Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone

Moester, M. J. C.; Schoeman, Monique A.E.; Oudshoorn, Ineke B.; Beusekom, Mara M. van; Mol, Isabel M.; Kaijzel, Eric L.; Löwik, Clemens W.G.M.; Rooij, Karien E. de

doi:10.1016/j.bbrc.2013.11.055

Cited by 13 publications

(15 citation statements)

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“…Mineralization as a marker of osteoblast differentiation was measured after 3, 7, 14 or 21 days of hBMSC‐TERT cell differentiation using a labelled calcium chelating agent, BoneTag™, as described elsewhere . In brief, 24 hour before measurements, BoneTag™ (final concentration 2 pmol/mL) was added to the cells.…”

Section: Methodsmentioning

confidence: 99%

“…Mineralization as a marker of osteoblast differentiation was measured after 3, 7, 14 or 21 days of hBMSC-TERT cell differentiation using a labelled calcium chelating agent, BoneTag™, as described elsewhere. 37 In brief, 24 hour before measurements, BoneTag™ (final concentration 2 pmol/mL) was added to the cells. The next day, the conditioned media was removed and the cells were washed with PBS before adding 500 µL PBS in each well and scanning at 800 nm using Odyssey-CLx Imaging System (LI-COR).…”

Section: Quantification Of Matrix Mineralizationmentioning

confidence: 99%

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Lansoprazole inhibits the cysteine protease legumain by binding to the active site

Bosnjak

Solberg

Hemati

et al. 2019

Basic Clin Pharma Tox

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Proton pump inhibitors (PPIs) are prodrugs used in the treatment of peptic ulcer diseases. Once activated by acidic pH, the PPIs subsequently inhibit the secretion of gastric acid by covalently forming disulphide bonds with the SH groups of the parietal proton pump, that is the H+/K+‐ATPase. Long‐term use of PPIs has been associated with numerous adverse effects, including bone fractures. Considering the mechanism of activation, PPIs could also be active in acidic micro‐environments such as in lysosomes, tumours and bone resorption sites. We suggested that the SH group in the active site of cysteine proteases could be susceptible for inhibition by PPIs. In this study, the inhibition by lansoprazole was shown on the cysteine proteases legumain and cathepsin B by incubating purified proteases or cell lysates with lansoprazole at different concentrations and pH conditions. The mechanism of legumain inhibition was shown to be a direct interaction of lansoprazole with the SH group in the active site, and thus blocking binding of the legumain‐selective activity‐based probe MP‐L01. Lansoprazole was also shown to inhibit both legumain and cathepsin B in various cell models like HEK293, monoclonal legumain over‐expressing HEK293 cells (M38L) and RAW264.7 macrophages, but not in human bone marrow‐derived skeletal (mesenchymal) stem cells (hBMSC‐TERT). During hBMSC‐TERT differentiation to osteoblasts, lansoprazole inhibited legumain secretion, alkaline phosphatase activity, but had no effects on in vitro mineralization capacity. In conclusion, lansoprazole acts as a direct covalent inhibitor of cysteine proteases via disulphide bonds with the SH group in the protease active site. Such inhibition of cysteine proteases could explain some of the off‐target effects of PPIs.

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Section: Methodsmentioning

confidence: 99%

Section: Quantification Of Matrix Mineralizationmentioning

confidence: 99%

Lansoprazole inhibits the cysteine protease legumain by binding to the active site

Bosnjak

Solberg

Hemati

et al. 2019

Basic Clin Pharma Tox

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“…Instead of using conventional staining techniques like Alizarin Red S, we have chosen Bonetag to assess the level of mineralization in our cultures, since we previously showed Bonetag to be more sensitive to small changes in mineralization 27. In the current study, we observed an increase in mineralization when cells were cultured with OM.…”

Section: Discussionmentioning

confidence: 52%

“…Mineralization of the cultures was assessed using the fluorescent dye Bonetag (Li‐COR Biosciences, Lincoln, NE), as described previously 27. Briefly, 24 h before fixation with 3.7% buffered formaldehyde, 2 nM Bonetag 800 was added to the culture medium.…”

Section: Methodsmentioning

confidence: 99%

“…Mineralization of the cultures was assessed using the fluorescent dye Bonetag (Li-COR Biosciences, Lincoln, NE), as described previously. 27 Briefly, 24 h before fixation with 3.7% buffered formaldehyde, 2 nM Bonetag 800 was added to the culture medium. The fixed cells were scanned with the Odyssey Infrared Imaging System (Li-COR) at a resolution of 42 mm, intensity 6.5 and medium quality.…”

Section: Mineralizationmentioning

confidence: 99%

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Peri-prosthetic tissue cells show osteogenic capacity to differentiate into the osteoblastic lineage

et al. 2017

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During the process of aseptic loosening of prostheses, particulate wear debris induces a continuous inflammatory‐like response resulting in the formation of a layer of fibrous peri‐prosthetic tissue at the bone‐prosthesis interface. The current treatment for loosening is revision surgery which is associated with a high‐morbidity rate, especially in old patients. Therefore, less invasive alternatives are necessary. One approach could be to re‐establish osseointegration of the prosthesis by inducing osteoblast differentiation in the peri‐prosthetic tissue. Therefore, the aim of this study was to investigate the capacity of peri‐prosthetic tissue cells to differentiate into the osteoblast lineage. Cells isolated from peri‐prosthetic tissue samples (n = 22)−obtained during revision surgeries−were cultured under normal and several osteogenic culture conditions. Osteogenic differentiation was assessed by measurement of Alkaline Phosphatse (ALP), mineralization of the matrix and expression of several osteogenic genes. Cells cultured in osteogenic medium showed a significant increase in ALP staining (p = 0.024), mineralization of the matrix (p < 0.001) and ALP gene expression (p = 0.014) compared to normal culture medium. Addition of bone morphogenetic proteins (BMPs), a specific GSK3β inhibitor (GIN) or a combination of BMP and GIN to osteogenic medium could not increase ALP staining, mineralization, and ALP gene expression. In one donor, addition of GIN was required to induce mineralization of the matrix. Overall, we observed a high‐inter‐donor variability in response to osteogenic stimuli. In conclusion, peri‐prosthetic tissue cells, cultured under osteogenic conditions, can produce alkaline phosphatase and mineralized matrix, and therefore show characteristics of differentiation into the osteoblastic lineage. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1732–1742, 2017.

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Inhibition of GSK3β Stimulates BMP Signaling and Decreases SOST Expression Which Results in Enhanced Osteoblast Differentiation

Schoeman

Moester

Oostlander

et al. 2015

J of Cellular Biochemistry

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Both bone morphogenetic protein (BMP) and Wnt signaling have significant roles in osteoblast differentiation and the interaction between BMP and Wnt signaling is well known. Sclerostin is an important inhibitor of bone formation, inhibiting Wnt signaling and downstream effects of BMP such as alkaline phosphatase activity and matrix mineralization in vitro. However, little is known about the effect of BMP and Wnt signaling interaction on the regulation of SOST, the gene encoding sclerostin. Possibly, uncoupling of osteoblast differentiation regulators and SOST expression could increase osteoblast differentiation. Therefore, we investigated the effect of BMP and Wnt signaling interaction on the expression of SOST and the subsequent effect on osteoblast differentiation. Human osteosarcoma cells (SaOS-2) and murine pre-osteoblast cells (KS483) were treated with different concentrations of Wnt3a, a specific GSK3β inhibitor (GIN) and BMP4. Both Wnt3a and GIN increased BMP4-induced BMP signaling and BMP4 increased Wnt3a and GIN-induced Wnt signaling. However, the effect of GIN was much stronger. Quantitative RT-PCR analysis showed that SOST expression dose-dependently decreased with increasing Wnt signaling, while BMP4 induced SOST expression. GIN significantly decreased the BMP4-induced SOST expression. This resulted in an increased osteoblast differentiation as measured by ALP activity in the medium and matrix mineralization. We conclude that GSK3β inhibition by GIN caused an uncoupling of BMP signaling and SOST expression, resulting in an increased BMP4-induced osteoblast differentiation. This effect can possibly be used in clinical practice to induce local bone formation, for example, fracture healing or osseointegration of implants.

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Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone

Cited by 13 publications

References 20 publications

Lansoprazole inhibits the cysteine protease legumain by binding to the active site

Lansoprazole inhibits the cysteine protease legumain by binding to the active site

Peri-prosthetic tissue cells show osteogenic capacity to differentiate into the osteoblastic lineage

Inhibition of GSK3β Stimulates BMP Signaling and Decreases SOST Expression Which Results in Enhanced Osteoblast Differentiation

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