Fransson OD Muep

Swedish Dental Journal Supplement 226, 2012
on the repair oF the dentine barrier
205 06 malm, sweden
www.mah.se
m a l m u n i v e r s i t y 2 012
malm university
s w e d i s h d e n ta l j o u r n a l s u p p l e m e n t 2 2 6 , 2 012 . d o c t o r a l d i s s e r tat i o n i n o d o n t o l o gy
helena Fransson
isbn/issn 978-91-7104-390-0/0348-6672
helena Fransson
on the repair oF
the dentine barrier
ON THE REPAIR OF THE DENTINE BARRIER
Helena Fransson 2012

ISBN 978-91-7104-390-0
ISSN 0348-6672
Holmbergs, Malm 2012
HELENA FR ANSSON
ON THE REPAIR OF
THE DENTINE BARRIER
Departments of Endodontics and Oral Biology,

Faculty of Odontology,
Malm University, Malm, Sweden 2012
The summary of this publication can be downloaded from

se www.mah.se/muep
CONTENTS
ABSTRACT .................................................................... 9
POPULRVETENSKAPLIG SAMMANFATTNING .................. 11
(Summary in Swedish)
PREFACE ..................................................................... 13
THESIS AT A GLANCE ................................................... 14
VOCABULARY/ABBREVIATIONS ..................................... 15
INTRODUCTION .......................................................... 17
AIMS ......................................................................... 28
MATERIAL AND METHODS ............................................ 29
Systematic review (I) ............................................................. 29
Clinical trial (II)..................................................................... 30
Immunohistochemical studies (II and III) ................................... 32
Cell culture study (IV) ............................................................ 34
RESULTS ..................................................................... 37
Systematic review (I) ............................................................. 37
Effect of EMDgel on experimentally performed
pulp exposures (II) ................................................................ 47
Characterization of the newly formed hard tissue (III) ................ 50
Effects of bacterial products on the activity of odontoblastlike
cells and their formation of Col I (IV) ....................................... 54
DISCUSSION ............................................................... 57
CONCLUSIONS ........................................................... 67
CONTRIBUTORS TO THE STUDIES ................................... 69
ACKNOWLEDGEMENTS ............................................... 70
REFERENCES ............................................................... 72
PAPERS I-IV ................................................................. 85
ABSTRACT
The overall aim of this thesis was to study some aspects of the repair of the dentine barrier, especially in conjunction with dental
pulp capping. Understanding the events leading to the healing of
the dentine and pulp, and hence successfully preserving the vitality
and functions of the tooth, would lead to a scientific basis for a less
invasive treatment of pulp exposures than performing root canal
treatments.
The surfaces of the body have physiological barrier functions
aimed at protecting the body from external noxious agents. In the
tooth, the odontoblasts, which line the outermost part of the pulp
and are responsible for the formation of dentine, play a central role
in the barrier function and thus in the defence mechanisms of the
tooth. The micro-organisms in the caries lesion can reach the pulp
via the dentinal tubules. However, the barrier function helps to
prevent microbial invasion and thereby avoid deleterious inflammation and subsequent necrosis of the pulp. Dentine repair is an
important part of the barrier function. There are however doubts
as to whether the repair also leads to restitution of the function
and the ability to withstand bacterial influx over the longer term.
Pulp capping is a treatment method used when the pulp has been
exposed in order to stimulate healing of the pulp and dentine. The
evidence for repair of the dentine after pulp capping in humans has
been studied by means of a systematic review. The focus of the literature search was studies performed in humans where hard tissue
formation had been studied with the aid of a microscope. We con-
cluded, based on the limited evidence available, that calcium hydroxide based materials but not bonding agents promote formation
of a hard tissue bridge. Scientific evidence was lacking as to
whether MTA was better than calcium hydroxide based materials
in this regard.
A gel (Emdogain Gel) containing amelogenin, known to be involved in dentinogenesis, was evaluated with regard to formation
of hard tissue in a clinical study. A greater amount of hard tissue
was formed after application of the gel compared to the control.
Characterization of the tissue concluded it to be dentine, based on
its content of type 1 collagen and dentine sialoprotein, although it
was not formed as a continuous bridge covering the pulp wound.
Beneath a deep caries lesion an important part of the barrier function is the odontoblasts response to bacteria with the formation of
new dentine. A cell model with odontoblasts was used to study the
effects of clinical isolates from a deep carious lesion on their viability and production of type 1 collagen, the major component of the
dentine in the early stages of its formation. There were bacteria
that negatively affected the viability of the odontoblast-like cells
and different bacteria varied in their effects on type 1 collagen production, suggesting that some bacteria may have a direct influence
on the odontoblasts ability to form dentine.
In summary; Emdogain Gel initiated dentine formation, though

not in a form that could constitute a barrier and there are indications that bacteria may differentially affect the odontoblasts ability
to repair the dentine barrier.
10
POPULRVETENSKAPLIG SAMMANFATTNING (SUMMARY IN SWEDISH)
Det vergripande mlet fr avhandlingen har varit att studera ngra aspekter av lkningen av tandens huvudsakliga hrdvvnad,
dentinet. Vid mycket djupa kariesangrepp dr dentinet frstrts
och pulpan drmed blottats, rotbehandlas ofta tanden vilket innebr att pulpan tas bort och att rotkanalen fylls med ett rotfyllningsmaterial. Djupare kunskaper om dentinets lkningsfrmga
kan leda till att andra mindre invasiva och kostsamma behandlingsmetoder n rotbehandlingar skulle kunna anvndas vid mycket djupa kariesangrepp.
Kroppens ytor har barrirfunktioner fr att skydda kroppen mot
skadliga mnen. I tanden svarar pulpans yttersta cell-lager fr en
viktig del i barrirfunktionen. Dessa celler, odontoblasterna, bildar
dentinet och verkar spela en central roll i de skyddsmekanismer
som tanden har. Nr ett kariesangrepp bryter ned tandens hrdvvnader kan bakterier eller deras produkter f mjlighet att
trnga in till pulpan vilket leder till ett inflammatoriskt och immunologiskt svar som kan leda till vvnadsdd av pulpan. Under vissa
omstndigheter verkar dock pulpan ha frmga att reparera hrdvvnadsbarriren p ett sdant stt att den fysiologiska funktionen
kvarstr s att vvnadsdd och drmed invasion av mikroorganismer undviks. Det finns emellertid studier som antyder att den reparerade hrdvvnadens barrirfunktion ger vika och att den inte kan
st emot ny mikrobiell belastning.
11
Pulpaverkappning r en behandling som anvnds nr pulpan blivit blottad i ett frsk att bibehlla pulpans vitalitet och funktion.
Faktorer som pverkar hrdvvnadsbildningen vid pulpaverkappningar har studerats i en systematisk litteraturversikt. Baserat p det begrnsade vetenskapliga stdet visade resultaten att
kalciumhydroxidbaserade material men inte bondingmaterial ger
en hrdvvnadsbildning som tcker pulpasret d de anvnds som
verkappningsmaterial. Det finns inget vetenskapligt std fr att
kunna fastsl att mineraltrioxidaggregat (MTA) skulle ge mer
hrdvvnadsbildning jmfrt med kalciumhydroxidbaserade material nr dessa anvnds som verkappningsmaterial.
En gel (Emdogain Gel) som innehller amelogenin som man vet r

inblandat i processen d dentinet brjar bildas, utvrderades i en
klinisk studie med syfte att studera hrdvvnadsbildningen. En
strre mngd hrdvvnad bildades efter appliceringen av gelen
jmfrt med kontrollmaterialet. Hrdvvnaden kunde karaktriseras som att vara likt det ursprungliga dentinet, men den bildades
inte i en struktur som skulle kunna utgra en fysiologisk barrir.
Under ett kariesangrepp br odontoblasterna svara p nrvaro av
bakterier med frsvarsreaktioner ssom bildande av nytt dentin,
men kvalitn p det dentinet verkar ibland bli smre n det ursprungliga dentinet. Produkter frn bakterier tagna frn ett djupt
kariesangrepp anvndes fr att studera dess effekter p odontoblastliknande cellers aktivitet och frmga att bilda en typ av kollagen som r den huvudsakliga bestndsdelen i nybildat dentin. Vissa
bakterier hade en negativ pverkan p odontoblasternas aktivitet
och bakteriernas effekt p kollagenproduktionen varierade, vilket
skulle kunna tyda p att bakterier kan ha en direkt effekt p odontoblasternas frmga att upprtthlla dentinets barrirfunktion.
Sammanfattningsvis kan man sga att Emdogain Gel initierade

dentinbildning, men inte i en struktur som skulle kunna utgra en
barrir och det frefaller som om bakterier i olika grad kan pverka odontoblasternas frmga att bilda en dentinbarrir.
12
PREFACE
This thesis is based on the following papers, which are referred to

in the text by their Roman numerals.
I
Olsson H, Petersson K, Rohlin M. Formation of a

hard tissue barrier after pulp cappings in humans. A
systematic review. Int Endod J 2006; 39: 429-442.
II
Olsson H, Davies JR, Holst KE, Schrder U, Peters

son K. Dental pulp capping: effect of Emdogain Gel
on experimentally exposed human pulps. Int Endod
J 2005; 38: 186-194.
III
Fransson H. Petersson K, Davies JR. Dentine sialoprotein and Collagen I expression after experimental
pulp capping in humans using Emdogain Gel. Int

Endod J 2011; 44: 259-267.
IV
Fransson H. Petersson K, Davies JR. Effects of bacterial products on the activity of odontoblast-like
cells and their formation of type 1 collagen. Submitted to J Endod.
The papers I, II and III are reprinted with permission of the copyright holder John Wiley & Sons Ltd.
13
14
14
THESIS AT A gLANCE
VOCABULARY/ABBREVIATIONS
There are several terms used in the literature concerning dentine

formation and different modes of pulp capping. The following applies to this thesis:
Dentine
Primary
Secondary
Tertiary
Pulp capping
Partial pulpotomy
dentine formed by primary odontoblasts until the root is fully formed

dentine that is formed at a highly reduced rate after the root has been fully
formed
dentine formed in response to injury.
This might further be characterized as
reactive when the primary odontoblasts have been triggered or reparative when the primary odontoblasts
have been replaced by odontoblastlike cells
application of a material to an exposed pulp (direct capping) in order to
allow the pulp to recover and maintain its normal vitality and function
a superficial pulp amputation and application of a pulp capping material as
in pulp capping
15
Indirect pulp capping
a treatment in which infected dentine

is intentionally left under a restoration
in order to avoid pulp exposure
CENTRAL
the Cochrane Controlled Trials
Col I
DAB
DMEM
DSP
DSPP
ELISA
EMD
EMDgel
type 1 collagen
3, 3-Diaminobenzidine
Dulbeccos Modified Eagle Medium
dentine sialoprotein
dentine sialophosphoprotein
enzyme-linked immunosorbent assay
enamel matrix derivative
enamel matrix derivative in a formulation with propylene glycol alginate
foetal bovine serum
lipopolysaccharide
lipoteichoic acid
a spontaneously immortalized cell line
from the dental papillae of mouse molars
Index Medicus: Medical Subject
Headings
methol-thiazolyl-diphenyltetrazolium
bromide
propylene glycol alginate
randomized controlled trial
standard error
Small Integrin-Binding LIgand Nlinked Glycoprotein
tris-buffered-saline
Visual Analogue Scale
FBS
LPS
LTA
MDPC-23
MeSH
MTT
PGA
RCT
SE
SIBLING
TBS
VAS
16
INTRODUCTION
The barrier function of dentine

The surfaces of the body including the skin, the gastro-intestinal
tract and the oral mucosa have physiological barrier functions
aimed at protecting the body from external noxious agents (Turner
2009; Elias 2008). In the tooth the enamel is, by virtue of its hardness and structure, an effective mechanical barrier while the
physiological barrier function of the tooth lies mainly within the
pulp-dentine complex.
In the pulp-dentine complex, the dentine is traversed by tubules
filled with fluid in which the odontoblast processes are located.
The odontoblasts are the cells responsible for the formation of dentine. They align at the periphery of the pulp in close proximity to
the dentine with their cellular processes situated within the dentine
tubules. As dentine is being formed, a layer of collagenous network
with associated non-collagenous proteins is laid down by the odontoblasts. This zone of so-called predentine is thus to be found
between the fully mineralised dentine and the odontoblast.
The physiological barrier function of the pulp-dentine complex
consists of several key elements:
Dentinal fluid
Formation of peri-tubular dentine
Formation of tertiary dentine
There is a continuous outward flow of dentinal fluid through the
dentine tubules and in exposed dentine, the outward fluid flow is
17
the first barrier against the inward diffusion of noxious agents such
as bacteria or bacterial components (Pissiotis & Spngberg 1994).
Consequently, any noxious agent migrating towards the pulp must
do so against a pressure gradient (Mjr 2009) and in addition, the
dentinal fluid dilutes the noxious agents. The dentinal fluid, which
is considered to be plasma-derived, contains serum proteins and
globulins which can agglutinate or bind noxious agents and thus
play a protective role in the barrier function (Knutsson et al. 1994).
A range of cytokines and chemokines such as IL-1, IL-6, IL-8, IL10 and TNFcan be found in the dentinal fluid (Geraldeli et al.
2012), though their role has not yet been elucidated. In addition,
the dentinal fluid also contains beta-defensins which have antimicrobial properties and can kill bacteria directly (Dommisch et al.
2005). The substances found in the dentinal fluid do not fully correspond to those in plasma and thus dentinal fluid composition
and flow thus appear to be regulated by the odontoblasts (Geraldeli et al. 2012).
The formation of peri-tubular dentine by the odontoblasts is another part of the physiological barrier function. This reduces the
diameter of the dentinal tubules and reduces the chance of noxious
agents penetrating into the pulp (Bjrndal & Mjr 2002). The secretion of peri-tubular dentine by the odontoblasts as well as the
precipitation of crystals dissolved during the caries process may
block the dentinal tubules, reducing the transport of fluids and
matter (Tagami et al. 1992).
Slow formation of secondary dentine is seen throughout life. It is a
continuum of the primary dentine, though the secondary dentine is
formed at a much reduced rate once the root has been fully
formed. In cases of disruption of the hard tissues, the odontoblasts
may again, very locally, increase the rate of dentine production to
form what is referred to as tertiary dentine. If the primary
odontoblasts have been triggered, the tertiary dentine is denoted as
reactive, whereas reparative dentine is the term used for tertiary
dentine that has been formed by differentiated stem cells often
called odontoblast-like cells, which have replaced the original
odontoblasts. As reparative dentine is not formed by the same
18
odontoblasts as those which formed the primary dentine, the continuum of the tubules is interrupted and the structure of reparative
dentine thus described as atubular (Barber & Massler 1964; Mjr
2009). The formation of tertiary dentine is part of the physiological barrier function through the increase of the dentine thickness
between the site of the insult and the pulp. In case of reparative
dentine, the atubular structure may reduce the transport of fluids
and matter to the pulp. However the function of the dentinal fluid
in the dentinal tubules and thereby the odontoblasts is affected.
These barrier functions of the dentine are considered to be under
control of the odontoblasts. Strategically positioned, the odontoblasts with their processes within the dentinal tubules are the first
cells to encounter and respond to noxious agents such as bacteria
or bacterial components within the dentinal tubules. Odontoblasts
are highly specialized cells and are involved in a similar defence
system to that of epithelial cells regulating the hosts response to
injury (Dommisch et al. 2008; Goldberg et al. 2008). Apart from
forming tertiary dentine, the odontoblasts are proposed to be involved in several defence mechanisms which protect the tooth from
bacterial invasion. The odontoblasts are known to express several
Toll-like receptors which recognise bacterial antigens (Staquet et al.
2011). Upon activation of Toll-like receptors the odontoblasts may
release substances that will affect angiogenesis and the regulation
of blood flow, key elements of the inflammatory process (Botero et
al. 2006; Soden et al. 2009). Through activation of Toll-like receptors it seems possible that the odontoblasts, by releasing different
substances, may negatively or positively regulate the inflammatory
and immune response within the pulp and thus affect the formation of dentine (Durand et al. 2006; Farges et al. 2011; Hahn &
Liewehr 2007). Odontoblasts may also release antibacterial peptides which can kill bacteria directly (Dommisch et al. 2005) and
appear to amplify the responses to the presence of bacteria through
a self-feedback system (Horst et al. 2011). To sum up; the physiological functions of the odontoblast-dentine unit play an important
role in protecting the pulp from microbial influence when exposed
to the oral environment and hence constitute an active barrier
function as described for the skin and gastro-intestinal tract.
19
Injury to the dentine

Apart from trauma, attrition or restorative procedures, caries is the
most evident process in which the dentine barrier function is activated and if overloaded, the dentine will eventually be destroyed
leading to exposure of the pulp. Movement of bacterial products
within the dentinal tubules will affect the odontoblasts, even
though the bulk of the micro-organisms are situated some distance
from the pulp.
Investigations of the micro-flora in deep caries lesions have proposed that the prevailing species are Gram-positive organisms such
as Lactobacillus, Actinomyces, Eubacterium, Proprionibacterium
and Streptococcus species even though Gram-negative organisms
including Fusobacterium, Veillonella and Bacteroides may be
found at lower levels (Edwardsson 1974; Hoshino 1985). More recent studies using 16S rRNA gene sequencing have shown a dominance of Lactobacillus species in some caries lesions (Chhour et al.
2005; Munson et al. 2004). Bacteria have a strong tendency to attach to surfaces and form biofilms (Costerton et al. 1995) and several oral bacteria express surface adhesins which allows them to
bind to collagen within the dentine matrix (McGrady et al. 1995;
Love et al. 1997). Secondary colonizers that do not express these
surface adhesins can co-aggregate to the primary colonizers allowing the formation of polymicrobial biofilms within the dentinal tubules (Love & Jenkinsson 2002). Bacteria in biofilms may express
phenotypes which differ from those of the same species growing in
planktonic state due to the closeness of the bacterial cells and the
presence of micro-niches in the biofilm (Stewart & Franklin 2008;
Svenster et al. 2001). Physiological adaptations to surface attachment include changes in the production of extracellular polymeric
substances as well as modifications in cell morphology.
In in-vitro models of biological events within the pulp due to caries, it is common to use commercially available solutions of known
bacterial pathogenic components such as lipoteichoic acid (LTA) or
lipopolysaccharide (LPS) (Durand et al. 2006; Farges et al. 2011;
Oliveira & Santos 2011; Soden et al. 2009; Telles et al. 2003). The
use of such solutions may be beneficial as comparisons between
20
studies are made easy, but is unlikely to reflect the situation during
the caries process as the pathogenic effect of the bacteria may not
solely be attributed to LTA or LPS. There is scant information concerning the biological events within the pulp using fresh isolates in
biofilm models.
Dentine reactions and repair

As mentioned above, the odontoblasts should be seen as a barrier
entity such as the epithelium lining of the oral cavity, the gastrointestinal tract and the skin. The odontoblasts may be injured and
die during restorative procedures or as a sequel to a rapidly progressing caries lesion. Unlike epithelial cells, odontoblasts are postmitotic and are therefore incapable of cell division in order to replace the lost cells (Arana-Chavez & Massa 2004; Tziafas &
Kodonas 2010). The fact that the odontoblast is post-mitotic might
be of importance since the death of other post-mitotic cells within
the brain or heart can lead to irreversible loss of function of the tissues. The ultimate aim of repair is to restore the original structure
and the biological functions of the damaged tissues (Schmalz & Galler 2011); however as the dead odontoblasts need to be replaced by
differentiated odontoblast-like pulpal stem cells, the repair process
does not completely fulfil this ultimate aim. How the recruitment of
the odontoblast-like cells actually takes place is not fully elucidated,
though it seems likely that multipotent pulpal stem cells migrate to
the site of the injury, where they proliferate and differentiate into
odontoblast-like cells (Balic et al. 2010; Shi & Gronthos 2003). In
cases of exposure of the pulp, healing with hard tissue has been observed although the morphology is similar to reparative dentine with
an atubular structure and with cellular inclusions (Arana-Chavez &
Massa 2004). It is not known whether this reparative hard tissue actually constitutes a functional recovery as it is questionable whether
the odontoblast-like cells responsible for the formation of the hard
tissue possess the same protective barrier functions as those seen in
primary odontoblasts. Since the tubular structure of dentine is
mostly missing, the pathway for agents to induce reactions in the
odontoblasts is more or less hindered. Nevertheless, healing of the
hard tissue structure with the physiological functions seems to be of
great importance in keeping the integrity and vitality of the tooth.
21
The odontoblasts may be affected to various extents during the

caries process. Theoretically this means that the dentine reaction
beneath a caries lesion may involve anything from up-regulating
primary odontoblasts to the replacement of the entire odontoblast
layer. In the reaction to minor injuries, the pulp tissue is not exposed but dentine production is up-regulated underneath what is
clinically observed as an intact layer of dentine. Beneath some caries lesions it seems to be rather similar to the primary dentine,
whereas beneath others it appears to be structurally different or
even absent (Bjrndal 2001). One explanation for these varying
states might be that different consortia of micro-organisms lead to
different degrees of repair. Thus invasion of the pulp by microorganisms is probably dependent on both the nature of the bacteria
present and the barrier function of the pulp-dentine complex.
When the pulp has been exposed, and thus the odontoblasts have
been destroyed, a pulp capping procedure in which a protective
agent is placed on the pulp tissue may result in the recruitment of
odontoblast-like cells and hard tissue repair (Nyborg 1958;
Schrder & Granath 1972).
As reparative dentine is sometimes quite irregular with cellular inclusions and tunnel defects not seen in the primary dentine, it is
sometimes referred to as osteodentine. As a consequence of the
morphological differences observed, the use of the terms dentinelike and odontoblast-like have gained popularity when denoting hard tissue and cells involved in the repair, especially since the
repair involves replacement of post-mitotic cells. Using a biochemical approach to characterize the odontoblast phenotype, a wide
range of differentiation markers such as members of the SIBLINGfamily (Small Integrin-Binding LIgand N-linked Glycoprotein) has
been suggested. Dentine sialoprotein (DSP) is a member of this
family and together with type 1 collagen (Col 1) is suggested to be
a late marker for odontoblast formation. These markers are frequently used to characterize the odontoblast and the dentine (Butler & Ritchie 1995; Nakashima et al. 2002; Narayanan et al.
2001). However members of the SIBLING-family are also expressed to some extent by osteoblasts (Qin et al. 2002). Using a
marker for the odontoblast may be considered to be a complement
22
to solely morphological observations aimed at increasing the possibility of identifying whether newly formed hard tissue is dentine.
However it does not actually elucidate whether all cellular functions are identical to those of the primary odontoblasts.
Treatment of pulp exposures

A pulp exposure is defined in the MeSH browser (Index Medicus:
Medical Subject Headings) as the result of pathological changes in
the hard tissue of a tooth caused by carious lesions, mechanical
factors, or trauma, which render the pulp susceptible to bacterial
invasion from the external environment. Two basically different
types of treatment of teeth with pulp exposures are used in order to
avoid extraction: pulp capping with the ultimate goal of preserving
the pulp and thereby the vitality of the tooth and pulpectomy
where the inflamed or injured pulp is removed and the root canal
obturated with a root filling material.
During the pulp capping procedure, a protective agent is applied to
an exposed pulp in order to allow it to recover and maintain its vitality and function. It is difficult to state the survival or success rate
of teeth that have been pulp capped as most studies are performed
retrospectively. The concepts pulp survival or success are
typically used for teeth that are sensitive to electric pulp testing and
show no signs of apical periodontitis. Partial pulpotomies, a type
of deep pulp capping, have been carried out in traumatized teeth
with complicated crown fractures with calcium hydroxide as a
pulp capping agent and success was reported in 96% of the teeth
after an average observation period of 31 months (Cvek 1978).
Partial pulpotomies performed in young permanent teeth with
carious pulp exposures were reported as successful in 93% of cases
after an average observational period of 56 months (Mejre &
Cvek 1993). Al-Hiyasat et al. (2006) found a success rate of 92.2%
after mechanical pulp exposures and 33.2% after caries pulp exposures at least three years after pulp capping with calcium hydroxide
performed by dental students. In a study by Hrsted et al. (1985) it
was demonstrated that the survival of the pulp after capping with
calcium hydroxide in teeth that had pulp exposures mainly due to
cavity preparation and some due to caries decreased over time. The
23
survival rate was 82% after 5 years and 73% after 10 years.
Barthel et al. (2000) has published a study with a long observation
period of pulp capping performed with calcium hydroxide in teeth
that had carious pulp exposures. Five years after the pulp capping
procedure had been performed there was a 37% success rate and
after 10 years the success rate had decreased to 13%. Results from
another recent study using MTA as a pulp capping material in
teeth with carious pulp exposures show a one-year pulp survival of
67.7% and a two-year survival of 56.2% (Miles et al. 2010). In a
prospective, multi-centre study in which patients with very deep
caries (involving 75% or more of the dentine thickness) leading to
pulp exposures were randomized to direct pulp capping or partial
pulpotomy. After 1 year no difference in pulp survival (31.8% and
34.5% respectively) was seen (Bjrndal et al. 2010). The observation that the failure rate increases over time might imply that the
newly formed hard tissue is not able to act as a functional barrier
protecting the pulp against bacterial micro-leakage along the restoration margins (Barthel et al. 2000; Hrsted et al. 1985; Miles et
al. 2010). The preoperative pulp status of the teeth also seems to
influence the pulp survival since high success rates have been observed for pulp capping of traumatic or mechanical pulp exposures
where most of the pulps are not inflamed, compared to some very
low success rates observed for caries pulp exposures with sometimes severely inflamed pulps (Al-Hiyasat et al. 2006; Barthel et al.
2000; Bjrndal et al. 2010).
Pulpectomy means that pulp tissue is irreversibly removed and the
root canal obturated with a root filling material. Because of the
varying outcome of pulp capping, pulpectomy with a subsequent
root filling that has a well-documented high success rate (Petersson
et al. 1982; Gesi et al. 2006) is a common treatment for teeth with
pulp exposures due to caries in adults. It is also the treatment recommended by The National Board on Health and Welfare in Sweden (National Guidelines for Adult Dental Care) in spite of this
treatment being more invasive and more technically difficult to perform than pulp capping. Even if high success rates are observed for
pulpectomy, the root filled tooth may be considered to be a tooth
at risk of developing complications. Studies show that 10 - 19% of
24
root filled teeth will be extracted within 5 years after treatment

(Alley et al. 2004; Tilashalski et al. 2004) and extractions of root
filled teeth are more common than extractions of teeth without
root fillings (Eckerbom et al. 1992).
Nearly a quarter of a million root canal treatments were reported
to the Swedish Health Service (Frskringskassan) during 2010
with reference costs from the Dental and Pharmaceutical Benefits
Agency (Tandvrds- och lkemedelsverket) varying from 3180 to
5095 SEK per root filled tooth depending on the number of canals
treated in each tooth. Thus, considerable resources are spent on
root canal treatments in Sweden. A low estimation would mean
that at least one billion SEK is spent solely on the fee for root canal
fillings reported to the Swedish Health Service. Avoidance of some
of these treatments in favour of pulp capping would be of benefit
for the individual, oral health and society. Understanding the biological events leading to the healing of functional dentine and the
successful preservation of the vitality of the tooth would give a scientific basis for a less invasive and more cost-effective treatment of
human pulp exposures.
Dentine repair by mimicry

There have been many attempts to find a technique and an agent
that will predictably induce repair of the hard tissue barrier (Hellner 1930; Nyborg 1958; Schrder & Granath 1972). Much attention has been given to calcium hydroxide as a pulp capping agent
as it is considered to promote a superficial necrosis which will act
as scaffold for the odontoblast-like cells (Schrder & Granath
1971). The cellular events in this process are poorly understood,
but it is thought that bio-active dentine matrix components are released from the dentine in contact with calcium hydroxide which
stimulate new hard tissue formation (Graham et al. 2006). There
have been reports of irregularities in the newly formed hard tissue
after pulp capping with calcium hydroxide (Schrder & Granath
1972). Cox has studied the formation of tunnel defects in dentine
bridges on rhesus monkeys and reported that 90% of the bridges
showed defects and subsequently inflamed pulps (Cox et al. 1996).
25
Much effort has been placed on finding strategies other than the
ones in clinical use for the induction and regulation of the reparative or regenerative process after pulp exposure. These include the
application of growth or cell differentiation factors to try to mimic
dentinogenesis during embryonic development in the hope of
achieving healing that resembles the original structure and functions of the lost dentine (Nakashima et al. 2002; Rutherford et al.
1993; Sloan et al. 2000; Smith et al. 2001).
Amelogenins and amelin are proteins that have been proposed to
participate in the final differentiation of odontoblasts and subsequent dentine formation during dentinogenesis (Inai et al. 1991;
Papagerakis et al. 2003; Spahr et al. 2002; Tompkins et al. 2005).
Amelogenins are detectable in the odontoblasts during tooth formation at the time that they are synthesizing and secreting dentine
sialophosphoprotein (DSPP), a protein involved in the mineralisation of dentine (Inai et al. 1991). An amelogenin-rich fraction of
porcine enamel matrix derivative (EMD) that also contains amelin
has been used to induce cementum formation and periodontal
ligament regeneration in monkeys (Hammarstrm et al. 1997). The
mechanism(s) by which EMD promotes periodontal ligament regeneration is still largely unknown, although the amelogenins are
thought to self-assemble into nanospheres that create an extracellular matrix. Within the body, this matrix will slowly be digested by
specific extracellular proteolytic enzymes (matrix metalloproteinases) releasing bioactive peptides to the surrounding tissues for
weeks after application. These will stimulate the secretion of local
growth factors and cytokine expression in the treated tissues,
prompting a regenerative process mimicking odontogenesis (Lyngstadaas et al. 2009). Nakamura et al. (2001; 2002; 2004) used a
formulation of EMD as a pulp capping agent in two studies performed on miniature swine. The amount of hard tissue formed in
the EMD-treated teeth was twice that of the control group capped
with calcium hydroxide cement. The biological responses to various treatments may differ between species, and prior to this thesis
the effects of EMD used as a pulp capping agent on the formation
of hard tissue and pulp status in humans had not been studied.
26
Today it is not known whether the unfavorable long-term results

of pulp capping in teeth with deep caries are due to a dysfunctional
barrier and a secondary infection at the exposure site or a withstanding inflammation originally caused by the caries lesion. Nevertheless, as the barrier function in the dentine seems to be important in protecting the pulp from microbial influence it would be
beneficial to gain knowledge about factors that influence the repair
of the dentine after dental pulp capping.
27
AIMS
Repair of the hard tissue after pulp capping has been observed in
human experimental studies; however the clinical outcome of pulp
capping is regarded as uncertain. The overall aim of this thesis was
to study some aspects of the repair of the dentine barrier, especially
in conjunction with dental pulp capping and to ascertain if, and
under what conditions, the exposed pulp is able to heal with a hard
tissue barrier. This, in order to obtain a scientific basis for a less
invasive and perhaps more cost-effective treatment of pulp exposures.
The specific aims were:
To evaluate the evidence for the formation of a hard tissue
barrier after pulp capping in humans by conducting a systematic review
To study the effect of an enamel matrix derivative (EmdogainGel) as a pulp capping agent on experimentally
exposed human dental pulps with regard to hard tissue
formation and pulp inflammation
To characterize the hard tissue formed after pulp capping
with EmdogainGel using DSP and type 1 collagen as dentine markers
To investigate how extracellular products from biofilms of
bacteria found in a deep caries lesion affect the activity of
odontoblast-like cells and their formation of type 1 collagen.
28
MATERIALS AND METHODS
Systematic review (I)

A systematic review of the English literature on the formation of a
hard tissue barrier after dental pulp capping was performed using
specific indexing terms. In order to achieve a systematic approach,
the literature search was conducted in four major steps using the
method described by Goodman (1993). These steps were to: (1)
specify the problem; (2) formulate a plan for the literature search;
(3) conduct a literature search and retrieve publications; and (4)
interpret and assess the evidence from the literature retrieved.
The following questions defined the problem:
Can a pulp exposure heal, i.e. form a hard tissue barrier
with pulp tissue that is free of signs of inflammation after
pulp capping?
Under which circumstances does a hard tissue barrier
form?
What happens to the pulp and the newly formed hard tissue over time?
Literature search
Publications were retrieved from PubMed with publication dates
from 1 January 1966 to 1 January 2005. As several new articles on
the subject have been published since the publication of the review
in 2006, the systematic review was supplemented with a literature
search extended until 1 October 2010. The searches were performed using the MeSH (Medical Subject Heading) term Dental
Pulp Capping and was limited to publications with abstracts.
29
Animal studies and case reports were excluded. An additional

search was performed in CENTRAL (Cochrane Controlled Clinical
Trials Register) and the reference lists of included publications
were hand searched to find additional original scientific articles
that had not been found through PubMed.
Interpretation and assessment

The interpretation and the assessment of the quality of the studies
were performed independently by the authors using pre-tested protocols. The quality of each included original scientific article was
rated as high, moderate or low according to criteria modified after
Guyatt et al. (1993; 1994). The data extraction and assessment
were performed without blinding and disagreements were solved
by discussion to reach consensus. The overall evidence grade, based
on the quality of each included article, was rated to be strong,
moderately strong, limited or insufficient as proposed by Centre
for Evidence Based Medicine, University of Oxford.
Clinical trial (II)

Subjects and pulp capping procedure
The subjects were recruited from the Public Dental Health Service;
Skne County Council, Sweden and were eligible to be included in
the study if they had contralateral premolars without clinically evident caries scheduled for extraction on orthodontic grounds. Eight
subjects, aged between 12 and 16 years, with a total of 9 pairs of
premolars were included in the study.
Following an experimental superficial pulp amputation, either EmdogainGel (EMDgel) or a mix of calcium hydroxide and saline
was placed at random in contact with the pulp wound. A disc of
Teflon was placed over the pulp capping material and the tooth
was restored with a temporary restoration. Each subject received
both test and control in a split mouth design. After 12 weeks the
teeth were extracted. Adverse events were recorded.
30
Outcome measures
Frequency of any symptoms

Evaluation of pulpal status
Evaluation of hard tissue formation
The subjects made records of any symptoms using a Visual Analogue Scale (VAS) (Huskisson 1983) over the first 10 days after the
pulp capping procedure. They were also asked to report any experience of spontaneous pain or use of analgesics. A blinded examiner carried out telephone interviews with the subjects about pain
or discomfort. The structured interviews were performed at 1 day,
2 weeks, 6 weeks and 12 weeks after the treatment. At 12 weeks,
just prior to extraction, the teeth were clinically examined for:
tenderness to percussion
tenderness to palpation
mobility
and the temporary filling was assessed for risk of leakage. The
teeth had been examined similarly prior to the experimental treatment.
Histological preparations and analysis

The extracted teeth were fixed in formaldehyde for 7 days and
thereafter demineralized in EDTA and subsequently embedded in
paraffin. After longitudinal serial sectioning (5 m), every fifth section was stained with haematoxylin and eosin. All stained sections
covering the wound area were studied with the aid of a light microscope equipped with a camera and computer with a software
programme in order to perform histometry. The sections were
blinded regarding the subject and type of treatment that had been
performed and were analysed by the operator performing the pulp
capping procedure. The inflammation in the pulp was analysed and
classified after criteria modified after Heyeraas et al. (2001) (Table
1). The inflammation was classified in three different locations
within the pulp; in the central part, just apically to where the pulp
had been amputated or within the proliferated pulp (if such a proliferation had occurred). The newly formed hard tissue was analysed with regard to: if and how it covered the wound area, the
31
area of hard tissue formed and the thickness of the hard tissue
bridge if such a hard bridge had been formed covering the wound
area.
Table 1 Criteria used for classification of the pulp status with regard to inflammation, modified after Heyeraas et al. (2001).
0 None: Fibroblasts but no inflammatory cells are found. Capillaries but no extravasated red blood cells can be found.
1 Slight: Increased number of cells, predominately fibroblasts. A

few inflammatory cells are involved. An increased number of capillaries are noted, and a few extravasated red blood cells may be
found.
2 Moderate: Predominantly characterised by more cells in the area

than in the slight reaction. Increased number of capillaries and vessels are found.
3 Severe: Marked cellular infiltration, including local abscess formation. Numerous blood vessels are found in the tissue surrounding the intense cellular infiltration.
4 Abscess formation or extended lesions not localised only to the
tissue beneath the cavity floor.
Ethical approval and informed consent

The study was approved by the Committee on Investigations Involving Human Subjects at Lund University, Sweden (LU 297-01)
and all the participants and their guardians completed a consent
form.
Immunohistochemical studies (II and III)

Localisation of EMD in EMDgel treated teeth
As the control material in the clinical study was calcium hydroxide
and not the propylene glycol alginate (PGA) which was used as the
vehicle for EMD, there were concerns regarding whether the effects
32
seen could be attributed to EMD. In order to see if the hard tissue

formed in the teeth treated with EMDgel was located in close proximity to the EMD, immunohistochemical localisation was carried
out using a polyclonal rabbit anti-EMD antibody. The antibody
has been described previously (Gestrelius et al. 1997b) and was
supplied by Biora AB. Endogenous peroxidase activity was
quenched and non-specific binding was blocked using normal goat
serum. Endogenous biotin was blocked with the DAKO biotin
blocking kit (Dako, Glostrup, Denmark) according to the enclosed
instructions. Sections were incubated overnight at 4C using the
antibody. Antibody binding was visualized using DAB in TBS containing hydrogen peroxide and sections were counterstained with
haematoxylin. Using a light microscope, evaluation was made by
the same operator who assessed the histological sections stained
with haematoxylin and eosin.
Localisation and assessment of DSP and Col I

In order to characterize the newly formed hard tissue in the teeth
pulp capped with EMDgel or calcium hydroxide, two relatively
specific markers for dentine were used; DSP and Col I. Representative sections from the clinical study were used, and at least one section from each tooth was used for each antiserum. The polyclonal
DSP antibody, raised in rabbit, was provided by Dr. Qin, Department of Endodontics, University of Texas-Houston Health Science
Center Dental Branch, Houston, TX, USA and the polyclonal rabbit anti-Col I was purchased from Nordic Biosite AB, Tby, Sweden. The same procedures were undertaken as for the localisation
of EMD. Using a light microscope, two calibrated observers independently evaluated the reactivity of anti-DSP and anti-Col I in different locations within the area that had been pulp capped:
newly formed hard tissue
areas with less mineralised hard tissue, corresponding to
predentine
lining cells
areas of inflammation
The immunostaining was classified as moderate when the intensity
was comparable to the staining of dentine and predentine in the
33
root of the same tooth examined, as well as in a stained tooth

which had been extracted prior to placement of a pulp capping material. In other cases the staining was classified as not detectable
or strong. The sections were coded during the evaluation and
disagreements among the observers were solved by discussion so
that consensus was reached.
Staining of micro-organisms
In order to examine the possible occurrence of micro-organisms in
the wound area, a modified Brown and Brenn technique (Churukian & Schenk 1982) was used to stain sections as well as the
LIVE/DEAD BacLight Bacterial Viability Kit according to the

manufacturers instructions (Molecular Probes Inc., Eugene, OR,
USA).
Cell culture study (IV)

An established cell-line of spontaneously immortalized cells from
the dental papillae of mouse molars was provided by Professor
Nr, University of Michigan, Ann Arbor, USA and was used in this
study. The cells of MDPC-23 express both DSP and Col I and are
therefore considered to be of the odontoblast lineage. The cells
were grown in Dulbeccos Modified Eagle Medium (DMEM) containing 10% foetal bovine serum (FBS) and supplemented with
penicillin, streptomycin and L-glutamine at 37C in a humidified
atmosphere of 5% CO2 in air.
Collection and isolation of bacteria

A dentine sample was taken from a tooth with a very deep caries
lesion. Further excavation lead to a pulp exposure, with bleeding
pulp tissue. Bacteria were cultured and identified on the basis of
aerobic and anaerobic growth. Isolated strains were Gram stained
and classified with regard to colony morphology into genus. Subculturing of Lactobacillus spp was done using Rogosa SL medium
and the isolates were kept frozen. Further identification using 16S
rRNA gene sequencing was done for the isolates used further in
the study. The gene sequencing was performed by Associate Professor Ann-Catherine Petersson, Skne University Hospital, Lund,
Sweden.
34
Biofilm supernatants
Fresh isolates from the caries lesion (Lactobacillus rhamnosus, Lactobacillus casei and Shuttleworthia satelles) and from a clinical isolate of Enterococcus faecalis from an infected root canal were allowed to form biofilms in tissue flasks containing DMEM supplemented with 1% FBS and L-glutamine. After 96 hours of incubation in 5% CO2 in air, the supernatants were collected and centrifuged to remove the bacteria. After concentration, the supernatant
was filtered using a 0.2 m filter. The total protein content was
measured to lie between 2.8 and 3.2 mg/ml.
Cell activity
In order to investigate whether the biofilm supernatants influenced
the MDPC-23 cells activity, a methol-thiazolyl-diphenyltetrazolium
bromide (MTT) assay was used. MDPC-cells were grown and then
exposed to the different biofilm supernatants or to the known bacterial antigens lipoteichoic acid (LTA) or lipopolysaccharide (LPS).
After 96 hours of exposure, MTT was added. Yellow MTT is reduced to purple formazon crystals by enzymes in the mitochondria
of cells and may therefore be used for assessment of metabolic activity but is also frequently used to assess viability or proliferation
of cells. After incubation, the reduced MTT was dissolved and the
absorbance was read. Absorbance values were divided by the value
for the control and expressed as a percentage.
Col I production
To determine whether bacterial components affected the production of Col I, the major component of the non-mineralised matrix
of tertiary dentine, MDPC-23 cells were grown as above. After 96
hours the cell layers were dissolved using guanidium hydrochloride, containing 0.1% CHAPS. The amount of Col I was assessed
using enzyme-linked immunosorbent assay (ELISA) with a biotinylated monoclonal rat-anti-mouse Col I antibody (Chondrex Inc.,
Redmond, WA, USA). Absorbance values were divided by the
value for the control and expressed as a percentage.
35
Statistical method
All data from study IV were analysed using GraphPad Prism 5
(GraphPad Software Inc., La Jolla, CA, USA). A one-sample t-test
was used to determine whether the mean percentage changes were
significantly different from the control value of 100%. Values p
<0.01 were considered to be significant.
36
RESULTS
Systematic review (I)

The search strategy yielded 107 articles that were retrieved in full
text of which 21 original scientific studies were included in the
published systematic review. The reasons, in descending order, for
exclusions were:
animal experiment
no histology
full pulpotomy
no pulp exposure
review
no light microscopic evaluation
no intervention
impossible to assess data
no examination of the hard tissue
case reports
non-English language
The extended literature search is presented in fig 1. Twenty articles
in full text were included in the extended literature. Hand searching of the reference lists of the reviews and articles included in this
extended literature search did not result in any additional publications. Table 2-4 shows the summary of data from the extended systematic review, corresponding tables for the previously published
systematic review can be found in paper I.
37
Figure 1 The extended literature search, reported according to

PRISMA (Moher et al. 2009).
Assessment of quality
The results from the published systematic review are combined
here with data from the new literature search. One of the studies
included was judged to have a high quality (Olsson et al. 2005), six
were judged to be of moderate quality (Accorinte et al. 2008; Hrsted-Bindslev et al. 2003; Kiatwateeratana et al. 2009; Lu et al.
2008; Min et al. 2008; Silva et al. 2006) and the remaining 34
studies were considered to have a low quality. The large number of
studies with low quality was predominantly due to deficiencies in
the study design, sample size or the manner in which the microscopic evaluation had been executed or reported.
Study designs
The studies were heterogeneous regarding their designs and the
pulp capping procedures. Sixteen were classified as being prospective and randomised (Accorinte et al. 2009; Accorinte Mde et al.
2005; Brnnstrm et al. 1979; deLourdes Rodrigues Accorinte et
38
a 2006; Demarco et all. 2001; Eliaas et al. 200

al.
07; Iwamoto
o et al.
2
2006;
Kiatwaateeratana et al. 2009; Lu
L et al. 2008; Min et all. 2008;
N et al. 20
Nair
008; Olsson et al. 2005; Sawicki et al.
a 2008; Silvva et al.
2
2006;
Sbay et al. 1995; Hrsted-Bindslev et al. 2003) aand an
eq
qual numbeer of studies,, sixteen, weere prospecttive and con
ntrolled
b without a randomissation proceedure (Acco
but
orinte et al. 2008;
2
2008;
Accoriinte Mde et al. 2005; 20
008; Aeineh
hchi et al. 20
003; de
Souza Costa et al. 2001; do Nascimeento et al. 2000;
2
Ersin & Eron 2005; Hebling
nat
H
et al.
al 1999, Jerrrell et al. 1984;
1
Negm
m et al.
1980; Paroliaa et al. 2010
0; Pereira et al. 2000; S
bay & Ascii 1993,
Sbay & Dem
mirci 2005; Turner et al.
a 1987). Th
he remaindeer, nine
sttudies, weree solely obseervational studies with several
s
singlle cases
(B
Bhaskar et al. 1972; Caicedo
C
et al
a . 2006; Ceehreli et al. 2000;
C
Clarke
1971; Cowan 196
66; Fitzgerald & Heys 19
991; Kashiw
wada &
T
Takagi
1991;; Sari et al. 1999;
1
Stanley
y & Lundy 1972).
1
Several studiees had divid
ded the samp
ple size into,, up to, 6 ob
bservatiional period
ds which meeant that theere were sam
mple sizes rranging
frrom one to 20
2 teeth per observational period.
P
Pulp
capping procedurres
Most studies used a direect pulp cap
M
pping proced
dure, perform
med in
younger healthy teeth wiithout signs of caries, allthough therre were
reeports of paartial pulpotomy and a few studies were condu
ucted in
teeeth with carrious exposu
ures.
A variety of materials was
w used forr the pulp capping
c
proccedure;
th
hough the most
m
frequen
nt were calccium hydrox
xide based o
or resin
b
bonding
matterials. The use of diffeerent calcium
m hydroxidee based
m
materials
ofteen resulted in
i hard tissu
ue bridges, though
t
obserrvation
p
periods
of sh
horter than 3 weeks rev
vealed no haard tissue brridging.
H
However,
thee use of bon
nding materials as the pulp
p
cappingg agent
reesulted in haard tissue formation only in single cases.
c
In the studies
w
where
bondiing materiall was comp
pared to a calcium
c
hyd
droxide
b
based
materiaal, the calciu
um hydroxid
de based maaterial was su
uperior
to
o the bondin
ng material in all studies.
39
40
Method
CCT
60 days
Reference
Accorinte Mde
et al. 2005
Study design
Observation time
Test (T), No of
teeth
Control (C), No
of teeth
No of subjects
Bleeding controlled with

T1: Saline solution n=5
T2: Ferric sulfate n=5
T3: 2.5% NaOCl n=5
T4: calcium hydroxide solution
n=5
followed by pulp capping with
Scotchbond Multi Purpose Plus
(3M ESPE, St Paul, MN, USA)
C: Bleeding controlled with saline
calcium hydroxide powder
covered with Dycal (DentsplyCaulk, Milford, DE, USA) n=5
Subjects n=?
Material
Dentine bridge formation in:

T1: 0%
T2: 0%
T3: 0%
T4: 0%
C: 100%
No inflammation in C, and
inflammation to different extent in
the majority of T
Reported results
Test (T)
Control (C)
Low
Study quality
Table 2 Data from the extended literature search regarding studies using other pulp capping materials
than bonding or MTA, or studying certain operative procedures.
41
RCT
2 observation intervals: 30 and 60
days
CCT
2 observation intervals: 15 and 45
days
RCT
6 months
RCT
12 weeks
de Lourdes
Rodrigues
Accorinte et al.
2006
Parolia et al.
2010
Kiatwateeratana
et al. 2009
Olsson et al.
2005
T: Emdogain Gel (Biora AB,

Malm, Sweden) n=9
C: Calcium hydroxide mixed with
saline n=9
Subjects n=8
T: Emdogain Gel (Biora AB,

Malm, Sweden) n=15
water n=15
Subjects n=15
T: Propolis (Ecuadorian Rainforest

LLC, USA) mixed with ethyl alcohol
n=12
C1: ProRootMTA (Dentsply Caulk,
Milford, DE, USA) n=12
C2: Dycal (Dentsply Caulk) n=12
Subjects n=?
T1: adhesive resin, rubber dam

n=10
C1: adhesive resin, no rubber dam
n=10
T2: Calcium hydroxide powder
covered with Dycal (DentsplyCaulk, Milford, DE, USA), rubber
dam n=10
C2: Calcium hydroxide powder
covered with Dycal, no rubber
dam n=10
Subjects n=?
Complete hard tissue bridge

T: 0 of 9
C: 9 of 9
More inflammation in T than in C,
though one tooth in C was necrotic
T: No complete hard tissue bridge,

inflammation
C: 10 of 13 complete hard tissue
bridge, no or minimal
inflammation
At 45 days:
T: 6 of 6 hard tissue bridge
C1: 6 of 6 hard tissue bridge
C2: 5 of 6 hard tissue bridge
More inflammation in C2 than in T
and C1
Dentine bridge formation in:

T1: 0 of 10
C1: 0 of 10
T2: 10 of 10
C2: 10 of 10
No inflammation in T2 or C2 and
some inflammation in the groups
treated with adhesive resin
High
Moderate
Low
Low
42
Test (T), No of
teeth
Control (C), No
of teeth
No of subjects
CCT
T: MTA (Angelus, Londrina, PR,
2 observation intervals: 30 and 60 days Brazil) n=20
C: Calcium hydroxide powder
covered with Life (Kerr Romulus, MI,
USA) n=20
Subjects n=?
Accorinte
Mde et al.
2008
Material
RCT
T1: ProRoot MTA (Dentsply Tulsa
2 observation intervals: 30 and 60 days Dental, Tulsa, OK, USA) n=20
T2: MTA (Angelus, Londrina, PR,
Brazil) n=20
Subjects n=?
Study design
Observation time
Accorinte et
al. 2009
Method
Reference
Test (T)
Control (C)
T: 11 of 20 total dentine bridge. Less

inflammation than in C
C: 14 of 20 total dentine bridge
Complete hard tissue bridge at 30

days:
T1: 5 of 8
T2: 5 of 8
Complete hard tissue bridge at 60
days:
T1: 5 of 8
T2: 6 of 10
No difference in inflammatory
response. Teeth in which microorganisms were found were excluded
from the analysis
Reported results
Low
Low
Study quality
Table 3 Data from the extended literature search regarding studies using mineral trioxide
aggregate as the pulp capping material.
43
Series of cases
Typically after 6 months
RCT
136 +/- 24 days
RCT
2 months
RCT
3 observation intervals: 1 week, 1 and
3 months
RCT
47-609 days, in average 138 days
Caicedo et
al.
2006
Iwamoto et
al. 2006
Min et al.
2008
Nair et al.
2008
Sawicki et al.
2008
T: White ProRoot MTA (Dentsply

Tulsa Dental, Tulsa, OK, USA) n=32
C: Life n=16
Subjects n=23
C: Dycal Ivory (Dentsply Caulk,

Subjects n=23
T: White MTA (Proroot , Dentsply,

Tulsa Dental, OK, USA) n=20
T: MTA ProRoot (Dentsply, Tulsa, OK,

USA) n=10
C: Dycal (Dentsply-Caulk, Milford, DE,
USA) n=10
Subjects n=16
T: White ProRoot MTA (patent

pending) mixed with sterile saline
n=22
C: Dycal (Dentsply-Caulk, Milford, DE,
USA) n=23
Subjects n=?
ProRoot MTA (Dentsply Tulsa Dental,

Tulsa, OK, USA) n=10
Subjects: n=12
CCT
T: MTA (Dentsply Caulk, Milford, DE,
2 observation intervals: 30 and 60 days USA) n=20
C: Life (Kerr, Romulus, MI, USA) n=20
Subjects: n=?
Accorinte et
al. 2008
T: 28 of 30 complete dentine bridge

and less inflammation than in C
C: 11 of 14 complete dentine bridge
At 1 month:
T: 3 of 6 complete bridge
C: 1 of 5 complete bridge
At 3 months:
No or minimal inflammation in T,
more inflammation in C
No significant difference in regard to
inflammation, though 1 of 10 in C
showed severe inflammation
T: 20 of 22 bridging, 17 of 22 no
superficial inflammation
C: 18 of 23 bridging, 14 of 23 no
superficial inflammation
7 of 8 dentine bridge. The majority of

teeth show inflammation.
At 60 days:
No difference in inflammation
between T and C
Low
Low
Moderate
Low
Low
Low
44
CCT
T1: Clearfil Liner Bond 2V (Kuraray
2 observation intervals: 30 and 60 days Medical, Osaka, Japan) n=12
T2: Clearfil SE Bond (Kuraray
Medical, Osaka, Japan) n=12
C1: Dycal (Dentsply, Petrpolis, Rio
de Janeiro, Brazil) covered by
Clearfil Liner Bond 2V
C2: Dycal covered by Clearfil SE
Bond
Test (T), No
of teeth
Control (C),
No of teeth
No of
subjects
Accorinte
et al. 2008
covered by Dycal (Dentsply Caulk,

Subjects n=?
T: different steps in Scotchbond

Multi Purpose Plus (3M ESPE, St
Paul, MN, USA) n=20 divided into 4
subgroups
Material
RCT
60 days
Study design
Observation time
Accorinte
Mde et al.
2005
Method
Reference
Test (T)
Control (C)
At 30 days hard tissue bridge in:

T1: 1 of 6
T2: 0 of 5
At 60 days hard tissue bridge in:
T1: 1 of 6
T2: 1 of 6
C1: 4 of 5
C2: 4 of 4
No difference with regard to
inflammation between T1 and T2,
though more than in control groups
Complete bridging in:

T: 0 of 20
C: 5 of 5
No or minimal inflammation in C,
more inflammation in T
Reported results
Moderate
Low
Study
quality
Table 4 Data from the extended literature search regarding studies using bonding adhesives
as pulp capping materials.
45
CCT
2 observation intervals: 7 and 90 days
RCT
3 observation intervals: 7, 30 and 90
days
RCT
4 observation intervals: 1, 3, 7 and 30
days
CCT
40 days
Ersin &
Eronat
2005
Lu et al.
2008
Silva et al.
2006
Sbay &
Demirci
2005
C: Dycal (Dentsply Caulk, Milford,

DE, USA) n=6
Subjects n=4
T1: Scotchbond Multi Purpose Plus

(3M ESPE, St Paul, MN, USA) n=10
T1: 37% phosphoric acid n=26

T2: 10% phosphoric acid n=26
Single Bond Adhesive System (3M
Dental Products, St Paul, MN, USA)
covered with Dycal (Dentsply Ind e
Com Ltda, RJ, Brazil) n=26
Subjects n=?
T: Clearfil SE Bond (Kuraray

Medical, Osaka, Japan) n=21
C: Dycal (Dentsply, Weybridge, UK)
n=20
Subjects n=27
T: Prime&Bond 2.1 (Caulk, Dentsply,

water n= 10
Subjects: n=?
RCT
T: Clearfil SE Bond (Kuraray
2 observation intervals: 30 and 90 days Medical, Osaka, Japan) n=16
C: Calcium hydroxide powder n=10
Subjects n=?
Elias et al.
2007
Dentine bridge in:

T: 0 of 10
C: 3 of 6
More inflammation in T than C.
At 30 days dentine bridge deposition

in:
T1: 0 of 5
T2: 0 of 5
C: 5 of 5
Somewhat more inflammation in T1
and T2 than in C
At 90 days:
T: 0 of 7 full bridge
C: 5 of 6 full bridge
No difference in inflammation
between T and C
At 90 days:
T: 0 of 5 dentine bridge,
inflammation
C: 5 of 5 dentine bridge, no
inflammation
At 30 days dentine barrier

formation in:
T: 0 of 8
C: 5 of 5
At 90 days dentine barrier
formation in:
T: 1 of 8
C: 5 of 5
Less inflammation in C than T.
Low
Moderate
Moderate
Low
Low
Concluding evidence grade

Combining the results from the published systematic review with
the new literature search leads to these conclusions regarding the
pulp capping materials:
The use of calcium hydroxide based materials as pulp
capping agents frequently results in the formation of hard
tissue covering the pulp exposure (limited scientific evidence).
The use of Emdogain Gel as a pulp capping agent does

not result in the formation of hard tissue covering the pulp
exposure (limited scientific evidence).
The use of bonding materials as pulp capping agent does
not result in the formation of hard tissue covering the pulp
exposure (limited scientific evidence).
Scientific evidence is lacking as to whether teeth treated
with MTA as a pulp capping material show hard tissue
formation more frequently than calcium hydroxide based
materials.
46
Effect of EMDgel on experimentally performed pulp

exposures (II)
All subjects completed the study and no adverse events related to
the treatment were recorded.
Symptoms and clinical assessment

No teeth were reported to give severe symptoms during the telephone interviews. The teeth treated with EMDgel gave fewer symptoms compared to the teeth treated with calcium hydroxide. At the
examination, just prior to extraction, all temporary fillings were
judged to be adequate. Two teeth treated with EMDgel were sensitive to percussion. The reported symptoms in each tooth are shown
in table 5.
Table 5 Pulpal status with regard to inflammation, classified from

0 (None) to 4 (Abscess formation). The mean number of classifications from the representative central sections (n = 5) from each experimental tooth is given. The inflammation was judged above the
exposure in the proliferated pulp tissue, directly below the exposure and in the centre of the pulp. Mild () and moderate () symptoms reported at any of the telephone interviews after the pulp
capping procedure. No severe symptoms were reported. At the
clinical assessment only mild sensitivity to percussion was reported.
47
Pulpal status
The pulp had proliferated into the space initially occupied by the
EMDgel (fig 2). Therefore the soft tissue reactions were judged in
three different areas of the pulp; the proliferated pulp, just below
the amputation site and in the central part of the pulp. Within the
proliferated pulp tissue there were areas where the inflammation
was judged to be moderate or severe. One tooth showed an abscess. Two teeth showed abscesses or extended lesions not only localized to the proliferated pulp. In the calcium hydroxide treated
teeth there was no proliferation of the pulp tissue (fig 3). The majority of teeth showed no or minimal inflammation, though one
tooth exhibited total necrosis. The pulpal status in each tooth is
shown in table 5.
Figure 2 Micrographs of teeth from subject no. 1 and subject no.

4a who twelve weeks earlier had received a partial pulpotomy and
had been treated with EMDgel. Staining with haemotoxylin and
eosin.
48
Figure 3 Micrograph of a tooth from subject no. 8 who twelve

weeks before had received a partial pulpotomy and had been
treated with calcium hydroxide. Staining with haemotoxylin and
eosin.
Hard tissue formation

The hard tissue in the EMDgel treated teeth was not formed as a
bridge covering the pulp tissue, but hard tissue was formed alongside the dentine walls which restricted the area which the pulp had
proliferated into. There was also hard tissue formed in patches
within the proliferated pulp tissue. In one tooth treated with EMDgel that showed an abscess, there was no hard tissue formed. In
the calcium hydroxide treated teeth the hard tissue was formed as a
bridge covering the pulp tissue.
The amount of hard tissue in the EMDgel treated teeth was considerably larger than in the teeth treated with calcium hydroxide. The
thickness of the hard tissue bridge that had been formed in the calcium hydroxide treated teeth was on average 148 m, though the
dentine bridge was considerably less thick in the junction with the
predentine of the primary dentine.
49
Localization of EMD
Detection of EMD was made in all EMDgel treated teeth. It was
localized within the patches of newly formed hard tissue as well as
alongside the dentine walls which restricted the area into which the
pulp had proliferated.
Characterization of the newly formed hard tissue (III)

EMDgel treated teeth
In the teeth treated with EMDgel there was hard tissue formed
alongside the dentine walls which restricted the area into which the
pulp had proliferated, however this did not appear to be attached
to the dentine wall. The areas corresponding to predentine in this
hard tissue did appear to be continuous to the predentine and
odontoblastic layer in the pulp. Staining with the DSP and Col Ispecific antibodies revealed the proteins to be both present in the
primary dentine and more abundantly in the predentine. They were
also localized in diffuse areas of the newly formed hard tissue. The
cells located in the close proximity to the hard tissue formed were
also stained for DSP and Col I (fig 4, table 6).
Figure 4 Micrographs of a tooth from subject no. 7 who twelve

weeks earlier had received a partial pulpotomy and been treated
with EMDgel. (a) New hard tissue is observed alongside the exposed dentine surfaces and in isolated masses within the proliferated pulp (arrows). A dentine chip (DC) is seen below the amputation site. Staining with haematoxylin and eosin. (b) Detail of (a) at
a higher magnification showing the dentine (D), predentine (PD),
the hard tissue formed alongside the exposed dentine surfaces
(HTA) and patches within the proliferated pulp (HTP). (c) DSP
staining (brown) is observed in the newly formed hard tissue and is
marked in areas corresponding to predentine (arrow). (d) Col I
staining (brown) was observed in the newly formed hard tissue, especially in the areas corresponding to predentine (arrow). The dentine (D) and predentine (PD) were used as positive controls for
each antibody in each tooth section.
50
51
Calcium hydroxide treated teeth

In the teeth treated with calcium hydroxide the same appearance
regarding the staining with DSP and Col I in the root was seen; it
was stronger in the predentine than in the primary dentine. Diffuse
Col I staining was seen throughout the hard tissue bridge. The DSP
staining was also observed in the bridge, though with a more
patchy appearance and with the same intensity as seen in the predentine areas. A layer of columnar cells, positive for DSP and Col I
lined the hard tissue bridge (fig 5, table 6).
No bacteria could be detected in the wound area after staining
with the modified Brown and Brenn technique or with the
LIVE/DEAD BacLight Bacterial Viability Kit.
Figure 5 Micrographs of a tooth from subject no. 4a who twelve

weeks earlier had received a partial pulpotomy and been treated
with calcium hydroxide. (a) The hard tissue was formed as a bridge
(B) covering the pulp. (b) DSP staining (brown) is observed in the
bridge (B) and is marked in areas corresponding to predentine (arrow). (c) Col I staining (brown) was observed in the bridge (B).
The dentine (D) and predentine (PD) were used as positive controls
for each antibody in each tooth section.
52
Table 6 Evaluation of the staining of DSP or Col I antibody in the

EMDgel or calcium hydroxide treated teeth. In two teeth, one
treated with EMDgel and one with calcium hydroxide, it was difficult to find one section representing the pulpal status and two different sections were chosen. In all; a total of 40 sections from 18
teeth were used for immunohistochemical staining. The primary
dentine in each section was used as reference standard (= moderate). The table presents number of sections with moderate or
strong staining of total number of sections in which recordings
were possible.
53
Effects of bacterial products on the activity of odontoblastlike cells and their formation of Col I (IV)
Identification of bacteria
The carious dentine sample contained in total 15 different bacterial
strains. The predominant species were Lactobacilli. Gram-positive
pleomorphic rods, most probably Actinomyces spp and Grampositive cocci were also present. A selection was made to represent
different strains according to morphology and growth conditions.
The three strains that were able to grow in DMEM supplemented
with FBS and glutamine were used in the experiments and were
identified by 16S rRNA gene sequencing to be:
L. rhamnosus
L. casei
S. satelles
Effects of bacterial products on the activity of MDPC-23 cells

When the MDPC-23 cells were exposed to the biofilm supernatants
from the three strains (L. rhamnosus, L. casei, S. satelles) taken
from the deep caries lesion, no significant effect on the activity
could be noted. However, the root canal isolate E. faecalis reduced
the activity of the MDPC-23 cells significantly (mean % of control
SE = 80 2.7; p < 0.05) (fig 6). Consequently, different bacterial
species differentially affected the activity of odontoblast-like cells.
54
Figure 6 Eff
F
ffects of thee biofilm su
upernatant on
o the activ
ivity of
M
MDPC-23
ceells over 96 hours.
h
The absorbance
a
v
values
were d
divided
by the absorrbance of th
he controls and
a expresseed as a perccentage
(1
100%). Thu
us values greeater than 10
00% represeent an increaase and
th
hose below 100%, a decrease
d
in activity. Vaalues presentted are
m
mean
% SE of values from
f
three independent
i
t experiments
ts (** =
p < 0.01).
150
% of control
100
50
is
al
ec
te
co
ro
te
En
Sh
ut
tl
ew
or
cc
th
us
ia
fa
sa
us
ill
ac
ob
ct
La
lle
se
ca
su
no
m
ha
sr
llu
ci
ob
a
ct
La
LTA, the celll wall comp

L
ponent of Gram-positiv
G
ve bacteria, significaantly reduceed the activitty of the MD
DPC-23 cells (mean % o
of contrrol SE = 73.4 3.5; p < 0.001). LPS,
L
the cell wall compon
nent of
G
Gram-negativ
ve bacteria did not influence th
he activity of the
M
MDPC-23
ceells.
55
Effects of bacterial products on the production of Col I by

MDPC-23 cells
To evaluate whether the extracellular bacterial products can influence the production of Col I by the MDPC-23 cells, the cell layers
were subjected to ELISA. The biofilm supernatant from L. rhamnosus as well as the S. satelles taken from the deep caries lesion
had no significant effect on the Col I production by the MDPC-23
cells. The bacterial supernatant from the carious sample containing
L. casei did however significantly increase the production of collagen (134% 12.1%, p < 0.05) while the biofilm supernatant from
E. faecalis caused an approximately 50% reduction in collagen
production (p < 0.05). Again, different bacterial strains, as well as
different bacterial species, differentially affected the collagen production.
LTA did not affect the production of Col I; however LPS significantly increased the production by almost doubling the amount
compared to the control (195.4% 12.2%, p < 0.01).
56
DISCUSSION
The aim of this thesis has been to study some aspects of the repair
of dentine, especially in conjunction with dental pulp capping.
The main findings from the systematic review combined with the
new literature search were (i) that based on the limited scientific
evidence available the use of calcium hydroxide based pulp capping
materials frequently results in the formation of hard tissue covering
the pulp exposure and (ii) the use of Emdogain Gel or bonding materials as a pulp capping agents does not result in the formation of
hard tissue covering the pulp exposure. (iii) Scientific evidence is
lacking as to whether teeth treated with MTA as a pulp capping
material show hard tissue formation more frequently than calcium
hydroxide based materials. The results from the clinical study testing EMDgel as a pulp capping agent showed that greater amounts
of hard tissue were formed in the EMDgel treated teeth, although
not in an arrangement that could constitute a barrier. The dentine
functions seem to be important for the defence of the pulp tissue.
Our studies revealed that the cells aligned with the newly formed
hard tissue as well as the hard tissue itself contained DSP and Col
I. This indicated that the cells were of the odontoblast lineage and
the newly formed tissue could be regarded as dentine. The main
findings from the experiments using a cell-line of mouse odontoblasts that formerly had been shown to express DSP and Col I, indicated that the responses with regard to activity and initial formation of tertiary dentine varied upon exposure to different exoproducts from bacteria grown in biofilms.
57
Endpoint measures
As stated in the introduction, repair of the exposed pulp should
ideally restore the original structure and the biological functions of
the damaged soft and hard tissues, however this is a difficult task
to evaluate and use as an end-point measure. Instead, different surrogate end-points such as whether the hard tissue covered the pulp
wound and whether the pulp showed any signs of inflammation
are often used and this is the case in this thesis. To the patient, the
outcome of pulp capping that probably matters is to have a functional tooth without any symptoms and with a low risk for future
pain. This is only an assumption, since no studies on patients preferences regarding pulp capping procedures currently exist.
The measures used here have primarily been based on histological
studies and cell culture, even though some clinical information
such as pain or discomfort from the treated teeth has been collected.
Systematic review
The systematic review included studies with a variety of study designs and only case reports were excluded since we wished to make
an assessment of all available articles on the subject. In order to
minimize the risk of missing relevant publications the search strategy used in PubMed was broad and the reference lists of included
articles as well as reviews were hand searched in order to find articles that had not been identified by the search in PubMed. The fact
that the new literature search only identified additional publications through the search in PubMed indicated that the search strategy was comprehensive. No additional publication could be found
through the hand searching of reference lists or searching CENTRAL when performing the new literature search. We have chosen
to include and present the results from all studies, even those with
low quality, in order to aid the planning of future studies of pulp
capping that will be assessed histologically.
The included studies were mostly performed in teeth without any
signs of caries which is understandable since there may be difficulties in finding teeth and subjects that are suitable for such a study.
58
However choosing such a strategy affects the external validity of

the results as the most common reason for exposure of pulps is caries. It is possible that different results may have been obtained if
the experiments had been performed in teeth with pulps inflamed
due to caries.
The majority of the studies included in the published systematic review were judged to have low quality. At the time of the publication of the systematic review only a few such reviews within the
field of endodontics could be found through searches in PubMed,
six years later an identical search yielded several hundred. The result of the systematic review was surprising at the time in that the
quality of the retrieved publications was assessed to be so low. The
same findings have however been the case in many systematic reviews. It is obvious that there is an evolving awareness of the importance of a proper study design and there was indeed a positive
difference between the studies included in the published systematic
review and those identified through the new literature search. The
majority of the newer studies were performed as randomised controlled trials and 6 out of 20 were judged to have at least a moderate quality compared to 1 out of 21 studies in the published systematic review. Some of the improvement in the execution and reporting of the studies identified through the new literature search
can probably be attributed to the growing awareness of evidencebased health care.
Nonetheless, there are still some major issues that should be considered; such as performing a power analysis prior to conducting
the study based on the anticipated difference in effect that would
be clinically significant. Such power analyses were lacking in the
studies identified. Moreover, in many studies the material was divided into subgroups of different time intervals with very few teeth
in each group, which means that an even larger sample size is
needed to get valid data from the subgroups. In this context it is
also obvious that the choice of control material affects the sample
size. For instance; testing a MTA-based material and having a
composite resin material as the control would mean that a very
small sample would be needed to detect a difference since a consid-
59
erable difference can be anticipated. This is not to say that such a

control material should be used. It seems most adequate to use a
calcium hydroxide containing material such as a slurry of calcium
hydroxide powder mixed in water, since calcium hydroxide has
frequently been associated with hard tissue formation covering the
pulp exposure and is the control material most often used in pulp
capping studies. Another quality issue is the criteria used when
evaluating the histological sections; many different criteria are used
and if our hypothesis that healing with tissue with the functions of
normal dentine is crucial for the long-term success of pulp cappings, a criterion such as attempted bridging seems difficult to
assess. It is also obvious from the clinical study (II) within this thesis that serial sectioning and assessment of all sections is important
since the hard tissue formation and the status of the pulp may vary
throughout the exposure site of the pulp.
It is interesting to note the difference with regard to pulp tissue reactions seen in the human compared to other species. In the systematic review that was limited to human material, hard tissue
formation was seldom observed in teeth that have been pulp
capped with dentine adhesive materials, in contrast to the frequent
hard tissue formation described in animal studies (Hafez et al.
2002, Kitasako et al. 1999; Olmez et al. 1998). A possible explanation for the occurrence of some cases of hard tissue formation in
humans may be that the odontoblast layer has been left partially
undisrupted during the pulp capping procedure.
Effects of EMDgel
In the literature there are reports of the use of different proteins
such as growth factors in order to mimic dentinogenesis (Lovschall
et al. 2001; Li & Sae-Lim 2007; Rutherford et al. 1993,
Nakashima 1994, Decup et al. 2000, Zhang et al. 2008). These are
all performed in animal models. Study I in this thesis identified two
studies Kiatwateeratana (2009) and Olsson (2005) (the latter being
Study II in this thesis), performed in humans in which dentine repair after pulp capping was induced by using material that would
theoretically have the potential to mimic a part of the
dentinogenesis process. These studies could be performed in hu-
60
mans since the experimental pulp capping material was already

approved for use in patients with periodontal disease and had also
been piloted in animal studies (Nakamura et al. 2001; 2002). The
animal studies showed however a slightly different result to the
ones performed in humans in that there was extensive hard tissue
formation, not only localized to the pulp wound, but narrowing
the whole pulp chamber.
Study II was designed as a pilot study, and had a limited sample
size. However, care was taken to avoid unnecessary methodological faults. The materials were placed according to a randomization
procedure that was performed immediately before placement of the
pulp capping material. The person assessing the teeth for symptoms as well as the person assessing the histologic sections were
blinded. On evaluation it was found that the pulp tissue reactions
differed markedly in all EMDgel treated teeth from those in the
control group treated with calcium hydroxide, and these reactions
were therefore considered representative in spite of the limited
sample size.
Calcium hydroxide should probably be the control material of
choice in most pulp capping studies since the appearance in the histological sections treated with calcium hydroxide is quite characteristic when performed correctly. Nevertheless, in Study II it might
be argued that PGA, which was the vehicle for EMD, should have
been used as a negative control. Even more so since a similar appearance of the pulp and the newly formed hard tissue was seen in
experimentally pulp capped monkey teeth after a disc of Teflon
was placed on the pulp exposure. However there was no hard tissue formed in patches as seen in Study II (Heys et al. 1990). A
study conducted in rats using PGA as a negative control showed
that reparative dentine was formed both in the teeth treated with
EMDgel and the negative control, though the formation was more
extensive in the EMDgel group (Igarashi et al. 2003). We used an
antibody to detect the EMD in the histological sections and it was
present in close proximity to the newly formed hard tissue, giving
some support to the idea of EMD being able to promote dentine
repair in humans.
61
The hard tissue formed after placement of EMDgel cannot act as a

barrier, as it was formed in patches and did not cover the pulp
wound. The amount of hard tissue was however substantial and
questions about how these patches of hard tissue would progress
over time arose. In a similar study to ours, Kiatwateeratana et al.
(2009) used EMDgel as a pulp capping material and had a longer
observation period than the one used in Study II. Their results
showed that the hard tissue formation was comparable to ours and
even after 6 months the hard tissue masses did not close the pulp
exposure.
The teeth used in Study II were free from caries which means that
at the time of the pulp capping procedure all pulps were considered
to be free from inflammation. Thus, the inflammatory response
seen in the histologic sections could be due to the pulp capping materials themselves or oral micro-organisms that reached the wound
area through micro-leakage along the restoration margins. It
should be stressed that the classification of the pulpal status was
made in the area which showed the most severe class of inflammation. In the EMDgel treated teeth where the pulp had proliferated
into the space initially occupied by the EMDgel there was also a
higher density of cells resembling pulp fibroblasts with no other
signs of inflammation. In an attempt to study if the presence of micro-organisms could be associated with the inflammation observed
in some of the histologic sections, bacterial staining was carried
out. In spite of the fact that some of the sections showed areas of
necrotic pulp, in which one would have expected to detect bacteria
with the techniques used, no micro-organisms were discovered.
The reason for this is not known but it cannot be ruled out that
bacteria have been removed as the temporary fillings were removed
with a bur before serial sectioning or during the actual processing
of the sections (Wijnbergen et al. 1991). Thus, no direct cause of
the inflammation could be established in Study II.
The hard tissue was characterized using DSP and Col I. A wide
range of differentiation markers has been suggested, but there
seems to be some consensus among researchers dealing with formation of dentine that SIBLING proteins, such as DSP, in combi-
62
nation with Col I are suitable phenotypic markers for this tissue
and the odontoblast (Chen et al. 2008, Hao et al. 2009). Staining
with the DSP and Col I -specific antibodies was also present in diffuse areas of the newly formed hard tissue. The cells located in
close proximity to the formed hard tissue were also stained with
DSP and Col I indicating that they could be of the odontoblast lineage. These findings are in accordance to the ones reported by
Nakamura et al. (2004) who had performed a similar study of pulp
cappings in pigs. Finding a positive control may be difficult, but we
had the advantage of being able to use primary dentine and
odontoblast from other parts of each tooth section for this purpose. The staining with the DSP and Col I-specific antibodies revealed the proteins to be presented both in the primary dentine and
more abundantly in the predentine, which perhaps could be explained by the higher content of extracellular matrix in the
predentine. DSP can also be detected in bone, however in considerable smaller amounts than in the dentine (Qin et al. 2002). Since
the staining of DSP in the newly formed hard tissue was equal to or
stronger than the staining of the primary dentine in each section,
we consider our results to be valid. Characterizing the hard tissue
repair with regard to the morphology and the markers DSP and
Col I is a way to approach the goal of evaluating whether the original structure and biological function of the dentine still remains.
Lesser symptoms were reported in teeth that had been treated with
EMDgel than teeth treated with calcium hydroxide. The children
who participated in the study were interviewed by a blinded examiner about any pain or discomfort from the teeth and they were also asked to report any pain on a VAS. The validity of VAS used in
young children has been shown to be low (Shields et al. 2003). As
reported by the Swedish Council on Health Technology Assessment (2010) the scientific evidence for the relation between symptoms and the assessments of inflammation in histologic sections is
lacking, as only one study with moderate quality could be found
(Hasler & Mitchell 1970). In the study by Hasler & Mitchell there
is no direct correlation between symptoms and histological appearance and this was also the case in Study II. The reports of pain did
not directly relate to the assessment of inflammation in the histo-
63
logic sections, though the two teeth classified with the most severe
grade of inflammation also presented with tenderness on percussion.
Effects of bacterial products on odontoblast-like cells

In order to investigate how extracellular products from biofilms of
bacteria found in a deep caries lesion affect the viability of
odontoblast-like cells and their formation of Col I a fresh microbial
sample was taken from a deep caries lesion. This sample was shown
to be dominated by Gram-positive rods and the majority of the isolates were identified as Lactobacillus spp which is in accordance to
others who have studied the microbiota of deep carious lesions
(Chhour et al. 2005; Gross et al. 2010). Further identification using
16S rRNA of the three species used in the experiment showed that
the identified lactobacilli both belonged to the L. casei group which
together with L. salivarius and L. fermentum have previously been
found to be prominent at the advancing front of a progressive caries
lesion (Byun et al. 2004). Munson et al. (2004) identified L. casei
and L. rhamnousus to be prominent in teeth with carious lesions
that had spread to the inner third of the dentine as seen on x-ray by
using 16S rRNA analysis. Lactobacilli have also been identified as
prominent members of the microbial community in the initial stages
of root canal infection (Nadkarni et al. 2010). The third species
from the caries lesion was identified as S. satelles. The
Shuttleworthia spp was identified in 2002 by Downes et al. amongst
isolates from periodontal pockets and subsequently also been isolated from an infected root canal (Jacinto et al. 2007). Little is known
about the role of this genus in the oral cavity. Shuttleworthia satelles
have been identified as a pathogen as it was found forming a monoculture on a prosthetic mitral valve in a patient suffering from endocarditis (Shah et al. 2010) but has also been identified in carious lesions (Chhour et al. 2005; Gross et al. 2010; Munson et al. 2004). It
is not solely Gram-positive species, as identified here, which may be
found in deep caries. Gram-negative micro-organisms such as
Prevotella intermedia and Porphyromonas endodontalis have also
been found at low levels in deep caries (Massey et al. 1993, Martin
et al. 2002). Therefore both LPS and LTA were used for comparison
in the experiments.
64
The supernatants from the three strains isolated from the deep caries lesion and grown in biofilms had no effect upon cell activity.
This suggests that under these experimental conditions, none of the
strains released substances that led to cell death or cell cycle arrest
in the odontoblast-like cells. In contrast, the supernatants from
biofilms of the root canal isolate of E. faecalis caused a decrease in
total activity of the MDPC-23 cells. Visual observation indicated
that the number of cells present after exposure was lower than that
in the control cultures and extracellular substances from biofilms
of E. faecalis thus appeared to inhibit proliferation. E. faecalis have
a number of virulence factors (Kayaoglu & rstavik 2004) that
could cause the effects seen here.
The Col I production was used as a marker to study the effects of
bacterial exo-products upon the odontoblasts synthesis of tertiary
dentine since collagen is abundant during the formation of
predentine. Cells exposed to the extracellular products from E.
faecalis demonstrated a lower production of Col I, even though
this effect can partially be attributed to a reduction in cell activity,
E. faecalis appears to have a small inhibitory effect on the production of Col I. In contrast, the isolate of L. casei from the deep caries lesion caused a significant up-regulation of the production of
Col I. The mechanisms underlying these effects are unknown but
since no change was seen with LTA, it appears unlikely that activation of toll-like receptor 2 is involved in these processes. LPS had a
significant up-regulatory effect upon the production of Col I, indicating that upon activation of toll-like receptor 4, the formation of
tertiary dentine may be enhanced.
The cell line used in the experiments, MDPC-23, is a wellestablished model. The cells are deemed to be of the odontoblast
lineage by virtue of their expression of both DSP and Col I (Hanks
et al. 1998). As pointed out before, there seem to be a difference in
pulp reactions between different species and it cannot be ruled out
that other results may be acquired using human odontoblasts.
65
Future studies
During the work with this thesis several ideas of future work have
been evoked, here a few brief thoughts are presented:
66
Before conducting any other pulp capping studies using

EMDgel, the formula of EMDgel should be altered as the
formula with PGA does not seem to promote dentine repair
which would be able to act as a barrier. One study of EMD
used in conjunction with other pulp capping materials has
recently been published (Al-Hezaimi et al. 2011).
There is a lack of health economic evaluations within the

field of endodontics. Root canal treatments seem to be difficult to perform in children and young persons (Ridell et
al. 2006). Even though recent studies of pulp cappings in
carious teeth (Bjrndal et al. 2010; Miles et al. 2010) have
shown somewhat discouraging results, pulp capping procedures performed in children and young persons may have
a better prognosis. This has generated an idea for a future
study with a health economic evaluation of pulp cappings
performed on caries exposures in children.
Another area of interest would be to study the patients

preferences. In the study of Bjrndal et al. (2010) a large
proportion of teeth with very large caries lesions that received a pulp capping procedure did in fact develop painful
conditions and the patients had to undergo emergency root
canal treatment before the 1-year clinical evaluation. It
would be valuable to know the patients treatments preferences in this perspective.
As the result from Study IV indicates differences in how the

odontoblasts are affected by the products from the biofilms, it would be interesting to develop this model further
with polymicrobial biofilms under different environmental
conditions.
CONCLUSIONS
Based on the limited scientific evidence available, calcium hydroxide based pulp capping materials frequently result in the formation
of hard tissue covering the pulp exposure, something the use of
Emdogain Gel or bonding materials as a pulp capping agents do

not. Scientific evidence is lacking as to whether teeth treated with
MTA as a pulp capping material show hard tissue formation more
frequently than calcium hydroxide based materials. In order to be
able to detect treatment effects, future studies should be designed
as RCTs with a prior sample size calculation.
Using EMDgel as a pulp capping agent in experimentally exposed
human pulps gave greater amounts of hard tissue, but more pulp
inflammation compared to the controls which were treated with
calcium hydroxide. However the hard tissue was not formed as a
bridge and thus could not provide a barrier in contrast to the hard
tissue formed in the calcium hydroxide treated teeth in which the
hard tissue covered the pulp wound.
The hard tissue formed after treatment with EMDgel or calcium
hydroxide as well as the cells lining it, contained the dentine markers DSP and Col I, indicating that the hard tissue formed could be
regarded as dentine and the cells to be odontoblasts.
The extracellular products from biofilms of bacteria found in a
deep caries lesion did not affect the activity of odontoblast-like
cells, as was seen with extracellular products from E. faecalis taken
67
from an infected root canal. However the synthesis of type 1 collagen was affected to various extents, suggesting that the bacterial
species in a caries lesion may influence dentine repair.
68
69
ACKNOWLEDGEMENTS
I wish to express my gratitude to all those who have helped and

encouraged me during the doctoral studies. I particular wish to
thank:
Kerstin Petersson, my primary supervisor. I really cannot express
enough appreciation for you and all the time and effort you have
put into my progress. You are a remarkable person and I will always think of you with gratitude.
Julia Davies, my other supervisor. I truly appreciate your expert
knowledge and support, your time and devotion. I would not have
had the courage to go into the lab without your friendly attitude.
Madeleine Rohlin and Gunnel Svenster for valuable scientific
guidance throughout the studies.
Kristina E Holst, Biora, who introduced me to the concept of Good
Clinical Practice. It was certainly an appreciated start of my doctoral studies.
Ulla Schrder for your time and devotion during the work with
paper II.
Eva Wolf, head of The Department of Endodontics and co-worker
in the up-dating of the systematic review, for being so distinct
about the importance of having time to do the thing that almost
always is so enjoyable research.
70
Staff at The Department of Endodontics for support and friendship. I am so grateful to all of you for making the department such
a great workplace.
Staff at The Department of Oral Biology, and especially Madeleine
Blomqvist, for your patience and for making the time spent at the
lab so enjoyable.
Staff at The Department of Oral Pathology for assisting with the
histologic processing.
The children and their parental guardians who courageously accepted to participate in the clinical study and the orthodontists and
dentists who recruited them.
My family, you are all so dear to me. Mamma and Pappa - for
seemingly never-ending love and support. Robert - for being such a
remarkable generous and supportive person.
Research grants were received from:
The European Society of Endodontology
The Edith Agrell Memorial Fund, the South Swedish Dental
Society
The Faculty of Odontology, Malm University
The Sven Sellman Memorial Fund, the Faculty of Odontology,
Malm University
The Swedish Dental Society
Te-Pe Munhygienprodukter AB, Malm
71
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on the repair oF the dentine barrier
205 06 malm, sweden
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m a l m u n i v e r s i t y 2 012
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Swedish Dental Journal Supplement 226, 2012

on the repair oF the dentine barrier

205 06 malm, sweden

ON THE REPAIR OF THE DENTINE BARRIER

Swedish Dental Journal Supplement 226, 2012

Helena Fransson 2012

Departments of Endodontics and Oral Biology,

The summary of this publication can be downloaded from

In summary; Emdogain Gel initiated dentine formation, though

POPULRVETENSKAPLIG SAMMANFATTNING (SUMMARY IN SWEDISH)

En gel (Emdogain Gel) som innehller amelogenin som man vet r

Sammanfattningsvis kan man sga att Emdogain Gel initierade

This thesis is based on the following papers, which are referred to

Olsson H, Petersson K, Rohlin M. Formation of a

Olsson H, Davies JR, Holst KE, Schrder U, Peters

pulp capping in humans using Emdogain Gel. Int

There are several terms used in the literature concerning dentine

dentine formed by primary odontoblasts until the root is fully formed

Indirect pulp capping

a treatment in which infected dentine

the Cochrane Controlled Trials

The barrier function of dentine

Injury to the dentine

Dentine reactions and repair

The odontoblasts may be affected to various extents during the

Treatment of pulp exposures

root filled teeth will be extracted within 5 years after treatment

Dentine repair by mimicry

Today it is not known whether the unfavorable long-term results

MATERIALS AND METHODS

Systematic review (I)

Animal studies and case reports were excluded. An additional

Interpretation and assessment

Clinical trial (II)

Frequency of any symptoms

Histological preparations and analysis

1 Slight: Increased number of cells, predominately fibroblasts. A

2 Moderate: Predominantly characterised by more cells in the area

Ethical approval and informed consent

Immunohistochemical studies (II and III)

seen could be attributed to EMD. In order to see if the hard tissue

Localisation and assessment of DSP and Col I

root of the same tooth examined, as well as in a stained tooth

LIVE/DEAD BacLight Bacterial Viability Kit according to the

Cell culture study (IV)

Collection and isolation of bacteria

Systematic review (I)

Figure 1 The extended literature search, reported according to

a 2006; Demarco et all. 2001; Eliaas et al. 200

Bleeding controlled with

Dentine bridge formation in:

T: Emdogain Gel (Biora AB,

T: Emdogain Gel (Biora AB,

T: Propolis (Ecuadorian Rainforest

T1: adhesive resin, rubber dam

Complete hard tissue bridge

T: No complete hard tissue bridge,

Dentine bridge formation in:

T: 11 of 20 total dentine bridge. Less

Complete hard tissue bridge at 30

T: White ProRoot MTA (Dentsply

C: Dycal Ivory (Dentsply Caulk,

T: White MTA (Proroot , Dentsply,

T: MTA ProRoot (Dentsply, Tulsa, OK,