Preservation of immunoexpression of type I collagen, BSP and BMP4 in the dentin-pulp complex of head and neck cancer patients after radiotherapy

Abstract

This study tested the hypothesis that head and neck radiotherapy (HNRT) impacts the immunoexpression of type I collagen, bone sialoprotein (BSP) and bone morphogenetic protein 4 (BMP4), thereby leading to micromorphological changes in the dentin-pulp complex (DPC), and promoting the onset and progression of radiation caries (RC). Twenty-two demineralized sections of carious teeth (a group of 11 irradiated teeth and a control group of 11 non-irradiated teeth) extracted from 19 head and neck cancer patients were analyzed by conventional optical microscopy and immunohistochemistry to investigate the micromorphology (cellular layer hierarchy, blood vessels, odontoblasts, fibroblasts, extracellular matrix, calcification, necrosis, reactionary dentin formation, and chronic inflammation), and the patterns of staining/immunolocalization of type I collagen, BSP and BMP4 in the dental pulp of irradiated and control samples. No significant differences attributable to the direct impact of radiotherapy were detected in DPC micromorphology between the groups. In addition, the patterns of immunohistochemical staining and immunolocalization of the proteins studied did not differ between the irradiated and the control samples for type I collagen, BSP or BMP4. This study rejected the hypothesis that HNRT directly damages dentition by changing the organic components and the microstructure of the DPC, ultimately leading to RC.

Collagen Type I; Integrin-Binding Sialoprotein; Bone Morphogenetic Protein 4; Immunohistochemistry

Introduction

Radiotherapy is one of the main treatment choices for head and neck cancer. Although considered highly effective, head and neck radiotherapy (HNRT) causes several acute and chronic toxicities that affect non-targeted tissues, such as oral mucositis, hyposalivation, recurrent oral infections, trismus, radiation caries (RC), and osteoradionecrosis, among other disorders.¹,²

RC is a chronic side effect that affects approximately 29% of the patients who undergo HNRT, with a 37% increased risk of developing RC within two years’ time.³ RC has a high potential to cause generalized dentition breakdown and clinical patterns of progression that differ from conventional caries, and can be characterized by widespread cervical demineralization, incisal edges, cusp tips lesions and diffuse brownish to black discoloration in the enamel surface.⁴ RC progresses rapidly, causing enamel cracks, delamination, and amputation of tooth crowns, and leading to tooth destruction in more severe cases. In addition, generalized infection in the oral microenvironment in advanced cases frequently requires dental extractions, increases the risk for developing osteoradionecrosis, and has a negative impact on the quality of life of cancer survivors.⁵,⁶ Atypical clinical progression patterns and lack of symptomatology associated with RC encourage research and investigation into its etiology and pathogenesis. Even though RC is commonly attributed to the indirect effects of radiotherapy, such as mucositis and hyposalivation,⁷,⁸ in vitro studies have suggested that the direct effects of HNRT on mineralized tooth structures might also be a significant causal factor for RC.⁹,¹⁰

The dentin-pulp complex (DPC) is a large and sophisticated component of the tooth structure,¹¹in which collagen and non-collagenous organic components, such as dentin sialoproteins, phosphoproteins, and proteoglycans, play an important role in preserving the mechanical and structural integrity of the tooth tissue.¹²,¹³ In this context, there are some proteins that participate in the molecular events underlying the DPC responses to caries progression, such as type I collagen – the predominant collagen in dentin – which plays a decisive role in the dentin mineralization process.¹⁴ In addition, bone sialoprotein (BSP) is synthesized by odontoblasts and preameloblasts, and accounts for 5-8% of the dentin extracellular matrix.¹⁴,¹⁵ Moreover, bone morphogenetic protein 4 (BMP4) – a growth factor related to tooth development – and type I collagen seem to be found specifically in the dental pulp, and may be related to the formation of the dentin matrix.¹⁶,¹⁷

Therefore, the present study aimed to test the hypothesis that HNRT impacts the immunoexpression of type I collagen, BSP and BMP4, leads to detectable micromorphological changes in the DPC, and has the potential to impact RC development.

Methodology

Patients and specimen collection

This study was approved by the local ethics committee (protocol number: CAAE 12837819.0.0000.5418), and was conducted in accordance with the Declaration of Helsinki. Eleven irradiated teeth with RC (irradiated group) and eleven non-irradiated carious teeth (control group), extracted from 19 head and neck cancer patients were included in this study. Dental extractions due to advanced caries or periodontal disease were performed for reasons not pertaining to the scope of this study. The teeth were identified immediately after being extracted, placed in plastic containers with 10% buffered formalin solution, and fixed for at least 72 h at 4^oC.⁵

The electronic medical records of each patient were consulted to gain clinical information on the patients, and were used to collect the following data: age, gender, tumor location, alcohol consumption and smoking habit, histological tumor type, clinical cancer stage according to the American Joint Committee on Cancer,¹⁸ total radiation dose prescribed for tumor treatment and final dose delivered to each tooth studied (Gy),¹⁹ and the anatomic origin of the extracted teeth.

Inclusion criteria

Inclusion comprised teeth extracted from patients with head and neck squamous cell carcinomas (SCC) subjected to clinical radiation protocols with tridimensional conformal radiotherapy (3DRT) in 6-mV linear accelerators on the Synergy Platform (Elekta AB, Stockholm, Sweden), with cumulative doses that ranged from 60 to 70 Gray (Gy) (2 Gy/day, five days per week). The 3DRT plan of the patients was retrieved from CMS XiO version 4.60 (Elekta CMS software, St. Louis, MS, USA) to study the radiation field and the total dose to the teeth.¹⁹

Exclusion criteria

Exclusion comprised teeth extracted from patients who had SCC occurring outside the head and neck region, who did not receive dental treatment prior to radiotherapy, who were submitted to radiotherapy regimens other than those included in the inclusion criteria, or whose demographic and clinicopathological information were not fully available from the medical records.

Demineralization and histological preparation

Each sample was codified and classified according to its anatomic group of origin. All the specimens were cleaned with manual periodontal curettes to remove residual soft tissues, decalcified in Ana Morse’s solution (equal volumes of 20% sodium citrate and 50% formic acid), at 4^oC for three weeks, refreshed every two days. The decalcification was monitored and confirmed by weekly periapical radiographs. Subsequently, all specimens were radiographed using an X-ray machine (Toshiba XR 6010 [127 V, 60 kV, 10 mA, and 60 Hz], Tokyo, Japan) with an exposure time of 0.3 seconds and focus-film distance of 15 cm, and following a standardized procedure. The teeth were placed individually on pieces of periapical radiographic film for adults (Kodak Ultra-speed; Eastman Kodak Company, Rochester, USA), with the crown facing the perforation of the film. All radiographic films were processed with the same automatic processor. The samples were embedded in Paraplast Plus® (Leica Biosystems Richmond, Richmond, USA) to produce 5-µm-thick sections on a microtome (Leica, Nussloch, Germany), in silanized slides for hematoxylin and eosin (H&E) morphological evaluation and immunohistochemical analysis.²⁰

Micromorphological analysis

An optical light microscope (OLM) (DM4000 B Leica, Wetzlar, Germany) was used to perform the micromorphological study. A previously calibrated oral pathologist analyzed three demineralized H&E-stained histological sections per sample descriptively, by examining the entire stained section of each sample on each slide, at both 200 X and 400 X magnifications.²¹

The microscopic analysis of the DPC focused on the presence and morphological preservation of parameters, such as hierarchy of dental pulp cellular layer, blood vessels, odontoblasts, fibroblasts, dental pulp extracellular matrix components, calcification, necrosis, reactionary dentin formation and chronic inflammation. ²²

Immunohistochemical preparation

The immunohistochemical reactions were conducted on 4-mm-thick histological sections cut from paraffin-embedded tissue blocks, and mounted on silane-coated glass slides. Antigen retrieval was performed in a water bath for 20 minutes at 95ºC in pH 6.0 citrate buffer, and endogenous peroxidase activity was blocked by 5% hydrogen peroxide in methanol for 20 minutes. Protein blocking was performed with powdered skim milk diluted in 5% PBS for 20 minutes. All the slides were incubated in a refrigerator at 2°C to 8^°C overnight with primary antibodies, and subsequently incubated with secondary antibody: goat anti-rabbit IgG, peroxidase conjugated, Millipore, code: AP132P, for 1 hour and 30 minutes at room temperature. Visualization of the reaction was performed with Liquid Dab (DAKO, K3468, Santa Clara, USA), according to the manufacturer’s recommendations. The sections were counterstained using Mayer’s hematoxylin and coverslips. Adequate positive control sections were used for each antibody, and the negative control was obtained by omitting the specific primary antibody.²⁰ Information on the antibodies, and the dilution protocol are described in Table 1.

Table 1
Antibodies and protocol used in the immunohistochemical analysis.

Antibody	Clone	Dilution	Source
Type I Collagen	ab34710	0,319444444	Abcam
Bone Sialoprotein (BSP)	ab125227	0,180555556	Abcam
Bone Morphogenetic Protein 4 (BMP4)	ab39973	0,111111111	Abcam

Immunohistochemical analysis

Two calibrated observers evaluated the reactivity of each antibody. The oral pathologist knew the antibodies but not the samples they were observing. The reactivity was evaluated qualitatively and semi-quantitatively, considering the following five regions for each sample: dentin-enamel junction - DEJ, sound dentin, predentin, dental pulp, and odontoblastic layer. The evaluation was graded as follows: 1 for positive staining in the total area of each region evaluated, and 2 for absence of staining in the total area of each region evaluated, whereas partial staining was considered absence of positivity ²⁰.

Statistical analysis

The data was analyzed statistically using PASW Statistics 18 © 2015 SPSS, all rights reserved (SPSS, Quarry Bay, Hong Kong), using the Fisher test, with the significance level set at α = 0.05.

Results

Patients and specimen collection

The demographic features and the clinicopathological data obtained from the 19 patients included in this study are described in Table 2. Eleven irradiated tooth samples were obtained from a total of 10 post-HNRT patients, and consisted of 5 (45.5%) molars, and 6 (54.5%) premolars. In the control group, 11 samples obtained from 9 non-irradiated head and neck cancer patients were included, and consisted of 5 (45.5%) molars, and 6 (54.5%) premolars.

Table 2
Clinicopathological profile of the patients included in the study.

Patient	Age	Tooth*	Gender	Tobacco	Alcohol	Site	T	N	M	CH	RT	Dose (Gy)	Dental dose (Gy)
Irradiated group
1	63	34	F	Yes	Yes	Tongue	1	2b	0	Yes	3D	60	59.42
2	44	35	M	Yes	Yes	Tongue	4	2b	X	Yes	3D	60	59.42
3	63	25	M	Yes	Yes	Oropharynx	3	2a	X	Yes	3D	70	51.81
4	54	36	M	Yes	Yes	Tongue	3	3	X	Yes	3D	70	67.83
5	62	15	M	Yes	Yes	Tongue	4	2c	X	Yes	3D	70	34.62
6	49	36	F	No	N	Nasopharynx	4	2	X	Yes	IMRT	69.96	66.90
7	-	15	M	Yes	Yes	Oropharynx	2	0	0	Yes	3D	70	51.81
8	47	36,37	M	Yes	Yes	Tongue	3	2	0	Yes	3D	60	58.14
9	56	37	F	No	No	Nasopharynx	4	1	X	Yes	IMRT	69.96	66.90
10	61	25	M	Yes	Yes	Oropharynx	2	1	X	Yes	3D	70	51.81
Control group
1	53	25	M	Yes	Yes	Oropharynx	3	2	X	No	-	-
2	56	45	M	Yes	Yes	Tongue	4a	3	X	No	-	-
3	56	25,34	M	Yes	Yes	Oral Cavity	4b	2b	X	Yes	-	-
4	52	26	M	Yes	Yes	Oral Cavity	4	2b	X	No	-	-
5	62	36	M	Yes	Yes	Pyriform sinus	3	3	X	Yes	-	-
6	57	36	F	No	No	Oral Cavity	3	0	X	No	-	-
7	46	27	M	No	No	Oropharynx	3	2a	X	Yes	-	-
8	57	26,27	M	Yes	Yes	Tongue	4a	3	X	No	-
9	46	34	M	Yes	No	Pyriform sinus	4a	3	X	Yes	-	-

• M: Male; F: Female; CH: Chemotherapy; RT: Radiotherapy; 3D: Tridimensional Conformal Radiotherapy; IMRT: Intensity Modulated Radiation Therapy. *According to FDI World Dental Federation notation. Dental Dose: mean radiation dose delivered to each tooth according to Morais-Faria et al.¹⁹

Micromorphological analysis

The microscopic analysis revealed the presence and the preservation of the hierarchy of the dental pulp cellular layers in 6 (54.5%) cases of the irradiated group, and 6 (54.5%) cases of the control group (p = 1.0), (Figure 1A, 1B). In the other samples of both groups, both the presence and the preservation of the hierarchy of dental pulp cellular layers were similarly and mildly altered, because of the presence of small dental pulp calcifications, diffuse chronic inflammation represented by mononuclear cells, and necrosis associated with bacterial invasion. Blood vessels and preserved vascular architecture were observed in all (100%) the samples of both groups.

Figure 1
Microscopic overview of control and irradiated samples, displaying preservation of the dental pulp layer hierarchy (hematoxylin and eosin-stained sections). A.B. (X 200) Dentin (D), predentin (PD), odontoblasts (O), dental pulp central region (C) in a control specimen and irradiated sample, respectively. C. (X 200) Inner demineralized layer with affected dentin showing normal patterns of bacterial invasion of the control dentin. D. (X 200) Caries-infected dentin composed of bacterial colonies and disorganized dentin in an irradiated sample. E.F. (X 400) Predominantly chronic inflammation represented by mononuclear cells affecting the dental pulp tissue of a control and irradiated specimen, respectively.

Odontoblasts were detected in all the samples of both groups; furthermore, the hierarchical layers of dental pulp cells were preserved in both groups, in a total of 6 samples (54.5%), and were characterized by tall columnar cells arranged in palisade pattern, and located at the periphery of the dental pulp. Cell processes arising from the odontoblast cell body could be observed penetrating the dentin, and in close contact with the fibroblasts of all the studied samples. The fibroblasts were preserved in all (100%) the samples of both groups.

Superficial caries-infected dentin composed of disorganized dentin and bacterial colonies, as well as an inner demineralized layer with affected, but not disrupted, dentin was consistently observed in all the specimens of both groups (Figure 1C, 1D).

Dental pulp extracellular matrix components were detected similarly among the samples of both groups, and were characterized by focal areas of dental pulp calcification among 6 (54.5%) control cases vs. 5 (45.4%) irradiated cases (p = 1.0); necrosis was detected in 5 (45.4%) control cases vs. 1 (9.0%) irradiated case (p = 0.14); chronic inflammation represented by mononuclear cells was reported in 2 (18.1%) control cases vs. 2 (18.1%) irradiated cases (p = 1.0), (Figure 1E, 1F); reactionary dentin was not observed in cases of the control group, whereas 3 (27.2%) cases were seen in the irradiated group (p = 0.21).

No significant difference was found between the irradiated and non-irradiated groups in any of the parameters analyzed. The summary of the histological analysis of the specimens is presented in Table 3.

Table 3
Micromorphological analysis of dentin-pulp complex components of the irradiated and control samples.

Morphological component	Control	Irradiated	p-value
Dental pulp hierarchy	6/11(54.5%)	6/11(54.5%)	1.0
Blood vessels	11/11(100%)	11/11(100%)	NA*
Odontoblastic layer	6/11(54.5%)	6/11(54.5%)	1.0
Fibroblasts	11/11(100%)	11/11(100%)	NA*
Extracellular Matrix	11/11(100%)	11/11(100%)	NA*
Calcification	6/11(54.5%)	5/11(45.4%)	1.0
Necrosis	5/11(45.4%)	1/11(9.09%)	0.14
Inflammation	2/11(18.1%)	2/11(18.1%)	1.00
Reactionary dentin	0/11(00%)	3/11(27.2%)	0.21

• NA*: not available. No statistics are computed because this variable is a constant. Values in parentheses are percentages of positivity.

Immunohistochemical analysis

Type I collagen expression was positive at the DEJ in all 22 (100%) specimens (Figure 2A, 2B). In sound dentin, type I collagen expression was positive for both groups (n = 22, 100%). The predentin region also showed positivity for all specimens (n = 22, 100%). In the odontoblastic layer, no cases of any group were positive (n = 0, 00%) (Figure 2C, 2D). In the extracellular matrix of the dental pulp, 10 (90.9%) cases of the control group were positive vs. 7 (70%) cases of the irradiated group (p=0.31), (Figure 2E, 2F).

Figure 2
A. (X 200) Type I collagen expression at the dentin-enamel junction (DEJ) in a control specimen. B. (X 200) The same pattern of positivity of the DEJ in an irradiated tooth. C. D. (X 200) Dentin (D), pre-dentin (PD), odontoblastic layer (O) and dental pulp central region (C) expression of type I collagen in a similar pattern of immunostaining in the dentinal tubules, pre-dentin, fibroblasts and extracellular pulp matrix regions, in a control and irradiated sample, respectively. E.F. (X 400) Immunoexpression of collagen type I in the central region of the dental pulp (extracellular matrix). Blood vessels of varying caliber and size showing discrete positivity for type I collagen in endothelial cell cytoplasm in a control and irradiated sample, respectively.

As for BSP, the immunoexpression of the DEJ was negative for all the cases in the control group (n = 0.00%), and was positive for only 1 case in the irradiated group (20%) (p = 0.33). In sound dentin, no case was positive in the control group (n = 0.00%) vs. 1 (20%) in the irradiated group (p = 0.33). The predentin region showed positivity in 4 (40%) cases of the control group vs. 1 (20%) of the irradiated group (p=0.60). In the odontoblastic layer, 6 (60%) cases indicated positivity vs. 3 (60%) from the irradiated group (p = 1.0), (Figure 3A, 3B). In the extracellular matrix of the dental pulp, BSP immunoexpression was found to be positive for 7 (70%) cases of the control group vs. 5 (100%) of the irradiated group (p=0.50), (Figure 3C, 3D).

Figure 3
A.B. (X 200) Dentin (D), pre-dentin (PD), odontoblasts (O) and dental pulp central region (C). BSP immunoexpression in the odontoblastic layer and components of the extracellular pulp matrix. Discrete positivity in dentinal tubules in a control and irradiated specimen respectively. C.D. (X 400) Preservation of blood vessels of varying caliber and size and calcifications displaying a negative labeling pattern. Homogeneous immunostaining in the extracellular dental pulp matrix in a control and irradiated sample, respectively.

The immunoexpression of the DEJ was negative for the BMP4 protein in all the cases of the control and irradiated groups. In sound dentin, 3 (27.2%) cases were positive in the control group vs. 2 (20%) in the irradiated group (p = 1.0). In the odontoblastic layer, no cases of either group showed positivity (n = 0.00%). The predentin region showed positivity for 5 (45.4%) cases of the control group vs. 5 (50%) of the irradiated group (p = 1.0), (Figure 4A, 4B). The extracellular matrix of the dental pulp was found to be positive in 7 (63.6%) cases of the control group vs. 7 (70%) cases of the irradiated group (p = 1.0), (Figure 4C, 4D).

Figure 4
A.B. (X 200) Dentin (D), predentin (DP), odontoblasts (O) and dental pulp central region (C). BMP4 immunostaining was similar between the groups for the extracellular matrix of the pulp and predentin, in the control and the irradiated tooth, respectively. C.D. (X 400) Immunoexpression of BMP4 in the extracellular matrix of the pulp. Blood vessels of varying caliber and size show discrete BMP4 immunopositivity in the endothelial cell cytoplasm in a control and irradiated specimen, respectively.

There were no significant differences between the test and the control groups regarding any of the features analyzed (Table 4).

Table 4
Immunohistochemical analysis of type I collagen, BSP and BMP4 in the irradiated and control samples.

Variable	Control	Irradiated	p-value
Type I collagen
DEJ	11/11(100%)	10/10(100%)	NA*
Sound dentin	11/11(100%)	10/10(100%)	NA*
Predentin	11/11(100%)	10/10(100%)	NA*
Dental pulp	10/11(90.9%)	7/10(70%)	0.31
Odontoblastic layer	0/11(00%)	0/10(00%)	NA*
BSP
DEJ	0/10(00%)	1/5(20%)	0.33
Sound dentin	0/10(00%)	1/5(20%)	0.33
Predentin	4/10(40%)	1/5(20%)	0.60
Dental pulp	7/10(70%)	5/5(100%)	0.50
Odontoblastic layer	6/10(60%)	3/5(60%)	1.0
BMP4
DEJ	0/11(00%)	0/10(00%)	NA*
Sound Dentin	3/11(27.2%)	2/10(20%)	1.0
Predentin	5/11(45.5%)	5/10(50%)	1.0
Dental pulp	7/11(63.6%)	7/10(70%)	1.0
Odontoblastic layer	0/11(00%)	0/10(00%)	NA*

• NA*: not available. No statistics were computed because this variable is a constant. Values in parentheses are percentages of positivity. DEJ: dentin-enamel junction.

Discussion

In this study, histological (H&E staining) and immunohistochemical analyses were used to determine the influence of the HNRT in the micromorphological response of the DPC in conventional and irradiated carious teeth. Apparently, this is the first study to make a simultaneous investigation of the distribution of immunomarkers such as type I collagen, BSP, and BMP4 in human teeth affected by RC, compared with conventional caries.

Some limitations of this study should be pointed out. A limited number of samples were included, since the present study sought to select carious human teeth from head and neck cancer patients. The chosen teeth had an anatomic origin similar to that of the extracted teeth, and were preserved well enough to perform the histological (H&E staining) and immunohistochemical analyses. Although the strict inclusion criteria allowed only a limited number of samples to be included, homogeneity was guaranteed. Moreover, standardization of the immunomarkers represented a limitation resulting from the challenge of preserving the dental pulp tissue after demineralization and histological preparation of the samples.

The profile of the patients in this study was in line with the traditional clinicopathological features of oral and oropharyngeal SCC patients observed worldwide.²³ The sample was mainly composed of elderly male individuals, smokers and drinkers with a poor oral health status, and diagnosed at late stages of tumor progression.²⁴

Some authors suggest that the direct effects of radiation on the dental pulp can negatively affect the metabolism of odontoblasts and damage the response of DPC to radiation caries progression.²,²⁵,²⁶ However, other studies disagree with the statement that radiation may be detrimental to the dental pulp, by negatively affecting odontoblast viability. In addition, some studies claim that the ability to react to caries progression is preserved in the dental pulp, thereby suggesting that the pulp structure and mechanism are not affected by the direct impact of the radiation. These studies posit that there is a cluster of oral symptoms, such as hyposalivation, oral microbiota alterations, and insufficient fluoride, which represent indirect causative factors for the increased risk of RC.⁵,⁸,²⁷,²⁸ Nevertheless, the literature has yet to confirm or refute this information.

No significant difference was found between the irradiated and non-irradiated groups in any of the histologically analyzed parameters of this study. This infers that the dentin structure and the hierarchy of the dental pulp cellular layers was preserved, hence low involvement of radiotherapy in the DPC. This finding corroborates that described in the literature,⁵,²⁹ which characterizes the pulp cellular layers as a central region (rich in blood vessels, fibroblasts, and neural bundles), and as the subodontoblastic region or Weil zone (the third most peripheral dental pulp layer with notable low cell density).²⁰,²⁷ Moreover, all the specimens showed normal extracellular matrix organization

In addition, the immunoexpression of type I collagen, BSP and BMP4 was carried out as a complementary assay to validate the morphological findings. One study demonstrated that the type I collagen distribution pattern in carious dentin may be associated with mineral loss in dental caries progression.³⁰ It reported that carious specimens stained for collagen type I showed a typical breakdown of the organic matrix, presenting areas where the collagenous network was completely degraded, evidenced by the evident lack of staining.³⁰ Unlike the present study, it did not indicate any differences in the immunoexpression of type I collagen, either in the control or the irradiated groups. This finding could be associated with the presence of more preserved epitopes for antibody recognition within the formic acid-demineralized tubules, where the arrangement of the collagen fibrils was preserved.¹³,³⁰

Type I collagen is an important part of the extracellular matrix of the dental pulp connective tissue, and was found to be preserved in the dental pulp of both the control and the irradiated teeth of this study.³¹ Some investigators suggest that this protein may be one of the factors involved in odontoblast differentiation, or else, just a component of the predentin secreted by polarized odontoblasts.¹⁷,³² Just like the BSP, this protein has been found in odontoblasts, predentin and the extracellular matrix of the dental pulp, with only slight differences in expression.³³,³⁴

Another factor possibly supporting the presence of BSP in mineralized tissues, such as sound dentin, cement and periodontal ligament, is that this protein was found not only in the cellular body of odontoblasts, but also in the cytoplasmic processes inside the dentin.³⁵ These findings strongly suggest that these cells secrete the proteins that ultimately compose the dentin matrix, and probably participate in the organization and maintenance of mineralized dentin structure.³⁶,³⁷ The presence of these proteins in the predentin fragments adjacent to the dental pulp of erupted teeth corroborates this hypothesis.¹⁴

CONCLUSION

Regarding BMP4, this protein may be expressed by odontoblasts and also at the region near the predentin, thus suggesting that it plays an important role in dentin matrix formation.¹⁶,¹⁷ In addition, BMP4 is a growth factor related to tooth development, hence the reason why it was chosen for evaluation in the present study. The current outcomes suggest that BMP4 expression is sensitive to the alterations induced by infection and inflammation.³⁸ Böttcher et al.³⁹ demonstrated that when dental pulp necrosis was induced, there was no BMP4 expression, indicating that inflammation of the apical periodontium arrests events associated with dentin formation. Similarly, this study sought to determine BMP4 immunoexpression changes in the DPC of control and irradiated carious teeth, but failed to determine any statistically significant differences between groups.

For all the foregoing reasons, it can be inferred that HNRT may not impair the DPC metabolism by changing the immunoexpression of type I collagen, BSP, or BMP4 of in vivo human teeth affected by RC. Therefore, the hypothesis that radiotherapy is able to directly modify the micromorphological response of the DPC to RC progression can be considered as rejected.

Acknowledgments

The authors are grateful to Fabiana Facco Cassarotti and Adriano Luis Martins for their technical support and contribution to the experimental development of the study.

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Publication Dates

• Publication in this collection
  14 Jan 2022
• Date of issue
  2022

History

• Received
  17 Jan 2021
• Accepted
  14 July 2021
• Reviewed
  14 Sept 2021