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Pentosan polysulfate promotes proliferation and chondrogenic differentiation of adult human bone marrow-derived mesenchymal precursor cells.

Ghosh P, Wu J, Shimmon S, Zannettino AC, Gronthos S, Itescu S - Arthritis Res. Ther. (2010)

Bottom Line: The relative outcome of PPS, HA, heparin or dextran sulfate (DS) on PG synthesis was compared in 5-day MMC.In the presence of 1 to 10 microg/ml PPS, a 38% reduction in IL-4/IFNgamma-induced MPC apoptosis was observed.Real-time PCR results were consistent with the protein data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Proteobioactives Pty Ltd, 27/9 Powells Road, Brookvale, New South Wales 2100, Australia. peterghosh@proteobioactives.com.au

ABSTRACT

Introduction: This study was undertaken to determine whether the anti-osteoarthritis drug pentosan polysulfate (PPS) influenced mesenchymal precursor cell (MPC) proliferation and differentiation.

Methods: Human MPCs were maintained in monolayer, pellet or micromass cultures (MMC) for up to 10 days with PPS at concentrations of 0 to 20 microg/ml. MPC viability and proliferation was assessed using the WST-1 assay and 3H-thymidine incorporation into DNA, while apoptosis was monitored by flow cytometry. Proteoglycan (PG) biosynthesis was determined by 35SO42- incorporation and staining with Alcian blue. Proteoglycan and collagen type I and collagen type II deposition in pellet cultures was also examined by Toluidine blue and immunohistochemical staining, respectively. The production of hyaluronan (HA) by MPCs in MMC was assessed by ELISA. The relative outcome of PPS, HA, heparin or dextran sulfate (DS) on PG synthesis was compared in 5-day MMC. Gene expression of MPCs in 7-day and 10-day MMC was examined using real-time PCR. MPC differentiation was investigated by co-culturing with PPS in osteogenic or adipogenic inductive culture media for 28 days.

Results: Significant MPC proliferation was evident by day 3 at PPS concentrations of 1 to 5 microg/ml (P < 0.01). In the presence of 1 to 10 microg/ml PPS, a 38% reduction in IL-4/IFNgamma-induced MPC apoptosis was observed. In 5-day MMC, 130% stimulation of PG synthesis occurred at 2.5 microg/ml PPS (P < 0.0001), while 5.0 microg/ml PPS achieved maximal stimulation in the 7-day and 10-day cultures (P < 0.05). HA and DS at > or = 5 microg/ml inhibited PG synthesis (P < 0.05) in 5-day MMC. Collagen type II deposition by MMC was significant at > or = 0.5 microg/ml PPS (P < 0.001 to 0.05). In MPC-PPS pellet cultures, more PG, collagen type II but less collagen type I was deposited than in controls. Real-time PCR results were consistent with the protein data. At 5 and 10 microg/ml PPS, MPC osteogenic differentiation was suppressed (P < 0.01).

Conclusions: This is the first study to demonstrate that PPS promotes MPC proliferation and chondrogenesis, offering new strategies for cartilage regeneration and repair in osteoarthritic joints.

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Effects of pentosan polysulfate on collagen type II deposition by micromass culture mesenchymal precursor cells. Bar graphs showing the concentration-dependent effects of pentosan polysulfate (PPS) on collagen type II deposition by mesenchymal precursor stem cells (MPCs) in micromass cultures (MMC) for (a) 5 days, (b) 7 days and (c) 10 days as determined by digital analysis of colour-intensity images of the 5-bromo-4-chloro-3'-indoyl phosphate/buffer + nitroblue tetrazolium salt stained conjugate to collagen type II applied to fixed MMC. n = 3 for each PPS concentration. Data expressed as mean ± standard deviation. (d) Digital photograph of the stained 7-day MMC cultures in 24-well culture plates after processing as described in the text showing the staining intensity in relation to PPS concentration. ‡P < 0.05, *P < 0.01; **P < 0.001; ***P < 0.0001 relative to control.
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Figure 5: Effects of pentosan polysulfate on collagen type II deposition by micromass culture mesenchymal precursor cells. Bar graphs showing the concentration-dependent effects of pentosan polysulfate (PPS) on collagen type II deposition by mesenchymal precursor stem cells (MPCs) in micromass cultures (MMC) for (a) 5 days, (b) 7 days and (c) 10 days as determined by digital analysis of colour-intensity images of the 5-bromo-4-chloro-3'-indoyl phosphate/buffer + nitroblue tetrazolium salt stained conjugate to collagen type II applied to fixed MMC. n = 3 for each PPS concentration. Data expressed as mean ± standard deviation. (d) Digital photograph of the stained 7-day MMC cultures in 24-well culture plates after processing as described in the text showing the staining intensity in relation to PPS concentration. ‡P < 0.05, *P < 0.01; **P < 0.001; ***P < 0.0001 relative to control.

Mentions: Deposition of collagen type II in the fixed matrices of MPC MMC was successfully demonstrated and semi-quantified using the immunostaining technique described by Denker and colleagues [29]. For the 7-day and 10-day culture periods, a progressive stimulation of collagen type II synthesis was observed with PPS concentrations up to 5 μg/ml followed by a marked decline at 10 and 20 μg/ml PPS (Figure 5b, c, d). In the 5-day cultures, lower but significant (P < 0.001 to 0.05) stimulation of collagen type II synthesis by MPCs was still evident at PPS concentrations up to 20 μg/ml (Figure 5a).


Pentosan polysulfate promotes proliferation and chondrogenic differentiation of adult human bone marrow-derived mesenchymal precursor cells.

Ghosh P, Wu J, Shimmon S, Zannettino AC, Gronthos S, Itescu S - Arthritis Res. Ther. (2010)

Effects of pentosan polysulfate on collagen type II deposition by micromass culture mesenchymal precursor cells. Bar graphs showing the concentration-dependent effects of pentosan polysulfate (PPS) on collagen type II deposition by mesenchymal precursor stem cells (MPCs) in micromass cultures (MMC) for (a) 5 days, (b) 7 days and (c) 10 days as determined by digital analysis of colour-intensity images of the 5-bromo-4-chloro-3'-indoyl phosphate/buffer + nitroblue tetrazolium salt stained conjugate to collagen type II applied to fixed MMC. n = 3 for each PPS concentration. Data expressed as mean ± standard deviation. (d) Digital photograph of the stained 7-day MMC cultures in 24-well culture plates after processing as described in the text showing the staining intensity in relation to PPS concentration. ‡P < 0.05, *P < 0.01; **P < 0.001; ***P < 0.0001 relative to control.
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Figure 5: Effects of pentosan polysulfate on collagen type II deposition by micromass culture mesenchymal precursor cells. Bar graphs showing the concentration-dependent effects of pentosan polysulfate (PPS) on collagen type II deposition by mesenchymal precursor stem cells (MPCs) in micromass cultures (MMC) for (a) 5 days, (b) 7 days and (c) 10 days as determined by digital analysis of colour-intensity images of the 5-bromo-4-chloro-3'-indoyl phosphate/buffer + nitroblue tetrazolium salt stained conjugate to collagen type II applied to fixed MMC. n = 3 for each PPS concentration. Data expressed as mean ± standard deviation. (d) Digital photograph of the stained 7-day MMC cultures in 24-well culture plates after processing as described in the text showing the staining intensity in relation to PPS concentration. ‡P < 0.05, *P < 0.01; **P < 0.001; ***P < 0.0001 relative to control.
Mentions: Deposition of collagen type II in the fixed matrices of MPC MMC was successfully demonstrated and semi-quantified using the immunostaining technique described by Denker and colleagues [29]. For the 7-day and 10-day culture periods, a progressive stimulation of collagen type II synthesis was observed with PPS concentrations up to 5 μg/ml followed by a marked decline at 10 and 20 μg/ml PPS (Figure 5b, c, d). In the 5-day cultures, lower but significant (P < 0.001 to 0.05) stimulation of collagen type II synthesis by MPCs was still evident at PPS concentrations up to 20 μg/ml (Figure 5a).

Bottom Line: The relative outcome of PPS, HA, heparin or dextran sulfate (DS) on PG synthesis was compared in 5-day MMC.In the presence of 1 to 10 microg/ml PPS, a 38% reduction in IL-4/IFNgamma-induced MPC apoptosis was observed.Real-time PCR results were consistent with the protein data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Proteobioactives Pty Ltd, 27/9 Powells Road, Brookvale, New South Wales 2100, Australia. peterghosh@proteobioactives.com.au

ABSTRACT

Introduction: This study was undertaken to determine whether the anti-osteoarthritis drug pentosan polysulfate (PPS) influenced mesenchymal precursor cell (MPC) proliferation and differentiation.

Methods: Human MPCs were maintained in monolayer, pellet or micromass cultures (MMC) for up to 10 days with PPS at concentrations of 0 to 20 microg/ml. MPC viability and proliferation was assessed using the WST-1 assay and 3H-thymidine incorporation into DNA, while apoptosis was monitored by flow cytometry. Proteoglycan (PG) biosynthesis was determined by 35SO42- incorporation and staining with Alcian blue. Proteoglycan and collagen type I and collagen type II deposition in pellet cultures was also examined by Toluidine blue and immunohistochemical staining, respectively. The production of hyaluronan (HA) by MPCs in MMC was assessed by ELISA. The relative outcome of PPS, HA, heparin or dextran sulfate (DS) on PG synthesis was compared in 5-day MMC. Gene expression of MPCs in 7-day and 10-day MMC was examined using real-time PCR. MPC differentiation was investigated by co-culturing with PPS in osteogenic or adipogenic inductive culture media for 28 days.

Results: Significant MPC proliferation was evident by day 3 at PPS concentrations of 1 to 5 microg/ml (P < 0.01). In the presence of 1 to 10 microg/ml PPS, a 38% reduction in IL-4/IFNgamma-induced MPC apoptosis was observed. In 5-day MMC, 130% stimulation of PG synthesis occurred at 2.5 microg/ml PPS (P < 0.0001), while 5.0 microg/ml PPS achieved maximal stimulation in the 7-day and 10-day cultures (P < 0.05). HA and DS at > or = 5 microg/ml inhibited PG synthesis (P < 0.05) in 5-day MMC. Collagen type II deposition by MMC was significant at > or = 0.5 microg/ml PPS (P < 0.001 to 0.05). In MPC-PPS pellet cultures, more PG, collagen type II but less collagen type I was deposited than in controls. Real-time PCR results were consistent with the protein data. At 5 and 10 microg/ml PPS, MPC osteogenic differentiation was suppressed (P < 0.01).

Conclusions: This is the first study to demonstrate that PPS promotes MPC proliferation and chondrogenesis, offering new strategies for cartilage regeneration and repair in osteoarthritic joints.

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