Mutanobactin-D, a Streptococcus mutans Non-Ribosomal Cyclic Lipopeptide, Induces Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells and Human Bone Marrow Stem Cells
<p>Concentration-dependent effects of Mub-D on hDPSC and hBMSC viability. (<b>A</b>) Experimental setup of Alamar Blue (AB) reduction tests for evaluating hDPSC and hBMSC viability in cultures supplemented with Mub-D. Four different concentrations of Mub-D were tested (5, 10, 20, and 50 μM). (<b>B</b>,<b>C</b>) AB reduction assay was performed in cultures of hDPSCs (<b>B</b>) and hBMSCs (<b>C</b>) for 24 h and 48 h. Bright-field images of the different culture conditions. Plots present the percentage of AB reduction. Data are presented as average values ± standard deviation. Two-way ANOVA and Tukey post hoc were used to analyse the differences between the experimental groups for each time point. Asterisks represent statistically significant differences between experimental groups for each time point (* <span class="html-italic">p</span> value < 0.05; ** <span class="html-italic">p</span> value < 0.01). Scale bars: 20 μm.</p> "> Figure 2
<p>Effects of Mub-D on hDPSC and hBMSC viability. (<b>A</b>) Experimental setup of AB reduction test for cell viability evaluation in hDPSCs and hBMSCs after a single or double Mub-D administration within 48h of culture. Four different concentrations of Mub-D were tested (5, 10, 20, and 50 μM). (<b>B</b>) Bright-field microscopy images of hDPSCs and hBMSCs after double Mub-D administration. (<b>C</b>) AB reduction assay in hDPSC and hBMSC cultures upon Mub-D addition. (<b>D</b>) Comparison between the two time points (24 h and 48 h) following a single or double Mub-D administration in cultured hDPSCs and hBMSCs. Data are presented as average values ± standard deviation. Two-way ANOVA and Tukey post hoc were used to analyse the differences between the experimental groups for each time point. Asterisks represent statistically significant differences between experimental groups for each time point (* <span class="html-italic">p</span> value < 0.05; ** <span class="html-italic">p</span> value < 0.01). Scale bars: 20 μm.</p> "> Figure 3
<p>Cytotoxic and proliferative effects of Mub-D in hDPSCs and hBMSCs. (<b>A</b>) Experimental setup of crystal violet assays for the evaluation of the cytotoxicity of Mub-D (single or double administration) and its proliferation effects on cultured for 48 h hDPSCs and hBMSCs. Four different Mub-D concentrations were tested (5, 10, 20, and 50 μM). (<b>B</b>) Cytotoxicity calculated by crystal violet assay in hDPSC and hBMSC cultures upon Mub-D addition. (<b>C</b>) Proliferation of hDPSCs and hBMSCs quantified via crystal violet assay upon Mub-D addition. Comparison between a single or double Mub-D administration in cultured hDPSCs and hBMSCs. Data are presented as average values ± standard deviation. Asterisks represent statistically significant differences between experimental groups for each time point (* <span class="html-italic">p</span> value < 0.05).</p> "> Figure 4
<p>Effects of Mub-D on hDPSC and hBMSC proliferation. Cell proliferation visualised upon BrdU immunostaining. (<b>A</b>,<b>B</b>) Inverted fluorescence microscopy images showing BrdU-positive hDPSCs (<b>A</b>) and hBMSCs (<b>B</b>) cultured in the presence of 20 μM Mub-D in DMSO. Four time points were analysed (1, 12, 24, and 36 h). (<b>C</b>) Plots present the percentage of BrdU-positive cells in hDPSCs and hBMSCs cultures after Mub-D administration. Two concentrations of Mub-D (10 and 20 μM) were analysed. Data are presented as average values ± standard deviation. Two-way ANOVA and Tukey post hoc tests were used to compare time points for each cell type. Asterisks represent statistically significant differences between experimental groups for each time point (* <span class="html-italic">p</span> value < 0.05). Scale bar: 100 μm.</p> "> Figure 5
<p>Effects of Mub-D on hDPSC and hBMSC mineral deposition. (<b>A</b>) The experimental setup for the Alizarin red staining (ARS) assay was used to evaluate mineralisation in cultures of hDPSCs and hBMSCs supplemented with 20 μM Mub-D for 7, 14, and 21 days. (<b>B</b>,<b>C</b>) Bright-field images of ARS on cultured hDPSCs (<b>B</b>) and hBMSCs (<b>C</b>) in the presence of 20 μM Mub-D, DMSO, or odontogenic/osteogenic medium alone. The right-side plots present the mineralisation rates between hDPSCs (<b>B</b>) and hBMSCs (<b>C</b>) cultured with Mub-D and DMSO. Data are presented as average values ± standard deviation. Asterisks indicate statistically significant differences between experimental groups for each time point (* <span class="html-italic">p</span> value < 0.05; ** <span class="html-italic">p</span> value < 0.01).</p> "> Figure 6
<p>Effects of Mub-D on gene expression in hDPSCs and hBMSCs. (<b>A</b>–<b>C</b>) <span class="html-italic">CD90, OCT4</span> (<b>A</b>), <span class="html-italic">CCDN1</span> (<b>B</b>), <span class="html-italic">RUNX2</span>, and <span class="html-italic">COLIA1</span> expression in hDPSCs and hBMSCs cultured in the presence of Mub-D for 0, 7, 14, and 21 days in odontogenic/osteogenic medium. The relative expression value on comparison graphs is normalised for each gene relative to Mub-D-treated hDPSCs at culture day 0. Statistical analysis data are presented as average values ± standard deviation. Two-way ANOVA and Tukey post hoc tests were used to compare hDPSCs and hBMSCs and time points for each cell type. Asterisks represent statistically significant differences between distinctive time points and day 0 as control (* <span class="html-italic">p</span> value < 0.05; ** <span class="html-italic">p</span> value < 0.01; *** = <span class="html-italic">p</span> value < 0.001).</p> "> Figure 7
<p>Effect of Mub-D on odontogenic/osteogenic genes expression in hDPSCs and hBMSCs. (<b>A</b>,<b>B</b>) <span class="html-italic">DMP-1</span> and <span class="html-italic">DSPP</span> (<b>A</b>) and <span class="html-italic">OCN</span>, <span class="html-italic">OSX</span>, and <span class="html-italic">BMP2</span> (<b>B</b>) relative mRNA expression in hDPSCs and hBMSCs cultured in the presence of Mub-D at 0, 7, 14, and 21 days. The relative expression value on comparison graphs is normalised for each gene relative to hDPSCs treated with Mub-D at T0. Statistical analysis data are presented as average values ± standard deviation. Two-way ANOVA tests, followed by the Tukey post hoc test, were used to compare hDPSCs and hBMSCs and time points for each cell type. Asterisks represent statistically significant differences between various time points and day 0 as control (* <span class="html-italic">p</span> value < 0.05; ** <span class="html-italic">p</span> value < 0.01; *** = <span class="html-italic">p</span> value < 0.001; **** <span class="html-italic">p</span> value < 0.0001).</p> "> Figure 8
<p>Mub-D induces odontogenic/osteogenic differentiation of hDPSCs and hBMSCs. Schematic representation of the effects of Mub-D in proliferation, differentiation, and mineral deposition of hDPSCs and hBMSCs cultured in odontogenic/osteogenic conditions. The expression of various genes during these processes is indicated. Gene upregulation is indicated by green arrows, and downregulation is indicated by red arrows.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Concentration-Dependent Mub-D Effect on hDPSC and hBMSC Viability
2.2. Mub-D Effect on hDPSC and hBMSC Proliferation
2.3. Osteogenic/Odontogenic Effect of Mub-D in hDPSCs and hBMSCs
2.4. Effect of Mub-D on Gene Expression in hDPSCs and hBMSCs
3. Discussion
4. Materials and Methods
4.1. Collection and Culture of Cells
4.2. Mutanobactin-D Synthesis and Use
4.3. Cell Viability
4.4. Cell Proliferation Analyses
4.5. Cell Differentiation Assays
4.6. Mineralisation Assessed via Alizarin Red Staining
4.7. RNA Extraction, Reverse Transcription, and Quantitative Real-Time PCR (qRT-PCR)
4.8. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Accession No. | Forward Primer 5′-3′ | Reverse Primer 3′-5′ |
---|---|---|---|
GAPDH | NM_002046.5 | AGGGCTGCTTTTAACTCTGGT | CCCCACTTGATTTTGGAGGGA |
BMP2 | NM_001200.2 | ATGGATTCGTGGTGGAAGTG | GTGGAGTTCAGATGATCAGC |
CCDN1 | NM_053056.3 | GCTGCGAAGTGGAAACCATC | CCTCCTTCTGCACACATTTGAA |
CD90 | NM_006288.3 | GAAGGTCCTCTACTTATCCGCC | TGATGCCCTCACACTTGACCAG |
COL1A1 | NM_000088.1 | GATTCCCTGGACCTAAAGGTGC | AGCCTCTCCATCTTTGCCAGCA |
DSPP | NM_014208.3 | GCATCCAGGGACCAAGTAAGCA | CTTGGACAACAGCGACATCCT |
OCT4 | NM_001285986.2 | CTTTCTCAGGGGGACCAGTG | GGGACCGAGGAGTACAGTGC |
RUNX2 | NG_008020.1 | GCCAGGGTCTAGGAGTTGTT | ACCCACCACCCTATTTCCTG |
OSX | NM_152860.1 | CCTCTGCGGGACTCAACAAC | AGCCCATTAGTGCTTGTAAAGG |
DMP-1 | NM_004407.1 | GAGCAGTGAGTCATCAGAAGGC | GAGAAGCCACCAGCTAGCCTAT |
OCN | NM_199173 | CGCTACCTGTATCAATGGCTGG | CTCCTGAAAGCCGATGTGGTCA |
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Nikolic, S.; Alastra, G.; Pultar, F.; Lüthy, L.; Stadlinger, B.; Carreira, E.M.; Bugueno, I.M.; Mitsiadis, T.A. Mutanobactin-D, a Streptococcus mutans Non-Ribosomal Cyclic Lipopeptide, Induces Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells and Human Bone Marrow Stem Cells. Int. J. Mol. Sci. 2025, 26, 1144. https://doi.org/10.3390/ijms26031144
Nikolic S, Alastra G, Pultar F, Lüthy L, Stadlinger B, Carreira EM, Bugueno IM, Mitsiadis TA. Mutanobactin-D, a Streptococcus mutans Non-Ribosomal Cyclic Lipopeptide, Induces Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells and Human Bone Marrow Stem Cells. International Journal of Molecular Sciences. 2025; 26(3):1144. https://doi.org/10.3390/ijms26031144
Chicago/Turabian StyleNikolic, Sandra, Giuseppe Alastra, Felix Pultar, Lukas Lüthy, Bernd Stadlinger, Erick M. Carreira, Isaac Maximiliano Bugueno, and Thimios A. Mitsiadis. 2025. "Mutanobactin-D, a Streptococcus mutans Non-Ribosomal Cyclic Lipopeptide, Induces Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells and Human Bone Marrow Stem Cells" International Journal of Molecular Sciences 26, no. 3: 1144. https://doi.org/10.3390/ijms26031144
APA StyleNikolic, S., Alastra, G., Pultar, F., Lüthy, L., Stadlinger, B., Carreira, E. M., Bugueno, I. M., & Mitsiadis, T. A. (2025). Mutanobactin-D, a Streptococcus mutans Non-Ribosomal Cyclic Lipopeptide, Induces Osteogenic/Odontogenic Differentiation of Human Dental Pulp Stem Cells and Human Bone Marrow Stem Cells. International Journal of Molecular Sciences, 26(3), 1144. https://doi.org/10.3390/ijms26031144