13- Sabreen F.docx J Bagh College Dentistry Vol. 28(3), September 2016 Expression of Syndecan 1 Oral Diagnosis 82 Expression of Syndecan 1 on Periodontium treated with Topical Application of Aloe-Vera Sabrin Sami Abed, B.D.S. (1) Athra'a Y. Al-Hijazi, B.D.S., M.Sc. Ph.D. (2) ABSTRACT Background: Periodontium mainly exposed to injury by trauma or pathologic diseases, Aloe vera is a plant has many basic ingredients in its extracted gel that acts as wound healing accelerator in addition to that it's safe, and economical and without recordable of side effect. This study aimed is to evaluate the effect of topical application of Aloe vera on expression of syndecan -1 by periodontium tissue. Materials and methods: Thirty six male Albino rats were subjected for periodontium defect by electric scaler on the distal sides of both lower anterior teeth. The animals divided into two groups; control group (without treatment) and the experimental group treated with 1µLAloe vera gel/normal saline. Periodontal healing was examined at periods (3, 7, 14 days) for immunohistochemical localization of syndecan 1. Results: Immunohistochemical examination of this study revealed that the aloe vera treatment increase expression of syndecan 1 by epithelial cell, osteoblasts, fibroblast, stromal cells and with highly significant differences in comparison with control and saline. Conclusion: Aloe vera gel may affect the expression of syndecan 1 which seems to play a role in periodontium healing. Key words: Aloe vera, periodontium, syndecans. (J Bagh Coll Dentistry 2016; 28(3):82-86). INTRODUCTION Periodontium injury healing is a series of biological processes; include migration, adhesion, proliferation, and differentiation of several cell types. All these activities are triggered by chemo- attraction of the cells; polypeptide mediators bind to their cell-surface receptors, integrins bind to extracellular matrix components, and different growth factors regulate different cell functions. The process is ending with the formation and maturation of a new extracellular matrix (1-3). Syndecan 1 is type-I-transmembrane cell surface Heparansulphat proteoglycans (HSPGs) (4). Syndecan -1 has an essential role in mediating cell proliferation, cell migration, and cell-matrix interactions by binding various extracellular matrix proteins via its heparansulphate chains during wound healing (5-6). Aloe vera is a plant has many health benefits such as anti-inflammatory, antibacterial, antiviral, and wound healing acceleration, used in different fields like cosmetology, dermatology, and dentistry (7). Aloe vera gel consists of many ingredients such as vitamins, enzymes, minerals, hormones and polysaccharides (acemannan),these contents play important roles in wound healing process. The present study has been prepared to illustrate the effect of local application of aloe vera gel on expression of syndecan -1 in periodontium wound healing. (1) Master Student. Department of Oral Diagnosis, College of Dentistry, University of Baghdad. (2)Professor, Department of Oral Diagnosis, College of Dentistry, University of Baghdad. MATERIALS AND METHODS Animals Thirty six albino rats weighting (250-400) gram, aged (6-8) months were used in the present study, maintained under control conditions of temperature, drinking and food consumption. All experimental procedures were carried out in accordance with the ethical principles of animal experimentation. The animals were divided into control and experimental group: A. Control group the periodontium defect left without any treatment and its number represented the all number of the following experimental groups as the right side of each animal considered to be the control. B. Experimental group was subdivided into following groups according to the applicable of biomaterials includes: • Group 1 contains (18) rats, the periodontium defect treated with 1µL normal saline. • Group 2 contains (18) rats, the periodontium defect treated with 1µL of A. vera gel. Materials - Aloe vera Gel 87.399%, Phytocare Company. - Anti-Syndecan– 1 monoclonal from Abcamcompany UK (ab34164) Methods Immunohistochemical evaluation The animals were sacrificed at 3,7,14 days (six rats for each period). The specimens were fixed by 10% buffered formalin for 3 days. The samples J Bag 1 J Bagh College Dentistry Oral Diagnosis then demineralized, paraffin. Five prepared for localization of immune syndecan 1. Determination of immunohistochemical results for syndecan 1 For each specimen, the number of positive expression of syndeca evaluating 100 cells for each power fields (HPF) counting the positive one, then the mean of count for eight slides for each sample(8) Statistic Analyse A. Descriptive data analysis. B. Inferential data analysis Figure 1: Control group with immunohistochemical expression of syndecan 1 (DAB At 3rd X20. (B) At 7 (arrow heads) .DAB stain X20. (C) At 14 bone (AB) shows positive immune reaction for syndecan A h College Dentistry Oral Diagnosis then demineralized, dehydrated, and embedded in . Five µm prepared for localization of immune syndecan 1. Determination of immunohistochemical results for syndecan 1 For each specimen, the number of positive expression of syndeca evaluating 100 cells for each power fields (HPF) counting the positive one, then the mean of count for eight slides for each ). Statistic Analyses Descriptive data analysis. Inferential data analysis Figure 1: Control group with immunohistochemical expression of syndecan 1 (DAB rd day, Keratinocyte cell (arrow)expressed positive DAB stain for syndecan X20. (B) At 7th day, Woven bone (WB) shows positive expression of syndecan (arrow heads) .DAB stain X20. (C) At 14 bone (AB) shows positive immune reaction for syndecan h College Dentistry dehydrated, and embedded in µm thickness of prepared for localization of immune Determination of immunohistochemical results For each specimen, the number of positive expression of syndecan 1 was determined by evaluating 100 cells for each slide in five high power fields (HPF) counting the positive one, then the mean of count for eight slides for each Descriptive data analysis. Inferential data analysis Figure 1: Control group with immunohistochemical expression of syndecan 1 (DAB day, Keratinocyte cell (arrow)expressed positive DAB stain for syndecan day, Woven bone (WB) shows positive expression of syndecan (arrow heads) .DAB stain X20. (C) At 14 bone (AB) shows positive immune reaction for syndecan fibroblast shows negative (arrows) .DAB stain X20. C h College Dentistry Vol. 2 dehydrated, and embedded in thickness of section was prepared for localization of immune-reaction of Determination of immunohistochemical results For each specimen, the number of positive n 1 was determined by slide in five high power fields (HPF) counting the positive one, then the mean of count for eight slides for each Descriptive data analysis. Figure 1: Control group with immunohistochemical expression of syndecan 1 (DAB day, Keratinocyte cell (arrow)expressed positive DAB stain for syndecan day, Woven bone (WB) shows positive expression of syndecan (arrow heads) .DAB stain X20. (C) At 14th bone (AB) shows positive immune reaction for syndecan fibroblast shows negative (arrows) .DAB stain X20. Vol. 28(3), September 83 dehydrated, and embedded in section was reaction of Determination of immunohistochemical results For each specimen, the number of positive n 1 was determined by slide in five high power fields (HPF) counting the positive one, then the mean of count for eight slides for each RESULT Result for i syndecan 1 in each group osteoblast group records a strong expression shows epithelial cell layers of results was illustrated by lymphocyte in lamina properia ligament shows 3). highly significant differences for the expression of syndecan 1 epithelial cells, cell, bone cells saline groups. Figure 1: Control group with immunohistochemical expression of syndecan 1 (DAB day, Keratinocyte cell (arrow)expressed positive DAB stain for syndecan day, Woven bone (WB) shows positive expression of syndecan th day,View for periodontal ligament (PDL) , alveolar bone (AB) shows positive immune reaction for syndecan fibroblast shows negative (arrows) .DAB stain X20. B September 2016 RESULTS Result for immuno syndecan 1 Expression of syndecan 1 in each group was osteoblast cell, group records a strong expression shows a negative expression of Syndecan epithelial cell layers of results was illustrated by lymphocyte in lamina properia. Some f ligament shows 3). Table (1) shows that highly significant differences for the expression of syndecan 1 epithelial cells, cell, bone cells saline groups. Figure 1: Control group with immunohistochemical expression of syndecan 1 (DAB day, Keratinocyte cell (arrow)expressed positive DAB stain for syndecan day, Woven bone (WB) shows positive expression of syndecan day,View for periodontal ligament (PDL) , alveolar bone (AB) shows positive immune reaction for syndecan -1 by cementoblast (arrow heads) fibroblast shows negative (arrows) .DAB stain X20. 2016 mmunohistochemical Expression of syndecan 1 was detected by cementoblast cell, cell, and keratinocyte group records a strong expression negative expression of Syndecan epithelial cell layers of gingivae, while results was illustrated by lymphocyte in lamina Some fibroblasts cell in periodontal ligament shows negative expression shows that A. highly significant differences for the expression of in periodontium cells include epithelial cells, stromal cell, cell, bone cells in comparison Figure 1: Control group with immunohistochemical expression of syndecan 1 (DAB day, Keratinocyte cell (arrow)expressed positive DAB stain for syndecan day, Woven bone (WB) shows positive expression of syndecan day,View for periodontal ligament (PDL) , alveolar 1 by cementoblast (arrow heads) fibroblast shows negative (arrows) .DAB stain X20. Expression of Syndecan histochemical findings Expression of syndecan 1for over all periods detected by cementoblast cell, and keratinocyte. Experimental group records a strong expression .Saline group negative expression of Syndecan gingivae, while a positive results was illustrated by lymphocyte in lamina ibroblasts cell in periodontal negative expression (figure vera group records a highly significant differences for the expression of in periodontium cells include stromal cell, periodontal ligament comparison to control and Figure 1: Control group with immunohistochemical expression of syndecan 1 (DAB Stain) ( day, Keratinocyte cell (arrow)expressed positive DAB stain for syndecan -1.DAB stain day, Woven bone (WB) shows positive expression of syndecan -1by osteoblast day,View for periodontal ligament (PDL) , alveolar 1 by cementoblast (arrow heads) Expression of Syndecan findings of for over all periods detected by cementoblast cell, xperimental aline group negative expression of Syndecan-1 in a positive results was illustrated by lymphocyte in lamina ibroblasts cell in periodontal figures1, 2, group records a highly significant differences for the expression of in periodontium cells include dontal ligament to control and Stain) (A) 1.DAB stain 1by osteoblast day,View for periodontal ligament (PDL) , alveolar 1 by cementoblast (arrow heads) while Expression of Syndecan J Bag 1 J Bagh College Dentistry Oral Diagnosis Figure 2 1 (DABStain). ( expression for syndecan (arrow).DAB stain X20. (B) Magnifying view for lymphocyte cell (arrows) with positive expression of syndecan saline group by sulcular epithelia (red arrow) ,inflammatory cell (pink arrows).DAB stian X20 Figure (DAB basal cell (red arrow heads),prickle cell (redarrows),inflammatory cell (pink arrows).DAB stain X 10. (B) A A h College Dentistry Oral Diagnosis 2: experimental group (normal saline) with immunohistochemical expression of syndecan 1 (DABStain). (A) At 3 expression for syndecan (arrow).DAB stain X20. (B) Magnifying view for lymphocyte cell (arrows) with positive sion of syndecan saline group by sulcular epithelia (red arrow) ,inflammatory cell (pink arrows).DAB stian X20 Figure 3: experimental group (DAB Stain). (A) At 3 basal cell (red arrow heads),prickle cell (redarrows),inflammatory cell (pink arrows).DAB stain X 10. (B) At 7th day, Lamina propria (LP) shows positive expression of syndecan A h College Dentistry experimental group (normal saline) with immunohistochemical expression of syndecan A) At 3rd day, Epithelial cell (EP) of gingiva of saline group shows negative expression for syndecan -1 ,while lamina properia (LP) shows positive expression by lymphocyte (arrow).DAB stain X20. (B) Magnifying view for lymphocyte cell (arrows) with positive sion of syndecan -1 .DAB stain X40. (C) At 14 saline group by sulcular epithelia (red arrow) ,inflammatory cell (pink arrows).DAB stian X20 experimental group A) At 3rd day, Positive expression of syndecan basal cell (red arrow heads),prickle cell (redarrows),inflammatory cell (pink arrows).DAB stain day, Lamina propria (LP) shows positive expression of syndecan C C h College Dentistry Vol. 2 experimental group (normal saline) with immunohistochemical expression of syndecan day, Epithelial cell (EP) of gingiva of saline group shows negative 1 ,while lamina properia (LP) shows positive expression by lymphocyte (arrow).DAB stain X20. (B) Magnifying view for lymphocyte cell (arrows) with positive 1 .DAB stain X40. (C) At 14 saline group by sulcular epithelia (red arrow) ,inflammatory cell (pink arrows).DAB stian X20 experimental group (Aloe vera) with day, Positive expression of syndecan basal cell (red arrow heads),prickle cell (redarrows),inflammatory cell (pink arrows).DAB stain day, Lamina propria (LP) shows positive expression of syndecan C C Vol. 28(3), September 84 experimental group (normal saline) with immunohistochemical expression of syndecan day, Epithelial cell (EP) of gingiva of saline group shows negative 1 ,while lamina properia (LP) shows positive expression by lymphocyte (arrow).DAB stain X20. (B) Magnifying view for lymphocyte cell (arrows) with positive 1 .DAB stain X40. (C) At 14th saline group by sulcular epithelia (red arrow) ,inflammatory cell (pink arrows).DAB stian X20 (Aloe vera) with immunohistochemical expression of syndecan 1 day, Positive expression of syndecan basal cell (red arrow heads),prickle cell (redarrows),inflammatory cell (pink arrows).DAB stain day, Lamina propria (LP) shows positive expression of syndecan B B September 2016 experimental group (normal saline) with immunohistochemical expression of syndecan day, Epithelial cell (EP) of gingiva of saline group shows negative 1 ,while lamina properia (LP) shows positive expression by lymphocyte (arrow).DAB stain X20. (B) Magnifying view for lymphocyte cell (arrows) with positive th day, Positive expression of syndecan saline group by sulcular epithelia (red arrow) ,inflammatory cell (pink arrows).DAB stian X20 immunohistochemical expression of syndecan 1 day, Positive expression of syndecan -1 by keratinocyte cells include basal cell (red arrow heads),prickle cell (redarrows),inflammatory cell (pink arrows).DAB stain day, Lamina propria (LP) shows positive expression of syndecan 2016 experimental group (normal saline) with immunohistochemical expression of syndecan day, Epithelial cell (EP) of gingiva of saline group shows negative 1 ,while lamina properia (LP) shows positive expression by lymphocyte (arrow).DAB stain X20. (B) Magnifying view for lymphocyte cell (arrows) with positive day, Positive expression of syndecan saline group by sulcular epithelia (red arrow) ,inflammatory cell (pink arrows).DAB stian X20 immunohistochemical expression of syndecan 1 1 by keratinocyte cells include basal cell (red arrow heads),prickle cell (redarrows),inflammatory cell (pink arrows).DAB stain day, Lamina propria (LP) shows positive expression of syndecan Expression of Syndecan experimental group (normal saline) with immunohistochemical expression of syndecan day, Epithelial cell (EP) of gingiva of saline group shows negative 1 ,while lamina properia (LP) shows positive expression by lymphocyte (arrow).DAB stain X20. (B) Magnifying view for lymphocyte cell (arrows) with positive day, Positive expression of syndecan saline group by sulcular epithelia (red arrow) ,inflammatory cell (pink arrows).DAB stian X20 immunohistochemical expression of syndecan 1 1 by keratinocyte cells include basal cell (red arrow heads),prickle cell (redarrows),inflammatory cell (pink arrows).DAB stain day, Lamina propria (LP) shows positive expression of syndecan -1 by plasma Expression of Syndecan experimental group (normal saline) with immunohistochemical expression of syndecan day, Epithelial cell (EP) of gingiva of saline group shows negative 1 ,while lamina properia (LP) shows positive expression by lymphocyte (arrow).DAB stain X20. (B) Magnifying view for lymphocyte cell (arrows) with positive day, Positive expression of syndecan -1 in saline group by sulcular epithelia (red arrow) ,inflammatory cell (pink arrows).DAB stian X20 immunohistochemical expression of syndecan 1 1 by keratinocyte cells include basal cell (red arrow heads),prickle cell (redarrows),inflammatory cell (pink arrows).DAB stain 1 by plasma Expression of Syndecan J Bagh College Dentistry Vol. 28(3), September 2016 Expression of Syndecan 1 Oral Diagnosis 85 cell (red arrows), endothelial cell (pink arrows ), lining blood vessel (BV). DAB stain X40. (C) At 14th day, Osteoclast (arrow) illustrates positive. DAB stain X20 Table1: Statistic analysis of positive expressed cells for syndecan 1 in the studied groups with comparisons significant Type of Pos. Groups No. M SD SE Min. Max. Levene's test ANOVA test L P-value F P-value Epithelial cell Saline 6 2 0.52 0.21 1 2 0.308 0.740 NS 81.47 0.000 HS Control 6 4 0.89 0.37 3 5 Aloe Vera 6 8 1.10 0.45 6 9 Stromal cells (Ging.) Saline 6 4 0.98 0.40 3 5 4.308 0.033 S 20.21 0.000 HS Control 6 7 1.67 0.68 5 9 Aloe Vera 6 8 0.52 0.21 8 9 Periodontal ligament Saline 6 8 0.82 0.33 7 9 5.281 0.018 S 59.69 0.000 HS Control 6 9 0.41 0.17 8 9 Aloe Vera 6 13 1.37 0.56 12 15 Bone cells Saline 6 6 0.63 0.26 5 7 8.015 0.004 HS 74.31 0.000 HS Control 6 11 1.72 0.70 9 13 Aloe Vera 6 20 2.88 1.17 17 24 (*) HS: Highly Sig. at P<0.01; S: Sig. at P<0.05; NS: Non Sig. at P>0.05 DISCUSSION The present study used Aloe vera gel in periodontium defect related to its ability to accelerate wound healing. Aloe vera increased syndecan 1 expression in epithelial cells, precursor progenitor cells and in early stage of cell proliferation of mesenchymal cell, and in inflammatory cells, and cementoblast. These results were in agreement with Filatova et al. (9) study, who show that syndecan-1 is distributed in distinct areas of the epithelium and mesenchyme during the early and late stages of odontogenesis, and with Bernfield et al. (10) and Kero et al. (11) that reported syndecan-1 is the major syndecan in epithelial cells. Positive expression of syndecan-1 in keratinocyte was reported in the present study and this is in agreement with Stepp (12) who suggested that syndecan -1 has a role as a regulator of gene transcription in keratinocytes and keratinocyte activation after injury. The present results illustrates positive immune reaction for syndecan- 1 by osteoclast in their active stage, these results coincide with Pap and Bertrand (13) who found that syndecan-1 present in both osteoblasts and osteoclasts of the alveolar bone that make syndecan-1 may play a role in alveolar bone formation and remodeling. Present findings for saline group which associated with high inflammatory score, shows negative expression of Syndecan-1 in epithelial cell layers of gingiva, while lamina properia shows positive expression by lymphocyte.This result coincides with Götte et al.(14) who found that expression of syndecan-1 in suprabasal keratinocytes of the epithelium was weak or absent in inflamed tissue. Some fibroblast cells show negative expression of syndecan -1, these findings may explained by some cells have the same histological feature of fibroblast and could be myofibroblast cell that appeared mostly in wound tissue. In conclusion, Aloe vera gel increased expression of syndecan -1 in periodontium wound as a result to its property in accelerating biological processes of wound healing. REFERENCES 1. Cillo C, Cantile M, Faiella A, Boncinelli E. Homeobox genes in normal and malignant cells. J Cell Physiol 2001; 188:161-9. 2. Conway EM, Collen D, Carmeliet P. Molecular mechanisms of blood vessel growth. Cardiovasc Res 2001; 49: 507-21. 3. Alpiste-Illueca FM, Buitrago-Vera P, de Grado- Cabanilles P, Fuenmayor-Fernandez V, Gil-Loscos FJ. Periodontal regeneration in clinical practice. Med Oral Patol Oral Cir Bucal 2006; 11: E382-92. 4. Kero D, KalibovicGovorko D, Vukojevic K, Cubela M, Soljic V, Saraga-Babi M. Expression of cytokeratin 8, vimentin, syndecan-1 and Ki-67 during human tooth development. J Mol Histol 2014; 45: 627–40. 5. Stepp MA. Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse. J Cell Sci 2002; 115: 4517–31. 6. Teng YHF, Aquino RS, Park PW. Molecular functions of syndecan-1 in disease. Matrix Biol 2012; 31: 3–16. 7. Sambhav J, Rohit R. Aloe-Vera: A boon in management of dental disease. Int J Pharm Res Sci 2014; 2(1): 18-24. 8. Piatelli A, Rubini C, fioroni M, TripodiD, Strocchi R. Transforming growth factorbeta 1 expression in normal healthy pulps and in those with irreversible pulpitis. Int Endod J 2004; 37:114-9. 9. Anna F, Thimois PM. Distribution of syndecan-1 protein in developing mouse teeth. Front Physiol 2015; 5: 518. J Bagh College Dentistry Vol. 28(3), September 2016 Expression of Syndecan 1 Oral Diagnosis 86 10. Merton B, Martin G, Woo PP, Ofer R, Marilyn L, et al. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 1999; 68:729–77 11. Kero D, Govorko D, Vukojevic K, Cubela M, Soljic V, Saraga-Babic M. Expression of cytokeratin 8, vimentin, syndecan-1 and Ki-67 during human tooth development. J Mol Histol 2014; 45: 627–40. 12. Stepp MA. Defects in keratinocyte activation during wound healing in the syndecan-1-deficient mouse. J. Cell Sci 2002; 115: 4517–31. 13. Pap T, Bertrand J. Syndecans in cartilage breakdown and synovial inflammation. Nat Rev Rheumatol 2013; 9; 43–55. 14. Martin G, Christian K, Isabel R; Kiesel, Pia LW. An expression signature of syndecan-1 (CD138), E- cadherin and c-met is associated with factors of angiogenesis and lymphangiogenesis in ductal breast carcinoma in situ. Breast Cancer Res 2007; 9(1): R8.