Human Dental Pulp Stem Cell Osteogenic Differentiation Seeded on Equine Bone Block with Graphene and Melatonin
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ammonia Graphene-Oxide (G-N) Equine Bone Block Coating
2.2. hDPSCs Extraction and Cell Cultures
2.3. Experimental Study Design
- Group 1: hDPSCs + equine bone block (Ctrl);
- Group 2: hDPSCs + equine bone block + melatonin (Ctrl + Mel);
- Group 3: hDPSCs + equine bone block G-N-coated 2 μg/mL (G-N2);
- Group 4: hDPSCs + equine bone block G-N-coated 2 μg/mL + melatonin (G-N2 + Mel);
- Group 5: hDPSCs + equine bone block G-N-coated 10 μg/mL (G-N10);
- Group 6: hDPSCs + equine bone block G-N-coated 10 μg/mL + melatonin (G-N10 + Mel).
2.4. Live/Dead Test
2.5. RNA Isolation and Quantification
2.6. Quantitative Real-Time PCR for MicroRNAs Expression
- -
- has-miR-133a (UUUGGUCCCCUUCAACCAGCUG; #002246);
- -
- has-miR-133b (UUUGGUCCCCUUCAACCAGCUA; #002247);
- -
- hsa-miR-135a-5p (UAUGGCUUUUUAUUCCUAUGUGA; #000460);
- -
- hsa-miR-206 (UGGAAUGUAAGGAAGUGUGUGG; #000510);
- -
- hsa-miR- 29b (CUGGUUUCACAUGGUGGCUUAG; # 002166);
- -
- hsa-miR-let-7b (UGAGGUAGUAGGUUGUGUGGUU; #002619);
- -
- hsa-miR-16-5p (UAGCAGCACGUAAAUAUUGGCG; # 000391).
2.7. Gene Expression Analysis
2.8. Osteocalcin Levels Measurement
2.9. Histological Evaluations
2.10. Statistical Analysis
3. Results
3.1. Chemico-Physical Characterization of Ammonia Graphene-Oxide-Coated Collagenated Bone Block
3.2. UV-VIS Spectroscopy
3.3. Raman Spectroscopy
3.4. TGA Analysis
3.5. Cell Viability Assessment on Blocks
3.6. Gene Expression
3.7. MicroRNAs Expression
3.8. Osteocalcin Assay
3.9. Histological Evaluation
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene Symbol | Days | Growth Medium | Differentiation Medium | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CTRL | G-N2 | G-N10 | CTRL + MEL | G-N2 + MEL | G-N10 + MEL | CTRL | G-N2 | G-N10 | CTRL ù + MEL | G-N2 + MEL | G-N10 + MEL | ||
SMAD5 | 7 | 6.7 ± 0.092 | 6.9 ± 0.03 | 6.52 ± 0.04 | 6.58 ± 0.007 | 6.2 ± 0.007 *** | 6.42 ± 0.05 | 6.18 ± 0.06 | 6.8 ± 0.014 *** | 6.46 ± 0.07 * ## | 6.01 ± 0.06 | 6.56 ± 0.06 *** | 7.06 ± 0.012 *** |
14 | 5.5 ± 0.05 | 6 ± 0.03 | 5.4 ± 0.08 | 6 ± 0.05 | 5.8 ± 0.03 | 5.6 ± 0.1 | 7.4 ± 0.1 | 6.6 ± 0.006 *** | 5.8 ± 0.08 *** | 6.4 ± 0.035 | 6 ± 0.01 *** | 6.15 ± 0.015 *** | |
21 | 7.2 ± 0.02 | 6.3 ± 0.1 *** | 6.7 ± 0.04 * ## | 7.2 ± 0.07 | 6.2 ± 0.02 *** | 6 ± 0.04 *** | 8.25 ± 0.03 | 8.4 ± 0.14 | 8 ± 0.05 ** | 8.1 ± 0.06 | 8.4 ± 0.025 ** | 8.3 ± 0.05 | |
RUNX2 | 7 | 7.1 ± 0.08 | 7.1 ± 0.017 | 6.6 ± 0.08 *** | 6.6 ± 0.13 | 6.2 ± 0.04 *** | 6.55 ± 0.092 | 6.2 ± 0.01 | 6 ± 0.03 | 5.8 ± 0.04 | 6.1 ± 0.6 | 5.7 ± 0.06 | 5.9 ± 0.05 |
14 | 5.36 ± 0.015 | 5.6 ± 0.04 ** | 5.26 ± 0.07 | 5.2 ± 0.07 | 5.53 ± 0.036 *** | 4.96 ± 0.035 *** | 8.5 ± 0.06 | 6.1 ± 0.02 *** | 6 ± 0.07 *** | 6.5 ± 0.09 | 5.5 ± 0.07 *** | 5.7 ± 0.1 *** | |
21 | 6.5 ± 0.05 | 6 ± 0.0 *** | 6 ± 0.01 *** | 6.7 ± 0.007 | 6.2 ± 0.02 *** | 6 ± 0.04 *** | 7.4 ± 0.075 | 7.6 ± 0.08 | 7.25 ± 0.04 | 7.25 ± 0.007 | 7.4 ± 0.09 | 7.7 ± 0.035 *** |
miR Symbol | Days | Growth Medium | Differentiation Medium | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CTRL | G-N2 | G-N10 | CTRL + MEL | G-N2 + MEL | G-N10 + MEL | CTRL | G-N2 | G-N10 | CTRL + MEL | G-N2 + MEL | G-N10 + MEL | ||
miR-133a | 7 | 11.6 ± 0.2 | 12.5 ± 0.4 ** | 12.8 ± 0.1 *** | 13.1 ± 0.1 | 13.1 ± 0.2 | --- | 10.9 ± 0.05 | 11.6 ± 0.7 | 10.6 ± 0.1 | 10.8 ± 0.8 | 9.7 ± 0.3 | 12.3 ± 0.1 * |
14 | 11.2 ± 0.2 | 11.5 ± 0.7 | 11.9 ± 1.01 | 11.3 ± 0.7 | 12.1 ± 0.3 | 11.3 ± 0.8 | 9 ± 0.3 | 10.7 ± 0.1 | 11.5 ± 0.5 * | 10 ± 0.2 | 11.7 ± 0.9 | 12.3 ± 0.8 ** | |
21 | 11.3 ± 0.2 | 11.5 ± 0.7 | 12.7 ± 0.8 | 11.3 ± 0.7 | 12.5 ± 0.3 | 12.3 ± 0.1 | 12.2 ± 0.05 | 12.2 ± 0.6 | 13.2 ± 0.6 | 11.6 ± 0.4 | 11.9 ± 0.2 | 11.7 ± 0.5 | |
miR-133b | 7 | 10.1 ± 0.4 | 12.3 ± 0.2 | --- | 11.4 ± 0.8 | 13 ± 0.8 | --- | 11.4 ± 0.05 | 11.9 ± 0.4 | 11.9 ± 0.2 | 11.7 ± 0.6 | 12.4 ± 0.02 | --- |
14 | 11 ± 0.4 | 12 ± 0.4 | 12.2 ± 1.3 | 11.2 ± 0.1 | 12.3 ± 0.4 | 12.8 ± 0.8 | 11.1 ± 0.6 | 11.5 ± 0.8 | 11.5 ± 0.8 | 10.8 ± 0.2 | 11.1 ± 0.9 | 12.5 ± 0.8 | |
21 | 12.6 ± 0.4 | 12.4 ± 0.4 | 12.8 ± 0.05 | 11.5 ± 0.7 | 12.7 ± 0.2 | 12.6 ± 0.1 | 11.8 ± 0.05 | 12 ± 0.9 | 10.5 ± 0.5 | 10.5 ± 0.3 | 12.2 ± 0.1 * | 14.4 ± 0.4 *** | |
miR-135a | 7 | 11 ± 0.5 | 12.7 ± 0.1 * | 12.3 ± 0.6 | 11.1 ± 0.1 | 12.4 ± 0.8 | 12.4 ± 0.4 | 11.2 ± 0.05 | 13.4 ± 0.7 *** | 12.2 ± 0.1 | 11.8 ± 0.1 | 10.4 ± 0.4 * | --- |
14 | 10.2 ± 0.1 | 10.9 ± 0.7 | 11.03 ± 0.2 | 10.6 ± 1.1 | 10.6 ± 0.6 | 10.8 ± 0.3 | 12 ± 0.1 | --- | --- | 12.2 ± 0.2 | --- | --- | |
21 | 12.1 ± 0.5 | 11.7 ± 0.3 | 12.3 ± 0.5 | 12.5 ± 0.6 | 12.2 ± 0.2 | 12.1 ± 0.4 | 10.9 ± 0.5 | 12 ± 1.04 | 12.8 ± 0.6 * | 11.8 ± 0.6 | 12.1 ± 0.5 | 11.3 ± 0.7 | |
miR-206 | 7 | 10.8 ± 0.8 | 12 ± 0.1 | 11.8 ± 1.1 | 11.4 ± 0.8 | 12 ± 0.7 | 11 ± 0.3 | 11.9 ± 0.6 | 11.9 ± 0.9 | 12.4 ± 0.2 | 11.7 ± 0.8 | 11.2 ± 0.5 | 11.6 ± 0.6 |
14 | 9.9 ± 0.2 | 12.1 ± 0.4 *** | 11.5 ± 0.6 ** | 11.7 ± 0.4 | 11.4 ± 0.4 | 12.4 ± 0.3 | 11.5 ± 0.3 | 11.1 ± 0.04 | 11.2 ± 0.4 | 11.6 ± 0.7 | 11 ± 0.8 | 11.9 ± 0.3 | |
21 | 12 ± 0.9 | 12.6 ± 0.5 | 11.3 ± 0.8 | 12.5 ± 0.7 | 11.5 ± 0.1 | 11.8 ± 0.3 | 12 ± 0.8 | 11.3 ± 0.5 | 12.3 ± 1.1 | 11.4 ± 0.4 | 11.8 ± 0.2 | 11.2 ± 0.07 | |
miR-29b | 7 | 3.1 ± 0.2 | 2.7 ± 0.2 | 2.4 ± 0.1 ** | 2.4 ± 0.05 | 3 ± 0.2 ** | 1.9 ± 0.1 * | 4.62 ± 0.02 | 4.99 ± 0.04 ** | 4.85 ± 0.1 | 4.14 ± 0.03 | 4.87 ± 0.02 *** | 3.81 ± 0.08 ** |
14 | 2.9 ± 0.2 | 2.5 ± 0.1 | 1.7 ± 0.3 *** | 1.9 ± 0.2 | 1.6 ± 0.1 | 2.1 ± 0.2 | 3 ± 0.3 | 3.4 ± 0.05 | 3.9 ± 0.2 ** | 2.7 ± 0.1 | 2.9 ± 0.1 | 3.3 ± 0.2 * | |
21 | 3.3 ± 0.08 | 2.5 ± 0.1 *** | 3.8 ± 0.2 ** | 2.6 ± 0.1 | 2.6 ± 0.1 | 3 ± 0.04 ** | 2.9 ± 0.06 | 2.7 ± 0.04 ** | 2.7 ± 0.09 * | 3 ± 0.06 | 2.9 ± 0.04 | 2.6 ± 0.07 *** | |
miR-let-7b | 7 | −1.8 ± 0.02 | −3.1 ± 0.3 *** | −3.7 ± 0.2 *** | −1.7 ± 0.1 | −4.6 ± 0.3 *** | −5 ± 0.2 *** | −1.46 ± 0.07 | −1.80 ± 0.03 | −2.03 ± 0.14 ** | −1.51 ± 0.14 | −1.8 ± 0.12 | −1.96 ± 0.06 * |
14 | −2.3 ± 2 | − 2.02 ± 0.2 | −2.3 ± 0.3 | −2.5 ± 0.1 | −2.5 ± 0.2 | −3 ± 0.3 | −1.3 ± 0.07 | −1.5 ± 0.08 | −1.6 ± 0.2 | − 2.1 ± 0.08 | − 1.7 ± 0.08 * | −1.6 ± 0.1 ** | |
21 | −2.1 ± 0.1 | −2.6 ± 0.1 *** | −1.9 ± 0.1 | −2.1 ± 0.1 | −2.2 ± 0.04 | −2.4 ± 0.08 * | −1.4 ± 0.05 | −1.7 ± 0.07 *** | −0.7 ± 0.06 *** | −1.05 ± 0.08 | −1.04 ± 0.08 | − 1.6 ± 0.05 *** |
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Mancinelli, R.; Di Filippo, E.S.; Tumedei, M.; Marrone, M.; Fontana, A.; Ettorre, V.; Giordani, S.; Baldrighi, M.; Iezzi, G.; Piattelli, A.; et al. Human Dental Pulp Stem Cell Osteogenic Differentiation Seeded on Equine Bone Block with Graphene and Melatonin. Appl. Sci. 2021, 11, 3218. https://doi.org/10.3390/app11073218
Mancinelli R, Di Filippo ES, Tumedei M, Marrone M, Fontana A, Ettorre V, Giordani S, Baldrighi M, Iezzi G, Piattelli A, et al. Human Dental Pulp Stem Cell Osteogenic Differentiation Seeded on Equine Bone Block with Graphene and Melatonin. Applied Sciences. 2021; 11(7):3218. https://doi.org/10.3390/app11073218
Chicago/Turabian StyleMancinelli, Rosa, Ester Sara Di Filippo, Margherita Tumedei, Mariangela Marrone, Antonella Fontana, Valeria Ettorre, Silvia Giordani, Michele Baldrighi, Giovanna Iezzi, Adriano Piattelli, and et al. 2021. "Human Dental Pulp Stem Cell Osteogenic Differentiation Seeded on Equine Bone Block with Graphene and Melatonin" Applied Sciences 11, no. 7: 3218. https://doi.org/10.3390/app11073218