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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

Elevated mRNA levels of TRAP, DC-STAMP and GM-CSF in day 14 co-cultures.Particles were added at the time of gel polymerization at the ratio of 10:1 particles to PBMCs. Endothelial cells (EA) were grown on the gel to form a monolayer. Peripheral blood mononuclear cells (PBMCs) were seeded either on top of the monolayer. Cells were harvested by digesting the gel with RNA extraction buffer and proceeded for RT-PCR as described in method. Each bar is represented as mean +/- S.E. of two experimental samples from two independent experiments, n = 2. *Asterisks indicate statistically significant TRAP and GM-CSF increase in particles exposed cells at day 14 compared with day0, (** = p< 0.005, * = p < 0.05)
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pone.0124389.g005: Elevated mRNA levels of TRAP, DC-STAMP and GM-CSF in day 14 co-cultures.Particles were added at the time of gel polymerization at the ratio of 10:1 particles to PBMCs. Endothelial cells (EA) were grown on the gel to form a monolayer. Peripheral blood mononuclear cells (PBMCs) were seeded either on top of the monolayer. Cells were harvested by digesting the gel with RNA extraction buffer and proceeded for RT-PCR as described in method. Each bar is represented as mean +/- S.E. of two experimental samples from two independent experiments, n = 2. *Asterisks indicate statistically significant TRAP and GM-CSF increase in particles exposed cells at day 14 compared with day0, (** = p< 0.005, * = p < 0.05)

Mentions: Total RNA was harvested from the cells at days 0, 1, 3, 5 and 14 and performed real time qPCR to quantify the markers of osteoclast, TRAP and DC-STAMP. Granulocyte macrophage colony stimulating factor (GM-CSF) is an inducer of MGCs and was also measured in this assay. The results showed that A13 particles enhanced the expression levels of TRAP and GM-CSF to 140 fold and 8 fold, respectively, compared to culture controls at day 0. Although the mRNA levels for GM-CSF did not increase significantly, we observed a trend in the up-regulation of GM-CSF transcript levels, Fig 5.


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)

Elevated mRNA levels of TRAP, DC-STAMP and GM-CSF in day 14 co-cultures.Particles were added at the time of gel polymerization at the ratio of 10:1 particles to PBMCs. Endothelial cells (EA) were grown on the gel to form a monolayer. Peripheral blood mononuclear cells (PBMCs) were seeded either on top of the monolayer. Cells were harvested by digesting the gel with RNA extraction buffer and proceeded for RT-PCR as described in method. Each bar is represented as mean +/- S.E. of two experimental samples from two independent experiments, n = 2. *Asterisks indicate statistically significant TRAP and GM-CSF increase in particles exposed cells at day 14 compared with day0, (** = p< 0.005, * = p < 0.05)
© Copyright Policy
Related In: Results  -  Collection

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

pone.0124389.g005: Elevated mRNA levels of TRAP, DC-STAMP and GM-CSF in day 14 co-cultures.Particles were added at the time of gel polymerization at the ratio of 10:1 particles to PBMCs. Endothelial cells (EA) were grown on the gel to form a monolayer. Peripheral blood mononuclear cells (PBMCs) were seeded either on top of the monolayer. Cells were harvested by digesting the gel with RNA extraction buffer and proceeded for RT-PCR as described in method. Each bar is represented as mean +/- S.E. of two experimental samples from two independent experiments, n = 2. *Asterisks indicate statistically significant TRAP and GM-CSF increase in particles exposed cells at day 14 compared with day0, (** = p< 0.005, * = p < 0.05)
Mentions: Total RNA was harvested from the cells at days 0, 1, 3, 5 and 14 and performed real time qPCR to quantify the markers of osteoclast, TRAP and DC-STAMP. Granulocyte macrophage colony stimulating factor (GM-CSF) is an inducer of MGCs and was also measured in this assay. The results showed that A13 particles enhanced the expression levels of TRAP and GM-CSF to 140 fold and 8 fold, respectively, compared to culture controls at day 0. Although the mRNA levels for GM-CSF did not increase significantly, we observed a trend in the up-regulation of GM-CSF transcript levels, Fig 5.

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