SEM Study of Apical Morphological Alterations in Primary Teeth with Vital and Necrotic Pulps

Journal ID: UUSP Article ID: 427738 SEM Study of Apical Morphological Alterations in Primary Teeth with Vital and Necrotic Pulps Le a Assed Bezerra da Silva, Patrı cia Motta Fernandes, Raquel Assed Bezerra da Silva, Marcos A. Rossi, Paulo Nelson-Filho, and Lenaldo Branco Rocha No Query QUERY SHEET

nesxml GNES_A_206661 February 13, 2008 15:53 0

Ultrastructural Pathology, 33:1 6, 2009 Copyright r Informa Healthcare USA, Inc. ISSN: 0191-3123 print /1521-0758 online DOI: 10.1080/01913120903275642 SEM Study of Apical Morphological 5 Alterations in Primary Teeth with Vital and Necrotic Pulps Léa Assed Bezerra da Silva, DDS, PhD, Patrícia Motta Fernandes, DDS, and Raquel Assed Bezerra da Silva, DDS, PhD Department of Pediatric, Preventive and Community Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil Marcos A. Rossi, MD, PhD Department of Pathology, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil Paulo Nelson-Filho, DDS, PhD Department of Pediatric, Preventive and Community Dentistry, School of Dentistry of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil Lenaldo Branco Rocha, DDS, PhD Department of Pathology, Medical School of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil ABSTRACT 10 This study evaluated, by SEM, the morphology of human primary teeth roots. Twenty-four teeth were divided into 3 groups: pulp vitality (group I) and pulp necrosis without (group II) and with apical periodontitis (group III). Roots were analyzed by the presence of periodontal ligament (PDL) fibers and resorption areas. In groups I and II, presence of PDL fibers and absence of resorption were observed in all cases (100%), while all specimens (100%) of group III showed no 15 PDL fibers and resorption areas. In conclusion, there are morphological differences in the apical region of primary teeth with different pulpal and periapical pathologies. Keywords: apical morphological alterations, periapical pathosis, periodontal ligament fibers, primary teeth, scanning electron microscopy 20 In pathological conditions, particularly in permanent teeth with necrotic pulps and radiographically evident periapical pathosis, the outer surface of the root apex may present areas of resorption on cementum and 25 dentin that favor the retention of microorganisms and colonization by pathogenic bacteria [1]. Pathological root resorption from bacterial origin occurs secondary to pulp infection by microorganisms [2], which initially occupy the root canal lumen and subsequently invade the root canal system, i.e., the dentinal tubules, lateral canals, accessory canals, secondary canals, and apical root cementum surface. The microorganisms reach the periapical tissues, leading to the development of extraradicular infection and bone resorption [3 5]. 30 1 Received 18 August 2009; accepted 20 August 2009. Address correspondence to Profa. Dra. Léa Assed Bezerra da Silva, DDS, PhD, Departamento de Clinica Infantil, Odontologia Preventiva e Social, Faculdade deodontologia deribeirão Preto, Universidade de São Paulo, Avenida do Café S/N, CEP, 14040-904 Ribeirão Preto, SP, Brasil. E-mail: lea@forp.usp.br

Morphology of Human Primary Teeth Roots 35 Scanning electron microscopic studies [5 10] have assessed the presence of microorganisms in the root canal system, on the outer surface of the root apex and in the periapical region of permanent teeth. In addition to the presence of periapical pathologies, root apex 40 topography has also been examined by scanning electron microscopy (SEM) [5]. This knowledge has served as the basis for the development of endodontic materials and root canal preparation techniques and has contributed to increase the success rates in endo- 45 dontically treated permanent teeth [1]. Up to now, there have been no studies evaluating the morphological alterations occurring in the apical region of primary teeth with and without periapical pathosis. Therefore, the objective of this study was to evaluate, by 50 SEM, root apex morphology (presence of periodontal ligament (PDL) fibers and areas of pathological root resorption on the outer root surface) in human primary teeth with pulp vitality or pulp necrosis (with and without radiographically evident periapical pathosis). 55 MATERIAL AND METHODS Specimen Selection The research proposal was independently reviewed by the Ethics in Research Committee of the School of Dentistry of Ribeirão Preto, University of São Paulo 60 (Process x2006.1.1310.58.2) and the study design was approved. The apical root third of recently extracted human primary incisors and molars without previous dental treatment were evaluated in three situations: teeth with pulp vitality (sound) (group I; n ¼ 8), teeth with pulp 65 necrosis without periapical pathosis (group II; n ¼ 8), and teeth with pulp necrosis and well-defined radiographically evident periapical pathosis (group III; n ¼ 8). Eligible participants were selected from patients of both sexes aged 4 8 years who had been referred for dental 70 treatment at the Pediatric Dentistry Clinic of the School of Dentistry of Ribeirão Preto, University of São Paulo, Brazil. Children who had received antibiotics within the previous 3 months were excluded from the trial. The study purposes were fully explained to the parents/ 75 guardians, who signed a written informed consent form authorizing the enrolment of the children in the study. The patients were submitted to a clinical interview, review of dental/medical history, and clinical/ radiographic examination for tooth selection. The peri- 80 apical radiographs were taken according to the parallel radiographic technique using a pediatric film holder to minimize image distortions. Overretained teeth with pulp vitality confirmed by pulp sensitivity tests should present intact crown, absence of fistula, less than 85 two-thirds of physiological root resorption, periodontal probing depth <3 mm, and normal periodontal ligament space. The teeth with pulp necrosis (with and without well-defined periapical pathosis) should present pulp exposure due to carious lesion, presence/absence of fistula, less than two-thirds of root resorption, and periodontal probing depth <3 mm. They were scheduled for extraction because of extensive crown destruction not amenable to restoration. Specimen Preparation After tooth extraction, the roots were gently rinsed with sterile saline. Care was taken not to damage the apical root surface. In the teeth with pulp necrosis and periapical pathosis, the chronic periapical lesion was removed using a scalpel with care not to cause any damage to the apical root surface. Thereafter, the teeth were placed in sterile glass receptacles for 20 min containing 0.15 g trypsin (Sigma Chemical, St. Louis, MO, USA), weighed in a precision analytical balance (model FA2104N, Bioprecisa, Curitiba, PR, Brazil), and diluted in 5 ml distilled water to reach a concentration of 0.03 g/ml. Next, the teeth were washed in 0.1 M sodium cacodilate buffer and placed in individual receptacles containing modified Karnovsky solution (8% glutaraldehyde, 12% paraformaldehyde, in 0.2 M sodium cacodylate, ph 7.2 7.4) for a minimum of 5 days. After this period, using a carborundum disk mounted in a low-speed handpiece, a groove was made perpendicular to the long axis of the root at a distance of 3 mm from the root apex. This groove served as a guide for the placement of a surgical chisel that was used together with surgical hammer to sever the apical portion from the tooth. In molars, the root that was least affected by physiological resorption was used. All procedures were performed in a laminate flow chamber with care taken not to damage the apical root. Specimen Processing for SEM The root apexes (apical 3 mm) were dehydrated in an increasing ethanol series (70, 95, and 100%), each solution being changed at 15-min intervals within 1 h per concentration. The specimens were critical-point dried with CO 2 (Bal-Tec CPD 030, Fürstentum, Liechtenstein), sputtercoated with gold to obtain an approximately 200-mmthick layer (Emitech K650 Sputter Coater, London, UK), and examined with a scanning electron microscope (DSM 940A; Zeiss, Oberkochen, Germany), operating at 15 kv. Specimen Analysis For analysis of the specimens, an initial magnification of 50 was used to locate the apical root third in order to obtain an overview of the surface morphology. The areas corresponding to the main apical foramen were examined under 100, 200, 350, and 500 magnification, using 90 95 100 105 110 115 120 125 130 135 2

L. Assed Bezerra da Silva et al. the apical region as a landmark. Greater magnifications ( 1000, 2000, 3000, and 5000) were used to identify the presence of PDL fibers and resorption areas on root 140 dentin and cementum, whose prevalence in the different groups was recorded as percentage. Representative SEM micrographs were obtained from each specimen. RESULTS Presence of PDL fibers and absence of resorption areas 145 were observed in 100% of the teeth with vital pulps (group I) (Figure 1) and teeth with necrotic pulps without periapical pathosis (group II) (Figure 2). In these groups, root apex surface was covered by a large number of remaining PDL fibers, arranged in different directions, without areas of exposed cementum, which indicates normal conditions. On the other hand, all teeth of group III (100%) showed absence of PDL fibers and presence of large resorption areas in the region close to the apical foramen, sometimes reaching almost all its extension and exposing cementum (Figure 3). In 2 specimens of this 150 155 3 Figure 1. SEM micrographs of group I (primary teeth with pulp vitality). (A) Panoramic view of the intact root apex; bar ¼ 400 mm. (B) Greater magnification of the root apex covered by PDL fiber remnants; bar ¼ 200 môçm. (C, D) Interwoven PDL fiber bundles arranged on cementum surface, indicating normal apical tissue; bars ¼ 20 and 10 mm, respectively.

Morphology of Human Primary Teeth Roots Figure 2. SEM micrographs of group II (primary teeth with pulp necrosis without periapical pathosis. (A) Root apex with normal anatomic characteristics; apical foramen; bar ¼ 400 mm; (B) Presence of PDL fibers covering the entire apical cementum surface; bar ¼ 10 mm. (C, D) Greater magnification of the apical surface, showing interwoven PDL fibers; bar ¼ 10 mm. group, there were areas of resorption with exposure of dentinal tubules. DISCUSSION 160 Knowledge of the morphological aspects of the cementum and PDL fibers is of paramount importance for the endodontic treatment because the inflammatory process develops initially in the periodontal ligament, leading to the occurrence of pathological 165 periapical alterations [11]. In the present study, the outer surface of the apical 3 mm of primary incisors and molars was examined by SEM. It was observed that PDL fibers and root cementum were intact in all teeth with pulp vitality and pulp necrosis without periapical pathosis (groups I and II). These findings do not agree with those of Malueg et al. [12], who observed cemental resorption areas in the apical region of permanent teeth with pulp vitality, but are consistent with those of Leonardo et al. [5] and Leonardo et al. [1], who also observed intact root cementum and PDL fibers in permanent teeth with vital pulps and teeth with necrotic pulps without apical periodontitis. All teeth with necrotic pulps and periapical pathosis showed absence of PDL fibers and presence of cemental resorption areas, demonstrating that the inflammatory process and infection were not restricted to the root 170 175 180 4

L. Assed Bezerra da Silva et al. Figure 3. SEM micrographs of group III (primary teeth with pulp necrosis and radiographically evident periapical pathosis). (A) Panoramic view of the root apex, showing absence of PDL fibers and area of apical cemental resorption adjacent to the foramen; bar ¼ 100 mm. (B) Greater magnification of (A) showing absence of PDL fibers and presence of extensive resorption areas on the apical cementum; bar ¼ 40 mm. (C, D) Region close to the apical foramen showing resorption areas of different sizes and depths with exposed dentinal tubules; bars ¼ 40 and 20 mm, respectively. canal lumen, but also extended to the apical region, reaching the outer root surface (extraradicular infec- 185 tion). These results are consistent with those of other authors [5, 10, 12 14], who examined extracted permanent teeth by SEM and observed absence of PDL fibers and presence of cemental resorption areas on the outer apical surface of these teeth. According Leonardo 190 et al. [1], pulp pathology can lead to severe changes in the apical structure, destruction of PDL fibers, and areas of resorption represented by lacunae of variable depths. In these areas, apical biofilm can develop and persist after root canal treatment. 5 The morphological differences observed on the outer apical root surface of the primary teeth in all groups may influence the healing process of in cases of periapical pathosis after root canal treatment because this region is inaccessible to the biomechanical preparation. Also, the altered necrotic surface is a favorable environment for microbial colonization, which delays apical and periapical healing in endodontically treated teeth. Knowledge of the existence of morphological differences on the root apex of teeth with different degrees pulp and periapical pathology can contribute to a more efficient and predictable treatment planning. 195 200 205

Morphology of Human Primary Teeth Roots Further studies are necessary to evaluate the efficacy of different endodontic treatment techniques and materials that allow the healing of root surface areas 210 with morphological alterations of pathological origin in primary teeth. Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. 215 REFERENCES 1. Leonardo MR, Rossi MA, Bonifácio KC, Silva LAB, Assed S. Scanning electron microscopy of the apical structure of human teeth. Ultrastruct Pathol. 2007;31:321 325. 2. Fuss Z, Tsesis I, Lin S. Root resorption, diagnosis, classification 220 and treatment choices based on stimulation factors. Dent Traumatol. 2003;19:175 182. 3. Tronstad L, Barnett F, Riso K, Slots J. Extraradicular endodontic infections. Endod Dent Traumatol. 1987;3:86 90. 4. Tronstad L, Barnett F, Cervone F. Periapical bacterial plaque in 225 teeth refractory to endodontic treatment. Endod Dent Traumatol. 1990;6:73 77. 5. Leonardo MR, Rossi MA, Silva LAB, Ito IY, Bonifácio KC. EM evaluation of bacterial biofilm and microorganisms on the apical external root surface of human teeth. J Endod. 2002;28:815 818. 6. Molven O, Olsen I, Kereses K. Scanning electron microscopy of bacteria in the apical part of root canals in permanent teeth with periapical lesions. Endod Dent Traumatol. 1991;7:226 229. 7. Perez F, Rochd T, Lodter JP, Calas P, Michel G. In vitro study of the penetration of three bacterial strains into root dentin. Oral Surg Oral Med Oral Pathol. 1993;76:97 103. 8. Lomçali G, Sem BH, C ankaya H. Scanning electron microscopic observations of apical root surfaces ofteethwithapical periodontitis. Endod Dent Traumatol. 1996;12:70 76. 9. Sen BH, Piskin B, Demirci T. Observation of bacteria and fungi in infected canals and dentinal tubules by SEM. Endod Dent Traumatol. 1995;11:6 9. 10. Gutiérrez JH, Brizuela C, Villota E. Human teeth with periapical pathosis after overinstrumentation and overfilling of root canals: a scanning electron microscopy study. Int Endod J. 1999;32:40 48. 11. Lamers AC, Mullen PJ, Simon M. Tissue reaction to sodium hypochlorite and iodine potassium iodide under clinical condition in monkey teeth. JEndod. 1980;6:788 792. 12. Malueg LA, Wilcox LR, Johnson W. Examination of external apical root resorption with scanning electron microscopy. Oral Surg Oral Med Oral Pathol. 1996;82:89 93. 13. Vier FV, Figueiredo JAP. Prevalence of different periapical lesions associated with human teeth and their correlation with the presence and extension of apical external root resorption. Int Endod J. 2002;35:710 719. 14. Noiri Y, Ehara A, Kawahara T, Takemura N, Ebisu S. Participation of bacterial biofilms in refractory and chronic periapical periodontitis. JEndod. 2002;28:679 683. 230 235 240 245 250 255 6