microRNA-200c Mitigates Pulpitis and Promotes Dentin Regeneration

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General comments :

1. MicroRNA-200c (miR-200c) was downregulated in inflamed pulp tissues and primary human dental pulp-derived cells (DPCs). Overexpression of miR-200c by transfecting plasmid DNA (pDNA) in human DPCs decreased proinflammatory cytokines IL-6 and IL-8 and enhanced odontogenic differentiation markers. It was concluded that miR-200c can modulate pulpal inflammation and facilitate dentin repair. The overall findings appear preliminary due to the difficulty to assess the reliability of the findings. Further details are indicated below in III), IV), V), and VI).
2. The ethical information concerning patients was provided. However, ethical information associated to the rat experiments was lacking.

• The number of healthy patients and that of and patients with pulpitis was not indicated to support significant statistical significance. The number of rats (n=3) appears to be a bit too low to assess the reliability of the statistical significance.

1. The number of independent measurements associated to cellular investigations was not reported anywhere (Fig .1D-F, Fig 2A-D, Fig 3A-D, Fig 4A-D). I can’t assess the reliability of the findings.
2. Full western blot shall be shown instead of fragmented ones (Fig. 2C, and Fig. 4C). Quantitative values of proteins shall be added to support the western blot findings.
3. While qualitative histological staining is valuable (Fig 5 and 6), quantitative determination of specific immunological characteristics are preferred in most Journals. The number of independent sets of immunological staining was not provided to support the significance of the findings.

Minor comments:

• Abstract, p1: Specify IL abbreviation in “Through the overexpression of miR-200c via transfecting plasmid DNA (pDNA), we observed a substantial downregulation of proinflammatory cytokines IL-6 and IL-8 in human DPCs.”
• Abstract, p1: Specify Runx2, OCN, DMP1, and DSPP abbreviations in “Furthermore, this overexpression significantly enhanced the transcript and protein levels of odontogenic differentiation markers, including Runx2, OCN, DMP1, and DSPP.”
• Introduction, p1: Add references to support “A pulpectomy is the traditional therapy of removing the pulp tissue to eliminate pulpal infection, inflammation, and pain.”
• Introduction, p1-2: Add references to support “However, patients cannot perceive pain in the tooth after a pulpectomy procedure, and as a result, the tooth becomes fragile and prone to root fracture due to a lack of resistance to infection and disease.”
• Introduction, p2: Justify, and add references to support “The success rate of calcium silicate-based material for partial and pulpotomy, including mineral trioxide aggregate (MTA), the most advanced material in VPT, remains similar to currently used calcium hydroxide.”
• Introduction, p2: Unclear which types of modifications, explain and add references to support “Modification of let-7c-5p, miR-146, -497-5p, -143-3p, - 508-5p, -675, and -34a have been reported to promote odontogenic differentiation of DPSCs in vitro.”
• Introduction, p2: Specify abbreviation Wnt/BMP in “Notably, miR-200c plays a crucial role in dentin development of Wnt/BMP signaling by targeting noggin [13].”
• Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, Title, p2: Replace hDPCs by its full name in the Title.
• Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, p3:It was unclear if N=11 corresponds to independent measurements or replicates in Fig 1A,B legend to support “Total RNA was extracted from the pulp tissues and quantitatively analyzed using qRT-PCR. The transcripts of IL-6 and IL-8 increased intensely in inflamed tissues relative to healthy pulps (Fig. 1A, B).”
• Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 1D, E legend to support “After hDPCs were treated with P. gingivalis lipopolysaccharide (Pg-LPS) at 0.1 and 1µg/ml to induce inflammation, the transcripts of IL-6 and IL-8 were significantly increased (Fig.1D, E).”
• Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 1F legend to support “Notably, miR-200c expression was significantly reduced in inflamed hDPCs 92 (Fig.1F).”
• Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, Title, p3: Replace DPSCs and hDPCs by their full name in the Title.
• Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2A legend to support “The increased miR-200c expression was confirmed in a dose- dependent manner (Fig.2A).”
• Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2B legend to support “miR-200c treatment at 0.1 and 0.3 µg significantly increased the transcripts of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and DMP1 compared to untreated control and treatment with empty vector (EV) after seven days (Fig.2B).”
• Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Replace fragmented western blot by full length western blot in Fig 2C, and quantify relative amount of proteins to support “These odontogenic differentiation markers were also improved after 14 days, as confirmed by western blots (Fig.2C).”
• Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2D legend to support “However, pre- treatment with pDNA miR-200c significantly downregulated IL-6 and IL-8 transcripts (Fig. 2D)”
• Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, Title, p4: Replace DPSCs by its full name in the Title.
• Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3A legend to support “As illustrated in Figure 3, the diameters of CaCO3/ miR- 200c nanocomplexes at CaCO3 : protamine sulfate (PS) of 1:0.25 were 185.59 ± 27.2 nm, and their Zeta potential was +19.3 (Fig.3A).”
• Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3B legend to support “Interestingly, the CaCO3:PS ratio impacts transfection efficiencies of pDNA encoding miR-200c, and the expression of miR-200c was upreg- ulated as PS concentrations increased. CaCO3:PS at ratios of 1:0.25 and 1:0.5 showed the best transfection efficiencies (Fig.3B).”
• Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3C legend to support “PEI and CaCO3 significantly increased the miR-200c expression than naked pDNA miR-200c. However, CaCO3 transfected significantly higher amounts of miR-200c than did PEI (Fig.3C).”
• Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3D legend to support “Notably, the delivery using PEI upregulated IL-6 transcripts; however, the delivery using CaCO3-based nanoparticles has significantly lower IL-6 expression (Fig. 3D).”
• Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, Title, p5: Replace DPSPs by its full name in the Title.
• Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, p5: Indicate number of independent measurements in Fig.4A-B legend to support “As illustrated in Figure 4, transfection of pDNA miR-200c using CaCO3 significantly increased transcripts of DMP1 (Fig.4A) and dentin sialophosphoprotein (DSPP) (Fig.4B).”
• Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, p5: Replace fragmented western blot by full length western blot in Fig 4C, and quantify relative amount of proteins to support “The western blot showed that the band intensities of DSPP and DMP1 were increased in DPSCs treated with miR-200c at different concentrations (Fig.4C).”
• Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, p6: Fig 4C-D legend was confusing. Rephrase “C-D: western blot of DMP1 and DSPP (C) and Alizarin Red staining (D) of DPSCs with different treatments of CaCO3/miR-200c.”
• Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, Fig. 4D, p6: Specify NA, NA+OS, abbreviations in Fig 4D.
• Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: It is unclear. Explain Fig5A-B in “A modified rat model of pulpitis was used to determine the functions of miR-200c in pulp inflammation and dentin regeneration[36, 37](Fig. 5A-B).”
• Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5D to support “Compared to healthy dental pulp tissues (Fig. 5C), the typical inflammation in rat pulpal tissues challenged with Pg-LPS was observed in a rat model while treated with EV after seven days, presenting a greater density of inflammatory infiltrates in the connective tissue underneath the restorative material (blue arrowed) (Fig. 5D).”
• Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5E to support “However, the miR-200c treatment effectively re- duced inflammatory cell infiltration (Fig.5E),”
• Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5E to support “Additionally, the IHC staining using an anti-IL-6 antibody indicated that treatment with miR-200c effectively reduced IL-6 (stained in brown color) in the pulpal tissues induced by Pg-LPS more so than EV (Fig. 5F,G)”
• Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p7: Add quantitative histological features in Fig 6A, B to support “After 3 weeks, we found new tissue formation con- sistent with reparative dentin adjacent to collagen with CaCO3/miR-200c (Fig. 6A, B).”
• Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p7: Add quantitative histological features in Fig 6C, D to support “However, the treatment with CaCO3/EV has limited dentin formation (Fig.6C, D).”
• Discussion, p8: Move into the introduction “While mild inflammation enhances odontogenic differentiation and dentin repair, excessive inflammation worsens pulpitis recovery[38, 39].”
• Discussion, p8: Add references, and move into the introduction “Upon binding to Toll-like receptors, NOD-like receptors, and inflammasomes in pulp cells, bacterial ligands activate proinflammatory signaling pathways, leading to amplified secretion of extracellular cytokines, chemokines, and inflammatory molecular mediators.”
• Discussion, p8: Move into the introduction “Consequently, these cytokines and mediators recruit and activate immune system cells, releasing reactive oxygen species and potent enzyme proteases, ultimately causing substantial pulp cell death and tissue damage[40].”
• Discussion, p8: Move into the introduction “Therefore, mitigating inflammation is imperative to protect the pulp- dentin complex from irreversible damage and foster odontogenic differentiation[3, 36].”
• Discussion, p8: The number of patients was unspecified, which makes the statement unconclusive. Rephrase “Our investigation revealed the downregulation of miR-200c in irreversible pulpitis patient pulp tissues and inflamed human pulp cells.”
• Discussion, p9: Add references to support “This contrasts with the potential for supraphysiological levels of mature miRNAs induced by mimics, which may lead to non- specific changes in gene expression.”
• Discussion, p9: Add references to support “Regarding potential mechanism(s) associated with miR-200c in dentin regeneration and pulpitis mitigation, prior studies have demonstrated that miR-200c exhibits the re- markable ability to target and inhibit the BMP antagonist noggin, a crucial factor known for its role in upregulating endogenous BMP signaling activities in dentin development.”
• Discussion, p9: It was not demonstrated in this work. Delete “Moreover, miR-200c can effectively enhance Wnt/β-catenin activities by targeting Sox2 and Klf4 in osteogenic differentiation.”
• Discussion, p9: It was not demonstrated in this work. Delete “This is noteworthy as these pathways align with the signaling mechanisms involved in odontogenic differentiation and dentin regenera- tion. Consequently, the existing robust evidence strongly suggests that miR-200c promotes odontogenic differentiation and dentin regeneration, potentially by upregulating BMP and Wnt signaling through the targeted inhibition of noggin, Sox2, and KLF4.”
• Discussion, p9: Add references to support “Earlier studies have also illustrated the effectiveness of miR-200c in mitigating inflammation by targeting IL-6 and IL-8 and down- regulating NF-κB signaling, elucidating the mechanism(s) underlying the overexpression of miR-200c in inhibiting inflammation in pulpitis.”
• Materials and Methods, Characterizing miR-200c and Proinflammatory Cytokines Within Inflamed Human Pulp Tissues and DPCs, Title, p9: Replace DPCs by its full name in the title.
• Materials and Methods, Characterizing miR-200c and Proinflammatory Cytokines Within Inflamed Human Pulp Tissues and DPCs, p10:Indicate number of patients in “The inclusion criteria are patients who are older than 14 years old and have American Association of Anesthesiologist status I or II:”
• Materials and Methods, Characterizing miR-200c and Proinflammatory Cytokines Within Inflamed Human Pulp Tissues and DPCs, p10:Indicate number of healthy patients in “For healthy controls, the pulp was harvested from healthy third molars with complete root formation or teeth extracted for orthodontic purposes.”
• Materials and Methods, Characterizing miR-200c and Proinflammatory Cytokines Within Inflamed Human Pulp Tissues and DPCs, p10: Specify abbreviation qRT-PCR in “Transcripts of proinflamma- tory cytokines, including IL-6 and IL-8, and miR-200c from the patient pulp tissues were quantified using qRT-PCR and compared to healthy controls.”
• Materials and Methods, Determining Odontogenic Differentiation Enhancement and Anti-Inflammation of miR- 200c Overexpression in Human DPSCs and DPCs, Title, p10: Replace DPSCs and DPCs by their full name in the Title.
• Materials and Methods, Determining Odontogenic Differentiation Enhancement and Anti-Inflammation of miR- 200c Overexpression in Human DPSCs and DPCs, p10: Add composition of the medium, and indicate time of incubationin “Human DPSCs were purchased (PT5025, Lonza, Walkersville, MD) and cultured with the DPSC BulletKit TM Medium.”
• Materials and Methods, Determining the Functions of pDNA miR-200c Delivered by CaCO3-Based Nanoparticles on Odontogenic Differentiation and Anti-Inflammation in Human DPSCs, Title, p10: Replace DPSCs by its full name in the Title.
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Add ethical information to support “All in vivo animal experiments were performed under the approval of the Office of Animal Resources at our university, and all animal surgeries were performed under sterile conditions.”
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Add references to support “A rat model of pulpitis was created by local application of Pg-LPS according to previous studies.”
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Indicate number of rats in “Briefly, after anesthesia of eight-weeks-old Sprague-Dawley (SD) pathogen-free rats (Charles River Laboratories, Wilmington, MA), Class I cavities of 0.5 mm depth were prepared on the mesial pit of occlusal surfaces on maxillary first molars using ¼ round carbide burs.”
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Change PBS buffer by another one since it is osteogenic in “To determine the pulpal inflammation induced by Pg-LPS and the anti-inflammatory function of pDNA miR-200c, after 10 µg Pg-LPS suspended in 1µl PBS was added to the exposed pulps, a collagen sponge (1 × 1 × 1 mm3) incorporated with either naked EV (1 µg) or pDNA miR-200c (1 µg) were implanted.”
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Specify abbreviation EV in “To determine the pulpal inflammation induced by Pg-LPS and the anti-inflamma- tory function of pDNA miR-200c, after 10 µg Pg-LPS suspended in 1µl PBS was added to the exposed pulps, a collagen sponge (1 × 1 × 1 mm3) incorporated with either naked EV (1 µg) or pDNA miR-200c (1 µg) were implanted.”
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Add more information how histology was performed to support “The anti-inflammatory function of miR-200c in pulp inflammation was determined after seven days using histological examination.”
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Specify treatments in “There were 3 rats for each treatment.”
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Consider to increase the number of rats to obtain valid statistical significances in “There were 3 rats for each treatment.”
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Add more information to support “To determine if miR-200c promotes dentinogenesis, CaCO3/miR-200c nanocomplexes with CaCO3:PS ratio of 1:0.25 were prepared and subsequently incorporated into trimmed collagen sponges and lyophilized.”
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Explain how histology was performed to support “Histological assessment of dentin repair with different treatments was performed after three weeks. ”
• Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Consider to increase the number of rats to obtain valid statistical significances in “There were 3 rats for each treatment.”
• Materials and Methods, qRT-PCR, Title, p11: Replace qRT-PCR by its full name in the Title.
• Materials and Methods, qRT-PCR, p11: Add more information how total RNA was extracted and indicate from which samples in“Total RNA was extracted using Qiagen miRNeasy microKit (Qiagen, Hilden, Germany).”
• Materials and Methods, qRT-PCR, p11: Replicates from one sample are considered as a one independent measurement. Perform at least three independent measurements in “Comparative real- time PCR was performed in replicate, and relative expression was obtained using the comparative Ct (ΔΔCt) method.”
• Materials and Methods, Statistical Analysis, p12: Indicate number of independent measurements to support “All statistical tests completed for the in vitro and in vivo quantifications used a significance level of 0.05, and each graphic depicts mean values and associated standard deviations.”

Author Response

Reviewer 1

Comment 1: MicroRNA-200c (miR-200c) was downregulated in inflamed pulp tissues and primary human dental pulp-derived cells (DPCs). Overexpression of miR-200c by transfecting plasmid DNA (pDNA) in human DPCs decreased proinflammatory cytokines IL-6 and IL-8 and enhanced odontogenic differentiation markers. It was concluded that miR-200c can modulate pulpal inflammation and facilitate dentin repair. The overall findings appear preliminary due to the difficulty to assess the reliability of the findings.

RE: We appreciated the reviewer’s positive comments and valuable suggestions. We have revised the manuscript based on the comments.

 

Comment 2: The ethical information concerning patients was provided. However, ethical information associated to the rat experiments was lacking.

RE: We have added information of the animal protocol in the revised manuscript.

Comment 3: The number of healthy patients and that of and patients with pulpitis was not indicated to support significant statistical significance.

RE: We have added patient sample size for each measurement in the manuscript.

Comment 4: The number of rats (n=3) appears to be a bit too low to assess the reliability of the statistical significance.

RE: We appreciate the reviewer's comments and agree with their assessment. We recognize the limitations of the project mentioned. However, combining qualitative analysis of in vivo animal models with solid quantitative measurements from human samples and in vitro studies can provide reliable outcomes, as stated in the paper. We have also addressed these limitations in the discussion section.

 

The number of independent measurements associated to cellular investigations was not reported anywhere (Fig .1D-F, Fig 2A-D, Fig 3A-D, Fig 4A-D). I can’t assess the reliability of the findings.

RE: We have added the number of measurements in the caption section of each figure.

Full western blot shall be shown instead of fragmented ones (Fig. 2C, and Fig. 4C). Quantitative values of proteins shall be added to support the western blot findings.

RE: We appreciate the reviewer's suggestion and have included the full blot images along with quantitative measurements in the supplementary data.

While qualitative histological staining is valuable (Fig 5 and 6), quantitative determination of specific immunological characteristics are preferred in most Journals. The number of independent sets of immunological staining was not provided to support the significance of the findings.

RE : We understand the reviewer’s concern and suggestion. See above

 

Minor comments:

 

Abstract, p1: Specify IL abbreviation in “Through the overexpression of miR-200c via transfecting plasmid DNA (pDNA), we observed a substantial downregulation of proinflammatory cytokines IL-6 and IL-8 in human DPCs.”

Corrected.

Abstract, p1: Specify Runx2, OCN, DMP1, and DSPP abbreviations in “Furthermore, this overexpression significantly enhanced the transcript and protein levels of odontogenic differentiation markers, including Runx2, OCN, DMP1, and DSPP.”

Corrected.

Introduction, p1: Add references to support “A pulpectomy is the traditional therapy of removing the pulp tissue to eliminate pulpal infection, inflammation, and pain.”

RE: Added

Introduction, p1-2: Add references to support “However, patients cannot perceive pain in the tooth after a pulpectomy procedure, and as a result, the tooth becomes fragile and prone to root fracture due to a lack of resistance to infection and disease.”

Added

Introduction, p2: Justify, and add references to support “The success rate of calcium silicate-based material for partial and pulpotomy, including mineral trioxide aggregate (MTA), the most advanced material in VPT, remains similar to currently used calcium hydroxide.”

Added

Introduction, p2: Unclear which types of modifications, explain and add references to support “Modification of let-7c-5p, miR-146, -497-5p, -143-3p, - 508-5p, -675, and -34a have been reported to promote odontogenic differentiation of DPSCs in vitro.”

Detailed of modification has been added.

Introduction, p2: Specify abbreviation Wnt/BMP in “Notably, miR-200c plays a crucial role in dentin development of Wnt/BMP signaling by targeting noggin [13].”

RE: Added

Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, Title, p2: Replace hDPCs by its full name in the Title.

Corrected

Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, p3:It was unclear if N=11 corresponds to independent measurements or replicates in Fig 1A,B legend to support “Total RNA was extracted from the pulp tissues and quantitatively analyzed using qRT-PCR. The transcripts of IL-6 and IL-8 increased intensely in inflamed tissues relative to healthy pulps (Fig. 1A, B).”

Information has been added.

Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 1D, E legend to support “After hDPCs were treated with P. gingivalis lipopolysaccharide (Pg-LPS) at 0.1 and 1µg/ml to induce inflammation, the transcripts of IL-6 and IL-8 were significantly increased (Fig.1D, E).”

 Added

Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 1F legend to support “Notably, miR-200c expression was significantly reduced in inflamed hDPCs 92 (Fig.1F).”

Added

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, Title, p3: Replace DPSCs and hDPCs by their full name in the Title.

Corrected

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2A legend to support “The increased miR-200c expression was confirmed in a dose- dependent manner (Fig.2A).”

Corrected

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2B legend to support “miR-200c treatment at 0.1 and 0.3 µg significantly increased the transcripts of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and DMP1 compared to untreated control and treatment with empty vector (EV) after seven days (Fig.2B).”

RE Information has been added.

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Replace fragmented western blot by full length western blot in Fig 2C, and quantify relative amount of proteins to support “These odontogenic differentiation markers were also improved after 14 days, as confirmed by western blots (Fig.2C).”

Information has been added.

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2D legend to support “However, pre- treatment with pDNA miR-200c significantly downregulated IL-6 and IL-8 transcripts (Fig. 2D)”

Addressed.

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, Title, p4: Replace DPSCs by its full name in the Title.

Corrected

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3A legend to support “As illustrated in Figure 3, the diameters of CaCO3/ miR- 200c nanocomplexes at CaCO3 : protamine sulfate (PS) of 1:0.25 were 185.59 ± 27.2 nm, and their Zeta potential was +19.3 (Fig.3A).”

Added

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3B legend to support “Interestingly, the CaCO3:PS ratio impacts transfection efficiencies of pDNA encoding miR-200c, and the expression of miR-200c was upreg- ulated as PS concentrations increased. CaCO3:PS at ratios of 1:0.25 and 1:0.5 showed the best transfection efficiencies (Fig.3B).”

Added

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3C legend to support “PEI and CaCO3 significantly increased the miR-200c expression than naked pDNA miR-200c. However, CaCO3 transfected significantly higher amounts of miR-200c than did PEI (Fig.3C).”

RE: Added

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3D legend to support “Notably, the delivery using PEI upregulated IL-6 transcripts; however, the delivery using CaCO3-based nanoparticles has significantly lower IL-6 expression (Fig. 3D).”

 RE: Added

Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, Title, p5: Replace DPSPs by its full name in the Title.

Corrected

Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, p5: Indicate number of independent measurements in Fig.4A-B legend to support “As illustrated in Figure 4, transfection of pDNA miR-200c using CaCO3 significantly increased transcripts of DMP1 (Fig.4A) and dentin sialophosphoprotein (DSPP) (Fig.4B).” Corrected.

Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, p5: Replace fragmented western blot by full length western blot in Fig 4C, and quantify relative amount of proteins to support “The western blot showed that the band intensities of DSPP and DMP1 were increased in DPSCs treated with miR-200c at different concentrations (Fig.4C).”

RE: Added in the supplementary data

Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, p6: Fig 4C-D legend was confusing. Rephrase “C-D: western blot of DMP1 and DSPP (C) and Alizarin Red staining (D) of DPSCs with different treatments of CaCO3/miR-200c.”

The legend has been rewritten.

Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, Fig. 4D, p6: Specify NA, NA+OS, abbreviations in Fig 4D.

The legend has been rewritten.

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: It is unclear. Explain Fig5A-B in “A modified rat model of pulpitis was used to determine the functions of miR-200c in pulp inflammation and dentin regeneration[36, 37](Fig. 5A-B).”

Rewritten

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5D to support “Compared to healthy dental pulp tissues (Fig. 5C), the typical inflammation in rat pulpal tissues challenged with Pg-LPS was observed in a rat model while treated with EV after seven days, presenting a greater density of inflammatory infiltrates in the connective tissue underneath the restorative material (blue arrowed) (Fig. 5D).”

RE: We have addressed the issue in the manuscript. See the response above.

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5E to support “However, the miR-200c treatment effectively re- duced inflammatory cell infiltration (Fig.5E),”

We have addressed the issue in the manuscript.

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5E to support “Additionally, the IHC staining using an anti-IL-6 antibody indicated that treatment with miR-200c effectively reduced IL-6 (stained in brown color) in the pulpal tissues induced by Pg-LPS more so than EV (Fig. 5F,G)”

We have addressed the issue in the discussion section of the manuscript.

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p7: Add quantitative histological features in Fig 6A, B to support “After 3 weeks, we found new tissue formation con- sistent with reparative dentin adjacent to collagen with CaCO3/miR-200c (Fig. 6A, B).”

 RE: We have addressed the issue in the manuscript.

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p7: Add quantitative histological features in Fig 6C, D to support “However, the treatment with CaCO3/EV has limited dentin formation (Fig.6C, D).”

We have addressed the issue in the manuscript.

Discussion, p8: Move into the introduction “While mild inflammation enhances odontogenic differentiation and dentin repair, excessive inflammation worsens pulpitis recovery[38, 39].”

Corrected.

Discussion, p8: Add references, and move into the introduction “Upon binding to Toll-like receptors, NOD-like receptors, and inflammasomes in pulp cells, bacterial ligands activate proinflammatory signaling pathways, leading to amplified secretion of extracellular cytokines, chemokines, and inflammatory molecular mediators.”

Corrected as suggested.

Discussion, p8: Move into the introduction “Consequently, these cytokines and mediators recruit and activate immune system cells, releasing reactive oxygen species and potent enzyme proteases, ultimately causing substantial pulp cell death and tissue damage[40].”

RE: Corrected as suggested.

Discussion, p8: Move into the introduction “Therefore, mitigating inflammation is imperative to protect the pulp- dentin complex from irreversible damage and foster odontogenic differentiation[3, 36].”

Corrected as suggested.

Discussion, p8: The number of patients was unspecified, which makes the statement unconclusive. Rephrase “Our investigation revealed the downregulation of miR-200c in irreversible pulpitis patient pulp tissues and inflamed human pulp cells.”

Corrected.

Discussion, p9: Add references to support “This contrasts with the potential for supraphysiological levels of mature miRNAs induced by mimics, which may lead to non- specific changes in gene expression.”

Added as suggested.

Discussion, p9: Add references to support “Regarding potential mechanism(s) associated with miR-200c in dentin regeneration and pulpitis mitigation, prior studies have demonstrated that miR-200c exhibits the re- markable ability to target and inhibit the BMP antagonist noggin, a crucial factor known for its role in upregulating endogenous BMP signaling activities in dentin development.”

RE: Added as suggested.

 

Discussion, p9: It was not demonstrated in this work. Delete “Moreover, miR-200c can effectively enhance Wnt/β-catenin activities by targeting Sox2 and Klf4 in osteogenic differentiation.”

Addressed

Discussion, p9: It was not demonstrated in this work. Delete “This is noteworthy as these pathways align with the signaling mechanisms involved in odontogenic differentiation and dentin regenera- tion. Consequently, the existing robust evidence strongly suggests that miR-200c promotes odontogenic differentiation and dentin regeneration, potentially by upregulating BMP and Wnt signaling through the targeted inhibition of noggin, Sox2, and KLF4.”

 Addressed

 

Discussion, p9: Add references to support “Earlier studies have also illustrated the effectiveness of miR-200c in mitigating inflammation by targeting IL-6 and IL-8 and down- regulating NF-κB signaling, elucidating the mechanism(s) underlying the overexpression of miR-200c in inhibiting inflammation in pulpitis.”

 RE: Added

Materials and Methods, Characterizing miR-200c and Proinflammatory Cytokines Within Inflamed Human Pulp Tissues and DPCs, Title, p9: Replace DPCs by its full name in the title.

Corrected

Materials and Methods, Characterizing miR-200c and Proinflammatory Cytokines Within Inflamed Human Pulp Tissues and DPCs, p10:Indicate number of patients in “The inclusion criteria are patients who are older than 14 years old and have American Association of Anesthesiologist status I or II:”

Corrected

 

Materials and Methods, Characterizing miR-200c and Proinflammatory Cytokines Within Inflamed Human Pulp Tissues and DPCs, p10:Indicate number of healthy patients in “For healthy controls, the pulp was harvested from healthy third molars with complete root formation or teeth extracted for orthodontic purposes.”

RE: Corrected

Materials and Methods, Characterizing miR-200c and Proinflammatory Cytokines Within Inflamed Human Pulp Tissues and DPCs, p10: Specify abbreviation qRT-PCR in “Transcripts of proinflamma- tory cytokines, including IL-6 and IL-8, and miR-200c from the patient pulp tissues were quantified using qRT-PCR and compared to healthy controls.” . Corrected

Materials and Methods, Determining Odontogenic Differentiation Enhancement and Anti-Inflammation of miR- 200c Overexpression in Human DPSCs and DPCs, Title, p10: Replace DPSCs and DPCs by their full name in the Title.

Corrected

Materials and Methods, Determining Odontogenic Differentiation Enhancement and Anti-Inflammation of miR- 200c Overexpression in Human DPSCs and DPCs, p10: Add composition of the medium, and indicate time of incubationin “Human DPSCs were purchased (PT5025, Lonza, Walkersville, MD) and cultured with the DPSC BulletKit TM Medium.”

 Corrected

Materials and Methods, Determining the Functions of pDNA miR-200c Delivered by CaCO3-Based Nanoparticles on Odontogenic Differentiation and Anti-Inflammation in Human DPSCs, Title, p10: Replace DPSCs by its full name in the Title.

Corrected

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Add ethical information to support “All in vivo animal experiments were performed under the approval of the Office of Animal Resources at our university, and all animal surgeries were performed under sterile conditions.”

Information added

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Add references to support “A rat model of pulpitis was created by local application of Pg-LPS according to previous studies.”

 Corrected

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Indicate number of rats in “Briefly, after anesthesia of eight-weeks-old Sprague-Dawley (SD) pathogen-free rats (Charles River Laboratories, Wilmington, MA), Class I cavities of 0.5 mm depth were prepared on the mesial pit of occlusal surfaces on maxillary first molars using ¼ round carbide burs.”

Corrected

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Change PBS buffer by another one since it is osteogenic in “To determine the pulpal inflammation induced by Pg-LPS and the anti-inflammatory function of pDNA miR-200c, after 10 µg Pg-LPS suspended in 1µl PBS was added to the exposed pulps, a collagen sponge (1 × 1 × 1 mm3) incorporated with either naked EV (1 µg) or pDNA miR-200c (1 µg) were implanted.”

Corrected

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Specify abbreviation EV in “To determine the pulpal inflammation induced by Pg-LPS and the anti-inflamma- tory function of pDNA miR-200c, after 10 µg Pg-LPS suspended in 1µl PBS was added to the exposed pulps, a collagen sponge (1 × 1 × 1 mm3) incorporated with either naked EV (1 µg) or pDNA miR-200c (1 µg) were implanted.” . Corrected

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Add more information how histology was performed to support “The anti-inflammatory function of miR-200c in pulp inflammation was determined after seven days using histological examination.”

Corrected

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Specify treatments in “There were 3 rats for each treatment.”

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Consider to increase the number of rats to obtain valid statistical significances in “There were 3 rats for each treatment.”

We have addressed in the discussion section

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Add more information to support “To determine if miR-200c promotes dentinogenesis, CaCO3/miR-200c nanocomplexes with CaCO3:PS ratio of 1:0.25 were prepared and subsequently incorporated into trimmed collagen sponges and lyophilized.”

RE: We added the reference.

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Explain how histology was performed to support “Histological assessment of dentin repair with different treatments was performed after three weeks. ”

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Consider to increase the number of rats to obtain valid statistical significances in “There were 3 rats for each treatment.”

 We have addressed in the discussion.

Materials and Methods, qRT-PCR, Title, p11: Replace qRT-PCR by its full name in the Title.

Corrected.

Materials and Methods, qRT-PCR, p11: Add more information how total RNA was extracted and indicate from which samples in“Total RNA was extracted using Qiagen miRNeasy microKit (Qiagen, Hilden, Germany).”

Corrected.

Materials and Methods, qRT-PCR, p11: Replicates from one sample are considered as a one independent measurement. Perform at least three independent measurements in “Comparative real- time PCR was performed in replicate, and relative expression was obtained using the comparative Ct (ΔΔCt) method.”

Materials and Methods, Statistical Analysis, p12: Indicate number of independent measurements to support “All statistical tests completed for the in vitro and in vivo quantifications used a significance level of 0.05, and each graphic depicts mean values and associated standard deviations.”

RE: We have addressed in the discussion

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The manuscript provides investigations into the potential therapeutic of microRNA-200c (miR-200c) in vital pulp therapy (VPT). The findings suggest miR-200c could improve outcomes in VPT by both mitigating pulp inflammation and stimulating dentin repair. This is a relevant area of research, particularly considering the increasing interest in miRNAs as potential therapeutic strategies in various diseases. However, I’ve some considerations and I’m going to take account again this manuscript after minor revision.

 

1. The introduction is well written, presenting an informative background on pulpitis, its pathophysiology, and the consequences. The introduction spends a considerable amount of time on miRNAs by discussing their potential therapeutic strategies for pulpitis. However, the introduction should provide background and context, define the problem or knowledge gap, summarize relevant literature, state the study's objectives or hypothesis, but the results belong in a separate section and should not appear in the introduction. So, I suggest eliminating lines 76-82, and extending the objectives.

 

2. Results are well established and described. Figures well sustain the result excepting for western blots. I suggest improving these images from western blots analyses.

 

 

3. Overall, the materials and methods section present a robust and well-structured experimental framework. The thoroughness of the study's design, along with appropriate statistical analysis, strengthens the validity of the findings.

 

4. Discussion and conclusion: I suggest introducing the limits of this work and the clinical relevance.

 

Overall, this paper has a good quality. It has an interesting subject considering that the management of pulpitis is critical. The manuscript is well written in each part and easily accessible for readers. The choice of experiments was in line with the purposes, and the data are supported by a good statistical analysis. The results are supported by experimental techniques, by clear images. It’s my opinion this manuscript is suitable for the publication after a minor revision.

 

I look forward to receiving the revised manuscript.

Sincerely,

Author Response

Reviewer 2

The manuscript provides investigations into the potential therapeutic of microRNA-200c (miR-200c) in vital pulp therapy (VPT). The findings suggest miR-200c could improve outcomes in VPT by both mitigating pulp inflammation and stimulating dentin repair. This is a relevant area of research, particularly considering the increasing interest in miRNAs as potential therapeutic strategies in various diseases. However, I’ve some considerations and I’m going to take account again this manuscript after minor revision.

 

1. The introduction is well written, presenting an informative background on pulpitis, its pathophysiology, and the consequences. The introduction spends a considerable amount of time on miRNAs by discussing their potential therapeutic strategies for pulpitis. However, the introduction should provide background and context, define the problem or knowledge gap, summarize relevant literature, state the study's objectives or hypothesis, but the results belong in a separate section and should not appear in the introduction. So, I suggest eliminating lines 76-82, and extending the objectives.

 RE: We really appreciate the reviewer’s positive comments and valuable suggestions. We have revised the introduction section based on the comments.

 

1. Results are well established and described. Figures well sustain the result excepting for western blots. I suggest improving these images from western blots analyses.

RE: We appreciate the reviewer’s comments. We have included quantitative measurement in the supplementary data.

 

 

1. Overall, the materials and methods section present a robust and well-structured experimental framework. The thoroughness of the study's design, along with appropriate statistical analysis, strengthens the validity of the findings.

RE Thank you

1. Discussion and conclusion: I suggest introducing the limits of this work and the clinical relevance.

 RE: we totally agree with the reviewer. We have included a limitation of this work in the discussion section.

Overall, this paper has a good quality. It has an interesting subject considering that the management of pulpitis is critical. The manuscript is well written in each part and easily accessible for readers. The choice of experiments was in line with the purposes, and the data are supported by a good statistical analysis. The results are supported by experimental techniques, by clear images. It’s my opinion this manuscript is suitable for the publication after a minor revision.

RE: Thank you.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Overall comments:

Most of my concerns remain unaddressed.

 

Reviewer 1

Comment1: MicroRNA-200c (miR-200c) was downregulated in inflamed pulp tissues and primary human dental pulp-derived cells (DPCs). Overexpression of miR-200c by transfecting plasmid DNA (pDNA) in human DPCs decreased proinflammatory cytokines IL-6 and IL-8 and enhanced odontogenic differentiation markers. It was concluded that miR-200c can modulate pulpal inflammation and facilitate dentin repair. The overall findings appear preliminary due to the difficulty to assess the reliability of the findings.

RE: We appreciated the reviewer’s positive comments and valuable suggestions. We have

revised the manuscript based on the comments.

Reviewer’s answer: My concerns concerning the reproducibility of animal and cellular findings were not addressed. The only statistically significant finding is centered on the analysis of human pulpitis and healthy tissues (Fig 1A-C). Findings corresponding to Fig 1 D-E, Fig 2, Fig 3, Fig 4, Fig 5, and Fig.6 remain unconclusive, since it is not possible to assess their reproducibility and statistical significance. The overall manuscript remains preliminary.

 

 

Comment 3: The number of rats (n=3) appears to be a bit too low to assess the reliability of the statistical significance.

RE: We appreciate the reviewer's comments and agree with their assessment. We recognize the limitations of the project mentioned. However, combining qualitative analysis of in vivo animal models with solid quantitative measurements from human samples and in vitro studies can provide reliable outcomes, as stated in the paper. We have also addressed these limitations in the discussion section.

Reviewer’s answer: It is correct that findings from human samples (Fig 1A-C) are solid. However, it was not the case for animal findings. The findings associated to rats (Fig 5 and Fig 6) are histological staining performed only once. No firm conclusion can be obtained from these figures.

 

Comment 4: The number of independent measurements associated to cellular investigations was not reported anywhere (Fig .1D-F, Fig 2A-D, Fig 3A-D, Fig 4A-D). I can’t assess the reliability of the findings.

RE: We have added the number of measurements in the caption section of each figure.

Reviewer’s answer. It remains unaddressed. What was added is “triplicates” in Figure-legend (Fig. 1D-F, Fig. 2A-D, Fig. 3A-D, Fig. 4A-D). Triplicates from the same sample are considered as one independent measurement. At least three independent measurements shall be performed to obtain statistical significance. No firm conclusions can be obtained from these figures. 

 

Comment 5: Full western blot shall be shown instead of fragmented ones (Fig. 2C, and Fig. 4C). Quantitative values of proteins shall be added to support the western blot findings.

RE: We appreciate the reviewer's suggestion and have included the full blot images along with quantitative measurements in the supplementary data.

Reviewer’s answer: It was insufficiently addressed. Full western blot associated to Fig 2C is of good quality, while that associated to Fig 4C is not well resolved. Fig 2C and Fig 4C are based from one set of samples. Several independent measurements shall be performed to obtain quantitative determination. There were no bar errors on the quantitative data. It is still unclear if the differences in the protein levels were statistically significant.

 

Comment 6: While qualitative histological staining is valuable (Fig 5 and 6), quantitative determination of specific immunological characteristics are preferred in most Journals. The number of independent sets of immunological staining was not provided to support the significance of the findings.

RE : We understand the reviewer’s concern and suggestion. See above

Reviewer’s answer: It was not addressed. No quantitative measurements were performed. It appears that there were only one set of immunological finding. No firm conclusions can result from Fig 5 and Fig 6.

 

Minor comments:

 

Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 1D, E legend to support “After hDPCs were treated with P. gingivalis lipopolysaccharide (Pg-LPS) at 0.1 and 1µg/ml to induce inflammation, the transcripts of IL-6 and IL-8 were significantly increased (Fig.1D,E).”

Added

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 1F legend to support “Notably, miR-200c expression was significantly reduced in inflamed hDPCs 92 (Fig.1F).”

Added

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

 

 

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2A legend to support “The increased miR-200c expression was confirmed in a dose- dependent manner (Fig.2A).”

Corrected

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

 

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2B legend to support “miR-200c treatment at 0.1 and 0.3 µg significantly increased the transcripts of runt-related transcription factor 2 (Runx2),osteocalcin (OCN), and DMP1 compared to untreated control and treatment with empty vector (EV) after seven days (Fig.2B).”

Information has been added.

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

 

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Replace fragmented western blot by full length western blot in Fig 2C, and quantify relative amount of proteins to support “These odontogenic differentiation markers were also improved after 14 days, as confirmed by western blots (Fig.2C).”

Information has been added.

Reviewer’answer: It was insufficiently addressed. Full western blot associated to Fig 2C is of good quality, while Fig 2C is based from one set of samples. Several independent measurements shall be performed to obtain quantitative determination. There were no bar errors on the quantitative data. It is still unclear if the differences in the protein levels were statistically significant.

 

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2D legend to support “However, pre- treatment with pDNA miR-200c significantly downregulated IL-6 and IL-8 transcripts (Fig. 2D)”

Addressed.

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

 

 

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3A legend to support “As illustrated in Figure 3, the diameters of CaCO3/ miR- 200c nanocomplexes at CaCO3 : protamine sulfate (PS) of 1:0.25 were

185.59 ± 27.2 nm, and their Zeta potential was +19.3 (Fig.3A).”

Added

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

 

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3B legend to support “Interestingly, the CaCO3:PS ratio impacts transfection efficiencies of pDNA encoding miR-200c, and the expression of miR-200c was upregulated as PS concentrations increased. CaCO3:PS at ratios of 1:0.25 and 1:0.5

showed the best transfection efficiencies (Fig.3B).”

Added

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

 

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3C legend to support “PEI and CaCO3 significantly increased the miR-200c expression than naked pDNA miR-200c. However, CaCO3 transfected significantly higher amounts of miR-200c than did PEI (Fig.3C).”

Added

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

 

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3D legend to support “Notably, the delivery using PEI upregulated IL-6 transcripts; however, the delivery using CaCO3-based nanoparticles has significantly lower IL-6 expression (Fig. 3D).”

Added

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

 

 

Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, p5: Indicate number of independent measurements in Fig.4A-B legend to support “As illustrated in Figure 4, transfection of pDNA miR-200c using CaCO3 significantly increased transcripts of DMP1 (Fig.4A) and dentin sialophosphoprotein (DSPP) (Fig.4B).”

Corrected.

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

 

Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, p5: Replace fragmented western blot by full length western blot in Fig 4C, and quantify relative amount of proteins to support “The western blot showed that the band intensities of DSPP and DMP1 were increased in DPSCs treated with miR-200c at different concentrations (Fig.4C).”

Added in the supplementary data

Reviewer’answer: It was insufficiently addressed. Full western blot associated to Fig 4C is of insufficient quality, and Fig 4C is based from one set of samples. Several independent measurements shall be performed to obtain quantitative determination. There were no bar errors on the quantitative data. It is still unclear if the differences in the protein levels were statistically significant.

 

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5D to support “Compared to healthy dental pulp tissues (Fig. 5C), the typical inflammation in rat pulpal tissues challenged with Pg-LPS was observed in a rat model while treated with EV after seven days, presenting a greater density of inflammatory infiltrates in the connective tissue underneath the restorative material (blue arrowed) (Fig. 5D).”

RE: We have addressed the issue in the manuscript. See the response above.

Reviewer’answer: It was not addressed. The findings associated to rats (Fig 5) are histological staining performed only once. There were no quantitative determinations. No firm conclusion can be obtained from these figures.

 

 

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5E to support “However, the miR-200c treatment effectively re- duced inflammatory cell infiltration (Fig.5E),”

We have addressed the issue in the manuscript.

Reviewer’answer: It was not addressed. The findings associated to rats (Fig 5E) are histological staining performed only once. There were no quantitative determinations. No firm conclusion can be obtained from these figures.

 

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5E to support “Additionally, the IHC staining using an anti-IL-6 antibody indicated that treatment with miR-200c effectively reduced IL-6 (stained in brown color) in the pulpal tissues induced by Pg-LPS more so than EV (Fig. 5F,G)”

We have addressed the issue in the discussion section of the manuscript.

Reviewer’answer: It was not addressed. The findings associated to rats (Fig 5F,G) are histological staining performed only once. There were no quantitative determinations. No firm conclusion can be obtained from these figures.

 

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p7: Add quantitative histological features in Fig 6A, B to support “After 3 weeks, we found new tissue formation consistent with reparative dentin adjacent to collagen with CaCO3/miR-200c (Fig. 6A, B).”

We have addressed the issue in the manuscript.

Reviewer’answer: It was not addressed. The findings associated to rats (Fig 6A,B) are histological staining performed only once. There were no quantitative determinations. No firm conclusion can be obtained from these figures.

 

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p7: Add quantitative histological features in Fig 6C, D to support “However, the treatment with CaCO3/EV has limited dentin formation (Fig.6C, D).”

We have addressed the issue in the manuscript.

Reviewer’answer: It was not addressed. The findings associated to rats (Fig 6C,D) are histological staining performed only once. There were no quantitative determinations. No firm conclusion can be obtained from these figures.

 

Discussion, p9: It was not demonstrated in this work. Delete “Moreover, miR-200c can effectively enhance Wnt/β-catenin activities by targeting Sox2 and Klf4 in osteogenic differentiation.”

Addressed

Reviewer’s answer: It was not deleted.

 

Discussion, p9: It was not demonstrated in this work. Delete “This is noteworthy as these pathways align with the signaling mechanisms involved in odontogenic differentiation and dentin regenera- tion. Consequently, the existing robust evidence strongly suggests that miR-200c promotes odontogenic differentiation and dentin regeneration, potentially by upregulating BMP and Wnt signaling through the targeted inhibition of noggin, Sox2, and KLF4.”

Addressed

Reviewer’s answer: It was not deleted.

 

 

Materials and Methods, Determining Odontogenic Differentiation Enhancement and Anti- Inflammation of miR- 200c Overexpression in Human DPSCs and DPCs, p10: Add composition of the medium, and indicate time of incubationin “Human DPSCs were purchased (PT5025, Lonza, Walkersville, MD) and cultured with the DPSC BulletKit TM Medium.”

Corrected

Reviewer’s answer: It was not addressed. No medium composition, neither incubation time were added.

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Change PBS buffer by another one since it is osteogenic in “To determine the pulpal inflammation induced by Pg-LPS and the anti-inflammatory function of pDNA miR-200c, after 10 µg Pg-LPS suspended in 1µl PBS was added to the exposed pulps, a collagen sponge (1 × 1 × 1 mm3) incorporated with either naked EV (1 µg) or pDNA miR-200c (1 µg) were implanted.”

Corrected

Reviewer’s answer: It was not addressed. There were no justification of the use of PBS buffer, especially it is osteogenic.

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Add more information how histology was performed to support “The anti-inflammatory function of miR-200c in pulp inflammation was determined after seven days using histological examination.”

Corrected

Reviewer’s answer: It was insufficiently addressed. More details how histology was performed shall be given (extraction of rat maxilla, buffer in which it was stored, dehydration process, clearing, etc…).

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Consider to increase the number of rats to obtain valid statistical significances in “There were 3 rats for each treatment.”

We have addressed in the discussion.

Reviewer’s answer: It was insufficiently addressed.

 

Materials and Methods, qRT-PCR, p11: Add more information how total RNA was extracted and indicate from which samples in“Total RNA was extracted using Qiagen miRNeasy microKit (Qiagen, Hilden, Germany).”

Corrected.

Reviewer’s answer: It was not addressed. No further information was given.

 

Materials and Methods, qRT-PCR, p11: Replicates from one sample are considered as a one independent measurement. Perform at least three independent measurements in “Comparative real- time PCR was performed in replicate, and relative expression was obtained using the comparative Ct (ΔΔCt) method.”

Reviewer’s answer: It was not addressed.

 

Materials and Methods, Statistical Analysis, p12: Indicate number of independent measurements to support “All statistical tests completed for the in vitro and in vivo quantifications used a significance level of 0.05, and each graphic depicts mean values and associated standard deviations.”

RE: We have addressed in the discussion

Reviewer’s answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. At least three independent measurements shall be performed to obtain valid statistics.

 

 

Author Response

Reviewer 1

Comment1: MicroRNA-200c (miR-200c) was downregulated in inflamed pulp tissues and primary human dental pulp-derived cells (DPCs). Overexpression of miR-200c by transfecting plasmid DNA (pDNA) in human DPCs decreased proinflammatory cytokines IL-6 and IL-8 and enhanced odontogenic differentiation markers. It was concluded that miR-200c can modulate pulpal inflammation and facilitate dentin repair. The overall findings appear preliminary due to the difficulty to assess the reliability of the findings.

RE: We appreciated the reviewer’s positive comments and valuable suggestions. We have revised the manuscript based on the comments.

Reviewer’s answer: My concerns concerning the reproducibility of animal and cellular findings were not addressed. The only statistically significant finding is centered on the analysis of human pulpitis and healthy tissues (Fig 1A-C). Findings corresponding to Fig 1 D-E, Fig 2, Fig 3, Fig 4, Fig 5, and Fig.6 remain unconclusive, since it is not possible to assess their reproducibility and statistical significance. The overall manuscript remains preliminary.

RE: We appreciate the reviewer’s points and have added the sample size to all quantitative measurements to the revised manuscript.

 Comment 3: The number of rats (n=3) appears to be a bit too low to assess the reliability of the statistical significance.

RE: We appreciate the reviewer's comments and agree with their assessment. We recognize the limitations of the project mentioned. However, combining qualitative analysis of in vivo animal models with solid quantitative measurements from human samples and in vitro studies can provide reliable outcomes, as stated in the paper. We have also addressed these limitations in the discussion section.

Reviewer’s answer: It is correct that findings from human samples (Fig 1A-C) are solid. However, it was not the case for animal findings. The findings associated to rats (Fig 5 and Fig 6) are histological staining performed only once. No firm conclusion can be obtained from these figures.

RE: We appreciate the reviewers' feedback regarding the preliminary nature of our in vivo animal model studies, which are indeed less robust when compared to our human studies. Currently, there is no standard or well-established animal model that accurately simulates patient pulpitis and vital pulp therapy (VPT). Furthermore, there are technical challenges in replicating patient VPT scenarios in rodent models.

However, to address the reviewer’s concerns, we have scored the severity of pulpal inflammation mediated by CaCO3/miR-200c in a double-blinded manner using previous published criteria. We have provided the scores for treatment with CaCO3 delivered miR-200c and EV. Furthermore, we included representative images of pulp tissue from various rat treatments in the supplementary data. We have also discussed the limitations of our in vivo studies in the discussion section of our paper.

 

Comment 4: The number of independent measurements associated to cellular investigations was not reported anywhere (Fig .1D-F, Fig 2A-D, Fig 3A-D, Fig 4A-D). I can’t assess the reliability of the findings.

RE: We have added the number of measurements in the caption section of each figure.

Reviewer’s answer. It remains unaddressed. What was added is “triplicates” in Figure-legend (Fig. 1D-F, Fig. 2A-D, Fig. 3A-D, Fig. 4A-D). Triplicates from the same sample are considered as one independent measurement. At least three independent measurements shall be performed to obtain statistical significance. No firm conclusions can be obtained from these figures.

RE: We totally agree with the reviewers. Our in vitro measurements were carried out through three independent measurements. The sample size has been added.

 

Comment 5: Full western blot shall be shown instead of fragmented ones (Fig. 2C, and Fig. 4C). Quantitative values of proteins shall be added to support the western blot findings.

RE: We appreciate the reviewer's suggestion and have included the full blot images along with quantitative measurements in the supplementary data.

Reviewer’s answer: It was insufficiently addressed. Full western blot associated to Fig 2C is of good quality, while that associated to Fig 4C is not well resolved. Fig 2C and Fig 4C are based from one set of samples. Several independent measurements shall be performed to obtain quantitative determination. There were no bar errors on the quantitative data. It is still unclear if the differences in the protein levels were statistically significant.

RE: The images used for the Western blot in Figures 2C and 4C were captured using different devices. The image in Figure 4C was obtained with the Azure 500 (Azure Biosystems; Dublin, CA), which is a more advanced imaging system for newer blots. Additionally, our Western blots were not intended for quantitative measurements, so they cannot be subjected to statistical analysis.

 

Comment 6: While qualitative histological staining is valuable (Fig 5 and 6), quantitative determination of specific immunological characteristics are preferred in most Journals. The number of independent sets of immunological staining was not provided to support the significance of the findings.

RE : We understand the reviewer’s concern and suggestion.

Reviewer’s answer: It was not addressed. No quantitative measurements were performed. It appears that there were only one set of immunological finding. No firm conclusions can result from Fig 5 and Fig 6.

Re: We have included quantitative measurements and additional information for Figure 6 in the revised manuscript, as noted above. However, the studies presented in Figure 5, which involved treating pulpitis with naked pDNA miR-200c for a very short duration, were designed for qualitative analysis. As a result, these studies cannot be subjected to statistical analysis.

 

Minor comments:

Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 1D, E legend to support “After hDPCs were treated with P. gingivalis lipopolysaccharide (Pg-LPS) at 0.1 and 1µg/ml to induce inflammation, the transcripts of IL-6 and IL-8 were significantly increased (Fig.1D,E).”

Added

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

Re: We have added the sample size.

 Results, miR-200c Participates in the Pathogenesis of Pulpitis and Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 1F legend to support “Notably, miR-200c expression was significantly reduced in inflamed hDPCs 92 (Fig.1F).”

 

Added

 

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

Re: We have added the sample size.

 

 Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2A legend to support “The increased miR-200c expression was confirmed in a dose- dependent manner (Fig.2A).”

 

Corrected

 

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

Re: We have added the sample size.

 

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2B legend to support “miR-200c treatment at 0.1 and 0.3 µg significantly increased the transcripts of runt-related transcription factor 2 (Runx2),osteocalcin (OCN), and DMP1 compared to untreated control and treatment with empty vector (EV) after seven days (Fig.2B).”

 

Information has been added.

 

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

Re: We have added the sample size.

 

 

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Replace fragmented western blot by full length western blot in Fig 2C, and quantify relative amount of proteins to support “These odontogenic differentiation markers were also improved after 14 days, as confirmed by western blots (Fig.2C).”

 

Information has been added.

 

Reviewer’answer: It was insufficiently addressed. Full western blot associated to Fig 2C is of good quality, while Fig 2C is based from one set of samples. Several independent measurements shall be performed to obtain quantitative determination. There were no bar errors on the quantitative data. It is still unclear if the differences in the protein levels were statistically significant.

 

RE: Our Western blots were not designed to perform quantitative measurements, and they cannot be subjected to statistical analysis. 

 

Results, miR-200c Overexpression Promotes Odontogenic Differentiation in Human DPSCs and Mitigates IL-6 and IL-8 in Inflamed hDPCs, p3: Indicate number of independent measurements in Fig. 2D legend to support “However, pre- treatment with pDNA miR-200c significantly downregulated IL-6 and IL-8 transcripts (Fig. 2D)”

 

Addressed.

 

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

Re: We have added the sample size.

 

 Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3A legend to support “As illustrated in Figure 3, the diameters of CaCO3/ miR- 200c nanocomplexes at CaCO3 : protamine sulfate (PS) of 1:0.25 were

185.59 ± 27.2 nm, and their Zeta potential was +19.3 (Fig.3A).”

 

Added

 

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

Re: We have added the sample size.

 

 

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3B legend to support “Interestingly, the CaCO3:PS ratio impacts transfection efficiencies of pDNA encoding miR-200c, and the expression of miR-200c was upregulated as PS concentrations increased. CaCO3:PS at ratios of 1:0.25 and 1:0.5 showed the best transfection efficiencies (Fig.3B).”

 

Added

 

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

Re: We have added the sample size.

 

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3C legend to support “PEI and CaCO3 significantly increased the miR-200c expression than naked pDNA miR-200c. However, CaCO3 transfected significantly higher amounts of miR-200c than did PEI (Fig.3C).”

Added

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

Re: We have added the sample size.

 

Results, Biocompatible CaCO3-Based Nanoparticles Significantly Improve the Transfection Efficiency of miR-200c in DPSCs, p4: Indicate number of independent measurements in Fig.3D legend to support “Notably, the delivery using PEI upregulated IL-6 transcripts; however, the delivery using CaCO3-based nanoparticles has significantly lower IL-6 expression (Fig. 3D).”

 

Added

 

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

Re: We have added the sample size.

 

 

Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, p5: Indicate number of independent measurements in Fig.4A-B legend to support “As illustrated in Figure 4, transfection of pDNA miR-200c using CaCO3 significantly increased transcripts of DMP1 (Fig.4A) and dentin sialophosphoprotein (DSPP) (Fig.4B).”

 

Corrected.

 

Reviewer’answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. Several independent measurements shall be performed to support statical significance.

 

Re: We have added the sample size.  

 

 Results, miR-200c Delivered by CaCO3 Promotes Odontogenic Differentiation in Human DPSCs, p5: Replace fragmented western blot by full length western blot in Fig 4C, and quantify relative amount of proteins to support “The western blot showed that the band intensities of DSPP and DMP1 were increased in DPSCs treated with miR-200c at different concentrations (Fig.4C).”

 

Added in the supplementary data

 

Reviewer’answer: It was insufficiently addressed. Full western blot associated to Fig 4C is of insufficient quality, and Fig 4C is based from one set of samples. Several independent measurements shall be performed to obtain quantitative determination. There were no bar errors on the quantitative data. It is still unclear if the differences in the protein levels were statistically significant.

RE: See above.

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5D to support “Compared to healthy dental pulp tissues (Fig. 5C), the typical inflammation in rat pulpal tissues challenged with Pg-LPS was observed in a rat model while treated with EV after seven days, presenting a greater density of inflammatory infiltrates in the connective tissue underneath the restorative material (blue arrowed) (Fig. 5D).”

 

RE: We have addressed the issue in the manuscript. See the response above.

Reviewer’answer: It was not addressed. The findings associated to rats (Fig 5) are histological staining performed only once. There were no quantitative determinations. No firm conclusion can be obtained from these figures.

 

Re: We have added the sample size.

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5E to support “However, the miR-200c treatment effectively re- duced inflammatory cell infiltration (Fig.5E),”

 

We have addressed the issue in the manuscript.

 

Reviewer’answer: It was not addressed. The findings associated to rats (Fig 5E) are histological staining performed only once. There were no quantitative determinations. No firm conclusion can be obtained from these figures.

 

RE: See above.

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p6: Add quantitative histological features in Fig 5E to support “Additionally, the IHC staining using an anti-IL-6 antibody indicated that treatment with miR-200c effectively reduced IL-6 (stained in brown color) in the pulpal tissues induced by Pg-LPS more so than EV (Fig. 5F,G)”

 

We have addressed the issue in the discussion section of the manuscript.

 

Reviewer’answer: It was not addressed. The findings associated to rats (Fig 5F,G) are histological staining performed only once. There were no quantitative determinations. No firm conclusion can be obtained from these figures.

RE: See above

 

 

Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p7: Add quantitative histological features in Fig 6A, B to support “After 3 weeks, we found new tissue formation consistent with reparative dentin adjacent to collagen with CaCO3/miR-200c (Fig. 6A, B).”

 

We have addressed the issue in the manuscript.

 

Reviewer’answer: It was not addressed. The findings associated to rats (Fig 6A,B) are histological staining performed only once. There were no quantitative determinations. No firm conclusion can be obtained from these figures.

RE: We have added quantitative measurements for the inflammation mediated by CaCO3/miR-200c. See above.

 

 Results, miR-200c Mitigates Pulp Inflammation and Improves Dentin Regeneration In Vivo, p7: Add quantitative histological features in Fig 6C, D to support “However, the treatment with CaCO3/EV has limited dentin formation (Fig.6C, D).”

 

We have addressed the issue in the manuscript.

 

Reviewer’answer: It was not addressed. The findings associated to rats (Fig 6C,D) are histological staining performed only once. There were no quantitative determinations. No firm conclusion can be obtained from these figures.

RE: See above

 

 

Discussion, p9: It was not demonstrated in this work. Delete “Moreover, miR-200c can effectively enhance Wnt/β-catenin activities by targeting Sox2 and Klf4 in osteogenic differentiation.”

 

Addressed

 

Reviewer’s answer: It was not deleted.

RE:  We agree with the reviewer's comments. However, these previous findings indicate potential mechanisms through which miR-200c regulates pulpitis. We have clarified this in our revision.

 

Discussion, p9: It was not demonstrated in this work. Delete “This is noteworthy as these pathways align with the signaling mechanisms involved in odontogenic differentiation and dentin regenera- tion. Consequently, the existing robust evidence strongly suggests that miR-200c promotes odontogenic differentiation and dentin regeneration, potentially by upregulating BMP and Wnt signaling through the targeted inhibition of noggin, Sox2, and KLF4.”

 

Addressed

 

Reviewer’s answer: It was not deleted.

RE:  These previous findings indicate potential mechanisms through which miR-200c regulates pulpitis. We have clarified this in our revision.

 

 

Materials and Methods, Determining Odontogenic Differentiation Enhancement and Anti- Inflammation of miR- 200c Overexpression in Human DPSCs and DPCs, p10: Add composition of the medium, and indicate time of incubationin “Human DPSCs were purchased (PT5025, Lonza, Walkersville, MD) and cultured with the DPSC BulletKit TM Medium.”

 

Corrected

 

Reviewer’s answer: It was not addressed. No medium composition, neither incubation time were added.

RE: We have more information into the manuscript per reviewer’s request.

 

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Change PBS buffer by another one since it is osteogenic in “To determine the pulpal inflammation induced by Pg-LPS and the anti-inflammatory function of pDNA miR-200c, after 10 µg Pg-LPS suspended in 1µl PBS was added to the exposed pulps, a collagen sponge (1 × 1 × 1 mm3) incorporated with either naked EV (1 µg) or pDNA miR-200c (1 µg) were implanted.”

 

Corrected

 

Reviewer’s answer: It was not addressed. There were no justification of the use of PBS buffer, especially it is osteogenic.

RE: Phosphate Buffered Saline (PBS) is a widely used solution in biological and biomedical research due to its isotonicity and non-toxicity. PBS treatment is extensively used a negative control of inflammatory stimulation.

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Add more information how histology was performed to support “The anti-inflammatory function of miR-200c in pulp inflammation was determined after seven days using histological examination.”

Corrected

 

Reviewer’s answer: It was insufficiently addressed. More details how histology was performed shall be given (extraction of rat maxilla, buffer in which it was stored, dehydration process, clearing, etc…).

RE: Information has been added in the manuscript per reviewer’s request.

 

Materials and Methods, Determining the Functions of pDNA Encoding miR-200c in Rat Models of Pulpitis, p11: Consider to increase the number of rats to obtain valid statistical significances in “There were 3 rats for each treatment.”

 

We have addressed in the discussion.

 

Reviewer’s answer: It was insufficiently addressed.

RE: We have addressed the weakness of a small sample size in vivo.

 

Materials and Methods, qRT-PCR, p11: Add more information how total RNA was extracted and indicate from which samples in“Total RNA was extracted using Qiagen miRNeasy microKit (Qiagen, Hilden, Germany).”

 

Corrected.

 

Reviewer’s answer: It was not addressed. No further information was given.

RE: These are routine procedures as per the manufacturer's protocol.

 

Materials and Methods, qRT-PCR, p11: Replicates from one sample are considered as a one independent measurement. Perform at least three independent measurements in “Comparative real- time PCR was performed in replicate, and relative expression was obtained using the comparative Ct (ΔΔCt) method.”

 

Reviewer’s answer: It was not addressed.

RE: We have added the sample size.

 

 

Materials and Methods, Statistical Analysis, p12: Indicate number of independent measurements to support “All statistical tests completed for the in vitro and in vivo quantifications used a significance level of 0.05, and each graphic depicts mean values and associated standard deviations.”

 

RE: We have addressed in the discussion

 

Reviewer’s answer: It was not addressed. Triplicates from one sample are considered as one independent measurement. At least three independent measurements shall be performed to obtain valid statistics.

RE: We have added the sample size.

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Comments and Suggestions for Authors

The authors addressed all the points.