Basic ResearchBiology

The Critical Apical Diameter to Obtain Regeneration of the Pulp Tissue after Tooth Transplantation, Replantation, or Regenerative Endodontic Treatment
Wim G.M. Laureys, DDS, MS,* Claude A. Cuvelier, MD, PhD, Luc R. Dermaut, DDS, MS, PhD,* and Guy A.M. De Pauw, DDS, MS, PhD*
Abstract
Introduction: Regeneration of pulp-like tissue in the pulp chamber after tooth transplantation, replantation, or in regenerative endodontic treatment is only possible if the apical foramen is open. According to the literature, the success of regeneration decreases considerably if the foramen is smaller than 1 mm when measured on radiographs. The aim of this study was to study histologically the relation between the width of the apical foramen and regeneration of tissue in the pulp chamber after autotransplantation. Methods: Fifteen single-rooted mature teeth of 3 adult beagle dogs were used. All experimental teeth were extracted and underwent apicoectomy. The teeth were photographed from the apical side, and the width of the foramen was calculated. The foramen width ranged from 0.241.09 mm. All teeth were replanted in infraocclusion. The observation period was 90 days after transplantation. Results: The 10 teeth with the smallest apical diameter, ranging between 0.24 and 0.53 mm, showed vital tissue in at least one third of the pulp chamber. The 6 most successful teeth showing vital tissue in the entire pulp chamber had an apical diameter between 0.32 and 0.65 mm, and 80% of the experimental teeth with a diameter varying between 1.09 and 0.31 mm showed vital tissue in at least one third of the pulp chamber 90 days after transplantation. Conclusions: The size of the apical foramen seems not to be the all decisive factor for successful revascularization and ingrowth of new tissue after transplantation. The minimum width of the apical foramen has not been determined, but a size smaller than 1 mm does not prevent revascularization and ingrowth of vital tissue. In this animal study an apical foramen of 0.32 mm did not prevent ingrowth of new tissue in two-thirds of the pulp chamber 90 days after transplantation. (J Endod 2013;39:759763)

Key Words
Apical foramen, histology, pulp tissue, regenerative endodontics, tooth transplantation

utotransplantation of immature teeth is a clinical procedure with a success rate of almost 98% when teeth are transplanted atraumatically and when the extraoral time is kept to a minimum (15). The developmental stage of the tooth highly determines the potential of pulpal repair after transplantation. To obtain a vital pulp in an autotransplanted or replanted tooth, the apical foramen should not be smaller than 1 mm in diameter (4). In a radiographic study, Andreasen et al showed that the revascularization of the pulp tissue was unpredictable if the apical foramen of the tooth is smaller than 1 mm (15). Only 15% of mature teeth (with closed apices) had a vital pulp after transplantation (4). Endodontic treatment is considered a standard procedure after transplantation of mature teeth, and therefore to avoid endodontic treatment, immature teeth are preferably the rst choice for transplantation. In an animal study on dogs, Skoglund et al (69) showed that if the apical foramen is enlarged by apicoectomy, revascularization of mature teeth after transplantation is possible and has a high success rate. When using apicoectomized teeth, it is interesting to know what the minimal apical diameter is for optimal pulp revascularization. To date, it is unknown to what diameter the apical foramen has to be widened to allow pulp revascularization. Moreover, so far no data on the minimal size of the apical foramen of teeth after apicoectomy are reported in the literature. Also in regenerative endodontic therapy, the migration of dental pulp stem cells into the pulp chamber might be inuenced by the width of the apical foramen. This new clinical procedure relies on creating a blood clot in the pulp chamber, delivering and activating growth factors and stem cells passing through the apical foramen (1013). In the 3 different procedures of autotransplantation, replantation, and regenerative endodontic therapy, success is highly determined by the possibility of revascularization of the pulp chamber by ingrowth from the surrounding periapical tissues, passing through the apical foramen. In 3 former studies, revascularization of an empty pulp chamber after transplantation was demonstrated (1416). In a former pilot study in our school that was carried out in beagle dogs, an empty pulp chamber was articially created by means of catheter tubes with variable diameters (17). The catheter tubes with a cylindrical lumen and different diameters (0.61.5 mm) were placed in an extraction alveolus, xed, and covered by gingival tissue. No scaffold or growth factors were used in the catheter tubes to stimulate ingrowth of new tissue. The dogs were killed after 9 weeks, and ingrowth of new tissue was histologically observed in the catheter tubes.

From the *Department of Dentistry (Orthodontics) and Department of Anatomic Pathology, Faculty of Medicine, University of Ghent, Ghent, Belgium. Address requests for reprints to Dr Wim G. M. Laureys, Department of Dentistry (Orthodontics), Faculty of Medicine, University of Ghent, University Hospital-P8, De Pintelaan, 185 9000 Ghent, Belgium. E-mail address: wim.laureys@ugent.be 0099-2399/$ - see front matter Copyright 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2013.02.004

JOE Volume 39, Number 6, June 2013

Critical Apical Diameter to Allow Pulp Regeneration

759

Basic ResearchBiology
That study indicated that revascularization may be possible with foramina smaller than 1 mm, indicating that the width of the apical diameter may not be the most important parameter to allow revascularization in teeth after transplantation. The aim of our new study was to demonstrate histologically the possible ingrowth of new tissue in the pulp chamber of autotransplanted teeth with an apical foramen smaller than 1 mm.

Materials and Methods

The ethical committee for animal research of the University of Ghent approved the use of beagle dogs in both the pilot study and the animal study. The experimental material consisted of 15 single-rooted mature teeth (incisors and rst premolars) from 3 adult beagle dogs (Fig. 1). This group was chosen to compare the results of this study with those of the studies on tooth transplantation published in the literature. The experimental teeth were extracted and underwent apicoectomy with a wire cutter to enlarge the apical foramen, holding the teeth by the crown with extraction forceps (Figs. 2 and 3). The apical side of all teeth was digitally photographed, including a measuring rod as reference (Fig. 4). Each tooth was photographed twice. On each photograph the number of pixels in 1 mm was compared with the number of pixels counted in the widest and smallest diameters. The widest and smallest diameters of each oval foramen were measured on a computer, and after calibration (Adobe Photoshop; Adobe Systems, San Jose, CA), the main diameter (mm) of the foramen was calculated for each tooth. Afterwards, all teeth were atraumatically replanted to their original site and positioned in infraocclusion because of the shorter root length after apicoectomy. No enlargement of the alveolus was carried out to prevent extra surgical manipulation, and no pressure was exercised on the teeth during replantation to avoid damage to the periodontal ligament. Only the lower incisors needed splinting by means of a exible wire (Dentaex 0.014 inch; Dentaurum, Ispringen, Germany) during the experimental period. The observation period for all teeth was 90 days after replantation. The dogs were killed, and the jaws were excised and xed in 4% neutral buffered formaldehyde. After demineralization

Figure 2. Apicoectomy of the tooth by means of a wire cutter.

(25% EDTA), histologic parafn sections were made and stained with hematoxylin-eosin. Serial sections (5 mm) were made as parallel as possible to the long axes of the transplanted teeth. Because of the curved roots, it was not always possible to have the entire length of the pulp chamber in one section. The consecutively prepared central sections of the pulp chamber were used to evaluate the amount of vital tissue. The histologic sections were examined under light microscope (magnication, 2.5 and 10). According to the amount of vital tissue lling the pulp chamber 90 days after replantation, 4 different categories were distinguished: 1. 2. 3. 4. More than two thirds of the pulp chamber lled Between one third and two thirds of the pulp chamber lled Less than one third of the pulp chamber lled (apical area only) No ingrowth

The periodontal healing of the replanted teeth was not measured in this study.

Results
The longitudinal sections of 15 teeth were analyzed histologically, and the amount of vital tissue in the pulp chamber was measured. The amount of tissue in each tooth in relation to the apical diameter of the tooth is shown in Table 1. The diameter of the apical foramen of the transplanted teeth varied between 0.24 and 1.09 mm. Only 1 of the 15 teeth had a diameter larger than 1 mm.

Figure 1. Schematic view of the used teeth in the dogs dentition.

Figure 3. Apicoectomy holding the tooth by the crown to prevent damage to the periodontal ligament.

760

Laureys et al.

JOE Volume 39, Number 6, June 2013

Basic ResearchBiology
TABLE 1. Amount of Vital Tissue in the Pulp Chamber in Relation to the Diameter of the Apical Foramen 90 Days after Transplantation Apical diameter (mm)
0.24 0.31 0.32 0.33 0.34 0.38 0.39 0.42 0.42 0.53 0.57 0.65 0.70 0.81 1.09

Area with vital tissue in pulp chamber

Apical area 1/3 to 2/3 of pulp chamber More than 2/3 of pulp chamber More than 2/3 of pulp chamber More than 2/3 of pulp chamber 1/3 to 2/3 of pulp chamber More than 2/3 of pulp chamber More than 2/3 of pulp chamber Apical area 1/3 to 2/3 of pulp chamber 1/3 to 2/3 of pulp chamber More than 2/3 of pulp chamber 1/3 to 2/3 of pulp chamber Apical area 1/3 to 2/3 of pulp chamber

Figure 4. Digital photograph of the apical foramen, including a ruler.

The amount of vital tissue in the pulp chamber of the 15 experimental teeth differed. Forty percent of the experimental teeth (6 of 15) showed more than two thirds of the pulp chamber lled with vital tissue 90 days after replantation. In another 40% of the teeth (6 of 15), one third to two thirds of the pulp chamber was lled with vital tissue. Twenty percent of the teeth (3 of 15) showed vital tissue only in the apical area, whereas no teeth were found with no ingrowth of tissue at all. This means that 90 days after replantation, 80% of the experimental teeth with an apical diameter varying between 1.09 and 0.31 mm showed vital tissue in at least one third of the pulp chamber. When we looked at the results of the 10 teeth with the smallest apical diameter (varying from 0.240.53 mm), 50% of these teeth showed vital tissue in more than two thirds of the pulp chamber. Another 30% showed vital tissue in one third to two thirds of the pulp chamber. In Figure 5, a selection of different histologic sections are described in detail. In the apical part of the pulp chamber, migration of the surrounding periapical tissue passing through the apex was observed (Fig. 5C1 and C2). The new vital tissue in the pulp chamber could be described as well-vascularized cell-rich connective tissue (Fig. 5B2). In some parts of the pulp chamber, a thick layer of tertiary dentin aligned with odontoblasts was visible (Fig. 5B1). However, in the more coronal part of the pulp chamber, necrotic remnants of the original pulp tissue, together with new connective tissue, were found (Fig. 5A). Only a few inammatory cells were observed in some of the pulp chambers.

Because of the site-specic and age-related restrictions, the indication area for tooth autotransplantation is rather small. To enlarge the indication area, 2 procedures can be used. 1. Cryopreserved immature teeth with an open apex and two-thirds of root development can be used to be transplanted at a later age. The cryopreservation technique allows more time for orthodontic treatment and provides enough time to create a suitable receptor site (15, 1820). By using the cryopreservation technique, tooth transplantation becomes less site-related and time-related, as was mentioned by Temmerman et al (2124). 2. Also, apicoectomized mature teeth with complete root development can be used. After apicoectomy, the teeth have an open apex and can be transplanted immediately at any age, keeping the possibility of revascularization (610, 15). By using mature apicoectomized teeth, the technique of tooth transplantation becomes less agerelated. According to the literature, an important parameter for revascularization of the pulp tissue seems to be the width of the apical foramen. The foramen allows the ingrowth of blood vessels and cell migration of the surrounding periapical tissues into the pulp chamber after replantation or transplantation. On a radiologic basis, Andreasen et al (14) concluded that revascularization of the pulp tissue after transplantation was unpredictable if the apical foramen of the tooth was smaller than 1 mm. They found a vital pulp in only 15% of transplanted mature teeth after transplantation (4). For that reason, endodontic treatment is mostly indicated after transplantation of mature teeth. Research by Skoglund et al (69) revealed that if the foramen is enlarged, the original pulp tissue of an autotransplanted tooth undergoes necrosis after transplantation, and migration of the surrounding periapical tissue passing through the apical foramen takes place. Furthermore, their study on beagle dogs demonstrated that apicoectomy of mature teeth is causing similar effects as in the case of transplanted immature teeth; the original pulp gets necrotic, and proliferation and migration of the surrounding tissue into the pulp chamber as well as the formation of bone-like and cementumlike hard tissue occur, which creates pulp obliteration. Our results show on a histologic basis that also foramina smaller than 1 mm allow revascularization and ingrowth of periapical tissue in the pulp chamber. Eighty percent of the teeth had vital tissue in at least one third of the pulp chamber 90 days after transplantation. Similar results were found in the pilot study of Kartsten 761

Discussion
Autotransplantation of teeth has many indications in dentistry. For example, in case of traumatic injuries with loss of 1 or more upper incisors, transplantation of a developing premolar can be a treatment modality in young growing patients. In case of agenesis of a second lower premolar, this tooth can be replaced by a developing second upper premolar, or a developing third molar can be transplanted to the area of a decayed rst molar. Autotransplantation can be highly successful if the root development has reached not more than two thirds of its nal length, and if the transplanted teeth can be moved atraumatically to a suitable receptor site in a minimum of time (15). Therefore, the receptor site must be wide and large enough and free of inammation at the moment of transplantation.
JOE Volume 39, Number 6, June 2013

Critical Apical Diameter to Allow Pulp Regeneration

Basic ResearchBiology

Figure 5. Histologic longitudinal sections of the apical (C), mid (B), and coronal (A) parts of the teeth at magnication 2.5 (A1, B1, C1) and 10 (A2, A3, A4). b, bone; bv, blood vessel; d, dentin; o, odontoblast; pc, pulp chamber; pt, pulp tissue; td, tubular dentin or tertiary dentin.

et al (17); the foramina of the empty catheter tubes in this pilot study were smaller than 1 mm, and ingrowth of new tissue was observed. In a developing tooth, root length and the size of the apical foramen are closely related to each other. The question arises whether the effect of root length and size of the apical foramen inuences revascularization and ingrowth of new pulp-like tissue after transplantation in a different way. It is important for the healing process that the tooth is not traumatized during the healing (25). The root length determines whether the tooth can be placed in infraocclusion after transplantation. Andreasen et al stated that reducing the root length of fully developed teeth before transplantation increases the chance of pulp healing (15, 20). Reducing the root length by apicoectomy creates a tooth that can be placed in infraocclusion, and simultaneously the apical foramen is widened. Therefore, one cannot conclude that a wider apical foramen is responsible for the success rate after tooth transplantation or replantation. If the tooth can be placed in infraocclusion during the healing process, then apicoectomy may not be necessary. Considering dental pulp engineering and regenerative treatment in endodontics, it is interesting to know that these new technologies are applicable not only in immature teeth but also in teeth with an apical foramen smaller than 1 mm. 762

Recently, the possibility of regeneration of new pulp-like tissue in mature teeth of a dog after removal of the original pulp tissue was demonstrated by Koichiro (12) by using stem cells and by opening the apical foramen to a diameter of 0.7 mm. These ndings support our conclusion that an apical foramen smaller than 1 mm does not prevent ingrowth of new tissue in the pulp chamber. The authors of this study want to emphasize that the conclusions of our study are based on results obtained from animal experiments. Some reservations must be made in extrapolating the results to the human situation. Clinical human studies are needed in the future to test the clinical relevance of our ndings.

Conclusion
On the basis of the results of this animal study, it can be concluded that the size of the apical foramen before transplantation is not the all decisive factor for successful revascularization and ingrowth of new tissue in the pulp chamber after transplantation in dogs. The minimum diameter of the apical foramen to allow ingrowth is not determined, but a diameter much smaller than 1 mm does not prevent revascularization and ingrowth of new vital tissue.

Acknowledgments
The authors deny any conicts of interest related to this study.
JOE Volume 39, Number 6, June 2013

Laureys et al.

Basic ResearchBiology
References
1. Andreasen JO, Paulsen HU, Yu Z. A long-term study of 370 autotransplanted premolars: part Isurgical procedures and standardized techniques for monitoring healing. Eur J Orthod 1990;12:313. 2. Andreasen JO, Paulsen HU, Yu Z, Schwartz O. A long-term study of 370 autotransplanted premolars: part IItooth survival and pulp healing subsequent to transplantation. Eur J Orthod 1990;12:1424. 3. Andreasen JO, Paulsen HU, Yu Z, Schwartz O. A long-term study of 370 autotransplanted premolars: part IIIperiodontal healing subsequent to transplantation. Eur J Orthod 1990;12:2537. 4. Andreasen JO, Paulsen HU, Yu Z, Bayer T. A long-term study of 370 autotransplanted premolars: part IVroot development subsequent to transplantation. Eur J Orthod 1990;12:3850. 5. Paulsen HU, Andreasen JO, Schwartz O. Pulp and periodontal healing, root development and root resorption subsequent to transplantation and orthodontic rotation: a long-term study of autotransplanted premolars. Am J Orthod Dentofacial Orthop 1995;108:63040. 6. Skoglund A, Hasselgren G, Tronstad L. Oxidoreductase activity in the pulp of replanted and autotransplanted teeth in young dogs. Oral Surg Oral Med Oral Pathol 1981;52:2059. 7. Skoglund A, Tronstad L, Wallenius K. A microangiographic study of vascular changes in replanted and autotransplanted teeth of young dogs. Oral Surg Oral Med Oral Pathol 1978;45:1728. 8. Skoglund A, Tronstad L. Pulpal changes in replanted and autotransplanted immature teeth of dogs. J Endod 1981;7:30916. 9. Skoglund A. Pulpal changes in replanted and autotransplanted apicoectomized mature teeth of dogs. Int J Oral Surg 1981;10:11121. 10. Howard C, Murray P, Namerow K. Dental pulp stem cell migration. J Endod 2010;36: 19636. 11. Lovelace T, Henry M, Hargreaves K, Diogenes A. Evaluation of the delivery of mesenchymal stem cells into the root canal space of necrotic immature teeth after clinical regenerative endodontic procedure. J Endod 2011;37:1338. 12. Koichiro I. Complete pulp regeneration after pulpectomy by transplantation of CD105 + stem cells with stromal cell-derived factor-1. Tissue Engineering Part A 2011;17:156. 13. Vojinovic O, Vojinovic J. Periodontal cell migration into the apical pulp during the repair process after pulpectomy in immature teeth: an autoradiographic study. J Oral Rehabil 1993;20:63752. 14. Laureys W, Dermaut L, Cuvelier C, De Pauw G. Does removal of the original pulp tissue before autotransplantation inuence ingrowth of new tissue in the pulp chamber? Dental Traumatology 2010;26:3015. 15. Laureys W, Beele H, Cornelissen R, Dermaut L. Revascularization after cryopreservation and autotransplantation of immature and mature apicoectomized teeth. Am J Orthod Dentofacial Orthop 2001;119:34651. 16. Claus I, Laureys W, Cornelissen R, Dermaut L. Histologic analysis of pulpal revascularization of autotransplanted immature teeth after removal of the original pulp tissue. Am J Orthod Dentofacial Orthop 2004;125:939. 17. Karsten AC, Laureys W, Dermaut L. Abstract 78: Ingrowth of blood vessels in implanted catheter tubes with varying diameter. Abstracts of Posters from the European Orthodontic Society 80th Congress at Aarhus, Denmark 711 June 2004. Eur J Orthod 2004;26:e3940. 18. Schwartz O. Cryopreservation as long-term storage of teeth for transplantation or replantation. Int J Oral Maxillofac Surg 1986;15:302. 19. Schwartz O, Rank CP. Autotransplantation of cryopreserved tooth in connection with orthodontic treatment. Am J Orthod Dentofac Orthop 1986;90:6772. 20. Schwartz O, Andreasen JO, Greve T. Cryopreservation before replantation of mature teeth in monkeys: IIeffect of preincubation, different freezing and equilibration rates and endodontic treatment upon periodontal healing. Int J Oral Surg 1985;14:35061. 21. Temmerman L, Vral A, Meire M, et al. Pulpal regeneration and root development after subcutaneous transplantation of cryopreserved immature teeth in rats. Cryobiology 2011;64:8190. 22. Temmerman L, Beele H, Dermaut L, et al. Inuence of cryopreservation on the pulpal tissue of immature third molars in vitro. Cell Tissue Bank 2010;11:2819. 23. Temmerman L, Dermaut L, De Mil M, et al. Inuence of cryopreservation on human periodontal ligament cells in vitro. Cell Tissue Bank 2008;9:118. 24. Temmerman L, De Pauw G, Beele H, Dermaut L. Tooth transplantation and cryopreservation: state of the art. Am J Orthod Dentofacial Ortop 2006;129:6915. 25. Hasegawa T, Suzuki H, Yoshie H, Ohshima H. Inuence of extended operation time and occlusal force on determination of pulpal healing pattern in replanted mouse molars. Cell Tissue Res 2007;329:25972.

JOE Volume 39, Number 6, June 2013

Critical Apical Diameter to Allow Pulp Regeneration

763