Abstract
IntroductionThis study was undertaken to determine whether the anti-osteoarthritis drug pentosan polysulfate (PPS) influenced mesenchymal precursor cell (MPC) proliferation and differentiation.MethodsHuman MPCs were maintained in monolayer, pellet or micromass cultures (MMC) for up to 10 days with PPS at concentrations of 0 to 20 μg/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.ResultsSignificant MPC proliferation was evident by day 3 at PPS concentrations of 1 to 5 μg/ml (P < 0.01). In the presence of 1 to 10 μg/ml PPS, a 38% reduction in IL-4/IFNγ-induced MPC apoptosis was observed. In 5-day MMC, 130% stimulation of PG synthesis occurred at 2.5 μg/ml PPS (P < 0.0001), while 5.0 μg/ml PPS achieved maximal stimulation in the 7-day and 10-day cultures (P < 0.05). HA and DS at ≥ 5 μg/ml inhibited PG synthesis (P < 0.05) in 5-day MMC. Collagen type II deposition by MMC was significant at ≥ 0.5 μg/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 μg/ml PPS, MPC osteogenic differentiation was suppressed (P < 0.01).ConclusionsThis is the first study to demonstrate that PPS promotes MPC proliferation and chondrogenesis, offering new strategies for cartilage regeneration and repair in osteoarthritic joints.
Highlights
This study was undertaken to determine whether the anti-osteoarthritis drug pentosan polysulfate (PPS) influenced mesenchymal precursor cell (MPC) proliferation and differentiation
MPC viability and proliferation in monolayer culture Using the WST-1 mitochondrial dehydrogenase cleavage assay at day 6, viable MPC numbers were significantly increased in the cultures containing 1 to 10 μg/ml PPS (P < 0.01) (Figure 1a)
Effects of PPS on proteoglycan synthesis in 5-day, 7-day and 10-day MPC micromass cultures (MMC) The incorporation of PPS in MPC MMC stimulated PG biosynthesis in a concentration-dependant manner that varied with the duration of culture (Figure 2)
Summary
This study was undertaken to determine whether the anti-osteoarthritis drug pentosan polysulfate (PPS) influenced mesenchymal precursor cell (MPC) proliferation and differentiation. Provide evidence for the existence within this niche of smaller precursor stem cell populations that exhibit extensive proliferative and multilineage differentiative capacity and can be distinguished by their expression of certain cell surface antigens [11,12] These undifferentiated mesenchymal precursor cells (MPCs) can be isolated from bone marrow aspirates using magnetic activated cell sorting in combination with antibodies that identify STRO-1, VCAM-1 (CD106), STRO-3 (tissue nonspecific alkaline phosphatase), STRO-4 (HSP-90b) and CD146 [11,12]. Using this approach, a homogeneous population of quiescent MPCs can be obtained that lack the phenotypic characteristics of leukocytes and mature stromal elements and exhibit extensive proliferative capacity while retaining the ability to differentiate into bone, cartilage and adipose tissues. Subcutaneous implantation of STRO-1+, STRO-3+, STRO-4+, CD106+ and CD146+ MPCs together with an osteoinductive hydroxy apatite/tricalcium phosphate carrier into NOD/SCID mice resulted in the successful deposition of bone at the site of implantation [11,12,13]
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