In Vitro Modeling of Diurnal Changes in Bone Metabolism
Abstract
1. Introduction
2. Results
2.1. Lack of Rhythmicity in Expression of Circadian Clock Genes in Conventional Osteoblast-Osteoclast Co-Culture
2.2. Adaptation of the Bone Cell Co-Culture
2.3. HSP Obtained in the Evening (7–8 Pm) Induced Cell Growth Stronger than That with HSP Obtained in the Morning (7–8 Am) or Noon (1–2 Pm)
2.4. Highest Osteoblast Function Observed in Co-Cultures Differentiated in the Presence of HSP Obtained in the Evening (7–8 Pm)
2.5. Composition of M-CSF, RANKL, and OPG in the HSP Favors Osteoclastogenesis in Co-Cultures Differentiated in the Presence of HSP Obtained in the Morning (7–8 Am)
2.6. Three-Dimensional Co-Culture Showed Highest Bone Mineral Density and Stiffness When Differentiated in the Presence of HSP Obtained in the Evening (7–8 Pm)
2.7. Replacement of FCS with HSP in the Osteogenic Differentiation Medium Affects the Expression of the Genes Involved in the Circadian Rhythm Core Loop
3. Discussion
4. Materials and Methods
4.1. Human Serum Pool
4.2. Osteoblast–Osteoclast Co-Culture
4.3. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
4.4. Resazurin Conversion Assay
4.5. Sulforhodamine B (SRB) Staining
4.6. Alkaline Phosphatase (AP) Activity
4.7. Von Kossa Staining
4.8. Alizarin Red Staining
4.9. Dot Blot Analysis
4.10. Carbonic Anhydrase II (CAII) Activity
4.11. Tartrate-Resistant Acidic Phosphatase (TRAP) Activity Assay
4.12. Mineral Content of the PRP Scaffolds
4.13. Stiffness of the PRP Scaffolds
4.14. Statistics
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
2D | two-dimensional |
3D | three-dimensional |
ALP | alkaline phosphatase |
APS | ammonium persulfate |
BAAm | N,N-methylene(bis)acrylamide |
BMD | bone mineral density |
CAII | carbonic anhydrase II |
CTX | collagen type I C-telopeptide |
FCS | fetal calf serum |
HSP | human serum pool |
M-CSF | macrophage colony-stimulating factor |
MSC | mesenchymal stem cells |
NTX | N-terminal cross-linked telopeptide of type I collagen |
OPG | osteoprotegerin |
pHEMA | poly-2-hydroxyethyl methacrylate |
PICP | carboxy-terminal propeptide of type I procollagen |
PRP | platelet-rich plasma |
qRT-PCR | quantitative reverse-transcription polymerase-chain reaction |
RANKL | receptor activator of nuclear factor kappa-Β ligand |
TEMED | N,N,N,N-tetramethyl-ethylenediamine |
TRAP5b | tartrate-resistant acidic phosphatase 5b |
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Gene | Role | Accession Number | Forward Primer (5′–3′) | Reverse Primer (3′–5′) | Amplicon Size [bp] | Efficiency [%] | Annealing Temp. [°C] | Cycles [N] |
---|---|---|---|---|---|---|---|---|
B2M | HKG | NM_004048.2 | AGATGAGTAT GCCTGCCGTG | GCGGCATCTT CAAACCTCCA | 105 | 101 | 60 | 40 |
CLOCK | Circadian Rhythm | NM_001267843.2 | ACGCACACAT AGGCCATCTT | ATTATGGGTG GTGCCCTGTG | 177 | 102 | 66 | 40 |
BMAL1 | NM_001030272.3 | TCCTTTGTTG TAGGTGGCCC | GCGATGACCC TCTTATCCTGT | 139 | 112 | 66 | 40 | |
NPAS2 | NM_002518.4 | ACACTCGGTG GTCAGTTACG | CCGATGGCGA ATGACTGGTA | 188 | 110 | 66 | 40 | |
CRY1 | NM_001413458.1 | CCCAGGTTGT AGCAGCAGTG | AGGACGTTTC CCACCACTTG | 111 | 135 | 60 | 40 | |
PER1 | NM_002616.3 | GGGGACCAAG AAAGATCCGC | GCTACACTGA CTGGTGACGG | 145 | 97 | 64 | 40 | |
PER2 | NM_022817.3 | CATCGACGTG GCAGAATGTG | ACGTCTGCTC TTCGATCCTG | 161 | 80 | 60 | 40 | |
MKI67 | Proliferation | NM_002417.5 | CGTCCCAGTG GAAGAGTTGT | CGACCCCGCT CCTTTTGATA | 143 | 102 | 63 | 40 |
TPX2 | NM_012112.5 | GGAAGCACCA GCTGGAAGA | GAACTAGAGA ACCAGAAAGGCCC | 147 | 110 | 63 | 40 | |
TOP2A | NM_001067.4 | GTTCTTGAGC CCCTTCACGA | ACCCACATTT GCTGGGTCA | 216 | 123 | 63 | 40 | |
RUNX2 | Transcription Factor | NM_001024630.4 | CTGTGGTTAC TGTCATGGCG | GGGAGGATTT GTGAAGACGGT | 170 | 118 | 60 | 40 |
SP7 | NM_152860.1 | CCCAGGCAAC ACTCCTACTC | GGCTGGATTA AGGGGAGCAAA | 175 | 91 | 62 | 40 | |
NFATC1 | NM_172390.2 | TGCAAGCCGA ATTCTCTGGT | CTTTACGGCG ACGTCGTTTC | 228 | 87 | 64 | 40 |
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Ehnert, S.; Gao, X.; Heßlinger, M.; Braun, N.R.; Schulz, K.A.; Jahn, D.; Springer, F.; Nussler, A.K. In Vitro Modeling of Diurnal Changes in Bone Metabolism. Int. J. Mol. Sci. 2025, 26, 7699. https://doi.org/10.3390/ijms26167699
Ehnert S, Gao X, Heßlinger M, Braun NR, Schulz KA, Jahn D, Springer F, Nussler AK. In Vitro Modeling of Diurnal Changes in Bone Metabolism. International Journal of Molecular Sciences. 2025; 26(16):7699. https://doi.org/10.3390/ijms26167699
Chicago/Turabian StyleEhnert, Sabrina, Xiang Gao, Maximilian Heßlinger, Niklas R. Braun, Kevin A. Schulz, Denise Jahn, Fabian Springer, and Andreas K. Nussler. 2025. "In Vitro Modeling of Diurnal Changes in Bone Metabolism" International Journal of Molecular Sciences 26, no. 16: 7699. https://doi.org/10.3390/ijms26167699
APA StyleEhnert, S., Gao, X., Heßlinger, M., Braun, N. R., Schulz, K. A., Jahn, D., Springer, F., & Nussler, A. K. (2025). In Vitro Modeling of Diurnal Changes in Bone Metabolism. International Journal of Molecular Sciences, 26(16), 7699. https://doi.org/10.3390/ijms26167699