Occlusal Forces Promote Periodontal Healing of Transplanted Teeth and Prevent Dentoalveolar Ankylosis: An Experimental Study in Rats

The autotransplantation of human teeth has been carried out since ancient times.1 The reliability of a successful result, however, has been unsatisfactory for just as long, primarily because of infection, followed by root resorption or loss of attachment.2 Advancements in modern medicines based on prevention of infection, the healing process of periodontal tissue, and the mechanism of root resorption have greatly improved the prognosis of autotransplantation. Although the surviving rate of autotransplanted teeth has increased in recent years,3–14 a review of clinical trials and other literature indicated a number of seated teeth with unfavorable results,5,11,13,15 primarily caused by dentoalveolar ankylosis.

Periodontal ligaments (PDL) are filled with vessels,16 collagen fiber,17,18 extracellular substances19 and cells and play a vital role in tooth stability.20 Also, other populations of mesenchymal-originated cells, such as fibroblasts, osteoblasts and osteoclasts, as well as odontoblasts and odontoclasts, have properties that model and remodel periodontal fibers, cementum, and alveolar bone, which require proper mechanical stimuli including occlusal forces.16,19,21–23 Accordingly, dentoalveolar ankylosis, evaluated as replacement resorption of the root, may cause a hypofunctional condition for the remaining PDL, and PDL features such as viscoelasticity, bony metabolism, and perceptual acceptance may be lost.

It has been reported that dentoalveolar ankylosis occurs because of PDL injury of the donor tooth during extraction24 or when the root does not fit normally in the socket. Moreover, the absence of mechanical stimuli to the PDL is also considered a cause of ankylosis.25–28 Clinically transplanted teeth may be rigidly splinted to adjacent teeth for weeks, eliminating occlusal contact so as to protect the teeth from traumatic occlusion. Although inflammatory resorption of the root or alveolar bone could be avoided by splinting, the probability of ankylosis might be increased. For better periodontal healing, prevention of dentoalveolar ankylosis may be essential, but there are few reports focusing on the effective application of mechanical stimuli during the healing process.

Recently, we experienced successful autotransplantation of permanent teeth without dentoalveolar ankylosis, when orthodontic forces were applied through an arch wire during the early stages of recovery. In this study, we examined the healing process of the PDL after transplantation and the role of mechanical stimuli in inhibiting dentoalveolar ankylosis, using an animal model of tooth transplantation with histologic observation and evaluation of PDL cell proliferation.

Twenty-eight five-week-old Sprague-Dawley male rats were used in this study. All animals had access to powder form fodder (Rodent Diet CE-2; Japan Clea Inc., Shizuoka, Japan) and drinking water ad libitum. All procedures followed the guidelines of the Tokyo Medical and Dental University for Animal Research. The experimental protocols were approved by the local Ethics Committee.29,30 

To examine the influence of occlusal stimuli on PDL healing, the animals were initially divided into occluded and nonoccluded groups. All experiments were performed under anesthesia by intraperitoneal injection with ketamine hydrochloride (KETARAL 50; Sankyo Co Ltd, Tokyo, Japan) containing 20% xylazine hydrochloride (Celactal 2% injections; BAYER-Japan Co Ltd, Tokyo, Japan). The right maxillary first molars of all animals were replanted according to the method described by Kvinnsland et al.31 The teeth were extracted with a tissue forceps, rotated once anteriorly so that all roots came out of the socket while leaving part of the attached mesial gingiva intact (Figure 1), and then immediately repositioned. No postoperative splinting was used. The contralateral first molar served as a control.

In the nonoccluded group, an anterior bite plate and a metal cap made of band material (0.180 × 0.005 inches, Rocky Mountain, Denver, Colo) were attached to the maxillary and mandibular incisors, respectively, using light-curing composite resin (Clearfil Liner Bond II; Kuraray Co Ltd, Okayama, Japan) (Figure 2). These appliances prevented occlusion at the molar region,21 whereas in the occluded group, occlusal contact was maintained.

The study period was two weeks. An additional experimental group, the recovery group, started with no occlusal force for one week and occlusal force was applied the next week. The animals were sacrificed under anesthesia at one or two weeks after replantation. The maxillary specimens were removed and fixed in 10% neutral buffered formalin (WAKO Pure Chemical, Osaka, Japan), decalcified in 10% ethylenediaminetetraacetic acid solution (pH 7.4) at 4°C for four weeks, and embedded in paraffin by conventional methods. Then, five-μm-thick serial sagittal sections of the root of M1 were cut (RM2155; LEICA Co Ltd, Nussloch, Germany), including the surrounding tissue. The distobuccal root was selected for observation because preliminary experiments demonstrated uniform morphology from this root after replantation. For histologic and histochemical examinations, the sections were stained with hematoxylin and eosin and tartrate-resistant acid phosphatase (TRAP) and observed with light microscopy.

The proliferative activity of PDL cells was evaluated by immunohistochemical reactivity of 5-bromo-2′-deoxyuridine (BrdU) on recently divided cells.9,32–34 The rats were injected intraperitoneally with 1 mL of BrdU per 100 g of body weight at a concentration of 10 mmol/L in phosphate-buffered saline (PBS) solution, pH 7.4, two to three hours before fixation. The sections were deparaffinized in two changes of xylene of five minutes each and rehydrated in descending grades of ethanol. The slides were washed three times in PBS, pH 7.4, for two minutes each and then digested in 0.1% trypsin in PBS for 30 minutes at 37°C and immersed in 2 N HCl for one hour at 37°C and twice in 0.1 M boric acid buffer for 10 minutes. After pretreatment, the sections were immersed in methanol containing 0.1% hydrogen peroxide for 30 minutes and exposed to 10% rabbit serum for 10 minutes to block endogenous peroxide activity and nonspecific reactions. The sections were incubated with 1:50 anti-BrdU antibody (anti-bromodeoxyuridine formalin grade, Roche, Indianapolis, Ind, USA) in PBS for 60 minutes at room temperature using a streptavidin-biotin method (Histofine SAB kits; Nichirei, Tokyo, Japan). The slides were finally washed three times in PBS buffer immunoreactive sites and observed using a liquid DAB substrate kit (Zymed, San Francisco, Calif).

The number of BrdU-positive cells in the entire PDL area surrounded by root and alveolar bone was counted and reported per square millimeter, with the area being measured by Image-Pro Plus Software (version 4.1; Media Cybernetics, Silver Spring, Ga).

To evaluate the differences between each group, a statistical analysis was performed using Tukey's honestly significantly different test. The significance level was set at P = .05.

Rats in the nonoccluded control group exhibited a narrower PDL and less TRAP activity (data not shown), compared with the occluded control group, where bone resorption due to physiologic molar drifting could be seen at the distal side of the root (Figure 3a,b, arrowheads). The recovery of the PDL was observed in all experimental groups at one week after replantation, and attachment between those teeth and alveolar bone occurred in all animals.

For rats in the occluded group, after one week the PDL was enlarged and thickened, with inconsistent width along the entire ligament length (Figure 3c), and the roots were extensively resorbed, especially on the mesial side (Figure 3d, arrowheads). These observations were more dramatic after two weeks (Figure 3e), where root resorption extended to the distal side (Figure 3f, arrowheads), but no ankylosis was detected.

At one week, rats in the nonoccluded group showed stricture of the PDL, but root resorption or ankylosis was not observed (Figure 4a). TRAP activity was not definitive, and even the area of physiologic resorption observed in the control disappeared (Figure 4b). After two weeks, dentoalveolar ankylosis was clearly noticeable on the mesial side, but no severe bone or root resorption could be detected (Figure 4d).

In the recovery group, even though bone and root resorption was partially observed, neither severe root nor severe bone resorption was seen in the remaining area (Figure 5a,b). The width of the PDL was consistent but thinner than that of the control group, and no dentoalveolar ankylosis was detected. (Figure 5a,c).

The proliferative activity of PDL cells in the occluded group was generally higher than in the nonoccluded group, and the activity of both groups was greatest at one week after replantation (Figure 6). The activity of cells in the recovery group was also higher than that of the nonoccluded group, but the deviation was relatively large.

Autotransplantation has been widely performed to replace missing teeth, but recently the prognosis has greatly improved. Compared with other treatment modalities, such as bridgework or implants, transplanted teeth have the advantages of functional adaptation and preservation of the alveolar ridge. This method is also favorable when combined with orthodontic therapy, where teeth are occasionally extracted for problems of discrepancy so the teeth are suitable for donation to the recipient region of the missing tooth.

Many publications have reported that the survival rate of transplanted teeth may be as high as 90%, but some still had dentoalveolar ankylosis.5,11,13,15 Ankylosis is the most frequent PDL healing complication and is evaluated as replacement resorption of the root, which may disturb normal periodontal healing. Even if the ankylosis region was only a small area of the root surface, the remaining PDL might be subjected to a hypofunctional condition, identified in many reports as a reduction in the number of vessels, a decrease in the thickness of PDL, disturbance of the functional arrangement of connective tissue fibers, and the loss of the perceptual acceptance.16,19,21–23 Therefore, prevention of ankylosis may assist the PDL to regenerate periodontal tissues and lead to a successful prognosis.

The animal model used in this study is a well-known replantation model.31 Although replantation is different from transplantation because of the possibility of retained connective tissue adjacent to the bony surface of the socket, the difference does not appear to alter the focus of our investigation, which was aimed at clarifying the influence of mechanical stimuli on the healing process of the PDL as measured by the incidence of ankylosis.

The PDL has a high natural ability of regeneration and plays a large role in the healing process of replantation or transplantation.35–37 Herr et al (1995)38 reported that fibroblasts originating from both the remaining PDL and alveolar bone compartments functioned to repair periodontium, but cells migrating to the root surface most likely originated from the PDL. Hence, the more severely the PDL was injured, the thinner the regenerated tissue. Under such circumstances, a poorly regenerated PDL may retain a high proliferating capacity. Some reports revealed that mechanical stimuli like occlusal forces were important in the migration or proliferation of PDL cells, and the lack of stimuli might cause dentoalveolar ankylosis.

In this study, the elimination of occlusal stimuli for two weeks inevitably caused ankylosis in the replanted teeth. However, teeth in the first week after replantation still had gaps between the root and alveolar bone, even if the width of PDL was much thinner than normal, indicating that dentoalveolar ankylosis required a period of time to develop. Some reports suggested that a prolonged period of rigid splinting of transplanted teeth might be a factor causing ankylosis.25,26 Our study results also suggest specific timing for the application of mechanical stimuli on replanted/transplanted teeth to prevent ankylosis.

Extensive inflammatory root and bone resorption were observed in the occluded group. This might be due to traumatic occlusion on the replanted teeth. Nevertheless, the PDL was thickest in this group and no dentoalveolar ankylosis was observed. These findings suggested that occlusal forces might assist in the regeneration of the PDL, as shown by the high proliferative activity of cells in the occluded group. However, too much force at the beginning of the PDL healing process might cause occlusal trauma, resulting in severe root or bone resorption. Based on these study findings, the replanted teeth were given one week of healing before occlusion in the recovery group, and this resulted in decreased ankylosis and lesser root resorption. Although no ankylosis was detected, the replanted teeth in this group retained some root resorption and an enlarged PDL width similar to that found in other animals after slight trauma (data not shown). Occlusal forces may have a certain ability to aid in the regeneration of PDL and to prevent ankylosis, despite the delay of intentional force.

In orthodontic practice, tooth movement is typically controlled by archwires that apply mechanical forces on the teeth. Because the archwire also holds the teeth together, it may be used to distribute, or even deliver, the appropriate level of force to a transplanted tooth to avoid root resorption and ankylosis. Our study suggested a better prognosis of periodontal healing in transplanted teeth using a combination of autotransplantation and orthodontic therapy.

Dentoalveolar ankylosis inevitably occurred in replanted/transplanted rat molars when occlusal stimuli were removed for two weeks, whereas only a narrowed PDL without ankylosis was found after one week. On the other hand, occlusal stimuli on the teeth immediately after replantation caused a severe traumatic condition with extensive root and bone resorption, yet the proliferative activity of PDL cells was vigorous and the width of the PDL was thicker than those in the nonoccluded rats. These results suggest a procedure for preventing ankylosis with a better prognosis for periodontal healing of autotransplanted teeth. The transplanted teeth need protection from traumatic forces for a certain period of time to let the PDL heal, and then, before ankylosis develops, appropriate forces should be applied to assist regeneration of the PDL. Orthodontic therapy might have the ability to provide proper mechanical stimuli on those transplanted teeth and contributes to a better prognosis.

This work was partially supported by Grant-in-Aids for Scientific Research (14370688, 15791203) from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Ministry of Education, Culture, Sports, Science and Technology of Japan.