Tooth autotransplantation is a potential treatment option for recovering a missing tooth [1,2]. Autotransplantation of tooth has several advantages such as bone induction, orthodontic movement, and normal proprioception [3]. Nevertheless, both healthy donor teeth and appropriate recipient site should be available simultaneously as limited indications of tooth autotransplantation which represent a major disadvantage [4].

To apply tooth autotransplantation more broadly in the clinic, there is a need to overcome such limited indications. Occasionally, there are situations in which tooth transplantation cannot be carried out immediately. In such cases, indication of transplantation could be expanded by cryopreservation of donor teeth to store the donor teeth until it is needed for transplantation.

Despite its limitations, tooth autotransplantation after cryopreservation could be a better treatment option than other treatment options such as dental implant in particular clinical situation. Cryopreservation of tooth could allow us to expand the storage period so that the tooth can be used for a later transplantation into the site with damaged bony socket that requires a period of healing [5].

However, autotransplantation of a tooth after cryopreservation has somewhat conflicting results in various studies. Some studies have reported that autotransplantation of tooth after cryopreservation shows periodontal healing similar to immediately autotransplanted tooth [5,6]. The first clinical case of autotransplantation of cryopreserved mature teeth was reported by Schwartz [5] in 1986. The first premolar cryopreserved for 18 months (connection with orthodontic treatment) was autotransplanted to a recipient site. Results showed clinically and radiographically normal periodontal healing without root resorption or alveolar bone loss at a 2-year follow-up. It has been reported that teeth cryopreserved for four weeks show almost the same level of periodontal regeneration as immediately transplanted teeth, although the healing with cryopreserved teeth proceeds much slower in rats [6]. However, survival of periodontal cells show a slightly increased ankylosis and inflammatory root resorption after cryopreservation in monkey experiments [7]. Therefore, many factors need to be overcome for autotransplantation after cryopreservation procedure as a treatment option.

The selection of the optimum cryopreservation media has a major effect in maintaining the viability of periodontal ligaments (PDLs) during cryopreservation of teeth for a long-term. Cryopreservation media and nutrients in media are important factors for the viability of PDLs [4]. Several studies have focused on the cryopreservation media, such as Hank’s balanced salt solution (HBSS) [8], autogenous serum, dextran and chondroitin sulfate-based corneal storage medium [9], ViaSpan (liver and kidney cold preservation media) [10], fetal bovine serum (FBS) [11], and soon. Exogenous growth factors and previous nutrients for cryopreservation such as FBS can induce immunological responses with a risk of transinfection such as hepatitis and bovine spongiform encephalopathy [4,12]. Platelet-rich plasma (PRP) and autogenous serum from autogenous venous blood contain lots of growth factors without inducing immunologic responses [13.14]. In addition, serum from an autogenous blood sample has the same osmotic pressure and lots of growth factors. Thus, cryopreservation media containing autogenous growth factors have many advantages than exogenous nutrients [15]. Cryopreservation media with self serum or PRP may protect the viability of PDLs [16]. If so, autotransplantation after cryopreservation will be more predictable. Recently, it has been reported that PRP could improve the healing period and cell regeneration [16]. We hypothesized that cryopreservation media with PRP could promote and preserve viability of PDLs after cryopreservation and thawing without inhibiting the activity of PDLs viability. If cryopreservation media with self serum or PRP could preserve cementum and protect the viability of PDLs, autotransplantation after cryopreservation will be more predictable and indication will be more extended.

Thus, the objective of this study was to evaluate the outcome of autotransplantation of cryopreserved teeth with self serum, PRP, or FBS as cryopreservation media (DMEM) additive nutrients histologically and histometrically in beagle dogs.

Dog serum, PRP preparation

Beagle dog was anesthetized with pentobarbital (30 mg/kg) and 10 mL blood sample was taken from the internal carotid vein. Total blood sample was centrifuged at 3,000 rpm for 15 minutes. The prepared dog serum and PRP were used as α-MEM additive nutrients for cell proliferation assay (Fig. 1).

Fig. 1. Preparation of self serum and PRP. PRP, platelet-rich plasma.

Cryopreservation procedure

This study included seven male beagle dogs (24–36 months old) randomly allocated to four groups. The protocol of this study was approved by the Ethics committee of Chonnam National University for animal experiments (no. CNU IACUC-YB-2011-16). Blood samples (10 mL) were taken from both internal carotid veins before surgery and centrifuged immediately. After anesthesia with pentobarbital (30 mg/kg), bilateral central and lateral incisors (4 teeth each dogs) were scaled and extracted using extraction forceps and periotome atraumatically. Extracted incisors were rinsed with saline and serum-free DMEM (Sigma-Aldrich Company, St. Louis, MO, USA) to eliminate blood clots and bone particles. Teeth with root fracture were eradicated.

Donor teeth were immersed in each group with 15 mL Eppendorf tube (E-tube) as cryotube overnight at 4°C. Dimethyl sulfoxide (DMSO) (DMSO HYDRA-MAX; Sigma-Aldrich Company) was used to prevent osmotic shock for cells during cryopreservation. A four-step procedure involved 5 minutes in 2.5% DMSO, 5 minutes in 5% DMSO, 5 minutes in 7.5% DMSO, and 5 minutes in 10% DMSO in DMEM media at room temperature. Cryotubes were then cooled at a rate of –0.5°C/min to –80°C and transferred to liquid nitrogen at –196°C for 3 months until it could be transplanted to the recipient site [17-19].

Preparation of cryopreservation media

Depending on the cryopreservation media before transplantation, teeth were allocated into four groups as follows:

A. Group I (Control group): DMEM (Invitrogen, Carlsbad, CA, USA) with 10% DMSO, penicillin (10,000 units/μL), and streptomycin (10 mg/mL) without serum (serum free DMEM)

B. Group II: DMEM media with 20% self serum (without buffy coat), 10% DMSO, penicillin and streptomycin (DMEM+20% self serum)

C. Group III: DMEM media with 20% self serum (with buffy coat), 10% DMSO, penicillin, and streptomycin (DMEM+20% self serum+PRP)

D. Group IV: DMEM media with 20% FBS, 10% DMSO, penicillin, and streptomycin (DMEM+20% FBS).

Transplantation procedure

Three months after cryopreservation of donor teeth, beagle dogs were anesthetized with pentobarbital (30 mg/kg) and recipient socket was prepared. Thawing was started by immersing teeth in a warm water bath at 37°C. Before transplantation, DMSO was removed gradually from 10% to 2.5%. Teeth were stored in each transport medium (without DMSO) to the time of transplantation [20,21]. After thawing, teeth were rinsed with physiologic saline and transplanted to recipient sites immediately. Recipient sockets were prepared using a low-speed round and fissure bur with physiologic saline irrigation after incision with #15 blade on anterior maxilla. The depth and width of the socket were slightly larger than those of the donor teeth. Donor teeth were then transplanted at the same level of cemento-enamel junction of donor teeth and alveolar bone crest. Sutures were done with 4-0 silk (4-0 Mersilk; Ethicon, Somerville, NJ, USA). Resin wire splint from canine to canine was done immediately. Beagle dogs were fed with soft food for 3 months and resin wire splint was maintained throughout the three months (Fig. 2).

Fig. 2. Photograph at transplantation procedure. (A) Preparing the recipient socket, (B) donor teeth placement, (C) suture and resin-wire splinting, (D) 12 weeks after transplantation.

Histological analysis

Three months after transplantation, beagle dogs were anesthetized with pentobarbital (90 mg/kg) and sacrificed. Transplanted teeth were excised with surrounding soft and hard tissues. Samples were fixed in 4% paraformaldehyde and decalcified in 5% nitric acid at room temperature for four weeks. The sample was dehydrated with ascending ethanol series and embedded in paraffin. Serial section was done at 5 μm (longitudinal/horizontal). After sections were deparaffinized with xylene and dehydrated with ethanol, samples were stained with Hematoxylin & Eosin (H&E) and Masson’s Trichrome. Masson's trichrome staining could differentiate between fibrous and mineralized tissues.

Histometric analysis

Photographs were taken from histologic section under microscope at 40× zoom and transferred to jpg image files. These image files were then analyzed using Image-Pro 6.0 (Media cybernetics Inc., Rockville, MD, USA). By connecting the respective image file, the whole root was reconstructed. Normal periodontium, ankylosis, and resorption degree as a percentage of the total root were calculated and analyzed (n=4).

Statistical analysis

Histometirc evaluation results were subjected to statistical analysis. The average and standard deviation of measurements were calculated and compared among groups with one-way analysis of variance (ANOVA) using SPSS version 22.0 (IBM Corp., Armonk, NY, USA). Differences were considered significant at p<0.05.

Clinical observation

Sutures were removed at one week after transplantation. The splinting wire was maintained for 12 weeks. During experimental periods, the resin wire splint was found to be partially detached in some dogs. They were immediately reattached. No clinically important changes were detected (Fig. 2D).

Histologic observation

Beagle dog’s incisor had a single conical root with the apical end was closed. There was no bone chip on root surface during the cryopreservation periods. PDLs on root surface were remained.

In Group Ⅰ, regenerated PDLs on the cementum were observed partially. They aligned parallel to the root surface. Autotransplanted teeth were surrounded by newly formed blood vessels and thin connective tissues. Cementum was partially intact. Cemental thickness was not constant. Newly formed alveolar bone was observed with intervention of periodontal ligaments (Fig. 3A, B). In cross-sectional view, progressive root resorption such as inflammatory resorption on cementum was observed. However, root surface without resorption showed partially regenerated PDLs (Fig. 4A, B).

Fig. 3. Histological observation in 12 weeks experimental groups using MT+H&E staining, longitudinal section, (B, D, F, H): enlarged view of the circle in (A, C, E, G) (×40: A, C, E, G; ×100: B, D, F, H). (A) Group I, note ankylosis and cemental lacunae. (B) Group I. (C) Group II. (D) Group II, arrows: cementum resorption, arrowheads: ankylosis, asterisks: inflammatory cell infiltration. (E) Group III, cementum and PDLs showed normal pattern. (F) Group III, newly formed PDLs and blood vessels, arrows: blood vessel, asterisks: cementocyte. (G) Group IV, cementum and PDLs showed normal pattern. (H) Group IV, newly formed PDLs and blood vessels, vacant arrow: osteoblast, arrowheads: cementocytes. PDLs, periodontal ligaments; c, cementum; bv, blood vessel.

Fig. 4. Histological observation in 12 weeks experimental groups using MT+H&E staining, cross section, (B, D, F, H): enlarged view of the circle in (A, C, E, G) (×40: A, C, E, G; ×100: B, D, F, H). (A) Group I, note the cemental lacunae, (B) Group I, arrows: cementum resorption. (C) Group II, note the cemental lacunae. (D) Group II, arrows: cementum resorption. (E) Group III, cementum and PDLs showed normal pattern, arrows, cementum resorption. (F) Group III, thick cellular cemuntum and blood vessels in the regenerated periodontal ligaments are observed, arrows: cemuntum resorption. (G) Group IV, arrows: cementum and PDLs showed normal pattern, arrows: cementum resorption. (H) Group IV, newly formed PDLs and blood vessels. PDLs, periodontal ligaments; A, alveolar bone; C, cementum; D, dentin.

In Group II, PDLs aligned parallel to cementum surface and newly formed blood vessels were seen. Regeneration of PDLs was observed, although the thickness was not constant because the cementum was absorbed. Root resorption and ankylosis were also observed. Infiltration of a lot of inflammatory cells was observed at the site of root resorption (Fig. 3C, D). In cross-sectional view, progressive root resorption such as inflammatory resorption on cementum was observed which was similar to GroupⅠ. Root surface without resorption showed partially regenerated PDLs (Fig. 4C, D).

In Group III, root resorption and cemental lacunae were not seen. PDLs regenerated along the entire root surface. Regenerated blood vessels were apparent in newly formed PDLs. Root cementum showed a pattern which was increasingly thicker in thickness downward from the upper root. Cementocytes were embedded in the lower 1/3 of root cementum. Some part of the regenerated PDLs aligned parallel to root axis. Some part of the regenerated PDLs were perpendicular and oblique to root surface (Fig. 3E, F). In cross-sectional view, partial root resorption with cemental lacunae were observed. However, most part of root cementum showed normal pattern and thickness. PDLs were intervened between alveolar bone and cementum normally (Fig. 4E, F).

In Group IV, cemental lacunae were observed in the upper third of the root. Periodontal ligament regenerated normally along the entire root surface, similarly to Group III. Regenerated PDLs showed significant vascular proliferation. Root cementum showed a normal pattern. It was increasingly thicker in thickness downward from the upper root. Cementoblasts were embedded in the lower 1/3 of the root. Most regenerated PDLs aligned parallel to the long axis of teeth (Fig. 3G, H). In cross-sectional view, some part of the root surface showed root resorption with cemental lacunae. However, root ankylosis was not seen on the root surface. In most part of the root surface, root cementum maintained a normal thickness and PDLs were intervened between alveolar bone and cementum normally (Fig. 4G, H).

Histometric analysis

In histometric analysis, images were analyzed by the percentage of normal periodontium, cemental lacunae (root resorption), and ankylosis per entire root surface in longitudinal section at 3 months after transplantation procedure (n=4). At first, the percentage of normal periodontium was 36.9% for Group I, 45.8% for Group II, 66.2% for Group III, and 61.4% for Group IV. Of all groups, Group III showed the highest regeneration. Normal periodontium rates (%) in Groups III and IV were significantly higher than those in Groups I and II (p<0.05) (Fig. 5).

Fig. 5. Percentages of normal periodontium at each groups (n=4). *Statistically significant (n=4).

Root resorption rate was 46.4% for Group I, 40.4% for Group II, 24.6% for Group III, and 27.6% for Group IV, with Group I showing the highest root resorption rate. Groups III and IV showed significantly (p<0.05) lower root resorption rate than Groups I and II (Fig. 6). The ankylosis rate was 16.7% for Group I, 14.2% for Group II, 10.2% for Group III, and 11% for Group IV showing no significant difference among the four groups (Fig. 7).

Fig. 6. Percentages of root resorption at each groups (n=4). *Statistically significant (n=4).

Fig. 7. Percentages of ankylosis at each groups (n=4). No significantly different (n=4).

Tooth autotransplantation has more advantage than dental implants, such as bone induction, orthodontic movement, normal proprioception, and no age-related requirements. The possibility of bone induction and proprioception is a major advantage of tooth autotransplantation. These characteristics are irreplaceable [4]. However, patients might not have available donor tooth sometimes because the tooth might have been previously extracted. Since both healthy donor teeth and appropriate recipient site must be available simultaneously for tooth autotransplantation [5], it is a major disadvantage. To solve this problem, tooth cryopreservation systems have been developed and many clinical reports and animal experiments have shown the efficacy of cryopreservation [5,7]. Periodontal healing after cryopreservation is a critical factor for successful tooth autotransplantation [20].

If PDLs show damage or necrosis during a transplantation procedure, inflammatory root resorption or replacement root resorption (ankylosis) will be inevitable. The most important factor for a successful autotransplantation is the viability of PDLs of transplanted teeth because cells can be easily damaged by ice crystal formation during the cryopreservation process, which can lead to tooth-bone ankylosis and root resorption. Therefore, it is necessary to preserve the vitality of PDLs of transplanted teeth during the cryopreservation process.

Cooling & freezing during cryopreservation should be programmed slow with a cooling technique using a cryopreservation medium with 10% DMSO (–0.3°C/min until –80°C and keep in liquid nitrogen). Slow and controlled-rate freezing with 10% DMSO (cryoprotectant) can induce an outflow of intracellular water and eliminate ice crystal formation and reduce ice injury. Based on a previous study on cryopreservation of teeth, cooling methods and cryopreservation media are essential factors during long-term cryopreservation [21].

Conventional cell cryopreservation medium was DMEM (with 10% DMSO) with 10% FBS. Recently, HBSS [8], dextran and chondroitin sulfate-based corneal storage medium [9], and ViaSpan (liver and kidney cold preservation media) [10] have been tested as cryopreservation media to preserve viability of PDLs. In addition, recent studies have reported that higher concentration of FBS (from 10% to 20%) could maintain higher cellular activity [12]. However, more attention should be paid to the use of exogenous ingredients in place of autogenous ingredients since FBS is not negligible of the occurrence of an immunological response or disease trans-infection (such as hepatitis, AIDS, bovine spongiform encephalopathy) [4,12].

Autogenous ingredients are taken from self blood and complete isotonic solution. In addition, autogenous ingredients have advantages in that they do not induce immune responses. PRP is a commonly used autogenous ingredient. It contains lots of growth factor. PRP may reduce recovery time, surgery related swelling, and pain. In addition, PRP can increase soft tissue healing and short-term bone regeneration [22]. Thus, we considered various cryopreservation media with growth factor such as DMEM with self serum and PRP for improving the viability of PDLs and long-term cryopreservation.

In this study, beagle dog’s teeth were transplanted after thawing following a long-term cryopreservation in media with PRP. Histologic features about PDLs and cementum were observed and analyzed. We attempted to assess the applicability of cryopreservation media with PRP and determine whether it could preserve the viability of PDLs and cementum better than other media.

Three months after transplantation, beagle dogs’ transplanted teeth were analyzed histologically and histometrically. In Group III, root resorption and cemental lacunae were not seen with PDLs regenerated along the root surface. Regenerated blood vessels appeared in newly formed PDLs. Sharpey’s fiber was embedded in the cementum and alveolar bone. In Group IV, PDLs regenerated normally along the root surface same as Group III. They showed significant vascular proliferation. In most parts of the root surface, root cementum had normal thickness. Sharpey’s fiber was embedded in the cementum and alveolar bone normally. However, Groups I and II showed progressive root resorption such as inflammatory resorption on most parts of cementum. The cementum without resorption showed partially regenerated PDLs. Moreover, regenerated PDLs aligned parallel to root surface. In some tissue sections, surface resorption appeared. These surface resorptions could be caused by unperformed root canal treatment. However, in most cases, inflammatory cells were rarely found in the apical portion. Even if apical root resorption and surface root resorption could be caused by inflammatory cells due to necrotic pulp tissues, the viability and activity of PDLs, cementum, and no crack in the relationship between the alveolar bone and cementum are crucial to successful transplantation after cryopreservation. Therefore, this study focused on the PDL-cementum-alveolar bone relationship. The necessity of endodontic therapy after transplantation has been reported in previous studies. In general, root canal treatment is necessary for transplantation of mature teeth in 2 weeks [23]. Further evaluation on immature teeth transplantation after cryopreservation is needed to determine whether pulp cell membranes can be preserved by cryopreservation using cryoprotectant DMSO. Previous research has reported that cells in the pulp are severely damaged during preservation due to DMSO penetrating into the pulp through the apical foramen insufficiently [24].

Mechanical properties such as cracks and fractures of cryopreserved tooth are also critical after autotransplantation. Namely, maintenance of physical and mechanical properties is essential to recover the function of transplanted teeth. Previous studies have reported that teeth with autotransplantation after thawing following cryopreservation can lead to weak mechanical properties and increase the prevalence of cracks [25]. Another research has reported that the hardness of dental hard tissues is not impaired by the freezing and thawing process [20]. Intracellular ice crystal formation can increase the width of cells in the pulp and size of odontoblastic dendrites localized in dentinal tubules during cryoperservation. The expanded pulp tissue might directly cause cracks in dentin and enamel [25]. In this study, we could observe cementum and dentin crack on some root surface in all groups (data not shown). These phenomena could be due to trauma from extraction forceps and ice crystal in dental pulp tissues. To decrease the prevalence of crack on enamel/dentin, force-controlled extraction forceps and orthodontic force before extraction could be helpful [25]. In addition, orthodontic force prior to transplantation procedure could increase the amount of donor teeth’s PDL cells and make the donor teeth extraction force down due to increased teeth mobility. If possible, extirpation of pulp tissue before donor teeth extraction should be done to reduce the prevalence of dentin crack and inflammatory reaction on apical area. Endodontic treatment makes the transplantation procedure more complex. It might increase root fracture during extraction. However, elevated donor teeth mobility due to orthodontic force can prevent such risk [26].

In histometric analysis, percentages of normal periodontium in Groups III and IV were significantly (p<0.05) higher than those in Groups I and II. Root resorption rates in Groups III and IV were significantly (p<0.05) lower than those in Groups I and II. Ankylosis rate showed no significant difference among Groups. These results indicate that the vitality of PDLs of tooth stored in autoserum with PRP is similar to that stored in FBS. FBS contains many nutrients and growth factors. Thus, it can reserve and improve the activity of PDL cells [12]. In this study, the vitality of PDLs in teeth stored in the media with autoserum was not sufficiently maintained. However, the vitality of PDLs in teeth stored in the media with autoserum and PRP was well maintained. This suggests that various nutrients and growth factors such as transforming growth factor-β exist in PRP to maintain and improve the vitality of PDLs. In previous studies, teeth were stored for a long time in media containing a high concentration of FBS to preserve and improve the vitality of PDLs [5,6]. In the present study, it was demonstrated that teeth stored in media containing autoserum with PRP were similar to those stored in media containing FBS.

In conclusion, our results showed that DMEM contained 20% self serum and PRP (with 20% self serum and PRP, Group III) could preserve viability of PDLs. Although DMEM with 20% self serum and FBS (Group IV) also showed similar histologic and histometric results to group III, autogenous additive ingredients had more advantages than exogenous in physiopathological and immunological aspects. Thus, PRP might be a positive factor in the cryopreservation process and the viability of PDLs. Thus, cryopreservation media with PRP could be a good option for long-term cryopreservation to achieve good periodontal tissue regeneration after autotransplantation.

None.

The authors declare that they have no competing interests.