An experimental study on periodontal regeneration after subcutaneous transplantation of rat molar with and without cryopreservation: An in vivo study q Sarah Staels a,⇑ , Peter De Coster b , Anne Vral c , Liesbeth Temmerman a , Guy De Pauw a a Department of Orthodontics, Dental School, University of Ghent, Belgium b Department of Oral Biology, Dental School, University of Ghent, Belgium c Department of Anatomy, Embryology, Histology and Medical Physics, Ghent University Hospital, Belgium article info Article history: Received 25 September 2012 Accepted 11 March 2013 Available online 3 April 2013 Keywords: Cryopreservation Tooth autotransplantation Rat molar In vivo Periodontal regeneration Bone formation abstract This study analysed the effects of cryopreservation on periodontal regeneration of autotransplanted rat molars. First and second maxillary molars (n = 92) of 24 four-week-old Wistar rats were gently extracted and autotransplanted into the abdominal tissue immediately (control group n = 44) or after cryopreser- vation in liquid nitrogen for 7 days (experimental group n = 48). At 1, 2, 4 and 10 weeks after transplan- tation, the transplanted molars were excised and regeneration of the periodontal tissues was analysed on histological sections stained with routine H&E and Goldner method. Different tissue responses were scored on a tooth basis: inflammation, regeneration of the periodontal ligament, resorption/apposition of cementum, and alveolar bone formation. Sixty-two teeth were available for histological evaluation, including 30 experimental and 32 control samples. One week after transplantation, both control and test teeth were surrounded by granulation tissue and some areas of root resorption could be seen. After 2 weeks, signs of regeneration of the periodontal ligament, cementum apposition, and new bone forma- tion roughly coincided in both groups, however markedly retarded in the experimental group. After 4 weeks, regeneration progressed equally in both groups, presenting fewer areas of cementum apposition in experimental samples. Finally, 10 weeks after baseline transplantation, no significant differences between both groups could be observed. Cryopreservation followed by autotransplantation of extracted teeth in rats appears to have minimal detrimental effects on regeneration of periodontal tissues after integration periods of 1–10 weeks. However, the present findings indicated that the regeneration process in general is retarded for cryopreserved teeth, as compared to their immediately transplanted homologues. Ó 2013 Elsevier Inc. All rights reserved. Introduction Autotransplantation of unerupted teeth in their root formation stage is a clinically accepted method for tooth replacement with high survival and success rates [4,7]. This procedure is often ap- plied in young adolescents after traumatic avulsion of front teeth or in case of congenitally absent teeth. Previous studies [3–5] have shown that monoradicular teeth with root development advanced to ½ to 3 = 4 of the eventual length are best suited as donor elements. In this late stage of root development, revascularization of the pulp and regeneration of the periodontal ligament following transplan- tation appear to be optimal, and the risk for root resorption drops to acceptable standards. Indications for tooth autotransplants can be extended significantly with cryopreservation, i.e. a procedure involving storing of the explanted tooth in liquid nitrogen at À196 °C until it can be transplanted to the receptor site. Moreover, this procedure makes transplantation less time-dependent. Few studies have, however, yet been published exploring the influence of cryopreservation on pulp survival and regeneration of the peri- odontal ligament, both of which phenomena appear to determine the success of tooth autotransplantation [1,6,9–11,19,20]. The aim of this histological study is to examine the influence of cryopreservation on regeneration of the periodontal ligament as- sessed at 1–10 weeks after transplantation, by performing an experimental in vivo study in rats. Materials and methods Sample This experiment was approved by the Ethics Committee of Ghent University for animal experiments (ECD 08/03). Twenty-four, 0011-2240/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cryobiol.2013.03.008 q Statement of funding: The authors declare that this study has been completed without any external financial support. ⇑ Corresponding author. Address: Department of Orthodontics P8, De Pintelaan 185, 9000 Ghent, Belgium. Fax: +32 93323851. E-mail address: sarah.staels@ugent.be (S. Staels). Cryobiology 66 (2013) 303–310 Contents lists available at SciVerse ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo