A histomorphometric and radiographic study of replanted human premolars

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1 European Journal of Orthodontics 36 (2014) doi: /ejo/cjt088 Advance Access publication 19 December 2013 The Author Published by Oxford University Press on behalf of the European Orthodontic Society. All rights reserved. For permissions, please A histomorphometric and radiographic study of replanted human premolars Jon Egil Tønnevold Fiane*, Martin Breivik** and Vaska Vandevska-Radunovic* *Department of Orthodontics, Institute of Clinical Dentistry, University of Oslo, and **Private Orthodontic Practice, Arendal, Norway Correspondence to: Vaska Vandevska-Radunovic, Department of Orthodontics, Institute of Clinical Dentistry, University of Oslo, PO Box 1109, Blindern, N-0317 Oslo, Norway. Summary Objectives: The purpose of this study was to investigate time-related dental tissue reactions to tooth replantation in mature and immature human premolars by histomorphometric and radiographic methods. Material and methods: Sixty premolars were extracted, immediately replanted (T0), and left without intervention for 1, 2, 3, 6, 12, and 24 weeks before re-extraction (T1). Periapical radiographs were taken at T0 and T1 in all groups except for the 24 week group. Paraffin-embedded teeth were sectioned buccolingually at 5 µm, stained with haematoxylin-eosin, and prepared for histological analysis. Root resorption, tertiary dentine, and root and crown length were recorded and compared on both radiographs and paraffin sections. Results: Initial degeneration of the odontoblast layer and disturbance of vasculature and normal architecture of the pulp were seen in both mature and immature teeth within the first 3 weeks. Revascularization and tertiary dentine formation was visible on histological sections in immature teeth at 6 weeks. After 24 weeks, most immature teeth had pulps partly or completely obliterated. Abundant tertiary dentine observed on histological slides was not easily seen on periapical radiographs. Likewise, surface root resorption found in both immature and mature teeth was not visible on the radiographs up to 6 weeks after replantation. Conclusion: Within the limits of this study it can be concluded that the dental pulp of immature teeth has the ability to regenerate and produce tertiary dentine after replantation. Root resorptions and tertiary dentine seen on histological sections are not visible on corresponding radiographs 6 and 12 weeks after replantation. Introduction Replantation of teeth after trauma, experimental replantation, and autotransplantation are closely comparable procedures, where an exarticulated, luxated, or a donor tooth is repositioned in its original alveolar socket, or in another surgically prepared site. Replanted teeth lose their vitality due to severed nerve and vascular supply, resulting often in pulp tissue degeneration. However, re-innervation and revascularization may be possible and has been verified by clinical and radiological methods in human studies (Kling et al., 1986; Andreasen et al., 1990; Andreasen et al., 1995). Comprehensive histological studies of replanted teeth are mainly based on animal material (Skoglund et al., 1978; Skoglund, 1981; Tsukamoto-Tanaka et al., 2006; Ferreira et al., 2010), while corresponding human studies are scarce (Andreasen and Hjorting-Hansen, 1966; Breivik, 1981; Breivik and Kvam, 1990). Studies in humans have demonstrated that extraction and immediate replantation of premolars leads to pulpal revascularization in almost all cases, given an open apical foramen and/or incomplete root formation (Öhman, 1965; Breivik, 1981). Both factors seem to be closely related to the frequency of pulpal healing not only in autotransplanted teeth but also in teeth transplanted after trauma (Andreasen et al., 1995). However, the authors point out that in cases of avulsed teeth and their treatment, the extra-alveolar period and storage medium may also be of importance for a successful outcome. Clinical and radiographic studies have given us important insight into tooth survival and the pulpal reactions subsequent to replantation and make the basis for our current clinical management of tooth avulsion and orthodontic problems related to tooth agenesis (Paulsen et al., 1995). However, the underlying biological mechanisms determining the healing process after tooth replantation remain unclear. Difficulties in studying tissue reactions after replantation of human teeth are obvious. Histological material of sufficient size is difficult to obtain mainly due to ethical implications. Furthermore, in a clinical setting, only those teeth failing to regain vitality are extracted. Observation of successful cases is only possible in animal studies, or in humans using radiographic techniques and/or clinical parameters. To our

2 642 knowledge, observation of histological dental tissue reactions in replanted teeth and their comparison with corresponding radiographs has never been previously reported. Therefore, the aim of this investigation was to study and semi-quantify time-related, dental tissue changes after replantation of mature and immature human premolars and to compare these histological findings with corresponding radiographs, thus assessing their reliability as a clinical tool. Materials and methods Premolars from 24 patients, 12 girls ( years) and 12 boys ( years), were collected for investigation. All teeth had been scheduled for extraction in connection with orthodontic treatment. Collection of the material was carried out from 1973 to 1976 at the Department of Orthodontics, University of Oslo. The following procedures are described in detail by Breivik and Kvam (1977): Sixty experimental teeth were extracted and immediately replanted into the original socket (T0) and left without any other intervention for 1, 2, 3, 6, 12, or 24 weeks before re-extraction (T1) and fixation for histological sectioning. Extraoral period was less than 1 minute without using a storage medium. Mild finger pressure was applied for five minutes to keep the tooth in place subsequent to replantation. One premolar was extracted from each subject at T1 to serve as a control. Forceps with straight beaks were used to reduce pressure below the enamel cementum junction. Citanest 2 per cent Octapressin was administered at the primary extraction and Carbocaine Dental at the secondary extraction and the extraction of control teeth. Following extraction, the teeth were fixed for 48 hours in neutral buffered formaldehyde before demineralization in 5.2 per cent nitric acid. The specimens were embedded in paraffin, prepared in serial 5 µm bucco-lingual sections, and stained with haematoxylin and eosin (H&E). Routine intraoral periapical radiographs were taken at T0 and T1 in all extraction groups except for the 24 week group. Damaged slides and slides with missing identification number were excluded from the investigation. Radiographs with obvious distortions and radiographs taken only at T0 were also excluded. The current selection comprises 77 teeth and 110 radiographs (Table 1). The maturity of experimental and control teeth was evaluated according to the index of Moorrees et al. (1963) and the method proposed by Kling et al. (1986). Teeth with an apical opening of 1 mm or less were considered mature (Table 2). The histomorphometric evaluation was carried out using two microscopes: a standard binocular microscope (Carl Zeiss, Jena, Germany) was used for the qualitative histological evaluation and a Leica microscope (Leica DMRBE Research Microscope, Leica Microsystems, Wetzlar, Germany) with Olympus DP50 camera for the metrical measurements and photographs. The metrical measurements were made on the longest sections cut through the T. E. T. FIANE ET AL. Table 1 The distribution of control and experimental teeth in the material. Control teeth Experimental teeth Total Maxilla Mandible Maxilla Mandible Female Male Total Table 2 The distribution of mature and immature teeth in each observation group. Observation time in weeks Control Total Mature Immature Total pulp. Every fifth slide, in total five per tooth, were measured and the mean values used for analysis. Observations were made in three separate zones equivalent to the method by Breivik and Kvam (1977): 1. apical zone, apical 5 mm of the roots; 2. middle zone, between apical and coronal zone; and 3. coronal zone, coronal to a line drawn between the buccal and lingual/palatal enamel cementum border. Metrical recordings from the radiographs were obtained using a digital sliding caliper (CappaµSystem, Mauser, Switzerland) with an accuracy of two decimals. Crown length, root length and apical opening were registered in accordance with Myrlund et al. (2004). Any signs of root resorption, obliteration, further root development, apical closure, or periapical pathology were recorded. Statistical analysis The statistical analysis was performed using version 17.0 of the SPSS package (SPSS Inc., Chicago, Illinois, USA). Potential differences or similarities between mature and immature teeth related to external root resorption, apical root resorption, and presence of tertiary dentine were assessed using Fisher`s exact test. The McNemar s test was used to test potential differences or similarities in observations of time-related changes between radiographs and slides. A sample of 30 periapical radiographs and 30 histological slides were chosen at random to measure reproducibility. Measurements of crown height, root length, and apical opening were repeated twice within 1 month by the same observer (JETF). The method error (ME) was calculated 2 2 using the formula of Dahlberg (1940): ME = d / 2n, where d is the difference between two measurements and n is

3 A STUDY OF REPLANTED HUMAN PREMOLARS the number of double determinations. The ME for the various measurements on intraoral radiographs was 0.25 mm for crown heights, 0.31 mm for root lengths, and 0.12 mm for the apical opening. Corresponding values for measurements on the histological slides were 0.25, 0.26 and 0.11 mm. Paired t-test was used to assess differences between crown and root length measurements and crown-root ratios on histological slides and radiographs. Results Histological evaluation Control teeth. Almost all teeth in the control group exhibited normal histological characteristics: predentine layer of uniform thickness, an intact odontoblast layer with regularly arranged columnar cells, and a loose, highly vascular, fibrous connective tissue that was rich in fibroblasts (Figure 1A C). Moderately filled arterioles and empty venules with intact, thin endothelial covering were seen throughout the pulp. The apical areas of immature control teeth showed the typical anatomic pattern of a developing tooth. The Hertwig`s epithelial root sheath was readily seen, and in some instances the layered arrangement of the sheath was visible (Figure 1D). Experimental teeth: 1 3 weeks. The differences between teeth extracted after 1, 2, and 3 weeks were negligible, and therefore, the teeth from these groups are described together. 643 The general appearance of the pulp tissue was noticeably different from the control teeth, with reduced number of fibroblasts, irregular fibrous matrix, loss of normal vasculature, and missing or abnormal odontoblast layer (Figure 2A D). Arterioles in the apical and especially the middle part of the pulp often showed signs of stasis, and extravascular erythrocytes were found in several teeth. Most immature and all mature teeth lost their normal odontoblast layer in the middle and coronal areas during the first two postoperative weeks. The distinct predentine layer and globular dentine disappeared in most teeth. No tertiary dentine was found in these observation periods (Table 3). A dense, cementum-like fibrous tissue was seen across the apical foramen in some of the extracted teeth (Figure 3A and 3B). External surface resorption was obvious in most of the teeth; however, there were no corresponding pulpal reactions and no signs of repair of the resorption lacunae (Table 4). Experimental teeth: 6 weeks. The histological appearance of the pulp tissue was consistent with pulp necrosis in six out of nine mature teeth. It was characterized by absence of normal vasculature, considerable vacuolization or loss of odontoblast layer, and large empty spaces throughout the pulp (Figure 4A and 4B). The immature teeth demonstrated a normal odontoblast layer in the apical zone and one in the Figure 1 Haematoxylin and eosin sections from an immature first premolar control tooth. (A) Coronal pulp (P) with highly vascular connective tissue. (B) Middle part of the pulp with large central blood vessels (arrows). (C) Higher magnification of the area marked in B. Intact odontoblast layer (OB) and predentine layer of uniform thickness (arrows). (D) Apical area with intact Hertwig`s epithelial root sheath. Three-cell layers of the root sheath are readily seen (arrows). D, mature dentine; PDL, periodontal ligament. Figure 2 Haematoxylin and eosin sections from a mature first premolar extracted after 2 weeks. (A) Middle/coronal pulp (P) with an irregular appearance. (B) Higher magnification of the area marked in A. Missing odontoblast layer and predentine. (C) Apical zone. Fibrous matrix with few cells and large empty blood vessels (arrows). (D) Higher magnification of the area marked in C. Disintegration of the pulp tissue with few fibroblasts and missing odontoblast layer. D, mature dentine.

4 644 T. E. T. FIANE ET AL. Table 3 Number of teeth with no reparative dentine, or different qualities of tertiary dentine in the three zones of the pulp. Observation time Area Tertiary dentine No reparative (rep.) dentine Irregular rep. dentine Regular rep. dentine Abundant rep. dentine Immature Mature Immature Mature Immature Mature Immature Mature 1 3 weeks Coronal Middle Apical weeks Coronal Middle Apical weeks Coronal Middle Apical weeks Coronal Middle Apical Figure 3 Haematoxylin and eosin sections from an immature first premolar extracted after 3 weeks. (A) Apical area. A dense, cementum-like fibrous tissue (*) seems to seal off the apex. (B) Higher magnification of the area marked in A. Cells and blood vessels are trapped in the dense fibrous matrix (arrowheads). The fibrous tissue appears to be continuous with dentine in some areas (arrows). P, pulp; D, mature dentine; C, cementum; PDL, periodontal ligament. middle zone. Blood-vessel distribution appeared normal in all areas of the pulp. In one tooth, initial hard tissue deposition had taken place and a thin layer of tertiary dentine was seen along the pulp walls. Tertiary dentin formation was more pronounced in immature than in mature teeth (Table 3). External root resorption was a common finding (Table 4). No visible further root development was seen in any of the teeth. Experimental teeth: 12 weeks. Vital pulps were observed in all but one immature tooth, while all mature teeth demonstrated necrotic pulps. Half of the immature teeth demonstrated excessive hard tissue formation in the coronal and middle areas of the pulp (Figure 5A). Postoperatively formed dentine lining the pulp walls and free islands of dentine were seen. In areas of abundant dentine, no distinct odontoblast layer was visible. Many immature teeth displayed a normal odontoblast layer accompanied by regular tertiary dentine in the apical region (Figure 5B; Table 3). One vital tooth demonstrated a fibrous, bone-like tissue bridging the apical foramen, with sparse tertiary dentine formation in the adjacent area (Figure 6A D). External surface resorption lacunae were seen on both buccal and lingual surfaces of all mature, and on majority of the immature teeth (Table 4). In most of the lacunae, repair had not taken place. Further root development was seen in all but one immature tooth. The newly formed apical hard tissue was generally irregular in shape with a marked demarcation line separating it from the preoperative apex. In the vital teeth, Hertwig`s root sheath was not easily seen, and in the three necrotic teeth, no further root development was observed. Experimental teeth: 24 weeks. At this stage, all teeth demonstrated tertiary dentine formation that, sometimes, tended to occupy the entire pulp (Figure 7A; Table 3). The structure of the tertiary dentine in the pulp and especially in the pulp horn was irregular, clearly atubular, with scattered cellular inclusions, and the hard tissue boundaries were not limited by a distinct odontoblast layer. Blood vessels were frequently entrapped in the newly formed hard tissue. In the middle and apical zones, tertiary dentine was more regular and resembled postoperatively formed dentine to a larger degree. All teeth had vital pulp tissue in the apical and middle zones, but the odontoblast layer was not always seen (Figure 7B D). External resorption lacunae were partially or completely repaired by apposition of cellular cementum; however, several small loci with active surface resorption could still be found. No resorptions extended to the pulp. Apical root resorption had occurred in three immature teeth (Table 4), but the resorption was repaired, and a demarcation line was clearly evident. Further root development was found in all immature teeth (Figure 7C and 7D).

5 A STUDY OF REPLANTED HUMAN PREMOLARS Table 4 Distribution of external root resorption, apical root resorption and tertiary dentine among mature and immature teeth. Significant or non-significant (NS) correlation between the variables (yes/no mature/immature). Number of mature teeth Number of immature teeth Fisher`s exact test External root resorption Yes No Yes No Week NS Week NS Week NS Week NS Apical root resorption Yes No Yes No Week NS Week NS Week NS Tertiary dentine Yes No Yes No Week P Figure 4 Haematoxylin and eosin sections from a mature first premolar extracted after 6 weeks. (A) The pulp (P) in the coronal/middle area displays necrotic appearance. Few cells are visible except from the area close to the odontoblast layer. (B) Higher magnification of the area marked in A. Vacuolization and degeneration of the odontoblast layer. Necrotic pulp tissue with few cells and barely visible empty blood vessels (arrows). No predentine is seen along the pulp wall. D, mature dentine. Histological versus radiographic observations A statistically significant difference between the degree of detection of external surface resorption on radiographs and slides was found at all experimental stages (Table 5). External surface resorptions seen on half of the histological slides were undetectable on the corresponding radiographs at 1, 2, and 3 weeks. At 6 and 12 weeks postoperatively, external root resorption was seen on most of the histological sections, but only those with severe apical root resorption were detected on the radiographs. Tertiary dentin was easily seen on histological slides at weeks 6 and 12, but not on the radiographs, and these differences were significant (Table 5). Length measurements on radiographs and slides The mean differences for both crown (0.11 ± 0.13 mm) and root length (0.52 ± 0.92 mm) measurements and crown-root 645 ratios (0.82 ± 1.25) between radiographs and histological slides were highly significant (P < 0.001). Discussion The dental pulp and supporting tissues have the ability to regenerate and regain normal function after a complete severance of the neurovascular supply. The results from the present study demonstrate that after replantation the pulpal tissues have the ability to regenerate, based on the histological evaluation, formation of tertiary dentin and root development. These changes were time dependent as signs of pulp vitality and root formation increased with the length of the observation period. Replanted premolars at 12 and 24 weeks demonstrated greater external surface resorption, and also signs of root repair. The presence of resorption loci was not significantly different between mature and immature teeth at any stage. However, tertiary dentin formation was significantly higher in immature than mature teeth. Autotransplantation of immature teeth is a well-established procedure in the treatment of multiple agenesis and traumatic loss of teeth. Long-term survival rates of more than 90 per cent have been reported after replantation of immature (Andreasen et al., 1990; Paulsen et al., 1995; Czochrowska et al., 2002; Jonsson and Sigurdsson, 2004) and also of mature teeth (Andreasen et al., 1990). However, the reported survival success rate does not always represent the true percentage of teeth with regained vitality. It was shown that pulp healing, as evaluated by sensitivity test and radiography, was much higher in teeth with incomplete, rather than complete root formation (Kristerson, 1985; Andreasen et al., 1990; Paulsen et al., 1995; Jonsson and Sigurdsson, 2004). Initial sensitivity response and initial pulp canal obliteration could not be clinically detected in more than 12 and 16 per cent of the teeth 12 weeks after transplantation (Paulsen et al., 1995). The present findings show that tertiary dentine formation started at 6 weeks postoperatively and gradually increased to 24 weeks, being significantly higher in immature than mature teeth. These histological data indicate that reparative dental tissue processes start much earlier than the clinical signs of pulp healing. Revascularization of the pulp is a process in which ingrowth of highly vascularized connective tissue takes place, or in which blood vessels already present in the pulp anastomose with blood vessels from the periodontium (Skoglund et al., 1978). The latter may explain the rapid revascularization of the mainly immature pulps that started as early as 3 weeks after replantation and was clearly visible at 6 weeks. In the mature teeth, revascularization and normalization of pulp anatomy were only occasionally seen, most often in the apical area and only in teeth with an apical opening smaller than 1 mm. This is in accordance with several other studies in humans, dogs, and monkeys (Skoglund, 1981; Kristerson and Andreasen, 1984; Andreasen et al., 1990; Schwartz and Andreasen, 2002) and

6 646 T. E. T. FIANE ET AL. Figure 5 Haematoxylin and eosin sections from an immature first premolar extracted after 12 weeks. (A) Coronal pulp (P) with abundant atubular tertiary dentine (TD). The remaining soft tissue seems normal and blood vessels are seen through the pulp (arrows). (B) A more regular TD is seen in the apical zone. Dentine tubuli are seen in the newly formed hard tissue and a distinct predentine layer (PD) is visible. The cells of the odontoblast layer (OB) have normal appearance. A border is seen between the original and newly formed dentine (arrows). D, mature dentine. Figure 7 Haematoxylin and eosin sections from an immature first premolar (A and B) and two immature first premolars (C and D) extracted after 24 weeks. (A) Coronal pulp horn filled with atubular tertiary dentine (TD). Strings of pulp tissue (P) with blood vessels (black arrows) are visible, as well as multiple cellular inclusions (white arrows). (B) In the middle zone, a normal odontoblast layer (OB) is seen. TD is formed in the pulpal direction (P) covered with a layer of predentine (PD). The arrows in picture B are indicating the border between pre- and postoperatively formed dentine. (C) Apical area with abnormal further root development. The length of the original apex can be seen (white arrow) and the border between pre- and postoperatively formed dentine (D) is readily visible (black arrows). Apical root resorption has taken place (black arrowheads) alongside tertiary dentine (TD) formed on the pulpal side (asterisk). (D) The apical opening is almost sealed of by postoperatively formed dentine (asterisk). Notice the border between pre- and postoperatively formed dentine (D; arrows). Wide but shallow areas of surface resorption are visible (arrowheads). P, pulp; PDL, periodontal ligament; C, cementum; OB, odontoblast layer; DT, denticle. Figure 6 Haematoxylin and eosin sections from an immature first premolar extracted after 12 weeks. (A) Apical area with a mineralized tissue crossing the apical foramen (asterisk). Sparse tertiary dentine formation (TD) has taken place along the pulp wall. The tooth lacks a normal odontoblast layer and the pulp tissue appears to be inflamed. Areas of active external root resorption are seen on both the buccal and lingual aspects of the root (arrows). (B) Higher magnification of the area marked in A. The hard tissue contains entrapped cells (arrows) and is penetrated by apparently vital pulp tissue (P). No regular anatomical features such as tubules, or Haversian channel systems are seen. D, mature dentine. Intraoral periapical radiographs from the same tooth at T0 (C) and T1 (D). Notice the more radiopaque area apically (between dashed lines). indicates that, under ideal replantation conditions, a vital pulp in a tooth with an apical opening less than 1 mm can survive replantation. Teeth that regain vitality after trauma respond by deposition of mineralized hard tissue along the pulp walls. In time, obliteration of the pulp is recognizable on intraoral radiographs and serves as an indication of pulp vitality (Andreasen and Bakland, 2012). In the present material, tertiary dentine was seen in significantly more immature than mature teeth, and had varied distribution and morphology. Some teeth displayed thin layers of regular, tubular tertiary dentine, while in others, irregular, bone-like tissue occupied the entire pulp. This pleomorphism coincides well with the results obtained from replanted animal teeth (Anderson et al., 1968; Skoglund and Tronstad, 1981; Tsukamoto- Tanaka et al., 2006). Regular, tertiary dentine was often associated with well arranged odontoblasts and highly vascular pulp tissue, while irregular dentine with cellular inclusions, often lacked a distinct odontoblast layer. Sometimes, hard tissue that resembled bone or cementum was observed particularly in the coronal pulp. The different morphological appearance of apical and coronal hard tissues can be attributed to the rate of revascularization and re-innervation, and the activity of the matrix producing cells. Several immunohistochemical studies have shown that resident pulp cells have the ability to differentiate into odontoblast- and osteoblast-like cells (Ohshima et al., 2001; Goldberg and Smith, 2004; Ogawa et al., 2006). Under pathological conditions, such as tooth injury and replantation, the regulatory mechanisms for suppression of bone formation can be disturbed (Zhao et al., 2007) and thus lead to formation of bone-like tissue instead of dentine.

7 A STUDY OF REPLANTED HUMAN PREMOLARS 647 Table 5 Number of teeth where external root resorption or tertiary dentine were or were not seen on slides and radiographs. Significant or non-significant (NS) difference between the two modalities (radiographs and histology). External root resorption Detection on slides (Number of teeth) Detection on radiographs (Number of teeth) McNemar s test Yes No Yes No Week P < 0.05 Week P < 0.05 Week P < 0.05 Tertiary dentine Yes No Yes No Week P < 0.05 More mature than immature teeth experienced external surface root resorption although the differences were not significant. The incidence of surface resorption generally increased from the second week on, but a small decrease was evident for immature teeth after 24 weeks. Healing by apposition of cementum was often seen in the long-term observation groups and has been previously reported (Andreasen, 1981). Jonsson and Sigurdsson (2004) conclude that external surface resorption seen following autotransplantation reflects normal biological responses and requires no treatment. The external root resorption registered on histological slides could not be detected on the radiographs during the first 3 weeks after replantation. Later on, only severe apical root resorptions could be seen on the radiographs and the differences between the observations were significant. Significant differences between histological and radiological observations of tertiary dentine were also noticed at 6 and 12 weeks postoperatively. This implies that radiographs are not a reliable source for the detection of external root resorption and tertiary dentine for the first 12 weeks after tooth replantation. Additionally, the reliability of radiographs for crown and root length measurements, and crown-root ratio is questionable, as mean differences between these measurements on slides and radiographs were statistically significant. The relatively short observation time and the lack of intraoral radiographs at Week 24 made it impossible to statistically analyse the root development and apical closure from T0 to T1. Nevertheless, further root growth was evident in most immature teeth on histological slides from Week 12 onwards. Some teeth, predominantly those with necrotic pulps, showed arrest of root formation. This is in accordance with previous studies (Slagsvold and Bjercke, 1974; Andreasen et al., 1988; Paulsen et al., 1995; Myrlund et al., 2004) and confirms that the Hertwig`s epithelial root sheath has the ability to resume normal activity after trauma, even if only a fraction of it is left intact (Andreasen et al., 1988). Conclusions Complete regeneration of normal vasculature occurred between 3 and 6 weeks after replantation in most immature teeth. The dental pulp of the immature teeth has the potential to produce tertiary dentine subsequent to replantation. The presence of external root resorption did not seem to be significantly correlated with the stage of root development. These histological responses are not readily seen on corresponding radiographs; therefore, observations from radiological investigations up to 12 weeks after replantation have to be interpreted with caution. However, the conclusions concerning the reliability of intraoral radiographs in the detection of surface root resorption have to be interpreted within the limits of this study due to the relatively short investigation period and small number of teeth. Acknowledgements The authors wish to thank Professor Leif Sandvik for help with the statistical analyses and Håkon Størmer for photographic assistance. References Anderson A W, Sharav Y, Massler M 1968 Reparative dentine formation and pulp morphology. Oral Surgery, Oral medicine, and Oral Pathology 26: Andreasen J O 1981 Relationship between cell damage in the periodontal ligament after replantation and subsequent development of root resorption. A time-related study in monkeys. Acta Odontologica Scandinavica 39: Andreasen J O, Bakland L K 2012 Pulp regeneration after non-infected and infected necrosis, what type of tissue do we want? A review. Dental Traumatology 28: Andreasen J O, Borum M K, Jacobsen H L, Andreasen F M 1995 Replantation of 400 avulsed permanent incisors. 2. Factors related to pulpal healing. Endodontics & Dental Traumatology 11: Andreasen J O, Hjorting-Hansen E 1966 Replantation of teeth. II. Histological study of 22 replanted anterior teeth in humans. Acta odontologica Scandinavica 24: Andreasen J O, Kristerson L, Andreasen F M 1988 Damage of the Hertwig s epithelial root sheath: effect upon root growth after autotransplantation of teeth in monkeys. Endodontics & Dental Traumatology 4: Andreasen J O, Paulsen H U, Yu Z, Bayer T, Schwartz O 1990 A long-term study of 370 autotransplanted premolars. Part II. Tooth survival and pulp healing subsequent to transplantation. European Journal of Orthodontics 12: 14 24

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