THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY In partial fulfilment for the Degree of

IN PERIODONTALLY HEALTHY AND DISEASED INDIVIDUALS BEFORE AND AFTER NON SURGICAL PERIODONTAL

THERAPY

Dissertation submitted to

THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY In partial fulfilment for the Degree of

MASTER OF DENTAL SURGERY

BRANCH II PERIODONTICS

2017 – 2020

CHENNAI

DECLARATION BY THE CANDIDATE

I, hereby declare that this dissertation titled “

EVALUATION OF SALIVARY AND SERUM PERIOSTIN LEVELS IN PERIODONTALLY HEALTHY AND DISEASED INDIVIDUALS BEFORE AND AFTER NON SURGICAL PERIODONTAL THERAPY

” is bonafide and genuine research work carried out by me under the guidance of Dr. S. Elanchezhiyan M.D.S., Professor & Head of the Department, Department of Periodontics, Vivekanandha Dental College for Women, Tiruchengode.

Dr. V. JANANIPPRIYA., Post Graduate Student, Department of Periodontics, Vivekanandha Dental College for Women, Tiruchengode.

CHENNAI

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation titled “

EVALUATION OF SALIVARY AND SERUM PERIOSTIN LEVELS IN PERIODONTALLY HEALTHY AND DISEASED INDIVIDUALS BEFORE AND AFTER NON SURGICAL PERIODONTAL THERAPY

” is a bonafide work done by Dr. V. JANANIPPRIYA, post graduate student, during the course of the study for the degree of MASTER OF DENTAL SURGERY in the speciality of BRANCH II PERIODONTICS, Vivekanandha Dental College For Women, Tiruchengode, during the period of 2017-2020

Dr. S. ELANCHEZHIYAN M.D.S., Professor and Head of Department, Department of Periodontics, Vivekanandha Dental College for Women, Tiruchengode.

CHENNAI

ENDORSEMENT BY THE HEAD OF THE DEPARTMENT AND HEAD OF THE INSTITUTION

This is to certify that Dr. V. JANANIPPRIYA, Post graduate student in the Department of Periodontics, Vivekanandha Dental College for Women, Elayampalayam, Tiruchengode, has done this dissertation titled “

EVALUATION OF SALIVARY AND SERUM PERIOSTIN LEVELS IN PERIODONTALLY HEALTHY AND DISEASED INDIVIDUALS BEFORE AND AFTER NON SURGICAL PERIODONTAL THERAPY

” under our guidance and supervision during her post graduate study period 2017-2020.

This dissertation is submitted to THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERSITY in partial fulfillment of the degree of MASTER OF DENTAL SURGERY, BRANCH II- Periodontics.

Dr. S. ELANCHEZHIYAN M.D.S. Dr. N. BALAN M.D.S Professor and Head, Principal,

Department of periodontics Vivekanandha Dental College for women, Vivekanandha Dental College for Women, Elayampalayam,

Elayampalayam, Tiruchengode.

Tiruchengode.

This is to certify that this dissertation work titled “

EVALUATION OF SALIVARY AND SERUM PERIOSTIN LEVELS IN PERIODONTALLY HEALTHY AND DISEASED INDIVIDUALS BEFORE AND AFTER NON SURGICAL PERIODONTAL THERAPY

” of the candidate Dr. V. JANANIPPRIYA, for the award of degree MASTER OF DENTAL SURGERY in the BRANCH II PERIODONTICS. I, personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file, from the introduction to conclusion pages and result shows of 8% plagiarism in the dissertation.

Guide & Supervisor with Seal.

First and foremost I would like to thank God Almighty, for his never-ending Grace.

I convey my sincere thanks and gratitude to Captain Professor Dr. S. Gokulanathan M.D.S, Dean, Vivekananda Dental College for Women for his

enormous support.

My sincere thanks and deep sense of gratitude to Dr. N. Balan M.D.S., Principal, Vivekananda Dental College for Women, for his support and permitting me to pursue this work.

With submissive ambition, I express my gratitude to my respected Professor &

Head of the Department, Guide, Dr. S. Elanchezhiyan M.D.S., Department of Periodontology and Implantology, Vivekananda Dental College for Women. From the bottom of my heart, I want to thank him for his constant support. He has been there providing his guidance and suggestions each and every day of my PG course. He had given me all the freedom to pursue my work and he made me to this level.

I have great pleasure in acknowledging my gratitude to Dr. Rajkumar Daniel

M.D.S, Professor, who always encouraged and supported me with smiling face;

Dr. Vennila M.D.S, Senior lecturer, for her timely advice, guidance and scrutiny;

contribution in completing my work. My special thanks to Dr. M. Suganya B.D.S, for her encouragement during my course.

I owe a lot to my Grandmother, Kaliyammal, my parents Mr. C. Viswanathan, Mrs. C. Muthulakshmi, and my sister Miss. V. Kavibharathi, who worked tirelessly to see my happiness who encouraged and helped me at every stage of my life.

I am very thankful to my amazing senior Dr. D. Surya and her husband Dr. Jayaprakash. I am very grateful for everything they did.

I express my heartfelt thanks to my friends Dr. M. Preethi, Dr. P. Niveditha for their endless support, care and appreciation.

I also extend my gratitude to my seniors Dr. T. Pavithra, Dr. P. S. Viola Esther, Dr. T. B. Meenalochani, Dr. S. Saranya, Dr. A. S. Abarnashree and to my juniors

Dr. S. V. Subhashini, Dr. C. Aarthi, Dr. T. Nirosa, and to my sub-juniors Dr. R. Priyadharshini, Dr. T. Anbarasi and Dr. M. Dharshana.

My sincere appreciation to our lab assisstant Mrs.Swarna sister and dental hygienist Deepshi, for their help and support.

S.NO INDEX PAGE NO

1 INTRODUCTION 1

2 AIM AND OBJECTIVES 3

3 REVIEW OF LITERATURE 4

4 MATERIALS AND METHODS 28

5 PHOTOGRAPHS 48

6 RESULTS 52

7 TABLES 54

8 GRAPHS 58

9 DISCUSSION 62

10 SUMMARY AND CONCLUSION 68

11 REFERENCES 69

S.NO TITLES PAGE NO.

1 ARMAMENTARIUM FOR NSPT 48

2 PERIOSTIN KIT REAGENTS 48

3 ELISA PLATE 49

4 SALIVARY SAMPLE COLLECTION 49

5 SERUM SAMPLE COLLECTION 49

6 CENTRIFUGE OF SAMPLES 50

7 SAMPLE STORAGE 50

8 PREOPERATIVE VIEW 50

9. POSTOPERATIVE VIEW 50

10 PERIOSTIN KIT ELISA PLATE WITH SAMPLES 51

11. ELISA READER 51

S.NO TITLES PAGE NO.

1

COMPARISON OF GENDER PROPORTIONS BETWEEN GROUPS

54

2

COMPARISON OF PRE GINGIVAL INDEX BETWEEN GROUPS

54

3

COMPARISON OF POST GINGIVAL INDEX BETWEEN GROUPS

55

4

COMPARISON OF PRE BLEEDING INDEX BETWEEN GROUPS

55

5

COMPARISON OF POST BLEEDING INDEX BETWEEN GROUPS

56

6

COMPARISON OF PRE OPERATIVE CLINICAL PARAMETERS BETWEEN GROUPS

56

7

COMPARISON OF POST OPERATIVE CLINICAL PARAMETERS BETWEEN GROUPS

57

8

COMPARISON OF PRE AND POST OPERATIVE CLINICAL PARAMETERS BETWEEN GROUPS

57

S.NO TITLES PAGE NO.

1 COMPARISON OF AGE BETWEEN GROUPS 58

2

COMPARISON OF GINGIVAL INDEX BETWEEN GROUPS

58

3

COMPARISON OF BLEEDING INDEX BETWEEN GROUPS

59

4

COMPARISON OF PROBING DEPTH BETWEEN GROUPS

59

5

COMPARISON OF CLINICAL ATTACHMENT LOSS BETWEEN GROUPS

60

6

COMPARISON OF SALIVARY PERIOSTIN LEVELS BETWEEN GROUPS

60

7

COMPARISON OF SERUM PERIOSTIN LEVELS BETWEEN GROUPS

61

INTRODUCTION

1

Loss of tooth-supporting structures affects up to 90% of the general worldwide adult population experiencing different types of periodontal diseases¹ and constitutes the primary cause of tooth loss. Gingivitis is an inflammatory condition characterized by the presence of symptoms and clinical signs of inflammation that are confined to the gingiva and associated with stable attachment levels on the periodontium². Periodontitis is defined as inflammation of gingival tissues together with a measurable loss of the attachment of the periodontal ligament and bony support³.

Periodontal diseases like gingivitis and periodontitis are chronic infectious diseases induced by bacteria, which interact with the host response and results in tissue destruction⁴. However, because this is a highly complex disease, it is unlikely that a single biomarker for disease detection and prediction can be found⁵.

Periostin is a secreted protein, which was originally identified from osteoblasts^6,7 related to the family of “matricellular proteins”. Originally termed osteoblast specific factor-2 (OSF-2), it was renamed periostin due to localized expression in the periosteum and the periodontal ligament⁸.

Periostin functions as a matricellular protein in cell activation by binding to their receptors on cell surface, thereby exerting its biological activities. In health, periostin plays an important role in collagen production, cell migration, adhesion, responses to mechanical stress, and wound repair by interacting directly with type I collagen and fibronectin⁹.

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The expression of periostin is most common in collagen rich connective tissues, where it appears essential for proper ECM synthesis, particularly with respect to collagen I fibrillogenesis¹⁰.

In bone, periostin binding to αvβ3 may also activate the downstream FAK and Akt/PKB pathway, one of most potent prosurvival signaling pathways that has been demonstrated in UMR-106 osteoblast-like cells, to regulate cell migration and survival^11,12,13.

Moreover, periostin-deficient mice show a wider periodontal ligament tissue, an inflammatory phenotype with a neutrophil infiltrate, enamel and dentin matrix defect as well as abnormal organization of alveolar bone, all of it resulting in a teeth unstable structure¹⁴. In a recent experimental study, periostin levels in rat PDL decreased after periodontitis induction, and inversely related to the extent of alveolar bone loss¹⁵.

TNF-α and Porphyromonas gingivalis lipopolysaccharide have been shown to decrease periostin levels in human periodontal ligament cell cultures¹⁶, suggesting that inflammation in the presence of bacterial biofilm may be one of the causes of periostin down-regulation.

These properties highlight the crucial role of periostin in tissue homeostasis and pathogenesis. So, reduction in periostin levels act as a co-factor for periodontal destruction. There were no studies on periostin levels before and after the non-surgical periodontal therapy. Therefore, the purpose of this study was to investigate the salivary and serum periostin levels in gingivitis and chronic periodontitis patients and to evaluate the effect of non surgical periodontal therapy.

AIM & OBJECTIVES

3

AIM

:

The purpose of this study was to investigate the salivary and serum periostin levels in gingivitis and chronic periodontitis patients and to evaluate the effect of non surgical periodontal therapy.

OBJECTIVES

:

• To compare the salivary and serum levels of periostin in periodontally healthy individuals, gingivitis and chronic periodontitis patients.

• To evaluate the effect of non-surgical periodontal therapy on periostin levels in gingivitis and chronic periodontitis patients.

• To assess whether periostin could be used as a marker for periodontal disease activity.

REVIEW OF LITERATURE

4

PERIOSTIN

Structure

1. According to Takeshita et al., 1993¹⁷ Periostin (Postn) is a 90kD extracellular matrix protein of 836 aa (in humans), originally named osteoblast specific factor 2 (OSF-2) when it was first cloned from a cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1.

2. Akira et al., 2018¹⁸ stated periostin is a secretory protein with a multi-domain Structure, comprising an amino-terminal cysteine-rich emi domain, four internal fas 1 domains, and a carboxylterminal hydrophilic domain. These adjacent domains bind to extracellular matrix proteins and bmp-1 that catalyzed cross- linking of type I collagen, and proteoglycans, which played a role in cell adhesion. The binding sites on periostin have been demonstrated to contribute to the mechanical strength of connective tissues, enhancing intermolecular interactions. In close proximity and their assembly into extracellular matrix architectures, where periostin played, further essential roles in physiological maintenance and pathological progression. Furthermore, periostin also binds to notch 1 and ccn3, which have functions in maintenance of stemness, thus opening up a new field of periostin action.

5 Expression

1. Takeshita et al., 1993¹⁷ in his study explained that Periostin derived from the periosteum of long bones, the aorta, stomach, lower gastrointestinal tract, placenta, uterus, thyroid tissue and breast. It was particularly expressed during ontogenesis and in adult connective tissues submitted to mechanical stimulation (stretch), such as heart valves, skin, periodontal ligaments, tendons and bones.

2. Merle and Garnero., 2012¹⁹ Stated Postn gene expression was up-regulated by platelet-derived growth factor (PDGF) and basic fibroblast growth factors (FGF) in cancer lines through different pathways including PI3K and p38MAPK. In bone, Postn is transcriptionally regulated by Twist, RUNX2, and C-Fos/ AP1. Its expression levels were maximal in the periosteum and osteocytes and further controlled by mechanical stimuli, hormones (PTH), growth factors (TGF-b, BMP2) and cytokines (TNFa, IL-4, IL-13, and likely PDGF), all known to have important roles in the determination and/or regulation of bone homeostasis.

6 Function

In bone homeostasis

1. According to Shimazaki et al., 2008²⁰

 Circulating levels were higher in men than in women

 Linked to fibronectin, tenascin-C and collagen 1

 Expressed in lining cells, osteoblasts and osteocytes

 Responsive to PTH, TGF-b and mechanical loading

 Binding to aVb3 integrin receptors

 Signaling through AKT/FAK and Wnt-beta-catenin pathways

 Down-regulates Sost

 May upregulate OPG

 Effects on osteoblasts migration, proliferation and differentiation

 Effects on osteocytes apoptosis

 Direct effects on osteoclasts remains to be investigated

2.

Taniguchi et al., 2014²¹ did a study to find whether the POSTN was involved in proliferation and differentiation of keratinocytes in the epithelial–mesenchymal interactions. It was already said that the release of interleukin (IL)-1α from keratinocytes and subsequent IL-6 production from fibroblasts were critical for keratinocyte proliferation and differentiation. This study found that POSTN secreted from fibroblasts was required for IL-1 α -induced IL-6 production and enhanced IL-6 production by activation of the NF-kB pathway synergistically with IL-1 α.

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Thus, the combination of an autocrine loop of POSTN and a paracrine loop composed of IL-1 α and IL-6 regulated keratinocyte proliferation and differentiation in the epithelial–mesenchymal interactions.

NSPT

1. Tunkel et al., 2002²² performed a systematic review of 27 articles to determine the efficacy of ultrasonic scalers in comparison to manual hand scalers in the treatment of chronic periodontitis. They concluded that though there was no difference between machine-driven and manual debridement in treatment of chronic periodontitis, the time required was less in ultrasonic/sonic subgingival debridement.

2. Ryan 2005²³ stated that SRP was considered the gold standard of non-surgical treatment of periodontitis, with its efficiency of reducing the microbial load, BOP and probing depths and improvement in CAL.

3. Smiley at al., 2015²⁴ in their systematic review stated that SRP resulted in 0.5 mm average improvement in CAL. SRP with adjuncts resulted in improvements of CAL by 0.2-0.6 mm over those achieved by SRP alone.

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PERIODONTAL INFLAMMATION

1. Padial-molina et al., 2015²⁵ done a case–control study to characterize periostin changes over time after periodontal surgery in tissue, oral fluids and serum. The histological analysis showed lower periostin in disease patients. After surgery levels of periostin in gingival Crevicular fluid increased over time for both groups, more noticeably in the periodontitis subjects. A transient and subtle change in circulating periostin levels was also noticed. This transient local increase in gcf periostin after eliminating the local etiology in periodontally affected sites suggested its importance in the maturation and stability of the connective tissue.

2. Balli et al., 2015²⁶ studied the periostin level in periodontal disease. 80 individuals were divided into healthy, gingivitis, and chronic periodontitis group.

Gingival crevicular fluid and serum samples were collected and periostin levels were determined using the enzyme-linked immunosorbent assay. Results showed the total amount and concentration of periostin decreased in gingival crevicular fluid with the progression of the disease from healthy controls to gingivitis and to chronic periodontitis groups. However, there was no significant difference in serum periostin concentration within all groups. This concluded that the periostin level in gingival crevicular fluid could be considered a reliable marker in the evaluation of periodontal disease susceptibility and activity.

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3. Aral et al., 2016 aimed to evaluate the levels of periostin in chronic periodontitis (CP) and aggressive periodontitis (AgP) compared to non- periodontitis (NP). Individuals were submitted to gingival crevicular fluid (GCF) and saliva sampling and other clinical parameters were measured. An enzyme- linked immunosorbent assay was performed. Result showed the mean GCF level of periostin was lowest in the AgP group as compared to the other groups and was lower in the CP group as compared to the NP group. Increased levels of periostin were observed in the saliva of patients with AgP as compared to the CP and NP groups. So in this study there was a negative relationship between GCF periostin levels and clinical parameters, whereas a positive correlation was observed between salivary periostin levels and full-mouth GI and CAL scores. This author suggested that subjects with CP and AgP exhibit a different periostin profile.

Periostin in GCF might have a protective role against periodontal disease.

Furthermore, salivary periostin concentrations might have a promising diagnostic potential for the aggressive forms of periodontal disease.

4. Akman et al., 2017²⁸ did a study to detect the levels of periostin in peri-implant sulcular fluid (pisf) and gingival crevicular fluid (gcf), and to evaluate the relationship between periostin, ictp (pyridinoline cross-linked carboxyterminal telopeptide of type 1 collagen) and ctx (c-terminal crosslinked telopeptide of type 1 collagen) levels.

For this study 9 women and 4 men with twenty ‘bone-level designed’

dental implants (di), without any signs of peri-implant bone loss and with a

10

restoration in function for at least 12 months and twenty contralateral natural teeth (nt) were included in the study as controls. Pisf and gcf ictp, ctx and periostin levels were evaluated using ELISA. Results showed ictp, ctx and periostin levels were similar between two groups. There were no statistically significant differences between pisf and gcf values.

5. Zeinab et al., 2019²⁹ did a study to compare the concentration of periostin in the saliva of patients with chronic Periodontitis and healthy controls. 45 individuals in which 25 patients with chronic periodontitis and 20 healthy controls were evaluated. After the saliva Sample collection, periostin levels were evaluated by standard enzyme‑linked immunosorbent assay. Results showed that the level of periostin in saliva in patients with periodontitis was significantly lower than healthy controls. This concludes that there was a significant relationship Between the level of periostin in saliva and chronic periodontitis. Periostin might be considered as an inflammatory marker in periodontal disease.

6. Fazal et al., 2018³⁰ in his study investigated GCF POSTN levels in patients with CP and AgP and compared them with GCF POSTN of healthy individuals. GCF from sites with the greatest CAL and maximum bleeding score in patients with CP and AgP; was collected using microcapillary pipettes and stored at −80°C.

Samples were analyzed using ELISA. The mean levels of total POSTN in GCF were 182.41 pg/μl, 79.87 pg/μl and 49.28 pg/μl for the healthy, CP, AgP groups respectively.

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The delay in tissue repair found in periodontal tissue inflammation might be explained by the reduced GCF POSTN levels seen in periodontitis groups in this study.

SYSTEMIC DISEASES

1. Li et al., 2004³¹ tested the hypothesis that expression of the novel adhesion molecule periostin (PN) and osteopontin (OPN) was increased in lung and in isolated pulmonary arterial smooth muscle cells (PASMCs) in response to the stress of hypoxia and explored the signaling pathways involved. Adult male rats were exposed to 10% O2 for 2 weeks, and growth-arrested rat PASMCs were incubated under 1% O2 for 24 h. Hypoxia increased PN and OPN mRNA expression in rat lung. In PASMCs, hypoxia increased PN but not OPN expression. The hypoxia- responsive growth factors fibroblast growth factor-1 (FGF-1) and angiotensin II (ANG II) caused dose- and time-dependent increases in PN and OPN expression in PASMCs. This suggested that PN and OPN might play different roles in pulmonary vascular remodeling under pathophysiological conditions.

2. Lindner et al., 2005³² Concluded Periostin expression was associated with smooth muscle cell differentiation in vitro and promoted cell migration. Unlike other mesenchymally derived cell lines, periostin expression was not regulated by FGF-2 in smooth muscle cells. This distinction might be useful in discriminating smooth muscle and fibroblast lineages.

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3. Erkan et al., 2007³³ evaluated the effect of periostin in cancer cells. The results showed periostin mRNA levels were elevated 42-fold in cancer, and patients with increased expression had a tendency toward shorter survival. It was concluded that periostin created a tumor-supportive microenvironment. So increased periostin expression might therefore reflect more aggressive tumor phenotype.

4. Baril et al., 2007³⁴ in their study, using an enzyme-linked immunosorbent assay (ELISA) assay, showed significantly increased levels of periostin in the sera of pancreatic cancer patients compared to non-cancer controls. This demonstrated periostin promoted the invasiveness of tumour cells by increasing the motility of cells without inducing expression of proteases, and enhanced the survival of tumour cells exposed to hypoxic conditions. These findings suggested an important role of periostin in pancreatic cancer and provided a rationale to study periostin for diagnostic and therapeutic applications.

5. Ma et al., 2009³⁵ studied to know how mechanical loading was known to trigger

proliferation of tumor cells. For this Lewis lung cancer cells were cultured and strained. Real-time PCR was used to quantify periostin and TGF-β1 mRNA levels at 6, 12, 18, and 24 h of loading. In addition, periostin and TGF-β1 neutralizing antibodies were added to the medium. The proliferation of cells were blocked by the neutralizing antibody. Periostin mRNA increased by 1.1-, 3.2-, 4.7-, and 9.2- fold while TGF-β1 mRNA increased by 5.3, 10.3-, 7.1-, and 6.5-fold at 6, 12, 18, and 24 h, respectively.

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This result suggested that periostin was a potent positive regulator of tumor growth in response to mechanical loading and was possibly a downstream factor of TGF-β1.

6. Min Zhu et al., 2011³⁶ newly developed a neutralizing monoclonal antibody to periostin, named MZ-1, and investigated its effects on human ovarian tumor growth and metastasis. This in vivo study showed significant growth inhibition by MZ-1 on both subcutaneous and intraperitoneal tumors derived from the ovarian cancer cell line A2780 which was found to secrete the highest amount of periostin into the culture medium. In addition, a reduction of the metastatic potential of the tumor was also found. This indicated that the periostin might display biphasic dose dependence in the tumor cell proliferation.

7. Sarita Sehra., 2011³⁷ stated that, in the lungs, periostin deficiency resulted in increased airway resistance and significantly enhanced mucus production.

Periostin also inhibited the expression of Gob5, a putative calcium-activated chloride channel involved in the regulation of mucus production, in primary murine airway epithelial cells. This study suggested that periostin might be part of a negative-feedback loop regulating allergic inflammation that reduced the asthma attacks. So this could be potential therapeutic in the treatment of allergic diseases.

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8. Nicolas Bonnet et al., 2012³⁸ conducted a study on the relationship of periostin expressed by bone cells and PTH on bone formation. They concluded that, the essential role of Periostin in the periosteal anabolic response to PTH was mediated by both a suppression of Sost expression in osteocyte and direct stimulatory effect of Periostin on Wnt signaling and osteoblast functions. In the absence of Periostin, bone formation was impaired and PTH was unable to effectively improve cortical structure and strength. This showed the definitive action of periostin on bone formation.

9. John et al., 2016³⁹ did a review which focused specifically on the role of Postn in wound healing and remodeling, an area of intense research in the last 10 years.

Postn interacted with cells through various integrin pairs and was an essential downstream effector of TGF-β superfamily signaling. Across different tissues, Postn was associated with pro-fibrogenic process, specifically, the transition of fibroblasts to myofibroblasts, collagen fibrillogenesis and ECM synthesis.

Although the complexity of Postn as a modulator of cell behavior in tissue healing was only beginning to be elucidated, its expression was clearly a defining event in moving wound healing through the proliferative and remodeling phases.

10. Jennifer M. Brown et al., 2018⁴⁰ the sections of bone tumors and tumor like lesions were subjected to immunohistochemistry and ELISA. They noted a strong periostin expression in benign and malignant lesions. Periostin was expressed in higher concentrations at the edge of the growing tumor mass.

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This represented the role of periostin in tumor cell growth. So in future periostin might be a potential therapeutic target to control bone tumor.

GENETIC STUDIES

1. Haertel-Wiesmann M et al., 2000⁴¹ compared gene profiles regulated by Wnt-3, β-catenin, and inhibition of glycogen-synthase kinase-3β. Cyclooxygenase-2 was up-regulated and POSTN was ten-fold down-regulated by both Wnt-3 and inhibition of glycogen-synthase kinase-3β.

2. Oshima et al., 2002⁴² investigated the mechanism of transcriptional regulation of periostin. In this study the in situ hybridization of mouse calvarial bones subjected to microarray Analysis, indicated that periostin and Twist mRNA were co-localized at the osteogenic fronts of calvarial bones. As Twist was an important transcription factor for cell type determination and differentiation, to determine whether Twist actually regulated the periostin expression, 293T cells were transiently co-transfected with the periostin promoter construct and the human Twist expression vector. Results indicated that the periostin promoter activities were enhanced by overexpression of Twist. This data suggested that Twist could bind to the periostin promoter in undifferentiated preosteoblasts and up-regulated periostin expression.

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3. Connerney et al., 2006⁴³ Twist-1 was a transcription factor played both positive and negative roles in cell differentiation. Twist-1 and POSTN were co-expressed by differentiating osteoblasts and fibroblasts at the osteogenic front of calvaria and at the alveolar bone surfaces in PDL. The occlusal force might have putative roles in POSTN and Twist gene expression in the PDL and the changes in their expression level during hypofunction might be considered a form of adaptation to environmental changes.

4. Bellido et al.,⁴⁴ studied the effects of PTH on Sost, a Runx2 target gene expressed in osteocytes, which antagonized the pro-osteoblastogenic actions of bone morphogenetic proteins and Wnts. They reported that continuous infusion of PTH to mice for 4 days decreased Sost mRNA expression in vertebral bone by 80–90%. This effect was accompanied by a comparable reduction of sclerostin, the product of Sost, in osteocytes, as determined by quantitative immunoblot analysis of bone extracts and by immunostaining. In contrast, a single injection of PTH caused a transient 50% reduction in Sost mRNA at 2 h, but four daily injections had no effect on Sost mRNA or sclerostin. These results, strongly suggested that suppression of Sost by PTH represents a novel mechanism for hormonal control of osteoblastogenesis mediated by osteocytes.

17

5. Li et al., 2006 did a study to understand mechanisms underlying periostin upregulation in vitro. For this the vascular smooth muscle cell culture was made.

Study showed periostin mRNA expression in cultured VSMCs was stimulated by growth factors like transforming growth factor-_1, fibroblast growth factors, PDGF-BB, and angiotensin II. This stimulatory effect was inhibited by the PI-3- kinase inhibitor. This concludeed Periostin protein secreted by vascular smooth muscle cells played a significant role in regulating vascular smooth muscle cells migration.

6. Wen et al., 2010⁴⁶ investigated whether periostin protein was expressed in the human PDL in situ and the mechanisms regulating periostin expression in PDL fibroblasts in vitro. With immunohistochemistry, periostin protein was identified in the PDL, with expression lower in teeth with reduced occlusal loading. In vitro application of cyclic strain to PDL fibroblasts and treatment with transforming growth factor-beta1 elevated periostin mRNA levels. FAK-null fibroblasts contained no detectable periostin mRNA, even after stimulation with cyclic strain.

In conclusion, periostin protein was strongly expressed in the human PDL. In vitro, periostin mRNA levels were modulated by cyclic strain as well as TGF-β1 via FAK-dependent pathways.

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7. Hoersch et al., 2010⁴⁷ combined genomic and transcriptomic sequence data from vertebrate organisms to study the evolution of periostin and particularly of its C- terminal region. Results found that the C-terminal part of periostin was markedly more variable among vertebrates and suggested that these beta strands might mediate binding interactions with other proteins.

8. Padial-Molina M et al., 2013⁴⁸ exposed human PDL cultures to inflammatory mediators (tumor necrosis factor (TNF)-

(Porphyromonas gingivalis liposachharides (LPS)) or a combination in a biomechanically challenged environment. Culture conditions were applied for 24 hours, 4 days, and 7 days. POSTN and TGF-ß inducible gene mRNA expression were analysed by Western blot. Result showed chronic exposure to pro- inflammatory cytokines and/or microbial virulence factors significantly decreased POSTN protein levels in the loaded cultures. Thus, inflammatory mediators TNF-

POSTN expression in human PDL fibroblasts.

9. Padial-Molina et al., 2014⁴⁹ evaluated whether periostin restored the regenerative potential of PDL cells in terms of proliferation, migration, and activation of survival signaling pathways after being challenged by Porphyromonas gingivalis lipopolysaccharides and tumor necrosis factor alpha.

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For this human PDL (hPDL) cells were cultured under different conditions to evaluate cell proliferation, cell migration and PI3K/AKT/mTOR pathway activation. A different set of cultures were challenged by adding tumor necrosis factor alpha and P. gingivalis lipopolysaccharides to evaluate the effects of periostin. Results showed periostin significantly increased cell proliferation, migration and activation of survival signaling pathway. Furthermore, periostin promoted similar cellular effects even after being challenged with proinflammatory cytokines and bacterial virulence factors. It concluded periostin act as an important modulator of hPDL cell–matrix dynamics and also helped in overcoming the altered biological phenotype.

10. Qingfeng et al., 2015⁵⁰ constructed a lentivirus vector containing the periostin gene and transduced it into PC3 and DU145 cells. After confirming periostin overexpression by PCR and Western blotting, they also measured the expression of EMT associated factors using Western blot analysis to test the effect of periostin on EMT in prostate cancer cells. Results confirmed that periostin was upregulated after infection with the periostin lentiviral vector. Periostin overexpression promoted increased cell proliferation, invasion, and migration. It concluded that periostin was an important mediator of TGF-β-induced EMT and suggested that periostin was a potential therapeutic target for suppressing the metastatic progression of prostate cancer.

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11. Afsaneh Rangiani et al., 2015⁵¹ Periostin played a stimulatory role in both SOST and TRAP responses to OTM in the compassion site, although it was not clear if this role was direct or indirect during orthodontic loading.

12. Hui-Li Liu et al., 2017⁵² investigated a molecular mechanism of periostin- mediated regulation of apoptosis. For this human periodontal ligament fibroblasts were cultured and periostin-expressing plasmid were constructed. Hypoxic conditions were obtained by anaero-pack system and using flow cytometry, the apoptosis was analysed. The result showed the apoptosis rate of fibroblasts was significantly increased after 48 hrs of hypoxia. At the same time the fibroblasts transfected with periostin expressing plasmids showed decreased apoptosis. The mechanism involved periostin activated the Akt/PKB signaling pathway through the αvβ3 integrins pathway. This study identified the action of periostin in cell survival and in regulation of apoptosis.

ANIMAL STUDIES

1. Horiuchi et al., 1999⁵³ in his study described the localization, regulation, and potential function of periostin. Immunohistochemistry revealed that in adult mice, the protein was preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure. Western blot analysis showed that periostin was a disulfide linked 90 kDa protein secreted by osteoblasts and osteoblast-like cell lines.

21

Nucleotide sequence revealed four periostin transcripts that differ in the length of the C-terminal domain, possibly caused by alternative splicing events.

Reverse transcription- polymerase chain reaction analysis revealed that these isoforms were not expressed uniformly but were differentially expressed in various cell lines. Together, these data suggested that periostin might play a role in the recruitment and attachment of osteoblast precursors in the periosteum.

2. Wilde et al., 2003⁵⁴ examined the expression of POSTN mRNA during experimental tooth movement. An experimental study in rats without tooth movement, the expression of POSTN mRNA was uniformly observed in the PDL surrounding the mesial and distal roots of the upper molars and was weak in the PDL of the root furcation area. The POSTN mRNA-expressing cells were mainly fibroblastic cells in the PDL and osteoblastic cells on the alveolar bone surfaces.

The expression of POSTN mRNA in the PDL began to be observed at 3 h and continued up to 96 h after tooth movement. The maximum changes were observed at 24 h. The expression of POSTN mRNA in the PDL 168 h after tooth movement exhibited a similar distribution to that of the control specimens. These results suggested that POSTN was one of the local contributing factors in bone and periodontal tissue remodeling following mechanical stress during experimental tooth movement.

22

3. Rios et al., 2005⁵⁵ investigated periostin role during development. They generated mice that lack the periostin gene and replaced with a lacZ reporter gene. Postnatally, however, 14% of the nulls died before weaning and all of the remaining perilacZ nulls were severely growth retarded. Skeletal analysis revealed that trabecular bone in adult homozygous skeletons was sparse.

Furthermore, by 3 months, the nulls developed an early-onset periodontal disease- like phenotype. Unexpectedly, these mice also showed a severe incisor enamel defect, although there was no apparent change in ameloblast differentiation. Also the periostin was required for the maintenance of periodontal ligament the periostin null mice could not withstand the mechanical stress.

4. Shimazaki et al., 2008⁵⁶ generated periostin null mice and after AMI, cardiac healing was noted. The results showed healing was impaired in these mice, resulting in cardiac rupture as a consequence of reduced myocardial stiffness.

Moreover, the inhibition of FAK or αv-integrin, which blocked the periostin- promoted cell migration, revealed that αv-integrin, FAK, and Akt were involved in periostin signaling. Their findings showed the effects of periostin which was a matricellular protein, on recruitment of activated fibroblasts through FAK- integrin signaling and on their collagen fibril formation specific to healing after AMI.

23

5. Rios HF et al., 2008 studied the role of periostin during occlusal loading. For this study, periostin-null mice were created. The effect of periostin on periodontal response to mechanical loading and unloading was compared by radiographs, cell culture and immunohistochemistry. Result showed severe periodontal defects in periostin-null mice and periostin expression was increased in strained PDL cells.

This indicated that mechanical loading maintained sufficient periostin expression to ensure the integrity of the periodontium in response to occlusal load.

6. Rani et al., 2010⁵⁸ studied to identify mechanisms that regulated tissue repair in work-related musculoskeletal disorders (wmsds). They examined the level of periostin like factor and periostin in Muscle, tendon, and nerve using immunohistochemistry and western blot analysis. Plf increased with continued task performance, whereas periostin was constitutively expressed. Plf was located in satellite cells and/or myoblasts, which increased in number with continued Task performance, supporting the hypothesis that periostin like factor played a role in muscle repair or regeneration. Periostin, on the other hand, was not present in satellite cells and/or myoblasts.

7. Kii et al., 2010⁵⁹ in their study showed that periostin, a matricellular protein, promoted incorporation of tenascin-c into the extra cellular matrix and organized a meshwork architecture of the ecm. They found that both periostin null mice And tenascin-c null mice exhibited a similar phenotype.

24

Periostin possessed Adjacent domains that binded to tenascin-c and other Ecm proteins: fibronectin and type I collagen, respectively. These adjacent domains functioned as a bridge between tenascin- C and the ecm, which increased deposition of tenascin-c on the ecm. This study suggested the role of periostin in adaptation of the ecm architecture in the mechanical environment.

8. Li et al., 2010⁶⁰ did an in-vitro study in which aortic SMCs isolated from PN null mice exhibited a significantly reduced ability to migrate and proliferate.

Endogenous PN protein was absent and very low in the culture medium from the primary cultures of PN null mice. Furthermore, in cultured human SMCs, specific integrin blocking antibodies showed that interactions of PN-3 and PN-5, but not PN-1 integrins, were required for SMC migration. These results revealed a mechanism whereby PN mediateed vascular SMC migration through an interaction with alpha V-integrins and subsequent activation of FAK pathway.

9. The study by Choi et al., 2011⁶¹ included 45 Wistar male rats (12 weeks of age) whose upper-right first molars were relieved from occlusion for 24 hours, 72 hours, 7 days or 21 days. The PDL was examined histologically, and changes in the gene and protein levels of POSTN and connective tissue growth factor were investigated. Result showed the PDL space width was reduced significantly.

Histologically, an initial reduction in the fiber number, thinning of PDL fibers, disarrangement of the PDL fibers and their attachments was noted.

25

Real-time PCR results revealed sharp down-regulation of the POSTN, which continued throughout the experiment. This concluded POSTN and connective tissue growth factor immunoreactivities became very weak without masticatory load.

10. Issei Takayama et al., 2012⁶² hypothesized the function of periostin in the PDL:

(1) activation of matrix metalloproteinases for collagen remodeling, and (2) stabilization of the Notch1 protein for anti-apoptotic signaling against stress conditions through the secretion pathway from the inside to outside of the PDL cells. Furthermore, recent observations have demonstrated that periostin functions in fibrillogenesis in association with extracellular matrix molecules.

11. Christopher G. Elliott et al., 2012⁶³ assessed the contribution of periostin in dermal healing. 6 mm full-thickness excisional wounds were created in the skin of periostin-knockout mice. In wild-type mice, periostin was potently induced 5–7 days after wounding. In the absence of periostin, day 7 wounds showed a significant reduction in myofibroblasts. But delivery of recombinant human periostin by electrospun collagen scaffolds restored a smooth muscle actin expression. So this study proposed that periostin promoted wound contraction by facilitating myofibroblast differentiation and contraction.

26

12. M. Padial-Molina et al., 2012⁶⁴ did an animal study in which the periodontal breakdown was induced by ligature. Periodontal tissue specimens were harvested at baseline, 2 weeks, and 4 weeks and prepared for histologic, immunofluorescence, and micro-CT examination. In conclusion, periostin PDL tissue levels significantly decreased under chronic inflammatory response and correlated with the detrimental changes to the periodontium over time.

13. John et al., 2016⁶⁵ did a review which focus specifically on the role of Postn in wound healing and remodeling, an area of intense research in the last 10 years.

Postn interacted with cells through various integrin pairs and was an essential downstream effector of TGF-β superfamily signaling. Across different tissues, Postn was associated with pro-fibrogenic process, specifically, the transition of fibroblasts to myofibroblasts, collagen fibrillogenesis and ECM synthesis.

Although the complexity of Postn as a modulator of cell behavior in tissue healing was only beginning to be elucidated, its expression was clearly a defining event in moving wound healing through the proliferative and remodeling phases.

14. Ziqiang Wu et al., 2017⁶⁶ conducted an in-vitro study to evaluate the effects of POSTN on human periodontal ligament mesenchymal stem cells. For this hPDLSCs were isolated. The cell counting kit-8 and scratch assay and alkaline phosphatase activity assay were used to analyze cell proliferation, migration and Osteogenic differentiation respectively.

27

The Result showed Isolated hPDLSCs were participated in Migration, Proliferation, and Differentiation of Human Periodontal Ligament. So this study concluded these findings to shed a new light on dental tissue–derived MSCs function and potential clinical applications.

MATERIALS AND METHODS

28

STUDY DESIGN & POPULATION:

60 Individuals were enrolled in this case-control study who were all visiting Vivekanandha Dental College for Women. They were divided into 3 groups: group A:

periodontally healthy individuals ( n=20), Group B: gingivitis patients (n=20), Group C:

chronic periodontitis patients (n=20). The study protocol was approved and supervised by the ethical committee of Vivekanandha Dental college for women, Tiruchengode.

Informed consent was obtained from the patients after being fully informed about the study protocol.

INCLUSION CRITERIA:

• Age = Above 20 yrs.

CONTROL:

GROUP A: Periodontally healthy individuals ( n=20)

 Gingival Index= 0

 Probing depth ≤ 3mm

 No signs of attachment loss and bone loss

29 STUDY :

GROUP B: Gingivitis patients (n=20)

 Clinical signs of inflammation,

 Gingival Index⁶⁷ ≥ 1

 Bleeding index⁶⁸ ≥ 1

 probing depth ≤ 3mm

 No signs of bone and clinical attachment loss.

GROUP C: Chronic periodontitis patients ( n=20).

 Patients with minimum of 20 teeth,

 Gingival Index⁶⁷ ≥ 1

 Bleeding index⁶⁸ ≥ 1

 Pocket depth ≥ 5mm

 Signs of attachment and Bone loss by clinical and radiographic examination

30

EXCLUSION CRITERIA

:

 Systemic diseases

 Smoking

 Pregnancy

 Steroid therapy

 Radiation therapy

 Immuno suppressive therapy

 History of periodontal therapy before 6 months

ARMAMENTARIUM:

(Figure-1)

o Gloves o Mouth mask o Head cap o Patient apron o Aluminium foil o Kidney tray o Cotton rolls o Gauze

31 o LA spray

o Saline o Betadine o Syringe o Mouth mirror o Explorer o Tweezer

o William’s periodontal probe o Ultrasonic scaler

o Hu- Friedy gracey curettes o Polypropylene tube

o Borosil funnel o Tourniquet o 2 ml syringe o Centrifuge o 5 ml aliquots o Deep freezer

o ELISA kid for detection of periostin

32

CLINICAL EXAMINATION

All individuals underwent a full mouth periodontal examination, which included evaluation of gingival index, bleeding index, probing pocket depth, clinical attachment level.

1. GINGIVAL INDEX (Loe and Silness, 1963)

The severity of gingivitis was scored on distal facial papilla, facial margin, mesial facial papilla, entire lingual margin of selected teeth.⁶⁷

Score Criteria

0 Normal gingiva.

1 Mild inflammation- - slight change in color, slight edema. No bleeding on probing.

2 Moderate inflammation -- redness, edema and glazing. Bleeding on probing.

3 Severe inflammation -- marked redness and edema. Tendency to spontaneous bleeding. Ulceration.

33

2. MODIFIED SULCULAR BLEEDING INDEX (Mombelli et. al, 1987)

A periodontal probe was passed along the gingival margin to provoke bleeding in all teeth and clinical findings were recorded according to the following criteria.⁶⁸

Score ^Criteria

0 No bleeding when probe is passed along the gingival margin 1 Isolated bleeding, spots visible

2 Blood forms a confluent red line on margins

3 Heavy or profuse bleeding

3. PROBING POCKET DEPTH (PPT)

Williams periodontal probe with calibration at 1,2,3,5,7,8,9,10 was used for the assessment of probing depth. It was measured from gingival margin to the base of the sulcus at six different sites around each tooth except third molars. [ mesiobuccal, distobuccal, midbuccal, mesiolingual, distolingual and midlingual]⁶⁹

4. CLINICAL ATTACHMENT LEVEL (CAL)

The clinical attachment level was recorded from cemento-enamel junction to the base of the gingival sulcus/periodontal pocket measured using William’s periodontal probe as mentioned for probing depth. ⁷⁰

34

COLLECTION OF SAMPLES

Clinical samples including saliva and serum were collected at baseline and after 1 month of non surgical periodontal therapy

SALIVA SAMPLING

Participants were asked to avoid eating or drinking 1 h before the collection of samples. Unstimulated whole saliva samples was collected by expectorating into polypropylene tubes every 30 s over a period of 5 min before clinical periodontal measurements. Saliva samples were centrifuged at 3000 rpm for 5 min. The resultant supernatants of the samples were stored at −80°C for further analysis. (Figure-4)

BLOOD SAMPLING

2 ml of blood was collected from the anticubital vein with the venipuncture method using 20 gauge needle with 2ml syringe. It was centrifuged at 3000rpm for 5 min and 2 ml of the extracted serum was transferred to aliquots and stored at -800C.

(Figure-5)

NON SURGICAL PERIODONTAL THERAPY

The non surgical periodontal therapy which included scaling and root planning were performed for group A (gingivitis) and group B (chronic periodontitis). For patients with gingivitis scaling was performed with the help of ultrasonic scalers (Woodpecker) and oral hygiene instructions given.

35

For patients with chronic periodontitis scaling along with root planning using hand curettes (Hu-Friedy curretes, Chicago, IL, U.S.A) was performed. Here scaling performed on the day of initial collection of samples and root planning was performed on the recall visit within 2 weeks following scaling. Both groups were instructed about the proper method of modified boss technique and flossing technique and asked to perform twice daily. (Figure- 8,9)

ENZYME-LINKED IMMUNOSORBENT ASSAY FOR PERIOSTIN

A commercially available enzyme-linked immunosorbent assay (ELISA) kit (RayBio Human Periostin ELISA Kit, RayBiotech, Norcross, GA) was used to measure periostin levels according to the manufacturer’s guidelines, as follows. (Figure-3)

ELISA ANALYSIS OF SAMPLES

The RayBio® Human Periostin ELISA kit was used to quantify the serum and salivary periostin concentrations. The following reagents and materials were used.

(Figure-2)

36

Component Size / Description

Periostin Microplate (Item A) 96 wells (12 strips x 8 wells) coated with anti- Human Periostin.

Wash Buffer Concentrate (20X) (Item B)

25 ml of 20X concentrated solution.

Standard Protein (Item C) 2 vials of Human Periostin. 1 vial is enough to run each standard in duplicate.

Detection Antibody Periostin (Item F)

2 vials of biotinylated anti-Human Periostin. Each vial is enough to assay half the microplate.

HRP-Streptavidin Concentrate (Item G)

200 μl 100X concentrated HRP-conjugated streptavidin

TMB One-Step Substrate Reagent (Item H)

12 ml of 3,3,5,5'-tetramethylbenzidine (TMB) in buffersolution.

Stop Solution (Item I) 8 ml of 0.2 M sulfuric acid.

Assay Diluent C (Item L) 30 ml of diluent buffer.

Assay Diluent B (Item E) 15 ml of 5x concentrated buffer.

37 ADDITIONAL MATERIALS REQUIRED

1. Microplate reader capable of measuring absorbance at 450 nm.

2. Precision pipettes to deliver 2 μl to 1 ml volumes.

3. Adjustable 1-25 ml pipettes for reagent preparation.

4. 100 ml and 1 liter graduated cylinders.

5. Absorbent paper.

6. Distilled or deionized water.

7. Log-log graph paper or computer and software for ELISA data analysis.

8. Tubes to prepare standard or sample dilutions.

REAGENT PREPARATION

1. All reagents and samples were brought to room temperature (18 - 25ºC) before use.

2. Assay Diluent (Item E2) was diluted 5-fold with deionized or distilled water before use.

3. Sample dilution: 1X Assay Diluent (Item E2) was used for dilution of serum and saliva samples. The suggested dilution for normal serum/plasma is 2 fold.

4. Preparation of standard: A vial of Item C was spun. 400 μl 1X Assay Diluent (Item E2) was added into Item C vial to prepare a 500 ng/ml standard solution.

The powder was dissolved thoroughly by a gentle mix. 60 μl Periostin standard was added into a tube with 440 μl 1X Assay Diluent Buffer to prepare a 60 ng/ml standard solution. 400 μl 1X Assay Diluent was pipetted into each tube.

38

Each tube was thoroughly mixed before the next transfer. 1X Assay Diluent served as the zero standard (0 ng/ml). Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1X Wash Buffer.

6. Detection Antibody vial (Item F) was briefly spun before use. 100 μl of 1X Assay Diluent (Item E2) was added into the vial to prepare a detection antibody concentrate. It was pipetted up and down to mix gently. The detection antibody concentrate was diluted 80-fold with 1X Assay Diluent (Item E2) and used in step 5 of Part VI Assay Procedure.

7. The HRP-Streptavidin concentrate vial (Item G) was spun and pipetted up and down to mix gently before use, as precipitates might form during storage. HRP- Streptavidin concentrate was diluted 100-fold with 1X Assay Diluent (Item E2) and mixed well.

39 ASSAY PROCEDURE

1. 100 μl of each standard and sample were added into appropriate wells. Wells were covered and incubated for 2.5 hours at room temperature with gentle shaking.

2. The solution was discarded and washed 4 times with 1X Wash Solution. Each well was washed by filling with Wash Buffer (300 μl) using a multi-channel Pipette or autowasher. After the last wash, any remaining Wash Buffer was removed by aspirating or decanting. The plate was inverted and blotted against clean paper towels.

3.100 μl of 1X prepared biotinylated antibody was added to each well and incubated for 1 hour at room temperature with gentle shaking.

4. The solution was discarded.

5. 100 μl of prepared Streptavidin solution was added to each well and incubated for 45 minutes at room temperature with gentle shaking.

8. Again the solution was discarded.

9. 100 μl of TMB One-Step Substrate Reagent (Item H) was added to each well and incubated for 30 minutes at room temperature in the dark with gentle shaking.

10. Finally 50 μl of Stop Solution (Item I) was added to each well and read at 450 nm immediately.

40 Standard curve:

SENSITIVITY

The minimum detectable dose of Human Periostin was determined to be 80 pg/ml.

SPECIFICITY

This ELISA antibody pair detected human Periostin. Other species not determined.

41

PROFORMA

VIVEKANANDHA DENTAL COLLEGE FOR WOMEN TIRUCHENGODE

DEPARTMENT OF PERIODONTICS

DISSERTATION TITLE:

EVALUATION OF SALIVARY AND SERUM PERIOSTIN LEVELS IN PERIODONTALLY HEALTHY AND DISEASED INDIVIDUALS BEFORE AND AFTER NON SURGICAL PERIODONTAL THERAPY

Name: Date:

Age/Sex: OP.No:

Address: Occupation:

HISTORY Chief Complaint:

Past Medical History:

42 Past Dental History:

Personal History:

General Examination:

Intra Oral Examination:

CLINICAL PARAMETER ASSESSMENT

INDICES

BASELINE

GINGIVAL INDEX (Loe&Silness 1963):

16 12 24 36 32 44

43

MODIFIED SULCULAR BLEEDING INDEX (Mombelli et. al, 1987):

18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28

48 47 46 45 44 43 42 41 31 32 33 34 35 36 37 38

PROBING POCKET DEPTH

CLINICAL ATTACHMENT LEVEL

44 DIAGNOSIS

AFTER 1 MONTH

GINGIVAL INDEX (Loe&Silness 1963):

16 12 24 36 32 44

MODIFIED SULCULAR BLEEDING INDEX (Mombelli et. al, 1987)

18 17 16 15 14 13 12 11 21 22 23 24 25 26 27 28

48 47 46 45 44 43 42 41 31 32 33 34 35 36 37 38

45 PROBING POCKET DEPTH

CLINICAL ATTACHMENT LEVEL

PERIOSTIN ANALYSES

BASELINE AFTER 1 MONTH

Serum periostin level Salivary periostin level

46

INFORMED CONSENT FORM

VIVEKANANDHA DENTAL COLLEGE FOR WOMEN TIRUCHENGODE

DEPARTMENT OF PERIODONTICS

Research student: V. Jananippriya.

Patient Name: Male/female:

Address : Age :

I have been explained the nature and purpose of the study in which I have been asked to participate. I have been given the opportunity to question about the study and other procedures. I hereby give the consent to be included in this study.

Place:

Date:

Signature of patient

47

தகவல்ஒப்புமைபடிவம்

விவவகானந்தாபல்ைருத்துவகல்லூரி

திருசெங்வகாடு

ஈறுவ ாய்ெிகிச்மெப்பிரிவு

ஆய்வாளர் : ஜனனிப்பிரியா.வி

வ ாயாளிசபயர்: ஆண்/சபண் :

முகவரி: வயது :

___________________________ ஆகிய எனக்கு பரிச ோதனன பற்றிய அனனத்து

விவரங்களும் ததளிவோக புரியும்படி எடுத்துனரக்கப்பட்டன. என்னுனடய

ந்சதகங்கள் அனனத்திற்கும் ததளிவோன பதில் அளிக்கப்பட்டது. எனது

பரிச ோதனனகளுக்குமுழுமனசதோடு ம்மதிக்கிசறன்.

இடம்: வததி:

ந ோயோளி கையயோப்பம்

PHOTOGRAPHS

48

ARMAMENTARIUM FOR NSPT

Figure -1

PERIOSTIN KIT REAGENTS

Figure - 2

49 ELISA PLATE

Figure - 3

SAMPLE COLLECTION AND STORAGE

Salivary sample collection Serum sample collection

Figure – 4 Figure - 5

50

Centrifuge of Samples Sample storage

Figure – 6 Figure - 7

PREOPERATIVE VIEW

Figure - 8

POSTOPERATIVE VIEW

Figure - 9

51

PERIOSTIN KIT ELISA PLATE WITH SAMPLES

Figure - 10

ELISA READER

Figure - 11

RESULTS

52

The Normality tests Kolmogorov-Smirnov and Shapiro-Wilks tests results reveal that some variables follow Normal distribution and some variables (BI, GI) did not follow Normal distribution. Therefore, to analyse the data both Parametric and Non parametric methods were applied.

To compare the mean values between groups one-way ANOVA was applied followed by Tukey’s HSD post hoc tests for multiple pairwise comparisons. To compare two mean values independent sample t-test was applied. To compare pre and post mean values paired t-test was applied.

To compare proportions between study and control groups Chi-Square test was applied, if any expected cell frequency was less than five then Fisher’s exact test was used. For variables which did not follow Normal distribution, to compare between school types Kruskal Wallis test was used followed by Bonferroni adjusted Mann Whitney test for multiple pair wise comparison.

To compare values between two-time points Wilcoxon Signed Rank test was used. Pearson correlation coefficient was calculated to assess the linear relationship between variables. To analyse the data SPSS (IBM SPSS Statistics for Windows, Version 25.0, Armonk, NY: IBM Corp. Released 2017) was used. Significance level was fixed as 5% (α = 0.05).