88

Pulp tissue inflammation and angiogenesis after pulp capping 
with transforming growth factor b1

Sri Kunarti
Department of Conservative Dentistry
Faculty of Dentistry, Airlangga University
Surabaya - Indonesia

abstract 
In	Restorative	dentistry	the	opportunity	to	develop	biomemitic	approaches	has	been	signalled	by	the	possible	use	of	various	biological	

macromolecules	in	direct	pulp	capping	reparation.	The	presence	of	growth	factors	in	dentin	matrix	and	the	putative	role	indicating	
odontoblast	differentiation	during	embryogenesis	has	led	to	the	examination	on	the	effect	of	endogenous	TGF-b1.	TGF-b1	is	one	of	the	
Growth	Factors	that	plays	an	important	role	in	pulp	healing.	The	application	of	exogenous	TGF-b1	in	direct	pulp	capping	treatment	
should	be	experimented	in	fibroblast	tissue	in-vivo	to	see	the	responses	of	inflammatory	cells	and	development	of	new	blood	vessels.		
The	increase	in	food	supplies	always	occurs	in	the	process	of	inflammation	therefore	the	development	of	angiogenesis	is	required	to	fulfil	
the	requirement.	This	in-vivo	study	done	on	orthodontic	patients	indicated	for	premolar	extraction	between	10–15	years	of	age.	A	class	
V	cavity	preparation	was	created	in	the	buccal	aspect	1	mm	above	gingival	margin	to	pulp	exposure.	The	cavity	was	slowly	irrigated	
with	saline	solution	and	dried	using	a	sterile	small	cotton	pellet.	The	sterile	absorbable	collagen	membrane	was	applied	and	soaked	in		
5	ml	TGF-b1.	It	was	covered	by	a	Teflon	pledge	to	separate	from	Glass	Ionomer	Cement	restoration.	Evaluation	was	performed	on	day	
7;	14;	and	21.	All	samples	were	histopathologycally	examined	and	data	was	statistically	analysed	using	one	way	ANOVA	and	Dunnet	
T3.There	were	no	inflammatory	symptoms	in	clinical	examination	on	both	Ca(OH)2	and	TGF-b1,	but	they	increased	the	infiltration	
of	 inflammatory	 cells	 on	 histopathological	 examination.	 There	 were	 no	 significant	 differences	 (p	 >	 0.05)	 between	 Ca(OH)2	 and		
TGF-b1	in	inflammation	cell	and	significant	differences	(p	<	0.05)	in	angiogenesis	on	day	7	and	14.	There	were	no	significant	differences	
(p	>	0.05)	in	inflammation	cell	with	in	TGF-b1	groups	and	significant	differences	(p	<	0.05)	with	in	Ca(OH)2	groups	on	day	7	and	14.	
It	is	concluded	TGF-b1	functions	as	direct	capping	medication	has	the	same	inflammatory	response	as	Ca(OH)2,	however,	TGF-b1	
developed	angiogenesis	earlier	than	Ca(OH)2.	

Key words:	calcium	hydroxide,	TGF-b1,	inflammation,	angiogenesis	

Correspondence: Sri Kunarti, c/o: Departemen Konservasi Gigi, Fakultas Kedokteran Gigi Universitas Airlangga. Jln. Mayjend. Prof. 
Dr. Moestopo No. 47 Surabaya 60132, Indonesia. E-mail: attybp@yahoo.com

introduction

Homeostatis condition in human’s tissue needs balanced 
interaction between cells and extra cellular matrix as protein 
supplier involving the role of various cytokines through 
specific receptor. Disease will consequently occur if the 
balance between cells and extra cellular matrix is disturbed. 
The improvement of damaged tissue will manifest if the 
body successfully maintains the homeostatic condition from 
the effect of harmful environment.1

Dentin perforation will initially cause several episodes 
such as: inflammation, synthesis of new collagen and the 
formation of dentinal bridge, finally, reparative dentin 
formation will be necessarily done.2,3 The inflammation is 
indicated by movement of fluid circulation, plasma protein 
and leukocyte to tissue responding to the presence of danger. 
Inflammation is essential mechanism which is required to 
improve structure and tissue functional disturbance as well 
as to defend itself from the danger such as: tissue damage, 
microorganism invasion and foreign body disturbing the 
balance interaction between cells and extra cellular matrix 

(ECM). Inflammatory response completely depends on 
blood vessel, cell as well as fluid circulating in blood 
vessel.4 The initial symptom of inflammation is indicated by 
secretion of various mediators such as transforming growth 
factor-b1 (TGF-b1) followed by activation of complement, 
coagulation system, inflammation cell and endothelial cell. 
Immediately after the injury is present in dentin, it will be 
followed by recovery process by releasing various growth 
factors, cytokine and accumulative molecule from serum 
from blood vessel rupture and platelet degranulation.5 
Inflammation cell is also the source of various growth factor 
which are needed to initiate proliferation phase in tissue 
recovery. The formation of reparative dentin is completely 
influenced by inflammation process, cell proliferation, cell 
migration, angiogenesis and production of extra cellular 
matrix. TGF-b1 regulates all of the episodes including 
chemotaxis of inflammation cell, angiogenesis, deposition 
of extra cellular matrix and the formation of new tissue.6

 Concentration of TGF-b1 increases in dentin caries 
condition comparing to healthy dentin. TGF-b1 in 
reparative dentin matrix is higher than primary dentin 



89Kunarti: Pulp tissue inflammation and angiogenesis after pulp capping

matrix. The increase of intracellular expression of TGF-b1 
and ECM in dentin carries is suspected that TGF-b1 has 
an essential role to response injury and tissue recovery.7 
The parameter of inflammation response in the present 
study is the number of inflammation cells and synthesis of 
new blood vessels (angiogenesis) in 7, 14 and 21 days of 
recovery process. The purpose of the present study was to 
observe the response of inflammation cell and angiogenesis 
of TGF-b1and material with calcium hydroxide [Ca(OH)2] 
as the comparation because up to the present the material 
of direct pulp capping.

materials and methods

The study was done on orthodontic patients indicated 
for premolar extraction. Anaesthesia in buccal fold region 
was done using 0.6 ml xylestesin F. Rubber dam, and saliva 
ejector were aplied. Alcohol 70% were used to disinfect 
the preparation at buccocervical. Preparation of the cavity 
on 1 mm buccal site above gingival margin, completed 
by intermittent bur with light pressure. Preparation was 
done declining toward apical using no. 3 round bur in 
1.5 mm diameter almost reaching the pulp, then no. 1 
round bur in 0.5 mm diameter was used to penetrate thin 
dentin layer reaching perforation in pulpa coronalis. Slow 
irrigation using 0.5 ml saline solution was performed in 
cavity region and dried by sterile cotton pellet. Visible light 
of Ca(OH)2 in tube (calcimol) was applied 1 mm on pulp 
for 40 seconds. In other groups, 20 ng/ml TGF-b1 caried by 
material collagen membrane3 was applied and completed 
by filling material of glass ionomer cement type 2. Teflon 
pledged8 was given to avoid the possible reaction between 
glass ionomer cement type 2 and material of pulp capping. 
Then extraction was classified into 3 extraction timing on 
day 7, 14 and 21 direct pulp capping. The preparation of 
histological preparat was began since the initial process 
of extraction. Fixation using 10% formalin buffer for 48 
hours followed by decalcification using AlCl, formic acid, 
37% HCl and aquadest continued by dehydration process 
to extract the water from the tissue and substituted by 
hardening media (paraffin) and clearance was done using 
xylene.

After paraffin hardening, the tissue could be cut using 
microtome in 4 µm thickness. The next step, hematoxylin 

and eosin were stained on the resulting preparat. Light 
microscope in 400 times magnification was used to count 
the number of inflammation cell (Figure 1) and blood 
vessel (Figure 2).

table 1. The mean and standard deviation of inflammation in control group of Ca(OH)2 and TGF-and TGF--b1 on day 7, 14, and 
21

Variable Material 
7 days 14 days 21 days

Mean SD Mean SD Mean SD

Inflammation Ca(OH)2
TGF-b1

19.3750
21.6250

6.2321
3.8522

19.1250
19.8750

4.1897
3.0909

16.5000
18.5000

4.5981
2.0000

Angiogenesis Ca(OH)2
TGF-b1

24.3750
16.6250

1.7678
4.6579

20.1250
12.8750

2.7484
4.7461

17.8750
12.3750

7.0191
3.3354

SD: Standar deviation

figure 1. Inflammation cell 

figure 2. Angiogenesis ( )

result

ANOVA test was performed to know the different 
response between inflammation cell and angiogenesis 
in Ca(OH)2 and TGF-b1. The result showed significant 
difference (p < 0.05) in control group, followed by LSD test 
to know the difference in the period of 7–14 days, 14–21 
days, and 7–21 days (Table 2).



90 Dent. J. (Maj. Ked. Gigi), Vol. 41. No. 2 April-June 2008: 88-90

table 2. LSD-test on inflammation cell and angiogenesis 
and Ca(OH) 2 and TGF-b1 in the period  
7–14 days, 14–21 days, and 7–21 days–21 days, and 7–21 days21 days, and 7–21 days–21 days21 days

Variable 
Period of 

Observation
P value

Ca(OH)2 TGF-β1

Inflammation cell 7–14 days
7–21 days

14–21 days

0.923
0.271
0.313

0.268
0.055
0.382

Angiogenesis 7–14 days
7–21 days

14–21 days

0.009*
0.093
0.785

0.096
0.061
0.818

* = Significant difference

p of Ca(OH)2 group showed significant difference in 
angiogenesis in comparison period of 7–14 days. The result 
of t-Test between Ca(OH)2 and TGF-b1 on day 7, 14 and 
21 showed significant different in angiogenesis on day 7 
and 14 (Table 3).

table 3. t-Test inflammation cell and angiogenesis 
comparison of Ca(OH)2 and TGF-b1 among of 
7 days, 14 days and 21 days

Variable

p value

Ca(OH)2 and TGF-β1

7 days 14 days 21 days

Inflammation cell
Angiogenesis

0.400
0.001*

0.690
0.002*

0.287
0.073

* = significant difference

discussion

In this study, there was no significant difference 
in infiltration of inflammation cell between Ca(OH)2 
and TGF-b1 on day 7, 14, and 21, however the mean 
showed that TGF-b1 was higher comparing to Ca(OH)2 
due to potential proinflammation character TGF-b1 
such as: affecting Leukocyte in the initial response of 
inflammation and strengthening inflammation response in 
low concentration by stimulating integrin expression, in 
addition, initiating chemotaxis process as well as increasing 
extravasation by enhancing useful enzyme in extravasation 
process. The concentration of TGF-b1 increased during 
inflammation process, by adding exogenic TGF-b1, thus, 
the concentration would be higher, monocyte/macrophage 
and lymphocyte would be activated by TGF-b1 to remove 
the pathogen. TGF-b1 has the most potential role in 
inflammation and immune response among TGF-b1 
isoform. Foreign molecules, tissue debris and accumulative 

leukocyte are no longer necessary prior to pathogen 
removal. Subsequently the number of plasmin production 
would also lower and the concentration of TGF-b15 would 
be similar.

In this study, the observation on day 7 and 14 showed 
significant difference in angiogenesis. The mean of  
TGF-b1 was lower compared to the mean Ca(OH)2 due 
to the presence of angiogenesis/neovascularization prior 
to day 7 in TGF-b1 group. The initial recovery started on 
day 3rd prior to injury meanwhile the granulation tissue is 
completely vascular. Capillary new blood vessel would 
penetrate into necrotic tissue in which inflammatory 
exudates is eradicated by macrophage. Observation on 
day 7 showed the presence of collagen synthesis type 
I and simultaneously showed the decrease of capillary 
blood vessel. The proceeding recovery process would be 
followed by decrease of ratio between vascular tissue and 
fibroblast tissue and subsequently collagen production 
would elevate.9 TGF-b1 is one of the growth factors which 
would influence angiogenesis activity and it is important for 
vascular maturization and remodelling.10 TGF-b1 not only 
increase fibrous tissue deposition, migration of fibroblast 
proliferation but also collagen synthesis.

Based on the above explanation it is concluded that 
angiogenesis decrease before day 7 in TGF-b1 group 
compared to Ca(OH)2 in which angiogenesis is higher. 
In the comparison between both materials the increase of 
inflammation cell is not present.

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