|Year : 2012 | Volume
| Issue : 1 | Page : 32-40
Bacterial intensity and localization in primary molars with caries disease
AP Beltrame1, M Bolan1, AC Serratine2, MJ Rocha1
1 Departament of Dentistry, Federal University of Santa Catarina, Brazil
2 Departament of Dentistry, South University of Santa Catarina, Brazil
|Date of Web Publication||3-May-2012|
Rua Delminda da Silveira, 740/403, Agronômica, Florianópolis, SC 88025500
| Abstract|| |
Aim: The aim was to assess the characteristics and outcomes of infections affecting the structures of carious primary molars. Materials and Methods: Forty primary molars were used and classified according to the following clinical situation: With profound caries lesion, with bone loss at the furcation region, with perforation of the pulp chamber floor, and residual roots. The teeth were demineralized, cut, and stained with both haematoxylin-eosin and Brown and Brenn staining techniques. Assessment was performed using optical microscopy. Results: Statistical analysis of the data by means of the Chi-square test suggests that there was a significant relationship (P<0.001) between the intensity and localization of infection and the level of destruction of dental structures. A significant difference was also observed in the intensity and localization of infection between the groups regarding crown, furca, and root (P<0.001). Conclusion: More intense and profound the infection, more severe is the dental destruction. The groups of residual roots showed the most severe bacterial infection compared to other groups.
Keywords: Caries, infection, inflammation, primary teeth
|How to cite this article:|
Beltrame A P, Bolan M, Serratine A C, Rocha M J. Bacterial intensity and localization in primary molars with caries disease. J Indian Soc Pedod Prev Dent 2012;30:32-40
|How to cite this URL:|
Beltrame A P, Bolan M, Serratine A C, Rocha M J. Bacterial intensity and localization in primary molars with caries disease. J Indian Soc Pedod Prev Dent [serial online] 2012 [cited 2013 May 21];30:32-40. Available from: http://www.jisppd.com/text.asp?2012/30/1/32/95576
| Introduction|| |
Caries disease progresses more quickly in primary teeth because of their anatomical characteristics. ,, Bacterial infection resulting from caries lesion contaminates the dentin and reaches the pulp and periradicular tissues, causing inflammatory reactions, tooth resorption, and periradicular lesions with abscesses. ,
The resorptions caused by chemical mediators and by-products of bacterial metabolism cause a devastating effect on primary teeth if not properly treated, leading to a rapid destruction of the tooth between cemento-enamel junction and furca. 
The consequences of such an infection in a primary tooth can have repercussions on the permanent dentition, ranging from enamel hypoplasia and partial or total interruption in the succeeding tooth formation ,,,,, to localized or generalized occlusal imbalance in the permanent dentition. , Also, the physiological resorption of primary teeth becomes usually accelerated,  leading to a precocious dental loss.  On the other hand, general conditions such as bacteremia, septicemia, and cerebral abscesses may be the result of dental infections not treated. ,
Most of the protocols used to treat endodontically compromised primary teeth simply reflect the non-observance of infection on the part of the practitioners, for they only consider pulpotomy for teeth with signs of pulp inflammation or necrosis without preparing the root canals biomechanically. ,,, Moreover, the biomechanical techniques have been successfully employed for removing or reducing dental infections since the second half of the past century.
However, due to the scanty studies on infection in primary teeth, ,,,,, some authors have established protocols in which all the steps for endodontic treatment are included, ,,, from the use of safe measurement techniques in several radiographic relationships between primary tooth and permanent tooth germ , to instrumentation of root canals. 
Some recent studies confirm the great diversity of bacteria present in the root canals of primary teeth with pulp necrosis and periapical lesion, predominantly the anaerobic bacteria. ,,, The presence of bacterial biofilm on the external root surface of these teeth was reported by Rocha et al.,  who used scanning electronic microscopy (SEM) in their study.
It is believed that determining the intensity and localization of the infection in primary molars at the different phases of destruction as well as their histopathologic characteristics will contribute to reviewing and improving the endodontic procedures for these teeth. This will allow the homeostasis to be maintained during the whole biological cycle. Therefore, the objectives of this research study were the following: 1) to assess intensity and localization of infection in primary molars with profound caries lesion, with bone loss at the furcation region, with perforation of the pulp chamber floor, and residual roots, and 2) to verify the histological aspects of the pulp and periradicular tissues.
| Materials and Methods|| |
This research study was approved by the Human Research Ethics Committee of the Federal University of Santa Catarina (number 171/2002).
Forty primary molars extracted from 4-13-year-old male and female children in the Pediatric Dentistry Clinic of the Federal University of Santa Catarina (UFSC) were selected for study. The teeth were divided into four experimental groups according to their clinical conditions: Group I, teeth with profound caries lesion without periradicular lesion (n=10); Group II, teeth with profound caries lesion and bone loss in the furca region without perforation of the pulp chamber floor (n=10); Group III, teeth with profound caries lesion and bone loss in the furca region with perforation of the pulp chamber floor; and Group IV, teeth with residual roots (n=10). Diagnosis was done based on clinical characteristics, radiographs, and direct visualization of the tooth after extraction.
The teeth of all four groups were submitted to demineralization in 10% ethylenediaminetetraacetic acid (EDTA). Next, teeth from Groups I, II, and III were cut transversally 1 mm above and 1 mm below the pulp floor, yielding four fragments. Group IV had the roots transversally cut, thus yielding two fragments [Figure 1].
|Figure 1: Illustration of the four experimental groups and their respective fragments (Groups I, II, and III: 1 – coronal portion; 2 – furca region; 3 and 4 – radicular portion. Group IV: 1 and 2 – root fragments. *Furca lesion)|
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After being cut, the specimens were washed with tap water, dehydrated with alcohol, diaphanized with xylol, and then immersed in paraffin. Serial sections of 5 mm in thickness by 50 mm, 100 mm, and 150 mm in depth were performed, and one section of each depth was prepared for slide. The sections were stained by using the hematoxylin-eosin (HE) and Brown and Brenn (BB) staining techniques and analyzed under optical microscopy. The use of HE stain was aimed at analyzing the histopathologic manifestations in pulp and periradicular tissues, whereas the BB staining method was employed to investigate infection intensity  and localization of bacteria in pulp chamber, root canals, dentinal tubules, cement, and Howship's lacunae. ,
The following scores were used to identify the intensity of bacteria within dental structures:
High - Presence of bacteria in 50% of the dental structures
Moderate - Presence of bacteria in 10-50% of the dental structures
Low - Presence of bacteria in less than 10% of the dental structures
The pulp tissue showed the following alterations: Hyperemia, acute inflammatory process, chronic inflammatory process, and necrosis.
The periradicular tissue showed the following alterations: Inflammatory process, acute abscess, chronic abscess, granuloma, epithelial granuloma, and cyst.
A descriptive analysis of the results was carried out and the groups were compared to each other by using Chi-squared test. A statistics software package was used for doing so (Epi Info, version 17).
| Results|| |
By comparing the groups, we have observed a statistically significant association between the intensity of infection and degree of destruction in dental structure as a whole (P<0.001).
Residual roots (Group IV) were the dental elements presenting the most severe infection compared to the roots of teeth with profound caries and bone loss at the furca region [Table 1].
|Table 1: Percentage of slides in each experimental group according to the infection intensity observed in the different parts of the tooth (crown, furca, and root)|
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[Table 2] shows the localization of bacteria in the different parts of the tooth.
|Table 2: Percentage of slides in each experimental group according to the localization of bacteria found in the different parts of the tooth (crown, furca, and root)|
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Histopathologic analysis of pulp and periodontal tissues
In Group I, nine of the specimens analyzed had their pulp tissue affected as follows: Four with hyperemia (being one with hydropic degeneration), one with acute inflammatory process, two with chronic inflammatory process, and two with pulp necrosis. With regard to the periodontum, four specimens had granulomatous tissue, including epithelial granuloma [Figure 2].
|Figure 2: Group I. (a and b) Histological sections of the coronal and radicular regions, respectively, showing the presence of biofilm and bacteria (in red, blue and purple) spread along the dentinal tubules (Brown and Brenn; ×33 and ×66 magnifi cations). (c) Abscess in the pulpal crown tissue and inflammatory reaction in the pulpal root tissue (HE, ×33), (d) hypermia and hydropic degeneration (HE, ×66)|
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In Group II, 10 of the specimens analyzed had their pulp tissue affected as follows: One with hyperemia, one with acute inflammatory process, three chronic inflammatory process, and eight with pulp necrosis. In the periodontum, two specimens had inflammatory process and nine had granulomatous tissue, including two with epithelial granuloma [Figure 3].
|Figure 3: Group II. (a) Radiographic aspect showing periradicular radiolucent area at the furca region. (b) Histological section of the furca region showing the presence of biofilm and bacteria in the inter-radicular tissue (Brown and Brenn; ×33 magnification). (c and d) Presence of biofilm in the root canal and periodontal ligament, including bacteria (in red) invading the dentinal tubules (Brown and Brenn and HE, respectively; ×66 and ×66 magnifi cations)|
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In Group III, 10 of the specimens analyzed had their pulp tissue affected as follows: Two with chronic inflammatory process and eight with pulp necrosis. In the periodontum, four specimens had inflammatory process, one had acute abscess, and four had granulomatous tissue, including two with epithelial granuloma [Figure 4].
|Figure 4: Group III. (a and b) Macroscopic and radiographic aspects, respectively, showing perforation in the pulp chamber floor. (c– f) Histological sections. (c and d) Presence of biofilm and infection on the external furca surface of the dentinal tubules (Brown and Brenn; ×66 and ×132 magnifications). (e and f) Bacteria within dentinal tubules and inflamed pulp tissue (HE, ×33 magnification). (g) Epithelial granuloma at the periapical region (HE, ×33)|
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In Group IV, 10 of the specimens analyzed had their pulp tissue affected as follows: Two with acute inflammatory process, one with chronic inflammatory process, and nine with pulp necrosis. In the periodontum, two specimens had inflammatory process, one had acute abscess, one had chronic abscess; and seven had granulomatous tissue, including two with epithelial granuloma and one with cyst [Figure 5].
|Figure 5: Group IV. (a) Macroscopic aspects of residual roots. (b-e) Histological sections. (b) Periodontal ligament region with presence of biofilm (Brown and Brenn; ×132 magnification). (c and d) Presence of bacteria in pulp tissue with necrosis and inner resorption (Brown and Brenn and H and E; ×33 magnification). (e) Inflammatory pathological resorption and inflammatory reaction in periodontum (H and E, ×33)|
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| Discussion|| |
Infectious diseases in pulp and periradicular tissues, like those occurring in other parts of the individuals' body, are the result of a poor immunological mechanism for eliminating foreign agents quickly, such as the bacteria.
When infection is severe and the phagocytic cells cannot eliminate the bacteria quickly, the foreign agents proliferate by using the nutrients of the invaded tissues. The resulting bacterial enzymes and toxins cause changes in the tissue cells and induce a host immune response which triggers an inflammatory process.
Cytokines are produced during this inflammatory process in order to eliminate the foreign agent, but this may lead to a destruction of tooth and periodontum.  Because of the anatomical characteristics of primary teeth ,, and the children's immunological mechanism process, ,, one can understand why this battle between infection and host is disproportionate most of the time.
Due to the methodology used in the present study, we have observed infections of low to moderate intensity at the furca region of teeth with deep caries (Group I) in the majority of the slides. The coronal pulp was also found to be infected, and the infection was already present in the radicular pulp in most teeth. Moreover, the low-intensity infection observed in the root region was associated with several pathological processes with the pulp. Interestingly, in most cases, the pulp had hyperemia despite the absence of bacteria within the root region. With regard to the periodontum, the histological sections showed formation of granulomatous tissue related to low-intensity infection in the roots of some teeth with inflamed pulp.
Hobson,  in a study on the presence of bacteria in extracted teeth with deep caries, found it to be 18.27% of infected furca. The author also reported that bacteria were present in dentinal tubules at the furca region whenever the pulp was necrotic. By comparing these results with ours, we observed that infections were present more in the furca region and not always periodontal changes were associated with necrotic pulp. Rayner and Southam  studied the bacterial proliferation within the tissues of deep carious primary teeth and found that dentine can neither impede the invasion of bacteria nor inhibit pulp reactions. This finding was also observed in the present work.
Based on the results obtained, teeth with deep caries lesions present, at least, hard-tissue infection at the furca region and coronal pulp areas, reaching the radicular pulp. These findings should not be ignored by the clinical practitioner when profound caries are found and the best treatment choice is to be selected. Rocha  has advised that any decision on conservative (pulpectomy) or radical (biopulpectomy) treatment is related to a direct visual examination of the pulp tissue. Therefore, clinical and radiographic evaluations should be carefully performed as well as the treatment itself. Most frequently, a vital pulp, associated with asymptomatic tooth, is not indicative of good health conditions and absence of infection.
With regard to the results regarding teeth with bone loss at the furca region (Group II), we found that infection was evident in both furca and root, but at low intensity. In addition, the histological sections of the furca region showed presence of bacteria in the coronal pulp as well as presence of biofilm on the walls of the pulp chamber. Presence of bacteria within the pulp forming biofilm on the wall of the root canals was observed when these teeth were analyzed. Godoy  and Bolan and Rocha  also found the presence of bacteria and biofilm on the walls and floor of the pulp chamber, walls of root canals and dentinal tubules, and even on the external surface of the root.
In the 1960s, Cohen et al.  showed evidence of the presence of microorganisms in the root canals of primary teeth. During the following four decades, other authors ,,,,,,, reported the presence of facultative anaerobic bacteria and strictly anaerobic bacteria (both gram positive and gram negative) invading the dentinal mass and accessory canals,  reaching the periodontum, , and forming bacterial biofilm at the apical region.  Sometimes, agglomerations of bacteria detach from the bacterial biofilm, thus resulting in the so-called planktonic bacteria that can cause distant infections. 
Despite the danger this infection can cause both locally ,,,,,,,, and to the child patient's general health, , some dental practitioners working with children still use protocols for endodontic treatment of primary teeth without biomechanical preparation. Consequently, the infection control depends on antiseptic substances only. ,,,
Through the present study, it was possible to demonstrate the presence of biofilm on the pulp chamber and root canals. This proves that endodontic treatments with no biomechanical preparation certainly fail in eliminating the infection completely from the mineralized structures of the primary teeth. However, there are authors who have reported a very successful treatment with these protocols  and others who observed an accelerated eruption of the permanent tooth.  In fact, the presence of residual infection within the root canals ends up maintaining the inflammation process, for the chemical mediators stimulate clastic cell activity and inhibit the blastic cells  existing in the periodontal ligament. As a result, there is an accelerated resorption of the primary tooth and precocious eruption of the permanent one. ,,
There are biomechanical protocols , in which odontometry, canal instrumentation, irrigation, delayed curative, and hermetic filling of the canal are required. , The protocol established by Rocha  and evaluated in two longitudinal studies , had a higher success rate for biopulpectomy compared to necropulpectomy. Treatment failure is less likely to occur when periradicular lesions are not present.  These outcomes depend on the infection progression through the dentinal tubules, which occurs more quickly in young teeth with immature dentine  and in necrotic teeth.  In vitro studies have shown that the bacterial penetration into the cervical, median, and apical thirds depends on the time of exposure to infection as well as the bacterial growth pattern. ,, Therefore, the longer an infection is, the greater is the chance of bacterial invasion into dentine and periodontum. Bone loss at the furca region and bacterial proliferation into the periodontum are signs that should alert the practitioner to the necessity for efficient instrumentation of necrotic root canals and use of delayed curative in order to eliminate the infection on the root walls and within the dentin tubules. It should be also emphasized that such endodontic procedures have to be rigorously performed; otherwise, the biofilm on the external root surface will not be completely eliminated. Phagocytic cells are not able to engulf the biofilm,  and in addition, they release cytokines next to the tissues surrounding the biofilm, causing pathological changes. 
In Group III, as expected, the moderate-high infection intensity at the furcation region was confirmed. Moreover, infection intensity was found to be high in the histopathologic sections of the roots. Some slides had biofilm on the external surface of the roots. In general, this biofilm is not eliminated by the individuals' immunological mechanism and need to be mechanically removed. 
In this same group, only two teeth had vital pulp, both with inflammatory process. Their periodontum had distinctive pathologies with resorption affecting the inner portion of the root, acute and chronic inflammatory processes, acute and chronic abscesses, granuloma, and epithelial granuloma. These findings are in accordance with those found by Winter and Kramer  , Valderhaug  , Savage et al.,  Myers et al.,  and Bolan and Rocha. 
Because these teeth cannot be repaired, their elimination from the oral cavity has to be done following diagnosis. Tomic-Karovic and Jelinek  had already adverted to the fact that necrotic molars should not remain in the child's mouth without adequate treatment only to fill the space for permanent teeth. These teeth should be extracted in order to avoid the harmful effects of infection on the permanent tooth germs, as has been observed by Winter and Kramer,  Valdenhaug,  Coll and Sandrian,  Cordeiro,  Roberts,  and Camp. 
The systemic effects of such an infection on the child's body in terms of dissemination through blood and lymphatic systems , should also be taken into account. The younger the children are, the lesser efficient is their immune response to bacteria invasion. ,, Moreover, nutritional deficiencies also decrease the immunological efficacy.
The results obtained in Group IV highlighted the constant presence of infection in the residual roots, both in their cervical portions and apical regions, mainly in periodontum. Infection-related pulp necrosis, which was observed in the majority of the cases, favored the development of anaerobic bacteria that are predominantly seen in this type of pathology.  Based on the studies by Tomic-Karovic and Jelinek  and Brook et al.,  one can suppose that the most predominant bacteria in these infections are Porphyromonas, Prevotella, Bacteroids, Peptococcus, and Peptostreotococcus. These bacteria genres have been associated with many pathologies diagnosed in children, such as respiratory infections, sinusitis, cerebral abscess, chronic mastoiditis, heart and joint infections. 
Based on the present study, the authors are unanimous in recommending more caution on the part of practitioners who treat primary molars with deep caries lesions. Clinical and radiographic examinations should be carefully performed before choosing the type of treatment and the cases always followed up. The endodontic treatment of primary molars should be performed according to a protocol similar to that for permanent teeth. Canal instrumentation should also be executed prior to root filling. Primary molars with perforation in their pulp chamber floor and residual roots should always be extracted in order to preserve the child's health. Further research studies on this issue are necessary so that the dental phenomena caused by bacterial infections can be fully understood. In addition, it is desirable to perform population studies on the impact these infections have on the child's body at a long-term basis.
It was concluded that the infection characteristics had differences regarding crown, furca, and roots in all four experimental groups. There was a gradual increase in intensity and dissemination of bacteria in the four groups, resulting in different levels of destruction of the dental structures according to severity and localization of the infection. The groups showed no characteristic histopathologic feature, although the presence of bacteria was always associated with changes in pulp and periodontum.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]