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Wear particles derived from metal hip implants induce the generation of multinucleated giant cells in a 3-dimensional peripheral tissue-equivalent model.

Dutta DK, Potnis PA, Rhodes K, Wood SC - PLoS ONE (2015)

Bottom Line: Our results show that endothelial cells induce the generation of MGCs to a level 4 fold higher in 3-dimentional PTE system as compared to traditional 2-dimensional culture plates.In sum, we have established a robust and relevant model to examine MGC and osteoclast formation in a tissue like environment using flow cytometry and RT-PCR.With endothelial cells help, we observed a consistent generation of metal wear particle- induced MGCs, which heralds metal on metal hip failures.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, CDRH, FDA, Silver Spring, Maryland, United States of America; Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.

ABSTRACT
Multinucleate giant cells (MGCs) are formed by the fusion of 5 to 15 monocytes or macrophages. MGCs can be generated by hip implants at the site where the metal surface of the device is in close contact with tissue. MGCs play a critical role in the inflammatory processes associated with adverse events such as aseptic loosening of the prosthetic joints and bone degeneration process called osteolysis. Upon interaction with metal wear particles, endothelial cells upregulate pro-inflammatory cytokines and other factors that enhance a localized immune response. However, the role of endothelial cells in the generation of MGCs has not been completely investigated. We developed a three-dimensional peripheral tissue-equivalent model (PTE) consisting of collagen gel, supporting a monolayer of endothelial cells and human peripheral blood mononuclear cells (PBMCs) on top, which mimics peripheral tissue under normal physiological conditions. The cultures were incubated for 14 days with Cobalt chromium alloy (CoCr ASTM F75, 1-5 micron) wear particles. PBMC were allowed to transit the endothelium and harvested cells were analyzed for MGC generation via flow cytometry. An increase in forward scatter (cell size) and in the propidium iodide (PI) uptake (DNA intercalating dye) was used to identify MGCs. Our results show that endothelial cells induce the generation of MGCs to a level 4 fold higher in 3-dimentional PTE system as compared to traditional 2-dimensional culture plates. Further characterization of MGCs showed upregulated expression of tartrate resistant alkaline phosphatase (TRAP) and dendritic cell specific transmembrane protein, (DC-STAMP), which are markers of bone degrading cells called osteoclasts. In sum, we have established a robust and relevant model to examine MGC and osteoclast formation in a tissue like environment using flow cytometry and RT-PCR. With endothelial cells help, we observed a consistent generation of metal wear particle- induced MGCs, which heralds metal on metal hip failures.

No MeSH data available.


Related in: MedlinePlus

Increase in particle to cell ratio increases frequency of dead cells.Particles were added at the time of gel polymerization at a ratio of 500:1, 100:1, or 10:1 or without particles (0:1) to PBMCs, as described previously, and incubated for 48h. Similar particle treated co-culture was set up in the absence of collagen gel in conventional 24 well plate. Cells were harvested with collagenase treatment from collagen gel, and stained with Live/Dead dye before acquisition on a flow cytometer. Cells from conventional culture plates were harvested by gentle pipetting and stained with Live/ Dead dye. 50,000 events were acquired on BD canto II and frequency of dead dye positive cells is represented in figure.
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pone.0124389.g007: Increase in particle to cell ratio increases frequency of dead cells.Particles were added at the time of gel polymerization at a ratio of 500:1, 100:1, or 10:1 or without particles (0:1) to PBMCs, as described previously, and incubated for 48h. Similar particle treated co-culture was set up in the absence of collagen gel in conventional 24 well plate. Cells were harvested with collagenase treatment from collagen gel, and stained with Live/Dead dye before acquisition on a flow cytometer. Cells from conventional culture plates were harvested by gentle pipetting and stained with Live/ Dead dye. 50,000 events were acquired on BD canto II and frequency of dead dye positive cells is represented in figure.

Mentions: The addition of a third dimension to the cell environment provides more surface area for cell adhesion, which is necessary for integrin ligation, cell contraction and even intracellular signaling [31]. The gene expression profile of cells grown in a 3D environment is different from those cells grown in 2D plastic or glass dishes [32]. As shown in Fig 2, we observed enhanced generation of MGCs in a 3D model compared to plastic plates. These results indicate that the cell-to-cell and cell-to-collagen interaction is a requisite for expression of biomolecules, which are necessary for MGC formation. An increase in the particle-to-cell ratio to 500:1 and 100:1 might have triggered apoptosis in the co-cultures and hence the 10:1 particle to cell ratio could possibly provide optimal conditions required for OC generation. As shown in Fig 7, an increase in particle to cell ratio induced more cell death for both the 2D and 3D collagen gel cultures. However the 3D collagen gel cultures were more resistant to cell death than the 2D culture system. For example, a 100:1 particle to cell ratio in the 2D system resulted in 100% cell death, whereas the 3D collagen gel cultures at the same ratio yielded 47% cell death. The reduced cell death effect might be due to dispersion of particles in 3D gel and thus few of the cells might have find a space (living environment) in the gel and in between the particles. In MoM bearing tissue, these non-apoptotic cells may directly or indirectly activated by pro-inflammatory cytokines, thus recruiting more lymphocytes. The activation and differentiation of non-apoptotic cells may lead to pseudo tumor like tissues as observed in MoM bearing patients with chronic pain. Molecular characterization of the giant cells in collagen gel will provide the important clues in our understanding of the generation of osteoclasts and pseudo tumor formation. The addition of IL-4 and GM-CSF induces the generation of MGCs from peripheral blood cells [19]. We measured IL-2, 4, -5, 10 and IFN- γ in the supernatants from 3D culture at different time intervals. IFN- γ levels in the supernatant were higher in early time points (day 4, 7 and 9) than at day 14. Conversely, IL-10 levels were lower in early time points than at day 14, which suggests a shift from a Th1 response to Th2 over time and we detected MGCs when IFN- γ levels were low. This is in accordance with the findings of Kohara H et. al. with regards to inhibition of multinucleated cell formation by IFN- γ [22].


Wear particles derived from metal hip implants induce the generation of multinucleated giant cells in a 3-dimensional peripheral tissue-equivalent model.

Dutta DK, Potnis PA, Rhodes K, Wood SC - PLoS ONE (2015)

Increase in particle to cell ratio increases frequency of dead cells.Particles were added at the time of gel polymerization at a ratio of 500:1, 100:1, or 10:1 or without particles (0:1) to PBMCs, as described previously, and incubated for 48h. Similar particle treated co-culture was set up in the absence of collagen gel in conventional 24 well plate. Cells were harvested with collagenase treatment from collagen gel, and stained with Live/Dead dye before acquisition on a flow cytometer. Cells from conventional culture plates were harvested by gentle pipetting and stained with Live/ Dead dye. 50,000 events were acquired on BD canto II and frequency of dead dye positive cells is represented in figure.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403993&req=5

pone.0124389.g007: Increase in particle to cell ratio increases frequency of dead cells.Particles were added at the time of gel polymerization at a ratio of 500:1, 100:1, or 10:1 or without particles (0:1) to PBMCs, as described previously, and incubated for 48h. Similar particle treated co-culture was set up in the absence of collagen gel in conventional 24 well plate. Cells were harvested with collagenase treatment from collagen gel, and stained with Live/Dead dye before acquisition on a flow cytometer. Cells from conventional culture plates were harvested by gentle pipetting and stained with Live/ Dead dye. 50,000 events were acquired on BD canto II and frequency of dead dye positive cells is represented in figure.
Mentions: The addition of a third dimension to the cell environment provides more surface area for cell adhesion, which is necessary for integrin ligation, cell contraction and even intracellular signaling [31]. The gene expression profile of cells grown in a 3D environment is different from those cells grown in 2D plastic or glass dishes [32]. As shown in Fig 2, we observed enhanced generation of MGCs in a 3D model compared to plastic plates. These results indicate that the cell-to-cell and cell-to-collagen interaction is a requisite for expression of biomolecules, which are necessary for MGC formation. An increase in the particle-to-cell ratio to 500:1 and 100:1 might have triggered apoptosis in the co-cultures and hence the 10:1 particle to cell ratio could possibly provide optimal conditions required for OC generation. As shown in Fig 7, an increase in particle to cell ratio induced more cell death for both the 2D and 3D collagen gel cultures. However the 3D collagen gel cultures were more resistant to cell death than the 2D culture system. For example, a 100:1 particle to cell ratio in the 2D system resulted in 100% cell death, whereas the 3D collagen gel cultures at the same ratio yielded 47% cell death. The reduced cell death effect might be due to dispersion of particles in 3D gel and thus few of the cells might have find a space (living environment) in the gel and in between the particles. In MoM bearing tissue, these non-apoptotic cells may directly or indirectly activated by pro-inflammatory cytokines, thus recruiting more lymphocytes. The activation and differentiation of non-apoptotic cells may lead to pseudo tumor like tissues as observed in MoM bearing patients with chronic pain. Molecular characterization of the giant cells in collagen gel will provide the important clues in our understanding of the generation of osteoclasts and pseudo tumor formation. The addition of IL-4 and GM-CSF induces the generation of MGCs from peripheral blood cells [19]. We measured IL-2, 4, -5, 10 and IFN- γ in the supernatants from 3D culture at different time intervals. IFN- γ levels in the supernatant were higher in early time points (day 4, 7 and 9) than at day 14. Conversely, IL-10 levels were lower in early time points than at day 14, which suggests a shift from a Th1 response to Th2 over time and we detected MGCs when IFN- γ levels were low. This is in accordance with the findings of Kohara H et. al. with regards to inhibition of multinucleated cell formation by IFN- γ [22].

Bottom Line: Our results show that endothelial cells induce the generation of MGCs to a level 4 fold higher in 3-dimentional PTE system as compared to traditional 2-dimensional culture plates.In sum, we have established a robust and relevant model to examine MGC and osteoclast formation in a tissue like environment using flow cytometry and RT-PCR.With endothelial cells help, we observed a consistent generation of metal wear particle- induced MGCs, which heralds metal on metal hip failures.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Chemistry and Materials Science, Office of Science and Engineering Laboratories, CDRH, FDA, Silver Spring, Maryland, United States of America; Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.

ABSTRACT
Multinucleate giant cells (MGCs) are formed by the fusion of 5 to 15 monocytes or macrophages. MGCs can be generated by hip implants at the site where the metal surface of the device is in close contact with tissue. MGCs play a critical role in the inflammatory processes associated with adverse events such as aseptic loosening of the prosthetic joints and bone degeneration process called osteolysis. Upon interaction with metal wear particles, endothelial cells upregulate pro-inflammatory cytokines and other factors that enhance a localized immune response. However, the role of endothelial cells in the generation of MGCs has not been completely investigated. We developed a three-dimensional peripheral tissue-equivalent model (PTE) consisting of collagen gel, supporting a monolayer of endothelial cells and human peripheral blood mononuclear cells (PBMCs) on top, which mimics peripheral tissue under normal physiological conditions. The cultures were incubated for 14 days with Cobalt chromium alloy (CoCr ASTM F75, 1-5 micron) wear particles. PBMC were allowed to transit the endothelium and harvested cells were analyzed for MGC generation via flow cytometry. An increase in forward scatter (cell size) and in the propidium iodide (PI) uptake (DNA intercalating dye) was used to identify MGCs. Our results show that endothelial cells induce the generation of MGCs to a level 4 fold higher in 3-dimentional PTE system as compared to traditional 2-dimensional culture plates. Further characterization of MGCs showed upregulated expression of tartrate resistant alkaline phosphatase (TRAP) and dendritic cell specific transmembrane protein, (DC-STAMP), which are markers of bone degrading cells called osteoclasts. In sum, we have established a robust and relevant model to examine MGC and osteoclast formation in a tissue like environment using flow cytometry and RT-PCR. With endothelial cells help, we observed a consistent generation of metal wear particle- induced MGCs, which heralds metal on metal hip failures.

No MeSH data available.


Related in: MedlinePlus