A Growth Factor-Free Co-Culture System of Osteoblasts and Peripheral Blood Mononuclear Cells for the Evaluation of the Osteogenesis Potential of Melt-Electrowritten Polycaprolactone Scaffolds
Abstract
:1. Introduction
2. Results
2.1. Scaffolds’ Morphology
2.2. Visualization of Cell Morphology
2.3. Visualization of the Mineralized Extracellular Matrix
2.4. ALP and TRAP Activity
2.5. Analysis of Gene Expression
3. Discussion
4. Materials and Methods
4.1. Scaffolds Fabrication and Post-Processing
4.2. Cell Isolation
4.3. Scaffold Sterilization and Cell Seeding
4.4. Visualization of Cell Morphology
4.5. Visualization of Mineralized Cell Matrix by Von Kossa Staining
4.6. Cell Proliferation
4.7. Alkaline Phosphatase (ALP) Assay
4.8. Tartrate-Resistant Acid Phosphatase (TRAP) Assay
4.9. Gene Expression by Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
TERM | tissue engineering and regenerative medicine |
EU-MDR | European Union medical device regulation |
AM | additive manufacturing |
MEW | melt electro-writing |
PCL | polycaprolactone |
OB | osteoblasts |
PBMC | peripheral mononuclear cells |
ECM | extracellular matrix |
ALP | alkaline phosphatase |
TRAP | tartrate-resistant acid phosphatase |
RT-qPCR | quantitative reverse transcription-polymerase chain reaction |
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Gene | Sequence | Tm (°C) |
---|---|---|
RunX2 | Forward: TGCCTAGGCGCATTTCAGGTGC Reverse: TGAGGTGACTGGCGGGGTGT | 60 |
ALP | Forward: ACGTGGCTAAGAATGTCATC Reverse: CTGGTAGGCGATGTCCTTA | 60 |
OCN | Forward: CCAGCGGTGCAGAGTCCAGC Reverse: GACACCCTAGACCGGGCCGT | 60 |
OPG | Forward: GGGACCACAATGAACAAGCTG Reverse: TGTTTTAGGGAGGTGCCAGG | 56 |
CTSK | Forward: GGCCCGAGTGGGACCTGTCT Reverse: CCCSCTGCCSSSSCCGCSTGG | 56 |
TRAP | Forward: CCCTCGGAGAAACTGCATCAT Reverse: CATGTCCATCCAGGGGGAGA | 60 |
RANK | Forward: TGCCTTGCAGGCTACTTCTC Reverse: CCTGCTGACCAAAGTTTGCC | 56 |
RANKL | Forward: GGGCCAGGTTGTCTGCAGCGT Reverse: ACCATGAGCCATCCACCACCAGG | 58 |
Col1A1 | Forward: GCCAAGAAGAAGACATCCCAGC Reverse: TCCCTTGGCACCATCCAA | 58 |
β-Tubulin | Forward: GAGGGCGAGGACGAGGCTTA Reverse: TCTAACAGAGGCAAAACTGAGCACC | 60 |
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Hammerl, A.; Diaz Cano, C.E.; De-Juan-Pardo, E.M.; van Griensven, M.; Poh, P.S.P. A Growth Factor-Free Co-Culture System of Osteoblasts and Peripheral Blood Mononuclear Cells for the Evaluation of the Osteogenesis Potential of Melt-Electrowritten Polycaprolactone Scaffolds. Int. J. Mol. Sci. 2019, 20, 1068. https://doi.org/10.3390/ijms20051068
Hammerl A, Diaz Cano CE, De-Juan-Pardo EM, van Griensven M, Poh PSP. A Growth Factor-Free Co-Culture System of Osteoblasts and Peripheral Blood Mononuclear Cells for the Evaluation of the Osteogenesis Potential of Melt-Electrowritten Polycaprolactone Scaffolds. International Journal of Molecular Sciences. 2019; 20(5):1068. https://doi.org/10.3390/ijms20051068
Chicago/Turabian StyleHammerl, Andreas, Carlos E. Diaz Cano, Elena M. De-Juan-Pardo, Martijn van Griensven, and Patrina S.P. Poh. 2019. "A Growth Factor-Free Co-Culture System of Osteoblasts and Peripheral Blood Mononuclear Cells for the Evaluation of the Osteogenesis Potential of Melt-Electrowritten Polycaprolactone Scaffolds" International Journal of Molecular Sciences 20, no. 5: 1068. https://doi.org/10.3390/ijms20051068