: cell. # p<0.05 vs. control 0:1, 500:1, 750:1 and 1000:1 latex particles: cells, ## p<0.05 vs. 0:1, 100:1, 250:1, and 500:1 latex particles: cells.
Figure 4
Figure 4
(A) Z-stacks of a synovial cell (red) cultured without (CTL) and with (CART) cartilage wear particles (yellow/green). (B) 3D reconstruction. Top (C) and side (D) views of the 3D reconstructions of actin staining from the cartilage particles are found on the cell surface (a) and internalized by the cells (b). Particles indicated by white arrows. Scale bar = 20μm.
Figure 5
Figure 5
Analysis of FLS cells sheets cultured ± IL-1α or TNF-α and ± cartilage wear particles. The DNA, ECM synthesis and media NO and PGE2 of the particles alone was subtracted from the total to determine the final values. DNA (A), FLS cell sheet GAG (B), FLS cell sheet collagen (COL) (C), media GAG/DNA (D), media NO/DNA (E) and media PGE2/DNA (F). Data presented as mean and 95% confidence interval. ND= not detected. * p<0.05 vs. non-particle treated control (CTL) with same cytokine treatment. # p<0.05 vs. − IL/-TNF, ## p<0.05 vs. + IL/-TNF within CTL or CART treated samples respectively.
Figure 6
Figure 6
(A) Z-stacks of a synovial cell (red) cultured with latex wear particles (yellow). B) Actin (red)-DAPI (blue) staining showing remodeling of the cytoskeleton around the ingested latex beads. Scale bar =20μm. Analysis of FLS cell sheets cultured ± latex particles for DNA (C), FLS cell sheet COL (D) and media NO/DNA (E), all normalized by corresponding control (CTL) values. Data presented as mean and 95% confidence interval. * p<0.05 vs. non-particle treated CTL.
Figure 7
Figure 7
DNA (A), FLS cell sheet GAG/DNA (B), and COL/DNA (C) for the conditioned media experiment. Data presented as mean and 95% confidence interval. * p<0.05 vs. Media only. # p<0.05 vs. Particles only.

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