EVALUATION OF HESPERIDIN [FLAVONOID] AS A DIRECT PULP CAPPING MATERIALPresented by: Ebtesam Osama Abo El-Mal

Under supervision of:

Prof. Dr. Salma Hassan El-Ashry

Prof. Dr. Ashraf Abd El-Rahman Abo Seeda

DEDICATION

ACKNOWLEDMENT

INTRODUCTION

• Pulp vitality is extremely important for tooth viability, maintaining the capacity for

limited dentin regeneration -which is particularly important in immature permanent

teeth- and dentinal wall development.

• Direct pulp protection after exposure due to caries or injuries permits preservation of

the pulp. Many experiments had been carried on several materials and new pulp

capping agents were introduced as MTA, Portlad cement & Bio-aggregate.

INTRODUCTION

• Nowadays there is a great trend to use natural materials as cure for many diseases.

During that last 10 years, considerable researches had been conducted on Propolis –a

resinous material collected from honey bees from various plants.

AIM OF THE STUDY

• The aim of the present study was to evaluate the effect of Hesperidin (flavonoid) -in

Egyptian honey- as a direct pulp capping material in terms of:-

Part I: the physical properties of Hesperidin in comparison to MTA-Angelusconcerning setting time, radio-opacity, solubility and pH determination.

Part II: Histopathological effect of the Hesperidin compared to MTA-Angelus asa pulp capping agent on dogs’ dental pulp tissue.

METHODOLOGY

Part I: Evaluating some physical properties of Hesperidin powder and MTA-Angelus:

SETTING TIME EVALUATION:

• The mixed materials were placed in molds designed according to the ANSI/ADA

specifications.

• Gilmore needle of 100 ± 0.5 gm in

weight and 2 ± 0.1 mm flat end

diameter was used to evaluate initial

setting time.

• Another Gilmore needle of 456 ± 0.5

gm weight and 1 ± 0.1 mm flat end

diameter was used for evaluation of the

final setting time.

RADIO-OPACITY EVALUATION USING DIGITAL RADIOGRAPHIC SYSTEM:

• An acrylic device that maintained thehead of the x-ray machine in the sameposition was prepared.

• 5 acrylic plates, 4 having 6 holes filledwith the materials and the other onehaving aluminium step-wedge, wereplaced in front of digital phosphorplate.

• Exposed images were scannedimmediately using Digora scanner andthe grey-pixel values of the materialswere obtained.

SOLUBILITY TEST:

• 20mm diameter & 1.5mm thick discs of

each material, with impermeable nylon

threads inserted in them, were immersed

and hung in wide mouthed bottles

containing 50ml distilled water.

• Materials’ solubility was calculated as the

% of changed mass to the initial mass.

PH EVALUATION:

• Recovered solutions, in which samples of solubility were stored, were taken formeasurement of pH changes using pH meter.

• Measuring pH was performed at 7, 14, 21 & 28 days from mixing.

Part II: Histopathological analysis of dogs’ dental pulp tissue capped with the materials

• After anaesthetizing the dogs, Class V

cavities were prepared on the cervical

third of the buccal surface.

• Cavities were deepened and the pulp

tissue was exposed in a standardized

manner.

• After controlling pulp haemorrhage, the

three capping materials were placed and

cavities were sealed.

• Animals were sacrificed after 2, 4 & 8

weeks. Labio-lingual sections passing

through the center of the exposure site were

evaluated for inflammatory cell response

and dentin bridge formation.

RESULTS I] Physical properties of the materials

SETTING TIME • Comparing initial & final setting

time of the 3 materials

SampleInitial setting time (min.) Final setting time (min.)

Mean ± SD Min Max Mean ± SD Min Max

Hesperidin15.64a±

0.38

15.1

016.13

48.26b±

3.4745.95 56.68

MTA13.33b±

2.18

11.0

317.62

72.83a±17.

2957.65

112.8

3

Dycal 0.85c± 0.15 0.50 0.98 1.58c± 0.26 1.38 2.20

F value 308.99 101.08

P value <0.0001* <0.0001*

• Comparing initial & final setting times within the same material

Sample

Setting time

Hesperi

dinMTA Dycal

Mean initial setting

time ±SD

15.64

±0.38

13.33

±2.18

0.85 ±

0.15

Mean final setting

time ±SD

48.26

±3.47

72.83

±17.29

1.58

±0.26

R value 0.176 0.861 0.304

P value 0.677 0.006* 0.463

RADIOPACITY:

Sample Mean ± SD Min Max

Hesperidin72.63a ±

7.3864.20 81.86

MTA179.85b ±

7.78171.07 188.89

Dycal135.56c ±

9.78122.27 146.92

F value 319.71

P value < 0.0001*

SOLUBILITY (% OF MASS CHANGE):

Sample Mean ± SD Min Max

Hesperidin-44.76c ±

11.5334.98 62.86

MTA 9.41a ± 1.36 7.89 12.11

Dycal -18.87b ± 5.17 7.58 28.11

F value 216.11

P value <0.0001*

PH EVALUATION:• Within the same group:

Evaluation

periods

Samples

pH

at 7

day

s

pH

at 14

days

pH

at

21

da

ys

pH

at

28

day

s

P

val

ue

(I)Hesperidin

5.71a

± 0.37

5.58a,b

± 0.07

5.43a,b

± 0.10

5.37b

± 0.170.0138*

5.26 5.40 5.25 5.23

6.18 5.63 5.60 5.76

(II)MTA

11.48b

± 0.11

11.56b

± 0.15

11.82a

± 0.21

11.12c

± 0.14<0.0001*

11.32 11.48 11.72 11.04

11.58 11.94 12.34 11.46

(III)Dycal

11.40a,b

± 0.06

11.49a

± 0.08

11.53a

± 0.11

11.33b

± 0.150.0028*

11.32 11.37 11.40 11.18

11.48 11.59 11.69 11.54

• Between groups at differentobservation periods:

Periods of

evaluation

Sample

pH at

7days

pH at

14day

s

pH

at

21

da

ys

pH at

28da

ys

(I)Hesperidin 5.71b

± 0.37

5.58b

± 0.07

5.43b

± 0.10

5.37b

± 0.17

(II)MTA11.48a

± 0.11

11.56a

± 0.15

11.82a

± 0.21

11.12a

± 0.14

(III)Dycal11.40a

± 0.06

11.49a

± 0.08

11.53a

± 0.11

11.33a

± 0.15

F value 1726.43 7987.04 4679.69 3833.95

P value <0.0001* <0.0001* <0.0001* <0.0001*

0

2

4

6

8

10

12

pH at 7days pH at 14days pH at 21days pH at 28days

pH

Hesperidin

MTA

Dycal

PART II: HISTOPATHOLOGIC EVALUATION:

i) Inflammatory response:

A)COMPARISON OF DIFFERENT SUBGROUPS WITHIN THE SAME TIME

INTERVAL:

• At 2 weeks interval:

• 14.3% of Hesperidin samples, 35.7% in

MTA-Angelus and 85.7% in Dycal showed

mild amount of inflammatory cells with

focal necrotic areas and massive

vasodilatation in non-necrotic areas.

A)COMPARISON OF DIFFERENT SUBGROUPS WITHIN THE SAME TIME

INTERVAL:

• At 2 weeks interval:

• 85.7% of Hesperidin subgroupand 64.3% of MTA-Angelusshowed moderate inflammation.

A)COMPARISON OF DIFFERENT SUBGROUPS WITHIN THE SAME TIME

INTERVAL:• At 2 weeks interval:

• In Dycal subgroup, 14.3% only revealed severe inflammation localized directly below the exposure site.

A)COMPARISON OF DIFFERENT SUBGROUPS WITHIN THE SAME TIME

INTERVAL:

• At 4 weeks interval:

• 85.7% of Hesperidin & MTA-Angelus subgroups exhibited mildamount of scattered chronicinflammatory cells.

A)COMPARISON OF DIFFERENT SUBGROUPS WITHIN THE SAME TIME

INTERVAL:• At 4 weeks interval:

• 64.3% of Dycal samples showedmoderate amount of inflammatoryinfiltrate with persistence of focalnecrosis and micro abscessformation.

A)COMPARISON OF DIFFERENT SUBGROUPS WITHIN THE SAME TIME

INTERVAL:

• At 8 weeks interval:

• Overall, all specimens ofsubgroups showed mild or noinflammatory cell infiltration.

A)COMPARISON OF DIFFERENT SUBGROUPS WITHIN THE SAME TIME

INTERVAL:

• At 8 weeks interval:

• Samples of both MTA-Angelus &Dycal were still having minutefocal necrotic areas.

A)COMPARISON OF DIFFERENT SUBGROUPS WITHIN THE SAME TIME

INTERVAL:

B) COMPARISON BETWEEN DIFFERENT TIME INTERVALS WITHIN THE SAME

MATERIAL’S SUBGROUP:

PART II: HISTOPATHOLOGIC EVALUATION:

ii) Dentin bridge formation:

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:

• At 2 weeks interval:

• Complete or even partial calcifiedbridge was not detected in any ofthe three subgroups, with noodontoblastic layer.

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:

• At 2 weeks interval:

• 28.6% of Hesperidin samplesonly showed a great deal ofthe material pushed into thepulp space with dystrophiccalcification around them.

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:• At 2 weeks interval:

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:

• At 4 weeks interval:

• 57.1% of Hesperidin samplesexhibited dense moderately thickstructure with scattered calcifieddeposits.

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:

• At 4 weeks interval:

• In MTA-Angelus subgroup,similar results were obtained inmost of the samples however withan interrupted thicker structure.

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:• At 4 weeks interval:

• Dycal samples showed verynarrow interrupted zone ofcondensed hard deposit.

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:• At 4 weeks interval:

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:

• In 8 weeks interval:

• Only 7.1% of samples inHesperidin subgroupdemonstrated dentin bridge withrecognizable continuous thickthickness and hyperplasticdisorganized odontoblast-like celllayer.

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:

• In 8 weeks interval:

• No samples in MTA-Angelussubgroup showed any dentinbridge, but 92.9% of the samplesrevealed randomly organizedodontoblast-like cell layer withirregular thin tubular dentin.

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:• In 8 weeks interval:

• 50% of the samples in Dycalsubgroup showed interruptedzone of hard deposit wider thanthat seen in the 4th week.

A) COMPARISON OF DIFFERENT MATERIALS (SUBGROUPS) IN THE SAME

TIME INTERVAL:• In 8 weeks interval:

B) COMPARISON OF DIFFERENT OBSERVATION PERIODS WITHIN THE

SAME SUBGROUP: Subgroup

Group

Item A (n=14) B (n=14) C (n=14)

1 2 3 1 2 3 1 2 3

I

(2 W)

Conti-

nuity

14

(100)

0 0 14

(100)

0 0 14

(100)

0 0

II

(4 W)

3 (21.4) 11

(78.5)

0 3 (21.4) 11

(78.5)

0 5

(35.7)

9

(64.3)

0

III

(8 W)

2

(14.3)

11

(78.5)

1

(7.1)

1

(7.1)

13

(92.9)

0 7

(50)

7

(50)

0

X2 27 28.583 13.529

P value 0.00002* 0.000009* 0.0089*I

(2 W)

Morpho-logy 14

(100)

0 0 14

(100)

0 0 14

(100)

0 0

II

(4 W)

14

(100)

0 0 14

(100)

0 0 14

(100)

0 0

III

(8 W)

2

(14.3)

11

(78.5)

1

(7.1)

2

(14.3)

12

(85.7)

0 2

(14.3)

12

(85.7)

0

X2 33.6 26.53 26.53

P value 0.00001* 0.000024* 0.000024*I

(2 W)

Thick-ness 14

(100)

0 0 14

(100)

0 0 14

(100)

0 0

II

(4 W)6

(42.9)

8

(57.1)

0 0 6

(42.9)

8

(57.1)

12

(85.7)

2

(14.3)0

III

(8 W)

2

(14.3)

11

(78.5)

1

(7.1)

11

(78.5)

3

(21.4)0 3

(21.4)

11

(78.5)

0

X2 22.39 35.04 22.95

P value 0.00017* 0.000014* 0.00013*

B) COMPARISON OF DIFFERENT OBSERVATION PERIODS WITHIN THE

SAME SUBGROUP:

Subgroup

Group

Item A (n=14) B (n=14) C (n=14)

1 2 3 1 2 3 1 2 3

I

(2 W)

Calci-fication 0 4

(28.6)

10

(71.4)

0 0 14

(100)

0 0 14

(100)

II

(4 W)

0 8

(57.1)

6

(42.9)

0 8

(57.1)

6

(42.9)

0 3

(21.4)

11

(78.6)

III

(8 W)

0 8

(57.1)

6

(42.9)

0 8

(57.1)

6

(42.9)

0 3

(21.4)

11

(78.6)

X2 3.055 12.923 3.5

P value 0.548ns 0.012* 0.4778ns

I

(2 W)

Odonto-blastic

layer

14

(100)

0 0 14

(100)

0 0 14

(100)

0 0

II

(4 W)

3

(21.4)

11

(78.6)

0 3

(21.4)

11

(78.6)

0 6

(42.9)

8

(57.1)

0

III

(8 W)

0 13

(92.9)

1

(7.1)

1

(7.1)

13

(92.9)

0 3

(21.4)

11

(78.6)

0

X2 33.43 28.58 18.645

P value <0.0001* 0.000009* 0.0009*

CONCLUSIONS

MTA-Angelus & Dycal had significantly higher radiopacity comparedto Hesperidin.

MTA-Angelus & Dycal had similar behaviour in pH analysis.

Hesperidin was a comparable material to MTA-Angelus in regard toinflammation and dentin bridge formation.

Direct pulp capping with Hesperidin delays pulp inflammation andstimulates dentin bridge formation.

Despite its slight acidic nature, Hesperidin is a promising pulp cappingmaterial.

RECOMMENDATIONS

Add radiopacifying material to the Hesperidin powder to be easily

distinguished radiographically.

Evaluation of other more physical properties of Hesperidin is recommended.

Further studies to improve solubility of Hesperidin is recommended.

Randomized clinical trial for Hesperidin material may be beneficial.