Calcium Polyphosphate Nanoparticles Act as an Effective Inorganic Phosphate Source during Osteogenic Differentiation of Human Mesenchymal Stem Cells
Abstract
:1. Introduction
2. Results
2.1. Inorganic PolyP-NP can Function As an Effective phosphate Source for Matrix Mineralisation during Osteogenic Differentiation of Human BM-MSCs
2.2. PolyP-NP Treatment Results in Comparable Effects on ALP Activity to BGP during Osteogenic Differentiation of hBM-MSCs
2.3. Osteogenic Differentiation of BM-MSCs, Supported by Either BGPor polyP-NP, Is Associated with Increased Intracellular Levels of ATP at Early TIME Points
2.4. BGP Results in High Free Phosphate Fluctuations in the Medium
2.5. PolyP-NP Induce Similar Changes to BGP-Containing Osteogenic Media in the Expression of BM-MSC-Relevant Transcription Factors
2.6. PolyP-NP-Supplementation of Ostegenic Medium Supports Expression of Early, Intermediate and Late Osteogenic Markers Comparable to BGP-Supplemented Osteogenic Media
3. Discussion
4. Materials and Methods
4.1. Preparation of Calcium-PolyP-NP
4.2. Cell Isolation and Culture
4.3. Osteogenic Differentiation
4.4. RNA Isolation and qRT-PCR
4.5. Alizarin Red Staining and Quantification
4.6. Quantification of ALP Activity and DNA Content
4.7. Quantification of ATP Content
4.8. Phosphate Quantification
4.9. Statistics
Author Contributions
Funding
Conflicts of Interest
Abbreviations
AA2P | L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate |
ALP | Alkaline phosphatase |
ANOVA | Analysis of variance |
ATP | Adenosine triphosphate |
BCP | 1-Bromo-3-chloropropane |
BGP | Β-Glycerolphosphate disodium salt hydrate |
BSP | Bone sialoprotein |
Dexa | Dexamethasone |
FBS | Fetal bovine serum |
hBM-MSCs | Human bone marrow-derived mesenchymal stem cells |
HUVEC | Human umbilical vein endothelial cells |
LG-DMEM | Low glucose (1 g/L) Dulbecco’s Modified Eagle’s Medium |
PBS | Phosphate-buffered saline |
PEN/STREP | Penicillin/Streptomycin |
Pi | Free phosphate |
polyP-NP | Polyphosphate nanoparticles |
PPARγ | Peroxisome proliferator-activated receptor gamma |
qRT-PCR | Quantitative reverse transcription PCR |
Runx2 | Runt-related transcription factor 2 |
Sox9 | Sex determining region Y box 9 |
TBS | Tris buffered saline |
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Osteocontrol | Osteocontrol + polyP-NP | Osteogenic (BGP) | Osteogenic (polyP-NP) | |
---|---|---|---|---|
LG-DMEM 1 | + | + | + | + |
PEN/STREP | + | + | + | + |
FBS | + | + | + | + |
Dexa | - | - | + | + |
AA2P | - | - | + | + |
BGP | - | - | + | - |
polyP-NP | - | + | - | + |
Gene | Forward | Reverse | Probe |
COL1A1 | 5′-CCC TGG AAA GAA TGG AGA TGA T-3′ | 5′-ACT GAA ACC TCT GTG TCC CTT CA-3′ | 5′-CGG GCA ATC CTC GAG CAC CCT-3′ |
RPLP0 | 5′-TGG GCA AGA ACA CCA TGA TG-3′ | 5′-CGG ATA TGA GGC AGC AGT TTC-3′ | 5′-AGG GCA CCT GGA AAA CAA CCC AGC-3′ |
RUNX2 | 5′-AGC AAG GTT CAA CGA TCT GAG AT-3′ | 5′-TTT GTG AAG ACG GTT ATG GTC AA-3′ | 5′-TGA AAC TCT TGC CTC GTC CAC TCC G-3′ |
Gene | Assay on Demand Ref No. (Applied Biosystems) | ||
ALPL | Hs00758162_m1 | ||
IBSP | Hs0017320_m1 | ||
PPAR-γ | Hs00234592_m1 | ||
SOX9 | Hs00165814_m1 |
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Phelipe Hatt, L.; Thompson, K.; Müller, W.E.G.; Stoddart, M.J.; Armiento, A.R. Calcium Polyphosphate Nanoparticles Act as an Effective Inorganic Phosphate Source during Osteogenic Differentiation of Human Mesenchymal Stem Cells. Int. J. Mol. Sci. 2019, 20, 5801. https://doi.org/10.3390/ijms20225801
Phelipe Hatt L, Thompson K, Müller WEG, Stoddart MJ, Armiento AR. Calcium Polyphosphate Nanoparticles Act as an Effective Inorganic Phosphate Source during Osteogenic Differentiation of Human Mesenchymal Stem Cells. International Journal of Molecular Sciences. 2019; 20(22):5801. https://doi.org/10.3390/ijms20225801
Chicago/Turabian StylePhelipe Hatt, Luan, Keith Thompson, Werner E. G. Müller, Martin James Stoddart, and Angela Rita Armiento. 2019. "Calcium Polyphosphate Nanoparticles Act as an Effective Inorganic Phosphate Source during Osteogenic Differentiation of Human Mesenchymal Stem Cells" International Journal of Molecular Sciences 20, no. 22: 5801. https://doi.org/10.3390/ijms20225801