Archives of Oral Biology 46 (2001) 275– 284

www.elsevier.com/locate/archoralbio

Expression of growth-factor receptors in normal and

regenerating human periodontal cells
M.H. Parkar a, L. Kuru a,b, M. Giouzeli a, I. Olsen a,*
a
Department of Periodontology, Room RL 16 Le6y Wing, Eastman Dental Institute, Uni6ersity College London,
256 Gray’s Inn Road, London WC1X 8LD, UK
b
Department of Periodontology, Faculty of Dentistry, Marmara Uni6ersity, Nisantasi 80200, Istanbul, Turkey

Accepted 15 August 2000

Abstract

Growth factors are biologically active mediators that bind to specific receptors on target cells and regulate genes
involved in cell growth, wound healing and regeneration. The expression of these receptors is thus of fundamental
importance for the response of the cells to the factors. The aim here was to examine, using immunohistochemistry and
flow cytometry, the expression of growth factor receptors in normal gingiva, periodontal ligament and in cells derived
from these tissues, and also in regenerated tissues following guided tissue regeneration (GTR). By immunocytochem-
istry platelet-derived growth factor receptor-a (PDGF-Ra) was not detected in any of the tissues, whereas the
PDGF-Rb and transforming growth factor-b receptor types I and II (TGF-b RI, RII) appeared to be upregulated in
regenerated tissues compared with gingival and periodontal ligament tissues. Epidermal growth factor receptor
(EGF-R) was also notably elevated in the regenerated tissue and was strongly expressed in the gingival epithelium but
not in the periodontal ligament. Neither were fibroblast growth factor receptor-I (FGF-RI) or insulin-like growth
factor receptor (IGF-R) detected in the periodontal ligament, nor in the gingiva, but they sometimes stained weakly
in the regenerated tissues. Flow cytometry (FCM) showed that all the cells derived from the normal gingiva and the
periodontal ligament expressed the PDGF-Rb, whereas the TGF-b RI and RII, FGF-RI and IGF-R were detected
in only a proportion of the total cells. In contrast, none of the cells expressed the PDGF-Ra or the EGF-R. These
observations show that the growth factor receptors are differentially expressed by the periodontal tissues and cells and
suggest that the corresponding factors may also be differentially involved in periodontal wound healing and
regeneration. © 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Growth factors; Receptors; Periodontal cells

1. Introduction tissues as well as in periodontal repair and regeneration

Growth factors are naturally occurring biological
mediators that play a fundamental part in the complex
wound healing events that control tissue repair and
remodelling (Clark and Henson, 1988). Such processes
have a major role in the normal turnover of periodontal
* Corresponding author. Tel./fax: +44-20-79151254.
E-mail address: iolsen@eastman.ucl.ac.uk (I. Olsen).
following the loss of tissue as a result of chronic
inflammatory periodontal disease (Ranney, 1993; Loe,
1993). Although studies using animal models have sug-
gested that platelet-derived growth factor (PDGF),
transforming growth factor-b (TGF-b), epidermal
growth factor (EGF), fibroblast growth factor (FGF)
and insulin-like growth factor (IGF) promote periodon-
tal regeneration (Terranova, 1993; Lynch, 1994; Gian-
nobile, 1996), it is still unclear which growth factors are

0003-9969/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 0 3 - 9 9 6 9 ( 0 0 ) 0 0 0 9 9 - 6
276 M.H. Parkar et al. / Archi6es of Oral Biology 46 (2001) 275–284
important in the wound healing and regeneration of the
human periodontium.
The response of target cells to growth factors de-
pends on the expression of their specific receptors.
These receptors are transmembrane antigens which, on
binding of the respective growth factors, produce a
cascade of intracellular signals that stimulate chemo-
taxis, cell growth, differentiation and the production of
extracellular matrix (Matsuda et al., 1992; Graves and
Cochran, 1994). The growth-factor receptors are there-
fore also likely to be of fundamental importance in
human periodontal growth and regeneration. We have
here used immunocytochemistry and flow cytometry to
examine growth factor receptor expression in intact
normal gingiva and periodontal ligament, and in cells
derived from these tissues, and also in regenerated
tissues following periodontal therapy by guided tissue
regeneration using artificial barrier membranes (Gott-
low, 1993).
2. Material and methods
2.1. Tissue collection
Six samples each of normal gingival and periodontal
ligament tissues were obtained from premolar teeth
extracted for orthodontic purposes. The gingival tissue
was removed from the tooth collar and the ligament
was scraped from the middle third of the root surface,
as described by Parkar et al. (1996). Precautions were
taken to avoid cross-contamination between the gingi-
val and ligamentous tissues.
Recently regenerated soft tissue was obtained from
four patients 6 weeks after undergoing surgery for
guided tissue regeneration using expanded polyte-
trafluoroethylene membranes (Gore-Tex; W.L. Gore &
Associates, Flagstaff, AZ, USA). These patients were
among those originally referred to the Periodontology
Clinic at the Eastman Dental Hospital with moderate
periodontal disease. They had first received treatment
comprising oral hygiene instruction, scaling and root
instrumentation. At reassessment, they had at least one
tooth with a residual probing pocket depth of ]5 mm
associated with attachment loss of ] 5 mm, radio-
graphic evidence of bone loss and either two- or three-
wall intrabony defects. For guided tissue regeneration,
full-thickness mucoperiosteal flaps were raised on both
the buccal and lingual/palatal surfaces of the teeth and
alveolar process from intracrevicular incisions. Vertical
releasing incisions were placed as required for proper
access to the defect. Granulation tissue associated with
the osseous defect and adherent to the alveolar bone
was removed and the exposed root surfaces were
planed. A membrane of appropriate size and shape was
placed so that it fully covered the defect area and
extended 2 – 3 mm beyond the margin of the defect,
then tightly adapted to the tooth with Teflon sutures.
The flaps were repositioned and sutured to cover the
membrane completely. Following surgery, the patients
were instructed to rinse with 0.2% chlorhexidine twice
daily for 2 weeks, at which time the sutures were
removed. The membranes were removed approximately
6 weeks after placement and, in the four patients in
which the extent of newly regenerated soft periodontal
tissue immediately below the membrane appeared to be
overabundant, a small portion was removed from the
most coronal part and placed immediately into sterile
medium, described below.
The participants signed voluntary informed consent
to the use of the tissue that was removed, which was
approved by the Joint Research and Ethics Committee
of the Eastman Dental Institute and Hospital.
2.2. Immunocytochemistry
The samples of gingiva, periodontal ligament and
regenerated tissue were washed in phosphate-buffered
saline (PBS), placed on cork discs and embedded in
Tissue-Tek OCT (Miles Inc., Elkhart, IN, USA). The
gingival samples were oriented so that each section to
be cut contained both epithelium and connective tissue.
Scrapings of periodontal ligament from the root sur-
faces of the extracted teeth and the samples of regener-
ated tissue were placed in the embedding material. The
samples were frozen immediately by immersing into
liquid nitrogen for 1 – 2 min, then stored at − 70°C
before use. Sections (7 mm) were cut on a Bright 5030
Microtome (Bright Instrument Co., Huntingdon, UK)
and transferred to glass slides (Merck, Poole, UK).
They were fixed with ice-cold acetone– methanol (1:1
v/v) for 10 min and washed three times with PBS for 5
min each, then treated with 0.3% H2O2 in methanol for
10 min to inhibit endogenous peroxidase.
The following incubations were carried out using
duplicate serial sections of each tissue sample, all in a
humidified chamber at room temperature. Sections
were treated with 20% normal goat serum (NGS; Life
Technology, Paisley, UK) in PBS for 30 min to block
non-specific binding of antibody. After washing three
times in PBS, the sections were incubated for 1 h with
the following primary antibodies, whose specificity and
reactivity have been reported in the literature, as de-
scribed by each of the manufacturers/suppliers as indi-
cated: mouse monoclonal antibodies against PDGF-Ra
and -Rb (Biogenesis, Poole, UK); TGF-b RI and RII
(R&D Systems, Abingdon, UK); EGF-R (Chemicon,
Harrow, UK); FGF-RI (Chemicon); and IGF-R (Bio-
genesis), diluted 1:100 in PBS containing 20% NGS.
Non-specific mouse immunoglobulin G (IgG) of the
same isotype as the monoclonals (Dako, High Wy-
combe, UK) was used as a negative control. They were
 M.H. Parkar et al. / Archi6es of Oral Biology 46 (2001) 275–284
then washed three times and incubated with
horseradish peroxidase (HRP)-conjugated goat anti-
mouse secondary antibody (diluted 1:100 in PBS con-
taining 20% NGS) for 1 h. After washing, they were
incubated with 3,3%-diaminobenzidine tetrahydrochlo-
ride (DAB; Sigma Chemical Co., Poole, UK) for 10
min and the reaction terminated by adding PBS. The
sections were counterstained with Mayer’s haema-
toxylin for 45 s and left under running tap water for a
further 10 min. They were dehydrated in ascending
concentrations of alcohol, dried for 2–3 min at room
temperature and mounted in DePex (Merck).
The sections were examined under a light microscope
(Olympus BX 50; Japan) and photomicrographs taken
on Kodak 64T film. Areas of the gingival epithelium,
gingival connective tissue, periodontal ligament and
regenerated tissue were scored as strongly positive ( +
+ ), weakly positive ( +), variably positive ( +/−) or
negative ( −), depending on the brown colour. The
data shown are the overall estimated level of antigen
expression observed on the replicate sections of all of
the various tissue samples.
2.3. Cell culture
The gingival and periodontal ligament tissues were
placed into Dulbecco Minimal Eagle Medium
(DMEM) supplemented with 100 U/ml penicillin, 100
mg/ml streptomycin, 2 mM L-glutamine, 25 mg/ml fungi-
zone (Life Technology) and 10% foetal calf serum
(FCS; PAA Labs, Linz, Austria). The ligament scrap-
ings, and the gingiva, which were cut into 2-mm3 pieces,
were washed twice with medium and placed into 24-
well plates in full DMEM without fungizone. These
explants were incubated at 37°C in a humidified atmo-
sphere of 5% CO2 in air, changing the medium twice
weekly. Outgrowths of adherent cells were obtained
from each of the six gingival tissues and three of the
ligament samples. When confluent, the monolayers
were detached by treatment with trypsin–EDTA for 10
min at 37°C, centrifuged and washed with culture
medium. The cells were recultured in 25-cm2 tissue
culture flasks until confluent monolayers were again
obtained. In some experiments, as noted, the cells were
also incubated in the absence of FCS. Cells between
passages 3 and 7 were subsequently used to examine the
expression of the growth factor receptors. Comparable
cells were not obtained from the samples of regenerated
tissue because of the very small quantity of biopsy
material available.
2.4. Flow cytometry
This is a technique in which certain physical and
chemical characteristics of cells in suspension are mea-
sured as they flow in a fluid stream past a laser beam,
277
a photomultiplier detecting scattered light and emitted
fluorescence light. If the cells are first treated with a
fluorescent-labelled reagent or antibody, the emitted
fluorescent light can be used to obtain information
about the relative levels of both cell surface and intra-
cellular components, immunoreactive antigens and
DNA, as we have previously shown (Sumner et al.,
1991; Bou-Gharios et al., 1994). It is thus a powerful
procedure for investigating multiple characteristics of
cells in a heterogeneous population without the need to
separate the individual types of cell.
Confluent cultures of gingival fibroblasts and peri-
odontal ligament cells grown for 3 days in the presence
and absence of 10% FCS were prepared for FCM as
described by Kuru et al. (1998). In brief, the cells were
washed with PBS and detached by treatment with
EDTA only, with no trypsin, so as to avoid possible
proteolytic destruction of cell surface antigens such as
growth factor receptors. The cells were then centrifuged
and fixed with 1% para-formaldehyde (Merck) in PBS
for 30 min (Bou-Gharios et al., 1994; Kuru et al., 1998).
They were washed with a buffer of 2% FCS in PBS
(PBS/FCS) and centrifuged at 400 × g for 10 min after
each step. Portions of the cell suspensions containing
1 ×105 cells were placed in separate tubes and incu-
bated for 60 min at room temperature with the same
primary antibodies (diluted 1:100 in PBS/FCS) as used
for immunocytochemistry above. Non-specific mouse
IgG was used as a negative control. After washing with
PBS/FCS, fluorescein isothiocyanate-labelled rabbit an-
timouse IgG, diluted 1:50 in PBS/FCS, was added for
30 min at room temperature.
The cells were suspended in 400 ml of PBS/FCS and
the fluorescence intensity of 10 000 individual cells
measured by flow cytometry using a FACScan flow
cytometer (Becton Dickinson, Cowley, UK). The data
were analysed using CELLQuest software and the level
of fluorescence is shown as the average fluorescence
intensity (AFI) of 10 000 individual cells. For each
antibody, the arbitrary AFI obtained, which is deter-
mined by the electronic input and output settings of the
FACScan, is proportional to the level of expression of
the specific antigen. Thus, differences in AFI between
cells after reaction with a specific antibody reflect the
relative levels of expression of that particular antigen
by each of the different cells. However, AFI values
obtained using different antibodies cannot be compared
with each other as they do not indicate the relative
levels of the different antigens, only differences in reac-
tivity of the antibodies with their respective antigens.
2.5. Statistics
The means and standard deviations were determined
for the results of the flow cytometry. Data for gingival
fibroblasts and periodontal ligament cells were com-
278 M.H. Parkar et al. / Archi6es of Oral Biology 46 (2001) 275–284

pared by Student’s t-test, with a probability value of Table 1

PB0.05 assigned as the level of statistical significance. Expression of growth-factor receptors in normal gingiva, nor-
mal periodontal ligament and regenerated tissuesa

Receptor Gingival CT Gingival EP PDL RT

3. Results
3.1. Receptor expression in tissues
The PDGF-Ra was not detected in any of the tissues
examined (data not shown). Biopsy samples of gingiva
exhibited moderate to strongly positive expression of
PDGF-Rb in connective tissue cells, and none in gingi-
val epithelium (Fig. 1a). This receptor was also hetero-
geneously distributed and expressed throughout the
normal periodontal ligament, although at a relatively
much lower level, (Fig. 1b). Strongly positive expression
of PDGF-Rb was observed in isolated areas within the
biopsy material from regenerated tissue (Fig. 1c).
TGF-b RI expression was weak in gingival connec-
tive tissue and almost undetectable in normal gingival
epithelium (Fig. 1d). The receptor was also only very
weakly expressed in the periodontal ligament (not
shown), whereas it was far more abundant and had a
highly characteristic localisation in the regenerated tis-
sue (Fig. 1e). The TGF-b RII had a very similar
distribution in replicate sections of the same specimens
(data not shown).
The EGF-R was readily visualised in normal gingival
epithelium but barely detected in gingival connective
tissue (Fig. 1f). It was not expressed by normal peri-
odontal ligament (not shown), but was readily visu-
alised in the regenerated tissue in highly localised areas
(Fig. 1g). Staining for the FGF and IGF receptors was
negative in both gingiva and periodontal ligament and
was very weak and variable, sometimes not detectable,
in the regenerated tissue (not shown). Control sections
of each type of tissue treated with non-specific IgG as
the primary antibody were all negative (not shown). A
summary of the results of these experiments is shown in
Table 1.
3.2. Receptor expression in cultured cells
The relative amounts of the growth factor receptors
in the cultured gingival fibroblasts and periodontal
PDGF-Ra − − − −
PDGF-Rb ++ − + ++
TGF-b RI + − + ++
TGF-b RII + − + ++
EGF-R − ++ − ++
FGF-RI − − − −
IGF-R − − − +/−
a
EP, epithelium; CT, connective tissue; ++, strongly posi-
tive; +, weakly positive; +/−, variably positive; −, negative.
ligament cells were measured by FCM are shown in the
representative histograms in Fig. 2. Neither type of cell
expressed the PDGF-Ra or the EGF-R, but both types
expressed the PDGF-Rb, TGF-b RI, TGF-b RII and
IGF-R, all of which had a narrow and uniformly
distributed peak of fluorescence. Although the positive
fluorescence profiles for PDGF-Rb were exhibited by
all the cells in the culture, as demonstrated by the shift
of the entire peak to a higher fluorescence intensity (to
the right compared with the negative control), the shift
of the peaks for TGF-b RI, TGF-b RII and IGF-R was
considerably less and encompassed only a proportion
of the total cells. In addition, unlike PDGF-Rb, TGF-b
RI and II and IGF-R, the fluorescence profile of FGF-
RI was not uniform but had a broader and more
extended distribution (Fig. 2).
Fig. 3 is a summary of four individual experiments,
using three cell lines each, in which the receptors and
the proportion of antigen expressing cells are compared
in the gingival fibroblasts and periodontal ligament cell
cultures. The density of PDGF-Rb was lower in the
gingival fibroblasts than in the ligament cells (AFIs of
37 and 50, respectively), and the PDGF-Rb-positive
cells was somewhat higher in the gingival fibroblasts
than in the ligament cells (90 and 75% of the total cells,
respectively), but these differences were not statistically
significant. TGF-b RI expression was higher in the
gingival fibroblasts than in the ligament cells (AFIs of
28 and 21, respectively), although approximately 50%

Fig. 1. Expression of growth factor receptors by gingival tissues, normal periodontal ligament (PDL), and regenerated tissues (RT).
In (a), while the gingival connective-tissue cells (CT) strongly expressed PDGF-Rb, the gingival epithelium (EP) was negative, as
seen by the absence of brown staining. Note the relatively weak expression of PDGF-Rb in the normal PDL (b) compared with the
high expression of this receptor in isolated areas of the RT (arrows) in (c). In (d), the gingival weakly expressed TGF-b RI and the
gingival epithelium (EP) was nearly negative for this antigen. However, note the relatively large amount and characteristic
distribution of this receptor in the RT (e). In gingiva, the connective tissue (CT) was almost negative for the EGF-R, as indicated
by the relative lack of brown colour in (f), whereas this receptor was clearly present throughout the gingival EP. The EGF-R was
also absent from the normal PDL (not shown), but was readily detected in relatively high amounts in certain localised groups of
cells in the RT (arrows) in (g). Original magnifications, × 200. Negative controls of tissue sections incubated with non-specific
mouse IgG had no detectable staining (data not shown).
M.H. Parkar et al. / Archi6es of Oral Biology 46 (2001) 275–284 279

Fig. 1. (Continued)
280 M.H. Parkar et al. / Archi6es of Oral Biology 46 (2001) 275–284

ligament cells than in the gingival fibroblasts (AFI of 60

and 22, respectively).
The same experiments were also carried out on repli-
cate cell cultures incubated for 3 days in the absence of
FCS, in order to examine the possible effects of a range
of unknown quantities of mediators, including the
growth factors themselves, which are likely to be
present in the FCS. It was notable, however, that in
cells incubated in the absence of FCS there was no
statistically significant change in the expression of the
receptors or in the proportion of receptor-positive cells
(data not shown).

4. Discussion

Growth factors initiate many of the events associated

with the orderly turnover, repair and regeneration of
periodontal tissues. The corresponding receptors are
thus of fundamental importance in maintaining peri-
odontal integrity and mediating periodontal wound

Fig. 2. Representative histograms of the fluorescence profiles

of growth factor receptors in cultured cells. The solid, bold
lines show the expression of the PDGF-Ra, PDGF-Rb, TGF-
b RI, TGF-b RII, EGF-R, FGF-RI and IGF-R in the gingival
fibroblasts (GF) and periodontal ligament (PDL) cells. The
negative controls (the same cells treated with non-specific
mouse IgG of the same isotype as the monoclonal antibodies),
are shown as dashed lines. GF and PDL cells did not express
either the PDGF-Ra or EGF-R, but all showed clear positive
staining for the PDGF-Rb. Only some GF and the PDL cells
expressed TFG-b RI, TGF-b RII, FGF-RI and IGF-R.

of both cell types expressed this receptor. In contrast,

although TGF-b RII was expressed very similarly in the
gingival fibroblasts and periodontal ligament cells
(AFIs of 19 and 24, respectively), only 18% of the
ligament cells were positive for this receptor compared
with 50% of the gingival fibroblasts (Fig. 3). As with
PDGF-Rb, the AFI for the FGF-RI was lower in the
gingival fibroblasts than in the periodontal ligament Fig. 3. Comparison of growth factor receptor expression by
cells (25 and 32, respectively), although the proportion gingival fibroblasts (GF) (dotted bars) periodontal ligament
of receptor-positive gingival fibroblasts was signifi- (PDL) cells (solid bars). The figure shows receptor expression
cantly higher (28%) than in the ligament cells (12%). In (AFI; top) and the percentage of receptor-positive cells (bot-
tom). 1, PDGF-Rb; 2, TGF-b RI; 3, TGF-b RII; 4, FGF-RI;
addition, although a similar proportion of both the
5, IGF-R, The values are the mean 9S.D. obtained for three
gingival fibroblasts and periodontal ligament cells was cell lines of each type, in four separate experiments. * Indicates
positive for the IGF-R (50–60% of the total cells), the significant difference of PDL cells compared with GF; P B
receptor density was approximately 3-fold greater in the 0.05.
 M.H. Parkar et al. / Archi6es of Oral Biology 46 (2001) 275–284
healing, but their expression in human gingiva, in the
periodontal ligament and in the regenerating periodon-
tium is not well defined. Moreover, although factors
such as PDGF and TGF-b and their receptors have
been examined during the early stages that follow tissue
injury (Clark and Henson, 1988; Green et al., 1997),
damaged tissue is rebuilt over a considerable period of
time and little definitive information is available on the
temporal sequence of complex events involved in this
process. Thus, the chronology of cell activation, prolif-
eration, synthesis of extracellular matrix and mineral-
ization, which follows the formation of complexes
between exogenous growth factors and their receptors
at the cell surface, underlies the successful restoration
of the structural and functional integrity of the peri-
odontium (Terranova et al., 1989b; Giannobile, 1996).
In the present study, however, tissue exhibiting regener-
ation was understandably not available during the
course of periodontal wound healing and the experi-
mental data are therefore restricted to the period of 6
weeks after surgery with guided tissue regeneration. At
this time, at which the barrier membrane is retrieved,
very substantial periodontal regeneration had already
occurred, which was particularly extensive in the four
patients selected. Despite this limitation, it was never-
theless possible to gain some insight into the potential
growth-factor ‘sensitivity’ of the regenerated tissue
compared with that of the normal gingival and peri-
odontal tissues, by measuring the relative amounts of
the corresponding receptors.
The ligand for PDGF-R, PDGF, is composed of two
polypeptide chains, A and B, which give rise to three
isoforms (AA, AB, BB) that differ in biological activity.
One receptor, PDGF-Ra, binds all three PDGF iso-
forms, while the PDGF-Rb receptor binds PDGF-AB
with low affinity and PDGF-BB with high affinity, but
not the PDGF-AA isoform (Raines and Ross, 1990). In
the present study, the a-receptor was not expressed by
any of the tissues examined whereas the b-receptor,
although only weakly expressed by the periodontal
ligament, was strongly expressed by the gingival con-
nective-tissue cells and by the regenerated tissues. This
apparent upregulation of the PDGF-Rb in the regener-
ated tissue, at 6 weeks after surgery, during which
extensive wound healing would already have taken
place, suggests that the corresponding ligands, PDGF-
AB and -BB, are likely to be present in this tissue at
this time. It is notable, however, that these factors and
their receptors are also involved at an early stage of
gingival wound healing (Green et al., 1997). Moreover,
that study also demonstrated the absence of the a-re-
ceptor in both healthy and healing gingival wound sites,
as we have now shown in gingival and normal and
regenerating periodontal tissue. It is thus likely that the
PDGF-Ra probably has only a limited role in peri-
odontal growth, whereas the strongly positive expres-
281
sion of the PDGF-Rb in isolated areas of the biopsy of
regenerated tissue examined here indicates that this
receptor is likely to have an important role in periodon-
tal regeneration 6 weeks postoperatively.
Our in vitro results using FCM also demonstrate that
cells derived from these tissues did not express the
PDGF-Ra, although the b-receptor was present in a
large proportion of the gingival fibroblasts and peri-
odontal ligament cells. However, previous work has
shown that, in addition to the AB and BB isoforms of
the growth factor, the PDGF-AA isoform is a function-
ally effective mitogen for periodontal ligament cells,
suggesting that the a-receptor as well as the b-receptor
is expressed by these cells (Matsuda et al., 1992; Boyan
et al., 1994), as also observed by Oates et al. (1995).
The discrepancy between these and the in vivo observa-
tions may have arisen because the functional studies are
carried out on cultured cells, and receptor expression
may be altered under different culture conditions, par-
ticularly by other growth factors (Oates et al., 1998).
Moreover, the degree of confluency of the cultured cells
also seems to affect receptor expression (Tiesman and
Rizzino, 1989), again highlighting the possibility that
the periodontal cells cultured in vitro may not precisely
reflect those present in vivo.
Six types of TGF-b R have been described in a
number of cells but only the types RI– RIII have been
extensively investigated thus far. Types I and II but not
type III are involved in intracellular signal transduction
processes, although the type III receptor plays a part in
the binding of the growth factor to TGF-b RI and
TGF-b RII (Massague et al., 1994). In addition, the
binding to these receptors of the three different iso-
forms of TGF-b expressed in mammalian tissues has
not yet been precisely delineated. In the present study,
TGF-b RI and TGF-b RII were both only weakly
expressed in the gingival connective tissue and peri-
odontal ligament and barely detected in the gingival
epithelium. In marked contrast, these receptors were
clearly upregulated in the regenerated tissue obtained 6
weeks after surgery, consistent with the continuing role
of TGF-b in cell proliferation and the production of
new extracellular matrix (Lawrence, 1996). The unique
morphological appearance of these TGF-b R-express-
ing cells suggests that they are the rests of Malassez, a
group of ‘residual’ cells of epithelial lineage but un-
known function in the periodontal ligament (Ten Cate,
1989). As with PDGF-Rb, the presence of these highly
localised cells, which are particularly prominent in ac-
tively growing tissue, suggests that they play an impor-
tant part in the regeneration of new periodontal tissue.
Moreover, our FCM studies of both the gingival
fibroblasts and periodontal ligament cells also demon-
strate that TGF-b RI and RII are expressed by a
subpopulation of cells, such that only this subset is
likely to be responsive to the corresponding growth
factors.
282 M.H. Parkar et al. / Archi6es of Oral Biology 46 (2001) 275–284
The EGF-R is expressed primarily by epithelial cells
and, although previous studies have not detected this
receptor in normal gingiva, positive staining has been
found in inflamed and hyperplastic human tissues, in
fibroblastic cells as well as in the epithelium (Irwin et
al., 1991; Saito et al., 1996). Moreover, in animal
studies the receptor has also been identified in certain
‘precursor’ cells of the periodontal ligament (Cho et al.,
1988, 1991). In contrast, the EGF-R is reportedly
present in normal human gingival epithelium but absent
from the periodontal ligament (Whitcomb et al., 1993).
In vitro, both gingival fibroblasts and periodontal liga-
ment cells were found to express only low amounts of
EGF-R, and the corresponding EGF had only limited
and variable mitogenic and chemotactic effects on these
cells (Modeer et al., 1990; Matsuda et al., 1992; Blom et
al., 1994). In the present study, EGF-R also appeared
to be present in only very low amounts in the gingival
connective tissue and was not detected in the cultured
gingival fibroblasts and periodontal ligament cells, but
was readily detected in the epithelium of the normal
gingival samples examined. Moreover, elevated
amounts of receptor were also clearly observed in cells
in isolated patches in the regenerated tissue, which, like
the PDGF-Rb- and TGF-b RI- and -RII-positive cells,
could be derived from the rests of Malassez, although
these findings do not unequivocally eliminate the possi-
bility that they are ‘contaminating’ epithelium present
in these samples. In addition, these observations also do
not preclude the possibility that the EGF-R-expressing
cells are the same as the undifferentiated precursor cells
for periodontal ligament identified in vivo (Cho et al.,
1988, 1991), as noted above. Such cells reportedly
downregulate their receptors on differentiation in vitro
(Matsuda et al., 1993), possibly also explaining the lack
of EGF-R expression we observed in the mature peri-
odontal ligament tissue.
The FGF-RI binds two related growth factors, acidic
FGF (aFGF) and the basic FGF (bFGF), which are
members of the heparin-binding family of growth fac-
tors that function in mitogenesis, the formation of
extracellular matrix and angiogenesis in vivo. The ex-
pression of FGF-RI was reportedly higher in hyper-
plastic gingival tissue than in normal gingiva (Saito et
al., 1996), but in the present study no expression of this
receptor was found in normal gingiva and the peri-
odontal ligament although a very small amount of
receptor appeared to be present in some samples of
regenerated tissue. In contrast, in vitro studies have
shown that FGF has a potent effect on gingival and
periodontal cells (Terranova et al., 1989a; Takayama et
al., 1997) and, moreover, that FGF-R of both high-
and low-binding binding affinity are present in peri-
odontal ligament cells (Takayama et al., 1998), al-
though the FGF-RI isoform was not identified
specifically. The FCM analysis of the gingival fibrob-
lasts and periodontal ligament cells reported here has
revealed a broad distribution of FGF-RI-positive cells,
the fluorescence profile showing that there is differential
expression of this receptor between the cells in the
population. In addition, although we also found that a
proportion of the total cells were negative for this
specific receptor, FGF can nevertheless bind to other
types of FGF-R in addition to FGF-RI and thereby
still exert an important influence on cells that are
FGF-RI-negative.
The present immunohistochemical results show that,
as with the FGF-RI, there was no detectable IGF-R
expression by normal tissues and only weak and vari-
able expression in the regenerated tissue. Although
IGF-1 alone appears to have only a limited effect on
periodontal regeneration in vivo (Lynch et al., 1991;
Rutherford et al., 1992), using this growth factor in
combination with PDGF-BB was found to accelerate
bone regeneration (Howell et al., 1997). It is thus
possible that IGF-R may have only a limited role in the
regenerated tissue examined in the present study, which
was obtained at membrane retrieval 6 weeks after
surgery for guided tissue regeneration. However, this
receptor may play a more prominent part in the subse-
quent formation of new bone at later stages of the
periodontal wound healing process, as IGF to enhances
DNA synthesis in osteoblasts and has a significant role
in osteogenesis (Hock et al., 1988). Alternatively, there
may have been factors present in vivo that suppress the
expression of the receptor, because, for example, it has
been shown that sex steroids downregulate the expres-
sion of this receptor (Fiorelli et al., 1991). In vitro,
IGF-I has a mitogenic effect on mesenchymal cells,
including periodontal cells (Matsuda et al., 1992; Blom
et al., 1992), and ligand-binding analysis has also
demonstrated the presence of the IGF-R on normal
periodontal ligament cells (Blom et al., 1992). Our
flow-cytometric results show that the receptor is ex-
pressed by a substantial proportion of the cultured
cells, suggesting that the culture conditions may have
either selected cells that were IGF-R-positive or could
have induced receptor expression in cells that had been
receptor-negative in vivo. These findings also show that
the particular monoclonal antibody used to detect IGF-
R was reactive against this antigen, as, despite the
previously reported reactivity of this reagent (Babajko
and Binoux, 1996; De Neubourg et al., 1998), we were
not able to demonstrate IGF-R expression in vivo by
immunohistochemistry. Nevertheless, the amount of
IGF-R in vitro was substantially higher in the peri-
odontal ligament cells than the gingival fibroblasts,
possibly because the ligament cells are considered to
exhibit far more functional heterogeneity, for example
the production of bone proteins such as collagen type I
and the secretion of the organic matrix of cementum
(Sodek and Berkman, 1987; Kawase et al., 1988; No-
 M.H. Parkar et al. / Archi6es of Oral Biology 46 (2001) 275–284
hutco et al., 1996). Studies are in progress to delineate
whether the higher overall amount of IGF-R expression
is due to the presence of an expanded and unique
subset of IGF-responsive cells in the periodontal liga-
ment cultures.
In conclusion, our finding of increased amounts of
PDGF-Rb, TGF-b RI and RII and EGF-R in regener-
ated tissue obtained 6 weeks after surgery suggests that
the corresponding factors play an important part in
periodontal wound healing. In addition, our observa-
tion that normal gingival and periodontal ligament
tissues express relatively lower amounts of the growth-
factor receptors may be a reflection of the quiescence of
these normal cells compared with those of the regener-
ated tissue, in which elevated amounts of the growth
factors themselves may be at least partly responsible for
the upregulation of the receptors. The FCM technique
has also highlighted the presence of diverse subpopula-
tions of cells that are likely to have functionally distinct
roles in periodontal regeneration.
Acknowledgements
We are indebted to the staff of the Periodontology
Department, Eastman Dental Hospital, for their assis-
tance in obtaining the clinical samples, and to S. Jyoti
Das for her helpful comments and suggestions about
the manuscript. L. Kuru is grateful for the award of a
scholarship from Marmara University.
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