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

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.

Figure 1

Mesenchymal precursor stem cell viability and proliferation in monolayer culture. (a) Bar graph showing the concentration-dependent effect of pentosan polysulfate (PPS) on mesenchymal precursor stem cell (MPC) proliferation. Primary MPCs were cultured in 24-well plates in growth media supplemented with PPS at the indicated concentrations (n = 3). On days 1 (black columns), 3 (white columns) and 6 (hatched columns), the cells were incubated with the tetrazolium salt WST-1 for 2 hours at 37°C to produce a formazan dye. Absorbance at 450 nm for each time point is shown for the indicated concentrations of PPS. Data expressed as mean ± standard error of the mean. A statistically significant increase in proliferation was observed on day 6 at concentrations of PPS in excess of 1 μg/ml (*P < 0.01). (b) Concentration effects of PPS on DNA synthesis in 3-day monolayer cultures of MPCs (n = 3). Data expressed as mean ± standard deviation. Significant elevation in DNA synthesis was observed at PPS concentrations of 1, 2.5 and 5 μg/ml relative to control cultures (P < 0.01). (c) Flow cytometric analysis profiles showing the inhibitory effects of different concentrations of PPS on MPC apoptosis induced by the addition of a combination of 30 ng/ml IL-4 plus 30,000 U/ml IFNγ. Following 5-day culture, cells were harvested by trypsinisation and viabilities assessed by Annexin V staining (n = 3 per PPS concentration). An average 38% reduction in apoptosis was observed when MPCs were cultured at PPS concentrations >1 μg/ml. DPM, Decays Per Minute.

Figure 2

Effects of pentosan polysulfate on proteoglycan synthesis in mesenchymal precursor stem cell micromass cultures. Bar graphs showing the temporal and concentration effects of pentosan polysulfate (PPS) on the biosynthesis of proteoglycans (determined as sulfated glycosaminoglycans (³⁵S-GAGs)) by mesenchymal precursor stem cells (MPCs) in micromass cultures for 5, 7 and 10 days. Data (mean ± standard deviation) presented as percentage of control calculated from the ³⁵S-GAG DPM/μg DNA values at each PPS concentration (n = 6). ^‡P < 0.05, *P < 0.01; **P < 0.001; ***P < 0.0001 relative to control cultures.

Figure 3

Effects of pentosan polysulfate, hyaluronan, dextran sulfate and heparin on mesenchymal precursor cell proteoglycan biosynthesis. Bar graphs showing the concentration-dependent effects of (a) pentosan polysulfate (PPS), (b) heparin, (c) hyaluronan (HA) and (d) dextran sulfate (DS) on prosteoglycan (PG) synthesis by mesenchymal precursor stem cells (MPCs) in micromass cultures for 5 days. Data (mean ± standard deviation) presented as percentage of control calculated from the sulfated glycosaminoglycan (³⁵S-GAG) Decays Per Minute/μg DNA values at each drug concentration (n = 3). ^‡P < 0.05, *P < 0.01; **P < 0.001; ***P < 0.0001 relative to control values.

Figure 4

Effects of pentosan polysulfate on proteoglycan deposition by mesenchymal precursor cells in micromass cultures. Photomicrographs of mesenchymal precursor stem cell micromass cultures maintained in the absence and presence of pentosan polysulfate (PPS) for 10 days then stained with Alcian blue as described in the text: (a) 0.0 μg/ml PPS, (b) 1.0 μg/ml PPS, (c) 2.5 μg/ml PPS, (d) 5.0 μg/ml PPS, (e) 10.0 μg/ml PPS, and (f) 20 μg/ml PPS. Note the more intense staining with the dye at concentrations ≥ 2.5 μg/ml PPS, indicating the presence of higher levels of proteoglycans in these cultures.

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.

Figure 6

Effects of pentosan polysulfate on proteoglycan, collagen type I and collagen type II deposition in pellet cultures. Photomicrographs of sections of mesenchymal precursor stem cell (MPC) pellet cultures showing the effects of pentosan polysulfate (PPS) on chondrocyte development. Representative 5 μm serial tissue sections are shown of cells cultured as pellets for 10 days in (a) the absence of PPS (top nine panels) or (b) the presence of PPS (bottom nine panels). Control pellet sections (a) appeared fragmented with less intense toluidine blue staining for proteoglycans in comparison with those pellet cultures treated with 2.5 mg/ml PPS (b). Immunohistochemical staining confirmed higher levels of the cartilage matrix protein and collagen type II, and lesser levels of collagen type I in pellet cultures treated with 2.5 μg/ml PPS (b) than in the corresponding control pellet cultures (a).

Figure 7

Effects of pentosan polysulfate on gene expression by mesenchymal precursor stem cells in micromass cultures. Bar graphs showing the concentration effects of pentosan polysulfate (PPS) on (a) SOX-9, (b) Aggrecan, (c) collagen type II, (d) collagen type I, (e) collagen type X, (f) RUNX2 (Cbfa1) and (g) Noggin gene expression by mesenchymal precursor stem cells (MPCs) in micromass cultures for 7 and 10 days as determined by real-time quantitative PCR. n = 3 for each PPS concentration. Data expressed as mean ± standard deviation for each PPS concentration after normalising for the expression of the housekeeping gene, β-actin. ^#P < 0.05, *P < 0.01, **P < 0.001, ***P < 0.0001 gene expression relative to control.

Figure 8

Effects of pentosan polysulfate on human mesenchymal precursor stem cell differentiation: mineralisation assay. Primary mesenchymal precursor stem cells (MPCs) were cultured in non-osteoinductive growth media (media control) or in osteoinductive conditions in the absence or presence of pentosan polysulfate (PPS) at the indicated concentrations. n = 3 for each PPS concentration studied. (a) Concentration of acid-solubilised calcium per well determined relative to the total amount of DNA per well. Data expressed as mean ± standard error of the mean for day 28. (b) Phase-contrast photomicrographs of cultures showing absence of mineralisation in the co-cultures with PPS (× 20). A statistically significant decrease (P < 0.01) in mineralised matrix formation was observed for PPS concentrations of 5 and 10 μg/ml. *P < 0.01 relative to controls.

Figure 9

Effects of pentosan polysulfate on human mesenchymal precursor stem cell differentiation: adipocyte formation. Bar graph showing the concentration effects of pentosan polysulfate (PPS) on human mesenchymal precursor stem cell (MPC) adipogenic differentiation when cultured for 28 days in an adipogenic inductive medium. Primary MPCs were cultured in nonadipogenic growth media (media control) or under adipogenic conditions in the presence of PPS at the indicated concentrations. n = 3 for each PPS concentration studied. On day 28: (a) levels of lipid determined relative to the total amount of DNA per well, and (b) phase-contrast photomicrography of Oil Red O-labelled adipocytes (× 20). Data expressed as mean ± standard error of the mean. A statistically significant increase (P < 0.01) in adipocyte number was observed at PPS concentrations > 1.0 μg/ml. *P < 0.01.