effect of green tea extract to angiogenesis in bone healing experimental study eri kriswanto
TRANSCRIPT
EFFECT OF GREEN TEA EXTRACT TO ANGIOGENESIS
IN BONE HEALINGEXPERIMENTAL STUDY
ERI KRISWANTO
Background:Non-union is a big problem in bone healingAngiogenesis plays important role in bone healingGreen tea is one of the strong anti-angiogenesis agentThere are no any research that study the effect of green tea to angiogenesis in bone healing
Green Tea(Camelia sinensis)
poliphenols in green tea : • flavanol• flavandiol• flavonol• phenolic acid, • catechin ( 70%) :Epigallocatehchin-3-
gallate (EGCG) about 67%, epigallocatehchin (EGC), epicatechin gallate (ECG), epicatechin (EC), dan gallocatechin
(Bokuchava, ..et al. 1980), (Kim, S,et,al.2000), (Fasina, G, et,al. 2004).
Bone Healing
Bone Healing in Mice Inflamation Soft callus (cartilage) day 7th
Chondrocytes presents VEGF Day 10th
Hard callus (bone) day 14th Remodelling
( Street, J,. et. al, 2002)
Effect of EGCG to Angiogenesis
fracture
Inflamation
Soft callus
Chondrocyte apoptosis
Cartilaginous matrix degradation and removal
AngiogenesisAngiogenesis
Rekrutmen osteogenic cell
Matrix production
Differentiation
Hard callus
Remodeling
Chondrocyte & osteoblast (Hypoxia)
Chondrocyte & osteoblast (Hypoxia)
HIFHIF VEGF, FGF, dan TGFVEGF, FGF, dan TGF
EndotelEndotelPericytePericyte
Survival, vascular permeability, migration, prolifera-tion, vascular guidance
Survival, vascular permeability, migration, prolifera-tion, vascular guidance
PDGFPDGF
VEGFVEGF
PDGFR, ERK 1/2PDGFR, ERK 1/2
FGFR, PI3K, ERK 1/2FGFR, PI3K, ERK 1/2
Green tea (EGCG)Green tea (EGCG)
Neo-vesselsNeo-vessels
ERK ½, PI3 K
Objective
1. To know the effect of green tea extract to amount of VEGF presenting cells in the callus
2. To know the effect of green tea extract to amount of vessels in the callus
3. To know the effect of green tea extract to cross-sectional-area of vessels in the callus
Materials and Method
DESIGN
Post Test Control Group Design.
S1 : sampleR : randomizationK1 : pure waterP1 : 15 mg green tea extractP2 : 20 mg green tea extractP3 : 25 mg green tea extractP4 : 30 mg green tea extract
Aclitimation ( 1 week)
P3
35 mices with rigth femur fracture
P2P1
20 mg15 mg 25 mg
Day 10th evaluation
Callus harversting
IHC staining
Analyzation
K1 P4
30 mgPure water
Aclitimation ( 1 week)
P7
35 mices with rigth femur fracture
P6P5
20 mg15 mg 25 mg
Day 14th evaluation
Callus harversting
HE staining
Analyzation
K2 P8
30 mgPure water
RESULTS
IHC stainingVEGF presenting cells
0 mg
25 mg
30 mg
15 mg
20 mg
Dose N Amount of VEGF presenting cells/15625 µ2
Mean SD Min Max
Pure water 5 4.8260
b,c 1.29475 3.30 6.13
15 mg 5 4.7000 b,c .90277 3.50 5.80
20 mg 5 3.6600 b .63087 3.00 4.40
25 mg 5 2.0200 a .31145 1.50 2.30
30 mg 4 2.3250 a .42720 1.80 2.80
a,b,c the different superscript shows there is significant difference ( p < 0.05 ) between groups ( Games – Howell)
Pure water
15 mg 20 mg 25 mg 30 mg 0
1
2
3
4
5
6
Amount of VEGF presenting cells/ 15625 μ2
Column1
HE staining- amount of vessels - cross-sectional area of vessels
0 mg
25 mg
30 mg
15 mg
20 mg
Dose N Amount of vessels / field
Mean SD Min Max
Pure water 5 48.8000 c,d 32.21335 14.00 98.00
15 mg 5 37.0000 a,b,c,d 23.18405 17.00 74.00
20 mg 5 26.4000 a,b,c 18.07761 15.00 58.00
25 mg 5 12.4000 a 7.09225 6.00 23.00
30 mg 5 16.6000 a,b 6.42651 6.00 23.00
a,b,c the different superscript shows there is significant difference ( p<0.05 ) between groups ( List Significant Data)
Purewater
15 mg 20 mg 25 mg 30 mg0
10
20
30
40
50
60
Amount of vessels / field
Column1
Dose N Cross-sectional area of vessels (µ2)
Mean SD Min Max
Pure water 5 139.8000
d22.0697
8 115.00 167.50
15 mg 5 101.1000 b,c,d
37.53065 75.00 165.50
20 mg 5 58.5000 b,c 7.20243 50.00 67.50
25 mg 5 48.5000 a,b 8.02340 37.50 60.00
30 mg 5 38.0000 a 7.58288 25.00 45.00
a,b,c the different superscript shows there is significant difference ( p<0.05 ) between groups ( Games – Howell)
Pure water
15 mg 20 mg 25 mg 30 mg0
20
40
60
80
100
120
140
160
Cross-sectional are of vessels (μ2)
Column1
DISCUSSION • Green tea or EGCG per orally shows significant
inhibition to angiogenesis (Fassina, G, et, al. 2004).
• 1.25 % (4,69 mg/ml) of green tea contains 708 µg/ml EGCG per orally will produce 0,1-0,3 µM EGCG in the blood, this dose is equal with concentration of EGCG in human after drinking 2 or 3 cups of green tea (Wang, Z.Y,et,al. 1992).
• Drinking 1.25% of green tea significantly inhibit neo-vascularization in mice’s cornea ( Cao Y,
Cao R, 1999).
Discussion
Drinking 1.25% (4.69 mg/ml) of green tea ad libitum in mice is equal with 15 mg of green tea extract single dose.
Results : • Significant decrease of cross-sectional area of vessels in
callus was found at minimal dose of 20 mg green tea extract .
• Significant decrease amount of VEGF presenting cells in callus was found at minimal dose of 25 mg green tea extract
• Significant decreases amount of vessels in callus was found at minimal dose of 25 mg green tea extract
Conclusion
• Drinking of green tea > 15 mg in mice equal with drinking
> 2 or 3 cups of green tea in human will decrease VEGF presenting cells, amount of vessels, and amount of cross-sectional area of vessels in bone healing.
• Green tea significantly inhibit angiogenesis in bone healing.
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