Keywords chitosan - hydroxyapatite - direct pulp capping - VEGF - blood vessel - fibroblast
Introductions
Caries, trauma, and iatrogenic factors are etiological factors for pulp tissue exposure.
When there is pulp tissue exposure, direct pulp caping is considered an efficient
conservative treatment option to maintain the vitality of the pulp. It is placed biomaterial
directly over an exposed coronal pulp after caries excavation to promote mineralization.
The ideal characteristics of a direct pulp capping material are biocompatibility,
antibacterial properties, sealing ability, insolubility in tissue fluids, and promotion
of the mineralization.[1 ]
[2 ] In previous decades, calcium hydroxide was the gold standard for direct pulp capping
treatment. Direct pulp capping materials such as calcium hydroxide and mineral trioxide
aggregate (MTA) can promote the migration, proliferation, and differentiation of human
dental pulp stem cells. It attempts to seal the pulp by enhancing tertiary dentine
deposition.[2 ]
[3 ] However, it has been found that calcium hydroxide can cause superficial coagulation
necrosis of the pulp, less adhesive to the dentin, and less successful in long studies.
Calcium hydroxide can be ionized into Ca and OH with strong alkali that trigger apical
abnormalities.[3 ]
[4 ] Tooth discoloration and high cost of operation are the drawbacks of MTA application.[4 ]
[5 ] Currently, there is no ideal direct pulp capping material. At present, in dentistry
various materials are being studied that have the ability to maintain the vitality
of the pulp and possess all the ideal characteristics of a direct pulp capping material.
Chitosan (CH) is a natural mucopolysaccharide with properties of biodegradability,
biocompatibility, nontoxicity, osteoinductivity, and bacteriostasis, which are suitable
for regenerative purposes in dentistry. CH has the potential to promote angiogenesis
of human dental pulp stem cells by increasing the expression level of vascular endothelial
growth factor (VEGF), fibroblast growth factor (FGF), and angiopoietin-1.[6 ]
[7 ] Therefore, CH is effective and promising for regenerative endodontic.[3 ]
CH is a natural polymer with low mechanical strength that could be blended with hydroxyapatite
(HA) and provides a synergistic effect. The physical and mechanical properties of
the calcium phosphate compound in HA could improve mechanical properties and osteoconduction
of the combination. CH-nanohydroxyapatite scaffold has potential as an effective pulp
capping agent to promote odontogenic differential dental pulp stem cells with significant
upregulation of VEGF, BMP-2, ALP, and Runx2.[8 ]
[9 ] The pulp is highly vascularized tissue; therefore, healing of injured pulp depends
on angiogenesis from angiogenic growth factor, particularly VEGF. Some studies observed
that VEGF plays an important role in dentin formation. VEGF can stimulate new blood
vessels to regulate migration, proliferation, and odontogenic differention.[3 ]
[6 ] Dental pulp fibroblasts are the most abundant cell type in dental pulp, which plays
an important role in dental pulp regeneration. Dental pulp fibroblast secretes VEGF,
FGF, and transforming growth factor β1 (TGFβ1), complements proteins when lipoteichoic
acid (LTA) stimulates it. These factors are important in promoting dental pulp stem
cell (DPSC) migration and growth factor participate in differentiation of DPSC into
odontoblastlike cells that generate reparative dentin.[10 ] The study aims to analyze the expression of VEGF, blood vessels, and fibroblast
cell proliferation in direct pulp capping treatment of Rattus norvegicus using a combination of CH and HA.
Materials and Methods
Preparation of CH, HA, and CH-HA Paste
CH powder with a deacetylation degree of 93% was used in this study. CH powder was
synthesized from the tiger prawn (Penaeus monodon ) through deproteinization, depigmentation, and deacetylation (DPA). Three percent
CH paste (w/p) was made by dissolving 1.5 g of CH powder in 50 mL of 2% acetic acid
(Merck). It was stirred using a magnetic stirrer and then neutralized with NaOH solution
(Merck), centrifuged at a speed of 2,000 rpm for 30 minutes, and filtered with filter
paper. The gel was added in 3 mL of 0.9% saline and hydroxypropyl methylcellulose
(HPMC) solution (Bate Chemical Co., Ltd.) until the consistency of a paste was reached.[11 ]
[12 ]
The HA paste was made by dissolving 1.5 g of HA powder (Sigma, Product number: 900204;
CAS Number: 1306-06-5) in 50 mL of 2% nitrate acid (Merck). It was then stirred using
a magnetic stirrer and added in 3 mL of 0.9% saline and HPMC solution (Bate Chemical
Co., Ltd.) until the consistency of a paste was reached. The CH-HA paste was made
by mixing CH paste and HA paste in a 50:50 ratio. The mixture was placed in a Petri
dish for 24 hours, then added in 3 mL of 0.9% saline and HPMC solution (Bate Chemical
Co., Ltd.) until paste consistency was reached. The paste was sterilized using gamma
ray radiation with a dose of 1 to 25 kGy.[12 ]
In Vivo Study
This study was experimental research with completely randomized design using samples
of molars of male R. norvegicus . Rats weighing 200 to 250 g and aged between 8 and 16 weeks were used. The rats were
acquired from an experimental animal laboratory of the Faculty of Dentistry, Universitas
Hang Tuah, Indonesia. Ethical approval was obtained from the Ethical committee of
the Faculty of Dentistry, Universitas Hang Tuah, with certificate number: 022/KEPK-FKGUHT/X/2022.
The rats were divided into five groups. There were six samples in each group with
3, 7, and 14 days of observation.
The occlusal surface of the molar of the R. norvegicus rat was prepared with class I cavity by using low-speed tapered round diamond burr,
and then perforated with the tip of the explorer under anesthesia using ketamine 10%
injection (Kepro Pharmacy, the Netherlands) at a dose of 0.1 mL/100 g body weight
and xylazine 2% injection (Xyla, Interchemie, the Netherlands) at 0.01 mL/100 g body
weight intramuscularly in the upper thigh.[13 ]
[14 ] In the KA control group, the cavity was filled with glass ionomer cement and the
cavity was filled with Ca(OH)2 in the KB group. In the treatment groups, the PA group, the cavity was filled with
CH; in the PB group, the cavity was filled with HA; and in the PC group, the cavity
was filled with CH and HA (CH-HA). In the treatment group, after the cavity was filled
with CH or HA, it was covered with type IX glass ionomer cement as the restorative
material. The R. norvegicus rats from each group were sacrificed after 3, 7, and 14 days. The mandibular bone
in the interdental region of the mandibular molar was cut and soaked in a fixation
solution using 10% formalin buffer. The decalcification process was carried out with
ethylenediaminetetraacetic acid (EDTA; J.T. Baker MDL number: MFCD00150037, Fisher
Scientific, UK). After that, it was embedded in paraffin blocks. The preparations
were made with a thickness of 4 to 5 μm and then continued with hematoxylin and eosin
(HE) staining (Sigma Aldrich) in order to observe blood vessels and fibroblast cells
in the groups with 3, 7, and 14 days of observation. Immunohistochemical examination
was conducted using VEGF monoclonal antibodies (Santa Cruz Biotechnology Inc, [C-1]:
sc-726) to observe the expression of VEGF on 3 day of observation. The preparations
were observed using a light microscope (Nikom H600L) with 400x magnification in five
fields of view.
Statistical Analysis
The data of the expression of VEGF, blood vessels, and fibroblast cells were statically
analyzed using the Shapiro–Wilk test to analyze the normally distributed data. Data
homogeneity was analyzed using the Levene test and the differences between the groups
were analyzed using one-way analysis of variance (ANOVA) and multiple comparison with
the least significant difference (LSD) test (p < 0.05).
Results
The result of the histopathological examination of the blood vessels and fibroblast
cells on days 3, 7, and 14 are shown in [Figs. 1 ] and [2 ]. The highest blood vessel and fibroblast cell proliferation on healing of direct
pulp capping was observed in the PC group with CH-HA, while the lowest was in the
KA control group filled with glass ionomer cement ([Tables 1 ] and [2 ]). The PA group using CH and the PB group using HA had a higher number of blood vessels
and fibroblast cells than the KA and KB control groups using Ca(OH)2 . The result of immunohistochemistry examination of VEGF expression on day 3 is shown
in [Fig. 3 ]. The highest expression of VEGF on healing of direct pulp capping was observed in
the PC group with CH-HA. It was followed by the PA, KB, PB, and KA groups.
Fig. 1 Blood vessels (black arrows ). KA1: glass ionomer cement (GIC) on day 3; KA2: GIC on day 7; KA3: GIC on day 14;
KB1: Ca(OH)2 on day 3; KB2: Ca(OH)2 on day 7; KB3: Ca(OH)2 on day 14; PA1: chitosan (CH) on day 3; PA2: CH on day 7; PA3: CH on day 14; PB1:
hydroxyapatite (HA) on day 3; PB2: HA on day 7; PB3: HA on day 14; PC1: chitosan and
hydroxyapatite (CH-HA) on day 3; PC2: CH-HA on day 7; PC3: CH-HA on day 14, with 400x
magnification.
Fig. 2 Fibroblast cell proliferation (black arrows ). KA1: glass ionomer cement (GIC) on day 3; KA2: GIC on day 7; KA3: GIC on day 14;
KB1: Ca(OH)2 on day 3; KB2: Ca(OH)2 on day 7; KB3: Ca(OH)2 on day 14; PA1: chitosan (CH) on day 3; PA2: CH on day 7; PA3: CH on day 14; PB1:
hydroxyapatite (HA) on day 3; PB2: HA on day 7; PB3: HA on day 14; PC1: chitosan and
hydroxyapatite (CH-HA) on day 3; PC2: CH-HA on day 7; PC3: CH-HA on day 14, with 400x
magnification.
Table 1
The average number of blood vessel on days 3,7 and 14
Groups
N
Mean of blood vessel ± standard deviation (SD)
Day 3
Day 7
Day 14
KA
6
1.33 ± 0.51a
2.83 ± 0.52b
3.5 ± 0.53c
KB
6
2.16 ± 0.75b
3.5 ± 0.54c
4.83 ± 0.75d
PA
6
3.33 ± 0.82c
4.33 ± 0.81d
5.98 ± 0.89e
PB
6
3.0 ± 0.63c
4.17 ± 0.74d
5.33 ± 0.51e
PC
6
4.17 ± 0.75d
6.33 ± 0.51f
7.17 ± 0.75g
Note: a,b,c,d,e,f,g Difference between groups with significance level of 5% (p < 0.05).
Table 2
The average number of fibroblast cell proliferation on days 3,7 and 14
Groups
N
Mean of fibroblast ± standard deviation (SD)
Day 3
Day 7
Day 14
KA
6
1.67 ± 0.52a
2.67 ± 0.52b
3.5 ± 0.35c
KB
6
2.5 ± 0.54b
3.33 ± 0.52c
4.5 ± 0.55d
PA
6
3.33 ± 0.82c
4.33 ± 0.82d
5.33 ± 0.52e
PB
6
3.83 ± 0.75c
5.0 ± 0.83d
5.77 ± 0.75e
PC
6
4.83 ± 0.75d
6.33 ± 0.52f
8.0 ± 0.89g
Note: a,b,c,d,e,f,g Difference between groups with significance level of 5% (p < 0.05).
Fig. 3 The expression of vascular endothelial growth factor (VEGF) on day 3 (black arrows ). (A ) KA: glass ionomer cement (GIC); KB: Ca(OH)2 ; PA: chitosan (CH); PB: hydroxyapatite (HA); PC: chitosan and hydroxyapatite (CH-HA),
with 400x magnification. (B ) The increase in VEGF expression was seen the most in the PC group, followed by the
PA, KB, PB, and KA groups.
The data were analyzed using normality test and homogeneity test. The result of analysis
was homogenous and had a normal distribution. Blood vessel and fibroblast cell proliferation
on healing of direct pulp capping showed significantly differences with the ANOVA
test value of p = 0.000 (p ≤ 0.05). There were significant differences in blood vessel and fibroblast cell proliferation
in the PA, PB, and PC groups compared to the KA and KB groups on days 3, 7, and 14.
Between the PA and PB groups, there was not significant difference in blood vessel
and fibroblast cell proliferation on days 3, 7, and 14. There was a significant difference
in the expression of VEGF in PC group compared to the PA, PB, KA, and KB groups (p = 0.018) on days 3. Between the PA and KB groups, there was no significant difference
in the expression of VEGF. The LSD test showed an increase in the expression of VEGF,
blood vessel, and fibroblast cell proliferation in the PA, PB, and PC groups.
Discussion
Direct pulp capping refers to placement of pulp capping material over an exposed coronal
pulp to protect the pulp against exposure to promote mineralization for tissue formation.
Many dental materials have been studied to preserve pulp vitality. However, currently
there is no ideal dental pulp material and it can be challenging to explore materials
for pulp treatment.[15 ] The paramount concern in pulp healing is angiogenesis. It plays a critical role
in efficiently transporting various nutrients, chemokines, inflammatory cells, and
cytokines. The formation and infiltration of new blood vessel depends on getting signals
from angiogenic growth factors, particularly VEGF.[6 ]
[16 ]
[17 ] In this study, the expression of VEGF, blood vessel, and fibroblast cell proliferation
in the treatment group using CH, HA, and CH-HA was higher than that in the control
groups using Ca(OH)2 or glass ionomer. There was a significant difference between the groups.
CH is a natural polysaccharide from chitin with properties of nontoxicity, biodegradability,
biocompatibility, and osteoinductivity. It also possesses antibacterial properties
and is suitable for regenerative purposes in dentistry. CH modulates inflammation
and regulates the function of inflammatory cells, macrophages, and fibroblast cells.
CH contains active N-acetyl-D-glucosamine dimer, which cross-links with glycosaminoglycan
and glycoproteins and activates macrophages to secret growth factors such as VEGF,
FGF, TGFβ1, and angiopontin.[3 ]
[14 ] Previous studies reported that loading angiogenic growth factor on biomaterials
could enhance pulp tissue healing. VEGF is an angiogenic growth factor that stimulates
the formation of new blood vessel. CH is able to stimulate the differentiation of
multipotent mesenchymal progenitor cells and growth factor such as FGF and TGFβ1,
increasing fibroblast proliferation.[2 ]
[7 ]
[18 ] Ca(OH)2 been the material of choice for direct pulp capping. However, this material has some
deficiencies. Ca(OH)2 releases hydroxyl ions that stimulate the expression of nuclear factor kappa β (NF-kβ)
and induces proinflammatory protein. Within a few years, the majority of mechanically
exposed and capped pulp indicate infection and superficial necrosis after its placement
on the exposed pulp. It was probably because of microleakage of the capping materials
and tunnel defect in the dentin bridge. It has incomplete setting reaction and ionized
into calcium and hydroxide that triggered the risk of internal root resorption and
apical lesion.[14 ]
[19 ] When compared to CH and Ca(OH)2 , blood vessel and fibroblast proliferations were the lowest in the control group
using glass ionomer. There were chronic inflammation and lack of dentin formation
caused by persistent acidity of the glass ionomer.[20 ]
Blood vessel and fibroblast cell proliferations in the treatment group using HA were
higher than those in the control group using Ca(OH)2 or glass ionomer. HA is biocompatible and provides good osseointegration to pulp
tissue. It is a porous material that accelerates the angiogenesis process. The calcium
phosphate compound in HA could improve the mechanical properties and osteoconduction.
It can reduce the production of intercellular fluid and increase the formation of
dentin reparative. The calcium phosphate component plays a role in the cell metabolic
process by increasing vascularization during pulp healing. It can stimulate the differentiation
of stem cell into fibroblast and odontoblastlike cells to promote tissue mineralization
and dentin reparative.[21 ]
[22 ]
[23 ]
CH has desirable characteristics and potential benefits to pulp healing; however,
CH has several limitations, such as the fact that it is a crystalline structure arising
from solid hydrogen and it has low mechanical strength.[24 ] It could be blended with HA to provide synergistic effect on the physical and mechanical
characteristics. The highest VEGF expression, new blood vessel, and fibroblast cell
proliferation on pulp healing were observed in the PC group using CH-HA. The combination
of CH-HA a has synergistic effect, resulting a material that has osteoconductive,
osteoinductive, and antibacterial properties that have the potential to reduce pulp
inflammation and necrosis.[23 ]
[25 ] The calcium phosphate compound of HA could improve mechanical properties and osteoconduction.
CH has characteristics of osteoinduction and antibacterial activity. The synergistic
effect of its combination promote the pulp healing. The polycationic nature of CH
in an acidic solution is provided by the presence of free amino groups within its
structure. In addition, the chemical modification of CH is feasible through its amino
and hydroxyl groups. The combination promotes mineralized tissue formation, controls
infections, prevents microleakage, and maintains the vitality of pulp.[2 ]
[7 ]
[23 ] Dental pulp fibroblasts interact with macrophage and modulate their differentiation
into M1 (proinflammatory) macrophage to control infection and M2 (anti-inflammatory)
to secrete of growth factor such as TGF-β1, FGF-2, VEGF, and complement proteins (C3a
and C5a), allowing dental pulp stem cell migration and odontoblastlike cell differentiation.
They also secrete angiogenic growth factor for pulp angiogenesis.[10 ] Thus, the combination of CH and HA can improve the pulp healing process and promote
mineralized tissue formation. However, further studies are required to observe the
proliferative assays with confocal laser scanning microscopy (CLSM) to approve the
degree of dentinal mineralization. Studies to approve the clinical application of
CH-HA are also required, exploring combination therapies such as coupling-derived
mesenchymal stem cells with growth factors or other regenerative agents to enhance
the angiogenic potential in pulp healing.[26 ]
[27 ]
Conclusion
It can be concluded that the combination of CH and HA could accelerate healing of
direct pulp capping treatment by increasing the expression of VEGF, blood vessel,
and fibroblast cell proliferation.
