effect of green tea extract to angiogenesis in bone healing experimental study eri kriswanto

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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