Ijdos volume3 issue10

International Journal of

Dentistry and Oral Science

ISSN: 2377-8075

scidoc.org/dentistry-and-oral-science

Volume -3 Issue -10

Aim and Scope International Journal of Dentistry and Oral Science (IJDOS) ISSN: 2377-8075 is a peer-reviewed, Open Access journal that publishes original research articles, review articles, and clinical studies in all areas of dentistry, including periodontal diseases, dental implants, oral pathology, as well as oral and maxillofacial surgery. IJDOS publishes high quality papers, rapid communications, original papers, research letters and case reports pertaining to clinical studies in the field of Dentistry.

IJDOS publishes original research, latest developments, review papers, scientific data, editorials from leading scientists and scholars around the world including but, not limited to the following fields:

• Dentistry

• Orthodontics

• Primary Care Dentistry • Oral Pathology

• Prosthodontics

• Endodontics • Periodontology • Human Disease • Oral Microbiology • Dental Pharmacology

• Oral Biology • Dental Biomaterials • Biochemistry • Oral & MaxillofacialSurgery

Editor In Chief Francesco Chiappelli, Professor, UCLA Center for the Health Sciences, USA.

Special Issue Themes We are delighted to present the Special Issues with a purpose to publish the research with respect to the subjects of Dentistry. Below you will find various proposals handled by eminent editors, who are experts in the respective field 1. Dental Biomaterials: Tools and Techniques

2. Prosthodontics and Maxillofacial Prosthetics

3. Temporomandibular Joint Disorders

4. Long-term effects of orthodontic treatment

5. Dental Implant Esthetics

6. Stem Cells in Craniofacial Development and Regeneration

7. Tooth Bleaching Agents

8. Research in Periodontology

9. Endodontics: Treatment & Technology

10. Evidence-Based preventive dentistry

11. Novel approaches for Restorative Dentistry

12. Facial Aesthetic Dentistry and Orthognathic Surgery

13. Future Trends in Dentistry

14. Oral Microbiology and Dental Infection

15. Pediatric dental caries

16. Dental Abnormalities and Oral Health

18. Biology of Oral Implants

17. Dental Trauma and Management

19. Biomedical and Tissue Engineering in Dentistry

Join As Editoral Board Member : editor.ijdos@scidoc.org For More Detail About IJDOS : scidoc.org/IJDOS Submit your Article : scidoc.org/submission or

articles.scidoc@scidoc.org Submit special Issue Article

: specialissues@scidocpublishers.com

Cytotoxicity of Different Degrees of De-Acetylated Chitosan on 3T3- and Two Human Tooth Fibroblast Cell-Lines - By VT Perchyonok

International Journal of Dentistry and Oral Science (IJDOS) ISSN: 2377-8075 Cytotoxicity of Different Degrees of De-Acetylated Chitosan on 3T3- and Two Human Tooth Fibroblast Cell-Lines

Research Article

SR Grobler1, A Olivier1, HW Kruijsse1, VT Perchyonok2* 1 2

Oral and Dental Research Institute, Faculty of Dentistry, University of the Western Cape, Private Bag X1, Tygerberg 7505, Cape Town, South Africa.

VTPCHEM PTY LTD, Glenhuntly, 3163, Melbourne, Australia.

Abstract Nowadays chitosan becomes more and more important as a drug carrier. In this study the influence of different degrees of de-acetylation of chitosan on the cytotoxicity were investigated.

Materials and Methods: Chitosan (87%, 70% and 40% de-acetylated) was analysed for their cytotoxic effect on mouse Balb/c 3T3 fibroblast cells as well as two different human tooth pulp fibroblast cell-lines. The cell survival rate was determined over a 24 hour period according to the standard MTT assay.

Results: The Univariate ANOVA test showed significant differences in the cell survival rates (p<0.01) amongst 87% and 70% and 40% de-acetylated chitosan products for the 3T3 as well as the two pulp fibroblast cell-lines.

Significant differences were also found between both tooth cell-lines and the 3T3 cell-line at all 3 different de-acetylated chitosan levels. The 3T3 cell line was mostly affected at 40% de-acetylation. The difference between both human cell-lines was not significant.

Conclusion: The lower the degree of chitosan de-acetylation the less the cell survival rate while an 87% de-acetylation degree improved the cell survival rate. Different cell lines react differently towards different degrees of de-acetylation. The relative survival rates of different cell lines changed at different degrees of de-acetylation.

Keywords: Chitosan; Degrees De-Acetylated; Cytotoxicity; 3T3; Tooth Fibroblasts.

Introduction Due to the increasing applications of nanoparticles in many fields’ becomes more and more under investigation. Especially, a growing concern is the possible adverse effects of exposure to nanoparticles. Therefore, cytotoxicity studies became more and more important. Of the most important factors to consider during such cytotoxicity studies are the cell type used, and in the case of chitosan, the degree of de-acetylation (DDA). Preferably the cell type under investigation should be the chitosan target cell.

Chitosan [1] is produced commercially by de-acetylation of chitin and is a natural cationic polysaccharide composed of randomly distributed β-1, 4-linked D-glucosamine and N-acetyl α-glucosamine.

This cationic nature is something special as most polysaccharides are neutral or negatively charged in an acidic environment [2]. The cationic nature allows it to form electrostatic complexes or multilayer structures with other negatively charged molecules. Chitosan is non-toxic, biocompatible, bio-degradable and has mucous-adhesive properties and as a result became widely used in the pharmaceutical field as a carrier system for drugs, hormones, proteins, enzymes and genes [3-6]. Chitosan can be successfully used as a drug carrier because it will solubilize and degrade in an acidic environment with the resultant release of the drug [7]. However

*Corresponding Author: VT Perchyonok, VTPCHEM PTY LTD, Glenhuntly, 3163, Melbourne, Australia.

E-mail: tamaraperchyonok@gmail.com.

Received: September 21, 2016 Accepted: October 13, 2016 Published: October 17, 2016 Citation: SR Grobler, A Olivier, HW Kruijsse, VT Perchyonok (2016) Cytotoxicity of Different Degrees of De-Acetylated Chitosan on 3T3- and Two Human Tooth Fibroblast Cell-Lines. Int J Dentistry Oral Sci. 3(10), 337-339.

Copyright: VT Perchyonok© 2016. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distr ibution and reproduction in any medium, provided the original author and source are credited.

SR Grobler, A Olivier, HW Kruijsse, VT Perchyonok (2016) Cytotoxicity of Different Degrees of De-Acetylated Chitosan on 3T3- and Two Human Tooth Fibroblast Cell-Lines. Int J Dentistry Oral Sci. 3(10), 337-339.

el error bars of the mouse Balb/c 3T3 fibroblast cell line, the human1 tooth pulp fibroblast cell-line and human2 tooth pulp fibroblast cell-line at 87% de-acetylated chitosan, 70% de-acetylated chitosan and 40% de-acetylated chitosan.

Mean Percentage Survival Rate

the low solubility in alkaline and neutral medium has a negative influence on the use. Chitosan is hypoallergenic and has natural antibacterial properties, which further support its use in the army as field bandages [8]. Furthermore, antioxidant-chitosan hydrogels (that of resveratrol, propolis and β-carotene) were found to significantly improve the bond strength to dentine with or without phosphoric acid pre-treatment [9] as many other hydrogels do [10].

Materials and Methods Chitosan (Aldrich, Australia) were used as received. The degree of de-acetylation of typical commercial chitosan used in this study was 87% and the molecular weight 2500 kDa. The isoelectric point was 4.0–5.0.

For the other de-acetylated products: Chitin was de-acetylated to ~70% (mw 45 kDa) and ~40% (mw 15 kDa) by the use of 45% sodium hydroxide solution in a 1:10 (W/V) ratio in a reaction vessel following Zhang’s method [11] and the reaction was under nitrogen atmosphere [12]. A Balb/c 3T3 mouse fibroblast cell line (The National Repository for Biological Materials, Sandringam) was maintained and cultured under standard conditions [13]. The two human dental pulp fibroblast cultures were established as previously described [14-16]. Briefly freshly extracted impacted 3rd molars were collected in Dubecco’s Modified Eagels Medium, the middle third of the pulp cultured and the cells between the 3rd and 5th passage used.

The cytotoxicity study was done as previously reported [13-16].

Briefly, the cells (mouse 3T3 Balb/c fibroblast, human tooth fibroblast 1 or human tooth fibroblast 2) were first grown to near confluency, diluted to a final cell suspension containing approximately 3 ×105 cells/ml and plated out in sets of 96 well plates. Chitosan 87% de-acetylated, 70% de-acetylated or 40% de-acetylated were then added to the growth medium at a concentration of 1 mg/ml. Two hundred μl of each group was added to 20 wells in the 96 well plates. Medium without any gels was used as controls. After 24 hours the well-known MTT colorimetric assay was used to evaluate the cell survival rate. Absorbance was measured at wavelength 540 nm on a spectrophotometer to determine the number of viable cells. Three replicates were done in each group.

Results The Figure shows the cell survival rates with 95% confidence lev-

Cell-line 3t3

H1cell

H2cell

123.00 119.00 115.00 111.00 107.00 103.00 99.00 95.00 91.00 87.00 83.00 79.00 75.00 71.00 67.00 63.00 59.00 55.00 51.00 47.00 43.00 39.00 35.00 31.00 27.00 23.00 19.00 15.00

Cell line 3t3 H1 cell H2 cell

40%

70% 87% Concentration Chitosan Error bars: 95% CI

The results of a Univariate ANOVA showed significant main effects and an interaction effect suggesting an effect of increase in the concentration of chitosan.

Cell-line: F(2,438) = 23.71; p<0.001 Concentration: F(2,4378) = 1120.8; p<0.001 Cell-Line*Concentration: F(4,438) = 233.1; p<0.001 Significant differences were found between the two human celllines and the 3T3 cell-line at all 3 different de-acetylated chitosan levels. The difference between both human cell-lines is not significantat all 3 different de-acetylated chitosan levels (see Table below).

Discussion We determined the survival rates at a growth medium concentration of 1mg/ml which is reported to be in line with other research and which may be considered as on the upper recommended level [16] of concentration. The three cell lines tested consisted of a standard cell line bought from the National Repository for Biological Materials and two self-established human tooth pulp fibroblast cell lines (H1 cell line and H2 cell line) from two different people. To our knowledge until now different degrees of de-acetylation towards the mentioned cell lines were not reported.

De-acetylation concentration

Mean

Std. Error

40% 70% 87% 40% 70% 87% 40% 70% 87%

27.028 106.894 115.399 72.370 89.883 107.714 74.807 90.260 103.127

1.406 1.179 1.456 1.422 1.473 1.198 1.208 1.362 1.492

95% Confidence Interval Lower Bound Upper Bound 24.264 29.791 104.577 109.212 112.538 118.26 69.574 75.165 86.987 92.779 105.360 110.069 72.433 77.181 87.584 92.936 100.196 106.059

SR Grobler, A Olivier, HW Kruijsse, VT Perchyonok (2016) Cytotoxicity of Different Degrees of De-Acetylated Chitosan on 3T3- and Two Human Tooth Fibroblast Cell-Lines. Int J Dentistry Oral Sci. 3(10), 337-339.

Different degrees of de-acetylation (DDA) of chitosan is normally obtained by the treatment of chitin with alkali and a higher DDA is obtained by increasing the time and temperature, while the molecular weight (mw) of chitosan is dependent on the initial source of material which could be crap, fungi, shrimp etc.

This mw normally decreases in the process to obtain a higher DDA [12]. In this study an 87% product with a mw of 2500 kDa was used as received, while the 70% (mw 145kDA] and 40% (mw 15kDA) were prepared. We did not investigate the effect of the molecular weight of the different chitosan’s on the cytotoxicity.

It was reported that the degree of de-acetylation (DDA) and the molecular weight are important parameters which determines many physiochemical and biological properties of chitosans [12].

Nunthanid [17] reported that chitosans with high mw have higher moisture adsorption and tensile strength than those with the same DDA but lower mw. Hidaka [18] reported that a 94% DDA chitosan membranes gave minimal film degradation, mild inflammatory reaction and minimal osteogenesis while between 65 to 80% gave the opposite response.This effect is more or less in line to our survival rate results for all three different cell lines (Figure) that the higher the degree of de-acetylation the more the survival rate and that 87% de-acetylation actually improved the cell survival rate to above 100%.

However, Hamilton [19] reported no relationship between the DDA of chitosan, mw or growth of cells. While a similar report by the growth of keratinocytes was noted [20]. From the above it is concluded that there is controversial statements as far as the effect of molecular weights, in general, are concerned. In our study, the mw of the three chitosan products differed from 2500 kDa through 145 kDa to 15 kDa but this effect was not investigated.

In this study (Figure) we found that a 40% de-acetylation chitosan affects the 3T3 cell line significantly more (only about 30% cell survival was found) than both the two pulp cell lines. However, the other way round was found both at 70% and 87% de-acetylation where the pulp cell lines were significantly more affected (Figure). This showed that the relative survival rates of different cell linescan also change at different degrees of de-acetylation.

The above finding is in line where it was reported that different cell lines have different levels of cytotoxicity towards a dentine bonding agent [14, 15]. In one study where 3 different human pulp fibroblast cell lines were studied it was found that one of these pulp cell lines were less cytotoxic than the 3T3 cell line but the other 2 were more sensitive.

Conclusion The lower the degree of chitosan de-acetylation the less the cell survival rate while an 87% de-acetylation degree improved the cell survival rate. Different cell lines react differently towards different degrees of de-acetylation. The relative survival rates of different cell lines changed at different degrees of de-acetylation.

References [1]. Shenderova OA, Hens SAC (2010) Detonation nanodiamondparticles processing, modification and bioapplications. In: Nanodiamonds. Springer Us. 79-116.

[2]. Cheung RCF, Ng TB, Wong JH, Yee Chan WY (2015) Chitosan: An Update on Potential Biomedical and Pharmaceutical Applications. Mar Drugs.

13(8): 5156–5186.

[3]. Ahn MR, Kunimasa K, Ohta T, Kumazawa S, Kamihira M, et al., (2007) Suppression of tumor-induced angiogenesis by Brazilian propolis: Major component artepillin C inhibits in vitro tube formation and endothelial cell proliferation. Cancer Lett. 252 (2): 235-243.

[4]. Amaral, RC, Gomes RC, Rocha Dos Santos WM, Abreu SLR, et al., (2006) Periodontitis treatment with Brasilian green propolis gel. Pharmacologyonline. 3: 336-341.

[5]. Bankova V, Christov R, Kujumgiev A, Marcucci MC, Popov S (1995) Chemical composition and antibacterial activity of Brazilian propolis. Z Naturforsch C. 50 (3/4): 167-172.

[6]. Bankova VS, Christov R, Popov S, Marcucci MC,Tsvetkova I, et al., (1999) Antibacterial activity of essential oils from Brazilian propolis. Fitoterapia 70 (2): 190-193.

[7]. Bankova V, Popova M, Trusheva B (2007) Plant origin of propolis: Latest developments and importance for research and medicinal use. Apicultura - De la stiinta la agribusiness si apiterapie, Editura Academic Press. 40-46.

[8]. Aoi W, Hosogi S, Niisato N, Yokoyama N, Hayata H et al., (2013) Improvement of insulin resistance, blood pressure and interstitial pH in early developmental stage of insulin resistance in OLETF rats by intake of propolis extracts. Biochem Biophys Res Commun. 432(4): 650-653.

[9]. Perchyonok VT, Reher V, Zhang S, Basson NJ, Grobler SR (2015) Bioinspired-Interpenetrating Network (IPNs) Hydrogel (BIOF-INPs) and TMD in Vitro: Bioadhesion, Drug Release and Build in Free Radical Detection and Defense. OJST. 5(3): 53-61.

[10]. Perchyonok VT, Reher V, Zhang S, Basson NJ, Grobler SR (2015) Bioactive-Functionalized Interpenetrating Network Hydrogel (BIOF-IPN): A Novel Biomaterial Transforming the Mechanism of Bio-Repair, Bio-Adhesion and Therapeutic Capability – An In Vitro Study. J Interdiscipl Med Dent Sci. 3: 1-5.

[11]. Zhang ZT, Chen DH, Chen L (2002) Preparation of two different serials of chitosan. J Dong Hua Univ. 19(3): 36-39.

[12]. Yuan Y, Chesnutt BM, Haggard WO, Bumgardner JD (2011) Deacetylation of chitosan: Material characterization and in vitro evaluation via albumin adsorption and pre-osteoblastic cell cultures. Materials. 4(8): 1399-1416.

[13]. Tamara VT, Grobler S, Zhang S, Olivier A, Oberholzer T (2013) Insights into chitosan hydrogels on dentine bond strength and cytotoxicity. OJST. 3: 75-82.

[14]. Grobler SR, Olivier A, Moodley D, Kotze TvW (2004) Cytotoxicity of two concentrations of a dentine bonding agent on mouse 3T3 and human fibroblast cell lines. SADJ. 59(9): 368-372.

[15]. Moodley D, Grobler SR, Olivier A (2005) Cytotoxicity of a dentine bonding agent on four different cell lines. SADJ. 60(6): 234-236.

[16]. Grobler SR, Olivier A, Perchyonok TV, Moodley D, Osman Y (2014) Cytotoxic effect of chitosan-H, resveratrol, b-carotene and propolis and their chitosan hydrogels on Balb/c mouse 3T3 fibroblast cells. Inter J Dent Oral Science.1(2): 10-14.

[17]. Zhang H, Neau SH (2001) In vitro degradation of chitosan by a commercial enzyme preparation: Effect of molecular weight and degree of deacetylation.

Biomaterials. 22(12): 1653-1658.

[18]. Hidaka Y, Ito M, Mori K, Yagasaki H, KafrawyAH (1999) Histpathological and immunohistochemical studies of membranes of deacetylated chitin derivatives implanted over rat calvaria. J Biomed Mater Res. 46(3): 418-423.

[19]. Hamilton V, Yuan Y, Rigney DA, Chesnutt, BM, Puckett AD, et al., (2007) Bone cell attachment and growth on well-characterized chitosan films.

Polym Int. 56(5): 641-647.

[20]. Chatelet C, Damour O, Domard A (2001) Influence of the degree of acetylation on some biological properties of chitosan films. Biomaterials. 22: 261268.

SR Grobler, A Olivier, HW Kruijsse, VT Perchyonok (2016) Cytotoxicity of Different Degrees of De-Acetylated Chitosan on 3T3- and Two Human Tooth Fibroblast Cell-Lines. Int J Dentistry Oral Sci. 3(10), 337-339.

A Possible Functional Role of HSP27 as a Molecular Chaperone of Wnt1 in Cell Differentiation of Pleomorphic Adenomas - By Toshiyuki Kawakami

International Journal of Dentistry and Oral Science (IJDOS) ISSN: 2377-8075 A Possible Functional Role of HSP27 as a Molecular Chaperone of Wnt1 in Cell Differentiation of Pleomorphic Adenomas

Research Article

Ueda Y1,2, Nakano K3,4, Ochiai T4, Yoshida W5, Sugita Y5, Kubo K5, Maeda H5, Hasegawa H4, Kawakami T1* Hard Tissue Pathology Unit, Matsumoto Dental University Institute for Oral Science, Shiojiri, Japan.

Department of Oral Surgery, KKR Hirakata Kohsai Hospital, Hirakata, Japan.

3 Department of Oral Pathology and Medicine, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

4 Department of Oral Pathology, Matsumoto Dental University School of Dentistry, Shiojiri, Japan.

5 Department of Oral Pathology, School of Dentistry, Aichi Gakuin University, Nagoya, Japan.

1

2

Abstract The study focused on the differentiation of parenchymal tumor cells in pleomorphic adenomas. According to our previous published data, Wnt1 is involved in squamous differentiation of basaloid cells in solid nests and small cuboidal cells in duct-like structures during cell differentiation in pleomorphic adenoma. Furthermore, HSP27 sometimes act as a molecular chaperone in neoplastic cells. Thus, immunohistochemistry was performed using Wnt1 and HSP27. A total of 30 cases of pleomorphic adenoma were re-evaluated histologically and categorized based on WHO classification. The mean age of the patients is 51.5 years consisting of 13 males and 17 females.

Fourteen tumors were located in the palate, 5 in the parotid gland, 4 in the mandibular gland, 3 in the upper lip, 3 in the buccal mucosa and 1 in other region. Wnt1 and HSP27 expressions were observed under a light microscope. Wnt1 was detected in almost all tumor cells. The basaloid cells with squamous metaplasia and small cuboidal cells forming duct-like structures were strongly positive to Wnt1. The immunofluorescent staining pattern of Wnt1 and HSP27 was consistent with previous results. The results suggest that Wnt1 and HSP27 are involved in tumor cell differentiation of basaloid cells in solid nests and small cuboidal cells in duct-like structures. Moreover, HSP27 is highly involved in cell differentiation such as the formation of squamous metaplasia in solid tumor nests. Since HSP27 expression was similar to Wnt1, it can be inferred that HSP27 works as possible molecular chaperone in Wnt1 signaling.

Introduction Pleomorphic adenoma (PA) is the most frequently occurring benign epithelial tumor of the salivary gland [1]. It has a wide spectrum of histopathological features even within the same tumor depending on the location [2, 3].

Wnt1 is known to be involved in cell differentiation and proliferation [4]. We showed that Wnt1 is involved in cell differentiation in 2 ways: proliferation of duct-like structures via the b-catenin pathway and differentiation of basaloid cells with squamous

metaplasia via the non b-catenin pathway. Furthermore, we also mentioned that Wnt1 is greatly involved in the differentiation of various tissues in PA [5].

HSP27 is a protein that belongs to the small family of heat shock proteins. Although it is generally present in the cytoplasm, the expression shifts to the nucleus in response to stress [6]. It works as molecular chaperone to other proteins during repair.

Hence, we hypothesized the possibility of HSP27 functioning as a molecular chaperone during cell differentiation. Immunohistochemistry was performed in sections of PA and the results were

*Corresponding Author: Toshiyuki Kawakami, Professor, Hard Tissue Pathology Unit, Department of Hard Tissue Research, Matsumoto Dental University Graduate School of Oral Medicine, Shiojiri, 399-0781, Japan.

Tel: +81-263-51-2035 E-mail: kawakami@po.mdu.ac.jp.

Received: September 26, 2016 Accepted: October 18, 2016 Published: October 19, 2016 Citation: Ueda Y, Nakano K, Ochiai T, Yoshida W, Kawakami T, et al., (2016) A Possible Functional Role of HSP27 as a Molecular Chaperone of Wnt1 in Cell Differentiation of Pleomorphic Adenomas. Int J Dentistry Oral Sci. 3(10), 340-343.

Copyright: Kawakami TŠ 2016. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Ueda Y, Nakano K, Ochiai T, Yoshida W, Kawakami T, et al., (2016) A Possible Functional Role of HSP27 as a Molecular Chaperone of Wnt1 in Cell Differentiation of Pleomorphic Adenomas.

Int J Dentistry Oral Sci. 3(10), 340-343.

observed under the light microscope.

Materials and Methods Samples were obtained from the Department of Oral Pathology, Faculty of Dentistry, Aichi Gakuin University. Histopathological re-evaluation of the files diagnosed as PA was done and 30 cases of PA with typical morphology based on WHO classification were utilized (Table 1). Briefly, the specimens were fixed in 4% neutral buffered formalin solution, embedded in paraffin blocks, serially sectioned into 4 micron thickness, dehydrated in series of alcohol, stained with HE and re-examined under the light microscope.

Fluorescence immunohistochemistry (FIHC) using rabbit polyclonal Wnt1 antibody (anti-Wnt1, Abcam, Cambridge, UK; 1/100) and mouse monoclonal HSP27 antibody (anti-HSP27, Abcam, Cambridge, UK; 1/100) were carried out. After deparaffinization, specimens were pre-treated in citrate buffer (citric acid buffer, pH 6.0, Mitsubishi Chemical Medience, Tokyo, Japan) placed in microwave for 1 min. Serum-free protein (Dako, Japan Co., Ltd, Tokyo, Japan) was used for blocking at room temperature for 30 min. The primary antibodies (rabbit polyclonal Wnt1 and mouse monoclonal HSP27 antibodies) were each diluted with CanGetSignal (Toyobo, Osaka, Japan) in 100-fold and incubated for 16 hours at 4°C. For secondary antibodies, donkey anti-rabbit IgG H&L (Alexa Fluor 594; Abcam, Cambridge, UK; 1/200) and donkey anti-mouse IgG H&L (Alexa Fluor 488, Abcam, Cambridge, UK, 1/200) were each diluted in CanGetSignal (Toyobo, Osaka, Japan) in 200-fold and allowed to react at room temperature for 1 hr. Nuclear stain was revealed using 1mg/ml DAPI incubated for 3 min. Then after, specimens were washed with TBS and mounted using Fluorescent Mounting Medium (Dako Japan Co., Ltd., Tokyo, Japan).

The study was approved by the Ethics Committee of Aichi-Gakuin University, School of Dentistry under the title ‘Basic, Clinical and Pathological Research in the Elucidation of Pathogenesis and Development of Diagnostic Method of Salivary Gland Tumors’ (Number 284).

Results Although Wnt1 was expressed by many tumor cells, strong positive expression was primarily seen in solid tumor nests. Moreover, increased expression was observed in areas were cells differentiated into squamous epithelial-like cells (Figure 1-a,b). Wnt1 was localized in small cuboidal cells in the outer lumen forming ductlike structures. The expression seemed to decrease in polygonal cells (Figure 2-a). Wnt1 was strongly expressed by basaloid cells showing squamous metaplasia however the expression decreased in squamous-like cells (Figure 3-a).

HSP27 was expressed by many tumor cells and the positive reaction was observed strongly in solid tumor nests (Figure 1-c, d). Tumor cells that form duct-like structures expressed intense HSP27 in the cytoplasm of tumor cells outside the lumen. Positive nuclear reactions were also noted (Figure 2-b). Positive reaction was observed in the cytoplasm of many squamous-like tumor cells inside the solid nests. Those tumor cells also showed partial positive reaction in the nucleus. In particular, intense expression was observed in basaloid cells during their transition into keratinocytes (Figure 3-b).

The expressions of Wnt1 and HSP27 tend to be similar and this was evident in the superimposition of the localization of Wnt1 and HSP27 as shown by double immunofluorescence staining (Figures 2-c, d, 3-c, d).

Table 1. Cases Examined.

Age Average 51.5

Sex Male 13 Female 17

Location Palate Parotid Mandibular gland Upper lip Buccal mucosa Other

14 5 4 3 3 1

Figure 1. FIHC expressions of Wnt1 (a, b) and HSP27 (c, d) in tumor tissues. a: Wnt1; b: Wnt1 + DAPI; c: HSP27; d: HSP27 + DAPI (scale bar=50 μm).

Ueda Y, Nakano K, Ochiai T, Yoshida W, Kawakami T, et al., (2016) A Possible Functional Role of HSP27 as a Molecular Chaperone of Wnt1 in Cell Differentiation of Pleomorphic Adenomas.

Int J Dentistry Oral Sci. 3(10), 340-343.

Figure 2. FIHC localization in duct-like structures. a: Wnt1; b: HSP27 + DAPI; c: Wnt1 + HSP27; d: Wnt1 + HSP27 + DAPI (scale bar=25 Îźm).

Figure 3. FIHC localization in squamous metaplasia area. a: Wnt1; b: HSP27 + DAPI; c: Wnt1 + HSP27; d: Wnt1 + HSP27 + DAPI (scale bar=25 Îźm).

Discussion HSP is a major protein expressed in various organs and tissues in response to cytotoxic stimuli and mechanical stress. HSPs are not only related to heat shock but are also stimulated by other various pathological conditions like radiation, enzyme, heavy metals, arsenic, ethanol and stress caused by active enzymes and amino acid derivatives [7]. It is also involved in cell injury, defense, repair and in homeostasis [8]. HSPs are classified according to molecular weight, size and structural type. In particular, HSP27 has been involved in various cellular differentiation [9]. Many of the HSPs are expressed in response to cellular stress. HSPs have been associated with suppression of protein breakdown and are know to function in the repair of degraded protein. The low molecular weight HSPs have been regarded as molecular chaperones associated in cell growth and differentiation [10-13]. Although Fujita et al., mentioned that HSP27 is highly involved in cell differentiation in ameloblastoma [14], our search did not reveal a study of HSP in tumors affecting the oral cavity.

Wnt1 has been investigated in various areas, especially in oral tumors such as calcified cystic odontogenic tumor, odontoma, calcifying epithelioma, ameloblastoma, craniopharyngioma, etc.

Wnt signaling pathway has been associated with ghost and shadow cells. Ghost cells showed strong positive reaction to antibodies against hair proteins localized in the nucleus and cytoplasm through b-catenin pathway. Epithelial cells also showed increased

expression and showed localization of Lef-1 in the nucleus. Involvement of Wnt signaling pathway is believed to be responsible for the appearance of these cells [12]. As described above, Wnt is a typical signal that controls the differentiation of cell and tissue growth. In addition, to homeostasis and cell proliferation, researches are on its way to determine its association in suppressing tissue differentiation in tumors [13].

Our results showed that Wnt1 is involved in cell differentiation in PA. In particular, Wnt1 was detected in peripheral cells in squamous metaplasia and in small cuboidal cells in duct-like structures. It was also believed that Wnt signaling is mediated by b-catenin pathway. Although a strong reaction was observed in basaloid cells surrounding squamous metaplasia, a weak reaction during the transition to prickle cell layer was also reported [5].

Wnt1 expression observed in this study is consistent with our previous report. Furthermore, HSP27 was also localized in the same sites with Wnt1. Fujita et al., [14] previously mentioned that HSP27 is expressed during the differentiation of cells into squamous metaplasia and said to be deeply involved in cell differentiation in ameloblastoma. Accordingly, the same expression in squamous metaplasia was also observed in PA. Since this is consistent with Wnt1 expression, HSP27 is presumed to have worked as a molecular chaperone of Wnt1. Furthermore, since Wnt1 and HSP27 were both expressed by cells forming duct-like structures and those that underwent squamous metaplasia, it is

Ueda Y, Nakano K, Ochiai T, Yoshida W, Kawakami T, et al., (2016) A Possible Functional Role of HSP27 as a Molecular Chaperone of Wnt1 in Cell Differentiation of Pleomorphic Adenomas.

Int J Dentistry Oral Sci. 3(10), 340-343.

possible that HSP27 worked as a molecular chaperone of Wnt1.

This research material is only limited 30 cases. However, the most staining results of almost cases indicated the same tendency. More examinations using large number of cases may be necessary, but I believe that the present result is effective as a temporary quarrel report. In near future date, we will present the large number cases examination results of the relation research.

Conclusions Pleomorphic adenoma (PA) has a wide spectrum of histopathological features even within the same tumor depending on the location. Regarding the cell differentiation, Wnt1 is known to be involved in PA. Further, HSP27 works as molecular chaperone to other proteins during repair. Thus, we hypothesized the possibility of HSP27 functioning as a molecular chaperone during cell differentiation. Our present results suggested that Wnt1 and HSP27 are involved in cell differentiation in squamous metaplasia and duct-like structures in PA, Number of cases of this subject of research was 30 and a limited number, but the above mentioned result believes indicated directionality not to be wrong. Thus, HSP27 may have function as a molecular chaperone of Wnt1 in PA.

Acknowledgement This research was supported by the Japan Society for the Promotion of Scientific Research, Basic Research C (# 23592951, #26463031).

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Recent Advances in Role of Matrix metalloproteinases in Some Dental Diseases - By Masood-ul-Hassan Javed,

International Journal of Dentistry and Oral Science (IJDOS) ISSN: 2377-8075 Recent Advances in Role of Matrix metalloproteinases in Some Dental Diseases

Review Article

Javed MU Professor of Basic Medical Sciences, College of Medicine King Saud bin Abdulaziz University, HS National Guards Health Affairs King Abdulaziz Medical City Jeddah, Saudi Arabia.

Abstract The common types of dental diseases are caries, periapical, pulpitis, gingivitis, oral infectious diseases and hereditary lesions. These chronic diseases are supposed to be due to interaction between the bacteria in oral cavity and the host. The progression of these diseases needs some major changes in the biochemistry and physiology of connective tissues where different types of collagens are present. In dental diseases the specific collagen is degenerated/lost in connective tissue probably due to matrix metalloproteinases (MMPs), reactive oxygen species (ROS), apoptosis and antioxidants. In this short review we discussed only the role MMPs in dental carries and periodontitis.

Keywords: Dental Caries, Periodontitis, MMP.

Introduction Dental health is not only essential but also an integral part of systemic health throughout the human life. Dental diseases are varied and may be of soft tissue origin (gingivitis) or may be of hard tissue (caries or periodontitis). Dental diseases may be simple like caries (cavitation) or complex one such as tumors and cyst of dental origin and may also cause more dangerous and life threatening problems [24, 41]. Dentition (teeth) plays an important role in mastication (chewing), speech (pronunciation of certain consonants and facial expression), cheek fullness(avoiding aged appearance) and also maintains facial height. The dental diseases may affect any of the above said functions of teeth [26]. The common types of humanâ&#x20AC;&#x2122;s dental diseases are caries, periapical and pulpal inflammation (pulpitis), gingival (gingivitis), periodontal (gumperiodontics) problems, oral infectious diseases, trauma from injuries, and hereditary lesions [41]. The inflammatory process is often associated with free radicaldamage and oxidative stress [39], [24, 29]. These diseases are considered as chronic problems and are supposed to be due to interaction between the bacteria in oral cavity (oral flora) and the host. The progression of these diseases

needs some major changes in the biochemistry and physiology of connective tissues because these tissues hold cells together to make tissues and organs in the body [42]. One of the important proteins in the connective tissue is the collagen, which is of different types and depends upon the tissues where it is present [19].

It is observed and suggested that in dental diseases the specific collagen is degenerated or lost in connective tissue probably due to matrix metalloproteinases (MMPs), reactive oxygen species (ROS), apoptosis and antioxidants. In this short review we discussed only the role MMPs in dental carries and periodontitis.

Matrix Metalloproteinases (MMPs) These endopeptidases enzymes belong to the M10A subfamily of metallopeptidases, which contain Znad methioninein their active site [1, 28]. They cut the extracellular matrix (ECM)/core matrisome proteins into various small peptides by hydrolyzing inner peptide bonds. Their activity depends upon Ca++ ions. MMPs are different from other endopeptidases because they do not function in the absence of metal ions [37]. These enzymes not only degrade collagens and ECM proteins but can also regulate

*Corresponding Author: Masood-ul-Hassan Javed, BSc, PhD, Professor of Basic Medical Sciences College of Medicine King Saud bin Abdulaziz University-HS National Guards Health Affairs King Abdulaziz Medical City Jeddah-21423, Saudi Arabia.

Tel: 00966592739044 Fax: 00966222 45769 E-mail: masoodjaved@hotmail.com Received: September 05, 2016 Accepted: October 13, 2016 Published: October 18, 2016 Citation: Javed MU (2016) Recent Advances in Role of Matrix Metalloproteinases in Some Dental Diseases. Int J Dentistry Oral Sci. 3(10), 344-347.

Copyright: Javed MUŠ 2016. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Javed MU (2016) Recent Advances in Role of Matrix Metalloproteinases in Some Dental Diseases. Int J Dentistry Oral Sci. 3(10), 344-347

the activity of many bioactive molecules, like cell surface receptors, the release of apoptotic ligands (FAS ligand) and cytokine/ chemokine inactivation [16, 35]. Thus MMPs are also called as hydrolyse components of the ECM because they have the tendency/activity to break matrix components. These MPPs modulate the activity of the above-mentioned factors either by direct cleavage, or release them from ECM bound stocks, like buffers [5]. These enzymes are also supposed to play a major role in cell behaviors during critical events (physiological or pathological) of life such as cell migration, proliferation (dispersion/adhesion), differentiation, development, embryogenesis, angiogenesis, apoptosis, cell defense, as well as wound healing [1]. In this way they coordinate remodelling of extracellular matrix by accurate synthesis and depletion of proteins like collagens and others in ECM which is promoted and controlled by various proteases [28].

These include aspartic protease, cysteine protease, MPPs and serine protease [19]. Up till now the researchers have identified 24 MMP genes in humans and 26 members have been listed for these MPPs [23]. These MMP enzymes have been classified in to 6 major groups namely collagenases, gelatinases, matrilysins, membrane type MMPs, other MPPs and stromelysins [9]. All six MMPs contain furin cleavage site in the pro-peptide.

In predentine (odontoblasts) and dentin, another class of MMPs are present called as collagenase MMP-1, the gelatinases MMP-2 and MMP-9, MMP-3 (stromelysin-1), the MMP-2 activator MT1MMP, and enamelysin (MMP-20) [9].

The collagenases are of different types depending upon their site of action and product formation. They degrade triple-helical fibrillar structure of collagens into various specific fragments and are the major components dentin (and cartilage and bone as well).

The different types of gelatinases mainly act on type-IV collagen and gelatin. The stromelysins act on proteins but unable to cleave the triple-helical fibrillar collagens [23].

It is known that all these MMPs are synthesized and secreted by the tissue cells as inactive form known as Zymogen (proenzymes) and then are activated by complex extracellular modulators [13].

As mentioned by Pardo, et al., (2016)[28], these MMPs are activated mostly in ECM or at the cell surface by molecules like α2-macroglobulin and the membrane-associated reversion-inducing Cys-rich protein with Kazal motifs (RECK). However, some of them can also be activated in side the intracellular organelles, including nuclear localization where they act on intracellular substrates, or may function as transcription factors [22]. These enzymes in turn are controlled by endogenous tissue inhibitors of (called as TIMPS) that inhibit the activity of MMPs and keep them at physiological desired levels (by the processes of homeostasis). At present the family of TIMPS consists of TIMP-1, TIMP-2, TIMP-3 and TIMP-4 [23, 38]. This remodelling of ECM is quite complex because these MPPs and their modulators are strongly regulated at multiple levels. Some are controlled at gene expression (transcriptional regulation), other by growth factors, cytokines, hormones, and cell-ECM, and some by cell-cell interactions. The stability of their mRNA, efficiency of protein translational and role played by microRNAs are thought to involve as post-transcriptional regulatory processes [13, 28]. Imbalance in the homeostasis of MMP and TIMP is responsible for the development of pathological conditions [1, 6, 28, 33]. Thus it is the dis-regulation of the balance between MMPs and TIMPs that

leads to many chronic systemic as well as dental diseases. It is observed that some nonspecific endogenous inhibitors can regulate the activity of MMP [9]. Similarly the search of exogenous modulators for these MMPs or TIMP can be used as drugs to treat various diseases of ECM including dental in origin. Most of the synthetic inhibitors have a chelating group that binds very strongly (like irreversible inhibitor) with the zinc atom at the active site of the MMP. The most common compounds in this category are hydroxamates, thiol, carboxylates and phosphinyls. Hydroxymates are particularly potent inhibitors of MMPs and other zinc-dependent enzymes, due to their bidentate chelation of the zinc atom. Other substitutents of these inhibitors are usually designed to interact with various binding pockets on the MMP of interest, making the inhibitor more or less specific for given MMPs. Clinical trials for many of these agents showed poor pharmacokinetic and lack of specificity as anticancer or for cardiovascular diseases.

Thus they showed many undesired side effects [2, 7, 27, 40]. The implication of MMPs and TIMPs in the dental disorders is vast and promising but due to complicated types very little is known the physiology of each of the MMPs. Therefore the understanding of the physiology & Biochemistry of these enzymes from the molecular, to the cellular and tissue levels, where these molecular complexes operate, will be necessary for the development of newtherapeutic agents [14].

Dental Caries It is one of the most common infectious diseases worldwide [29]. As quoted by the WHO (2012), “worldwide, 60–90% of school children and nearly 100% of adults have dental cavities, often leading to pain and discomfort”. It is an irreversible disease caused mostly by Streptococcus mutans, Streptococcus sobrinus and Lactobacillus species of the oral cavity (oral flora). These bacteria form a complex community, which adheres on surfaces of tooth in a gelatinous mat, orbiofilm, or dental plaque. These bacteria produce lactic acid (mostly) by degrading sugar component of diet. This acid then diffuses through dental calcified tissues and thus the pH at this point drops below 5.5. The acidic pH and probably heat activate “host” pro MMPs further from both dentin and saliva. This activation is supposed to be due to the conformational change of the propeptide and thus induce the cysteine switch, which is necessary for the activation process [9, 10]. It is suggested that although the MMPs are activated by the acidic pH but they cannot degrade components of organic matrix at this pH. As the pH drops the salivary buffer system neutralizes the acid and may increase in the pH towards 7.4 at the spot of demineralised dentin. This pH environment allows activated MMPs to degrade the organic matrix [9, 34]. The activated enzymes then dissolute mineral crystals in that local area and causes cavitation [8, 23]. It is known that dentin contains more organic material and water than enamel so this part of tooth acts, thus more preferable substrate for enzymatic degradation (due to host or bacterial proteinases). The organic components of dentine consist of type-I collagen (90%) and phosphorylated proteins called dentinphosphosialoprotein (DSPP) (10%). DSPP forms dentin sialoprotein (DSP) & dentin phosphoprotein (DPP) immediately after its secretion [23].

It is also observed that many Growth Factors (GFs) like Transforming GF-beta, fibroblast growth factor-2 (FGF-2) & Insulin like growth factor 1 and 2 (ILGF-1 &-2) are also present in

Javed MU (2016) Recent Advances in Role of Matrix Metalloproteinases in Some Dental Diseases. Int J Dentistry Oral Sci. 3(10), 344-347

dentin. According to researchers as dental caries starts and ECM degradation takes place, these GFs are then released and stimulate odontoblasts to form compensatory dentin materials [9, 12].

It is proposed that bacterial collagenases are the main “culprit” for the catabolism of organic matrix. However, now it has been found that MMPs derived from host have more important role to degrade organic matrix of dentin. These enzymes have been to be present both in dentin and saliva. It has been proposed that gingival crevecular fluid (GCF) is the main source of salivary MMPs [3, 9, 31].

Biochemical Basis of Periodontitis Periodontitis is an inflammatory condition of gingival tissue, due to which there is a loss of periodontal ligament that is attached with bones for functional support. To initiate and development of this disease, a plaque is observed at the affected areas. At the same time it is also believed that the physiological response of host to the pathogen is mainly responsible bone loss and breakdown of the respective connective. It is proposed that the MMPs, prostanoids (PGs) and cytokines originated from host are responsible for activation of the osteoclastic activity, which then destroys the periodontium tissue [36]. Due to exogenous and endogenous causative factors collagen degradation then starts in persons susceptible to this disease. Inflammatory process then extends laterally and apically and towards deeper connective tissues and bone as well [23]. Due to this inflammatory process, at the place of lesion, phagocytes and other defence cells are recruited (known as inflammatory cells). The macrophages secrete interleukins (ILs), PG-E2, MMPs and tumor necrosis factor alpha (TNFα) [25, 36]. For this disease, initial hypothesis was that collagenases that initiate the process of this disease are secreted from infected or normal oral flora. However now it is believed that the host collagenases, especially MMP-1 (MMP- 1) cut collagens of the connective tissue at a single point and thus only two fragments are produced [11]. These are the best substrates for microbial proteolytic enzymes that produce different types of short peptides because they hydrolyse these fragmented collagens at multiple sites [32, 36]. In an experimental study, Makela et al., (1994) [20] have demonstrated that compared to normal tissues there were higher level of MMP-2 & MMP-9 gelatinases in periodontitis tissues. At the same time as the inflammatory process moves towards the apical side then the levels of ILs, PGs, and TNFα become more compared to normal tissues. This altered micro-environmental activates the osteoclasts that initiate the resorption of alveolar bone.

It is also believed that MMP-2 plays vital role in bone remodelling while MMP-14 (present at border of osteoclasts) helps for the interaction between matrix and osteoclasts [17, 18]. The collagen remnants left over by osteoclasts in the lacunae of affected bones are cleared by MMp-13 already present in resorption lacunae [30].

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