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

1. 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

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

3. INTRODUCTION
The aim of regenerative
periodontal therapy is to
restore the tooth –
supporting apparatus lost
following periodontal
disease or trauma.

4. 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.

5. 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

6. 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

7. 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.

8. 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. )

9. 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.

10. 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.

11. 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.

12. 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.

13. 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 was
dissolved 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.

14. 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

15. 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.

16. 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.

17. 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.

18. 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.

19. 5. Alizarin Red Staining
Accumulation of calcium in the Periodontal ligament cells was analysed
by Alizarin red staining.

20. RESULTS

21. 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

22. 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.

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

24. 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.

25. DISCUSSION

26. 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.

27. 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 the
grafting 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.

28. 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.

29. 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.

30. 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.

31. 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

32. 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.

33. 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 not
differentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-like
MG63cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells. J Periodontol
2000;71:1287-1296.

34. 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.