Microbiological, pathological, inflammatory, immunological and molecular biological aspects of periradicular disease K. TAKAHASHI Department of Periodontology and Endodontology, Okayama University Dental School, Japan Summary Multimicrobial infection of the dental pulp triggers inflammatory responses and ultimately causes bone destruction in the periradicular tissues. Besides bacteria, noxious substances such as degraded protein components and cholesterol could also act as antigens and elicit a host response, which can be harmful to periradicular tissues. Histologically, a dense infiltration of immunocompetent cells is seen in periradicular lesions and their host reactions may induce bone resorption. Polymorphonuclear leucocytes (PMN) and macrophages migrate to the periapical lesions and phagocytose pathogens as a first line of defence. Dead PMN are quickly phagocytosed by macrophages and this disposal system plays a role in maintaining chronicity of the lesions. The pathological roles of periradicular T cells have been assessed through analysing phenotypic markers of cell types, especially CD antigens, but the results are still controversial. Recently, technical developments in immunology and molecular biology have made it possible to investigate the pathogenesis of many diseases at molecular level. The investigation of functional analysis of the immune cells and their regulatory molecules such as apoptosis-associated molecules and adhesion molecules, will lead to a better understanding of the pathogenesis of periradicular lesions. The role of inflammatory mediators including antibodies, cytokines, matrix metalloproteinases, growth factors and arachidonic metabolism is becoming known for these lesions. Knowledge from these investigations improve the understanding of the pathological mechanisms of periradicular infections. Keywords: chronic inflammation, host–parasite interaction, immune response, pathological mechanism, periapical diseases. Introduction Bacterial infection of the pulp may result in pulpal destruction and ultimately elicits a host defence reaction in periapical lesions. The pulpless tooth has a ‘dead space’ where there is no vascular circulation creating a suitable environment for bacterial colonization and degradation of protein components of body fluids. The host defensive reaction against the irritants from the infected root canal induces numerous inflammatory mediators from a variety of cells. These processes operate in the periradicular lesion to prevent the invasion of pathogens from infected root canals over a relatively long time and the host responses may paradoxically account for much of the tissue damage. Periradicular lesions are now treated successfully in many cases compared to periodontal disease. The basic concept of root canal treatment is based on removing the irritants from infected root canals both mechanically and chemically, and then obturating the root canal system to eliminate or reduce the ingless of microorganisms. The precise pathological mechanisms involved in the periradi- cular lesion remain unclear. Diagnostic tools are still not available to evaluate the progression of periapical period- ontitis nor to predict future exacerbation of lesions. The present review paper will summarize the major accom- plishments of studies on periradicular lesions over the past three decades. Furthermore, possible pathological mechanisms of periradicular lesions and some of the important issues remaining will be discussed. Pathogens Periapical infection. The major pathways of pulpal contami- nation are exposed dentinal tubules, direct pulp exposure, q 1998 Blackwell Science Ltd 311 International Endodontic Journal (1998) 31, 311–325 Correspondence: Dr Keiso Takahashi DDS PhD, Department of Periodontology and Endodontology, Okayama University Dental School, 25-1 Shikata-cho, Okayama 700, Japan. Review Article
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Microbiological, pathological, inflammatory,
immunological and molecular biological aspects of
periradicular disease
K . T A K A H A S H I
Department of Periodontology and Endodontology, Okayama University Dental School, Japan
Summary
Multimicrobial infection of the dental pulp triggers
inflammatory responses and ultimately causes bone
destruction in the periradicular tissues. Besides
bacteria, noxious substances such as degraded
protein components and cholesterol could also act
as antigens and elicit a host response, which can be
harmful to periradicular tissues. Histologically, a
dense infiltration of immunocompetent cells is seen
in periradicular lesions and their host reactions may
induce bone resorption. Polymorphonuclear
leucocytes (PMN) and macrophages migrate to the
periapical lesions and phagocytose pathogens as a
first line of defence. Dead PMN are quickly
phagocytosed by macrophages and this disposal
system plays a role in maintaining chronicity of the
lesions. The pathological roles of periradicular T
cells have been assessed through analysing
phenotypic markers of cell types, especially CD
antigens, but the results are still controversial.
Recently, technical developments in immunology
and molecular biology have made it possible to
investigate the pathogenesis of many diseases at
molecular level. The investigation of functional
analysis of the immune cells and their regulatory
molecules such as apoptosis-associated molecules
and adhesion molecules, will lead to a better
understanding of the pathogenesis of periradicular
lesions. The role of inflammatory mediators
including antibodies, cytokines, matrix
metalloproteinases, growth factors and arachidonic
metabolism is becoming known for these lesions.
Knowledge from these investigations improve the
understanding of the pathological mechanisms of
periradicular infections.
Keywords: chronic inflammation, host±parasite
interaction, immune response, pathological
mechanism, periapical diseases.
Introduction
Bacterial infection of the pulp may result in pulpal
destruction and ultimately elicits a host defence reaction
in periapical lesions. The pulpless tooth has a `dead space'
where there is no vascular circulation creating a suitable
environment for bacterial colonization and degradation of
protein components of body fluids.
The host defensive reaction against the irritants from
the infected root canal induces numerous inflammatory
mediators from a variety of cells. These processes operate
in the periradicular lesion to prevent the invasion of
pathogens from infected root canals over a relatively long
time and the host responses may paradoxically account
for much of the tissue damage.
Periradicular lesions are now treated successfully in
many cases compared to periodontal disease. The basic
concept of root canal treatment is based on removing the
irritants from infected root canals both mechanically and
chemically, and then obturating the root canal system to
eliminate or reduce the ingless of microorganisms. The
precise pathological mechanisms involved in the periradi-
cular lesion remain unclear. Diagnostic tools are still not
available to evaluate the progression of periapical period-
ontitis nor to predict future exacerbation of lesions. The
present review paper will summarize the major accom-
plishments of studies on periradicular lesions over the past
three decades. Furthermore, possible pathological
mechanisms of periradicular lesions and some of the
important issues remaining will be discussed.
Pathogens
Periapical infection. The major pathways of pulpal contami-
nation are exposed dentinal tubules, direct pulp exposure,
q 1998 Blackwell Science Ltd 311
International Endodontic Journal (1998) 31, 311±325
Correspondence: Dr Keiso Takahashi DDS PhD, Department of
Periodontology and Endodontology, Okayama University Dental
School, 25-1 Shikata-cho, Okayama 700, Japan.
ReviewArticle
lateral and apical foramina, and blood-transmitted bacter-
ia. It has been reported that dental extraction, periodontal
and orthodontic treatment and even brushing the teeth,
can cause bacteraemia because the oral cavity is septic
(Burket et al. 1937, Sconyers et al. 1973, Hobson & Clark
1995). Therefore, it is possible that during a bacteraemia,
circulating microorganisms could move and colonize pulp-
less teeth (anachoresis). Regardless of the infection path-
ways, it is difficult to distinguish `colonization' or
`infection' by bacteria in infected root canals and infection
modalities in periradicular lesions are still unknown.
Multibacterial infection. The important role of microbes in
pulp and periapical lesions was established by Kakehashi
et al. (1965) using an experimental rat model. Microor-
ganisms in the infected root canals may directly damage
cellular and structural components of the periapical bone
via the release of their proteolytic and noxious waste pro-
ducts (Nair et al. 1996). Bacterial byproducts also elicit an
immune response that could injure host tissues. For exam-
ple, endotoxin from Gram-negative bacteria are capable of
invoking inflammatory and immune responses (Meghji
et al. 1996). It is now generally accepted that `multibac-
terial infection' occurs in the infected root canals (Baum-
gartner 1991a, Trowbridge & Stevens 1992, Simon 1994,
Kettering & Trabinejad 1994). Many anaerobic bacteria,
such as Porphyromonas and Prevotella species, were de-
tected from infected root canals. Some anaerobic bacteria
were thought to be involved in acute periapical abscess
(Brook et al. 1981) and non-healing periradicular lesions
(Sundqvist et al. 1989). Therefore, rapid and reliable mi-
crobial identification methods have been developed to de-
tect specific microorganisms in the infected root canals.
Microbial identification. Current techniques for microbial
identification include culturing, immunological and nu-
cleic acid based methods. Culturing techniques are time-
consuming and expensive, and may fail to grow some
pathogenic organisms. In fact, depending on the culturing
methods used for bacterial identification, the types and
numbers of isolated microorganisms varied (Sundqvist
et al. 1989, Iwu et al. 1990, Barnett et al. 1990, Brook
et al. 1991, Wayman et al. 1992).
Immunological methods require specific antibodies
against targeted bacteria and then may result in false-
positive results because of a cross-reaction with non-
it has been reported that bacterial DNA containing un-
methylated CpG motifs induce B cell proliferation, immu-
noglobulin and cytokine production (Krieg et al. 1995,
Klinman et al. 1996, Schwartz et al. 1997). This evidence
may indicate that any bacteria in infected root canals
which contain the unmethylated CpG motifs can elicit in-
flammatory and immune responses like mitogens in
periapical lesions. The concept of `nonspecific multibacter-
ial infection' (Takahashi et al. 1996b) also suggests that
microbial identification from infected root canals may not
be so useful for diagnosis and hence the choice of
treatment regimens for periradicular lesions at present.
Overall, the variability in the outcome of bacterial
infection is determined by differences in the virulence of
the infecting pathogens, and the effectiveness of host
response against them. Therefore, bacterial characteriza-
tion from infected root canals seems to have limited value
in assessing the risk for exacerbation of periradicular
lesions at present. More clinical investigations are required
to confirm this.
Other pathogens. It has been reported that no bacteria were
detected in cyst fluids (Nair et al. 1993, Meghji et al.
1996). Yeasts were detected from therapy-resistant peria-
pical lesions (Nair et al. 1990). Kettering & Torabinejad
(1993) have speculated the possible involvement of
viruses in infected root canals because natural killer (NK)
cells infiltrate the periapical lesions. In fact, HIV was de-
q 1998 Blackwell Science Ltd, International Endodontic Journal, 31, 311±325
312 K. Takahashi
tected in the dental pulp of patients with AIDS (Glick et al.
1989). Shinoda et al. (1986) have reported that degraded
pulpal tissue components could act as antigens. Nair et al.
(1993) speculated that cholesterol crystals would be pre-
dominant antigens in dental cysts. These results support
the hypothesis that `nonmicrobial pathogens' could, in
some cases, be crucial to the pathogenesis of periradicular
lesions.
There would be numerous different pathogens in
infected root canals and the persistence of irritation may
cause inflammatory and immunological reactions in peri-
radicular lesions. It is still not known what the immuno-
dominant pathogens in infected root canals are.
Furthermore, additional attention should be paid to the
feature of host response that operates against the irritants
in infected root canals.
Host defence processes
The histopathological feature of periapical lesions is
largely the same as that seen in other granulation tissues
that develop from the connective tissue surrounding the
damaged area. A feature common to periapical lesions, ir-
respective of the possible underlying cause, is the
persistent exudation of large number of immunocompe-
tent cells, such as polymorphonuclear leucocytes (PMN),
macrophages, lymphocytes, plasma cells, giant cells, NK
cells and mast cells (Yanagisawa 1980, Stern et al. 1981,
Nevins et al. 1985, Perrini & Fonzi 1985, Piattelli et al.
1991, Kettering & Torabinejad 1993). PMN and
macrophages are important cell types that are involved in
cell-mediated innate immunity which phagocytose
opsonized microorganisms and dead cells.
T and B cells are predominant cellular components in
human periapical lesions (Stern et al. 1982, Nilsen et al.
1984, Skang et al. 1984, Torabinojad & Kettering 1985,
Matthews & Browne 1987, Gao et al. 1988, Barkhordar
et al. 1988, Lukic et al. 1990, Tani et al. 1992a, Marton
& Kiss 1993). These cell types play a central role in
antigen-specific immune response. A number of studies
suggest that both humoral and cellular immune responses
play a role in the pathogenesis of the lesions.
The differences in the proportions of immunocompetent
cells between granulomas and cysts have been studied.
Kopp et al. (1989) have reported that helper/inducer T
cells (Th/i), suppressor/cytotoxic T cells (Ts/c),
macrophages and Ia antigen-positive cells showed a
significant increase in cysts compared to granulomas. In
contrast, Matsuo et al. (1992) showed that there were no
significant differences in the proportion of T cell subsets,
Th/i, Ts/c and immunoglobulin-positive cells. Although
agreement on the cell proportions in the two lesions has
not been reached, no marked differences have yet been
found, hence the immune responses in both lesions may
not appear to be fundamentally different.
Antigen-presenting cells. Animal experiments suggest that
Ia antigen-expressing non-lymphoid cells can be observed
and that an antigen-specific immune defence system is ac-
tive in periapical lesions (Okiji et al. 1994). Although Lan-
gerhans and dendritic cells do not function directly in
antigen elimination, these cells are capable of capturing
antigen in the periphery and migrating to lymphoid or-
gans where they present the antigen to T cells. T helper
cells recognize the association of foreign antigens and self
major histocompatibility complex and then become stimu-
lated, proliferate and release cytokines (Fig. 1). However,
the expression of Ia antigen alone on the cell surface in
vivo is not enough to prove the antigen presentation from
antigen-presenting cells (APC) to CD4 positive helper T
cells because other accessory molecules are also essential
for the event. In fact, Ia antigen-positive cells do not al-
ways show antigen-presenting ability (Geppert & Lipsky
1987, Bal et al. 1990). Furthermore, B cells and activated
T cells are also positively stained for the Ia antigens. After
all, there is only circumstantial evidence about the anti-
gen presentation and cell-to-cell interaction between APC
and helper T cells in periradicular. Therefore, further stu-
dies focusing on cell events are necessary.
T cells. T cells play a central role in cell-mediated immu-
nity. The functional analysis of periradicular T cells has
been performed immunohistochemically and an immunor-
egulatory imbalance of periradicular T cells has been sug-
gested (Trowbridge 1990). Some investigators, have
reported that Ts/c are predominant in the lesions (Kontiai-
nen et al. 1986), whilst others have reported that Th/i are
predominant (Kopp et al. 1989). The majority of T cells in
the lesions are resting (Piattelli et al. 1991). Kawashima
et al. (1996) suggested that the progression of periapical
lesions, with bone resorption, required helper T cells,
whilst suppressor T cells and plasma cells are related to
the chronicity of inflammation. These results indicate that
the immune response is involved in the development of
these lesions. In contrast, Babal et al. (1987) have re-
ported that antibody-mediated immune reactions are pre-
dominant rather than cell-mediated immunity in
periapical granulomas. In addition, an animal study has
supported T cells having a minor role in the pathogenesis
of periradicular lesions (Wallstrom et al. 1993).
Taken together, it is clear that simple evaluation of
phenotypic markers of T cell subsets does not adequately
q 1998 Blackwell Science Ltd, International Endodontic Journal, 31, 311±325
Pathogenesis of periradicular disease 313
reflect the immune processes that may be occurring.
Therefore, the effector functions or cell activity of periradi-
cular T cells should be assessed. Immunological and
molecular biological techniques can be applied. Possible
approaches to investigate periradicular T cell functions
will now be discussed.
T cell functions can be estimated by their cytokine profiles
(Yamamura et al. 1991, Fresno et al. 1997), i.e. CD4�
helper T cells can be subdivided into T helper 1 (Th1) or T
helper 2 (Th2) cell types (Fig. 1). The humoral immune
response is prompted by Th2 cell types which produce char-
acteristic cytokines, IL-4, IL-5, IL-10 and IL-13. Th1 cells
release IL-2 and interferon-g that enhance cell-mediated
immune responses. In periodontal research, cytokine profiles
in adult periodontitis have been well investigated at both
protein and messenger RNA (mRNA) levels using immuno-
histochemistry, reverse transcription PCR and in situ hybri-
dization methodologies (Table 1). The role of periradicular T
cells can be assessed with the same methodologies.
However, cytokine profiles in periodontitis are still contro-
versial (Matsuki et al. 1992, Fujihashi et al. 1993, Yamazaki
et al. 1995, Fujihashi et al. 1996) and the cytokine profile in
inflamed tissues may be changeable because of different
antigenic challenges, treatment modalities and the stage of
the disease. Furthermore, it has been reported that different
methods for the detection and quantification of cytokines at
protein or mRNA level may give different results (Favre et al.
1997) and therefore we need to choose suitable methods for
our study aim.
Fig. 1 Th1/Th2 immune response. T helper cells recognize peptides that are bound to major histocompatibility complex (MHC) and expressed
on the cell surface of antigen-presenting cells (APC), such as macrophages and dendritic cells. The production of IL-12 by APC promotes thedevelopment of Th1 cells that activates macrophages. In contrast, Th2 cells produce IL-4, IL-5, IL-10 and IL-13, which are responsible for
antibody production. Abbreviations: Ag, antigen; B, B cell; IFN-g, interferon-g; IL, interleukin; Mf, macrophages; T, T cell; TCR, T cell receptor;
q 1998 Blackwell Science Ltd, International Endodontic Journal, 31, 311±325
314 K. Takahashi
T cell functions can also be assessed by determining the
T cell receptor (TCR) repertoire in the disease sites
(Forman et al. 1994, Caignard et al. 1994, Yamazaki et al.
1996, Hingorani et al. 1996, Mato et al. 1997). Antigen
recognition by T cells is mediated by the cell surface
receptors (Pannetier et al. 1995). The expression of the
variable region genes of the TCR alpha and beta chain
can be analysed to study the involvement of T cells in the
disease tissues. The evidence of limited repertoire usage of
T cells in disease sites suggests the clonal expansion and/
or migration of restricted antigen-specific T cells or super-
antigen activation in the inflamed sites.
By using immunohistochemical techniques, TCR
repertoire in situ can be analysed. In addition, quantita-
tive PCR and DNA sequence analysis for the third com-
plementarity-determining region of the TCR now permits
a more in-depth analysis of the repertoire of T cells
recovered from small biopsy samples (Cottrez et al.
1994). Oligoclonal expansion of local T cells in
autoimmune diseases has been suggested using RT-PCR
and single-strand conformational polymorphism method
(Hayashi et al. 1995, Struyk et al. 1995, Gulwani-
Akolkar et al. 1996). In periodontal research, Nakajima
et al. (1996) and Yamazaki et al. (1996, 1997) have
characterized the TCR repertoire usage in adult period-
ontitis and they have suggested that oligoclonal
expansion of limited repertoire of TCR-bearing T cells or
superantigen activation would occur in inflamed gingiva.
Thus, molecular techniques may be useful to understand
the functional role of periradicular T cells in the patho-
genesis of periradicular lesions.
Immunoglobulin-producing cells. There are numerous plas-
ma cells in the lesions, and immunoglobulin G (IgG)-con-
Fig. 2 Migration of leucocytes and their fate in periapical lesions. Apoptosis occurs predoninantly in PMN that are engulfed by macrophages.
This may be a disposal mechanism for dead and effete PMN in the periapical lesions. This disposal system may suppress the release of enzymesand inflammatory agents from dead PMN and thus regulate the chronicity of periapical inflammation. In contrast, lymphocytes were not
removed by apoptosis and apoptosis-suppressing molecules, bcl-2 and bcl-x could be involved in this mechanism. Abbreviations: Ag, antigen; IL,
et al. 1967, Shinoda et al. 1986, Marton & Kiss 1992). This
evidence provides a useful model for investigating the inter-
actions between chronic oral inflammation and homeostasis
of the host related to infected root canals.
Besides bacteria, potent soluble mediators, such as PGs,
cytokines and MMPs, can be produced by periapical in-
flammatory cells. It has been suggested that focal
infection in the oral cavity, such as marginal and
periapical periodontitis, may cause a bacteraemia and
ultimately exacerbate sepsis in compromised hosts; for
example, patients treated with chemotherapy following
organ transplantation. Strict infection control including
oral infection will be required in these patients and closer
cooperation between dentists and physicians is necessary.
It has been reported that certain features of rheumatoid
inflammation may occur, and free rheumatoid factor has
been detected in periapical lesions of patients with
rheumatoid arthritis (Malmstrom 1975, Malmstrom &
Natvig 1975). These reports raise the possibility that
systemic disease could have an influence on the pathological
changes of periapical lesions. Tani-Ishii et al. (1996) have
recently reported that leprosy periapical granulomas may
develop as a result of an immunological response to Myco-
bacterium leprae. Incidentally, we observed that patients with
Mycobacterium leprae, who had a dysfunction of monocyte
phagocytosis, showed a delayed healing after root canal
treatment (unpublished observation). These results suggest
that monocyte dysfunction may be a risk factor for
periapical periodontitis and that there is individual suscept-
ibility to periapical lesions depending on their host defense
and/or tissue repair ability.
Conclusions
It is apparent from the literature that immune responses
which are involved in the pathogenesis of periapical lesions
are complex and variable. There are still many questions
concerning the pathological mechanisms of the lesions. Does
host response against irritants work protectively or
harmfully? Do T cells play a minor or major role on the
pathogenesis of periapical lesions? Do periapical plasma cells
produce antigen-specific antibody, and if so, what are the
predominant antigens in infected root canals? In addition,
the processes of bone resorption and remodeling elicited by
the host-parasite interaction are still unknown for periapical
lesions as well as in other infectious diseases such as
periodontal disease. Therefore, molecular mechanisms of
bacterially induced bone destruction and bone formation,
tissue repair, growth factors for epithelial cells and the
cytokine network in periapical lesions, should be researched.
The application of microbiological, immunological and
molecular biological techniques may help us to resolve the
enigma of the pathogenesis of periapical lesions.
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