Apigenin Promotes Proliferation and Mineralization of Human Osteoblasts and Up-Regulates Osteogenic Markers

Round 1

Reviewer 1 Report

Well executed and written research work. 

Can be improved with the suggestions below:

- The Introduction section is too long. It can be made short by combining paragraphs 3,4,5 (Line 53-78) into one short paragraph as the information given is not very relevant to this paper.

- Should include more papers on the use of Apigenin in the oral apparatus.

 

Author Response

Well executed and written research work. 

Can be improved with the suggestions below:

- The Introduction section is too long. It can be made short by combining paragraphs 3,4,5 (Line 53-78) into one short paragraph as the information given is not very relevant to this paper.

AUTHOR’S ANSWER: Thank you for this comment. We have summarized part of this section to meet your suggestion.

- Should include more papers on the use of Apigenin in the oral apparatus

AUTHOR’S ANSWER: Thank you for this comment; we have added the following paragraph:

In the oral cavity APG accelerated the healing of oral mucositis induced in hamsters by the application of 5-fluorouracil [20]. Another important action of APG is the inhibitory effect on cell survival and apoptotic effect in oral cancer cells. The apoptosis in SCC-25 cells by APG, also determinated the cell cycle arrest acting as a CDK1 inhibitor and inducing a decrease in cyclin D1 expression[21]. Also Silvan et al. showed the inhibitory action of APG against induced oral carcinogenesis using topical application of 0.5% DMBA in hamsters[22]. The effects of APG were the improving of antioxidant defense mechanism and the modulation of the activities of phase I and phase II detoxification cascade toward increased excretion of active metabolite of DMBA.[22]

Reviewer 2 Report

Dear Authors, I appreciate the team for performing significant research in the field. The manuscript could be improved as per the suggested comments to make it suitable for publication.

Comments for author File: Comments.pdf

Author Response

#2

Dear Authors, Apigenin a natural flavonoid compound with anti-inflammatory and antioxidant properties seems to be efficient material in both osteogenic differentiation and formation. This study had evaluated the effect of apigenin on human osteoblast. Accordingly, apigenin has been used to assess the viability, the proliferation, the morphology, calcium deposition and the ALP activity of the cells. Additionally, the gene expression of key osteoblastic genes such as OCN, COL1, ALP has been further investigated. General comments: The justification for the purpose of the study is insufficient for the introduction section. More information on the lacunae in existing background literature would add strength.

Specific comments:

1. Keywords: a. Page 1 - Line 32 – Arrange the keywords in alphabetical order.

AUTHOR’S ANSWER: Thank you for your suggestion. We have arranged the keywords in alphabetic order

2. Introduction:
3. Page 1 - Para 1 – lines 7 to 8 – Rephrase the sentence.

AUTHOR’S ANSWER: Thank you for your suggestion. We have modified the sentence:

“APG is a substance contained in vegetables and plants in water-soluble glycosylated form, but after a purification process it appears as a chemically unstable yellow powder, insoluble in water, moderately soluble in hot alcohol and soluble in dimethyl sulfoxide (DMSO)”.

1. Page 2 - Pare 2 & 3 – These two paragraphs can be briefly mentioned, instead of elaborating the mechanism of apigenin role in lipid metabolism and diabetes because it seems to be irrelevant to the current context.

AUTHOR’S ANSWER: Thank you for your comment. We have briefed the paragraphs:

“Another positive effect of apigenin is found against metabolic diseases, in particular type 2 diabetes mellitus (DM2), obesity and inflammation [15]. APG inhibits pancreatic lipase, an enzyme that catalyzes the conversion of triglycerides into monoglycerides and fatty acids in the intestine [14]. In particular, APG is able to decrease insulin resistance, to reduce the abnormal metabolism of glycolipids and to alleviate oxidative stress [16] through regulation of miRNAs, inhibition of the insulin kinase receptor and the upregulation of the GLUT4 / AMPK signal [11], the inhibition of α-amylase, with the consequent reduction of the digestion of carbohydrates in the diet, and the delay in the absorption of glucose [17].”

1. Page 3 – Para 1- lines 101-103- The justification given for the novelty of the study can be more precise.

AUTHOR’S ANSWER: Thank you for your request. Nevertheless, Apigenin having different crucial implications on bone health, there are a limited number of in vitro studies on Apigenin, and even less on the effects of Apigenin in oral apparatus. Considering the implication of bone related diseases in dentistry fields and that the bone regeneration as well as a good osseintegration represent a challenge, a study on the effects of molecules such as Apigenin on oral osteoblasts might be a good starting point for developing new tools, considering that Apigenin is a natural safe molecule. Thus, the aim of this work was to evaluate the effects of different concentrations of APG on oral hOBs.

1. Page 3- Para 1- Line 104- Abbreviation must be expanded in the first appearance.

AUTHOR’S ANSWER: Thank you for your suggestion. We have introduced the acronym.

1. Page 3- Para 2- Line 107 & 108 - Abbreviations must be expanded.

AUTHOR’S ANSWER: Thank you for your suggestion. We have introduced the acronyms.

3. Materials and Experimental procedures
4. Page 3 - Para 3 – line 112-114 – Paraphrase the sentence

AUTHOR’S ANSWER: Thank you for your comment. We have modified the sentence:

“Human Oral Osteoblasts were extracted from bone biopsies, obtained from n°12 volunteers managed at the dental clinic of the G. D'Annunzio University in according to the protocol appproved by Ethics Committee of University of Chieti-Pescara (reference numbers: BONEISTO N. 22-10.07.2021).”

1. Page 3 – Para 3- line 113-114 – Add more precise information about the harvesting of osteoblast i.e., include the inclusion and exclusion criteria for selecting the sample

AUTHOR’S ANSWER: Thank you for your suggestion. We have added the inclusion and exclusion criteria:

“Inclusion criteria were as follows: patients aged between 18 and 60 with controlled oral hygiene subjected to routine dental practice, while exclusion criteria were patients with systemic diseases, with an history of osteoporosis and subjected to a therapy that interfere with bone metabolism and heavy smokers (more than 10 cigarettes/day).”

1. Page 4- Para 3 – In the experimental designs, how did you arrive at the number 12? Explain the sample size calculation and method if applicable.

AUTHOR’S ANSWER: Thank you for your comment. The sample size calculation was not applicable, we have enrolled a number of patients adequate for the number of cells required for the experiments. The number of cells used in each experiment was chosen on the basis of the previous protocols and scientific literature.

1. Page 4 – Para on cell culture and treatments- line 120- the consistency of the abbreviation mentioned should be constant throughout the manuscript.

AUTHOR’S ANSWER: Thank you for your suggestion. We have introduced the abbreviation used in the manuscript.

1. Page 4 - Para on cell culture and treatments- line 121- rephrase the sentence.

AUTHOR’S ANSWER: Thank you for your suggestion. We have rephrased this sentence:

“The protocol used to extract primary oral osteoblasts (hOBs) from bone biopsies was extracted based on the study of Pierfelice TV et al 2022”.

1. Page 4 - Para on cell culture and treatments- line 133-134- expand the abbreviations

AUTHOR’S ANSWER: Thank you for your suggestion. We have expanded the abbreviations.

4. Results
5. Page 5- line 199- Rephrase the sentence grammatically.

AUTHOR’S ANSWER: Thank you for your suggestion. We have modified the sentence

“Apigenin showed no-significant effects on cell viability and morphology of hOBs”

1. Page 6- Para 2 Line 224-225- rephrase the sentence to explain the clear outcome.

AUTHOR’S ANSWER: Thank you for your comment. We have reformulated the sentence:

“In addition, the response of the cells was inversely proportional to the concentrations. In particular, the highest and statistically significant rate was found when hOBs were treated with 5 µM compared to CTRL.”

5. Discussion
6. Page 9 – para 2- line 282-284- rephrase the sentence grammatically.

AUTHOR’S ANSWER: Thank you for your comment. We have rephrased the sentence:

“The aim of this study was to evaluate whether APG could be further considered as a bioactive agent capable of promoting bone regeneration for clinical applications in dentistry. In particular, APG could be beneficial to increase the osteoinductive properties of bone graft mixed with other heterologous biomaterials.”

 

1. Page 10 – Mention the limitations and strength.

AUTHOR’S ANSWER: Thank you for your request.

The beneficial effects of Apigenin, on oral osteoblasts, here demonstrated, in addition to already known other biological properties of Apigenin [Abid, R 2022; Salehi, B 2019] might potentially make Apigenin a multifunctional bioactive agent helping different situations in dentistry field, considering that Apigenin is a natural molecule, safe with very low risk of toxicity. Considering that the current treatment options for bone related diseases consisting of antiresorptive agents (such as bisphosphonates, hormone-replacement therapy, selective estrogen-receptor modulators and anti-RANKL antibodies) and/or anabolic agents (such as intermittent low doses of teriparatide and antisclerostin antibodies) are not free from adverse effects that limit their use [Wong, S.K 2020]. Thus, the beneficial properties on oral osteoblasts, of Apigenin that is a natural molecule safe represent the strength for a future use of this molecule in the dentistry field in which bone regeneration and new bone formation represent a challenge. The limit of APG clinical application is its poor solubility, at the present to overcome this limit, some approaches nanoparticles drug carriers have been formulated to improve the solubility, dissolution, and the oral bioavailability of APG [Nozhat, Z 2021]. In dentistry field this limit of Apigenin might not represent an obstacle, into the development of tools useful to improve osteoconductivity and osseointegration, for example as coatings on dental implants surfaces, or to favor dental restorations by incorporating Apigenin into dental composites. A strength of our study is that the lowest concentration here used, 5 µM, resulted the suitable concentration to improve the biological functions of oral osteoblasts and this represents a strength to prevent potential adverse side effects related to higher doses [Birkett, D.J. 1995; Singh, P 2012; Miyoshi, N 2007].

6. Conclusion
7. Page 10- Mention points related to future scope of research.

AUTHOR’S ANSWER: Thank you for your request.

The beneficial properties on oral osteoblasts, of Apigenin that is a natural molecule safe represent the strength for a future use of this molecule in the dentistry field, into the development of tools useful as coatings on dental implants surfaces to improve osteoconductivity and osseointegration. Thus, the next step in our research will be to test apigenin as a dental implant coating.

7. Reference
8. Make sure that all the references uniformly follow the recommended style of the journal.

AUTHOR’S ANSWER: Thank you for your comment; we have checked the references