Creeping Stroke - Venous thrombosis presenting with pc-stroke.pptx
Jc 7
1. EFFECT OF ENAMEL MATRIX
DERIVATIVE LIQUID ON OSTEOBLAST
AND PERIODONTAL LIGAMENT CELL
PROLIFERATION AND DIFFERENTIATION
Miron RJ et al.
JOP 2016;87:91-99.
Presented by,
Dr. Bibina George
3. ENAMEL MATRIX DERIVATIVERootConditioningAgent
Embryonic
Porcine enamel
Tooth dev.---HERs
EMP
Acellular Cementum
formation
REGENERATION
Cell adhesion
Gestrelius et al 1997
Palioto et al 2004
Rodrigues et al 2007
Proliferation
Differentiation
BoneRemodelling
Cell attachment(+)
Transcription
factors
GF
Okubo et al 2003
TNF,IL-8
Myhre et al 2006
4. • FDA approved EMD ------- EMDOGAIN (Viscous EMD in polypropylene liquid)
Constituents:
90 % Amelogenin
10 % Proline rich Nonamelogenin,
Tuftelin, Ameloblastin, Amelins
• Copacked with Prefgel
5. LIMITATIONS
• Viscous nature in its carrier gel
May not be sufficient to prevent flap
collapse in large pdl defects
< space available for regeneration
Limiting clinical outcomes
OVERCOME BY-----
• EMD + Bone Graft
Liquid formulation in acetic acid
Zhyang Y et al, 2015
Better surface coating
Protein adsorption(Amelogenin)
Better penetration of Amelogenin
Miron RJ
Bosshardt DD et al,
2015
6. AIM:
• To investigate the two carrier systems with EMD on primary human
osteoblasts and PDL cell behavior.
• Tested for their ability to induce osteoblast and PDL cell proliferation and
differentiation as assessed by real-time polymerase chain reaction (PCR)
and alizarin red staining.
7. MATERIAL 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.
Ethical clearance:
Reviewed and approved
by Ethics Committee for Human
Research, Wuhan University, China
8. 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.
2. OSTEOBLASTAND PDLCELLISOLATIONAND DIFFERENTIATION
9. 3. Real-Time PCR for Growth Factors and Differentiation Markers
Total RNA was isolated using : -
Reagent - Trizol
Kit - RNA easy Mini Kit ( Quiagen , Switzerland )
Primer & probe sequences for genes encoding bone morphogenetic protein
2 (BMP 2), transforming growth factor (TGF β1), IL-8 were purchased as
predesigned gene expression assays.
10. 4. Alizarin Red Staining
• Accumulation of calcium in the Periodontal ligament cells was analysed by
Alizarin red staining.
15. 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.
16. Discussion :
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 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.
17. 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.
18. • In the present study, the authors report that 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 EMD in liquid formulation when combined with a
bone grafting material compared to EMD in a gel formulation
19. RELATED STUDIES :
• 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
20. • 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
21. • 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
22. • 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
23. CONCLUSION :
The results from the present study demonstrate that Enamel matrix
derivative (EMD) in liquid formulation maintains osteopromotive properties
similar to those of EMD gel.
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.
Editor's Notes
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.
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.
24% EDTA to remove smear layer
Since there is no preliminary data are available testing this new formulation of EMD in a liquid carrier compared to EMD in a gel carrier,
Human alveolar osteoblasts and PDL cells were obtained from three human donors. All patients provided written informed consent
Both formulations significantly increased cell proliferation of both A) osteoblasts and B) PDL cells compared to control surfaces at 3 and 5 days after seeding. No significant difference in cell number was observed between samples treated with EMD in the gel formulation versus EMD in the liquid formulation.
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.
Influence of EMD in gel and liquid formulations on mRNA expression of BMP2 of primary human osteoblasts and PDL cells 8, 24, and 72 hours after seeding. Both EMD formulations were able to significantly alter gene expression of both osteoblast and PDL cell mRNA levels as assessed by real-time PCR.
Both the Enamel Matrix Derivative formulations significantly reduced the expression of IL – 8 in osteoblasts & periodontal ligament cells at all points.
