Lymphangiogenesis in human dental pulp
F. J. G. S. Pimenta, A. R. Sä & R. S. Gomez
Department of Oral Surgery and Pathology, School of Dentistry, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
Abstract
Pimenta FJGS, Sä AR, Gomez RS. Lymphangiogenesis in
human dental pulp. International Endodontic Journal, 36, 853^856,
2003.
Aim To investigate the impact of in£ammation on lym-
phangiogenesis in human dental pulp.
Methodology Eleven samples of dental pulp without
in£ammation and 11 dental pulps with moderate to
intense mononuclear cell in£ammatory in¢ltrate asso-
ciated with dentine caries were selected. The streptavi-
din^biotin complex stain was used to detect CD31,
vascular endothelial growth factor receptor-3 (VEGFR-
3) and a-smooth muscle actin. The number of lymphatic
vessels was obtained by counting the number of vessels
Introduction
The dental pulp is a loose connective tissue enclosed
within the rigid structure of the mineralized tissues of
the tooth. The low interstitial compliance restricts the
possibilities of £uid exchange with the neighbouring tis-
sues. Therefore, the dental pulp is more sensitive to
changes in tissue pressure than other tissues, and it
requires an active drainage of the extracellular matrix
to eliminate excess £uid and macromolecular sub-
stances. During in£ammation, changes in blood pres-
sure and capillary permeability result in an increase in
tissue volume and interstitial £uid pressure. However,
unless a severe in£ammatory state is induced, tissue
necrosis is normally not observed. Thus, it is evident that
an e¤cient drainage system operates in human dental
pulp limiting the initial e¡ects of in£ammation.
Correspondence: Prof. Ricardo Santiago Gomez, Department of
Oral Surgery and Pathology, School of Dentistry, Universidade
Federal de Minas Gerais, AvenueAntonio Carlos,6627, BeloHori-
zonte, MG, CEP 31270 901, Brazil (e-mail: rsgomez@ufmg.br).
ß 2003 Blackwell Publishing Ltd
positive for CD31 and VEGFR-3 and negative for a-
smooth muscle actin.
Results The results demonstrated that the mean num-
ber (SD) of vessels positive for CD31and VEGFR-3 (lym-
phatic vessels) in the group with in£ammation (6.09 
1.81) was statistically higher (P ˆ 0.0123) than the mean
number in the group without in£ammation (3.73  2.20).
Conclusion Increased co-immunostaining of CD31
and VEGF-3 in vessels associated with human dental
pulp in£ammation occurred, which suggests lymphan-
giogenesis.
Keywords: dental pulp, immunohistochemical meth-
ods, lymphangiogenesis, VEGFR-3.
Received 25 March 2003; accepted16 July 2003
Although the existence of lymphatic vessels in human
dental pulp was considered controversial, it has been
con¢rmed by histochemical, immunohistochemical
and electron microscopic techniques (Bernick1977, Kato
1990, Marchetti et al. 1991, 1992, Matsumoto et al. 1997,
2002, Marchetti & Poggi 2002). Despite the fact that
blood and lymphatic vessels were identi¢ed in these stu-
dies, the lack of reliable lymphatic endothelial markers
has hampered a consistent evaluation of the lymphatic
vasculature in human dental pulp. Recently, the vascu-
lar endothelial growth factor receptor-3 (VEGFR-3, also
indicated as Flt-4) was demonstrated to be a marker of
lymphatic endothelium (Enholm et al. 1998, Jussila et al.
1998, Breitened-Gele¡ et al. 1999, Clarijs et al. 2001).
VEGFR-3 is initially expressed in all embryonic endothe-
lia, but its expression in the blood vessel endothelium
decreases during development, and it becomes largely
restricted to the lymphatic endothelium in adult tissues
(Kaipanen et al. 1995, Kukk et al. 1996).
Considering that lymphatic vessels in the dental pulp
play an important role in the drainage of excess tissue
£uid, the purpose of the present study was to investigate
the impact of in£ammation on lymphangiogenesis in
human dental pulp.
International Endodontic Journal, 36, 853^856, 2003 853
 Lymphangiogenesis in human dental pulp Pimenta et al.
Materials and methods
Sample selection
The methodology was approved by the local ethics com-
mittee. Dental pulps from erupted teeth extracted for
orthodontic reasons or with dentine caries were col-
lected at dental clinics and were snap-frozen and stored
in liquid nitrogen. All the samples were evaluated micro-
scopically according to the presence of in£ammatory
in¢ltrate. Eleven samples of dental pulp without in£am-
mation and dentine caries and 11 dental pulps with
moderate to intense mononuclear cell in£ammatory
in¢ltrate with dentine caries were selected.The patients'
age ranged from 18 to 40 years in both the groups.
Immunohistochemical methods
Five-micrometer consecutive cryosections of the tissues
were air-dried and ¢xed in cold acetone for 10 min. The
sections were rehydrated in Tris^HCl bu¡er (pH 7.4)
and immersed in 3% hydrogen peroxide solution for
10 min to block endogenous peroxidase activity. Then,
the sections were incubated with the primary antibodies
for 18 h at 4 8C with1% bovine serum albumin. The pri-
mary sera were anti-VEGFR-3 (Flt-4; Clone C-20, Santa
Cruz Biotech., San Diego, USA) diluted 1 : 100; anti-a-
smooth muscle actin (Clone 1A4, DAKO, Carpinteria,
USA) diluted 1 : 50 and anti-cluster di¡erentiation 31
(CD31) (Clone JC/70A, DAKO, USA) diluted 1 : 40. After
washing in 20 mM Tris^HCl bu¡er (pH 7.4) containing
0.19 M NaCl, the sections were treated with the labelled
streptavidin^biotin (LSAB) kit (DAKO, USA). Peroxidase
activity was developed with diaminobenzidine tetra-
854 International Endodontic Journal, 36, 853^856, 2003
hydro-chloride (Sigma, St Louis, MI, USA). Finally, the
sections were stained with haematoxylin and were
covered.
Quantitative analysis of lymphatic vessels
Six high-power microscopic ¢elds (400) were ran-
domly selected on each of the three serial histological
sections labelled by the three primary antibodies. The
same microscopic ¢eld on the three serial sections was
examined simultaneously under three microscopes
(Zeiss, KF2). The number of lymphatic vessels was
obtained by counting the number of vessels positive for
CD31 and VEGFR-3 and negative for a-smooth muscle
actin in the same microscopic ¢eld.Vessel count was per-
formed by two investigators without knowledge of
whether the samples were control or experimental. The
mean number of lymphatic vessels in the group of dental
pulp without in£ammation was compared with the
group with moderate to dense in£ammatory in¢ltrate
by Student's t-test. Values with a P-value of 0.05 or less
were considered to be statistically signi¢cant.
Results
The mean number  SD of CD31‡ vessels in the group
without in£ammation (19.7  4.1) was signi¢cantly
higher than the group with in£ammatory in¢ltrate
(25.3  7.2; P ˆ 0.0357). Positive vessel for VEGFR-3 is
shown in Fig. 1. The mean number  SD of vessels posi-
tive for CD31 and VEGFR-3 (lymphatic vessels) in the
group with in£ammation (6.09  1.81) was signi¢cantly
higher (P ˆ 0.0123) than the mean number in the group
without in£ammation (3.73  2.20).
Figure 1 Lymphatic vessel in human
dental pulp labelled byVEFGR-3
antibody (brown colour)
(streptavidin^biotin ampli¢ed system,
400).
ß 2003 Blackwell Publishing Ltd

Discussion
As the human dental pulp is enclosed by the rigid struc-
ture of mineralized tissues of the tooth, it has a low com-
pliance, and it may be susceptible to tissue damage
when there is an increase in interstitial £uid pressure
in an in£ammatory state. Therefore, it is evident that
the dental pulp must have an e¤cient draining system
to remove the excess interstitial £uid.
Various reports have demonstrated the presence of
lymphatic vessels in human dental pulp (Bernick
1977, Kato 1990, Marchetti et al. 1991, 1992, Matsumoto
et al.1997,2002, Marchetti & Poggi 2002). Recently, lym-
phatic vessels have been demonstrated by the presence
of VEGFR-3 in immunohistochemical reactions
(Enholm et al. 1998, Clarijs et al. 2001). This ¢nding
prompted the investigation of its immunolocalization
in human dental pulp tissue and evaluation of the e¡ect
of in£ammation.
Increased co-immunolocalization of CD31 and
VEGFR-3 in the vessels of human dental pulp with mod-
erate to dense in£ammatory in¢ltrate compared with
dental pulp without in£ammation was observed sug-
gesting lymphangiogenesis in human dental pulp dur-
ing in£ammation. The new lymphatic vessels could
facilitate the removal of excess interstitial £uid, limiting
the e¡ect of tissue pressure on connective tissue. How-
ever, further studies are necessary to exclude the possibi-
lity that the lymphatic vessels in the group with
in£ammatory in¢ltrate were already present before
in£ammation and its identi¢cation was because of up-
regulation of the VEGFR-3 induced by the in£ammatory
reaction.
VEGF-C and VEFD-D are growth factors that stimu-
late blood and lymphatic vessels (Marchetti & Poggi
2002). When these growth factors bind and activate
VEGFR-3, it induces lymphangiogenesis (Valtola et al.
1999, Paavonen et al. 2000, Achen et al. 2001, Clarijs
et al. 2001,Yonemura et al. 2001). The origin of lympha-
tic vessels is not known. It is likely that lymphatic
endothelial cells originate from pre-existing lympha-
tics or from blood vasculature. The restriction of
VEGFR-3 expression to only the lymphatic endothelial
cells in adult tissues (Kaipanen et al. 1995, Kukk et al.
1996) and the induction of this receptor on angiogenic
tumour blood vessels (Partanen et al. 1999) support
the accepted assumption that lymphatics are derived
from blood vessels (Kaipanen et al. 1995, Clarijs et al.
2001). Further experiments are necessary to investi-
gate the origin of lymphatic vessels in the human den-
tal pulp.
ß 2003 Blackwell Publishing Ltd
Pimenta et al. Lymphangiogenesis in human dental pulp
Conclusion
Increased co-immunostaining of CD31 and VEGF-3 in
vessels associated with human dental pulp in£amma-
tion occurred, which suggests lymphangiogenesis. The
modulation of lymphatic vessel induction may be impor-
tant for human dental pulp regeneration and may be use-
ful in conservative endodontic procedures, such as
pulpotomy.
Acknowledgements
This study was supported in part by grants from PRO-
NEX, PADCT, FAPEMIG and CNPq. Dr R.S. Gomez is
research fellow of CNPq.
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