Journal club

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Effect Of Enamel Matrix Derivative Liquid On Osteoblast & Periodontal Ligament Cell Proliferation & Differentiation chard J. Miron, Fatiha Chandad ,Daniel Buser, Anton Sculean David L Journal Of Periodontology 2016;87(1):91-98 Dr Shivani Iyer PG II nd Year

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Effect Of Enamel Matrix Derivative Liquid On Osteoblast & Periodontal Ligament Cell Proliferation & Differentiation

Richard J. Miron, Fatiha Chandad ,Daniel Buser, Anton Sculean David L. Cochran

Journal Of Periodontology 2016;87(1):91-98

Dr Shivani Iyer PG IInd Year

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CONTENTS

1. INTRODUCTION 2. AIM OF THE STUDY 3. MATERIALS AND METHODS 4. RESULTS 5. DISCUSSION 6. RELATED STUDIES7. CONCLUSION

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INTRODUCTION

The aim of regenerative periodontal therapy is to

restore the tooth – supporting

apparatus lost following

periodontal disease or trauma.

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New Attachment:

The union of connective tissue or epithelium with a root surface that has been deprived of its original attachment apparatus. This new attachment may be epithelial adhesion and/or connective tissue adaptation or attachment and may include new cementum.

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Karring et al. (1985) studied the formation of new connective tissue attachment by inducing periodontitis in 4 monkeys.

The authors concluded that new connective tissue attachment forms on previously periodontitis- involved roots by coronal migration of cells originating from the periodontal ligament

ANIMAL STUDIES

HEALING BY NEW ATTACHMENT

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Nyman et al. (1988) evaluated the role of diseased cementum on new attachment formation. Eleven patients were treated surgically using a split mouth design. In 2 quadrants (control), the teeth were scaled and root planed to remove all cementum. In the remaining quadrants (test),calculus was removed without removal of cementum and the teeth were polished. The patients were followed for 24 months.

The results showed that the same degree of improvement was achieved following both types of treatment: there was some gain of probing attachment for both treatment modalities

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One modality that has been shown to facilitate periodontal wound healing and regeneration is the application of an Enamel matrix derivative (EMD) onto debrided root surfaces and into periodontal defects.

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ENAMEL MATRIX DERIVATIVE

1. Derived from enamel matrix proteins.

2. Enamel Matrix Derivative (EMD ) is extracted from developing embryonic enamel of porcine origin & contains hydrophobic enamel matrix proteins. ( Hammarstrom 1997 )

3. Enamel matrix proteins may be secreted by the Hertwigs epithelial root sheath (HERS) during tooth development.

4. It induces acellular cementum formation. ( Hence believed to favour periodontal regeneration. )

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EMDOGAIN

One enamel matrix protein derivative obtained from the developing porcine teeth has been approved by the U.S Food and Drug Administration (FDA ) and marketed under the trade name EMDOGAIN.

COMPOSITION : 90 % Amelogenin : Remaining percentage are proline rich non amelogenin , tuftelins , tuft proteins , ameloblastin , amelins.

Viscous gel consisting of EMD in a polypropylene liquid.

1 ml of vehicle solution mixed with powder – delivered with syringe.

It is copackaged with Prefgel – 24 % EDTA which removes smear layer.

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Enamel Matrix Derivative ( EMD ) – root conditioning agent known to accomplish healing by regeneration.

Hejil et al , 1997 compared EMD with placebo in 1/2 wall defects – showed significant radiographic bone gain ( 2.6 mm ) in EMD group.

Yukna and Mellonig, 2000 in a histological evaluation reported evidence of regeneration ( new cementum , bone and periodontal ligament ) with Enamel Matrix Derivative ( EMD ).

Tonneti et al , 2002 evaluated the clinical outcomes of papillary preservation flap surgery with or without the application of enamel matrix proteins and reported more CAL gain in EMD group.

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Additionally , the enamel matrix derivative is not osteoinductive , but it is “osteopromotive” in that it stimulates bone formation when combined with demineralized freeze-dried bone graft. ( Boyan BD , Weesner TC , Lohmann CH et al 2000 )

Enamel Matrix Derivative ( EMD ) has shown to upregulate the synthesis of growth factors such as TGF-β1 & insulin like growth factor ( IGF – 1). ( Okubo et al 2003 )

Enamel Matrix Derivative ( EMD ) causes an increase in PDL cell adhesion. ( Gestrelius et al 1997 , Lyngstadaas et al 2001 , Palioto et al 2004 , Rodrigues et al 2007 )

Myhre et al ,2006 have reported that EMD attenuates the release of tumour necrosis factor – α and IL – 8 from human blood cells challenged with lipopolysaccharide or peptidoglycan , indicating that EMD has anti inflammatory properties.

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Despite the many histologic and controlled clinical studies providing evidence for periodontal regeneration, concerns have been expressed regarding the viscous nature of EMD in its carrier gel, which may not be sufficient to prevent flap collapse in large periodontal defects.

A flap collapse may lead to a limitation of the space available for regeneration, thus limiting clinical outcomes.

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Recently, quantification of proteins contained in Enamel Matrix Derivative (mainly amelogenin) to adsorb to various bone grafting materials was analyzed. ( Miron RJ , Bosshardt DD et al , 2015 )

It was found that protein adsorption was superior when EMD wasdissolved as a soluble liquid formulation in acetic acid compared to the commercially available EMD gel.

Furthermore, results demonstrated that the combination of a liquid delivery system of EMD in slightly acidic conditions favored absorption of amelogenin proteins to the surface of the grafting particles as assessed by high-resolution transmission electron microscopy and protein quantification.

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Recently, a new delivery system has been explored to better optimize the physico-chemical properties of EMD in combination with bone grafting materials.

This new formulation is a slightly acidic liquid formulation of EMD, dissolved with a low concentration of acetic acid and maintaining its liquid formulation.( Zhang Y et al 2015 )

Combined with a bone grafting material, advantages include better surface coating and adsorption of amelogenin proteins to the bone grafting material surface and better penetration of amelogenin protein throughout certain grafting materials

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AIM OF THE STUDY : To analyze the effect on cell growth of Enamel Matrix Derivatives ( EMDs ) in a gel carrier in comparison to Enamel Matrix Derivatives (EMD) in a liquid carrier.

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MATERIALS AND METHODS

1. Enamel Matrix Proteins

EMDs were used either in a propylene glycol gel carrier (0.7-mL vials, working concentration 30 mg/mL)

Liquid carrier (0.3-ml vials, working concentration 30 mg/mL).

To reach the working in vitro concentration of 100 mg/mL, both formulations were diluted in a-minimal essential medium (a-MEM) containing 10% fetal bovine serum.

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2. OSTEOBLAST AND PDL CELL ISOLATION AND DIFFERENTIATION

Human Periodontal ligament cells were harvested from premolars extracted for orthodontic reasons & with no sign of periodontal disease.

Osteoblasts were harvested from alveolar bone collected during third molar extractions and with no signs of periodontal disease or other complication.

Primary osteoblast & periodontal ligament cells were seeded at a density of 5,000 cells in 96- well culture plates for cell proliferation and 300,000 cells per well for real - time PCR and alizarin red experiments.

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4. Real-Time PCR for Growth Factors and Differentiation Markers

Total RNA was isolated using : -

•Reagent – Trizol •Kit - RNAeasy Mini Kit ( Quiagen , Switzerland )

Primer & probe sequences for genes encoding bone morphogenetic protein 2 ( BMP 2 ) , transforming growth factor ( TGF β1 ) , IL-8 , osteocalcin were purchased as predesigned gene expression assays.

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5. Alizarin Red Staining

Accumulation of calcium in the Periodontal ligament cells was analysed by Alizarin red staining.

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RESULTS

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No significant difference in cell number was observed between samples treated with EMD in the gel formulation versus EMD in the liquid formulation.

1. Proliferation of Osteoblasts and Periodontal ligament cells

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2. Cytokine and Growth Factor Expression

Cells treated with either formulation had a slight non – significant increase in TGF –β1 mRNA expression in primary osteoblasts.

On the other hand , TGF – β1 expression was significantly upregulated at all time points for Periodontal ligament cells.

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Both the Enamel Matrix Derivative formulations significantly reduced the expression of IL – 8 in osteoblasts & periodontal ligament cells at all points.

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3. CELL DIFFERENTIATION

Osteoblasts and Periodontal ligament cells were next investigated for their mineralization potential after culture with either Enamel Matrix Derivative ( EMD ) formulation as assessed by alizarin red staining.

After 14 days of culture , alizarin red staining was significantly higher in both osteoblasts and periodontal ligament cells treated with either formulation.

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DISCUSSION

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Enamel Matrix Derivative (EMD) as an agent capable of periodontal regeneration has widely been used to improve new periodontal ligament, cementum, and alveolar bone formation.

It was recently found that the Enamel Matrix Derivative (EMD) gel significantly increased the thickness of coating of enamel matrix proteins to a bone grafting surface but were easily dissolved with a simple PBS rinse.*

*Miron RJ, Bosshardt DD, Buser D, et al. Comparison of the capacity of enamel matrix derivative gel and enamel matrix derivative in liquid formulation to adsorb to bone grafting materials. J Periodontol 2015;86:578-587.

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The use of EMD in a liquid formulation is able to provide better surface coating directly adjacent to the scaffold surface, with a significantly higher concentration of amelogenin proteins bound to the surface of thegrafting materials.

The liquid formulation of Enamel matrix derivative (EMD) is better able to increase surface loading of enamel matrix proteins to scaffold surfaces and improve the stability of this coating to the grafting material surface.

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This study was performed to specifically evaluate the cellular effects of Enamel Matrix Derivative ( EMD ) in liquid formulation and its subsequent influence on osteoblast and periodontal ligament cell behavior.

In the present study, the authors report that Enamel matrix derivative (EMD) in liquid formulation maintains an effect on cell behavior similar to EMD in gel formulation, and future investigations on cell behavior when seeded in the presence of a bone grafting material are now necessary.

Furthermore, in vivo study is also necessary to further evaluate the regenerative potential of Enamel matrix derivative (EMD) in liquid formulation when combined with a bone grafting material compared to EMD in a gel formulation.

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RELATED STUDIES

1. Hagewald S* et al investigated the effects of enamel matrix derivative ( EMD ) on proliferation , protein synthesis and mineralisation in primary mouse osteoblasts.

They concluded that EMD treatment enhanced cellular activities of primary osteoblasts and might support the regeneration of periodontal bony defects

* Hagewald S, Pischon N, Jawor P, Bernimoulin JP, Zimmermann B. Effects of enamel matrix derivative on proliferation and differentiation of primary osteoblasts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:243-249.

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2. He J , Jiang J , Safavi KE* investigated the effects of Enamel matrix derivative on the growth and differentiation of osteoblastic cells and on the expression of osteoprotegerin ( OPG ) , a key cytokine that inhibits osteoclastogenesis and osteoclast function.

They found that EMD promotes both proliferation and differentiation of osteoblastic cells and indirectly inhibits osteoclastogenesis and osteoclast function by stimulating the expression of OPG.

*He J, Jiang J, Safavi KE, Spangberg LS, Zhu Q. Emdogain promotes osteoblast proliferation and differentiation and stimulates osteoprotegerin expression. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:239-245.

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3. Weishaupt P et al in their study determined the effects of enamel matrix derivative on mRNA expression of markers related to periodontal healing.

The beneficial effects were seen in periodontal regeneration after treatment with EMD which may be related to an increase of the mineralisation markers.

*Weishaupt P, Bernimoulin JP, Trackman P, Hagewald S. Stimulation of osteoblasts with Emdogain increases the expression of specific mineralization markers. Oral Surg Oral Med Oral

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4. Miron RJ et al* in their study evaluated the influence of cell clustering on vital cell – cell communication and adhesion molecules.The results of their study further substantiates the clinical evidence that EMD increases the speed and quality of new bone formation in vivo.

*Miron RJ, Hedbom E, Ruggiero S, et al. Premature osteoblast clustering by enamel matrix proteins induces osteoblast differentiation through up-regulation of connexin 43 and N-cadherin. PLoS One 2011;6:e23375.

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5. Schwartz Z* et al in their study examined the response of osteoblasts at 3 stages of osteogenic maturation to porcine fetal enamel matrix derivative (EMD).

They concluded that EMD affects early stages of osteoblastic maturation by stimulating proliferation , but as cells mature in the lineage , EMD enhances differentiation.

*Schwartz Z, Carnes DL Jr., Pulliam R, et al. Porcine fetal enamel matrix derivative stimulates proliferation but notdifferentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-likeMG63cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells. J Periodontol2000;71:1287-1296.

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CONCLUSION

1. The results from the present study demonstrate that Enamel matrix derivative (EMD) in liquid formulation maintains osteopromotive properties similar to those of EMD gel.

2. The need for future research aimed at determining gene expression of cells exposed to EMD in the liquid formulation with a bone grafting material is now emphasized.