Keywords
microscope - biocompatibility - histology - root canal filling sealer
Introduction
The root canal filling sealer (RCFS) is an important component of the filling process
because it provides adhesion of gutta-percha to the dentinal walls.[1] These RCFSs must meet certain basic requirements to ensure safety and effectiveness,
such as biocompatibility,[1]
[2]
[3]
[4] ability to seal the root canal, radiopacity, dimensional stability, and microbicidal
and bactericidal properties.[1]
[2]
Mineral trioxide aggregate (MTA) is one of the best known RCFSs.[5] Its composition is approximately 80% by weight of Portland sealant and 20% by weight
of bismuth oxide (Bi2O3), included as a radiopacifying agent. However, Bi2O3 has been shown to interfere with hydration kinetics, reducing the precipitation of
calcium hydroxide in the hydrated paste,[6] affecting the compressive strength[7] and delaying hardening.[5] In addition, the toxicity due to the Bi2O3 compounds themselves and the associated arsenic impurities have raised concerns about
the safety of MTA.[8] RCFSs based on calcium aluminate represent a promising alternative, exhibiting physicochemical
characteristics such as a thermal coefficient and chemical composition similar to
human teeth and bone, controllable rheology and short setting time at room temperature,
high initial strength,[9] and bioactivity.[10] In particular, dodecacalcium hepta-aluminate has rapid hydration, and its configuration
can be controlled by adding additives to the mixture.[11]
In this line, strontium aluminates have also shown potential as RCFSs.[12] They can provide higher X-ray absorption and radiopacity than their Ca aluminate
counterparts and have shown the ability to significantly inhibit bone resorption and
improve bone regeneration in animals.[13]
[14] Tristrontium aluminate is the most hydraulic of this class of compound, as when
mixed with water, it becomes an RCFS paste with short handling and setting times.
Hydration begins immediately and progresses rapidly without any induction period but
with considerable production of heat.[12]
The combination of tristrontium aluminate and dodecacalcium hepta-aluminate has demonstrated
higher hydration rates, a shorter setting time that can better adapt to the time span
of clinical interventions, and an intense bioactive response.[15] However, no study has been performed to evaluate the in vivo biocompatibility of this new RCFS. Thus, this study aimed to evaluate the biocompatibility
of RCFSs based on tristrontium aluminate and dodecacalcium hepta-aluminate in living
tissue.
Materials and Methods
Animal Model and Experimental Groups
The study was approved by the Committee for Ethics in Animal Research, protocol CEP/CEUA/CSTR
No 032023. The sample size calculation was based on a pilot study. For a standard
deviation of 0.44 and a minimal intergroup difference of 1 for the inflammatory infiltrate
to be detected, a sample of five animals is required to provide a statistical power
of 80% with an alpha of 0.05. Forty-five male Wistar rats (Rattus norvegicus) weighing 200 to 250 g and aged between 3 and 4 months were obtained from the vivarium
of the Center for Education and Health of the Federal University of Campina Grande
(UFCG). The animals were housed in the INSIGHT vivarium cabinet of the Materials Bioassay
Laboratory (LBio) of the UFCG, with a controlled temperature of 23 ± 1°C and ambient
humidity of 41%, and maintained on chow and water ad libitum.[16]
[17]
[18]
The animals were randomly distributed into three groups according to the material
to be implanted: the Sealer group (Bio-C Sealer bioceramic filling sealant, Lot: 66041,
Angelus S.A., Londrina, PR, Brazil), the experimental group (filling sealant based
on tristrontium aluminate and dodecacalcium hepta-aluminate) according to Barbosa
et al[15] who evaluated the physicochemical and bioactivity properties of this compound previously,
and the control group (polyethylene tube), which were implanted with a biologically
inert tube without the presence of filling sealant ([Fig. 1]). Previously, the tubes were autoclaved at a temperature of 120° C for 20 minutes
and then used as inoculation vehicles for the tested materials.[19]
[20]
Fig. 1 Flow diagram of the included animals and experimental groups.
Each specimen was implanted in the subcutaneous tissue on the back of the animal bilaterally.
The side of the biopsy to be used was defined randomly, using the closed envelope
process; chosen, and opened by an examiner external to the study. In the absence of
material implanted in the biopsy on the side of choice, the opposite side was used.
The three groups were further distributed into three sampling times: 7, 15, and 30
days, for a total of nine groups.[21]
[22]
Manipulation of Cements
The experimental filling material underwent formulation and characterization based
on the tests performed by Barbosa et al.[15] The resulting cement consisted of a paste composed of tristrontium aluminate (80%)
and dodecacalcium hepta-aluminate (20%) mixed with aqueous polyethylene glycol (30%),
with a powder to liquid ratio equal to 0.39 mL/g. The filling material showed working
characteristics suitable for clinical application, with a setting time of 60 minutes
without excessive heat development, radiopacity equivalent to 3.1 mm of Al, and compressive
strength of 5 MPa in 24 hours after sufficient mixing for use in places with low mechanical
stress.[15] After 24 hours of preparation of the powder and liquid, they were manipulated as
recommended previously.[15]
The filling material was introduced into the openings at the extremities of the tubes
using a syringe (Centrix, Connecticut, United States) supported on a glass slide at
one end and a small glass slide at the other to flatten the material.[23]
[24]
[25] Each cement was placed in a polyethylene tube 10 mm in length and 1.5 mm in diameter,
with the exception of the tubes used in the control group, which were implanted and
left empty.[24] The sealer cement, based on calcium silicate, was manipulated according to the manufacturer's
specifications.
Surgical Procedure
For the surgical procedure, anesthesia was performed with intraperitoneal injections
of ketamine hydrochloride (0.10 mL/kg, Vetnil, Vetecia, SP, Brazil), 2% xylazine hydrochloride
(0.1 mL/kg, União Química, SP, Brazil), and 0.9% sodium chloride (0.15 mL/kg, Fresenius
Kabi, Barueri, SP, Brazil).[18] Antisepsis with 2% chlorhexidine solution (Riohex, Rioquímica, SP, Brazil) was used
for trichotomy of the bilateral scapular region to an anteroposterior length of 2 cm.
A 1-cm-long incision was made with a No. 15 scalpel blade for implantation of the
material for each group.[26]
With hemostatic forceps, the tissues were divided laterally, allowing insertion of
the tube into the surgical site. To facilitate localization of the area of histological
interest for the study, all tubes were implanted to the right of the incision and
with a single opening in the cranial direction. The surgical edges were closed by
simple three-point suture using 3.0 silk thread (Ethicon, Somerville, New Jersey,
United States). After the surgical procedure, two drops of sodium dipyrone (500 mg/mL,
Neo Química, Anápolis, GO, Brazil) were administered orally immediately after suturing
was completed. In the postoperative period, the rats were returned to the animal house,
where they were kept under daily observation until they underwent biopsy at 7, 15,
and 30 days.
All procedures in this study were performed in accordance with the guidelines of the
Canadian Council on Animal Care.[27] After the sampling periods, the animals were sedated with ketamine hydrochloride
(0.10 mL/kg, Vetnil, Vetecia, SP, Brazil), 2% xylazine hydrochloride (0.1 mL/kg, União
Química, SP, Brazil), and 0.9% sodium chloride (0.15 mL/kg, Fresenius Kabi, Barueri,
SP, Brazil) and sacrificed by cervical dislocation.[24]
[28] Euthanasia was confirmed by observing the absence of the eyelid reflex, chest movements,
and heartbeat for 3 minutes.
Next, the implanted specimen underwent tissue biopsy along with a safety margin of
adjacent connective tissue of 5 mm.[29] The samples were stored in individual, labeled containers and fixed in 10% buffered
formalin solution (pH 7.4) for 7 days.
Morphological Analysis—Biocompatibility
After fixation, the biopsy samples were prepared for histological processing, and
6-µm- thick serial sections were obtained and stained with hematoxylin (Proquimios,
Rio de Janeiro, RJ, Brazil) and eosin (Dinâmica, Química Contemporânea LTDA, Indaiatuba,
SP, Brazil) (HE) for evaluation under optical microscopy.[30]
All histological slides were photodocumented at a standardized magnification of 400x
with a Leica DM500 binocular optical microscope (Leica-Microsystems, Wetzlar, Germany)
using the rear digital camera of a smartphone (iPhone 10, Apple, Cupertino, California,
United States) attached to the microscope through an adapter custom built for this
function. For each sample, five sections representative of the histological condition
of the tissue adjacent to the implanted cements were analyzed[19]
[20] as well as five equal and equidistant areas in the tissue surrounding the implanted
specimen. The microscopic evaluation in this analysis was performed by a single calibrated
researcher (κ = 0.85).
For the cellular variables, including the number of fibroblasts, fibrocytes, multinucleated
giant cells, and blood vessels in each area,[31] counts were performed using ImageJ version 1.51 (National Institute of Health, United
States) by using its 10 × 10 mesh tool to create a field containing 100 equidistant
points, where only cells located in these intersections were counted.[32] The values obtained in each of these fields were added together, thus establishing
the total number of cells; subsequently, these data were used to calculate the average
value/per field of each group. The evaluator was previously calibrated (κ = 0.85) in a blinded evaluation.[31]
For the cellular variables inflammatory infiltrate, edema, necrosis, granulation tissue,
and collagen, the five sections representative of the histological condition of the
tissue were histologically evaluated according to the following scores: 0—absent (when
absent in the tissue); 1—sparse (when little was present, or in very small groups),
2—moderate (when densely present, or in a few groups), and 3—intense (when found throughout
the field, or present in large numbers).[19]
[20] These values represent the mean scores of the sum of five histological sections
representative of the evaluated tissue (n = 5, per group). The histological sections were evaluated randomly at five different
areas of the tissue adjacent to the specimen. The microscopic evaluation in this analysis
was performed by a single calibrated researcher (κ = 0.90).
This study was randomized and triple-blind; all experimental materials used in the
animals were placed in groups I to III, so that the examiner and the statistical evaluator
were not aware of the materials used.
Statistical Analysis
The distribution of the data was analyzed by the Kolmogorov‒Smirnov test (Graph Pad-Prism
5.0, San Diego, California, United States). The results for the cellular events did
not present a normal distribution; therefore, they were subjected to the Kruskal‒Wallis
and Dunn test. p-Value less than 0.05 was considered statistically significant.
Results
In the initial evaluation period, mild inflammatory infiltrate was demonstrated in
the control group, while both filling material groups demonstrated moderate inflammatory
infiltrate; the difference between them was not significant (p = 0.725; [Fig. 2A–C]). The intensity of the inflammatory infiltrate was inversely proportional to the
experimental time interval, with no significant difference among groups (p > 0.05; [Fig. 2A–C], [Table 1]).
Fig. 2 Photomicrographs of the histological samples. (A) Seven days after implantation, control group: mild inflammatory infiltrate (MII),
presence of congested blood vessels (CV), fibrocytes (F) and young fibroblasts (YF)
around the polyethylene tube (PT) (HE, 200× magnification, scale: 50 μm). (B) Seven days after implantation, Group Sealer: moderate inflammatory infiltrate (MII),
expressive presence of multinucleated giant cells (MGC), congested blood vessels (CV),
and young fibroblasts (YF) around the polyethylene tube (PT) (HE, 200× magnification,
scale: 50 μm). (C) Seven days after implantation, experimental group: moderate inflammatory infiltrate
(MII), presence of multinucleated giant cells (MGC), congested blood vessels (CV)
and young fibroblasts (YF) (HE, 400× magnification, scale: 25 μm). (D) Fifteen days after implantation, Group Control: presence of congested blood vessels
(CV), young fibroblasts (YF) and collagen fiber deposition (CFD) around the polyethylene
tube (PT) (HE, 100× magnification, scale: 100 μm). (E) Fifteen days after implantation, Group Sealer: presence of congested blood vessels
(CV), multinucleated giant cells (MGC), young fibroblasts (YF) and onset of collagen
fiber deposition (CFD) (HE, 200× magnification, scale: 50 μm). (F) Fifteen days after implantation, experimental group: mild inflammatory infiltrate
(MII), congested blood vessels (CV), expressive presence of multinucleated giant cells
(MGC), fibrocytes (F) and young fibroblasts (YF), onset of collagen fiber deposition
(CFD) around the polyethylene tube (PT). (HE, 100× magnification, scale: 100 μm).
(G) Thirty days after implantation, Group Control: presence of congested blood vessels
(CV), area of collagen fiber deposition (CFD) disposed in parallel bands, around the
polyethylene (PT) tube (HE, 100× magnification, scale: 100 μm). (H) Thirty days after implantation, Group Sealer: presence of congested blood vessels
(CV), expressive response by multinucleated giant cells (MGC), young fibroblasts (YF)
and collagen fiber deposition (CFD) disposed in parallel bands, around the polyethylene
(PT) tube (HE, 200× magnification, scale: 50 μm). (I) Thirty days after implantation, Experimental Control: presence of young fibroblasts
(YF), area of collagen fiber deposition (CFD) disposed in parallel bands, around the
polyethylene (PT) tube (HE, 100× magnification, scale: 100 μm).
Circulatory changes (edema) were most present at 7 days, but there was a significant
difference only between the control group and the sealer and experimental groups at
15 days (p = 0.006). Tissue degeneration (necrosis) and granulation tissue were not substantial
and did not show significant differences among the evaluated groups (p > 0.05).
The blood vessel count showed a higher amount at 30 days in the cement groups, especially
in the experimental group, which showed an increasing count over the observation intervals.
There was a significant difference between the experimental group and the control
group at 7 days (p = 0.020; [Fig. 2A and C]) and 30 days (p = 0.002) and between the experimental group and the sealer group at 15 days (p = 0.022; [Fig. 2D–F], [Table 1]).
Multinucleated giant cells were more abundant in the sealer and experimental groups,
and there was a significant difference between the sealer and control groups at 7
days (p = 0.001; [Fig. 2A–B]) and 30 days (p = 0.001; [Fig. 2G–I]) and between the experimental and control groups (p = 0.001) at 15 days ([Fig. 2D and F], [Table 1]).
All groups evaluated had greater numbers of young fibroblasts in the initial, 7-day
period, the result of the initial reaction for tissue repair ([Fig. 2A–C]), followed by a quantitative decrease proportional to the experimental time interval
([Fig. 2D–I]). The experimental group showed the highest cell quantity at all time intervals
([Fig. 2C], [2F] and [2I]), with no significant difference from the sealer group at 7 (p = 0.001) and 15 days (p = 0.002) or with the control group at 30 days (p = 0.001) [Table 1].
Fibrocytes were found in greater quantity in the control group at 7 days (p = 0.004) and 30 days (p = 0.115) ([Fig. 2A and G]). The sealer group had fewer fibrocytes at 15 days (p = 0.004) than the experimental group ([Fig. 2E and F]).
Regarding tissue repair events, the amount of collagen fibers increased over the experimental
intervals, with no significant difference between the cement groups and the control
group (p > 0.05; [Fig. 2A–I], [Table 1]).
Discussion
New materials for clinical use should be initially evaluated by histocompatibility
tests,[33] human tissue,[34] cell culture,[35] and implantation in living tissues.[19]
[36]
The healing of damaged tissue is initiated by signals sent by the immune cells present
in the inflammatory infiltrate.[37] In this study, an RCFS based on tristrontium aluminate and dodecacalcium hepta-aluminate
was compared with a bioceramic RCFS – Sealer Plus BC, which is based on calcium di-
and trisilicate and zirconium oxide, to evaluate its tissue response in living tissue.
Empty polyethylene tubes were used as controls in this study, as they are considered
biologically inert. The tissue damage caused was the response of the surgical procedure
to the implantation of the tubes, with lower inflammatory intensity than in the filling
material groups, corroborating other studies.[21]
[32]
[38]
At 7 days, both RCFSs showed a moderate inflammatory reaction[39] that was similar between the groups (p = 0.725). The intensity of the inflammatory infiltrate was inversely proportional
to the experimental time interval, with no significant difference between groups (p > 0.05). Studies[40]
[41] have suggested that the fixation reaction of RCFSs with similar compositions promotes
the formation of calcium and hydroxyl (OH2) ions, and the alkaline pH[3]
[40]
[41] stimulates the recruitment of inflammatory cells and the production of cytokines.
In addition, the proinflammatory cytokine interleukin-6 (IL-6), which can activate
and modulate specific cells, plays an important role in the inflammatory reaction
and bone resorption.[39]
[42] In RCFSs similar to the one in this study, a significant increase in the amount
of IL-6 was observed, greatest at 7 days followed by a gradual decrease.[43]
Circulatory changes (edema) were more present at 7 days, but there was a significant
difference only between the control group and the sealer and experimental groups at
15 days (p = 0.006). Tissue degeneration (necrosis) and granulation tissue were not substantial
and did not show significant differences between the evaluated groups (p > 0.05).
Angiogenesis is based on the formation of new blood vessels and partially driven by
inflammation because the creation of new vessels is related to increased nutrition,
oxygenation, and a greater number of immune cells[23]
[25]
[44] and repair of the damaged area.[45] The blood vessel count was higher at 30 days in the RCFSs, especially in the experimental
group, which showed increasing counts over the experimental intervals. There was a
significant difference between the experimental group and the sealer group at 15 days
(p = 0.022).
Multinucleated giant cells are formed from the union of macrophages[37] for the isolation and elimination of foreign body reactions.[2] The multinucleated giant cells were closer to the sealer particles and were more
present in the sealer group at 7 and 15 days (p < 0.05) and in the experimental group at 15 days (p < 0.05). This suggests that the RCFS may have released more irritants[46] in the initial intervals. The presence of RCFS remnants near the cavity observed
in some samples in later periods demonstrated that these biomaterials were not easily
digested by the multinucleated giant cells or quickly eliminated by local lymphatic
drainage.[47]
Fibrocytes, which play an important role in the recovery of damaged tissue, are able
to differentiate into fibroblasts.[37] These cells were found in greater quantity in the control group at 7 days (p = 0.004), having proliferated due to the increased need for tissue healing. A lower
amount of fibrocytes was observed in the sealer group at 15 days (p = 0.004) than in the experimental group, which could indicate later recruitment by
the RCFS based on calcium and strontium aluminates than by the others. The increase
in these cells at 30 days in the control and sealer groups indicates a decrease in
fibroblast differentiation, resulting from the stabilization of the scar tissue.
An important characteristic of cements is their bioactivity, wherein they induce repair
in damaged tissues in their surroundings,[3]
[39]
[42] stimulate tissue repair, and induce mineralization, promoting an integration of
the material with dental tissues.[1] However, this property was not evaluated in this study. Barbosa et al[15] showed the bioactive capacity of an experimental cement formed from apatite after
3 days in simulated body fluid solution.
Young fibroblasts were more numerous in all groups at 7 days, the result of the initial
reaction for tissue repair, followed by a quantitative decrease proportional to the
experimental time interval. The experimental group showed the highest cell quantity
at all time intervals but was not significantly different from the sealer group at
7 (p = 0.001) and 15 (p = 0.002) days. These results corroborate the fact that neither cement caused an intense
inflammatory process, since the presence of fibroblasts decreased over time due to
the process of tissue remodelling.[48] However, at 30 days, a significant difference in the number of fibroblasts was observed
between the cements, which suggests a slower healing response in this period for the
experimental cement.
Regarding tissue repair events, the amount of collagen fibers increased over the experimental
intervals, with no significant difference between the RCFSs and the control group
(p > 0.05). In addition, the presence of well-oriented collagen fibers arranged in bundles
in the capsules indicated that the inflammatory reaction was gradually replaced by
dense connective tissue. This hypothesis is reinforced by the reduction in the thickness
of the capsule at the 30-day evaluation period.
The Sealer Plus has calcium disilicate, nanoparticulate calcium trisilicate, and zirconium
oxide in its formulation; the experimental RCFS was formulated with tristrontium aluminate
and dodecacalcium hepta-aluminate. These differences may have affected the physicochemical
properties as well as the biocompatibility and bioactive potential of the RCFS.[3]
[39]
[42]
Calcium aluminates have been used as dental and bone fillers, exhibiting controllable
rheology and short setting time at room temperature, high initial strength, bioactivity,
and biocompatibility.[10]
[11] They exhibit certain physicochemical characteristics, such as their thermal coefficient
and chemical composition, which are similar to those of human teeth and bone.[49] For the strontium aluminates, since the Sr cross-section is larger than that of
Ca, it provides greater absorption of X-rays and radiopacity, and they are more radiopaque
than their Ca aluminate counterparts. In addition, they significantly inhibit bone
resorption and improve bone regeneration in animals with induced osteoporosis.[13]
From a clinical view, the experimental RCFS[15] did not cause relevant tissue damage, allowed normal tissue recovery, and proved
to be a bioinert biomaterial.[22] This corroborates other authors who showed regression of the inflammatory reaction
and scar repair in similar RCFSs.[3]
[39]
[42]
The results found in this study indicate that this new RCFS[15] has the potential to be applied in root canal treatments. However, for it to be
effectively used in clinical procedures, human clinical trials involving endodontic
canals should be conducted.[50]
Conclusion
The experimental filling sealant based on calcium aluminates and strontium proved
to be biocompatible for use in close contact with periapical tissue, inducing a low
inflammatory reaction and favoring rapid tissue repair.
Table 1
Mean of the scores[a] attributed to the groups, after the time intervals of 7, 15, and 30 days, for the
nine conditions evaluated
|
Condition
Time/Days
|
Groups
|
p-Value*
|
|
Control
|
Sealer
|
Experimental
|
|
Inflammatory infiltrate
|
|
7
|
1.00
|
1.20
|
1.40
|
0.725
|
|
15
|
0.20
|
0.40
|
1.00
|
0.067
|
|
30
|
0.00
|
0.00
|
0.40
|
0.285
|
|
Edema
|
|
7
|
1.40
|
1.60
|
1.60
|
0.999
|
|
15
|
0.00A
|
1.00B
|
0.80B
|
0.006
|
|
30
|
0.00
|
0.00
|
0.40
|
0.285
|
|
Necrosis
|
|
7
|
0.00
|
0.00
|
0.00
|
1.000
|
|
15
|
0.00
|
0.00
|
0.00
|
1.000
|
|
30
|
0.00
|
0.00
|
0.00
|
1.000
|
|
Granulation tissue
|
|
7
|
0.00
|
0.40
|
0.40
|
0.450
|
|
15
|
0.00
|
0.00
|
0.00
|
1.000
|
|
30
|
0.00
|
0.00
|
0.00
|
1.000
|
|
Blood vessel
|
|
7
|
2.55A
|
2.00AB
|
1.66B
|
0.020
|
|
15
|
2.44AB
|
1.44A
|
2.55B
|
0.022
|
|
30
|
2.55A
|
3.11AB
|
3.66B
|
0.002
|
|
Multinucleated giant cells
|
|
7
|
4.14A
|
7.20B
|
5.00AB
|
0.001
|
|
15
|
2.00A
|
5.75AB
|
6.28B
|
0.001
|
|
30
|
2.00A
|
5.20B
|
2.00A
|
0.001
|
|
Young fibroblasts
|
|
7
|
4.11A
|
4.55AB
|
5.55B
|
0.001
|
|
15
|
2.88A
|
3.44AB
|
4.00B
|
0.002
|
|
30
|
2.55AB
|
2.11A
|
3.88B
|
0.001
|
|
Fibrocytes
|
|
7
|
1.77A
|
0.66AB
|
0.44B
|
0.004
|
|
15
|
0.44AB
|
0.33A
|
1.11B
|
0.034
|
|
30
|
1.11
|
0.66
|
0.55
|
0.115
|
|
Collagen
|
|
7
|
2.00
|
1.80
|
1.57
|
0.230
|
|
15
|
2.75
|
2.40
|
2.00
|
0.090
|
|
30
|
3.00
|
3.00
|
2.66
|
0.285
|
a For each sample of the study, five representative sections of the histological condition
of the tissue were analyzed.
*p-Value indicates nonparametric Kruskal–Wallis test, followed by Dunn's multiple comparisons
test.
A or B Means followed by the same single letter do not express statistically significant
difference (p > 0.05).
AB Means followed by different letters express statistically significant difference
(p < 0.05).
