Effects of Photobiomodulation Using Near-Infrared Light on the Dentin and Periodontal Ligament in a Beagle Model

Round 1

Reviewer 1 Report (New Reviewer)

This manuscript conducts a study of NIR (810nm) PBM on the dentition of a single beagle dog. The manuscript uses "80.22mj/cm²  as the light dose in various sections but  Figure 2 p5 L169 indicates the dose was 80 mJ/cm². Which is it? Similarly, the methods state in lines 169-170 in section 2.2 that treatment was every other WEEK for 8 weeks and  D+Figure 2 indicates the treatment was every other DAY. Which is correct? The introduction intimates that NIR is a form of PBM. It is true that NIR wavelengths of light are used for PBM, but the current statement is incorrect.  Please clarify what is meant by p8 L 229 "Authors dental hypersensitivity". The authors describe the device used and the angle of light output ad center wavelength. However they must also include the fwhm (i.e. the bandwidth of the LED device).

Please check the manuscript for typographical errors

Author Response

We revised all the manuscript according to your appoints. 

Author Response File: Author Response.pdf

Reviewer 2 Report (New Reviewer)

The authors examined  the application of photobiomodulation(PBM) as a treatment strategy  to enhance odontoblast activity and improve fibrous connective tissue of the periodontal ligament.

The PBM was done by help of an LED array system placed between the gingival tissue and teeth to facilitate exposure to the beagle’s teeth with a central wave-length of 810 nm.

The LED array was operated in pulsed wave (PW) mode at a frequency of 300 Hz and a 30 % duty cycle. The selected radiated power density averaged 1.91 mW/cm2, producing an energy density of 80.22 mJ/cm2 towards the target over 42 seconds. 

 Light intensity was checked before running each experiment. 

Beagle’s gingival tissue and teeth were exposed to this light regimen every other week for 8 weeks to stimulate neodentin formation.

Similar areas of control and photobiomodulation-treated teeth were compared by help of micro-computed tomography, scanning electron mi-18 croscopy, and Hematoxylin and Eosin staining.

The authors observed increased odontoblast activity at the pulp-dentin interface in PBM-treated samples.

Structural analysis by scanning electron microscopy (SEM) indicates increased activity of odontoblasts in dentin formation after PBM treatment.

The microscopic examination revealed that the photobiomodulation-treated sample had denser fibrous connective tissue in the periodontal ligament than control teeth. 

Enhanced photobiomodulation of vascular activity in the odontoblast layer was observed. A great number of vascular structures were observed in PBM-treated tissue, compared with the untreated PBM tissue.

Unlike CW, which operate at higher power densities, typically in the range of 0.5 to 48 J/cm2, PW uses lower power densities, delivering photon energy in pulses. In this case, the PW setting of power density of 1.91 mW/cm2 at the duty cycle of 30 % was used to be energy density of 80.22 mJ/cm2.

Despite its lower energy density, PW as observed to be more effective than CW in stimulating tooth tissue regeneration, as evidenced in the evaluated study by enhanced activity of odontoblasts and improved condition of fibrous connective tissue within the 

periodontal ligament

The autors used  300 Hz-near-infrared PBM,  sugesting potential therapies in dentin hypersensitivity, in maintening periodontal ligament health.  Odontoblast activity was effectively improved in a beagle model, and fibrous connective tissue within the periodontal ligament was improved.

Although  the study model  was confined to a single animal subject, the article is interesting.

PLease check if the sentence is correct:

In the raw 281:

Although PW uses lower energy density, it brought more effective than PW. 

Author Response

We revised all according to your comments.

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

A very perfect study

Reviewer 2 Report

This paper, Hong Bae Kim et al, addressed about the effect of NIR-light on dentin.

The researchers used “normal, healthy” dentin in a beagle model. We have concerns about the model, and we suggest authors to re-examine their concept in the disease model.

Major Points

Introduction

In caries and pulp trauma, the pulp is replaced by an artificial prosthesis through root canal therapy, which is insufficient to restore full tissue functionality due to the lack of vascular and nerve elements. The introduction of exogenous cultured stem cells carries the risk of contamination. Therefore, the use of endogenous stem cells is ideal. Therefore, you have developed the following method. Near-infrared light is expected to contribute to the development of dentin and periodontal ligament fibroblasts by activating resident stem cells.

The article is nuanced in its description.

→The disease conditions assumed in this research are unclear.

The goal of regenerative medicine is self-healing by inducing self-healing capacity in lost tissues.

In dentistry, regenerative therapies include restoration of dentin lost to caries, regeneration of dental pulp, and regeneration of periodontal tissue destroyed by periodontal disease.

Is the purpose of regenerative therapy to revitalize healthy teeth for further health improvement, or to regenerate and protect teeth or pulp damaged by caries or other causes?

If the goal is to regenerate inactivated pulp tissue, it is difficult to activate dentinoblasts with near-infrared light because dentinoblasts are also inactivated in the inactivated pulp.

Material and Methods

　Animals

　As mentioned in the introduction, it is necessary to study the regenerative ability of tissues damaged by periodontal disease and caries.

For example, caries and periodontal disease models have been established and used in many previous works.

We need to study the activation of cells in disease models.

Photobiomodulation

In the case of near-infrared irradiation, how was the shielding applied to the Un-PBM when the left and right groups were divided into the Un-PBM and PBM groups?

We believe that it is important that the Un-PBM side is not exposed to near-infrared light in order to compare the treatment effects.

Result

As mentioned in the Discussion, a comparison by quantitative analysis is desirable.

Figure3

Please clearly indicate dentin, predentin, odontoblast, and pulp stem cells with arrows or symbols.

For clarity, use arrows or symbols, or immunohistochemistry.

Discussion

The advantages of secondary dentin formed by PBM are described here.

Compared to tertiary dentin formed by caries or irritation, it has a more predictable response.

To reiterate the points made in the introduction, it is necessary to clarify some of the assumptions made.

In dentistry, dentin regeneration is needed to protect the pulp from external stimuli such as caries and to replace lost dentin. However, the dentin formed in such cases is tertiary dentin.

Secondary dentin is added gradually as a physiological phenomenon with aging in caries-free teeth. Therefore, the need to increase the formation of secondary dentin in healthy teeth without external stimuli is not clear in this study.

Moderate English editting shoud be done.

Reviewer 3 Report

Dear authors,

Unfortunately, the experiment has some critical flaws and cannot be accepted.

a) the experiment was performed on one beagle, meaning that the sample size is one. Even though, split-mouth technique was applied and 8 teeth per groups were assigned, the concept of PBM is concerning the whole body as a unit. Thus, the experiment is highly compromised.

b) the parameters used were extremely low, e.g. 80.22 mJ/cm². It is known that for biostimulation on cells' level is required 2-10 J/cm². Also, for periodontal ligament stem cells, a recent systematic review stated the preferable fluence (Mylona, V.; Anagnostaki, E.; Chiniforush, N.; Barikani, H.; Lynch, E.; Grootveld, M. Photobiomodulation effects on periodontal ligament stem cells: A systematic review of in-vitro studies. Curr. Stem Cell Res. Ther. 2022, 17, 1–15, doi:10.2174/1574888x17666220527090321.)

c) the split-mouth applied technique presents the cross-over PBM effect, where both sides will be benefit.

Except from these critical flaws, there are some other comments:

a) Abstract is incomplete. Parameters and devices are missing.

b) The general knowledge about PBM seems to be low.

c) Extensive english editing is required, as many sentences are not clear.

d) At Discussion, there is no comparison with other studies and no scrutiny of parameters.

Extensive english editing is required, as many sentences are not clear.