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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

Particles at 10:1 ratio induced protein expression the DC-STAMP and TRAP in day 14 cultures.As described above, co-cultures were set up at 10:1 ratio and incubated for 14 days. Cells were harvested by collagenase treatment, washed and intracellular stained with propidium iodide, FITC conjugated- TRAP and APC conjugated-DC-STAMP. Both the dot plots (A and B) were first gated for giant cell i.e. high Forward Scatter and high PI positive cells, and then for DC-STAMP and TRAP. Cells without particles are represented in (A) and with particles in (B).
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pone.0124389.g006: Particles at 10:1 ratio induced protein expression the DC-STAMP and TRAP in day 14 cultures.As described above, co-cultures were set up at 10:1 ratio and incubated for 14 days. Cells were harvested by collagenase treatment, washed and intracellular stained with propidium iodide, FITC conjugated- TRAP and APC conjugated-DC-STAMP. Both the dot plots (A and B) were first gated for giant cell i.e. high Forward Scatter and high PI positive cells, and then for DC-STAMP and TRAP. Cells without particles are represented in (A) and with particles in (B).

Mentions: To examine the differentiation of MGCs into osteoclasts, the co-culture was set up on collagen gel in the presence of a particle-to-cell ratio of 10:1 and cells were harvested for evaluation of DC-STAMP and TRAP, the markers for osteoclasts. Cells were gated for high forward scatter and PI positive cells, i.e. giant cells, and further analyzed for osteoclast markers. Particle exposed cells showed a frequency of double positive expression of DC-STAMP and TRAP of 9% compared to 4.1% in cells without particles, (Fig 6).


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)

Particles at 10:1 ratio induced protein expression the DC-STAMP and TRAP in day 14 cultures.As described above, co-cultures were set up at 10:1 ratio and incubated for 14 days. Cells were harvested by collagenase treatment, washed and intracellular stained with propidium iodide, FITC conjugated- TRAP and APC conjugated-DC-STAMP. Both the dot plots (A and B) were first gated for giant cell i.e. high Forward Scatter and high PI positive cells, and then for DC-STAMP and TRAP. Cells without particles are represented in (A) and with particles in (B).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124389.g006: Particles at 10:1 ratio induced protein expression the DC-STAMP and TRAP in day 14 cultures.As described above, co-cultures were set up at 10:1 ratio and incubated for 14 days. Cells were harvested by collagenase treatment, washed and intracellular stained with propidium iodide, FITC conjugated- TRAP and APC conjugated-DC-STAMP. Both the dot plots (A and B) were first gated for giant cell i.e. high Forward Scatter and high PI positive cells, and then for DC-STAMP and TRAP. Cells without particles are represented in (A) and with particles in (B).
Mentions: To examine the differentiation of MGCs into osteoclasts, the co-culture was set up on collagen gel in the presence of a particle-to-cell ratio of 10:1 and cells were harvested for evaluation of DC-STAMP and TRAP, the markers for osteoclasts. Cells were gated for high forward scatter and PI positive cells, i.e. giant cells, and further analyzed for osteoclast markers. Particle exposed cells showed a frequency of double positive expression of DC-STAMP and TRAP of 9% compared to 4.1% in cells without particles, (Fig 6).

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