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GROWTH HORMONE AND INSULIN-LIKE GROWTH FACTOR-1
EFFECTS ON DENTINOGENESIS: IN VITRO AND IN VIVO
Michael Robert Stevens
Department of Oral Biology
University of Queensland
St.Lucia
Queensland
4067
A thesis submitted for the degree of Master of Dental Science (Research) 1999.
1.STATEMENT
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The work presented in this thesis is, to the best of my knowledge and belief, original; except as
acknowledged in the text. It has not been submitted, either in whole or in part for a degree at this
or any other University.
Signed ...........................................................
Michael Stevens
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2. ABSTRACT
Growth hormone and insulin-like growth factor-1 are polypeptides that have essential roles in
normal growth and development and have been shown to have important roles in the complex
and highly regulated process of dentine formation, or dentinogenesis. This study investigated
some aspects of their roles in in vitro and in vivo dentinogenesis.
The first experiment compared the effects of growth hormone, insulin-like growth factor-1 and
foetal calf serum to serum-free medium on in vitro dentinogenesis in mouse molar tooth germs.
Insulin-like growth factor-1 treated germs demonstrated significant volumetric growth and
differentiation over the other treatments while growth hormone also elicited advanced
differentiation, increased mitotic activity and cell density.
The second experiment compared the effects of growth hormone, growth hormone and insulin-
like growth factor-1 combination, bone morphogenetic proteins (BMP) 2 and 4 and saline in
calcium chloride-coated sodium alginate bead carriers as in vivo pulp-capping agents. Direct
pulp-capping was performed on 72 dog teeth comparing these treatments to two “traditional”
pulp-capping agents (calcium hydroxide and corticosteroid/antibiotic combination) over periods of
two and five weeks. Calcium hydroxide stimulated a strong inflammatory from the pulps which could
persist, however it also produced the greatest dentinogenic activity of all treatments with extensive
reparative dentine bridging. The corticosteroid-antibiotic treatment produced moderate to heavy
inflammation that remained for the length of the study and inhibition of dentinogenesis. The pulps
treated with the bone morphogenetic proteins 2 & 4 produced comparatively disappointing pulpal
results in this experiment compared to other studies. The use of growth hormone as a capping agent
resulted in a favourable inflammatory pulpal state and vital pulpal cell function with localized
stimulation of dentinogenesis. The growth hormone/insulin like growth factor-1 combination, although
producing more pulpal inflammation than the growth hormone treatment, was the only treatment,
apart from calcium hydroxide, to elicit closure of the exposure site and stimulated secondary and
reparative dentine production. These growth factors may have potential as natural pulp-capping
agents.
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Key words: growth hormone, insulin-like growth factor-1, dentinogenesis, direct pulp-capping,
bone morphogenetic protein, calcium hydroxide, corticosteroid/antibiotic
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3. ACKNOWLEDGEMENTS
I wish to specially thank Associate Professor William George Young (Department of Dentistry), my
supervisor, for his advice, guidance, support and direction over the years. Thanks also to Dr Michael
Waters (Department of Physiology and Pharmacology) my associate supervisor for his advice and
for supplying the growth factors.
Acknowledgment is given to my other co-authors on the paper published on mouse molar
odontogenesis – Bill Young, Jean-Victor Ruch, Catherine Bègue-Kirn, Chavis Zhang, Hervé Lesot
and Michael Waters.
To Doug Harbrow and Terry Daley special thanks for the invaluable technical and practical
assistance given.
Dr Richard Prankerd (Department of Pharmacy) is thanked for his time and efforts devoted to
developing growth factor carriers and patience in showing me how to produce them.
For the dog experiments grateful acknowledgment is made to Dr Helen Keates (Department of
Veterinary Science), Gary Godbold (Department of Anatomy) and Geraldine Mills (Wellcome
Research Institute -Royal Brisbane Hospital) - dental assistant and dog anaesthetist extraordinaire.
Thankyou, to my wife Michelle for her forebearance and tolerance.
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4.CONTENTS
1.Statement……………………………………………………………………………… 2
2.Abstract ……………………………………………………………………………….. 3
3.Acknowledgements…………………………………………………………………… 5
4.Contents ………………………………………………………………………………. 6
5.Figures ………………………………………………………………………………… 8
6.Tables…………………………………………………………………………………… 15
CHAPTER 1.
Growth Hormone (GH) and Insulin-like Growth Factor-1 (IGF-1): Roles in
dentinogenesis……………………………………………………………………………16
Hypothesis for Experiment 1………………………………………………………….… 21
Chapter 1 References ………………………………………………………………….. 22
CHAPTER 2.
Experiment 1. Comparison of the effects of growth hormone, insulin-like growth
factor-1 and foetal calf serum on mouse molar odontogenesis in vitro……………. 31
Chapter 2 References …………………………………………………………………... 49
CHAPTER 3.
Hypothesis for Experiment 2……………………………………………………………. 54
CHAPTER 4.
Reviews
I. Healing of the pulpo-dentinal complex following exposure…………………….. 57
II. Corticosteroid/antibiotic preparations and direct pulp-capping…………………68
III. Calcium hydroxide and direct pulp-capping……………………………………… 75
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IV. Bone morphogenetic proteins in odontogenesis and dentinogenesis………… 92
V. Carriers for delivering growth factors to the dental pulp…………………………100
CHAPTER 5.
Experiment 2. A histological comparison of growth hormone and growth factors
with calcium hydroxide and a steroidal-antibiotic combination as dental
pulp-capping agents in the dog………………………………………………………….103
CHAPTER 6.
Summary and conclusions……………………………………………………………….139
BIBLIOGRAPHY…………………………………………………………………………..143
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5. FIGURES
LEGENDS TO FIGURES
PLATE 1 (p33)
Mouse molar tooth germs were rated after various times and treatments in culture by the
degree of differentiation of the dental papilla and of the odontogenic epithelium.
Stain H & E. Bar = 50 µm.
Fig. 1 Rating 1 was assigned when non-polarised preameloblasts and non-differentiated cells of
the dental papilla were found astride the basement membrane.
Fig. 2 Rating 2 was assigned to a tooth germ when initial polarisation of the odontoblasts, without
dentine matrix formation, was observed. The nuclei of the preameloblasts were at different levels.
Fig. 3 Rating 3 was characterised by initial dentinal matrix production by well-polarised
odontoblasts. The preameloblasts assumed a taller configuration, however their nuclei remained in a
pseudostratified configuration.
Fig. 4 Rating 4 was achieved when a definitive band of dentine was produced and the
preameloblasts became tall columnar cells with proximally-polarised nuclei - the area below the
preameloblasts is an artefact, which was not due to loss of enamel matrix.
Ar-artefact, BM-basement membrane, DE-dentine, DM-dentinal matrix, DP-dental papilla, O-
odontoblasts, PA-Preameloblasts, PO-preodontoblasts, and SR-stellate reticulum.
PLATE 2 (p37)
Fig. 5 Comparison between the development of individual 16 day mouse molar tooth germs –
cultured for six days. The sections illustrated are from the greatest areas of each tooth germ in
sagittal section.
The serum-free control (-FCS) showed limited differentiation of the inner enamel epithelium and
preodontoblast layer with no dentinal matrix formation and a comparatively sparsely populated dental
papilla. The germ treated with foetal calf serum 20% (+FCS) shows dentinal matrix formation with
polarised odontoblasts and preameloblasts in a pseudostratified configuration. The growth hormone-
treated germs (+GH 50 ng/ml and +GH 100 ng/ml) also show dentinal matrix production with well-
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polarised preameloblasts and odontoblasts. The cells of the dental papilla are densely packed. The
insulin-like growth factor-I treated germs (+IGF-I 100 ng/ml and +IGF-I 200 ng/ml) show striking
dentine matrix production with well polarised odontoblasts and preameloblasts - no enamel
production was elicited, the space is an artefact.
Ar-artefact, D-dentine, DM-dentinal matrix, DP-dental papilla, O-odontoblasts, and PA-
Preameloblasts. Bar = 200 µm.
PLATE 3. (p41)
Fig. 6 Histograms of the effects of treatments on mice tooth germs with days of treatment, 16 days
post conception (vaginal plug = day 0 ) 4, 5, 6 days. Bars represent Standard Errors of the Means.
6(a). Effects on the average volumes of tooth germs: The serum-free treated germs and both growth
hormone treated groups showed little volumetric change over the three days of sampling. Foetal calf
serum-treated germs showed a comparatively high initial volume (Day 16+4) but show no significant
change over the subsequent days. Both insulin-like growth factor-I treated groups show volumetric
increases over the three days, with IGF-I 200 ng/ml treated germs having greatest average volumes
on all three days.
-FCS - Serum-free, +FCS - Foetal Calf Serum, GH 50 (GH1) - Growth Hormone 50 ng/ml, GH 100
(GH2)- Growth Hormone 100 ng/ml, IGF-I 100 (IGF1) - Insulin-like Growth Factor-I 100 ng/ml, +IGF-
I 200 (IGF2)- Insulin-like Growth Factor-I 200 ng/ml.
6(b). Effects on mitotic indices in odontogenic epithelium between treatments. Mitotic activity was
highest in the foetal calf serum and in the growth hormone treated-groups which were
significantly higher than the serum-free group. Values for the IGF-I treatments were intermediate.
Differences between days, within treatments were not significant (data not shown).
6(c). Effects on cell density within the dental papillae on treatment day 16+6: Significantly higher cell
density was recorded in the growth hormone-treated low dose group than for all other treatment
groups with the exception of the GH high dose group. Although marginally less dense than all other
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groups, IGF-I high dose treatment group was not significantly different from serum-free or serum-
supplemented treatments.
PLATE 4. (p44)
Fig. 7 This diagram represents the ratings assigned to mouse molar tooth germs according to their
degrees of differentiation with time and treatment (cf Fig. 1-4). The trend towards greater
differentiation with time within treatment groups is evident. The serum-free control germs were, in
general, the most poorly differentiated. Growth hormone 100 ng/ml and foetal calf serum 20%
treated germs showed similar differentiation over time. The insulin-like growth factor-I groups
showed the earliest differentiation with IGF-I 200 ng/ml treatment demonstrating three germs
producing dentine (Rating 3) and two germs with polarised preameloblasts (Rating 4) at Day 16+4.
-FCS - Serum-free treated germs; +GH - Growth Hormone treated germs; +FCS - Foetal Calf
Serum
treated germs and +IGF - Insulin-like Growth Factor-I treated germs.
PLATE 5. (p110)
Fig. 8 Fluorescent markings demonstrating continued incremental deposition of dentine following
exposure.
Both pictures are from sections close to the exposure, the prepared cavity can be seen (C). The first
dose of tetracycline was given at the time of the exposure.
a. Corticosteroid-antibiotic treated pulp at two weeks - the narrow nature of the fluorescent line under
the cavity signifies decreased early deposition. Note the fluorescence in the tubules (t) exposed to
the Ledermix and the increased width in the band peripherally.
b. Calcium hydroxide treated pulp at five weeks - there seems to be initial disruption with the first two
increments (1,2) which stabilizes as time progresses.
PLATE 6. (p114-115)
Fig. 9
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Comparison of the degrees of pulpal inflammation encountered in canine, premolar and molar dog
teeth shows that canine and molar teeth were more often lightly inflamed and a preponderance of
heavy inflammation and necrosis was encountered in premolar teeth following exposure and
capping.
Fig. 10
Comparison of the severity of inflammation found in pulps of different exposure widths. Light
inflammation was generally associated with smaller exposures, however heavy inflammation and
necrosis could not, predictably, be related to size.
PLATE 7. (p118-119)
Fig. 11
The severity of inflammation in dog tooth pulps after two weeks of treatment with either calcium
hydroxide (Calxyl), growth hormone (gh), growth hormone/insulin-like growth factor-1 combination
(gh/igf-1), antibiotic/antiinflammatory combination (Ledermix), or normal saline (control).
Fig. 12
The severity of inflammation in dog tooth pulps after five weeks of the treatments, detailed in Figure
11, with the addition of treatments utilizing bone morphogenetic proteins 2 and 4 (bmp). Note the
proportion of lightly inflamed pulps found in association with growth hormone and Calxyl
preparations.
PLATE 8. (p120)
Fig. 13
The effects of treatments on dentinogenesis. Partial indicates stimulation of matrix at the exposure
site. A bridge indicates a zone of reparative dentine. The highest rate of complete bridging was
found after Calxyl treatment. Growth hormone (GH) and GH/IGF-1 in combination treated pulps
showed stimulation of dentinogenesis, while the combination showed bridging in two instances.
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PLATE 9. (p121)
Fig. 14
Saline control pulp at 2 weeks. Dog pulp exposed and covered with an alginate bead containing
saline (Control) for 2 weeks. A dentine chip (DC) is displaced. An amorphous protein (AP) is
associated with the test material and moderate chronic inflammation and increased vascularity (V) is
present. No preodontoblast differentiation or new dentine is evident at the exposure site (E).
Predentine (PD) continues to be deposited either side of the wound.
Fig. 15
Dog pulp treated with corticosteroid-antibiotic combination at 2 weeks. The pulp shows a wide zone
of chronic inflammation (I). No identifiable odontoblasts have differentiated and there is no activity at
the exposure site (E) or on the dentinal chips (DC). The odontoblasts are disrupted peripherally to
the wound site (DO).
PLATE 10. (p124)
Fig. 16
Dog pulp treated with corticosteroid-antibiotic combination at 5 weeks. No odontoblastic activity was
found around the exposure site (E) or on the dentinal chip (DC). A zone of inhibition (Z) is present
along the left hand side corresponding to tubules that were exposed to the treatment. Normal
odontoblasts (O) are active on the opposite side of the pulp chamber. Note the increased vascularity
(V) and persistent chronic inflammation generally within the pulp chamber.
Fig. 17
A Growth Hormone/Insulin-like Growth Factor-1 treated pulp at 5 weeks. Some inflammation
persists in the pulp. Reparative dentine (RD) is found beneath the exposure and on dentine chips.
Secondary dentine (SD) has been stimulated in the odontoblasts whose tubules originate in the
primary dentine of the exposure (E). Glass ionomer cement (GIC).
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PLATE 11. (p125)
Fig. 18
Dog pulp with an exposure treated with Growth Hormone in an alginate bead for two weeks. A zone
of denatured protein (DP) spans the gap between dislodged dentine chip (DC). Diffuse chronic
inflammation (I) is present in the pulp. No preodontoblasts are evident under the gap however
predentine (PrD) formation can be seen on the adjacent dentine.
Fig. 19
Growth hormone-treated dog pulp at five weeks. There has been cellular organization around the
exposure site (E). Odontoblastic activity has attempted repair with new dentinal matrix (RD) joining
the dentinal chip (DC) to dentine, secondary dentine is evident on the surrounding dentine surface.
Glass ionomer cement restoration (GIC).
PLATE 12. (p126)
Fig. 20a+b.
Figure 20a.Dog pulp exposure treated with calcium hydroxide (CH) for two weeks. Pulp health is
good and odontoblastic activity (O) is uninhibited on the pulpal aspect of the dentine chips and
adjacent to the exposure. Plump, pre-odontoblasts (PO) have differentiated between the dentinal
chips (DC) and increased localized vascularity (V) can be seen subjacent to the new cells.
Figure 20b. Early bridging (RD) between dentinal chips (DC) in a calcium hydroxide treated pulp at
two weeks. Exposure (E), odontoblasts (O), calcium hydroxide (CH).
PLATE 13. (p127)
Fig. 21a+b.
Figure 21a. Dog pulp exposure treated with calcium hydroxide (CH) at five weeks. A continuous
bridge of reparative dentine (RD) extends well into the healthy pulp surrounding the capping agent.
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Exposure (E).
Figure 21b. Dog pulp exposure treated with calcium hydroxide (CH) at five weeks. A glass ionomer
cement (GIC) extrusion has evoked a small local inflammatory reaction (I) but no odontoblast or
dentine differentiation. Reparative dentine bridge (RD) is well formed.
PLATE 14. (p128)
Fig. 22
Calcium hydroxide treated pulp (5 weeks) Reparative dentine bridging (RD) has spanned the pulp
chamber and constricted vascularity. This has apparently resulted in necrosis (N) of the pulp tissue
(“strangulation necrosis”). Exposure (E), calcium hydroxide (CH).
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6.TABLES
TABLE 1. (p45)
ANOVA RESULTS : COMPARISON OF VOLUMETRIC DATA.
Significant interrelationships, established by analysis of variance, between the treatments and
days; tooth germs grown in serum-free medium (CONTROL), with the addition of foetal calf serum
(FCS).
Growth hormone 50 ng/ml treated germs, Growth hormone 100 ng/ml treated germs. Insulin-
like growth factor-I 100 ng/ml treated germs. Insulin-like growth factor-I 200 ng/ml treated
germs. NS - Not Significant.
TABLE 2. (p138)
INFLAMMATION, TREATMENTS AND TIME.
Inflammation and pulp-capping treatments compared over time (percentage of treatment and total
number (n)) in the dog model.
GH/IGF-1 - Growth hormone/insulin-like growth factor-1 combination, GH - Growth hormone, BMP -
Bone morphogenetic proteins 2 and 4.
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CHAPTER 1.
GROWTH HORMONE (GH) AND INSULIN-LIKE GROWTH FACTOR-1 (IGF-1):
ROLES IN DENTINOGENESIS
The presence of growth-promoting activities in an extract from the anterior lobes of bovine pituitary
glands was discovered in 1921 and human growth hormone, or somatotropin, was first isolated and
identified in 1954. Growth Hormone (GH) belongs to a family of polypeptide hormones and is
essential for normal growth and development of mammals. In particular it is required for structural
growth and maintenance of nitrogen, mineral, lipid and carbohydrate metabolism. The
somatomedin hypothesis predicted that the mitogenic effects of GH are not direct but rather indirect
effects mediated by somatomedins (Daughaday et al., 1972). Insulin-like Growth Factor-1 (IGF-1)
or somatomedin-C is a single chain polypeptide with a molecular weight of 7.6kDa that has a 47%
sequence homology with insulin. It is found in the plasma bound to carrier proteins (with levels
primarily controlled by GH), the liver, kidneys and fibroblasts. Its synthesis in foetal and adult
tissues is partially regulated by GH (Daughaday and Rotwein 1989). Schoenle et al. (1982) showed
that IGF-1 administration to hypophysectomized rats mimicked the effects of GH on important
indices of growth. Insulin-like growth factor-1 mediates GH in cell replication, differentiated function
in many tissues, synthesis of such substances as proteoglycans in cartilage (Hock et al., 1988) and
stimulates growth of fibroblasts in non-skeletal tissue (Zapf et al., 1978). Its receptors are found on
many cell types, such as endothelial cells and fibroblasts, with important roles in wound healing and
can interact with many cell types and tissue components to stimulate many wound-healing
responses. Wound fibroblasts produce high levels of IGF-1 in an autocrine fashion, which is more
biologically active than plasma IGF-1 (Spencer et al., 1988). Research combining platelet-derived
growth factor (PDGF) with IGF-1 as a treatment for hard and soft tissue chronic non-healing wounds
has shown improved healing brought about by the synergistic induction of a prolonged highly
controlled cascade of events (Lynch et al., 1989, Lynch et al., 1991) which stimulates recruitment
and proliferation of fibroblasts and increases collagen synthesis and maturation.
In several in vitro, serum-deprived systems, growth hormone (GH) and insulin-like growth factor-1
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(IGF-1) have been shown to induce proliferation and differentiation of cells equivalent to that
produced by serum-rich media (Smid, Steiner and Froesch 1984; McCarthy, Centrella and Canalis,
1989). There is evidence that the effects of GH are mediated by in vitro production of IGF-1 by the
cultured cells (Stracke et al.,., 1984; Ernst and Froesch 1988; Chenu et al.,., 1990). Many of the in
vivo effects of GH on cartilage and bone differentiation/growth are thought to be mediated by
circulating IGF-1 derived from the liver and under GH control (Daughaday 1989).
The dual effector hypothesis (Green, Morikawa and Nixon 1985) proposes that tissue growth
occurs in two stages with both GH and IGF-1 acting in a complementary fashion:
1. Differentiated cells are formed initially from their precursors by direct action of GH.
2. These differentiated cells proliferate by clonal expansion through the intermediating effector
IGF-1, acting as an autocrine/paracrine factor.
In adipocytes, GH differentiated cells were found to be much more sensitive to the mitogenic
effects of IGF-1 than the precursor stem cells. By itself IGF-1 had a small mitogenic effect on
precursor cells but no differentiation, while with GH there was a large mitogenic effect but mainly
on differentiating cells resulting in clonal expansion (Zezulak and Green 1986). Nilsson et al.
(1986) found the same pattern in cells involved in longitudinal bone growth.
Roles in odontogenesis and dentinogenesis
Circulating and autocrine/paracrine growth factors, and their specific receptors, interact in a
complex series of relationships resulting in inhibition, regulation, enhancement and stimulation,
which promote tooth bud growth and development. Thus, such factors exert epigenetic control of
odontogenesis in vitro and in vivo (Partanen and Thesleff 1989). Growth hormone has been shown
to increase cell proliferation and colony forming efficiency in cell cultures (Isaksson et al., 1987).
GH and GH receptor binding protein are detected at the tissue and developmental stages of control
shift from epithelium to mesenchyme (Joseph et al., 1994a) when the bone morphogenetic proteins
(BMP’s) change their expression. These BMP’s are important inductive morphogens in the epithelial-
mesenchymal interactions of the tooth germ differentiation, manifesting as a four to five-fold increase
in the BMP messenger ribonucleic acid (mRNA). Growth hormone was shown to induce this action
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even with inhibition of IGF-1. Some of the important actions of BMP in the induction of limb
patterning and morphogenesis may be the direct result of local GH action.
Growth hormone significantly restores dentine production in hypophysectomized rats, in contrast to
other pituitary hormones (Hansson, Stenstrom and Thorngren 1978 a,b ). Nakashima (1992b)
utilized bovine dental pulp cells to study the effects of growth factors such as platelet derived
growth factor (PDGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor
(bFGF), epidermal growth factor (EGF), transforming growth factor- (TGF-) and insulin-like
growth factor -I and II (IGF-I and -II) on DNA synthesis, proteoglycan synthesis and alkaline
phosphatase activity. The IGFs were shown to be potent mitogens for pulp cells (along with
PDGF, FGFs and EGF). They were shown to stimulate proteoglycan synthesis during the
proliferating, but not during the post mitotic stage of culture, thus its effects were dependent on
the degree of differentiation of the cells. Nakashima felt that proliferation of mesenchymal cells
was stimulated mainly by IGF-1 and PDGF and the production of extracellular matrix
proteoglycan may be enhanced by the IGFs and aFGF. However, he felt that TGF-, PDGF and
the FGFs were the possible regulators for the differentiation of pulp cells into odontoblasts.
Different combinations of growth factors result in transformations of cultured cells and this
transforming activity can be seen within the cultured teeth by their endogenous synthesis and
secretion of growth factors (Cam, Neuman and Ruch 1990).
Dentinogenesis is a complex, highly regulated process involving cell interaction and differentiation,
synthesis of organic matrix and formation of mineral crystals in the extracellular matrix. Growth
factors are proteins that influence processes such as cell recruitment and differentiation, amplify
cellular synthetic activities and thus have potential roles in dentinogenesis. In vivo, GH stimulates
DNA synthesis and mitotic activity in the odontogenic epithelia and mesenchyme of the developing
tip of the dwarf rat incisor (Young et al., 1992, 1993). Its role in odontogenesis has been reinforced
because dividing precursor cells are strongly reactive for GH-receptor, as are active odontoblasts
(Zhang, Young and Waters 1992).
Growth hormone may support the finite numbers of cell cycles necessary for terminal differentiation
of odontoblasts (Ruch 1990). Hansson et al., (1978 a,b) showed that GH could restore
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dentinogenesis in the incisor of hypophysectomized (HYPOX) rats while other pituitary hormones
had no effect.
Growth hormone has been shown to affect the population dynamics of the odontogenic epithelia with
lower indices of bromodeoxyuridine (BrdU) labelling, mitosis and cell compartment sizes in dwarf rat
incisors (Young et al., 1992). Labelling (BrdU) and mitotic indices of labial and lingual preodontoblast
cell populations was significantly lower in dwarf than normal rats These indices were returned
equivalent to normal after treatment with GH (Young et al., 1993). Growth hormone may have this
effect by its direct action on odontoblast differentiation and dentine matrix synthesis thus providing
mitogenic feedback to the preodontoblast population in the preodontoblast layer and dental papilla
(Young 1995). Studies of growth hormone receptor binding protein (GHrbp) and growth hormone
receptor (GHr) in the developing tooth (Joseph 1994c) reveal their presence on polarizing
preodontoblasts and on odontoblasts engaged in matrix formation (Zhang et al., 1992a, c). Zhang et
al., (1992a) demonstrated the presence of GH immunoreactivity in dividing cells, differentiating
preameloblasts and preodontoblasts and secretory ameloblasts and odontoblasts. Zhang felt GH
may influence cell proliferation, differentiation and differentiated function of cells independent of a
systemic IGF-1 mediator and may thus stimulate odontogenesis directly. GHrbp is not expressed
until there is polarization of the preodontoblasts and staining is absent once odontoblasts have
formed the full thickness of dentine (Zhang et al., 1992 c). Post-mitotic odontoblasts are sensitive to
GH stimulation of mRNA synthesis in HYPOX rats as measured by specific silver stained nucleolar
regions (Zhang et al., 1992b). GHrbp expression and IGF-1 immunoreactivity correlates identically
during foetal tooth development (Joseph et al., 1994a) and IGF-1 receptor is expressed strongly in
predentine as well as the odontoblasts (Joseph et al., 1994b). The growth factors present in non-
collagenous protein extracts from rabbit incisor dentine stimulate mesenchymal cells to elongate,
polarize and increase their metabolic activity (Lesot et al., 1986). Human dentine non-collagenous
protein extracts contain Transforming growth factor- (TGF-), insulin-like growth factor-1 (IGF-1)
and insulin-like growth factor-2 (IGF-2), although present in lower concentrations than in bone
(Finkelman et al., 1990).
A role for soluble growth factors in odontoblast differentiation has been suggested through
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immunohistochemical and/or in situ hybridization studies concerning the transforming growth factor -
family and the IGFs (Vaahtokari et al., 1991, Cam et al., 1992, Joseph et al., 1993). Within the
TGF superfamily are the BMPs (see below) which have important roles in development, epithelial-
mesenchymal interactions, stimulation of differentiated function and induction of expression of
transcription factors.
A study by Bègue-Kirn et al., (1994) showed growth factors combined with heparin (HN) had positive
differential effects on odontoblast-like cells in dental papillae on culture medium. TGF-1-HN in
combination induced gradients of cytological and functional differentiation, BMP-2-HN allowed some
polarized secretion while IGF-1-HN demonstrated extensive cytological differentiation without matrix
deposition. It was suggested that the lack of matrix deposition might be because the cells were not
able to express the TGF molecules. When IGF-1 was combined with BMP2 (without heparin)
initiation and propagation of odontoblast-like differentiation was seen and pulpal morphology was
well-maintained. TGF1 and IGF-1 together induced partial cytological and functional polarization
over extended areas and secretion of extracellular matrix. Interestingly, TGF1 and BMP2 did not
produce odontoblast-like cell differentiation. This study suggested the upregulation of msx2 (a
murine transcription factor) transcription and that members of the TGF superfamily are
prerequisites for terminal differentiation, polarization and apical accumulation of matrix by
odontoblasts. Physiologically this is triggered through a stage-specific inner dental epithelium via
matrix-mediated interactions. Ruch et al., (1995) have suggested that, in vivo, members of the TGF
superfamily are secreted by preameloblasts, trapped and activated by basement membrane-
associated components which initiate terminal differentiation of odontoblasts.
Insulin-like growth factor-1 (IGF-1) is known to be involved in the sulphation of the matrix
proteoglycans of cartilage and of dentine in vitro (Luyten et al., 1988; Nakashima 1992). It has been
shown in the dwarf rat, that GH, can regulate the production of an N-acetylgalactosamine (GalNAc)
rich matrix component in odontoblasts, predentine and other matrices, which may be a proteoglycan
or a glycoprotein necessary for normal tooth growth. Proteoglycans containing chondroitin and
dermatan sulfate and some glycoproteins have GalNAc as a principal component (Zhang et al.,
1994). This is the key sugar of the chondroitin-sulphate-rich proteoglycans, decorin and biglycan.
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Growth hormone modulates the expression of both decorin and biglycan (Zhang et al., 1995), thus
affecting matrix deposition in the rat tooth. Studies in the normal Lewis rat showed wide distribution
of decorin throughout the enamel organ, papilla and dental follicle of developing teeth while biglycan
was identified in predentine matrices. Dwarf Lewis rats with low circulating GH levels had markedly
decreased expression of both proteoglycans expression which was restored to almost normal levels
when growth hormone was administered (Zhang et al., 1995). It is probable that IGF-1 moderates
this dependency by its involvement in sulphation of predentine proteoglycans.
Extradental serum factors do affect early proliferation and perhaps influence the duration of the cell
cycle (Ahmad and Ruch 1987). Jowett and Ferguson (1991) have found that mouse molar tooth
buds explanted at 16 days and grown in chemically defined media (Yamada et al., 1980) showed
increases in area limited to the first four days in vitro. Thereafter expansion ceased. Bronkers,
Bervoets and Woltgens (1982) have found that such explants had a two-day lag phase before they
increased over two days and became static. The static phase was associated with the onset of
dentine synthesis. Both these sets of data indicate that volume increases of tooth buds in serum
free medium are, at best, minimal.
HYPOTHESIS
These considerations led to the hypothesis that GH and/or IGF-1, if substituted for the growth
factors present in foetal calf serum, would induce proliferation, growth and differentiation in mouse
molar tooth buds cultured in vitro. Chapter 2 is the report of the experiment to investigate this
hypothesis.
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CHAPTER 1. REFERENCES
Ahmad N and Ruch JV.
Comparison of growth and cell proliferation kinetics during mouse molar odontogenesis in vivo and in
vitro.
Cell Tissue Kinet 1987, 20:319-329.
Bronkers ALJJ, Bervoets TJM and Wöltgens JHM.
A morphometric and biochemical study of the pre-eruptive development of hamster molars in vitro.
Arch Oral Biol 1982, 27:831-840.
Cam Y, Neumann MR and Ruch JV.
Immunolocalization of transforming growth factor 1 and epidermal growth factor receptor epitopes
in mouse incisors and molars with a demonstration of in vitro production of transforming activity.
Arch Oral Biol 1990, 35:813-822.
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2
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29
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CHAPTER 2.
EXPERIMENT 1:-
COMPARISON OF THE EFFECTS OF GROWTH HORMONE, INSULIN-LIKE GROWTH
FACTOR-1 AND FOETAL CALF SERUM ON MOUSE MOLAR ODONTOGENESIS IN VITRO .
INTRODUCTION
In several in vitro, serum-deprived systems, growth hormone and insulin-like growth factor-I (IGF-I)
have been shown to induce proliferation and differentiation of cells equivalent to that produced by
serum-rich media (Smid, Steiner and Froesch, 1984; McCarthy, Centrella and Canalis, 1989). There
is evidence that the effects of growth hormone are mediated by in vitro production of IGF-I by the
cultured cells (Strake et al.,1984; Ernst and Froesch, 1988; Chenu et al.,1990). Many of the in vivo
effects of growth hormone on cartilage and bone differentiation and growth are thought to be
mediated by circulating IGF-I derived from the liver and under growth hormone control (Daughaday,
1989). Alternatively, growth hormone is considered to upregulate IGF-I production in growth
hormone-sensitive tissues, where IGF-I is thought to act as an autocrine/paracrine growth factor
(Nilsson et al.,1986). Thus, many of the effects of growth hormone or of IGF-I on cell cultures are
equivalent (Isaksson, 1987). However, no in vitro evidence exists that growth hormone increases
IGF-I synthesis in odontoblasts or that serum IGF-I can mimic the effects of growth hormone on
odontogenesis. This is despite the fact that IGF-I expression has been demonstrated in vivo by
immuno-histochemistry in the identical odontogenic cell populations that also express growth
hormone receptor/binding protein (Zhang, Young and Waters, 1992; Zhang et al.,1992; Joseph et
al.,1993). Moreover, Ferguson et al., (1992) have found increased reaction for IGF-I with a
monoclonal antibody in the epithelia and mesenchyme of developing molar tooth germs and for IGF-
I binding protein in the enamel organ and dental papilla mesenchyme.
Odontogenesis in mouse molars removed from 16 day old foetuses proceeds, during organ culture
in foetal calf serum-containing media, to the differentiation of polarised ameloblasts and functional
odontoblasts producing dentine matrix, after 6 days in culture (Ahmad and Ruch, 1987). In vitro,
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odontoblasts and ameloblasts are sustained towards terminal differentiation in an equivalent
temporal sequence to the in vivo state (Ahmad and Ruch, 1987). Culture of 16 day molars in
chemically-defined medium (Yamada et al.,1980) without foetal calf serum, has shown that ascorbic
and retinoic acids are required for dentinogenesis and crown morphogenesis in tooth germs (Amar,
Fabre and Ruch, 1992; Mark, Bloch-Zupan and Ruch, 1992). No data which specifically contrasts
the growth of 16 day molars cultured in serum-rich and serum-free media with growth factor
supplementation are available for this system. Accordingly, this study compared the size (volume),
mitotic indices, cell densities of the dental papillae and the degree of cytodifferentiation of 16 day
molars over 4-6 days in media in the presence or absence of foetal calf serum, IGF-I or growth
hormone to provide evidence that these growth factors could contribute to the ability of serum to
sustain tooth germ development.
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MATERIALS AND METHODS
The mandibular first molar tooth germs of foetal Swiss mice were removed at 16 days post-
conception (vaginal plug = day 0) and cultured in groups of six for 4, 5 and 6 days respectively. At
16 days, the germs are in late cap or early bell stage and morphogenesis of the cusps is just
beginning (Ahmad and Ruch, 1987; Jowett and Ferguson, 1991). All tooth germs were cultured on
semi-solid medium comprising essential medium, (RPMI-1640) ascorbic acid (180 µg/ml), L-
glutamine (2 mM), kanamycin (100 µg/ml) and retinoic acid (all-trans, Sigma, 1.5 x 10-7 M) to which
0.4% agar was added. All media were supplemented with ascorbic and retinoic acids because their
absence is known to influence odontogenesis adversely in this system (Amar, Fabre and Ruch 1992;
Mark, Bloch-Zupan and Ruch 1992). Transferrin was not added, as there is sufficient endogenous
transferrin in the germs, at 16 days post conception, to allow growth (Mark, Bloch-Zupan and Ruch,
1992) [in contrast to cultured 14 day foetal molars (Partanen et al.,1984; Cam, Boukari and Ruch,
1989)].
One control group of six germs was cultured in serum-free medium. The medium for the second
group contained 20% foetal calf serum. Recombinant bovine growth hormone (rbGH Monsanto)
was added to serum-free medium for the next two groups at concentrations of 50 and 100 ng/ml.
Recombinant insulin-like growth factor 1 (rIGF-I Genentech) was added to serum-free media of two
further groups at concentrations of 100 and 200 ng/ml. Cultures were incubated at 37oC, in the dark,
in an atmosphere of 5% carbon dioxide. The medium was changed every second day. After 4, 5 or
6 days of culture, the six tooth germs of each treatment group were fixed in Bouin-Hollande solution,
embedded in paraffin by standard procedures, serially sectioned at 5 µm and stained with Mallory's
phosphotungstic acid-haematoxylin.
The volume of each tooth germ was accurately estimated by digitizing (Houston Instrument Hipad
Plus) the entire circumference of each serial section (as per Ahmad and Ruch (1987) and unlike
Jowett and Ferguson (1991) who digitized twelve sections per germ). The sum of the areas,
multiplied by the average thickness of the sections (5 µm) gave the volume expressed in cubic
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millimetres (mm3.). The areas were recorded blind, by coding and randomising the slides, and the
code was broken for tabulation of the data. Significant differences between mean tooth germ
volumes were sought within and between treatments by one-way analysis of variance (ANOVA).
To compare the mitotic activity in the tooth germs, between treatments, mitotic figures (metaphase,
anaphase and telophase) were counted in the inner odontogenic epithelium from the cervical loop
along the epithelial mesenchymal interface up to fifty cells or until dentine matrix was apparent. At
least three hundred cells per tooth germ were counted from the region of greatest sectional area on
three germs per treatment day. To avoid counting the same mitotic figures twice, alternate sections
were counted which allowed a minimum of 10m between sections. The number of mitotic figures,
per total cells counted, was recorded for each treatment and was expressed as mean mitoses per
hundred cells (or mean mitotic index - MMI). These indices were subjected to analysis of variance
(ANOVA).
Cell densities were measured within the dental papillae of cultured tooth germs in longitudinal section
by counting the cells within a 0.1mm band along the longest vertical section of each papilla from the
odontoblast layer to the apical edge of the tooth germ. A projection microscope (Leitz-Wetzlar XI-C)
projected sections at a magnification of 470X. Counts were performed on five sections, a minimum
of 10m. apart, in the region of greatest cross-sectional areas ensuring maximal papilla inclusion on
each tooth germ. The sections counted were from Day 16+6 and were only included if they showed
an intact, artefact-free longitudinal section. Data was expressed as cells per 0.1mm square,
standard deviation and standard error of the mean were calculated and were compared between
treatments by one way analysis of variance (ANOVA).
To compare the degree of differentiation of the germs, an arbitrary rating system was devised based
around qualitatively appreciable differences in cytodifferentiation of both the odontoblastic layer of
the dental papilla and the inner enamel epithelium, utilising the following criteria (illustrated in Plate
1).
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PLATE 1.
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3
RATING 1: Absence of polarisation of cells of the dental papilla subadjacent to the inner enamel
epithelium and an inner enamel epithelium comprised of cuboidal or low columnar
cells with round or oval nuclei (Fig. 1).
RATING 2: Polarising odontoblasts were identifiable as low columnar cells of the dental papilla
whose nuclei were polarised away from the basement membrane. The inner
enamel epithelium comprised low columnar cells with oval or fusiform nuclei at
different levels in a pseudostratified arrangement (Fig. 2).
RATING 3: Odontoblast differentiation was evident with the formation of unequivocal dentinal
matrix. The odontoblast layer was well defined with polarisation of the oval nuclei
away from the forming matrix. The nuclei of the preameloblasts were fusiform and
this layer had a pseudostratified columnar configuration (Fig. 3).
RATING 4: Where sufficient dentine matrix formation had taken place, a transformation
occurred in the preameloblasts to tall columnar cells with fusiform nuclei at a level
towards the proximal pole (Fig. 4).
A rating of zero was assigned to germs in which degenerative changes or absence of tissue
precluded rating of the degree of differentiation.
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plate 2.
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RESULTS
Overview
Volume differences and qualitative differences in the degree of differentiation of the odontogenic
epithelium, were observed both within and between treatments. Differences were also seen at the
epithelial-mesenchymal interface, in dentine production and in the appearance of the dental papilla.
An overview of these differences can be gained from Plate 2, Figure 5. This illustrates
representative sections from one germ after each of the treatments, taken after six days of culture
from the level of greatest area of that germ. Thus the sections represent relative area differences
with treatments. These sections show that dentine differentiation and growth did not occur to any
significant extent in the absence of foetal calf serum (Fig. 5). Foetal calf serum supplementation
produced larger germs in which crown morphogenesis and dentine differentiation occurred to a
greater extent (Fig. 5). It is evident that dentine differentiation occurred with growth hormone
present, however smaller germ volumes were seen than those treated with foetal calf serum or IGF-I
(Fig. 5). In addition, most of the germs treated for 6 days with growth hormone showed a closely
packed distribution of cells in the papillae. This was rarely seen with any other treatment (Fig. 5).
Germs treated with IGF-I were characterised by sections of greatest area, with dental papillae of
similar cell distribution to foetal calf serum-treated germs and by high ratings of differentiation,
characterised by the presence of dentine and polarisation of the nuclei of the inner enamel
epithelium (Fig. 5).
Volumes
The changes in mean tooth germ volumes, with time, and between treatments, are illustrated in
Figure 6a. The tooth germs grown in the absence of foetal calf serum showed no appreciable
growth over the last three days of treatment (mean volume at 4 days 0.02686 mm 3, at 5 days
0.02686 mm3 and at 6 days 0.02727 mm3, SD 0.000236). Although larger at 4 days, germs grown in
the presence of 20% foetal calf serum did not continue to enlarge either (mean volume at 4 days
0.08631 mm3, at 5 days 0.09156 mm3 and at 6 days 0.07154 mm3, SD 0.0138). This was also the
case in both growth hormone treated groups (GH:50 ng/ml, mean volume at 4 days 0.05319 mm3, at
38
3
5 days 0.04139 mm3, and at 6 days 0.04668 mm3, SD 0.0059 and GH:100 ng/ml 0.0483 mm3 at 4
days, 0.0513 mm3 at 5 days and 0.04806 mm3 at 6 days, SD 0.0017). Volume changes within these
treatments were not significant.
In contrast, the IGF-I-treated germs showed strong growth over the three days (IGF-I:100 ng/ml
mean volume at 4 days 0.0457 mm3, 5 days 0.06027 mm3 and 6 days 0.1055 mm3, SD 0.0312;
IGF-I:200 ng/ml mean volume at 4 days 0.09699 mm3, at 5 days 0.11865 mm3 and 6 days 0.14858
mm3, SD 0.0259). Both of the IGF-I-treated groups showed significant changes between days
(ANOVA). Germs treated with IGF-I:100 ng/ml had a mean volume, at 6 days, which was greater
than those at 5 and 4 days (p<0.05 and 0.01 respectively), while the mean volume of IGF-I:200
ng/ml treated germs at 6 and at 5 days were significantly greater than at 4 days (p<0.05) in both
instances.
Analysis of variance, within days, (Table 1 p45) revealed that germs which received IGF-I treatment
(200 ng/ml), by 4 days, were significantly larger than those receiving all treatments, barring foetal calf
serum. Foetal calf serum-treated germs grew significantly larger than those in serum-free medium,
both growth hormone groups and, initially, IGF-I:100 ng/ml-treated germs at day 4. The lower dose
IGF-I:100 ng/ml supplanted foetal calf serum at 6 days to be significantly larger than the serum-free
control and both growth hormone groups. In this system it is evident that IGF-I, at both
concentrations, produced significant increases in volume growth.
Mitotic Activity
The means and standard error means for each treatment are shown in Figure 6b. The serum-free
treatment control group showed minimal mitotic activity over the three days of treatment (mean
mitotic index (MMI) 0.2, SD 0.178). Significantly higher mitotic indices than the control group were
recorded for the foetal calf serum and both GH-treated groups, which were not, however,
significantly different from one another (FCS MMI 1.88, SD 0.485; GH:50ng/ml 1.61, SD 0.35;
GH:100ng/ml 1.42, SD 1.04). In both IGF-I-treated groups mitotic indices of less than one were
recorded (IGF-I:100ng/ml MMI 0.68, SD 0.626; IGF-I:200ng/ml 0.97, SD 0.478).
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4
It appears that in this system GH but not IGF-I produces similar mitotic activity to that found in the
foetal calf serum treated germs.
PLATE 3. Overleaf
Fig. 6 Histograms of the effects of treatments on mice tooth germs with days of treatment, 16
days post conception (vaginal plug = day 0 ) 4, 5, 6 days. Bars represent Standard Errors of
the Means.
6(a). Effects on the average volumes of tooth germs: The serum-free treated germs and both
growth hormone treated groups showed little volumetric change over the three days of
sampling. Foetal calf serum-treated germs showed a comparatively high initial volume (Day
16+4) but show no significant change over the subsequent days. Both insulin-like growth
factor-I treated groups show volumetric increases over the three days, with IGF-I 200 ng/ml
treated germs having greatest average volumes on all three days.
6(b). Effects on mitotic indices in odontogenic epithelium between treatments. Mitotic activity
was highest in the foetal calf serum and in the growth hormone treated-groups which were
significantly higher than the serum-free group. Values for the IGF-I treatments were intermediate.
Differences between days, within treatments were not significant (data not shown).
6(c). Effects on cell density within the dental papillae on treatment day 16+6: Significantly
higher cell density was recorded in the growth hormone-treated low dose group than for all
other treatment groups with the exception of the GH high dose group. Although marginally
less dense than all other groups, IGF-I high dose treatment group was not significantly
different from serum-free or serum-supplemented treatments.
-FCS - Serum-free, +FCS - Foetal Calf Serum, GH 50 (GH1) - Growth Hormone 50 ng/ml,
GH 100 (GH2)- Growth Hormone 100 ng/ml, IGF-I 100 (IGF1) - Insulin-like Growth Factor-I 100 ng/ml,
+IGF-I 200 (IGF2)- Insulin-like Growth Factor-I 200 ng/ml.
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4
Plate 3
41
4
Cell densities in dental papillae
The relative cell densities in the dental papillae of the tooth germs from the different treatment
groups on Day 16+6 are presented in Fig. 6c.
Tooth germs grown in the presence of foetal calf serum showed similar cell density in the dental
papilla to those grown in the serum free control (+FCS 91.58 and -FCS 92.1 mean cells per 0.1mm2,
SD 4.15 and 4.05 respectively) and were denser than either of the tooth germs exposed to insulin-
like growth factor-I (IGF-I 100 ng/ml 86.84 and IGF-I 200 ng/ml 76.31 mean cells per 0.1mm 2, SD
3.44 and 2.77 respectively).
The growth hormone treated tooth germs showed the highest cell densities with GH 50ng/ml at
126.8 mean cells per 0.1mm2 and GH100 ng/ml at 117.9 (SDs 9.68 and 2.52).
The one way analysis of variance (ANOVA) showed significant variance between both GH groups
and the IGF-I treated tooth germs (p values <0.01-0.05) while only the low dose GH group (GH 50
ng/ml) showed a significant increase in density over the foetal calf serum treated cultures (p<0.05).
Thus GH and IGF-I appear to exert different influences on molar tooth germs in vitro to produce
higher cell densities after six days of GH treatment compared to IGF-I. However, the effects of IGF-I
did not appear to vary from those of foetal calf serum.
Differentiation
The results of the ratings ascribed to the 84 cultured tooth germs are shown diagrammatically in
Figure 7. Absence of tissue or degenerative change excluded nine germs (rated 0). Ratings of the
remaining 75 germs showed that germs grown on serum-free medium did not form predentine by
four days of culture, and never attained preameloblast polarisation (rating 4). In the presence of
growth hormone, predentine production was achieved by at least one germ by four days of culture,
however rating 4 was only achieved by two germs at six days of culture in the presence of the higher
dose [GH 100 ng/ml]. The frequency of rating 3 and 4 in germs cultured in the presence of GH 100
ng/ml was essentially the same as that achieved when 20% foetal calf serum was added to the
medium. All IGF-I-treated germs produced predentine by 5 days and the incidence of preameloblast
42
4
polarisation (rating 4) was increased over all other treatments, such that rating 4 was even recorded
in two out of six germs treated with IGF-I 200 ng/ml at 4 days. By five days, all germs, bar one, had
achieved rating 4 in the IGF-I 100 ng/ml treatment group. Preameloblast polarisation was seen in
the six IGF-I 200 ng/ml-treated germs at five days and in all IGF-I treated germs at six days. Thus,
in this system IGF-I induced the greatest degree of differentiation while growth hormone was
equivalent to foetal calf serum in this regard.
PLATE 4. Overleaf
Fig. 7 This diagram represents the ratings assigned to mouse molar tooth germs according to their degrees of
differentiation with time and treatment (cf Fig. 1-4). The trend towards greater differentiation with time within treatment
groups is evident. The serum-free control germs were, in general, the most poorly differentiated. Growth hormone 100
ng/ml and foetal calf serum 20% treated germs showed similar differentiation over time. The insulin-like growth factor-I
groups showed the earliest differentiation with IGF-I 200 ng/ml treatment demonstrating three germs producing dentine
(Rating 3) and two germs with polarised preameloblasts (Rating 4) at Day 16+4.
-FCS - Serum-free treated germs; +GH - Growth Hormone treated germs; +FCS - Foetal Calf Serum
treated germs and +IGF - Insulin-like Growth Factor-I treated germs.
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4
plate 4.
44
4
TABLE 1. Anova results summary: a comparison of volumetric data.
F.C.S. GH 50 ng/ml GH 100 ng/ml IGF 100 ng/ml IGF 200 ng/ml
-FCS 4d **
5d **
NS
NS
NS
NS
NS
NS
NS
NS
NS
6d **
4d ***
5d ***
6d ***
F.C.S. 4d *
5d **
NS
4d **
5d *
NS
4d *
NS
NS
NS
NS
6d **
GH 50 ng/ml NS
NS
NS
NS
NS
6d **
4d **
5d ***
6d ***
GH 100 ng/ml NS
NS
6d **
4d ***
5d ***
6d ***
IGF 100ng/ml 4d ***
5d **
6d *
Bonferroni p values p <0.001 *** extremely significant
p <0.01 ** highly significant
p <0.05 * significant
NS not significant
TABLE 1. ANOVA RESULTS SUMMARY: A COMPARISON OF VOLUMETRIC DATA.
Significant interrelationships, established by analysis of variance, between the treatments and days; tooth germs grown in serum-
free medium (CONTROL), with the addition of foetal calf serum (FCS). Growth hormone 50 ng/ml treated germs, Growth
hormone 100 ng/ml treated germs. Insulin-like growth factor-I 100 ng/ml treated germs. Insulin-like growth factor-I 200 ng/ml
treated germs.
NS - Not Significant.
45
4
DISCUSSION
This study shows that, in an in vitro model of odontogenesis, growth hormone and IGF-I induce
proliferation and differentiation at least equivalent to that produced in serum-rich medium. Omission
of foetal calf serum from the medium in this system clearly demonstrated a lack of sustained growth
as indicated by lack of mitotic activity, germs of small volume and of poorest differentiation over the
six days of culture (Figures 6 and 7). Ruch (1990) has proposed that a finite number of cell cycles is
necessary prior to post-mitotic terminal differentiation of odontoblasts and ameloblasts. Accordingly,
the difference in mitotic activity in tooth germs grown in serum-free as compared to serum, hormone
or growth factor-rich media could account for the lack of differentiation produced by this treatment.
Specifically, the lack of differentiation of dentinal matrix with this treatment suggests that factors in
foetal calf serum are essential for the synthesis of the matrix and that growth hormone and IGF-I
enhance matrix differentiation.
We have previously demonstrated in vivo that growth hormone stimulates DNA synthesis and mitotic
activity in the odontogenic epithelia and mesenchyme of the developing tip of the dwarf rat incisor
(Young et al.,1992, 1993). In vitro, growth hormone added to the serum-free medium supported
mitotic activity equivalent to that produced by foetal calf serum (Fig.6b). However, this level of mitotic
activity was not accompanied by a significant increase in the volume of the growth hormone-treated
tooth germs over the six days of culture (Fig. 6a). The finding that dental papillae of growth
hormone-treated germs had higher densities of closely-packed mesenchymal cells than any of the
other treatments (Fig. 5 and Fig. 6b) may explain this apparent contradiction. Interestingly, Ahmad
and Ruch (1988) have found that cell density is also higher than normal when 10% FCS is used to
support tooth germ growth in vitro, in contrast to the in vivo situation, where the density of dental
papilla cells decreases significantly from day 15 to day 24. This implies that although 10% foetal calf
serum, or growth hormone, in the medium support production of dental papilla cells, these cells do
not elaborate sufficient extracellular matrix to disperse them in the pattern associated with the fully
developed dental papilla of the early bell stage in vivo. This pattern, however, was achieved in 20%
foetal calf serum and in both IGF-I treated groups in this study. It is curious that lower mean mitotic
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indices were recorded in the IGF-I treatment groups and further measures of cell population
dynamics would be required to determine the detailed role of both growth hormone and IGF-I in cell
proliferation in vitro.
These studies have not resolved the question as to whether in vivo or in vitro increases in cell
replication are a direct effect of growth hormone, an effect of the growth hormone-dependent
somatomedin action of liver-derived IGF-I, or an effect of locally upregulated IGF-I (or other local
paracrine growth factors). A greater number of the growth hormone-treated germs achieved
terminal differentiation of ameloblasts and odontoblasts (rating 4) than germs grown in serum free
medium (Fig. 7). This degree of differentiation was equivalent to that produced by foetal calf serum
by Bronkers et al., (1982) and in this study. This implies that growth hormone can support the finite
numbers of cell cycles necessary for terminal differentiation (Ruch, 1990) and then enhance
differentiation of dentine matrix. Alternatively, growth hormone may be capable of upregulating
sufficient local IGF-I in vitro which, in turn, enhances matrix synthesis. By quantitative
immunohistochemistry growth hormone has been shown to upregulate local IGF-I expression in
odontoblasts of dwarf rats in vivo (Joseph et al.,1993) but this has not so far been confirmed by in
situ hybridization.
It should be noted that the addition of unbound IGF-I, at the doses employed in this study, does not
simulate the physiological action of humoral, bound IGF-I or of growth hormone-upregulated local
IGF-I production in vivo. In this system, the effects of IGF-I were significantly greater in terms of
tooth germ volume increases (Fig. 6) and significant enhancement of differentiation (Fig. 7)
compared to the growth hormone and foetal calf serum treatments. However, mitotic activity induced
by IGF-I was not as high as that found for growth hormone or foetal calf serum and the density of
cells in the dental papillae was the lowest for all treatments. These findings together suggest that the
effects of IGF-I were most appreciable as gross increases in tooth germ size due to increased
differentiation of dental papilla cell matrix and dentine matrix production.
These differences observed between the effects of growth hormone and IGF-I in vitro are possibly
due to a number of factors. The doses of both used in vitro were not physiological as growth
hormone and IGF-I exert their physiological effects at picogram levels and their affinities may change
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when bound to extracellular matrices. Luyten et al., (1988) has suggested that most IGF-I present in
the tissues is derived from the circulation. Paracrine effects of IGF-I are influenced by its short half-
life in the extracellular space and by its association with extracellular matrix and various growth-
factor-binding proteins (Pusztal et al., 1993). Thus if, in vitro, IGF-I was produced locally in response
to growth hormone, it may be insufficient to mimic the physiologic growth response to serum IGF-I.
The supply of IGF-I (100 or 200 ng/ml) in the unbound form, may have produced effects on cells
primed prior to explantation by in vivo growth hormone (Zezulak and Green, 1986; Cook, Haynes
and Werther, 1988).
Some of the in vitro effects of IGF-I on differentiation in this system may be explained by the known
involvement of IGF-I in the sulphation of matrix proteoglycans of cartilage (Luyten et al., 1988) and
of dentine in vitro (Nakashima, 1992). We have shown that growth hormone in the dwarf rat can
influence the production of N-acetyl galactosamine in predentine (Zhang et al.,., 1994). N-
acetylgalactosamine is the key sugar of the chondroitin-sulphate-rich proteoglycans, decorin and
biglycan. Further, we have shown that the expression of the core proteins of both decorin and
biglycan are growth hormone-dependent in the predentine matrix of the dwarf rat (unpublished). It is
probable that IGF-I moderates this growth hormone dependency by its involvement in sulphation of
predentine proteoglycans, thus explaining the marked differentiation of dentine observed after IGF-I
treatments in vitro.
This study has shown that both growth hormone and IGF-I have effects on developing tooth germs
in vitro and on the cells of odontogenesis therein. Growth hormone appears to affect odontogenic
cell proliferation and subsequent differentiation equivalent to foetal calf serum. Insulin-like growth
factor-I strongly promotes the differentiation and development of odontoblasts and their differentiated
cell functions in the form of dentinal matrix formation as well as promoting significant volumetric
growth. Studies are being pursued to determine the role of IGF-I in matrix production in isolated
dental papillae compared with other growth factors (Bègue-Kirn et al.,1992).
CHAPTER 2. REFERENCES
Ahmad N and Ruch JV.
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Comparison of growth and cell proliferation kinetics during mouse molar odontogenesis in vivo and in
vitro.
Cell Tissue Kinet 1987, 20, 319-329.
Ahmad N and Ruch JV.
Mouse molar cell proliferation kinetics in vivo and in vitro.
Bulletin de l'Association des Anatomistes 1988, 72, 3-13.
Amar S, Fabre M and Ruch JV.
Effects of ascorbate-deficiency on collagen secretion and resorption in cultured mouse incisor
germs.
Connective Tiss Res 1992, 28, 125-142.
Bègue-Kirn C, Smith AJ, Ruch JV, Wozney JM, Purchio A, Hartmann D and Lesot H.
Effects of dentine proteins, transforming growth factor 1 (TGF1) and bone morphogenetic protein
2 (BMP2) on the differentiation of odontoblasts in vitro.
Int J Dev Biol 1992, 36, 491-503.
Bronkers ALJJ, Bervoets TJM and Wöltgens JHM.
A morphometric and biochemical study of the preeruptive development of hamster molars in vitro.
Arch Oral Biol 1982, 27, 831-840.
Cam Y, Boukari A and Ruch JV.
Stimulating effect of transferrin on proliferation of mouse odontoblasts and preameloblasts in organ
culture.
Arch Oral Biol 1989, 34, 153-159.
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Chenu C, Valentin-Opran A, Chavassieux P, Saez S, Meunier PJ and Delmas PD.
Insulin-like growth factor-I hormonal regulation by growth hormone and by 1.25 (OH2) D3 and activity
on human osteoblast-like cells in short term cultures.
Bone 1990, 77, 81-86.
Cook J, Haynes KM and Werther GA..
Mitogenic effects of growth hormone in cultured human fibroblasts evidence for action via local IGF-I
production.
Journal Clin Invest 1988, 81, 206-212.
Daughaday WH.
Somatomedins: A new look at old questions. In: LeRoith D, Raizada MK (ed) Molecular and cellular
biology of insulin-like growth factor and their receptors.
Plenum Press, New York and London, 1989 1-4.
Ernst M and Froesch ER.
Growth hormone-dependent stimulation of osteoblast-like cells in serum-free cultures via local
synthesis of IGF-I.
Biochem Biophys Res Comm 1988, 151:142-7.
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Ferguson MWJ, Sharpe PM, Thomas BL and Beck F.
Differential expression of insulin-like growth factors I and II(IGF-I and II) mRNA, peptide and binding
protein I during mouse palate development:comparison with TGFB peptide distribution.
Journal of Anatomy 1992, 181:219-238.
Isaksson OGP, Isgaard J, Nilsson A, Lindahl A.
Direct actions of growth hormone.
In: Bercu B (ed) Basic and Clinical Aspects of growth hormone. Serono Symposia, Plenum Press,
New York, 1988, 199-211.
Joseph BK, Savage NW, Young WG, Gupta GS, Breier BM and Waters MJ.
Expression and regulation of Insulin-like growth factor-I in the rat incisor.
Growth Factors 1993, 8:267-275.
Jowett AK and Ferguson MWJ.
Morphometric analysis of the developing murine molar tooth in vivo and in vitro.
J Anat 1991, 177:135-144.
Luyten FP, Hascall VC, Nissley SP, Morales TI and Reddi AM.
Insulin-like growth factors maintain steady-state metabolism of proteoglycans in bovine articular
cartilage explants.
Arch Biochem Biophys 1988, 267:416-425.
McCarthy TL, Centrella M, and Canalis E.
Regulatory effects of insulin-like growth factors I and II on bone collagen synthesis in rat calvarial
cultures.
Endocrinology 1989, 124:301-309.
Mark MP, Bloch-Zupan A and Ruch JV.
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Effects of retinoids on tooth morphogenesis and cytodifferentiation in vitro.
Int J Dev Biol 1992, 36:517-526.
Nakashima M.
The effects of growth factors on DNA synthesis, proteoglycan synthesis and alkaline phosphatase
activity in bovine dental pulp cells.
Arch Oral Biol 1992, 37:231-236.
Nilsson A, Isgaard J, Lindahl A, Dahlström A, Skottner A and Isaksson O.
Regulation by growth hormone of number of chondrocytes containing IGF-I in rat growth plate.
Science 1986, 233:571-574.
Partanen AM, Thesleff I and Ekblom P.
Transferrin is required for early tooth morphogenesis.
Differentiation 1984, 27:59-66.
Pusztal L, Lewis CE, Lorenzen J and McGee J.
Growth factors: Regulation of normal and neoplastic growth.
Journal of Pathology 1993, 169:191-201.
Ruch JV.
Patterned distribution of differentiating dental cells: facts and hypotheses.
J Biol Buccale 1993, 18:91-98.
Smid JR, Steiner T and Froesch ER.
Insulin-like growth factor-I supports differentiation of cultured osteoblast-like cells.
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FEBS Lett, 1984 173, 1.
Stracke H, Shultz A, Moeller D, Rossol S and Shatz H.
Effect of growth hormone on osteoblasts and demonstration of somatodmedin C (IGF-I) in bone
organ culture.
Acta Endocrinologica 1984, 107:16-24.
Yamada KM, Bringas P, Grodin M, MacDougal M, Cummings E, Grimmett J, Weliky B and Slavkin
HC.
Chemically defined organ culture of embryonic mouse tooth germs, morphogenesis, dentinogenesis
and amelogenesis.
Journal de Biologie Buccale 1980, 8:127-139.
Young WG, Zhang CZ, Li H, Osborne P and Waters MJ.
The influence of growth hormone on cell proliferation in odontogenic epithelia by bromodeoxyuridine
immunocytochemistry and morphometry study in the Lewis dwarf rat.
J Dent Res 1992, 71:1807-1811.
Young WG, Zhang CZ, Li H, Lobie PE and Waters MJ.
Cell proliferation in odontogenic mesenchyme is influenced by growth hormone:
A bromodeoxyuridine immunocytochemistry and morphometry study in the Lewis dwarf rat.
Arch Oral Biol 1993, 93:207-214.
Zezulak KM and Green H.
The generation of IGF-I sensitive cells by Growth Hormone action.
Science 1986, 233:551-553.
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Zhang CZ, Young WG and Waters MJ.
Immunocytochemical localization of growth hormone receptor in rat maxillary teeth.
Arch Oral Biol 1992, 37:77-84.
Zhang CZ, Young WG, Li H, Garcia-Aragon J, Clayden AM and Waters MJ.
Expression of growth hormone receptor by immunocytochemistry in rat molar root formation and
alveolar bone remodelling.
Calcif Tiss Inter 1992, 50:541-546.
Zhang CZ, Young WG, Breipohl W, Doehrn S, Li H and Waters MJ.
Growth hormone regulates an N-acetylgalactosamine component in odontogenesis : a specific
lectin-binding study in the Lewis dwarf rat.
J Oral Pathol Med 1994, 23:193-199.
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CHAPTER 3
INTRODUCTION TO EXPERIMENT 2
The results from the first experiment in this thesis encouraged planning of a second experiment
designed to investigate the efficacy of GH and IGF-1 in stimulating the formation of reparative
dentine in the dog pulp-capping model. If GH and IGF-1 are capable of inducing proliferation,
differentiation and dentine formation by potential odontoblasts, as experiment 1 suggests (Young
et al., 1995), they may have potential for stimulating in vivo dentine repair.
It was decided to trial the growth factors in two forms. Growth hormone would be utilized by itself
for its mitogenic effects and in an attempt to prime pulpal cells to be more sensitive to circulating
IGF-1 action (Zezulak and Green 1986). A combination of IGF-1 and GH would also be used for
IGF-1’s role in the development and differentiation of odontoblasts and its mitogenic effects. This
was done with the knowledge that IGF-1 has a short half-life in the extracellular space; it also
associates with extracellular matrix and various growth factor- binding proteins (Pustzal et al.,
1993).
The literature on healing of the pulpo-dentinal complex following exposure was reviewed and the
factors influencing this response, particularly the importance of inflammation and the role of
bacteria. In addition, the agents traditionally used for pulp-capping, calcium hydroxide and a
corticosteroid/antibiotic combination, were reviewed for consideration as baseline comparisons.
Calcium hydroxide has been considered a successful capping agent since the 1920’s and is
known for commonly eliciting reparative dentine formation following placement on the dental pulp
(Foreman and Barnes 1990). Corticosteroid-antibiotic combinations, such as the Ledermix
compounds, have proved efficacious in the relief of clinical symptoms (Baume and Fiore-Donno
1970) but are less successful in producing pulpal healing (Langeland et al., 1977) when
evaluated histologically.
Non-setting medicaments were used in order to maximize contact with the pulp at the exposure
site and thus optimize the release of their active ingredients. The use of pastes also minimized
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the exposure of the pulp to extraneous and potentially irritating ingredients seen in cement type
medicaments, such as eugenol. By avoiding mixing two-part treatments the potential for
variations in the active ingredients exposed to the pulp were minimized.
Thus calcium hydroxide (Calxyl Blue®) was proposed as the positive, and Ledermix paste the
negative, histological controls for pulpo-dentinal healing. The bone morphogenetic proteins were
also included as they have important roles in normal growth and development, secondary
induction and terminal differentiation of cells and roles in odontogenesis and have shown success
as capping agents in animal models (Lianjia et al., 1993, Rutherford et al., 1993, 1994,
Nakashima 1994b). There appears to be growing evidence that coordinated effects of growth
factors are required for the induction and functional differentiation of odontoblasts in stage
specific patterns (Heikinheimo 1994). GH and IGF-1 increase the expression of BMP2 and BMP4
mRNA in cultured pulp fibroblasts and this suggests that BMP may mediate some of the local
actions of GH and IGF-1 (Li et al., 1998).
All these agents, excluding GH and IGF-1, had previously been utilized as capping agents in the
dog model. The dog model provides large teeth, with easy access for fine operator movement,
for normal instrumentation and for ease of material placement.
Also investigated were possible vehicles for pulp-capping with growth factors, required because
of the factor’s high in vivo solubility. A pilot study (unpublished) in the dog model had revealed
that carbopol gels were unsuitable for use as carriers of the growth factors, because of its
tendency to disperse through the pulp. A solid carrier of alginate coated with calcium chloride was
utilized because of its biocompatibility, handling characteristics, ease of manufacture and proven
release characteristics (Prankerd unpublished). Poly-maleinate glass ionomer cement was
chosen as the restorative cover because of its sealing ability to minimize microleakage (Kaplan et
al., 1992), its ability to be placed with minimal pressure and its compatibility with the pulp
(DeGrood et al., 1995).
HYPOTHESIS
The hypothesis for experiment 2 was that GH alone, or in combination with IGF-1 would have
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biological advantages over the traditional pulp-capping agents because of their roles as naturally
occuring components of growth and repair processes. It was proposed that these factors would
stimulate pulpal cell differentiation and function, resulting in the formation of dentine bridging at the
wound site, comparable to calcium hydroxide and produce pulpal health superior to
corticosteroid/antibiotic therapies. Bone morphogenetic proteins were also trialled for comparison.
The rationale for use of pulp-capping agents and their evaluation is reviewed in Chapter 4 and the
second experiment is dealt with in Chapter 5.
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CHAPTER 4
REVIEWS
REVIEW I. HEALING OF THE PULPO-DENTINAL COMPLEX FOLLOWING EXPOSURE
A. DENTINAL FACTORS INFLUENCING PULPAL HEALTH PRIOR TO EXPOSURE
Introduction
The dental pulp is afforded protection by the supragingival impervious enamel layer and underlying
semi-permeable dentinal layer. When the enamel layer is breached (or bypassed via loss of
cementum), dentinal trauma manifests as changes in the pulp. Fluid moves in or out of the dentinal
tubules, and the transport of noxious and natural substances has dynamic effects on the
odontoblasts, nerve endings of the subodontoblastic plexus, fibroblasts and other components of the
pulpal mesenchyme (Pashley 1996). Pulpal health is important, not only for the optimal function of
the tooth (Stanley 1989) but also for its inherent ability to respond to dentinal damage and exposure
(Torneck 1981). Some aspects of the effects of dentinal injury to the pulp, preceding exposure, that
may influence pulpal healing are covered below.
Reactions of dentine to caries
Caries destroys dentine by a combination of acid demineralisation, hydrolysis and enzymatic
breakdown. The pulpal reaction to this is variable and depends on its nature and depth (Trowbridge
1981). Teeth with only minor caries show slight or mild pulpal inflammation, with microorganisms
present only superficially in dentinal tubules (Langeland et al., 1987). The permeability of the dentine
is a key factor in the pulpal response, because it allows the passage of soluble irritants and
inflammatory stimuli (Brännström et al., 1965,1967). The first response of the dentine to caries is
sclerosis, the localised deposition of mineral as peritubular dentine which is thought to require the
presence of intact odontoblastic processes (Magliore et al., 1992). Therefore sclerosis is more likely
to occur in more chronic cases and to result in decreased dentine permeability. In an acute attack,
odontoblast processes are quickly destroyed and dead tracts are formed, these empty tubules are
sealed only by reparative dentine at the pulpal end of the tubules and are thus more permeable than
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sclerotic dentine.
Further barriers to permeability include "caries crystals". These are thought to represent the
recrystallization of calcium and phosphate ions that have been dissolved during demineralisation.
Caries then affects the pulp with a reduction in numbers, and changes in size and shape of
odontoblasts. Reparative (or tertiary) dentine represents a further defensive reaction of the pulp to
specific injury and is related to the size of the carious attack and is better stimulated by more chronic
lesions. The quality of this matrix is variable, it is generally less tubular and more irregular in form
and less mineralised than primary dentine. Langeland et al., (1977) found that the thickness of
reparative dentine afforded the pulp little, or no, protection from inflammation. Irregularities in the
formation of this layer of dentine may lead to inclusions of soft tissue within the matrix, these may
become necrotic contributing additional inflammatory stimuli (Trowbridge 1981). It has been
postulated that acidic conditions in dentine due to caries or inflammation are responsible for
liberating IGF-1 or TGF- from dentine matrix which stimulate the reactive dentine responses
(Finkelman et al., 1990, Bessho et al., 1991, Harada et al., 1990, Magliore et al., 1992, Lesot et al.,
1993).
Carious lesions are generally slow developing and the pulpal inflammatory response begins as a
low-grade chronic response - lymphocytes, plasma cells and macrophages. The plasma cells
frequently seen in chronic pulpitis secrete humoral antibodies acting as precipitins, opsonins,
agglutinins and lysins, all aiding to destroy the bacteria (Watts and Paterson 1981). The acute
response comes as bacteria invade the reparative dentine and directly affect the pulp.
Polymorphonuclear leukocytes (PMNL) are the first inflammatory cells to migrate in large numbers
into the damaged pulp. Regarded as the hallmarks of an acute inflammatory response, their normal
function is to engulf and destroy bacteria. However the enzymes contained in their intracellular
lysosomes can also degrade collagen, elastin, vascular basement membrane and stimulate
coagulation, fibrinolysis and production of kinins (Goldstein 1977). Tissue destruction by PMNL to
form an abscess in the pulp is inimical to repair. Macrophages are recognised as the predominant
cell of chronically inflamed tissues, but they are also present in acute inflammation, although
overshadowed by PMNLs because of their much slower migration rate. Consequently, fibroblast
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proliferation and localised deposition of collagen are seen.
The pulp is a highly vascular organ (despite the mean volume of each human tooth being only 0.02
ml) with many thin walled vessels (Avery 1981). Larger diameter arterioles and venules are seen
centrally with smaller vessels on the periphery, a capillary network supports the odontoblasts, some
of these blood vessels are continuous while others are fenestrated. Early inflammatory hyperaemia
is associated with proliferation of smaller vessels, pulp arteriole enlargement, capillary and venular
distension and congestion resulting in oedema and new blood vessel formation. If bacteria continue
to penetrate, more inflammatory cells are seen and neutrophils emerge from the adjacent venules
(Trowbridge 1981). As neutrophils only live for a few hours after leaving the bloodstream and their
death releases lysosomal enzymes, they not only digest phagocytosed bacteria but also destroy
pulp parenchyma. The accumulation of neutrophils results in suppuration, which may be diffuse or
localised in the form of a microabscess. Large numbers of bacteria are not usually seen until later in
total irreversible pulpitis, because of the effectiveness of neutrophils in destroying invading bacteria.
Unfortunately in the diagnosis of pulpitis, there seems to be little direct correlation between clinical
and histological findings, this has been known for some time (Greth 19331) and confirmed in later
studies (Langeland and Langeland 1968, Fiore Donno et al., 1969).
Reactions of dentine to trauma and restoration
Once the clinician has identified caries, the very act of removing it causes a pulpal reaction and
the deeper the cavity, the greater the inflammatory reaction (Stanley and Swerdlow 1964). As
soon as drilling commences in dentine the initial pulpal reaction is swift, with plasma proteins
from the circulation moving between the odontoblasts, up the tubules and to the cut dentine
surface within five minutes of cavity preparation (Chiego 1992). The trauma results in disruption
of the odontoblast cell-layer and junctional complexes. Odontoblasts become aspirated,
breakdown of odontoblasts and their processes suggest that cell death occurs. The initial
response (less than an hour after trauma), shows a breakdown of the junctional complexes between
1 As quoted in Langeland et al., 1977 Greth H (1933) 'Diagnostik der Pulpaerkrankungen' Hermann Meusser Verlag Berlin
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adjacent odontoblasts and underlying fibroblasts (Chiego 1992). The majority of odontoblasts under
the cavity preparation show signs of cellular injury (including mitochondrial swelling and rough
endoplasmic reticulum dilatation), and few contain intracellular collagen fibres.
Other immediate effects include destruction and injury of nerve fibres in the dentine and pulp, this
leads to the release of neuropeptides such as calcitonin gene related peptide (CGRP) and
substance P (SP) which create a local neurogenic inflammatory condition (Kimberley and Byers
1988, Byers et al., 1990, Byers 1996).
The dynamic shifting of fluids in the dentinal tubules induced by restorative procedures has direct
effects on the pulp (Brännström 1968, Brännström et al., 1968, Brännström et al., 1969, Pashley
1996). Cutting burs generate heat (inward fluid movement), air cooled burs cause evaporative water
loss (outward), water-cooling induces shifts through osmosis (inward). The use of air/water syringes
causes osmotic and evaporative fluid shifts with washing and drying. Conditioners, primers,
varnishes and bonding agents induce outward fluid shifts; the polymerisation of light curing and self-
curing restoratives produces heat that leads to inward fluid shifts. These fluid movements and their
subsequent pressure changes cause pain and damage to the pulp.
Restorative materials are no longer attributed with causing as much pulpal irritation, via diffusion
through the dentine, as they were before experiments with “irritant” materials on germ free animals
demonstrated the important role of bacteria (eg Kakehashi 1965, Cox et al., 1987, Watts and
Paterson 1987). Hume and Massey (1990), stress maintenance of pulpal health through sealing the
cavity surface to prevent bacterial ingress as opposed to assuming a chemical toxicity from materials
used. Brännström (1971,1973) has proposed that gaps exist between tooth structure and most
restorations. Such gaps are large enough to allow passage of bacteria or their metabolites and these
diffuse through the dentine to the pulp. There is a positive correlation between bacteria in the cavity
and inflammation (Brännström 1971,1973). Despite the influence of bacteria it is reasonable to
assume that diffusion of various chemicals from some restorative materials across the dentine may
damage the pulp cells. Most non-metallic restorations are cytotoxic in cell culture - but if they do not
diffuse across dentine the pulp should be safe (Cox et al., 1987). It appears that the acids present in
certain restorative materials, particularly strong acids, are extremely well buffered by intact dentine
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and do not provide hydrogen ions at the pulpal surface (Wang and Hume 1988). This explains why
acidic materials like zinc phosphate cement and glass ionomer cement are well tolerated by the pulp
when placed on intact dentine, even close to the pulp.
The structural variables in dentine.
Pashley (1996) has suggested some factors that may affect hydrodynamic interactions through
dentinal tubules to the pulp:-
i) The length of the odontoblastic process
Under normal conditions the odontoblast process does not extend any further than one third the
length of the tubule (considered a point of conjecture by Pitt Ford (1985) and opposed by authors
such as Sigal and Chernecky 1988 who found that in rats and humans the odontoblast processes
ended in a dilated sphere at, or near, the DEJ). This means deep restorations could sever the
cytoplasmic process and irritate the pulp cell body. Interestingly, Lesot et al., (1993) have proposed
that odontoblasts whose processes were cut, in cavities where the pulp was not exposed, did not die
but were stimulated.
ii) deeper dentine has a higher water content, which increases fluid movement through the tubules
closer to the pulp.
iii) the smear layer and plugs formed by the cutting of dentine occlude a significant fraction of tubule
luminal surface that could be occupied by water.
iv) the number of dentinal tubules per mm2 varies from 15000 at the DEJ to 65000 at the pulp (Fosse
et al., 1992). Not only does their density increase but also the diameter of the tubules thus leading to
increased permeability near the pulp.
v) freshly exposed dentine has an outward fluid flow, which acts as the first line of defence against
inward diffusion of noxious substances. This flow may be due to hydrodynamic stimulation of nerves,
releasing neuropeptides such as CGRP and SP, causing increased blood flow and tissue pressure
intrapulpally (Olgart et al., 1991, Olgart and Kerezoudis 1994 and Heyeraas et al., 1996). In
denervated teeth this phenomenon is not seen Matthews 1996). In vital dog teeth, permeability falls
following cavity preparation (Pashley 1985) and this may be due to plasma proteins like fibrinogen
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polymerising into fibrin at the pulpal termination of the fibrils.
vi) functionally, dentinal tubules have much less permeability than their luminal diameters would
suggest because of intratubular collagen and mineral constrictions which may resist bacterial
movement.
The pulpal neural system obviously plays an important role in response to injury because dentine
and pulp exposure experiments in denervated teeth have shown a much greater pulpal destruction
than in innervated teeth (Byers and Taylor 1993, Heyeraas et al., 1996).
A healthy pulpal vascular system is essential to the pulpal response to injury. Good blood supply is
required for the transport of the cells involved in the inflammatory response, regulation of oedema,
repair of damaged cells, disposal of the degenerated products of pulpal damage and repair, reaction
to bacterial invasion, the transport of materials for repair and support of the cells of repair
As the health of the pulp before exposure is important as a basis for its subsequent healing capacity,
pre-existing inflammation (from causes such as bacterial or iatrogenic trauma) is an essential
variable and should be minimised. Hume and Massey (1990) suggested that, to keep the pulp
healthy, clinicians should minimise the threat of caries by prevention, or debridement and therapy;
protect exposed dentine from the oral environment; minimise pulpal trauma when cutting and
preparing cavities, use materials of low pulpal toxicity and seal the cavity well to prevent bacterial
ingress.
The ability of the pulp to withstand insult is multifactorial and cannot be quantified. It varies between,
and within, species (Pitt Ford 1985), with age, pulpal and dentine structure/anatomy, the type of
insult, neural elements (Byers and Taylor 1996, Heyeraas et al., 1996, Avery 1990) and circulation
(Periera 1981, Horsted et al., 1985).
B. THE PULP FOLLOWING EXPOSURE
Pulpal injury following exposure
The two key components in pulpal inflammation are micro circulation and sensory nerve activity (Kim
1990). Mechanical, chemical or bacterial stimuli degranulate mast cells disrupt blood vessels,
damage cells and stimulate sensory nerves releasing inflammatory mediators such as histamines,
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kinins, prostaglandins and neurokinins like substance P (from c-fibre nerve terminals). These
mediators increase pulpal blood pressure by vasodilating arterioles and promoting venule leakage
and chemotaxis via leukocytes.
When the pulp is cariously, iatrogenically or traumatically exposed, the pulpal mesenchyme is
susceptible to bacterial invasion and an acute inflammatory reaction may result. Bacterial
contamination must be minimised, contained and reinfection prevented to allow the pulp to heal.
Ample evidence suggests mainly anaerobic bacteria are involved in pulpal and periapical infection
(obligate anaerobic non-sporulating and a lower proportion of facultatively anaerobic) as well as
some aerobic bacteria (Bergenholtz 1974, Fabricius et al., 1982, Sundqvist 1994).
The significance of bacteria to the healing process have been known since Kakehashi’s important
1965 study demonstrating pulpal healing and dentine-bridging in germ-free rats under various
restorative materials. This was reinforced by Watts and Paterson (1987) who found that even toxic
materials merely produced a superficial necrosis at the capping site in germ-free animals. Review of
the literature reveals that the majority of authors now believe the best way to allow pulpal healing is
to seal the pulp effectively against bacterial invasion through microleakage (Brännström and Nyborg
1971,1973; Cox et al., 1985,1987; Heide 1991; Milner-Snuggs et al., 1993). Although it remains
possible that bacteria could remain viable under a restoration by nutrient polysaccharides diffusing
through the dentinal tubules (Lado et al., 1986).
Pulpal reactions and repair following exposure
Exposure of the pulp results in destruction of the underlying segment of the odontoblast cell layer
and other underlying pulp cells. Large numbers of polymorphonuclear leukocytes (PMNL) rapidly
migrate to the site as part of an acute inflammatory response. The PMNLs are followed by
macrophages; these may already be present in the case of carious exposure where a chronic
inflammatory process pre-exists (in conjunction with lymphocytes and plasma cells). The PMNLs
and macrophages play important roles in the early process of healing and tissue repair (Fitzgerald
1979, Cox and Bergenholtz 1986, Ten Cate 1992). The PMNLs phagocytose and digest damaged
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tissue and bacteria, while the increasing numbers of macrophages phagocytose effete neutrophils
and debride the area in preparation for new tissue growth. Kinins and growth factors, particularly
Transforming Growth Factor- (TGF-) and Platelet Derived Growth Factor (PDGF), released from
the platelets, stimulate the influx of monocytes/macrophages to the wound area and are
subsequently important in initiating and augmenting the inflammatory phase of wound repair (Kiritsy
et al., 1993). Macrophages also stimulate the differentiation of new fibroblasts, from undamaged
fibroblasts at the wound periphery or from undifferentiated perivascular cells. These daughter cells
migrate to the defect, differentiate and deposit collagen (Ten Cate 1992).
Neuropeptides such as CGRP and substance P are released and cause a local neurogenic
inflammatory condition (Kimberley and Byers 1988, Byers et al., 1990, Byers 1996), the flow and
permeability of local blood vessels are affected. The damaged nerves also release growth factors
which may promote pulpal healing (Trantor 1996). Arterioles enlarge, capillaries and venules distend
and become congested. At a traumatic exposure site in the first 24 hours, a fibrin clot can be seen
containing red blood cells and various leukocytes. This clot contracts towards the pulp over the next
24 hours with clefts in it suggesting active fibrinolysis. Clot resolution continues over the next couple
of days with pulp vessels still congested and capillary infiltration at the exposure site. Fibroblasts
infiltrate and align around the exposure site towards the end of the week (Fitzgerald 1979, Mjor et
al., 1991). Thus the early sequence of mechanical pulp-exposure healing is firstly, clot resolution by
lysis and macrophage infiltration, secondly, fibroblasts and endothelial cells invasion of the clot area
to form granulation tissue and finally, as discussed below, the recruitment and differentiation of
odontoblast-like cells from the pulp that begin the process of reparative dentinogenesis.
Reparative dentinogenesis
Reparative dentinogenesis is the formation of a tertiary dentine matrix secreted by a new generation
of odontoblast-like cells, as opposed to reactive dentine which is the secretion of a tertiary dentine
matrix by surviving post-mitotic odontoblast cells in response to noxious stimuli (Lesot et al., 1993).
Reparative dentinogenesis requires a pulpal environment free from excessive inflammatory
mediators (Torneck 1983), lack of infection (Ten Cate 1992), adequate vascularity (Periera and
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Stanley 1981, Horsted et al., 1985) sufficient cell density, a sufficient concentration of morphogenic
tissue proteins (Tziafas 1994) and the release of growth factors.
Some authors consider that the “odontoblast-like” cells that produce reparative dentine differ
sufficiently from primary odontoblasts, morphologically and physiologically, to not be considered true
odontoblasts (Chiego 1992, Magliore 1992). However, like odontoblasts, they control matrix
formation. The odontoblast is the source of collagen and proteoglycans and is able to delete various
components from the dentine matrix prior to and during mineralisation of the reparative dentine.
Moreover, they have the important role of regulating the transcellular migration of various inorganic
salts necessary for initial mineralization (Kirk and Meyer 1992). Chiego (1992) found that the cells
forming the dentinal bridge were larger, had greater rough endoplasmic reticulum (RER) and
mitochondria than primary odontoblasts. The major difference seemed to be the random
arrangement of organelles within these odontoblast-like cells cytoplasm, with no clearcut
arrangement of the intracellular organelles, or of an odontoblast cell process. The few junctional
complexes resemble those between primary odontoblasts and these complexes increased as the
cells became more densely arranged. These odontoblast-like cells synthesize extracellular matrix
proteins at a rate 138% greater than control primary odontoblasts, even after construction of the
dentinal bridge (35 days after pulpal exposure) (Chiego 1992). They have a polarised nucleus,
cellular extensions, well developed RER and are arranged in an “epithelial” fashion. They generate
an organic matrix which later mineralises (Yamamura 1985). A calcium binding protein, 28kDa
calbindin, only found in odontoblasts, is found in these cells (Magliore et al., 1988a). However
Magliore et al., (1988b) felt they could not be considered fully differentiated odontoblasts as they
synthesised type I and type III collagen as well as fibronectin.
Fibronectin, a multifunctional morphogenic glycoprotein, seems to be implicated in healing of dental
tissues and can exert a direct effect on the odontoblastic cytoskeleton and may consequently
stimulate collagen gene-expression in these cells (Magliore et al., 1992). It is necessary for terminal
differentiation (Lesot 1985,1988,1990) and may have a mediating role during pulp biomatrix-cell
interactions because of its strong affinity for collagenous matrix (Tziafas et al., 1992, Tziafas et al.,
1994). Fibronectin is also involved in the process of odontoblast elongation and polarisation (Lesot et
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al., 1988) and has been shown to induce ecto-mesenchymal cells, in culture, to do the same. It
induces calcified tubular and atubular dentinal matrix in dog dental papillae (Tziafas et al., 1992).
Tziafas et al., (1992) supported Veis’ (1985) hypothesis that the critical requirement for odontoblast
differentiation is the creation of a surface for attachment and polarization. The origin of odontoblast-
like cells in the pulp tissue is open to some conjecture. In 1979 Fitzgerald proposed, from his
observations and 3H-thymidine studies, that fibroblasts may be the cells that replace odontoblasts.
Candidates for the new odontoblast-like cells may be undifferentiated cells from the pulp
parenchyma such as a proliferation of perivascular cells (Fitzgerald et al., 1990) or fibroblast-like
cells (Yamamura 1985) produced by a process similar in origin to that of new fibroblasts in soft
connective tissue repair (Ten Cate 1992). Lesot et al., (1993) proposed that odontoblast-like cells
were probably derived from neural crest papilla cells and that undifferentiated mesenchymal cells,
the precursors of fibroblasts, pericytes and endothelial cells were candidates, as the morphology of
the cells producing matrix was as variable as was the matrix itself. Tziafas (1994) has postulated
that the organisation of extracellular matrix within the pulp during reparative dentinogenesis can be
directed along different paths dependent on the stage of differentiation of the odontoblast-like cells
and their orientation. The adhesion of pulp cells to a suitable surface may be critical for the
appearance of elongated polarized cells (Veis 1985). Matrix subsequently formed is variable in
nature (osteotypic, tubular or an atubular fibrodentine).
Whatever the origin of these precursor cells, differentiated or undifferentiated, pulp cells apparently
differentiate into odontoblast-like cells after a number of cell cycles (Ruch 1990). In young pulps, with
many odontoprogenitor cells and a high concentration of morphogenetic factors, a surface containing
concentrated fibronectin is all that is required to express the odontoblastic phenotype (Tziafas 1994).
In older pulps the presence of other extracellular factors is required.
Dentine extracellular matrix contains growth factors such as IGF-1, IGF-2, TGFß (Finkelman et al.,
1990) and the BMP’s (Kawai and Urist 1989, Bessho et al., 1991). These affect cell recruitment and
differentiation and thus may amplify cellular synthetic activities and enhance tissue growth and
repair. It is known that growth factors influence soft tissue repair. Also factors like TGF-
chemotactically also stimulate an influx of macrophages (Kiritsy et al., 1993) and IGF-1 is able to
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induce a cascade of cellular and molecular events having profound effects on soft tissue healing. In
the absence of the odontogenic epithelium and its related basement membrane, in mature teeth the
odontoblast response to injury is probably closely linked to dentine extracellular matrix components
(Magliore et al., 1992). The role of the TGF-s in reparative dentinogenesis has been elaborated by
analysis of knock-out mice that produce no TGF-1 mRNA (TGF-1(-/-) mice). Histopathologic
analysis of the adult dentition in these mice show significant destruction in teeth and of periapical
tissues compared to heterozygote TGF-1 mice (+/-) suggesting important modulatory roles in
dental pulps (D’Souza and Litz 1996). It is known that members of the TGF- superfamily, the
BMPs, are involved in matrix induction events after pulpal injury (see below). Active fractions of
dentine have been used to initiate and maintain odontoblast differentiation in vitro (Lesot et al., 1986,
Bègue-Kirn et al., 1992).
Combinations with heparin or fibronectin and IGF-1 stimulate differentiation, polarisation and function
of odontoblast-like cells in isolated murine dental papillae in vitro (Bègue-Kirn et al., 1992, Lesot et
al., 1993). It has been suggested that during repair, fibrodentine could control odontoblast
differentiation and assume the role played by the basement membrane during odontogenesis (Ruch
1985). Vaahtokari et al., 1991 has suggested that TGFß is synthesised by these differentiated
odontoblasts and this may stimulate other dental papilla cells which have the right cell surface
receptors to change their phenotypes and give rise to more odontoblast-like cells.
There also appears to be a role for pulpal neural elements in dentinogenesis, with neuropeptides like
CGRP thought to stimulate the secretory function of odontoblasts (Heyeraas et al., 1996). Pulpal
injury causes pulpal fibroblasts to release nerve growth factor (NGF) (Byers et al.,1992), this leads to
sprouting of CGRP and SP containing nerves, which, on release, stimulate local fibroblast cell
division leading to increased NGF expression and other growth factors (Trantor et al., 1995).
The sensory fibres containing CGRP and SP grow towards surviving odontoblasts and the pulp
tissue associated with the lesion. These nerves accompany granulation tissue and the nerve
sprouting subsides as inflammation decreases and the injury site is covered by reparative dentine
(Byers et al., 1992). Zhang and Fukuyama (1999) found a large number of CGRP containing
nerve fibres in the residual pulp of pulpotomized rats seven days after exposure. Some of these
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nerve fibres appeared to be terminating in the differentiating odontoblasts and the initial matrix of
the dentine bridge. Over the next three weeks the residual pulp demonstrated decreased nerve
density, while regenerated axons terminated in the fibrous matrix layer of the calcified dentine
bridge suggesting that sensory neuropeptides may play a role in dentine bridge formation.
Summary
Successful healing of the pulp is the end product of many interacting and complex processes that
are not yet fully understood. Pulpal healing subsequent to exposure seems to depend on:-
a. the inflammatory state at the time of the exposure
b. the presence of bacteria
c. the inflammatory response to the exposure and
d. the generation of odontoblast-like cells requiring a sufficient concentration of essential extracellular
factors and proteins.
e. the neural response.
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REVIEW II. CORTICOSTEROID/ANTIBIOTIC PREPARATIONS AND DIRECT PULP CAPPING
The success of corticosteroids as anti-inflammatory agents in general medicine in the 1950s led to
their use in the treatment of pulpal inflammation. It was theorized that, as the adult dental pulp was
confined, it could not react to inflammation as other body tissues do and that the pressure from
inflammatory exudate could cause constriction of apical vessels with subsequent loss of vitality.
Their use was proposed to eliminate postoperative pain and pulpal inflammation following cavity
preparation (Mjor and Ostby 1966) and following direct pulp capping (Schroeder and Triadan 1962,
Ehrmann 1965).
Ledermix2 arose as a result of a 1962 study by Schroeder and Triadan utilizing a combination of
corticosteroid to provide anti-inflammatory relief for the pulp and an antibiotic to protect it from
bacterial attack in its immunosuppressed state (Clarke 1971a).
The Ledermix compounds are marketed as a paste, for the treatment of acute pulpal conditions and
a cement, for basing deep cavities and permanent pulp capping.
The two formulations are:-
LEDERMIX paste per gram:triamcinolone acetonide 10.0 mg
demethylchlortetracycline calcium 30.21 mg
LEDERMIX cement powder per gram: triamcinolone acetonide 6.7 mg
demethylchlortetracycline HCL 20.0mg
in combination with zinc oxide and
calcium hydroxide
Hardener Eugenol type N solution (normal) 850mg
Eugenol type F solution (rapid)
Polyethylene glycol 4000 100mg
Clarke (1971a) reported that the versions of Ledermix paste he was working with also contained
2 Lederle Pharmaceuticals, Wolfratshausen, FRG.
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triethanolamine NF, calcium chloride USP, zinc oxide, anhydrous sodium sulphate, polyethylene
glycol 4000 USP and distilled water. The cement powder used contained zinc oxide USP, Canada
balsam resin NF and calcium hydroxide USP while the liquid also contained 13% oil of turpentine!
The importance of bacteria in the healing response of the pulp to trauma is well recognized from
experiments with germ free animals (Kakehashi et al., 1965, Watts and Paterson 1987, Paterson
1976 and Kakehashi et al., 1969). Because of the immuno-suppressive effect on the pulpal tissues
by the corticosteroids, protection against bacterial ingress is especially important when exposed
pulps are capped with Ledermix. The antibiotic in Ledermix cement and paste is
demethylchlortetracycline, (demeclocycline) and this is a tetracycline-type antibiotic which is the
product of a mutant strain of Streptococcus aureofaciens. Tetracyclines are primarily bacteriostatic
broad-spectrum antibiotics with a greater effect on Gram-positive than Gram-negative bacteria.
Allergic reactions are rare and the dosage used in pulp-capping poses no anticipated systemic
threat (Heling and Pecht 1991).
Ledermix paste containing 3.21% demeclocycline has the ability to provide high local levels of
antibacterial effect, however this effect is shortlived (Abbott 1988). Not all organisms are sensitive
to the bacteriostatic effects of tetracycline, and all yeasts are resistant, so pulpal defence
mechanisms continue to be important (Ehrmann 1965, Barker and Ehrmann 1969) particularly if
infected dentinal chips have been displaced into the pulp. Ehrmann (1965) demonstrated five
carious cavities out of 19 showed tetracycline-resistant bacteria. In necrotic canals 25% of all
bacteria were resistant and yeasts were present in 8-20%. Its rapid diffusion means that it is
ineffective in killing Staphylococcus aureus in dentinal tubules at 24 hours (Heling and Pecht
1991). Langeland et al., (1977) found Streptococcus viridans, haemolyticus and faecalis to be
present in culture from four pulps out of nine that failed subsequent to Ledermix therapy. There was
no positive correlation between pain, the pulpal condition and bacterial growth. Thus if resistant
bacteria are present in the immuno-suppressed pulp, particularly in young teeth with good
vascularity and open apices, a corticosteroid-antibiotic combination may permit development of a
transient bacteraemia, particularly dangerous in patients with a bacterial endocarditis (Ehrmann
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1965, Laws 1967).
When tetracycline is applied by itself to intact pulps it is irritating and inflammatory (de Souza and
Holland 1974). A dense lymphocytic/plasmocytic infiltrate and extensive circulatory stasis
characterize the inflammation. The antibiotic by itself produces no bridging after 40 days despite the
presence of odontoblasts (Baume and Fiore-Donno 1970). Demethylchlortetracycline also inhibits
collagen synthesis by chelating ferrous iron - an important cofactor for the enzyme protocollagen
proline hydroxylase (Uitto et al., 1975). Work by Golub et al., (1983,1984) suggests that members
of the tetracycline family inhibit matrix metalloproteinases (MMP's), including mammalian
collagenase, by a mechanism independent of their antimicrobial activity (Golub et al.,1987). Dentine
collagenase has been demonstrated by immunotechnique in human predentine (Dumas et al.,
1989) and presumably plays an important role in dentinogenesis. Metalloproteinases are present in
porcine dentine and may degrade non-collagenous proteins during dentinogenesis. (Fukae et al.,
1991).
In summary, despite its broad spectrum bacteriostatic action, high initial levels and rapid diffusion,
the antibiotic's action is short, pulpal repair is partially inhibited by tetracyclines and resistant
organisms may survive and multiply in a steroid-suppressed pulp.
The corticosteroid in Ledermix is triamcinolone acetonide, an anti-inflammatory synthetic
corticosteroid that is much more potent than cortisol (5:1 effect/weight) and has fewer side effects
than the glucocorticosteroids (Fauci et al., 1976). The triamcinolone acts as a rapid release and
very effective short-term pulp sedative - it moves through the dentine and into the pulp space within
a few hours and disappears after two, to three, days (Abbott et al., 1988, Hume and Testa 1981).
Tests with 3H-triamcinolone found that 70% of the labelled triamcinolone is released by 24hrs from
dentine with more than 90% being released at 48 hrs from a Ledermix lining cement (Hume and
Testa 1981).
Ledermix paste contains triamcinolone acetonide at 1%, the cement contains 0.67% and as the
pulp is a highly vascular organ (Avery 1981) the question of potential systemic effects of the
corticosteroids is raised. Abbott's 1992 study concluded that the maximum amount of triamcinolone
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that could be released into the systemic circulation was equivalent to the following amounts of
cortisol during the first day (remembering that after 24hrs there is a rapid exponential decrease in
release):-
a) from Ledermix cement as an indirect capping agent - 1.85mg cortisol equivalent (1.3mg cortisol
equivalent in Hume and Testa 1981)
b) from Ledermix paste in root canals as an interim endodontic dressing - 1.2mg cortisol equivalent.
It must be noted that these are figures derived from in vitro studies with maximum utilizable
amounts of cement/paste. In a clinical situation figures are lower and concentrations are rapidly
reduced in vivo as the drug enters the systemic circulation through the pulpal or periradicular
tissues.
To bring these figures into focus, it should be considered that the human body produces 20-30 mg
of endogenous cortisol per day and that this may increase, in stressful situations, to 300-400mg per
day (Parnell 1964). It appears to be extremely unlikely that systemic effects would be produced
from the use of Ledermix products as direct capping agents (Barker and Ehrmann 1969, Abbott
1992, Hume and Testa 1981).
Researchers have long recognized the real differences between the clinical and histological
parameters presented by symptomatic and asymptomatic teeth (Clarke 1971a, Barker and
Ehrmann 1969, Baume 1966, Baume and Fiore-Donno 1970, Baume and Holz 1981). There is no
argument that Ledermix is very efficient at eliminating the symptoms of a painful pulpitis,
sometimes for years (Baume and Fiore-Donno 1970). However, it seems that the absence of
symptoms can occur in the presence of extensive pulpal inflammation and pulp destruction is the
rule rather than the exception (Langeland et al., 1977).
There is a histological pattern that seems to emerge when Ledermix products are used to cap
painful human teeth or previously intact human or animal teeth with the following features:-
I) Effects on dentinogenesis
Before pulpal odontoblasts resume the tasks of controlling matrix formation (sourcing collagen and
proteoglycans as well as removing components from the predentine matrix) and mineralizing the
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reparative dentine, they have to be recruited, divide, differentiate and resume function. Any of these
steps may be susceptible to chemical interference.
There has been conjecture over the effect of corticosteroids on dentinogenesis. The majority of
authors (Baume 1966 (cement and paste, indirect), Baume and Holz 1981, Kirk and Meyer 1992
(cement), Ivanovic et al., 1987 (cement, indirect), Clarke 1971a (paste followed by cement), 1971b
(cement), Fiore-Donno and Baume 1966 (paste followed by cement), Laws 1967(paste), Uitto et
al., 1975 (paste), Baratieri et al., 1981 (cement, indirect)) suggest inhibition and disruption of
dentinogenesis while another author does not see interference (Rowe 1967 paste).
Review of the literature suggests that teeth capped with Ledermix paste and cement show localized
disruption of odontoblasts, inhibition of mineralization, arrest of new predentine formation and, with
rare exceptions, a lack of a solid reparative dentine bridge.
Odontoblastic disruption and atrophy are common findings (Baume and Fiore-Donno 1970 (cement
and paste), Mjor and Ostby 1966 (cement and paste, indirect), Clarke 1971b (cement), Barker and
Ehrmann 1969 (cement), Ulmansky and Langer 1967(paste), Barker and Lockett 1972 (cement and
paste)), and it is quite clear that corticosteroids (and demeclocycline) have inhibitory effects on the
processes of dentinogenesis. Soft connective tissues, in response to injury, show an initial
polymorph response followed by a macrophage response which, in part, elicits the additional
differentiation of new fibroblasts. In soft tissue, the fibroblast response is to migrate to the defect,
differentiate and deposit collagen to form scar tissue - the hallmark of repair (Ten Cate 1992), in the
teeth, this tissue is mineralized by odontoblasts or odontoblast-like cells to produce reparative
dentine (Tziafas 1994). Corticosteroids interfere with increased collagen synthesis in response to
exposure. Uitto (1975) found collagen synthesis to be inhibited at concentrations of hydrocortisone
greater than 10-4M. Because triamcinolone is five times more active, concentrations of 2 x 10-5M
certainly affect healing and this concentration is present for most of the first day (Hume and Testa
1981). When collagen synthesis increases so to does the activity of protocollagen proline
hydroxylase (a critical enzyme in the intracellular synthesis of collagen), and, as mentioned above,
this is inhibited by the demeclocycline in Ledermix which chelates iron as an important cofactor
(Uitto 1975).
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The anti-anabolic effects of corticosteroids on protein metabolism seem to interfere with the
formation of dentinal matrix. Incorporation of amino acids into proteins is blocked thus inhibiting
collagen fibre formation and fibroblastic proliferation, the calcification of dentine is disturbed or
irregular, and the proteoglycans necessary for predentinal matrix formation are lacking (Baume
1966, Baume and Holz 1981).
These inhibitory effects on dentinogenesis are restricted to a limited zone in the pulp which extends
from the exposure (Kirk and Meyer 1992, Harris and Bull 1966 (glucocorticosteroid), Clarke 1971b)
rather than affecting the total organ. Baratieri (et al., 1981) found dentine apposition was slowed
and discontinued by corticosteroid effects (Ledermix) on odontoblast activity and collagen
synthesis, but was not completely arrested. Hume and Testa (1981) also believe that the inhibition
is only transitory and Clarke (1971b) found evidence that some reparative dentine deposition
occurred 4 to 48 weeks post-capping.
Transitory and localized inhibition of dentinogenesis or not, it is with rare and questionable
exceptions that solid reparative bridging occurs (Schroeder and Triadan 1962 (triamcinolone,
chloramphenicol and lignocaine), Ulmansky and Langer 1967).
II) Pulpal inflammation.
The persistence of pulpal inflammation is an unhealthy sequel to any dental treatment and many
studies have shown continued inflammation, subsequent to the application of corticosteroids onto
carious dentine or the exposed pulp (Fiore-Donno and Baume 1966, Harris and Bull 1966, Laws
1967, Baume and Fiore-Donno 1970, Langeland et al., 1977, Ulmansky and Langer 1967).
In Baume and Fiore-Donno’s 1970 Ledermix (cement and paste) study all 180 treated painful
human molar pulps showed a persistent chronic inflammation often leading to eventual necrosis
without symptoms (even after 12 months). Barker and Ehrmann (1969) quote unpublished data of
Ehrmann's which suggests that 50% of pulps treated with Ledermix succumbed after 3 years.
Again, acute conditions were merely converted to a chronic state. Clarke (1971a) showed that the
continued inflammation in human molar pulps ranged from mild chronic to severe after sequential
treatments with paste and cement over a 24 hour to 48 week time span. However, in a dog study,
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Clarke (1971b) found that chronic inflammation subsided after 4 weeks.
Persistence of a chronic inflammatory response in previously painful pulps may be due to:-
i) congestion and poor circulation of the inflamed pulp which may be further irritated by the
antibiotic in the medicaments
ii) the advanced state of the inflammation.
iii) microbial contamination (Baume and Fiore-Donno 1970).
Corticosteroids do not appear to eliminate inflammation from an involved pulp and pulpal
degeneration may continue asymptomatically.
III) Other pulpal effects.
Dilation of blood vessels in the pulp was noted by Harris and Bull (1966) and Barker and Lockett
(1972), perhaps due to corticosteroid control of permeability, it was suggested that this may be a
sign of pulpal degeneration.
Baume and Fiore-Donno (1970) went as far as to suggest that treatment with the Ledermix
compounds, applied to intact and painful human pulps, induced metaplastic changes manifesting as
atrophy of mesenchyme derivatives. These changes included reduction of fibroblasts and the
capillary network, odontoblastic atrophy and disappearance of Von Korffs fibres and the argyrophilic
network from the area. In contrast, Barker and Lockett (1972) felt that 80% of the dog pulps they
capped with Ledermix were “normal” after periods of 2-8 months and Clarke (1971b) felt repair
suppression in his dog study was temporary.
Some authors have suggested various degrees of attempt at fibrous repair in directly capped teeth
(Laws 1967, Clarke 1971a, Ulmansky and Langer 1967, Barker and Lockett 1972).
This review has shown that components of Ledermix cement and paste inhibit pulpal repair,
following direct capping of the pulp, through inhibitory effects on odontoblasts and collagen
synthesis. Pulpal inflammation is often inadequately suppressed, or converted to a symptom-free
chronic state, with hard tissue bridging repair not being seen. The components of Ledermix are
unlikely to have systemic effects, although bacterial contamination should be minimized because of
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the immunosuppressed state of the pulp and the limited antibacterial effect of the antiobiotic
component.
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REVIEW III. CALCIUM HYDROXIDE AND DIRECT PULP CAPPING.
Calcium hydroxide has been used as a pulp-capping agent since 1920 when it was successfully
used by Hermann to achieve solid biological closure of a pulpal exposure (Baume and Holz 1981).
It’s continued use is, at least in part, because of its perceived ability to stimulate the formation of a
reparative dentine bridge, which is supposed to offer the pulp protection against further insult and
thus minimize pulpal inflammation. Calcium hydroxide capping agents have the additional
advantages of being relatively inexpensive, having a long shelf life and possessing easy
manipulation and placement properties. Interestingly however, some sections of dental clinical
opinion feel capping with calcium hydroxide is no more advantageous than capping with other
agents, that no specific stimulation of reparative dentine occurs and that, if there is, the bridge is
ineffective barrier to chemical and bacterial insult.
Despite calcium hydroxide's widespread acceptance as a capping agent, little is definitely known
about its mechanisms of action on the pulp. This review will look at the properties of the various
forms calcium hydroxide and some of the proposed theories and explanations for cellular,
mineralizing, antibacterial effects observed when calcium hydroxide is used as a pulp-capping
agent.
The forms of calcium hydroxide
Non-setting pastes (at their simplest, analar calcium hydroxide and distilled water) are usually
dispersions of calcium hydroxide (with a radiopaque agent) in what is usually a hydrophilic base.
This simple composition makes for ready release of the active agents and dissolution in the
acceptor medium. Calcium hydroxide cements (like Dycal) set by some of the available Ca(OH)2
reacting with the salicylate ester chelating agent in the presence of a hydrophilic and soluble
toluene sulphonamide plasticiser. The weak secondary attractions of the chelates and the solubility
of the plasticiser allow release of calcium and hydroxide ions - however a greater number of
calcium ions are bound and unable to be released. There are also single paste setting agents
utilizing polymerization of a dimethacrylate by means of light (eg VLC Dycal).
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Calcium hydroxide pastes are more effective in inducing dentine bridge formation than cements
because of their calcium release profiles and antibacterial nature, however cements are more
efficient in producing healing in cases where microleakage may occur (Lim and Kirk 1987).
The hard setting formulations of calcium hydroxide tend to cause less chemical cell injury (1-2 cell
layers) while maintaining sufficient irritation and a sufficient hydroxyl environment to encourage
odontoblast differentiation and dentine formation up against the capping material. This occurs
because the zone of coagulation necrosis is rapidly removed by phagocytes and replaced with
granulation tissue that quickly organises and differentiates mature odontoblasts against the cap
surface (Stanley and Lundy 1972). This leads to a more uniform bridge and less pulpal obliteration
(Stanley 1989).
Tamburic et al., (1993) tested the in vitro release of calcium and hydroxyl ions of setting and non-
setting calcium hydroxide pastes, after diffusion through sintered glass by potentiometric method
utilising a Ca 2+ ion analyser and the hydroxyl ions with a pH meter. The maximum liberation of
calcium ions was exhibited with the non-setting calcium hydroxide (Calxyl Red®), 56.35% during
the first six hours. All the non-setting pastes showed maximal calcium ion release after six hours
and hydroxyl ions after eight hours. Dycal® showed the highest pH and the highest release from
the setting pastes tested, however the maximum was 10.92% of released calcium ions in 24 hours.
The release of calcium and hydroxyl ions from calcium hydroxide preparations has direct effects on
pulpal tissue (Tamburic et al., 1993) and it is difficult to separate beneficial from harmful ones
following the application of calcium hydroxide to the pulp (Lesot et al., 1993).
a) the effects of calcium hydroxide on pulpal cells
Because of variations in the pH of calcium hydroxide, generally between the basic pastes and the
newer less alkaline products, two different modes of pulpal healing following capping can be
considered (Stanley 1989):
1.High pH calcium hydroxide (pH 11-13 eg. Calxyl Red, Calxyl Blue, Calcipulpe)
A) Early changes
Within an hour calcium hydroxide produces a three layered zone of necrosis over healthy pulpal
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tissue (Schroder 1985):-
i. the superficial zone- necrosis due to the application pressure and from pressure due to oedema
from an intermediate zone.
ii. the intermediate zone- oedema and chemically induced liquefaction necrosis from the hydroxyl
ions.
iii. the apical zone -coagulation necrosis (tissue and plasma proteins from the intermediate zone,
with partially neutralized hydroxyl ions resulting in a weaker chemical effect).
Stanley (1989) has re-classified these early changes as :-
i.zone of obliteration- where the high pH calcium hydroxide chemically cauterizes the pulp tissue
directly in contact with it, leaving an area of dentinal, blood and cell debris; this zone is also due to
the pressure of application and is visible after an hour of contact (Schroder and Granath 1971). This
zone presumably contains the superficial and intermediate zones of Schroder (1985).
ii.zone of coagulation necrosis- this area of devitalized tissue below the obliterated zone is
approximately 0.3-0.7mm thick and shows coagulation necrosis and thrombosis. This zone appears
to correspond to Schroders apical zone.
iii.line of demarcation- this separates the subjacent vital tissue from the necrotic area and it is
across this layer that sufficient stimulation is provided to elicit a healing response from the healthy
tissues with vascular changes and inflammatory cell migration starting the process within six hours
(Schroder 1985).
B) Intermediate changes (Fitzgerald 1979)
i. two to three days after the injury, mesenchymal cell proliferation is seen subjacent to the line of
demarcation, there is a dense accumulation of connective tissue fibres (fine and coarse) and
concomitant increase in agyrophilic fibres.
ii. three to seven days following injury, collagen formation, agyrophilic fibre organisation and a
number of fibroblasts and mesenchymal cells have developed to present a modified cell rich layer in
which the cells proliferate and differentiate into odontoblasts. Below this area agyrophilic fibres
become organised perpendicular to the line of demarcation then they splay and take on the
characteristics of collagen.
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iii. By seven days the collagenous matrix differentiates and thickens, engulfing the more superficial
areas and the more alkaline environment favours odontoblastic differentiation and replication over
fibroblasts.
As soon as predentine has formed, calcification soon follows and in sound teeth (particularly young
teeth with open roots) tubular predentine formation may be laid down within two weeks however, in
older teeth irregular (reparative) atubular dentine is often found (Stanley 1989). Dystrophic
calcification of the coagulation zone (and degenerated cells in the adjacent tissue) starts the
process of mineralization in the vital tissue containing the newly formed collagen (Schroder 1985).
At one month, the barrier shows predentine with odontoblasts on the pulpal aspect lined by an
irregular osteodentine-like tissue, after 3 months the barrier is distinctly two layered as the pulpal
side of the barrier becomes more highly differentiated. The final barrier forms a pit around the
exposure site because it formerly contained the necrotic tissue that eventually degenerates.
2) Low pH calcium hydroxide (pH 9-11 eg. Life, Dycal, VLC Dycal)
Fitzgerald (1979) observed the following sequence of pulpal healing in monkeys:
i. first day- a large fibrin clot, containing many RBC and varying leukocytes (PMNLs) was seen
closely adapted to the pulp capping agent.
ii. two days- the clot contracts towards the pulp with clefts suggesting active fibrinolysis.
iii. three to four days- clot resolution continues with fibroblasts migrating towards the capping agent.
Nearby blood vessels are still congested and capillary infiltration increases adjacent to the capping
agent.
iii. five days- a one, to three cell-thick layer of fibroblasts aligns parallel and adjacent to the capping
agent.
Iv. six days- full resolution of the clot with viable fibroblasts within the cavity replacing the clot and
forming a layer 4-7 cells thick. The area immediately subjacent to the exposure shows an alignment
of cells resembling odontoblasts.
v. seven days- short processes were seen extending from the cells adjacent to the capping agent
towards it.
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vi. eight days- showed a zone of amorphous material between the fibroblasts and the cap.
vii. nine days- a thick zone of calcified dentine like material extended across the exposure site.
Inflammation and vascular congestion in adjacent tissues was minimal.
viii. ten days- increased dentine bridging and the tissues further approached normality.
Fitzgerald (1979) summarised the early sequence of pulp healing as:-
The clot is resolved by lysis and macrophage infiltration, fibroblasts and endothelial cells invade the
clot area to form granulation tissue and these cells organise and differentiate into functional
odontoblasts as early as nine days after exposure.
Fitzgerald’s study showed conformity with studies by Schroder (1973), Mjor et al., (1991) and
supported Cox and Bergenholtz (1986) in the concept that polymorphonuclear monocytes and
macrophages played important roles in inflammation and healing (also Reeves and Stanley 1966).
Plasma cells and lymphocytes are characteristic of chronically inflamed pulps (Torneck 1981) and
were not seen in this study.
Schroder’s (1985) study suggested the following summary of healing after the application of
calcium hydroxide to the pulp:-
i. proliferation, migration and differentiation of papilla cells
ii. elaboration of new collagenous matrix
iii. dystrophic calcification of the area of necrosis
iv. mineralization of the newly deposited collagen which led to the formation of fibro- or
osteodentine.
v. a new generation of odontoblast-like cells differentiated and deposited dentine.
Increased cellular activity is seen in connection with application of calcium hydroxide to the pulp
with increases in DNA synthesis in fibroblasts and endothelial cells in the monkey pulp (Fitzgerald
1979). From Fitzgerald’s observations, and 3H-thymidine studies, he proposed that fibroblasts may
be the cells that replace odontoblasts. The calcium ions are well tolerated by the tissues (Schroder
1985) and can increase cell proliferation (Das 1981). Calcium is necessary for cell migration,
differentiation and mineralization (Schroder 1985). It can activate adenosine triphosphatase (Guo
and Messer 1976) and is mitogenic to pulp fibroblasts (Torneck et al., 1983). Torneck also showed
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that certain concentrations of calcium hydroxide are mitogenic for pulp fibroblasts in vitro while
others were not.
The high pH of calcium hydroxide neutralizes the acidic nature of the infected and inflamed tissues
(Heithersay 1975, Jaber et al., 1992, Cox and Suzuki 1994) and stimulates various cell-enzyme
systems affecting positive fibroblast migration, proliferation and eventual tissue repair (Yamamura
1985, Cox et al., 1982, Torneck et al., 1983). Indirectly, calcium hydroxides alkalinity may also
result in the release of growth factors from dentine leading to stimulation of odontoblast
differentiation (Lesot et al., 1993).
The hydroxyl ions induce the chemical injury which results in the limiting necrosis responsible for
stimulating pulpal defence and repair (Schroder 1985). Unfortunately this ability to induce necrosis
means that when calcium hydroxide agents are placed against active haemorrhage there is the
possibility that some of the particles will be introduced to the venous system and will cause focal
necrosis and inflammation when they lodge in a vessel. If enough foci are formed the subsequent
coalescence may cause pulpal death (Stanley and Lundy 1972). The release of hydroxyl ions also
correlates strongly with calcium hydroxide's antimicrobial effects (Fisher and Shortall 1984, Lado et
al., 1986). Milosevic (1991) suggests a role for calcium could be to reduce the solubility of the
hydroxyl ion component and thus reduce the toxicity and pH. The chemical cautery produced by
calcium hydroxide penetrates from 0.3-0.7mm into the pulp. This is particularly relevant in some
anterior teeth where some diameters of labial-lingual thickness may be less than 0.5mm. The pulpal
tissue superior to the capping agent may be cut off from its blood supply causing necrosis and
potentially release sufficient toxin to cause total pulpal death (Stanley and Lundy 1972) this is
known as “strangulation necrosis”.
b) calcification and reparative dentine
Cellular events in calcification
There has been some speculation about the role of calcium hydroxide in the initiation of
calcification with some authors such as Cotton (1974) claiming that necrosis, not calcium hydroxide
was the important factor. The deposition of mineral in the newly formed collagen has its beginnings
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as dystrophic calcification of the zone of firm necrosis and degenerated cells in adjacent tissue
(Baume and Holz 1981). Matrix vesicles are seen at this time indicating initial mineralization
(Schroder 1985, Schroder and Sundstrom 1974, and Hayashi 1982). In contrast authors such as
Mjor et al., (1991) proposed that no necrotic layer was necessary for a stimulatory effect for dentine
bridging production (unlike Schroder 1985) and could not find the stage specific basement
membrane required for odontogenesis mentioned by Ruch (1982). It has since been suggested that
during repair, fibrodentine could control odontoblast differentiation and assume the role played by
the basement membrane during odontogenesis (Ruch 1985). In fact, some studies suggest there
are no unique biological or therapeutic properties of the calcium hydroxide preparations that
specifically stimulate reparative dentine and subsequent bridge formation (Cvek et al., 1978, Cox et
al., 1987, Cox and Suzuki 1994).
Secretion of tertiary dentine may be made by post-mitotic true odontoblast cells, or by odontoblast-
like cells differentiated from the pulpal cell population (undifferentiated mesenchymal cells,
fibroblasts, pericytes and endothelial cells), the morphology of the cells producing the matrix can be
quite variable as can the structure of the matrix itself (Lesot et al., 1993).
Tziafas et al., (1994) showed in vivo that serum fibronectin exhibits a high affinity for microcrystals
produced at the surface of calcium hydroxide-containing cements. After pulp exposure, a zone of
new collagen is deposited subjacent to the wound, attracting further calcium ions and creating a
pulp biomatrix secreted by, they believe, fibroblasts or "osteodentinoblasts". Initiation of tubular
dentine formation is thought to require further cell/matrix interactions. Tziafas suggests a mediating
role for fibronectin during the pulp biomatrix-cell interactions because of its strong affinity for
collagenous matrix. Fibronectin is involved in the process of odontoblast elongation and polarization
(Lesot et al., 1988) and has been shown to induce ecto-mesenchymal cells to do the same and
produce calcified tubular and atubular dentinal matrix in dog dental papillae (Tziafas 1992).
Vaahtokari et al., (1991) suggested that because TGFß is synthesized by differentiated
odontoblasts, this may stimulate other dental papilla cells which have the right cell surface
receptors to change their phenotypes and give rise to reparative odontoblasts.
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Theories of mineralization
Following the initial seeding of mineral in a collagenous tissue an epitactic mechanism operates to
enable mineralization to take place (Foreman and Barnes 1990). Hayashi (1982) studied the
healing in amputated dog pulps after the application of calcium hydroxide and found that initial
calcification was characterised by an abundance of extracellular matrix vesicles (0.1-0.2mm)
between the forming cells and the wound surface. As the vesicles matured, needle-like crystals
grew, the vesicular membrane disappeared and the crystals aggregated in calcified fronts. This
suggests that the calcification events that occur in the pulp are similar to those in other normal and
pathologic calcified tissues.
There are a number of theories regarding the role of calcium hydroxide in mineralization:-
- Free hydroxyl ions cause a rise in pH initiating or favouring mineralization (Tronstad et al., 1981, a
study with admitted errors in the measurement of pH). This study showed that diffusion through the
dentine could occur after a calcium hydroxide root filling (circumpulpal pH 8.0-11.1 through to
peripheral dentine of pH 7.4-9.6) and was thus useful for managing resorption. However, other
highly alkaline compounds fail to initiate mineralization. The high pH acts as a local buffer against
the acidity of inflammation (Heithersay 1975) and may neutralize the lactic acid produced by
osteoclasts preventing breakdown of mineralized tissue.
Milosevic (1991) suggests that there is a critical degree of alkalinity for dentinogenesis to take place
proposing the pH 10.2 found by Gordon (1985) to be optimal for mineralization. This may also
activate alkaline phosphatase activity (Guo and Messer 1976).
However, the pH of setting calcium hydroxide materials is reduced to almost neutral in contact with
dentine (Ida et al., 1989) thus minimizing the likelihood of beneficial effects from the high pH of
these materials.
- Torneck et al., 1983 proposed a mitogenic and osteogenic effect from the combination of high pH
and calcium and hydroxyl ions, thus affecting enzymatic pathways and mineralization.
- Once mineralization has started it is important that the process can be halted - one such factor is
the presence of pyrophosphate ions which normally act as inhibitors. Pyrophosphatase, a member
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of the alkaline phosphatase group, is necessary in any mineralizing tissue to breakdown
pyrophosphate (Foreman and Barnes 1990).
Heithersay (1975) has proposed that calcium ions reduced the permeability of new capillaries thus
decreasing intercellular serum and concentrating calcium ions at the mineralization site. The high
calcium content of the tissues would increase the concentration of calcium-dependent
pyrophosphatase, while the decreased permeability of the capillaries would restrict the amount of
pyrophosphate getting through, allowing a potential for uncontrolled mineralization and pulpal
obliteration.
- Pisanti and Sciaky (1964) sought to show that the calcium in the protective wall did not come from
the calcium hydroxide capping material but rather from the bloodstream by using labelled calcium
hydroxide injected intravenously into dogs and comparing radioautographs. This suggested that
calcium hydroxide was an initiator rather than a substrate for repair. Holland et al., (1982) challenge
Pisanti’s hypothesis, and again using the dog for comparison, they suggested that calcium
hydroxide may have a role in the healing process by helping to compose the birefringent
granulations (calcium carbonate as calcite) in the superficial layer of the hard tissue bridge. Calcium
carbonate formation is an immediate tissue response and should be considered with the initial
mineralization following calcium hydroxide application because of the large von Kossa-positive
granulations. The carbonate granulations possibly stimulate the precipitation of other calcium salts
and start the mineralization of collagen. These granulations may encourage pulpal tissue to
precipitate a calcium salt layer at the beginning of the healing process allowing more favourable
conditions for odontoblast differentiation and dentine bridging.
- Ca2+, Mg2+ -activated adenosine triphosphatases (ATPases) have been identified in tissues where
intracellular calcium regulation or transcellular calcium transport are important. ATPases have been
demonstrated in the dental pulp with greatest activity in the odontoblast and subodontoblast layers
and may play a role in mineralization of dentine (Guo and Messer 1976). Energy from adenosine
triphosphate breakdown could be used to pump calcium across concentration gradients. Guo and
Messer found that the ATPase in pulpal tissue appeared to be membrane bound and activated by
Ca2+ at low concentrations. Research has shown that extracellular vesicles are involved with the
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mineralization of dentine and bone. The proposed mechanism involves accumulation and
precipitation of calcium and phosphate within the vesicles, with ATP acting as the source of energy
of the calcium pump.
c) antibacterial properties of calcium hydroxide
Bacterial contamination is a major contributing factor to the success or failure of pulpal healing and
this can be seen by the dramatic healing capacity of germ-free animals, in which bridging can occur
without any treatment (Kakehashi et al., 1965), irrespective of the capping material (Paterson
1972), under irritants (Watts and Paterson 1987), or even under toxic materials (Cox et al., 1987).
Paterson (1976) states the impossibility of creating a completely sterile exposure. Thus the
importance of control of bacterial colonization cannot be understated for pulpal healing (Cox et al.,
1987, Fisher and Shortall 1984) and for reparative dentine bridging (Heide 1991).
The high pH of calcium hydroxide is thought to neutralize the infected tissues and disinfect carious
dentine (Paterson 1972, Fisher 1981, Milosevic 1991, Cox and Suzuki 1994, Leinfelder 1994) and
this function is thought to be linked to its ability to diffuse from the capping material (Fisher and
McCabe 1978). The antibacterial activity of calcium hydroxide products can be directly correlated to
the availability of their hydroxyl ions (Foreman and Barnes 1990). Safavi and Nichols (1993)
showed calcium hydroxide could mediate the degradation of bacterial lipopolysaccharide, in
particular the lipid component known as Lipid A, which is thought to be responsible for effects like
toxicity, pyrogenicity, macrophage and complement activation. Fisher (1972) found sterilization in
10 carious teeth following application of a calcium hydroxide/water paste. After 6 months bacterial
samples were taken and cultured - no viable organisms were detected but the calcium hydroxide
material was friable and unsuitable mechanically. Hard-setting calcium hydroxide agents such as
Dycal have shown dentine disinfection in vivo for periods of up to 6 months (Fisher 1977) and
strong in vitro inhibition of bacterial growth in organisms such as Lactobacillus casei and
Streptococcus mutans (Fisher and Shortall 1984). Lado et al., (1986) demonstrated that setting
calcium hydroxide compounds such as Dycal®, Life®, and Renew® were more effective
antibacterials against Lactobacillus, Actinomyces and Streptococcal species in vitro than reagent
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calcium hydroxide. Lado and Stanley (1987) showed that visible light-cured products are just as
effective as self-curing calcium hydroxide products (Life®, Dycal® etc) in inhibiting the growth of
bacterial organisms in vitro.
However the efficacy of calcium hydroxide as an antibacterial has been questioned by in vivo
studies in rats showing no hindrance of bacterial growth and colonization seen adjacent to the cap
(Cotton 1974). Watts and Paterson (1987) have found bacteria in intimate contact with and present
in the cavity and coronal pulp of rats treated with calcium hydroxide. Calcium hydroxide appears to
be ineffective against Streptococcus faecalis over 7 experimental days in infected dentinal tubules
(Heling et al., 1991). Calcium hydroxide does not appear to kill bacteria that have penetrated
necrotic tissue (Cox et al., 1982) and is therefore indicated only for the treatment of superficially
contaminated pulps (Watts and Paterson 1987). Cox et al., (1985) felt that cases of persistent
inflammation and long-term failure of teeth capped with calcium hydroxide were due to
contamination of the dental pulp by bacteria. The source of these bacteria may be from bacteria
entrapped at the time of cavity preparation which multiplied as the medicaments efficacy as an
antibacterial decreased. More plausibly, they came from microleakage progressing through the
medicament as it lost its antimicrobial properties, thus leading to ultimate clinical failure. These
bacteria also lower the pH by converting the capping material to calcium carbonate (Watts and
Paterson 1987) thus further decreasing its antibacterial efficacy. Milosevic (1991) has pointed out
that the persistence of bacteria in rats may be due to a possible species specificity of microbial
sensitivity to calcium hydroxide.
What is considered successful pulp-capping with calcium hydroxide?
Kopel (1991) considered successful pulp capping to have occurred with dentine bridging,
maintenance of pulp vitality, lack of undue sensitivity, minimal pulpal inflammatory response, ability
of the pulp to maintain itself without progressive degeneration and lack of internal resorption and or
intraradicular pathology. A simpler construct is “vitality without inflammation” (Pitt-Ford 1985).
Horsted et al., (1985) used the following as clinical indicators of successful calcium hydroxide pulp-
capping:-
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1.There was no history of pain or discomfort
2.Positive response to electric pulp tester
3.No tenderness to percussion
4.No periapical pathology observed radiographically.
Horsted et al., (1985) showed an 81.8% survival rate in 510 calcium hydroxide-capped human teeth
after five years with a fairly constant failure rate over time, they felt extended study times may
present complicating factors, such as repeated operative procedures, further damaging the pulp
and obscuring the reason for failure. Baume and Holz (1981) reported an 80 - 90% success rate in
calcium hydroxide capped teeth if the pulp was accidentally injured, showed no symptoms and was
hermetically sealed. In traumatically exposed teeth, with minimal contamination, success rates of
96% have been reported (Cvek 1978). Watts and Paterson (1981) suggest the dentine bridge may
be a good criterion for success in pulp capping studies because odontoblasts are delicate cells and
their continued function to produce dentine, in close proximity to an exposure, indicates healthy
pulpal function. Langeland et al., (1971) however believe bridging is not the criteria for success and
believe the tooth should be free from inflammation and resorption/apposition .
Despite the fact that there are some authors who feel there are no unique properties of the calcium
hydroxide preparations that specifically produce reparative dentine bridge formation (Cox et al.,
1987, Cox and Suzuki 1994), it is often generally held that repair does occur. This encourages the
use of calcium hydroxide as a capping agent.
Some substances, such as tricalcium phosphate, produce more reparative dentine than calcium
hydroxide (Chohayeb et al., 1991) but leave the pulp often severely inflamed. This calls into
question Stanley's (1972) contention that reparative dentine is essential for maintaining the health
of pulp tissue after exposure. Criticism has been levelled at the permeability and porosity of the
dentine bridge formed by calcium hydroxide (Langeland et al., 1971, Cox et al., 1985). Some
authors believe this is the reason for the ultimate clinical failure of calcium hydroxide as a direct
pulp capping agent - its inability to provide a long term seal against microleakage (Cox and Suzuki
1994).
However studies, such as those by Holland et al., (1979), have found that the hard tissue barriers
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formed proved reliable in affording pulpal protection and were more effective than the primary
tubular dentine of the cavity floor in protecting the pulp from irritant induced inflammation, possibly
because the coronal surface has no, or few, dentinal tubules. Incomplete bridges were seen in this
study and these did not provide pulpal protection. The dentine bridge may simply act as an
additional barrier to irritants from within the cavity as well as the oral environment (Periera and
Stanley 1981) and the very formation of the bridge demonstrates active pulpal cell function (Watts
and Paterson 1981).
Hume and Massey (1990) suggest that the long term sequelae of calcium hydroxide capping use is
a high incidence of late calcification or necrosis of the pulp. Toxicity screening data (Hume 1985)
suggest hard- setting calcium hydroxide cements provide a good chemical barrier but are capable
of releasing moderately toxic components that may adversely effect pulpal cells.
Factors modifying the success of calcium hydroxide capping
1. Bacteria
There is little doubt that the biggest modifying factor for the success of any form of pulp-capping is
the presence of bacteria, sterile pulp exposures tend to heal, whatever the capping agent, as
shown by the germ-free animal studies quoted earlier.
Cox et al., 1985 studied the long-term effects of calcium hydroxide direct pulp capping on the tissue
and found vitality may be preserved but that 50% of the teeth capped demonstrated varying
degrees of pulpal inflammation over periods of 1-2 years. This is at variance with studies utilizing
shorter time periods and it was felt that, after initial healing (as evidenced by hard tissue formation
seen in 86% of pulps), bacterial- based irritation occurred. The major causes of post-operative
inflammation are non-sterile procedures and bacterial infiltration due to inadequate sealing of the
exposure area (Brännström and Nyborg 1974). Clinical studies with 2338 human teeth have shown
that the risk of failure is increased if the pulp is diseased before capping (Baume and Holz 1981).
Interestingly, Horsted et al., (1985) showed in humans that the capping of carious exposures
showed a success rate similar to accidentally exposed pulps. However, these carious cases were
pain free, the exposures were less than 1mm square, the exposures were in the coronal third,
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bleeding occurred instantly and there was no periapical lesion evident on radiographs. Pulps can
still heal after exposure to bacteria in saliva if the contamination is superficial (Cox et al., 1982).
The importance of minimizing bacterial contamination of the pulp at, and subsequent to, capping
cannot be overemphasized for successful healing (Langer et al., 1970). The extent of bacterial
infiltration and the microbial species involved can modify the response of the pulp to calcium
hydroxide (Milosevic 1991).
2. Size of pulpal exposure
Cohen and Burns (1987) believe that pulp capping should only be performed on small traumatic or
mechanical exposures and other authors have found that the larger the exposure, the poorer the
prognosis because of increased bacterial contamination (Foreman and Barnes 1990). It is possible
that pulp exposures can be too small for effective pulp capping and this can result where the
calcium hydroxide fails to make contact with the living pulp tissue either due to pulpal shrinkage or
debris blockage. Heide (1991) suggests that lack of initial contact between the pulp and the capping
agent merely retarded fibrodentine and tubular dentine deposition rather than preventing complete
bridge formation altogether.
Some authors feel that size is immaterial (Cvek 1978, Periera and Stanley 1981) and Stanley
(1989) simply likens pulpotomies to large pulp caps.
3. Pulpal bleeding
It has been a long held view that calcium hydroxide should never be placed against a bleeding or
oozing (serum or plasma) pulp, otherwise the subsequent clot will prevent the desired chemical
necrosis and allow possible secondary infection (Schroder 1973, Schroder 1985, Stanley 1989).
Extrapulpal blood clots seriously impair healing and sustain chronic inflammation with incomplete
and poorly formed dentinal barriers (Schroder 1973). Blood clots can attract PMNs chemotactically,
because of their fibrin content, thus prolonging the inflammatory response, they may neutralize the
hydroxyl ions and may act as a bacterial substrate (Schroder 1985). If the surface of the pulp cap is
irregular, because of continued bleeding before setting, clot organization may be prevented (Pitt-
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Ford 1985).
Cotton (1982) advocated the use of Hemodent™ (Medical Products Laboratories, Philadelphia)
soaked sterile pellets (drained of excess liquid) for bleeding control, as it acts as an astringent and
protein precipitator on the pulpal surface cells. This is particularly important for the newer calcium
hydroxide materials do not tend to coagulate the pulp tissue, or cause capillary thrombosis, and
hence there may be no visible clot.
Pulpal bleeding acts to physically wash away bacteria and debris, however, a pulp stump that
continues to bleed after five minutes may indicate irreversible pulpitis (Webber RT 19813 as quoted
in Stanley 1989).
The vascularity of pulpal tissue is an important factor in pulpal healing because of the degenerative
changes that take place at the trauma site and the necessity of transport for the essential elements
of tissue repair (Periera and Stanley 1981). Their study found that blood clots did not appear to
influence the pulpal response to capping. Blood clots were not intentionally allowed to form as clots
were believed to contribute to a high failure rate (Schroder 1973).
4. Pre-existing pulpal inflammation
There is general consensus that the degree of inflammation, including cellularity and vascularity, at
the time of capping is a decisive factor in the ability of the pulp to heal following calcium hydroxide
capping (Langeland et al., 1971, Baume and Holz 1981, Torneck et al., 1983, Schroder 1985,
Foreman and Barnes 1990). Calcium hydroxide may have a beneficial effect on a superficially
inflamed pulp because its pH may modify the local pH, to levels favouring cellular activity and
repair. However, in cases of widespread pulpal inflammation, the inflammation will be increased, by
the initial effects of the calcium hydroxide, thus delaying healing (Schroder 1985). The success of
calcium hydroxide pulp-capping decreases markedly with the degree of inflammation, particularly
that of a chronic nature.
5. Extent of chemical necrosis
3Webber RT. Traumatic injuries and the role of calcium hydroxide. (Manuscript) 1981:59
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It has been reported that capping failure occurred when pulpal cell cautery was induced across the
pulp chamber cutting off the blood supply for more coronal tissues and also through embolized
particles of Dycal causing enough foci of necrosis to cause pulpal death (Stanley and Lundy 1972).
Hard-setting calcium hydroxide materials, with necrosis and organization directly adjacent to the
cap, have an obvious advantage in comparison to the more extensive changes induced by the soft
formulations. It is clear that the location of the exposure then makes a difference, particularly if the
calcium hydroxide is placed on an narrow area of tissue such as on the edge of a pulp horn, or on
radicular pulp or in a narrow chamber (such as that found in the lower incisor). Other studies have
shown that transference of materials from the site of direct pulp-capping had occurred. If these
materials were contained within phagocytic cells there was little pulpal response, however, if not,
severe inflammatory reaction or necrosis was found (Watts and Paterson 1982).
6. The species
Dog pulp is more sensitive to trauma and seems more prone to degeneration than the human pulp
but its pattern of healing seems similar following capping (Barker and Lockett 1971). Some authors
have found that the application of calcium hydroxide to dog pulps results in variable and sometimes
atypical, generally disappointing results, compared to humans (Mohammed et al., 1961, Barker and
Lockett 197, Periera et al., 1980). In the dog, transference of materials from direct pulp-capping
sites has been frequently observed, compared with no reports of significant transference in humans
(Watts and Paterson 1982). Pitt-Ford (1985) found lower success rates in dogs than in monkeys (in
which success was considered due to pulpal vitality without inflammation). Differences in
inflammation were also found within animals of the same species.
In Watts and Paterson’s (1981) study a less favourable response was seen in the dog than in the
rat with only 9 of 15 pulps capped (with Dycal®) demonstrating bridging, compared to 20 of 24
teeth in the rat (only two beagles were used in this study). This may have been due to a difference
in the bacterial flora - the dogs showed mainly gram negative coliform organisms while rats, with
high sugar-diets, demonstrate mainly streptococci. This perhaps leads to differences in the healing
response.
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The use of calcium hydroxide in rats has also produced variable results - from severe pulpal
inflammation to dense dentine formation (Jaber et al., 1992) and rat pulp is more reactive than
human pulp (Schroder 1985). Using such small animals for capping experiments increases the
variables of operator dexterity, instrumentation and effective restoration (to ensure prevention of
microbial contamination by microleakage) as well as the other biological factors that may influence
the success of capping.
7. Signs and symptoms
Contraindications to direct pulp-capping with calcium hydroxide include, toothaches at night,
spontaneous pain, tooth mobility, thickening of the periodontal membrane, an intraradicular or
periapical radiolucency, excessive bleeding at the exposure site and purulent or serous exudate
from the exposure site (Kopel 1991). Baume and Holz (1981) felt that the only teeth which could be
capped with calcium hydroxide were asymptomatic vital pulps that had sustained accidental injury
and demonstrated no symptoms.
8. Other factors
Older pulps seemed to have reduced healing potential (Horsted et al., 1985) perhaps due to their
decreased circulation. However Baume and Holz (1981) felt that age did not have an adverse
effect.
Horsted et al., (1985) found that over time pulp capping was more successful in molars than
premolars - this may be because of the greater pulpal volume allowing more opportunity for
collateral circulation. Also premolars because of their pulpal anatomy are fairly narrow mesio-
distally and that as most exposures occur on the approximal surfaces greater opportunities for
constriction were present.
When amalgam is subsequently used as a restorative material (Periera et al., 1980), high failure
rates have been reported possibly due to the condensation pressures required for insertion.
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Conclusions
Calcium hydroxide still enjoys widespread clinical usage as a direct pulp-capping agent. It has
dramatic and rapid effects on exposed pulpal soft tissue and seems to stimulate production of a
reparative dentinal bridge by mechanisms which are not yet fully understood. A number of factors
influence its success or failure and calcium hydroxide seems most effective at healing accidental,
uncontaminated exposures in teeth free of inflammation.
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REVIEW IV. BONE MORPHOGENETIC PROTEINS IN ODONTOGENESIS AND
DENTINOGENESIS.
During embryogenesis continuous cell and tissue interreactions result in the organization of cells
into the specialized tissues and organs.
In vertebrates, organs are formed through heterotypic cell interactions, known as induction,
between embryonal epithelial and mesenchymal cells. Recent studies have shown that growth
factors seem to play the central roles in mediating induction of the tooth organ forming cells in
odontogenesis (Lyons et al., 1991, Pelton et al., 1991, Vaahtokari et al., 1991, Vainio et al., 1993).
Other forms of mediation, such as interactions between extracellular matrix molecules and cell
surface receptors are important in differentiation (Thesleff et al., 1978, Ruch et al., 1983, Ruch
1987) but in this process, growth factors are also involved.
Odontogenesis encompasses all of the embryonal ectomesenchymal interactions necessary for the
formation of the tooth. The anlage of the odontogenic apparatus is an area of thickened oral
epithelium, the dental lamina that grows into the mesenchyme of the developing jaw under the
influence of neural crest cells. Along the dental laminae, at sites of the future primary teeth, individual
ectodermal buds grow and influence the adjacent mesenchyme to condense around them.
Interestingly, it is at this stage in early tooth development that the control of morphogenesis switches
from this presumptive dental epithelium to the mesenchyme (Lumsden 1988). The expression of a
number of genes can be seen in the dental mesenchyme at the time of its condensation including
the homeobox-containing transcription factors msx-1 and msx-2 (Hox-7 and Hox-8) (Jowett et al.,
1993) and others (for review see Thesleff et al., 1996). This array of gene products is thought to
determine the competence of the cells to respond to epithelial inductive signals. For example,
experiments separating epithelial and mesenchymal tissues, at a time when the cell-membrane-
proteoglycan syndecan-1 is about to be induced, and the subsequent evidence of its induction in the
freshly dissected mesenchyme, suggests that epithelial signals are diffusible and may act as growth
factors (Vainio and Thesleff 1992). The epithelium invaginates at the cap stage and the adjacent
mesenchyme continues to proliferate and differentiates into the dental papilla. The epithelium
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differentiates into an inner and outer enamel epithelium and the intermediate stellate reticulum. The
cap leads to the bell stage characterized by morphogenesis of the sites of future cusps, adjacent to
areas of epithelial proliferation and apoptosis known as the enamel knots. The cells of the enamel
knot express a number of important growth factors, eg. bone morphogenetic proteins and sonic
hedgehog, suggesting their importance in epithelial-mesenchymal interactions. The late bell stage
sees cytodifferentiation of odontoblasts and ameloblasts and secretion of their respective dentine
and enamel matrices.
Bone Morphogenetic Proteins (BMPs)
In 1965 at the UCLA School of Medicine, Marshall Urist induced bone formation by intramuscular
implantation of demineralized bone in rabbits. The ectopic bone formation observed closely
resembled that seen in embryonic skeletal development and, although Urist was unable to isolate
the ingredient responsible for the bone morphogenesis, he named it Bone Morphogenetic Protein
(BMP) (Urist 1965).
By 1988, four separate DNA clones for BMP were reported by Wozney and his team based on
amino-acid-sequence data of a highly purified preparation of bovine bone, the following were
characterized: BMP-1, BMP-2a (later BMP-2), BMP-2b (later BMP-4) and BMP-3 (Wozney et al.,
1988). Celeste et al., (1990) introduced BMP-5, BMP-6 and BMP-7 (or osteogenic protein-1 (OP-
1)) and finally Ozkaynak and his team (1992) found expression of BMP-8 (or OP-2). BMP-1 was
similar to a known protease, while the others were found to share structural features with the
Transforming Growth Factor-ß (TGF-ß) superfamily, based upon their primary amino-acid-
sequence homology, including the absolute conservation of seven cysteine residues between
TGF-ß and BMPs. They have a pre, pro and mature region which is dimerized through cysteine
disulphide bonds in much the same way as TGF-ß, with the pro region required for proper folding
and dimerization (Celeste et al., 1990). The BMPs could be divided into subgroups with BMP-2
and 4, 92% identical, and BMP-5,-6 and -7 which were on average about 90% identical (Wozney
1992). BMP-8 (OP-2) shows a 76% identity to BMP-5 and a 75% identity to BMP-7 (OP-1) in the
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TGF-ß domain and has a unique eighth cysteine residue in the c-terminal "seven cysteine
domain" of the superfamily. Moreover, the BMPs are structurally related to a family of gene
products that have important roles in the structural development of many diverse organisms.
i) BMP-2 and -4 share 92% homologous amino acids and 74% and 76% homology (BMP-3 43%)
with the Drosophila decapentaplegic (dpp) gene (Wozney et al., 1988) which is critical for the
development of dorsoventral patterning in the fly, suggesting an analogous role in vertebrate
development (Ripamonti 1994).
ii) The Drosophila 60A gene, which has a role in embryonic mesoderm and ectoderm
determination in the fly, shows a greater sequence similarity to BMP-5 (72%), BMP-6 (71%), BMP-
7 (69%) and BMP-8 (65%) than to its relative, dpp. This suggests an evolutionary conservation
predating the divergence of chordates and arthropods (Wharton et al., 1991).
iii) BMPs 2-7 share a strong homology with Xenopus Vg-1, a maternal RNA localized to the vegetal
hemisphere of eggs and affecting the development and formation of mesoderm (Weeks and Melton
1987).
iv) Murine Vgr-1, a protein related to Vg-1, found in a variety of embryonic, neonatal and adult
tissues (Lyons et al., 1989) has a 75% identity to BMP-8 (OP-2) (Ozkaynak et al., 1992).
v) BMPs 2-7 show considerable homology to the activins/inhibins which, in mammals, regulate
erythrocyte differentiation and modulate the release of follicle stimulating hormone (Ling et al.,
1986) while activin is also a mesoderm-inducing substance.
vi) BMP-1 is the human homologue of the Drosophila tolloid gene (Schimell et al., 1991) which
interacts with dpp to ensure proper embryonic patterning in flies and may have a role in the
developing embryonic skeleton.
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This striking evolutionary conservation across species suggests their critical importance for normal
growth and development and their fundamental role in epithelial-mesenchymal interactions and
secondary induction in embryonic tissues (Massague 1990, Rosen and Thies 1992, Alper 1994,
Ripamonti and Reddi 1994, Hogan 1996 (b)). Elimas (1993) paper proposed, from studies of
transgenic mice (with BMP gene constructs), that BMPs were strong signals, in epidermal growth,
for cessation of proliferation and terminal differentiation.
It has been long recognized that BMP-2 plays various roles in morphogenesis and induces cartilage
and bone formation (Wozney et al., 1988, Lyons et al., 1990, Wang et al., 1990). Lyons et al.,
(1990) showed that BMP-2 played multiple roles in pattern formation and morphogenesis in mouse
embryos by showing high levels of BMP-2 mRNA expression in developing limb buds, heart,
whisker follicles, toothbuds and craniofacial mesenchyme.
Roles for the BMP family in odontogenesis
Harada et al., (1990) had determined TGF- was present in dentine matrix and the localization of
TGF- in developing tooth germs (Vaahtokari et al., 1991; Jepsen et al., 1992) suggested a
possible role for the Transforming Growth Factor family in mediating epithelial-mesenchymal
interactions, growth and differentiation (Pelton et al., 1989,1990) during odontogenesis.
With its suggested roles in morphogenesis, induction and pattern formation the presence of BMP-2
in the primordial tooth is not surprising. In toothbuds, BMP-2 transcripts were first discovered at the
base of the bud, localized in a small population of epithelial cells, at 12.5 days and by 14.5 days
were detected, not only over the mesenchymal cells of the papilla, but also in the odontoblast layer
that differentiates from it (Lyons 1990, Thesleff et al., 1995b).
In contrast to Jones et al., (1991), who found no BMP-4 in developing tooth buds, Heikinheimo
(1994) detected low levels of BMP-4 in the human dental papilla mesenchyme at the cap and bell
stages suggesting a role in early tooth morphogenesis and proposed it as an early molecular
marker for odontogenic cells in humans.
Vainio et al., (1993) found BMP-4 expression in the thickened epithelium before it shifted to the
mesenchyme, where it remained until the bell stage (Vainio et al., 1993). Vainio et al., (1993) have
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shown, by introducing BMP-4-releasing agarose beads to dental mesenchyme in culture, that BMP-
4 is a biochemical signal, which mediates secondary induction between epithelial and mesenchymal
tissues during early murine tooth development.
The addition of BMP-4 produced:-
i) a translucent mesenchymal zone
ii) three transcription factors (Msx-1 (Chen et al., 1996 believe Msx-1 acts as an amplifier for BMP-
4), Msx-2 and Egr-1) and
iii) BMP-4s own mesenchymal expression.
BMP-4 could not substitute for presumptive dental epithelium, as it did not produce cellular
proliferation or induce syndecan-1 or tenascin. However the production of transcription factors and
its own autoregulated expression strongly suggest its role as a morphogen. In fact BMPs (2,4 & 7)
are thought to inhibit cell proliferation in the developing tooth bud and cap stages from their
domains in the enamel knot (Vaahtokari et al., 1996). This is in contrast to their actions in the
embryonic ectoderm, kidney and eye where they are required for cell proliferation and survival
(Hogan 1996 (a)). Induction of dental papillary mesenchyme and subsequent completion of tooth
morphogenesis requires transient expression of the transcription factor LEF1. The fact that the
expression of LEF1 can be activated by BMP-4 suggests a role in BMP mediated -inductive tissue
reactions (Kratchowil et al., 1996).
Thesleff et al., (1996) have supported the proposal that BMP-4 is an early epithelial signal helping
to shift odontogenic potential from the epithelial to the mesenchymal cells.
Heikinheimo (1994) proposed that BMP-6 may also mediate epithelial-mesenchymal interactions
controlling cytodifferentiation and may, with BMP-2, be involved in odontoblast secretory function.
BMP-7 was found to have similar distribution and expression to the other members of the family in
the developing tooth, but is not considered an essential factor for tooth development (Helder et al.,
1998).
BMP’s, odontoblasts and matrix secretion.
BMP-2 is expressed in the early dental epithelium (early bell stage, about 3 days later than BMP-4
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(Vainio et al., 1993) and subsequently in the dental papilla (progenitor cells) and odontoblasts (Lyons
et al., 1990). It is thought to act with TGF-ß (Begue-Kirn et al., 1992) or BMP-4 (Vainio et al., 1993)
to initiate odontoblast differentiation. Nakashima's 1994c study also supported the potential role of
BMP-2 in the differentiation of preodontoblasts. Lianjia and colleagues (1993) used
immunohistochemical staining with a monoclonal antibody against BMP on dental pulp tissue and
cell culture. They found BMP containing cells earlier and stronger in the inner enamel epithelium
than in odontoblasts, suggesting some role in induction.
Heikinheimo (1994) immunolocated human BMP-6 proteins in early enamel epithelium with a shift to
dental papilla mesenchyme predominantly within developing and functional odontoblasts, thus
perhaps also inducing the differentiation of the odontoblastic cell lineage.
It has been proposed that the in vivo induction of odontoblast terminal differentiation requires the
upregulation of msx2 transcription of TGF-ß1&3 and BMP-2&-4 to allow polarization and
accumulation of matrix respectively (Begue-Kirn et al., 1994). In 1992, Begue-Kirn et al. had shown
that both BMP-2 and TGF-1, when combined with ethylene diamine tetra-acetic acid (EDTA)-
soluble dentine proteins, not only stimulated matrix secretion but also promoted the cytological and
functional differentiation of odontoblast-like cells in vitro from dental papillae devoid of an inner
dental epithelium and a competent basement membrane. Lesot and colleagues (1993) also found
that odontoblast cytological and functional differentiation in isolated dental papillae could be initiated
and maintained by using BMP-2 in combination with an EDTA-soluble fraction of dentine. The
EDTA-soluble constituents could be replaced by heparin or fibronectin, interacting with BMP-2. This
biologically-active complex triggers odontoblastic functional differentiation. In bovine adult pulp cell
culture, BMP-4 increased expression of extracellular matrix proteins (Nakashima et al., 1994c)
while BMP-2 increased osteocalcin synthesis. Nakashima also found that the expression of type 1
collagen mRNA in pulp cells was increased when BMP-4 was expressed (BMP-2 and BMP-3 had
no effect). Increased collagen synthesis is a significant step in odontoblastic differentiation and
osteocalcin is expressed about the same time as predentine deposition. Heikinheimo (1994) had
located BMP-2 in functional human odontoblasts, by in situ hybridization, thus reinforcing the likely
role for this protein in human dentine matrix production. Bessho et al., (1991) had previously
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extracted bone morphogenetic protein from human dentine matrix.
Lianjia and colleagues (1993) found that BMP increases the level of alkaline phosphatase in dental
pulp cell culture which, as an enzyme, catalyses phosphorylative glycogenolysis and promotes the
storage of adenosine triphosphate, as well as promoting calcification. These results were supported
by Nakashima (1994c) who found BMP-4, BMP-2 and purified natural BMP-3 stimulate alkaline
phosphatase activity in bovine adult pulp cell culture. It is interesting to note that TGF-1 inhibited
activity at both the proliferative and matrix formation stages in vitro.
BMPs as pulp-capping agents
The use of BMPs as pulp capping agents to induce reparative dentine has been relatively
successful in dogs and monkeys (Lianjia et al., 1993, Rutherford et al., 1993, 1994, Nakashima
1994b, Gao et al., 1995, Jepsen et al., 1997) and it is thought that the BMP signals are probably
mediated by interaction of type I and II BMP receptors on cells that are yet to be identified (Guo
et al., 1996). The BMPs 2,4 and 7 in carrier are replaced by reparative dentine when directly
applied to a partially amputated pulp. This avoids encroachment on the remaining vital pulp tissue
(unlike calcium hydroxide) and allows the induction of a predetermined and controlled amount of
reparative dentine. The capping agent is resorbed and initially replaced by a connective tissue
which then mineralizes. Mineralization is 75 % complete at one month and 95% complete at four
months (Rutherford et al., 1994). The dentine formed is initially osteodentine but, with the right
carrier and time, tubular dentine is often deposited (Nakashima 1990,1994b; Lianjia et al., 1993).
BMP-7 has also been shown to form reparative dentine after application to a freshly cut but intact
layer of dentine (Rutherford 1995) and also in direct capping in miniature swine (Jepsen et al.,
1997).
The BMP seems to be most successful when complexed to a carrier such as “ceramic” dentine
(Gao et al., 1995), collagen matrix (Nakashima 1994b, Rutherford et al., 1993), although collagen
was not as successful in dogs as when BMP was combined with inactivated, enriched dentine
matrix (Nakashima 1994b).
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Clearly the research to date suggests that BMPs have important roles in odontogenesis and there
appears to be growing evidence that combinations of growth factors are required for the induction
and functional differentiation of odontoblasts (Heikinheimo 1994), with characteristic stage-specific
distribution patterns emerging for TGF-ß1-3, BMPs, EGF and TGF- in various studies (Lyons et
al., 1990, Pelton et al., 1990, Vaahtokari et al., 1991, Cam et al., 1990, Heikinheimo et al., 1993,
Vainio et al., 1993, Nakashima et al., 1994c). Recent work by Begue-Kirn et al., (1994) has found
that upregulation of transcription of TGF-1, TGF-3, BMP-2 and BMP-4, and transcription factor
msx2 (Jowett et al., 1993, Mackenzie et al., 1992) are essential for the terminal differentiation of
odontoblasts to enable polarization and matrix accumulation in vitro. A recent study has found that
both growth hormone (GH) and insulin-like growth factor-1 (IGF-1) increase BMP-2 and BMP-4
mRNA expression in cultured pulp fibroblasts (Li et al., 1998) and this suggests that BMP may also
mediate some of the influences of GH and IGF-1 in dentinogenesis.
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REVIEW V. CARRIERS FOR DELIVERING GROWTH FACTORS TO THE DENTAL PULP
To maximize the effects of growth factors, suitable carrier systems must be chosen to provide a
suitable medium for storage and delivery to the biological site.
Medical researchers looking for functional carriers of bone morphogenetic protein to bone have
considered the following factors in searching for an appropriate vehicle (Lindholm and Gao 1993):
1. good affinity with BMP and the biological site
2. no toxicity and immunogenicity
3. biodegradability
4. no interference with repair
5. promotion of delivery effect and function
6. mechanical strength
7. amenability to sterilization
8. practical in use
9. easy to work with
Medical researchers have delivered BMP to bone in hydroxyapatite and coralline hydroxyapatite,
tricalcium phosphate, true bone ceramics and organic carriers such as inactivated demineralized
bone matrix, collagen, autolyzed antigen-extracted allogeneic bone, fibrin sealant, polylactic acid-
polyglycolic acid copolymer (PLA) and synthetic composite carriers (from a review by Lindholm
and Gao 1993).
Carriers can modify the responses of the pulp to the growth factors. Rutherford et al., (1993)
found BMP-7 bound with a carrier of bovine type-1 collagen powder (CM) could stimulate
reparative dentine in monkeys where the teeth remained sealed. Nakashima (1994a) successfully
used enriched, inactivated dentine matrix as a carrier for BMP-2 and -4. Gao et al., (1995) found
BMP complexed to ceramic dentine acted as a useful delivery system. Lianjia et al., (1993),
however, successfully used BMP alone applied directly on the pulp despite its high solubility in
vivo (Bessho and Iizuka 1994).
For this present study, the following carrier systems were considered because of their
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biocompatibility, lack of in vivo inflammatory response and minimal chemical interaction with
growth factors (aqueous hydrogels 1-3, or bioerodable polymer matrices 4,5):
1. Sodium alginate gel
2. Polyvinylpyrrolidone gel
3. Carbopol gel
4. Poly-d,l-lactide-co-glycolide matrices
5. Cholesterol/lecithin matrices
A pilot study (unpublished), in collaboration with Prankerd, using carbopol gel found the gel
dispersed too easily into the pulp. Alternately, an alginate gel was trialled encapsulated in a
coating of calcium chloride. The resultant bead could be handled and placed in small exposures
while maintaining a relatively localized reservoir.
Alginic acid is a polyuronic acid whose biofunctional properties are determined by the relative
proportions of the residues of -D-mannuronic and -L-guluronic acids and its calcium-induced
gelation has been well studied (Yostsuyanagi et al., 1987). The alginate gel bead is
biodegradable and biocompatible and has been used for controlled release in oral drug delivery
models (Sugarawa et al., 1994).
Prankerd (unpublished) created the alginate beads by extruding sodium alginate from a needle
into a calcium chloride solution; the bead then hardened and separated from the needle. The
beads were physically stable and the ideal size (approximately 0.9mm) for capping use. The
release of bovine serum albumin (BSA) from the beads was measured. This happened quickly,
with 40% released within the first hour and 98% released within 24 hours.
The calcium chloride coating had the potential to be irritant to the pulp and no pilot study was
carried out to test its compatibility with the pulp. Studies with hypertonic solutions of calcium
hydroxide have shown that it does not induce nerve activity when applied to the exposed pulp of
beagles (Narhi and Hirvonen 1987) and that it decreases nerve excitability when applied to deep
cavities in cats (Panopoulos et al., 1983). Because of calcium chloride's unknown potential to
cause pulpal reaction, some beads were chosen to carry isotonic saline as a baseline
comparison to those carrying growth factors.
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The beads for this dog study were created by using a Gastight#75 50µl syringe with a 21 gauge
needle, 0.2µl of 4% (w/v) sodium alginate was extruded into 1ml of 0.1M calcium chloride
solution, the extrusion hardened and dropped off the end of the needle as a bead. The beads
were able to be handled with tweezers and showed a relatively uniform size of approximately
0.9mm. The growth factors were adsorbed into the dried beads at the concentrations given
below, under sterile conditions, and stored in sterile aliquots.
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CHAPTER 5.
EXPERIMENT 2.
A histological comparison of growth hormone and growth factors with calcium hydroxide
and a steroidal-antibiotic combination as dental pulp-capping agents in the dog.
INTRODUCTION
One objective in modern clinical dentistry is to maintain oral health through preservation of an intact,
vital dentition. Vital pulp tissue affords maximal functional protection from the forces of mastication in
teeth by ensuring the resilience and suppleness of the dentine. Moreover vital pulp has the ability to
produce secondary and reparative dentine (Stanley 1989) in response to damage.
When the pulp is exposed to the oral environment, its vitality is threatened and it must be protected
and allowed to heal - this is direct pulp capping. The degree of pulpal inflammation at the time of
exposure appears to determine its ability to heal. This can be modified by operative/mechanical
injury or traumatic exposure, caries, active or chronic, as well as contamination of the exposed pulp
by microorganisms. In fact, some authors believe that a pulp that is protected from infection will heal
without specific topical pulpal treatment (Kakehashi et al., 1965) and may heal even under toxic or
irritant materials (Cox et al., 1987, Watts et al., 1987).
Any pulpal damage elicits an inflammatory response that modifies healing. Polymorphonuclear
leukocytes (PMNL) are the first inflammatory cells to migrate in large numbers into the damaged
pulp and are often regarded as the hallmarks of acute inflammation. Their normal function is to
engulf and destroy bacteria and stimulate coagulation, fibrinolysis and release of kinins (Goldstein
1977). However the enzymes contained in their intracellular lysosomes can also degrade collagen,
elastin, vascular basement membrane and destroy local growth factors.
In the pulp underlying carious lesions, the usual infiltration consists of lymphocytes, plasma cells and
macrophages. The plasma cells frequently seen in chronic pulpitis secrete humoral antibodies acting
as precipitins, opsonins, agglutinins and lysins all aiding to destroy bacteria. Macrophages are
recognized as the predominant cell of chronically inflamed tissues, but they are also present later in
acute inflammation. Tissue repair cannot occur without macrophages which may fuse to form
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multinucleated giant cells.
Fitzgerald 1979 proposed that after mechanical pulp exposure, the early sequence of pulp healing
can be divided into 3 stages: -
1.The clot is resolved by lysis and macrophage infiltration
2.Fibroblasts and endothelial cells invade the clot area to form granulation tissue
3.These cells organize and differentiate into functional odontoblast-like cells.
Physiologically, odontoblasts act as a selective barrier protecting the pulp from fluid/molecular/ion
flow from the dentinal tubules affected by caries or restorative procedures and it is the junctional
complexes between the cells that modify this barrier (Magliore et al., 1992). Odontoblasts chronically
exposed to the irritants released from established carious lesions will be destroyed (Trowbridge
1981, Langeland 1987) and reparative dentine will be deposited subjacent to the affected tubules in
the pulp.
Reparative dentine is produced by odontoblast-like cells. They have a polarized nucleus, cellular
extensions, well-developed rough surfaced endoplasmic reticulum and are arranged in an epithelial
fashion. They generate the organic matrix which later mineralizes (Yamamura 1985). A calcium
binding protein, 28kDa calbindin, only found in odontoblasts, is found in these cells (Magliore et al.,
1988a). However they cannot be considered fully differentiated odontoblasts for they synthesize type
I and III collagen and fibronectin (Magliore et al., 1988b). Moreover, in rats, Chiego (1992) has
found that primary and replacement odontoblasts are morphologically and physiologically dissimilar.
Fitzgerald et al., (1990) has proposed that the replacement odontoblasts arise from a proliferation of
perivascular cells in a process similar to that of fibroblast formation in soft tissue repair. Chiego
(1992) used 125I labelled fibrinogen in rat molars to determine that operative trauma can effect rapid
changes in the pulp, with plasma proteins from the circulation moving between the odontoblasts, up
the tubules and to the cut dentine surface as early as five minutes after cavity preparation. Thus
trauma leads to disruption of the odontoblast cell layer and their junctional complexes, with
odontoblasts being aspirated and broken down by a process suggestive of cell death. It has been
suggested that such damage to the dentine matrix from operative procedures and dentinal caries
may release growth factors from extracellular matrix. Specifically the growth factors Insulin-like
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Growth Factor-1 (IGF-1), Insulin-like Growth Factor-2 (IGF-2), Transforming Growth Factor-ß (TGF-
ß) and Bone Morphogenetic Proteins (BMPs) (Finkelman et al., 1990, Harada et al., 1990, Bessho
et al., 1991, Kawai and Urist 1989) are thought to modify the odontoblasts' response to injury
(Finkelman et al., 1990, Magliore et al., 1992) and thus stimulate the formation of reactionary dentine
(Lesot et al., 1993). There is also abundant evidence that active fractions from dentine matrix and
growth factors can initiate and maintain odontoblast differentiation in isolated dental papillae in vitro
(Lesot et al., 1986, Bégue-Kirn et al., 1992).
Thus, in view of the possible role of growth factors in stimulating odontogenesis and hence dentine
repair, combinations of growth factors were empirically placed within cavities drilled into the dentine
of dogs teeth and into contact with pulp tissue through an exposure. A suitable vehicle for carrying
the growth factors was also trialled. These treatments were compared with calcium hydroxide, as a
capping agent of established ability to induce reparative dentine, and with a steroid-antibiotic
combination used in pulp capping to reduce inflammation and infection.
The treatments trialled were: -
a) Growth Hormone (GH), Insulin-like Growth Factor-1 (IGF-1) naturally occurring proteins found in
tooth buds and dentine matrix (Finkelman et al., 1990, Harada et al., 1990). GH and IGF-1 have
been shown by the authors to stimulate differentiation and growth in mouse molar tooth buds in vitro
(Young et al., 1995). IGF-1 stimulated the differentiation and development of odontoblasts in these
buds and stimulated dentinal matrix formation; GH priming may have potentiated its effect. Growth
Hormone stimulated DNA synthesis and mitotic activity in the odontogenic epithelia and
mesenchyme and increased cell proliferation within the tooth buds.
b) Bone Morphogenetic Proteins 2&4 (BMP2&4), these proteins are closely related to the
Transforming Growth Factor-ß (TGF-ß) family which has important roles in structural development, a
fundamental role in epithelial-mesenchymal interactions and secondary induction in embryonic
tissues. BMP2&4 are 92% identical and show important roles in toothbud development and in vitro
dentinogenesis (Lyons et al., 1990, Heikinheimo 1994, Vainio et al., 1993, Thesleff et al., 1996,
Nakashima 1992, 1994c and Begue-Kirn 1992). Bone Morphogenetic Proteins are found in dentine
(Bessho et al., 1991, Kawai and Urist 1989) and have been successfully trialled as pulp capping
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agents by Nakashima (1994 a,b) and Rutherford et al., (1993, 1994).
c) Calcium hydroxide paste, a traditional pulp-capping agent known to produce reparative dentine
bridging after chemically cauterizing the pulp.
d) An antibiotic/anti-inflammatory combination, these arose out of the success of steroidal anti-
inflammatory drugs in general medicine in the 1950s. It was believed that suppression of the
inflammatory response would allow pulpal healing to take place and the antibiotic was required to
protect the tissue in its immunosuppressed state.
It was hypothesized that the various growth factors would stimulate the production of dentine
comparable to the known effects of calcium hydroxide, and superior to the corticosteroid-antibiotic
combination, with accompanying pulpal health.
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MATERIALS AND METHODS
All animals used in this experiment were handled in accordance with National Health and Medical
Research Council guidelines and ethical clearance was granted by the Dental Animal Ethics
Committee, University of Queensland (AEEC renewal of DENT213/93/ADRF/SPG).
Six male dogs, each approximately two years old, were tranquilized with acepromazine (0.5 ml)
anaesthetized by intravenous general anaesthetic (Nembutal) (approx. 6 ml at onset, and added as
required), this was supplemented by the local anaesthetic prilocaine hydrochloride (66 mg/2.2 ml)
with fellypressin (0.066 IU/2.2 ml) (Citanest™3% with Octapressin, Astra Pharmaceuticals) of 1 ml
per quadrant. All teeth were swabbed with antiseptic solution (Betadine). Rubber dam was not
applied. Black's Class V cavities were prepared on the buccal aspect of molar, premolar and canine
teeth, with a diamond drill in a high speed handpiece under copious sterile water irrigation to remove
enamel and to provide retention form in the dentine. Then a tungsten carbide round bur was used at
slow speed, with sterile saline irrigation, for the final approach to the pulp. Rarely, the slow speed bur
exposed the pulp, however usually, when “pink” dentine was observed, the exposure was created
with a sharp sterile probe. Postoperative bleeding was controlled with sterile cotton pledgets placed
on the exposure, after gentle saline irrigation. Clots were intentionally not allowed to form, as they
have been associated with a high failure rate (Schroder 1973).
The vehicle for all of the growth factors was a permeable bead, 0.8-0.9 mm in diameter, comprising
an absorbent sodium alginate core surrounded by a shell of calcium chloride, developed by one of
the authors (Dr Richard Prankerd). From experiments carried out by Prankerd (unpublished) it was
known that the stored growth factors would be released in the first few days postoperatively. The
growth factors were adsorbed to each bead in the following combinations, concentrations and
estimated quantities per bead, as was sterile physiological saline.
.Bovine growth hormone (Dr Michael Waters, Department of Physiology, University of Queensland)
15 micrograms per bead at a concentration of 5 mg/ml.
.Recombinant human insulin-like growth factor-1 (Genentech, San Francisco, USA) 5 micrograms
plus bovine growth hormone 15 micrograms per bead at a concentration of 5mg/ml.
.Recombinant human bone morphogenetic protein-2 (227 g/ml) and BMP-4 (337 g/ml) (Genetics
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Institute, Cambridge, USA) were incorporated at 4 microlitres per bead.
.Calcium Hydroxide suspension (23% calcium hydroxide, 27% barium sulphate) in the form of Calxyl
Blue® paste (OCO Praparate GmbH, D-67246 Dirmstein) and
.Triamcinolone acetonide/demethylchlortetracycline calcium combination (10% Ledercort™/ 30%
Ledermycin™) in the form of Ledermix™ paste (Lederle Pharmaceuticals GmbH, Wolfratshausen),
were applied as pastes in sufficient quantity to cover the exposure and base of the cavity.
Teeth of differing sizes and in differing quadrants were allotted in a rotation so that the various pulp-
capping treatments were trialled in a variety of tooth sites. Each treatment site was sealed against
microleakage with polymaleinate glass ionomer cement (Ketac-Fil Aplicap®, Espe Dental-Medizin
GmbH, Seefeld), placed with minimal pressure.
The dogs were exercised daily, were closely observed for any signs of distress and the integrity of
the restorations were inspected grossly every two days. To delineate dentine (Boyne and Miller
1961, Crier 1970) formed preoperatively, and at two weeks postoperatively, all dogs received 3ml of
tetracycline intramuscularly on these two occasions (Plate 5).
Two dogs were kept for 14 days (week two group), two for 33 and two for 35 days (week five group)
before sacrifice. At sacrifice, the dogs were anaesthetized, the carotid arteries were cannulated and
were perfused with a lethal dose of sodium pentothal followed by Bouins solution infusion at
approximately 32° C. When the gingival tissues were well coloured by the fixative, the jaws were
removed and the teeth were separated and stored in buffered Bouins fixative. The teeth were further
reduced on a Leitz™ saw microtome 1600 (Leitz Wetzlar, GmbH) to remove extraneous bone and
gingival tissue, then dehydrated in alcohols of increasing strengths. The teeth were not decalcified.
The teeth were infiltrated with two changes of LR White resin and embedded in resin at 56.5° C
under argon gas and a slight vacuum for 24 hours. The blocks were trimmed, mounted and
sectioned at 100 µm sections around and through the exposure point using a Leitz™ microtome.
The sections were stained with Herovici’s polychrome stain and examined under light microscopy
(Photomicroscope III, Carl Zeiss, Oberkochen, West Germany), at magnifications ranging from 40-
400x for signs of pulpal inflammation and repair, without knowledge of the treatments.
Histologic examination of the sections enabled the size of each exposure to be measured, using an
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eyepiece micrometer (Carl Zeiss, Oberkochen, West Germany), as the distance in millimetres
between intact dentine and predentine on both sides of the exposure in the section of the tooth
which showed the widest diameter.
Pulpal inflammation was assessed as per the following criteria (Fitzgerald and Heys 1991) for
grading pulpal histology and comparisons between treatments, over time, and between teeth.
1. None-to-light inflammatory response in the coronal pulp. A few scattered lymphocytes may be
present.
2. Moderate cellular infiltrate of neutrophils and/or monocytes in the coronal pulp
3. Heavy inflammatory response with the presence of neutrophils and/or monocytes involving at
least one third of the coronal pulp
4. Total necrosis of the coronal pulp
The pulps were also assessed for dentine matrix deposition around the exposure and for repair of
the exposure defect, partial or complete. Chips of detached dentine and predentine impacted in the
pulp were also examined for evidence of dentine induction. To be categorized as partial repair,
matrix or reparative dentine had to be deposited at or around the wound site. Complete bridging was
recorded when there was a visible, continuous deposition of matrix across the wound site.
Unstained sections were examined under ultraviolet light on the Zeiss Photomicroscope III for
fluorescent incremental lines demonstrating continued, or disrupted, incremental deposition of
dentine in the area surrounding the exposure.
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plate 5 Fluoro
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RESULTS
General observations
All dogs remained healthy and active throughout the study with no signs of distress, weight loss or
impaired appetite.
At the time of operation, seven premolars were considered too small to treat and five teeth were
damaged early in gross sectioning. The remaining sixty teeth were processed and examined.
The glass ionomer cement dressing proved an effective seal for the capping treatments, with no
obvious signs of bacterial invasion, or microleakage, seen. Two restorations were lost from the 65
teeth treated; this no doubt contributed, partially or wholly, to the resultant pulpal condition of one
necrotic Ledermix-treated pulp (5 weeks) and one GH/IGF-1-treated pulp (2 weeks).
The alginate-calcium chloride carrier was easy to place over the pulpal exposure despite its small
size and seemed to remain in position histologically. In most cases the calcium chloride coating was
not obviously visible pulpally in the sections suggesting dissolution or dislodgement. The alginate
seems to have been tolerated by the pulp, there was no cell growth in apposition to it, nor does there
seem to be any inflammation, cellular organization or cell breakdown produced by extrusions into the
pulp. No pilot study had been done exposing dental pulps to this form of bead.
Pulpal damage is an inevitable sequelae to these invasive procedures on the teeth and in this study
a number of stimuli affected the pulps. The drilling of fresh dentine severed odontoblastic processes
visible as aspiration of odontoblasts from the pulp into the dentine. However as cavity preparation
was carried out with copious irrigation, to minimize heat-induced damage and injudicious desiccation
of the freshly cut surface, minimal damage occurred to odontoblasts peripheral to the exposure site.
Placement of medicaments over the exposure and cut dentinal tubules had direct and possibly
indirect effects on pulpal and odontoblast reactions. Moreover, the exposure of the pulp pushed
chips of dentine and predentine into the pulpal tissue.
The pulps examined in this study, selected two weeks post-operatively, in variable numbers showed
the hallmarks of chronic inflammation - macrophages, lymphocytes and plasma cells. Presumably
the acute inflammatory response, with predominance of polymorphonuclear leukocytes (PMNL) had
subsided.
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The status of the pulps in the treated teeth (over 2 and 5 weeks) fell into the following broad
categories: -
a) Pulps that succumbed to an overwhelming inflammatory response with no organization or repair
b) Pulps in which chronic inflammation persisted, with little or no attempt at hard tissue repair
c) Pulps that showed mild inflammatory response in conjunction with partial hard tissue repair
d) Pulps that were healthy and demonstrated recruitment, development and organization of
odontoblasts and complete hard tissue repair.
Teeth and pulpal inflammation
Both canine and molar teeth demonstrated very similar distributions of pulps in the light to heavy
inflammation range, with only the pulps of two canines of 24 (8.3%) and three molars in 22 (14%)
showing necrosis. In contrast, premolar teeth demonstrated six necrotic pulps out of 14 (43%) and
seven teeth (50%) with heavy pulpal inflammation (Figure 9) - over 90% of premolar pulps had
unfavourable pulpal inflammation.
Exposure size and inflammation
The average width of pulpal exposure size was 0.56 mm from a range that extended from 0.125 to
1.625 mm over 56 measured exposures. In four instances, the pulp was not found to be exposed
through the predentine in the plane of the section. As can be seen from the Figure 10 the majority of
exposures were in the range 0.2-0.6 mm with few exposures at the higher end of the range. In the
1.0-1.8 mm exposure range heavy inflammation was the most common finding although light
inflammation also occurred in the widest (1.6-1.8 mm) category. Interestingly, pulp necrosis was
found more commonly in association with narrower exposures.
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PLATE 6. Following pages
Figure 9. Tooth type and inflammation
Comparison of the degrees of pulpal inflammation encountered in canine, premolar and
molar dog teeth shows that canine and molar teeth were more often lightly inflamed and a
preponderance of heavy inflammation and necrosis was encountered in premolar teeth
following exposure and capping.
Figure 10. Exposure size vs. inflammation
Comparison of the severity of inflammation found in pulps of different exposure widths.
Light inflammation was generally associated with smaller exposures, however heavy
inflammation and necrosis could not, predictably, be related to size.
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Plate 6/9
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Plate 6/10
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Treatments, pulpal inflammation and dentinogenesis
The degrees of inflammation found in pulps, two and five weeks after the various treatments are
summarized in Table 2. and illustrated in Figures 11 and 12. Figure 13 illustrates the degree of
dentinal repair produced by the treatments.
Saline control pulps were found to have moderate or heavy inflammation at two weeks and light or
moderate inflammation at 5 weeks with no instance of necrosis. However, neither partial nor
complete dentine bridging was found associated with the saline treatment (Fig. 13). Figure 14
illustrates the histology of the moderate inflammation found at two weeks associated with an alginate
bead containing saline placed over the exposure. An amorphous protein-like precipitate was found
adjacent to the test material, but no differentiation of odontoblast-like cells or dentine was found
despite continued predentine formation peripheral to the wound. The adjacent pulp contained
inflammatory cells and was hypervascular.
Ledermix treated pulps also showed moderate to heavy inflammation at two weeks however, unlike
the saline controls at five weeks, no instance of light inflammation was found. The pulp chambers
were moderately or heavily inflamed in pulps after 5 weeks. One instance of necrosis was found at
two weeks and none at five weeks. Figures 15 and 16 show the degree of inflammation and lack of
dentine formation associated with this treatment at both two and at five weeks. In association with
the Ledermix paste no odontoblast-like cell differentiation was found. In the primary dentine
surrounding the exposure, odontoblasts whose tubules terminated in the excised dentine appeared
atrophic and had formed no new dentine by five weeks. Nor did chips of dentine in the pulp elicit any
dentinogenic response. There appeared to be a zone of inhibited odontoblastic activity in the pulp
around the exposure site.
In the pulps treated with GH/IGF-1 combination, moderate or heavy inflammation was found at 2
weeks and only light or moderate inflammation was found at 5 weeks (Table 2, Figures 11 and 12),
similar to the saline controls. Three pulps were necrotic in this treatment group (it should be
remembered that all three were in premolar teeth - see Teeth and inflammation above). As illustrated
in Fig. 17, new dentinal matrix was found beneath the exposure and in association with
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PLATE 7 (Following pages)
Figure 11. Pulpal inflammation (2 weeks)
The severity of inflammation in dog tooth pulps after two weeks of treatment with either
calcium hydroxide (Calxyl), growth hormone (gh), growth hormone/insulin-like growth
factor-1 combination (gh/igf-1), antibiotic/antiinflammatory combination (Ledermix), or
normal saline (control).
Figure 12. Pulpal inflammation (5 weeks)
The severity of inflammation in dog tooth pulps after five weeks of the treatments, detailed
in Figure 11, with the addition of treatments utilizing bone morphogenetic proteins 2 and 4
(bmp). Note the proportion of lightly inflamed pulps found in association with growth
hormone and Calxyl preparations.
PLATE 8. (Following pages)
Figure 13. Effect of treatments on dentinogenesis
The effects of treatments on dentinogenesis. Partial indicates stimulation of matrix at the
exposure site. A bridge indicates a zone of reparative dentine. The highest rate of complete
bridging was found after Calxyl treatment. Growth hormone (GH) and GH/IGF-1 in
combination treated pulps showed stimulation of dentinogenesis, while the combination
showed bridging in two instances.
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Plate 7/11
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Plate 7/12
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Plate 8
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Plate 9
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dentinal chips. Moreover this treatment was associated with more secondary dentine produced by
the odontoblasts whose tubules terminated in the primary dentine excision wound, suggesting an
effect by tubule permeation (Fig.17). Complete closure of the wound was found in 2 pulps (Figure
13).
The inflammatory responses of pulps to the growth hormone-containing beads were milder than to
saline or the other capping treatments, except calcium hydroxide at two weeks (Figs. 11 and 12).
However, unlike the combination, growth hormone alone produced only partial bridging of the
exposure by five weeks (Fig.13). Figures 18 and 19 illustrate the histology of the partial bridging
associated with the growth hormone treatment. An amorphous matrix of uncertain origin was evident
close to the exposure and new dentine was found in association with the dentinal chips. Secondary
dentine formation by the odontoblasts peripheral to the wound was evident but was not as marked
as that found with the GH/IGF-1 treatment.
No pulps were exposed to BMP in the two week group and, in those exposed to five weeks of
treatment, the pulpal response ranged from slight inflammation to necrosis. Partial bridging was only
found in 1 pulp and no instance of complete bridging was achieved in this study by BMP. No
qualitative differences were found between the pulpal effects of the bone morphogenetic proteins 2
and 4.
Calcium hydroxide elicited more examples of slightly inflamed pulps at two weeks than any other
treatment, although examples of heavy inflammation and necrosis were also encountered (Figures
11 and 12). It was by far the most successful agent in inducing dentine bridging in this study (Fig.13).
Figure 20a shows that preodontoblast differentiation occurred close to this agent in the area of an
exposure. Figure 20b illustrates that, with minimal exposure, reactive dentine can neatly bridge the
gap within two weeks. Figures 21a and b illustrate the relative capacity of Calxyl to induce reparative
dentinogenesis by five weeks. While not all instances of necrosis in calcium hydroxide treated pulps
were caused by strangulation necrosis, one example was found where a substantial dentine bridge
had spanned the chamber and impeded the vascularity of the pulp (Fig.22).
The fluorescence microscopy was unreliable in demonstrating tetracycline labelling as slides that
were stained did not fluoresce. However Plate 5 shows the effects of Ledermix inhibiting incremental
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dentine deposition, compared to Calxyl which stimulated deposition in areas away from the
exposure, as evidenced by the increased spacing of the fluorescent lines at the dentine-pulp
interface.
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Plate 10
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Plate 11
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Plate 12
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Plate 13
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Plate 14
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DISCUSSION
General
The experimental animals remained healthy throughout the study. It is impossible to state with any
certainty that the teeth treated in this study remained symptom-free; however, over the course of the
study the dogs remained healthy and active with no signs of distress or impaired function, activity or
appetite.
Dog pulp is more sensitive to trauma and seems more prone to degeneration than monkey (Pitt Ford
1985), rat (Watts and Paterson 1981) and human pulps (Barker and Lockett 1971). The dog
provides a tough model for pulp-capping agents. Dogs were also used because of their size; the
teeth were easy to isolate and prepare, and the dental instruments used in clinical practice were
suitable for cavity preparation and pulp therapy.
Rats were not used because, although readily available, they are more difficult to treat and restore
(Jaber et al., 1992). Pulps in the rat model show greater success rates following pulp-capping,
compared to humans, monkeys and dogs, perhaps because of the vascularity of the tooth and its
resistance to infection (Baume and Fiore-Donno 1970). Because its dentinogenesis is rapid and
continuous it has been suggested that it is not a representative model for pulp-capping experiments
(Kirk and Meyer 1992).
The beads and the traditional capping agents tested were relatively easy to place and handle. Non-
setting medicaments were used in order to maximize contact with the pulp at the exposure site
and thus optimize the release of their active ingredients. The use of pastes also minimized the
exposure of the pulp to extraneous and potentially irritating ingredients seen in cement type
medicaments, such as eugenol. By avoiding mixing two-part treatments the potential for
variations in the active ingredients exposed to the pulp were minimized. The calcium chloride
coating had the potential to be irritant to the pulp and no pilot study was carried out to test its
compatibility with the pulp. Studies with hypertonic solutions of calcium hydroxide have shown
that it does not induce nerve activity when applied to the exposed pulp of beagles (Narhi and
Hirvonen 1987) and that it decreases nerve excitability when applied to deep cavities in cats
(Panopoulos et al., 1983). Because of calcium chloride's unknown potential to cause pulpal
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reaction, some beads were chosen to carry isotonic saline as a baseline comparison to those
carrying growth factors.
The conditions for the placement of the glass ionomer cement were ideal because of the moisture
control achieved on the exposed dental quadrants by cotton wool roll isolation, high speed
evacuation, post-restorative varnish and with the dog heads parallel to the surgery table. Pressure of
placement was minimal, due to the good flow of the material, and the cement was simply finished
with a flat plastic instrument.
The process of perfusion in the dogs at the time of sacrifice posed some problems with the blood not
clearing effectively (due to carotid pressure) and the perfusion slowed rapidly, presumably because
of vasoconstriction induced by cold solution and the action of the fixative. The perfusion process that
allowed the most rapid and complete perfusion of the head was to cannulate both carotids, introduce
warmed heparinized saline followed by the warmed Bouins solution. Cannulating both carotids
increased the rapidity of blood loss and decreased intracranial pressure, the heparinized saline
helped clear the head of blood and minimized the amount of Bouins which had to be introduced for
perfusion. Warming the solutions helped decrease the rapidity of vasoconstriction.
The capping agents were evaluated for their compatibility with the pulp by assessment of the pulpal
inflammatory reaction and for their ability to stimulate dentinogenesis. Possible direct effects of the
capping agents on bacteria were not assessed. Nor was it possible to ascribe inflammation or
necrosis to trauma or bacterial infection.
It was hypothesized that the various growth factors would stimulate the production of dentine
comparable to the known effects of calcium hydroxide, and superior to the corticosteroid-antibiotic
combination, with accompanying pulpal health. Growth hormone alone and in combination with
insulin-like growth factor-1 did stimulate dentinogenesis, however not to the extent of calcium
hydroxide treated pulps. Both growth factor treatments resulted in a healthier pulpal inflammatory
state and increased dentinogenesis compared to the corticosteroid-antibiotic and bone
morphogenetic protein treatments.
Pulpal vitality
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Pulpal vitality was maintained, over the period of the experiment, in 52 of 60 pulps overall and the
ratio of necrotic to vital teeth was relatively the same at the two time periods, with 13.3% of pulps in
the two week group necrotic, compared to 13.1% in the five week group. The incidents of necrosis
seen in the dog pulps could be attributed to the following factors:-
1) Bacterial contamination
Ten Cate (1992) states that in the absence of infection, new odontoblast-like cells quickly
differentiate and elaborate dentinal repair after exposure, however the clinical reality is that achieving
capping without some bacterial ingress is unlikely. Sterilization of instruments, aseptic technique,
topical antisepsis, copious irrigation and minimization of postoperative microleakage, through use of
an adhesive restorative, may have all contributed to decrease contamination. The extent of
contamination is unknown as the slides were not stained for bacteria. Two teeth lost restorations,
one pulp was necrotic (Ledermix 2 week group) one pulp was heavily inflamed with likely
progression towards necrosis (GH/IGF-1 2 week group).
Differences in the bacterial flora of different species may influence pulpal outcomes. Dogs
demonstrate less favourable outcomes to capping than rats or humans and this may be because of
a predominance of gram negative coliforms rather than streptococci (Watts and Paterson 1981). The
diminished ability of the dog pulp to heal following capping, compared to other species, ensures that
the dog model provides a tough testing ground for potential capping agents.
2) The ability of the pulp to withstand insult
The type of tooth appears to be significant. In this study, premolars appeared less able to cope with
the trauma of pulp capping as over 90% of premolar pulps demonstrated heavy inflammation or
necrosis. This is likely to be due to the decreased pulp chamber volumes and smaller buccal-lingual
dimensions of premolars which result in decreased ability to cope with the acute inflammatory
response, compared to the molars and canines of dogs. Dog pulps have been found to be more
sensitive to trauma and seem more prone to degeneration than human pulps in capping experiments
despite the similarities in the healing patterns (Barker and Lockett 1972).
While the operative technique utilized was as atraumatic as possible and most of the exposures
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were created by the probe, larger exposures, caused by mechanical exposure with the bur, did not
result in a greater incidence of necrosis. In fact, exposure size did not appear to affect the
inflammatory state of the pulp. The largest exposure sizes produced variably light to heavy
inflammatory responses. This is perhaps not surprising, as the larger exposures would allow wider
exposure of active capping ingredient to the pulp. The clinical success rate of the largest of all
exposures, the pulpotomy, on vital asymptomatic teeth is high (Cvek 1978).
Inflammation of the pulps
Pulpal inflammation changed with time. The graphs in Figures 11 and 12 show a phase shift from
the medium to heavy inflammatory state seen at 2 weeks to generally a lighter, chronic inflammation
in the 5 week group that is consistent with the perception of an ongoing healing process in the pulp
with time.
This was seen most clearly in the reaction to the saline control beads where the 2-week group
demonstrated 66%/33% in the heavy to moderate inflammation range, shifting to 75%/25%
moderate to slight/no inflammation in the 5 week group. Because no pilot study specifically testing
the effects of the calcium chloride beads on pulpal inflammation was carried out, one cannot
eliminate the possibility that the beads had some effect on the persistence of inflammation.
Ledermix treated pulps did not appear to settle over time as all 5-week pulps remained moderately to
heavily inflamed and hyperaemic. This is a somewhat surprising result as it incorporates an anti-
inflammatory agent and given the clinical relief of symptoms seen with its use in humans. Many
studies have also shown continued inflammation, subsequent to the application of corticosteroids
onto carious dentine or the exposed pulp, with little anti-inflammatory effect (Fiore-Donno and
Baume 1966, Harris and Bull 1966, Laws 1967, Baume and Fiore-Donno 1968, Seltzer 1968, Fiore-
Donno, Baume and Fiore-Donno 1970, Langeland et al., 1977, Ulmansky and Langer 1967). Harris
and Bull (1966) and Barker and Lockett (1972) have also noted dilation of blood vessels in the pulp,
perhaps due to corticosteroid control of permeability.
The bone morphogenetic proteins 2 and 4 treated pulps demonstrated variable inflammation from
light to necrotic in the 5-week group with an overall inflammatory presentation more marked than the
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control group. It is difficult to suggest why this occurred. The BMP was human recombinant and may
have been incompatible for dogs, perhaps stimulating some sort of antigenic response, although the
high evolutionary conservation of BMPs across species makes this unlikely. The BMPs were not
tested for activity before use (owing to the small quantities available) and thus may have been
inactive or degraded in transit from the supplier.
The GH treatment produced much more satisfactory pulpal outcomes with an even distribution of
inflammation in the slight, moderate and heavy inflammatory categories in the 2 week group settling
to 66% light and 33% moderate inflammation in the 5 week group. No instance of a necrotic pulp
was recorded.
The combination of GH/IGF-1 produced a less satisfactory inflammatory result, with the 2-week
group demonstrating a mainly moderate to heavy inflammatory result and two necrotic pulps in the
5-week group. This is tempered by the fact that both these teeth were premolars but is perhaps
surprising given that IGF-1 is known to promote wound healing (Lynch et al., 1989, 1991).
Calcium hydroxide chemically cauterizes the pulp under the exposure point and creates zones of
necrosis, through its strong alkalinity. This violent stimulus invariably causes a strong inflammatory
reaction which may settle or persist depending on the pulp's ability to cope. This is found in the 5-
week group where just over half the pulps demonstrated mild inflammation, a third displayed severe
inflammation. One pulp was necrotic. Some studies in humans suggest an 80-90% success rate
following capping with calcium hydroxide (Horsted et al., 1985, Baume and Holz 1981 and Cvek
1978), the results of our experiment demonstrated a 15% failure rate in the dogs. Continued severe
inflammation (33%) in the 5-week group, however, did not offer a healthy long-term prognosis either.
Cox et al., (1985) has found that 50% of teeth capped with calcium hydroxide demonstrated varying
degrees of pulpal inflammation over 1 to 2 years. Calcium hydroxide introduced into the pulp can
cause small foci of necrosis which may coalesce. The chemical cautery induced, by calcium
hydroxide in paste form, can span narrow pulpal chambers restricting blood supply to areas superior
to the exposure leading to “strangulation necrosis” (Stanley and Lundy 1972). The stimulation of
reparative dentine may be so extensive that it too may span the pulpal cavity restricting vascularity
and inducing necrosis (see Figure 22).
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Dentinogenesis
While pulpal healing does not necessarily mean dentinal bridging across the wound site, the
presence of reparative dentine is a sign of healthy function of pulpal cells (Watts and Paterson
1981). This means that the pulp has been healthy enough to recruit, differentiate and divide
odontoblast-like cells and provide the materials necessary for matrix deposition and mineralization.
Despite criticisms in the past of the lack of continuity and permeable nature of such dentine bridges
(Langeland et al., 1971, Cox et al., 1985) they do provide some barrier to further ingress of bacteria
and toxins to the cavity (Periera and Stanley 1981). They also provide a framework for pulpal
regeneration and become increasingly impermeable with time (Holland et al., 1979).
The saline control beads produced no obvious stimulation of dentine production or bridging repair of
the defect in the pulp. Ledermix actively inhibited reparative dentinogenesis and pre-existing
odontoblast activity, consistent with other pulpal studies. This inhibition was present not only at the
exposure site, but also in a zone either side. This was most evident at the pulpal surface of severed
dentinal tubules exposed to the agent and is testament to the diffusion ability of Ledermix’s active
ingredients. Inhibition and disruption of dentinogenesis has been a common finding in Ledermix
studies (Baume 1966, Baume and Fiore-Donno 1970, Baume and Holz 1981, Kirk and Meyers
1992, Clarke 1971a, 1971b, Fiore-Donno and Baume 1966, Laws 1967, Uitto et al., 1975, Baratieri
et al., 1981, Ulmansky et al., 1981) and odontoblastic disruption and atrophy are also seen (Baume
and Fiore-Donno 1970, Mjor and Ostby 1966, Clarke 1971b, Barker and Ehrmann 1969, Ulmansky
and Langer 1967, Barker and Lockett 1972). Corticosteroids and the antibiotic in Ledermix interfere
with collagen synthesis in response to exposure (Uitto 1975). It has also been proposed that
glucocorticoids may inhibit growth by retarding IGF-1 gene expression (Luo and Murphy 1989). The
anti-anabolic effects of corticosteroids on protein metabolism seem to interfere with the formation of
dentinal matrix. Incorporation of amino acids into proteins is blocked thus inhibiting collagen
synthesis and fibroblastic proliferation, the calcification of dentine is disturbed or irregular, and the
acid mucopolysaccharides necessary for predentinal matrix formation are lacking (Baume and Holz
1981).
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Calcium hydroxide is a popular pulp-capping agent and was used here as the positive capping
control, at least in part because of its perceived ability to induce formation of reparative dentine. This
experiment reinforced its “reputation” with over 80% of pulpal wounds covered by a reparative
dentine bridge. Calcium hydroxide induces localized necrosis within an hour of placement on healthy
pulp tissue (Schroder 1985), over the following days mesenchymal cells proliferate in the area,
connective tissue fibres accumulate and agyrophilic fibres increase. Collagen forms, agyrophilic
fibres organize and cells proliferate and differentiate to form odontoblast–like cells which produce the
matrix that will become the reparative dentine bridge. This experiment demonstrated rapid
stimulation of dentinogenesis by calcium hydroxide as evidenced by complete reparative dentine
bridging in the two week group. As mentioned earlier, the reparative dentine bridge is a sign of
continued healthy pulpal cell function and may provide the pulp with protection, as such it is
considered a generally favourable sequelae to pulp-capping. The dentine bridges formed in the five
week group often extended well into the pulp around extrusions of calcium hydroxide and appeared
well formed and continuous with healthy pulpal tissue adjacent. Dentine bridging can, however,
compromise pulpal vascularity if intrusive enough; this is particularly so with non-setting calcium
hydroxide pastes such as Calxyl (Fig.22).
Bone Morphogenetic Proteins 2 and 4 produced a disappointing result compared with other studies
(Lianjia et al., 1993, Rutherford et al., 1993, 1994, Nakashima 1994b, Gao et al., 1995, Jepsen et
al., 1997). This may be due to a few factors. As only small quantities of both were available, we
were unable to test its activity and the beads were not tested for BMP release, hence little was
known about the active amount to which the pulp tissue was exposed and this seems the most likely
reason for failure. At the concentrations used, the maximum BMP-2 in the bead would have been
0.9 g, and BMP-4, 1.3 g, other studies have successfully used BMPs to stimulate dentine
formation in quantities greater than 2.0 g (Jepsen et al., 1997, Nakashima 1994). Moreover BMP’s
have a very short half-life in tissues. As mentioned earlier the BMP was human recombinant and
may have been incompatible for dogs (although the high evolutionary conservation of BMPs across
species makes this unlikely).
The growth hormone and insulin-like growth factor-1 combination produced reparative closure of the
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wound in two instances and stimulated odontoblasts in the area to produce secondary dentine, while
growth hormone by itself produced a much more satisfactory inflammatory result and stimulated
reparative and secondary dentine but with no complete closure of the wound. Insulin-like growth
factor-1 was not trialled by itself because of its short half-life in the extracellular space, it also
associates with extracellular matrix and various growth factor-binding proteins.
The rationale behind the use of GH and IGF-1, in combination, as a capping agent, stemmed from
their success in our in vitro model (Young et al., 1995). IGF-1 stimulated the differentiation and
development of odontoblasts and stimulated matrix formation; it was thought that GH priming might
have potentiated this effect. GH stimulated DNA synthesis and mitotic activity in the odontogenic
epithelia and mesenchyme and increased cell proliferation in the buds. It was theorized that the
GH/IGF-1 would act in a dual effector fashion with the GH forming differentiated cells from their
precursors, which would then be stimulated to expand, by clonal expansion, through the action of
IGF-1. The combination treatment did provide the only examples of complete closure of the
exposure area with some form of matrix, apart from the calcium hydroxide treatments and the
dentine in the area of the exposures appears to have been well stimulated.
Growth hormone treatment alone produced some reparative dentine in half the pulps, however
complete bridging of the exposure was not seen. It is possible that the GH has indirect and direct
effects on the pulp following capping. The somatomedin hypothesis (Daughaday et al., 1972)
proposed GH actions are mediated by somatomedins like IGF-1 (somatomedin-C). Both GH and
IGF-1 may come to the site from the circulation, which is increased following exposure. Moreover
the pulpal wound fibroblasts around the site of the exposure may produce high levels of IGF-1 in an
autocrine fashion in a form that is more biologically active than the plasma IGF-1 (Spencer et al.,
1988). Thus IGF-1 would be present in this in vivo model, at the site, to potentiate the effects of the
applied GH. The GH may also act to upregulate local IGF-1 production increasing its effects on the
pulp, thus increasing wound healing (Lynch et al., 1989, 1991), through its potent mitogenic effect on
pulp cells (Nakashima 1992a,b) and its involvement in the sulphation of predentine proteoglycans. It
is also known that GH may have direct effects on odontoblast differentiation, proliferation and
dentine matrix synthesis independent of a systemic IGF-1 mediator (Zhang et al., 1992a,b). The
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primacy of IGF-1 mediation has also been challenged by Li et al., (1998) who found that GH induced
the potent mitogenic and differentiating factors BMP-2 and BMP-4 even with IGF-1 inhibition. The
BMP’s would then induce their own positive effects on the pulpal healing process.
CONCLUSIONS
The hypothesis for experiment 2 was that GH alone, or in combination with IGF-1, would have
biological advantages over the traditional pulp-capping agents because these factors are
naturally occuring components of growth and repair processes.
Growth hormone and insulin-like growth factor-1 are naturally occurring growth factors, which play
important roles in growth and development. Classically it has been proposed that GH actions are
mediated by IGF-1 however recent evidence suggests GH may have direct effects on odontoblasts,
dentine matrix synthesis and upregulation of other factors like the BMP’s.
In this experiment GH and IGF-1, in combination, produced some reparative closure of the exposure
site and stimulated dentinogenesis, while GH produced a more satisfactory inflammatory result,
stimulated secondary and reparative dentine but produced no wound closure.
The growth factors GH and IGF-1 displayed obvious advantage over corticosteroid-antibiotic
combination which failed to produce any pulpal repair, inflammatory or dentinogenic. They were not
however as successful in stimulating reparative bridging as calcium hydroxide nor did they induce
the severe, sometimes overwhelming, stimulation of pulpal inflammation seen with this paste.
Further capping experiments utilizing growth hormone and insulin-like growth factor-1 for longer time
periods may be necessary to examine their full potential as natural agents in pulpal repair.
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TABLE 2. The numbers and percentages of dog tooth pulps affected by different degrees of inflammation after
the pulp-capping treatments of control (saline), Ledermix, GH/IGF-1 (Growth hormone/insulin-like
growth factor-1 combination), GH (Growth hormone), BMP (Bone morphogenetic proteins 2 and 4)
and Calxyl.
INFLAMMATION Control Ledermix GH/IGF-1 GH BMP Calxyl
2 Weeks Percent (n)
Mild 0 (0) 0 (0) 0 (0) 33 (1) NA 50 (2)
Moderate 33 (1) 33 (2) 50 (3) 33 (1) NA 0 (0)
Severe 66 (2) 50 (3) 33 (2) 33 (1) NA 25 (1)
Necrosis 0 (0) 17 (1) 17 (1) 0 (0) NA 25 (1)
5 WEEKS Percent (n)
Mild 25 (1) 0 (0) 66 (4) 66 (2) 14.3 (1) 55.5 (5)
Moderate 75 (3) 44.5 (4) 0 (0) 33 (1) 28.6 (2) 0 (0)
Severe 0 (0) 55.5 (5) 0 (0) 0 (0) 28.6 (2) 33.3 (3)
Necrosis 0 (0) 0 (0) 33 (2) 0 (0) 28.6 (2) 11.2 (1)
TOTAL Percent (n)
Mild 12.5 (1) 0 (0) 33 (4) 50 (3) As above 54 (7)
Moderate 50 (4) 40 (6) 25 (3) 33 (2) 0 (0)
Severe 37.5 (3) 53.3 (8) 17 (2) 17 (1) 31 (4)
Necrosis 0 (0) 6.7 (1) 25 (3) 0 (0) 15 (2)
n=60 100 (7) 100 (15) 100 (12) 100 (6) 100 (7) 100 (13)
CHAPTER 6
142
1
SUMMARY AND CONCLUSIONS
Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) are natural polypeptides that have
been found to have important roles in growth, proliferation, differentiation, development and
healing of cells and tissues.
In light of their potential roles in odontogenesis and dentinogenesis, growth hormone, insulin-like
growth factor-I and foetal calf serum were compared to serum-free medium as to their effects on
developing tooth germs in vitro. The study implied that growth hormone (at 100 ng/ml) and IGF-1
could support the finite number of cell cycles necessary for post-mitotic terminal differentiation of
odontoblasts and then elicit deposition of dentinal matrix.
The results from this first experiment suggested a potential role for GH and IGF-1 in in vivo
dentine repair. Traditional pulp-capping agents such as calcium hydroxide stimulate dentinal
bridging through chemical cautery while corticosteroid-antibiotic combinations have been used in
an attempt to minimize the inflammation and infection following exposure to allow healing to take
place. A relatively new capping agent is the inductive morphogen, bone morphogenetic protein,
which has demonstrated biological repair following capping of teeth in animal models.
In experiment 2, the pulps of 72 teeth on six, male, two year old dogs were exposed under general
and local anaesthesia. Direct pulp-capping was done with growth hormone (GH), growth hormone
and insulin-like growth factor-1 (GH/IGF-1), bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4)
or saline, incorporated in calcium chloride-coated sodium alginate beads as a vehicle; or with
calcium hydroxide or a steroidal-antibiotic as pastes. All pulp exposures and cappings were
performed with sterile instruments and preparations were sealed with glass ionomer cement.
Ledermix elicited persistent pulpal inflammation and inhibited predentine and dentine formation
correlating with published research. The BMP preparations elicited inflammation, little significant
matrix formation and no dentine bridge formation. The disappointing failure of the bone
morphogenetic proteins in comparison to other studies is unlikely to be related to interspecies
incompatibility because of the proteins high evolutionary conservation, however release may have
been impeded from the carrier beads or the proteins used may have been inactive or degraded.
In contrast, calcium hydroxide demonstrated marked initial inflammation which sometimes persisted
143
1
followed by the formation of reparative dentine bridges.
The growth hormone preparation elicited the mildest transient inflammation and reparative dentine
formation, however no complete dentine bridges were formed. The combination of growth hormone
and insulin-like growth factor-1 produced more persistent inflammation but also induced stimulation
of dentinogenesis and two cases of reparative wound closure.
Growth hormone and GH/IGF-1 combination were not as effective as calcium hydroxide in forming
reparative dentine bridging but were superior to Ledermix in stimulating dentinogenesis and without
the detrimental effects of the steroidal antibiotic on the pulpal tissues.
Growth hormone in combination with IGF-1 has potential as a pulp-capping agent, when delivered in
a suitable vehicle, because of the resultant favourable inflammatory pulpal state and vital pulpal cell
function observed, the GH may have had direct effects on the pulp as well as upregulating local IGF-
1 production. The combination of growth hormone and insulin-like growth factor-1 stimulated
dentinogenesis and, given longer periods of study, may result in more extensive wound closure and
pulpal repair following pulp-capping.
144
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