Object Following microdiscectomy, discs generally fail to undergo spontaneous regeneration and patients may experience chronic low-back pain and recurrent disc prolapse. In published studies, formulations of mesenchymal progenitor cells combined with pentosan polysulfate (MPCs+PPS) have been shown to regenerate disc tissue in animal models, suggesting that this approach may provide a useful adjunct to microdiscectomy. The goal of this preclinical laboratory study was to determine if the transplantation of MPCs+PPS, embedded in a gelatin/fibrin scaffold (SCAF), and transplanted into a defect created by microdiscectomy, could promote disc regeneration. Methods A standardized microdiscectomy procedure was performed in 18 ovine lumbar discs. The subsequent disc defects were randomized to receive either no treatment (NIL), SCAF only, or the MPC+PPS formulation added to SCAF (MPCs+PPS+SCAF). Necropsies were undertaken 6 months postoperatively and the spines analyzed radiologically (radiography and MRI), biochemically, and histologically. Results No adverse events occurred throughout the duration of the study. The MPC+PPS+SCAF group had significantly less reduction in disc height compared with SCAF-only and NIL groups (p < 0.05 and p < 0.01, respectively). Magnetic resonance imaging Pfirrmann scores in the MPC+PPS+SCAF group were significantly lower than those in the SCAF group (p = 0.0213). The chaotropic solvent extractability of proteoglycans from the nucleus pulposus of MPC+PPS+SCAF-treated discs was significantly higher than that from the SCAF-only discs (p = 0.0312), and using gel exclusion chromatography, extracts from MPC+PPS+SCAF-treated discs also contained a higher percentage of proteoglycan aggregates than the extracts from both other groups. Analysis of the histological sections showed that 66% (p > 0.05) of the MPC+PPS+SCAF-treated discs exhibited less degeneration than the NIL or SCAF discs. Conclusions These findings demonstrate the capacity of MPCs in combination with PPS, when embedded in a gelatin sponge and sealed with fibrin glue in a microdiscectomy defect, to restore disc height, disc morphology, and nucleus pulposus proteoglycan content.

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.

OBJECTIVE Disc degeneration and associated low-back pain are major causes of suffering and disability. The authors examined the potential of mesenchymal precursor cells (MPCs), when formulated with pentosan polysulfate (PPS), to ameliorate disc degeneration in an ovine model. METHODS Twenty-four sheep had annular incisions made at L2–3, L3–4, and L4–5 to induce degeneration. Twelve weeks after injury, the nucleus pulposus of a degenerated disc in each animal was injected with ProFreeze and PPS formulated with either a low dose (0.1 million MPCs) or a high dose (0.5 million MPCs) of cells. The 2 adjacent injured discs in each spine were either injected with PPS and ProFreeze (PPS control) or not injected (nil-injected control). The adjacent noninjured L1–2 and L5–6 discs served as noninjured control discs. Disc height indices (DHIs) were obtained at baseline, before injection, and at planned death. After necropsy, 24 weeks after injection, the spines were subjected to MRI and morphological, histological, and biochemical analyses. RESULTS Twelve weeks after the annular injury, all the injured discs exhibited a significant reduction in mean DHI (low-dose group 17.19%; high-dose group 18.01% [p < 0.01]). Twenty-four weeks after injections, the discs injected with the low-dose MPC+PPS formulation recovered disc height, and their mean DHI was significantly greater than the DHI of PPS- and nil-injected discs (p < 0.001). Although the mean Pfirrmann MRI disc degeneration score for the low-dose MPC+PPS–injected discs was lower than that for the nil- and PPS-injected discs, the differences were not significant. The disc morphology scores for the nil- and PPS-injected discs were significantly higher than the normal control disc scores (p < 0.005), whereas the low-dose MPC+PPS–injected disc scores were not significantly different from those of the normal controls. The mean glycosaminoglycan content of the nuclei pulposus of the low-dose MPC+PPS–injected discs was significantly higher than that of the PPS-injected controls (p < 0.05) but was not significantly different from the normal control disc glycosaminoglycan levels. Histopathology degeneration frequency scores for the low-dose MPC+PPS–injected discs were lower than those for the PPS- and Nil-injected discs. The corresponding high-dose MPC+PPS–injected discs failed to show significant improvements in any outcome measure relative to the controls. CONCLUSIONS Intradiscal injections of a formulation composed of 0.1 million MPCs combined with PPS resulted in positive effects in reducing the progression of disc degeneration in an ovine model, as assessed by improvements in DHI and morphological, biochemical, and histopathological scores.

ABSTRACT. Intervertebral disc degeneration is the main cause of lumbago disease, in which the extracellular matrix structure and moisture in the nucleus pulposus is lost continuously. In this study, we aimed to detect differential expression of silence mating type information regulation 2 homolog 1 (SIRT1) and matrix metalloproteinase-1 (MMP-1) in human intervertebral disc nucleus pulposus cells and to explore the effects of SIRT1 and MMP-1 on the development of the intervertebral disc degeneration. Intervertebral disc nucleus pulposus specimens from 41 patients who underwent lumbar protrusion resection at HuiZhou Municipal Central Hospital, during the period from October 2011 to December 2013, were studied in comparison with 23 control cases from patients who underwent fractured lumbar resection. In degenerated human intervertebral disc nucleus pulposus cells, the expression of SIRT1 is decreased and MMP-1 is increased compared with that of the control cells. Resveratrol could reverse these effects, thereby increasing the expression of SIRT1 (0.87 ± 0.07 vs 0.54 ± 0.04), Coll2α1 (0.90 ± 0.08 vs 0.38 ± 0.01), and aggrecan (0.69 ± 0.07 vs 0.42 ± 0.05) and decreasing the expression of MMP-1 (0.61 ± 0.03 vs 0.93 ± 0.08). These results suggest that resveratrol could possibly reverse the process of intervertebral disc degeneration and thus could be applied as a potential drug for the disease.

Glioblastoma (GBM) is one of the most aggressive tumors and its 5-year survival is approximately 5%. Fluorescence-guided surgery (FGS) improves the extent of resection and leads to better prognosis. Molecular near-infrared (NIR) imaging appears to outperform conventional FGS, however, novel molecular targets need to be identified in GBM. Proteoglycan glypican-1 (GPC-1) is believed to be such a target as it is highly expressed in GBM and is associated with poor prognosis. We hypothesize that an anti-GPC-1 antibody, Miltuximab®, conjugated with the NIR dye, IRDye800CW (IR800), can specifically accumulate in a GBM xenograft and provide high-contrast in vivo fluorescent imaging in rodents following systemic administration. Miltuximab® was conjugated with IR800 and intravenously administered to BALB/c nude mice bearing a subcutaneous U-87 GBM hind leg xenograft. Specific accumulation of Miltuximab®-IR800 in subcutaneous xenograft tumor was detected 24 h later using an in vivo fluorescence imager. The conjugate did not cause any adverse events in mice and caused strong fluorescence of the tumor with tumor-to-background ratio (TBR) reaching 10.1 ± 2.8. The average TBR over the 10-day period was 5.8 ± 0.6 in mice injected with Miltuximab®-IR800 versus 2.4 ± 0.1 for the control group injected with IgG-IR800 (p = 0.001). Ex vivo assessment of Miltuximab®-IR800 biodistribution confirmed its highly specific accumulation in the tumor. The results of this study confirm that Miltuximab®-IR800 holds promise for intraoperative fluorescence molecular imaging of GBM and warrants further studies.

BackgroundThe pharmaceutical agent pentosan polysulfate (PPS) is known to induce proliferation and chondrogenesis of mesenchymal progenitor cells (MPCs) in vitro and in vivo. However, the mechanism(s) of action of PPS in mediating these effects remains unresolved.In the present report we address this issue by investigating the binding and uptake of PPS by MPCs and monitoring gene expression and proteoglycan biosynthesis before and after the cells had been exposed to limited concentrations of PPS and then re-established in culture in the absence of the drug (MPC priming).MethodsImmuno-selected STRO-1+ mesenchymal progenitor stem cells (MPCs) were prepared from human bone marrow aspirates and established in culture. The kinetics of uptake, shedding, and internalization of PPS by MPCs was determined by monitoring the concentration-dependent loss of PPS media concentrations using an enzyme-linked immunosorbent assay (ELISA) and the uptake of fluorescein isothiocyanate (FITC)-labelled PPS by MPCs. The proliferation of MPCs, following pre-incubation and removal of PPS (priming), was assessed using the Wst-8 assay method, and proteoglycan synthesis was determined by the incorporation of 35SO4 into their sulphated glycosaminoglycans. The changes in expression of MPC-related cell surface antigens of non-primed and PPS-primed MPCs from three donors was determined using flow cytometry. RNA sequencing of RNA isolated from non-primed and PPS-primed MPCs from the same donors was undertaken to identify the genes altered by the PPS priming protocol.ResultsThe kinetic studies indicated that, in culture, PPS rapidly binds to MPC surface receptors, followed by internalisation and localization within the nucleus of the cells. Following PPS-priming of MPCs and a further 48 h of culture, both cell proliferation and proteoglycan synthesis were enhanced. Reduced expression of MPC-related cell surface antigen expression was promoted by the PPS priming, and RNA sequencing analysis revealed changes in the expression of 42 genes.ConclusionThis study has shown that priming of MPCs with low concentrations of PPS enhanced chondrogenesis and MPC proliferation by modifying their characteristic basal gene and protein expression. These findings offer a novel approach to re-programming mesenchymal stem cells for clinical indications which require the repair or regeneration of cartilaginous tissues such as in osteoarthritis and degenerative disc disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0723-y) contains supplementary material, which is available to authorized users.

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