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Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors.

Mo IF, Yip KH, Chan WK, Law HK, Lau YL, Chan GC - BMC Cell Biol. (2008)

Bottom Line: We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12.But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression.In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong, PR China. irene_hku@yahoo.com

ABSTRACT

Background: Human mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings.

Results: We selected lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Streptococcus pyogenes as our toxins of choice. Our findings showed both LPS and LTA did not affect MSC proliferation, but prolonged LPS challenge upregulated the osteogenic differentiation of MSCs, as assessed by alkaline phosphatase activity and calcium deposition. Because toll-like receptors (TLRs), in particularly TLR4 and TLR2, are important for the cellular responsiveness to LPS and LTA respectively, we evaluated their expression profiles serially from MSCs to osteoblasts by quantitative PCR. We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12. But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression. This peculiar response to LPS suggests a possible adaptive mechanism when MSCs are subjected to continuous exposure with bacteria.

Conclusion: In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.

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Related in: MedlinePlus

Effects of LPS and LTA on the osteogenic differentiation of MSCs. MSCs were cultured in osteogenesis-induction medium and exposed to toxin for 3 days or for the whole incubation period. According to ALP activity and calcium deposition assays performed on Day 10 (LPS) or 14 (LTA), short-term LPS challenge did not affect the osteogenic differentiation (A and B), but prolonged challenge to 1 and 10 μg/mL upregulated ALP activity and calcium deposition. Neither short-term nor prolonged LTA challenge affected the osteogenic differentiation (C and D). The data are from three independent experiments and expressed as mean ± SEM. *P < 0.05; ** P < 0.01 versus control.
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Figure 2: Effects of LPS and LTA on the osteogenic differentiation of MSCs. MSCs were cultured in osteogenesis-induction medium and exposed to toxin for 3 days or for the whole incubation period. According to ALP activity and calcium deposition assays performed on Day 10 (LPS) or 14 (LTA), short-term LPS challenge did not affect the osteogenic differentiation (A and B), but prolonged challenge to 1 and 10 μg/mL upregulated ALP activity and calcium deposition. Neither short-term nor prolonged LTA challenge affected the osteogenic differentiation (C and D). The data are from three independent experiments and expressed as mean ± SEM. *P < 0.05; ** P < 0.01 versus control.

Mentions: To study the effect of bacterial toxins on the osteogenic differentiation of MSCs, we exposed MSCs with osteogenesis-induction medium and toxin for a short-term of 3 days, or prolonged period of either 10 or 14 days as time points for respective quantitative assays. Alkaline phosphatase (ALP) activity was used as an early marker of osteogenic differentiation [33,34]. A period of 3 days was chosen as the duration for the short-term challenge because a significant difference in ALP activity between MSCs with and without osteogenic induction has been reported previously on Day 4 after osteogenic induction [9]. Prolonged toxin challenge lasted 10 to 14 days to allow optimal quantification of MSC osteogenic differentiation by ALP and calcium assays respectively based on our preliminary studies. Under short-term toxin challenge, neither LPS at concentrations of 0.1, 1, and 10 μg/mL (Figure 2A and 2B) nor LTA at 10 μg/mL (Figure 2C and 2D) had a significant effect on ALP activity or calcium deposition. After prolonged LPS challenge of higher doses (1 and 10 μg/mL), ALP activity and calcium levels were significantly higher than those in untreated controls (n = 3, P < 0.05) (Figure 2A and 2B). In contrast, prolonged LTA challenge had no effect on ALP activity or calcium level (Fig 2C and 2D). The increased calcium deposition after prolonged LPS challenge was further confirmed semi-quantitatively by von Kossa staining (data not shown).


Prolonged exposure to bacterial toxins downregulated expression of toll-like receptors in mesenchymal stromal cell-derived osteoprogenitors.

Mo IF, Yip KH, Chan WK, Law HK, Lau YL, Chan GC - BMC Cell Biol. (2008)

Effects of LPS and LTA on the osteogenic differentiation of MSCs. MSCs were cultured in osteogenesis-induction medium and exposed to toxin for 3 days or for the whole incubation period. According to ALP activity and calcium deposition assays performed on Day 10 (LPS) or 14 (LTA), short-term LPS challenge did not affect the osteogenic differentiation (A and B), but prolonged challenge to 1 and 10 μg/mL upregulated ALP activity and calcium deposition. Neither short-term nor prolonged LTA challenge affected the osteogenic differentiation (C and D). The data are from three independent experiments and expressed as mean ± SEM. *P < 0.05; ** P < 0.01 versus control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567970&req=5

Figure 2: Effects of LPS and LTA on the osteogenic differentiation of MSCs. MSCs were cultured in osteogenesis-induction medium and exposed to toxin for 3 days or for the whole incubation period. According to ALP activity and calcium deposition assays performed on Day 10 (LPS) or 14 (LTA), short-term LPS challenge did not affect the osteogenic differentiation (A and B), but prolonged challenge to 1 and 10 μg/mL upregulated ALP activity and calcium deposition. Neither short-term nor prolonged LTA challenge affected the osteogenic differentiation (C and D). The data are from three independent experiments and expressed as mean ± SEM. *P < 0.05; ** P < 0.01 versus control.
Mentions: To study the effect of bacterial toxins on the osteogenic differentiation of MSCs, we exposed MSCs with osteogenesis-induction medium and toxin for a short-term of 3 days, or prolonged period of either 10 or 14 days as time points for respective quantitative assays. Alkaline phosphatase (ALP) activity was used as an early marker of osteogenic differentiation [33,34]. A period of 3 days was chosen as the duration for the short-term challenge because a significant difference in ALP activity between MSCs with and without osteogenic induction has been reported previously on Day 4 after osteogenic induction [9]. Prolonged toxin challenge lasted 10 to 14 days to allow optimal quantification of MSC osteogenic differentiation by ALP and calcium assays respectively based on our preliminary studies. Under short-term toxin challenge, neither LPS at concentrations of 0.1, 1, and 10 μg/mL (Figure 2A and 2B) nor LTA at 10 μg/mL (Figure 2C and 2D) had a significant effect on ALP activity or calcium deposition. After prolonged LPS challenge of higher doses (1 and 10 μg/mL), ALP activity and calcium levels were significantly higher than those in untreated controls (n = 3, P < 0.05) (Figure 2A and 2B). In contrast, prolonged LTA challenge had no effect on ALP activity or calcium level (Fig 2C and 2D). The increased calcium deposition after prolonged LPS challenge was further confirmed semi-quantitatively by von Kossa staining (data not shown).

Bottom Line: We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12.But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression.In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong, PR China. irene_hku@yahoo.com

ABSTRACT

Background: Human mesenchymal stromal cells (MSCs, also known as mesenchymal stem cells) are multipotent cells with potential therapeutic value. Owing to their osteogenic capability, MSCs may be clinically applied for facilitating osseointegration in dental implants or orthopedic repair of bony defect. However, whether wound infection or oral microflora may interfere with the growth and osteogenic differentiation of human MSCs remains unknown. This study investigated whether proliferation and osteogenic differentiation of MSCs would be affected by potent gram-positive and gram-negative derived bacterial toxins commonly found in human settings.

Results: We selected lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid (LTA) from Streptococcus pyogenes as our toxins of choice. Our findings showed both LPS and LTA did not affect MSC proliferation, but prolonged LPS challenge upregulated the osteogenic differentiation of MSCs, as assessed by alkaline phosphatase activity and calcium deposition. Because toll-like receptors (TLRs), in particularly TLR4 and TLR2, are important for the cellular responsiveness to LPS and LTA respectively, we evaluated their expression profiles serially from MSCs to osteoblasts by quantitative PCR. We found that during osteogenic differentiation, MSC-derived osteoprogenitors gradually expressed TLR2 and TLR4 by Day 12. But under prolonged incubation with LPS, MSC-derived osteoprogenitors had reduced TLR2 and TLR4 gene expression. This peculiar response to LPS suggests a possible adaptive mechanism when MSCs are subjected to continuous exposure with bacteria.

Conclusion: In conclusion, our findings support the potential of using human MSCs as a biological graft, even under a bacterial toxin-rich environment.

Show MeSH
Related in: MedlinePlus