Changes in Human Foetal Osteoblasts Exposed to the Random Positioning Machine and Bone Construct Tissue Engineering
Abstract
:1. Introduction
2. Results
2.1. Morphological Examination of RPM-Exposed hFOB 1.19 Cells Reveal Signs of Organoid Formation
2.2. Alterations of the Cytoskeleton and the Extracellular Matrix
2.3. Changes in BMP2/TGF-β Signalling Pathways
2.4. Secretion of Soluble Factors in the Supernatant
2.5. Interactions of Proteins Involved in Bone Formation
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Random Positioning Machine
4.3. F-Actin, Tubulin and Laminin Staining
4.4. Microscopy
4.5. RNA Isolation
4.6. Quantitative Real Time PCR
4.7. Determination of Intracellular Protein Levels
4.8. Measurement of Secreted Bone Biomarkers
4.9. Histological Staining
4.10. Determination of Calcification of the MCS
4.11. Pathway Analysis
4.12. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
µg | Microgravity |
2D | Two- dimensional |
3D | Three- dimensional |
ACTB | Actin beta |
ACTH | Adrenocorticotrophic hormone |
AD | Adherent monolayers |
ALC | Alcian blue |
BMP2 | Bone morphogenic protein 2 |
CLSM | Confocal laser scanning microscopy |
CN | Clinostat |
COL1A1 | Collagen type 1 alpha 1 |
Dkk1 | Dickkopf- related protein 1 |
ECM | Extra cellular matrix |
FGF-23 | Fibroblast growth factor-23 |
FN1 | Fibronectin 1 |
HE | Haematoxylin–Eosin |
hFOB | Human foetal osteoblast |
IL-1β | Interleukin 1 beta |
ISS | International space station |
LAMA1 | Laminin subunit alpha 1 |
MCS | Multicellular spheroid |
MKI67 | Marker of proliferation Ki-67 |
OC | Osteocalcin |
OPG | Osteoprotegerin |
OPN | Osteopontin |
PTH | Parathyroid hormone |
qRT-PCR | Quantitative real-time PCR |
r- | Real |
RANK | Receptor activator of nuclear factor-κB |
RANKL | Receptor activator of nuclear factor-κB ligand |
RCC | Rotating Cell Culture |
RPM | Random Positioning Machine |
RWV | Rotating wall vessel |
s- | Simulated |
SOST | Sclerostin |
SOST | Sclerostin |
SOX9 | SRY-Box 9 |
SPP1 | Secreted Phosphoprotein 1 |
TGF-β1 | Transforming growth factor beta 1 |
TNF-1α | Tumour necrosis factor 1 alpha |
TUBB | Beta tubulin |
VIM | Vimentin |
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mRNA Levels [% of 1 g Control] | |||||||||||||||||||
Gene | |||||||||||||||||||
Sample | ACTB | BMP2 | COL1A1 | FN1 | SPP1 | LAMA1 | MKI67 | SOX9 | TGFB1 | TUBB | VIM | ||||||||
7 days | 1 g | 100.0 ± 11.0 | 100.0 ± 6.7 | 100.0 ± 24.3 | 100.0 ± 10.8 | 100.0 ± 14.8 | 100.0 ± 3.6 | 100.0 ± 20.6 | 100.0 ± 20.8 | 100.0 ± 12.1 | 100.0 ± 15.1 | 100.0 ± 7.2 | |||||||
AD | ▲181.2 ± 2.9 | ▼45.6 ± 9.7 | 124.0 ± 6.0 | ▲127.8 ± 6.2 | ▲1094.0 ± 89.0 | ▲197.3 ± 18.4 | 111.4 ± 10.3 | ▲160.1 ± 10.1 | 123.2 ± 3.5 | ▲297.4 ± 27.8 | ▲162.3 ± 8.5 | ||||||||
MCS | ▲154.7 ± 19.7 | ▲207.2 ± 69.9 | 112.6 ± 41.7 | 218.5 ± 73.9 | ▲1719.2 ± 709.5 | ▲244.4 ± 67.0 | 89.0 ± 35.9 | 140.1 ± 51.6 | 147.1 ± 32.3 | ▲236.2 ± 74.5 | ▲166.7 ± 38.5 | ||||||||
14 days | 1 g | 100.0 ± 8.6 | 100.0 ± 32.1 | 100.0 ± 8.6 | 100.0 ± 3.6 | 100.0 ± 3.4 | 100.0 ± 11.5 | 100.0 ± 9.6 | 100.0 ± 8.1 | 100.0 ± 3.8 | 100.0 ± 11.3 | 100.0 ± 11.1 | |||||||
AD | 113.1 ± 25.8 | ▼28.8 ± 6.5 | ▲234.0 ± 5.3 | ▲119.8 ± 2.7 | ▲813.4 ± 22.2 | 127.9 ± 8.1 | 132.1 ± 9.5 | ▲151.2 ± 5.9 | ▲133.8 ± 4.0 | ▲125.7 ± 4.8 | ▼76.1 ± 3.5 | ||||||||
MCS | 225.2 ± 61.7 | 100.1 ± 43.8 | 225.7 ± 11.4 | 107.7 ± 7.0 | 1098.2 ± 11.3 | 148.9 ± 17.6 | 109.7 ± 5.8 | 108.0 ± 4.3 | 87.7 ± 0.7 | 67.3 ± 2.7 | 72.1 ± 1.7 | ||||||||
Intracellular Protein Levels [% of 1 g Control] | |||||||||||||||||||
Protein | |||||||||||||||||||
Sample | β-actin | BMP2 | Col 1 | FN | OPN | laminin | Ki-67 | SOX9 | TGF-β1 | β-tubulin | vimentin | ||||||||
7 days | 1 g | 100.0 ± 7.5 | 100.0 ± 13.6 | 100.0 ± 10.4 | 100.0 ± 18.8 | 100.0 ± 16.6 | 100.0 ± 13.2 | 100.0 ± 14.8 | 100.0 ± 27.3 | 100.0 ± 20.1 | 100.0 ± 8.2 | 100.0 ± 6.9 | |||||||
AD | 105.8 ± 12.9 | 86.5 ± 5.4 | 91.4 ± 5.2 | 139.2 ± 19.2 | ▲154.4 ± 13.2 | 110.7 ± 11.1 | 107.8 ± 9.1 | 75.3 ± 17.0 | ▲312.1 ± 41.4 | ▼59.7 ± 23.9 | 107.4 ± 29.3 | ||||||||
MCS | 83.2 ± 10.4 | ▼55.7 ± 3.2 | ▼68.8 ± 1.1 | ▲186.9 ± 19.1 | ▼19.4 ± 7.5 | ▼72.1 ± 7.7 | ▼67.5 ± 6.5 | 80.9 ± 3.7 | ▲345.9 ± 30.4 | ▼44.3 ± 5.6 | ▼21.5 ± 6.3 | ||||||||
14 days | 1 g | 100.0 ± 13.1 | 100.0 ± 8.8 | 100.0 ± 9.8 | 100.0 ± 13.2 | 100.0 ± 24.6 | 100.0 ± 7.6 | 100.0 ± 7.5 | 100.0 ± 9.6 | 100.0 ± 27.3 | 100.0 ± 11.3 | 100.0 ± 19.2 | |||||||
AD | 84.8 ± 8.0 | ▲196.9 ± 13.6 | ▲148.1 ± 11.1 | ▲146.9 ± 18.0 | ▲2852.2 ± 964.4 | 83.5 ± 7.6 | ▼75.3 ± 12.0 | 79.6 ± 10.8 | ▼15.5 ± 2.4 | ▲188.8 ± 18.0 | 69.3 ± 4.3 | ||||||||
MCS | 103.9 ± 11.7 | ▼16.9 ± 1.0 | ▼25.5 ± 2.1 | 91.0 ± 34.3 | ▼14.8 ± 17.3 | ▲266.4 ± 44.4 | ▲300.8 ± 15.9 | ▲125.4 ± 12.8 | 39.8 ± 26.0 | ▼35.7 ± 3.8 | ▼1.6 ± 0.8 | ||||||||
Protein Secretion [% of 1 g Control] | |||||||||||||||||||
Protein | |||||||||||||||||||
Sample | DKK1 | IL-1β | OC | OPG | OPN | SOST | TNF-α | ||||||||||||
7 days | 1 g | 100.0 ± 1.4 | 100.0 ± 13.0 | 100.0 ± 32.6 | 100.0 ± 15.3 | 100.0 ± 14.7 | 100.0 ± 7.8 | 100.0 ± 8.7 | |||||||||||
RPM | 98.8 ± 2.5 | ▼34.8 ± 5.7 | 112.0 ± 47.8 | ▼42.6 ± 19.0 | ▲206.7 ± 38.1 | ▼49.4 ± 16.9 | ▼46.2 ± 12.2 | ||||||||||||
14 days | 1 g | 100.0 ± 6.6 | 100.0 ± 11.2 | 100.0 ± 24.9 | 100.0 ± 26.2 | 100.0 ± 14.8 | 100.0 ± 32.6 | 100.0 ± 7.5 | |||||||||||
RPM | 102.1 ± 6.0 | 125.6 ± 13.5 | ▲148.9 ± 28.0 | ▲154.1 ± 22.5 | ▲579.2 ± 20.8 | 74.4 ± 21.4 | 96.0 ± 11.0 |
Gene | Primer Name | Sequence |
---|---|---|
18S rRNA | 18S-F | GGAGCCTGCGGCTTAATTT |
18S-R | CAACTAAGAACGGCCATGCA | |
ACTB | ACTB-F | TGCCGACAGGATGCAGAAG |
ACTB-R | GCCGATCCACACGGAGTACT | |
BMP2 | BMP2-F | GACCTGTATCGCAGGCACTCA |
BMP2-R | TCGTTTCTGGTAGTTCTTCCAAAGA | |
COL1A1 | COL1A1-F | ACGAAGACATCCCACCAATCAC |
COL1A1-R | CGTTGTCGCAGACGCAGAT | |
FN1 | FN1-F | TGAGGAGCATGGTTTTAGGAGAA |
FN1-R | TCCTCATTTACATTCGGCGTATAC | |
LAMA1 | LAMA1-F | TGCTCATGGTCAATGCTAATCTG |
LAMA1-R | TCTATCAATCCTCTTCCTTGGACAA | |
MKI67 | MKI67-F | TGGGGAAAGTAGGTGTGAAAGAAG |
MKI67-R | CTCCTTAAACGTTCTGATGCTCTTG | |
SOX9 | SOX9-F | TTGAGACCTTCGACGTCAATGAG |
SOX9-R | TCTGGCCACGAGTGGCC | |
SPP1 | SPP1-F | CGAGGTGATAGTGTGGTTTATGGA |
SPP1-R | CGTCTGTAGCATCAGGGTACTG | |
TBP | TATA-F | GTGACCCAGCATCACTGTTTC |
TATA-R | GCAAACCAGAAACCCTTGCG | |
TGFB1 | TGFB1-F | CACCCGCGTGCTAATGGT |
TGFB1-R | AGAGCAACACGGGTTCAGGTA | |
TUBB | TUBB-F | CTGGACCGCATCTCTGTGTACTAC |
TUBB-R | GACCTGAGCGAACAGAGTCCAT | |
VIM | VIM-F | TTCAGAGAGAGGAAGCCGAAAAC |
VIM-R | AGATTCCACTTTGCGTTCAAGGT |
Antibody Target | Company/No | Host | Size (kDa) | Used Dilution |
---|---|---|---|---|
β-actin | Sigma/A5316 | Ms | 42 | 1:2000 |
BMP2 | Novus biological/ | Rb | 44 | 1:500 |
Collagen I | Sigma/C2456 | Ms | ~130 | 1:500 |
Cyclophilin | Abcam/AB178397 | Rb | 20 | 1:2000 |
Fibronectin | Invitrogen/MA1198 | Ms | 250 | 1:1000 |
Ki-67 | Santa cruz/c-15402 | Rb | 250 | 1:200 |
Laminin | Invitrogen/PA1-1673 | Rb | ~200 | 1:1000 |
Osteopontin | Sigma/07264 | Rb | 50 | 1:1000 |
SOX9 | Abcam/ab26414 | Rb | 56 | 1:1000 |
TGF-β1 | Abcam/ab92486 | Rb | 44 | 1:500 |
TNF-α | Abcam/ab9739 | Rb | 26 | 1:500 |
β-tubulin | Sigma/T5293 | Ms | 50 | 1:1000 |
Vimentin | Sigma/WH0007431 | Ms | ~53 | 1:1000 |
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Mann, V.; Grimm, D.; Corydon, T.J.; Krüger, M.; Wehland, M.; Riwaldt, S.; Sahana, J.; Kopp, S.; Bauer, J.; Reseland, J.E.; et al. Changes in Human Foetal Osteoblasts Exposed to the Random Positioning Machine and Bone Construct Tissue Engineering. Int. J. Mol. Sci. 2019, 20, 1357. https://doi.org/10.3390/ijms20061357
Mann V, Grimm D, Corydon TJ, Krüger M, Wehland M, Riwaldt S, Sahana J, Kopp S, Bauer J, Reseland JE, et al. Changes in Human Foetal Osteoblasts Exposed to the Random Positioning Machine and Bone Construct Tissue Engineering. International Journal of Molecular Sciences. 2019; 20(6):1357. https://doi.org/10.3390/ijms20061357
Chicago/Turabian StyleMann, Vivek, Daniela Grimm, Thomas J Corydon, Marcus Krüger, Markus Wehland, Stefan Riwaldt, Jayashree Sahana, Sascha Kopp, Johann Bauer, Janne E. Reseland, and et al. 2019. "Changes in Human Foetal Osteoblasts Exposed to the Random Positioning Machine and Bone Construct Tissue Engineering" International Journal of Molecular Sciences 20, no. 6: 1357. https://doi.org/10.3390/ijms20061357