Distraction osteogenesis (DO) is the formation of new bone between separated bone segments by applying a continuous and stable distraction force on the separated bone segments, which has the advantages of minimal surgical trauma, no bone grafting, and simultaneous expansion of the surrounding soft tissue. In 1998, Liou et al[1 ] proposed the method of periodontal membrane distraction osteogenesis, which can increase the tooth movement speed several times and shorten the treatment process greatly. Subsequently, Liou et al[2 ] and Kisnisci et al[3], foreign scholars gradually noticed that the rate of tooth movement within the bone regeneration zone obtained after distraction osteogenesis could also be significantly increased. However, the optimal timing and force values for moving the teeth into the new bone zone after distraction osteogenesis are still the main issues of discussion. In this experiment, we established an animal model for moving teeth into the new bone zone after mandibular distraction osteogenesis in dogs, and observed the speed of tooth movement and the changes of root and periodontal tissues under different force values. 1. Materials and methods 1.1 Experimental animals and grouping 8 healthy beagle dogs (provided by the Experimental Animal Center of Anhui Medical University), 10-18 months old, 10-12 kg, male and female. All dogs had permanent dentition in the mouth, and the formula for permanent dentition: I3/3.C1/1.P4/4.M2/3.×2 = 42, with intact dentition and healthy periodontium, and were kept in captivity for one week before surgery, injected with rabies vaccine, and developed into soft food habit. The teeth were randomly divided into two groups of AB, four in each group, numbered and identified, and the experimental teeth were moved by applying 50g and 100g force after 6 weeks of retraction of bone respectively. 1.2 Retractor Self-designed stainless steel intraoral retractor, using the rotation of the screw rod to drive the sliding part to move along the guide groove of the fixed part to produce retraction, the maximum extension of 25 mm, one week of rotation, the sliding part moved 0.5 mm. 1.3 Experimental methods 1.3.1 Mandibular retraction osteogenesis All experimental dogs were subjected to unilateral mandibular retraction osteogenesis between the third and fourth premolar teeth. On the operation day, the dogs were fasted and weighed, and general anesthesia was administered by intravenous infusion of 3% pentobarbital sodium 1 ml/kg. Lidocaine containing 1/300,000 epinephrine was used for submucosal infiltration anesthesia; the incision was located at approximately 62.5 px in the horizontal direction of the buccal vestibular groove of the third and fourth premolar teeth, and the buccal mucoperiosteal flap was turned up below the lower edge of the mandible to fully expose the body of the mandible; the jaw bone between the third and fourth premolar teeth was cut longitudinally with a 700# fissure drill under water jet conditions to form an artificial complete fracture, and the extra-oral angle of the mandible on the same side was made. An incision of about 5 mm was made in the skin to pierce the retractor screw rod, fix the retractor, and adjust the extra-oral screw rod to make the bone segments on both sides as tight as possible, so that the mandible was firmly fixed; after no active bleeding, the mandible was sutured in alignment and in layers. After the operation, for 5 consecutive days, 800,000 units of penicillin was injected intramuscularly for anti-infection, 2 times/ d. A semi-liquid diet was given to enhance nutrition, and the wound was cleaned with gentamicin solution. After 5 d of delay, the retractor was rotated once every 12 h, about 1 mm of retraction per day, and traction was applied for one week. 1.3.2 Experimental tooth movement and measurement method After 6 weeks of retraction osteogenic consolidation period, NiTi helical tension spring was fixed between the bone regeneration area between the third and fourth premolar teeth with orthodontic ligature wire under general anesthesia, and the fourth premolar teeth were ligated with the first molar 8 to increase the support resistance. A force gauge combined with vernier calipers was used to measure the length of the tension spring as an indicator to determine the force value, and 50g and 100g were applied to move the third premolar into the bone regeneration zone toward the distal center. The force was increased once every two weeks to reduce the stress fatigue of the tension spring and to slow the decay of the force value, so that the experimental tooth tension was more stable and reliable. The movement speed was measured by the same technician three times by vernier calipers (accuracy: 0.02mm), and the average value was taken and recorded. 1.3.3 X-ray film observation In the same program-controlled 500mA remote-controlled medical diagnostic X-ray machine (Beijing Wandong Medical Equipment Co., Ltd., Model FSK302-1A), the osteogenesis of the mandibular bone regeneration area was observed during the distraction osteogenesis. Meanwhile, after the 1st, 2nd, 3rd and 4th weeks of moving the experimental teeth, the apical films of the experimental teeth were taken using the digital apical X-ray machine to observe the apical resorption, changes of periodontal membrane and periodontal tissues. All the above were done by the same technician. 1.3.4 Taking material After the experimental teeth were moved for 4 weeks, 90~160 ml of air was injected into the experimental dogs intravenously to execute the animals, and the specimens including the experimental teeth, periodontal tissues, and some jawbones were intercepted. The specimens were immediately immersed in 10% neutral formalin fixative for 48 h and then prepared for use. The specimens were placed in a cyclotron oscillator (Jintan Huacheng Kaiyuan Experimental Instrument Factory, HY-5 type, amplitude 20 mm) vessel at 130 r/min for 10 days at room temperature. After decalcification, the specimens were rinsed with running water for 24 h. The specimens were dehydrated in ethanol solution step by step, placed in paraffin wax with a melting point of 60-62°C for 3-4 h (the temperature of wax immersion was 70-72°C) and then embedded. The specimens were sectioned by RM2135 Leica slicer (made in Germany) with a thickness of 4~5μm, routinely stained with HE, and observed under light microscope. 2. Results 2.1 General condition of animals All animals tolerated the whole experiment; no deformation, fracture or dislodgement of the traction device occurred; no infection occurred on the surgical wounds. Feeding and activities gradually returned to normal from 2 to 3 d postoperatively. The average extension of mandibular bone after retraction was 6.65 mm, and the masticatory function was mildly impaired and normal after proper dentition. The mandibular midline on the operated side of the experimental animals was slightly deviated to the opposite side, and the face was mildly skewed to the opposite side. Different degrees of gingival redness and swelling occurred when moving the dentition, which returned to normal after periodontal treatment. All the teeth were within the first degree of loosening, except for the first to second degree of loosening after four weeks of traction with 100g force value. The mandibular distraction osteogenesis achieved the expected effect, the gap in the distraction area was replaced by new bone tissue, the bone regeneration area of the isolated mandible was hard to touch, no bone discontinuity or bone defect was found, the bone cortex was flat and continuous, the old and new bone boundary was difficult to identify, and the lingual mandibular margin was slightly expanded and rough. 2.2 Condition of the experimental teeth The speed of the experimental teeth movement was judged by the distance the teeth moved each week. Where W0 indicates the initial distance between the experimental tooth (third premolar) and the fourth premolar after distraction osteogenesis; W1-4 indicate the distance between the experimental tooth and the fourth premolar after the first, second, third and fourth weeks of movement, respectively. The difference of the measured distance between the third and fourth premolar teeth in two adjacent weeks was the distance of the experimental teeth moving in two adjacent weeks. The data were expressed as mean ± standard deviation, and the cumulative mean movement distances of the experimental teeth were 1.98 ± 0.10 mm and 3.68 ± 0.09 mm after 4 weeks of movement at 50 g and 100 g force values, respectively. 2.3 X-ray observation It was found by X-ray observation of the mandible that the bone density of the new bone tissue in the distraction area increased gradually with the prolongation of the fixation period of distraction bone formation, and the bone density of the new bone formation was higher at the 6th week The bone density of the new bone was higher at the 6th week, and it was close to the normal bone tissue. The apical X-rays showed that when the experimental teeth were moved at different force values, the apices showed obvious resorption or only mildly indistinct and rounded apical contours; when the experimental teeth were moved at 100g force value, the periodontal membrane was mildly widened, the alveolar ridge was resorbed, and the teeth were tilted. 3.Discussion Retraction osteogenesis is gradually becoming an effective method for craniofacial skeletal malformation correction as well as improving the speed of orthodontic tooth movement. At present, domestic research reports are mainly on experiments of rapid tooth movement by periodontal or alveolar bone distraction osteogenesis. The present experiment is the first one in which a certain amount of bone tissue is extended to provide the necessary clearance for orthodontic teeth after direct distraction of bone formation in the mandible. 3.1 Selection of experimental animals There are many animal models for osteogenesis, such as goats, rabbits and rats. However, in this experiment, in addition to distraction osteogenesis, it is also necessary to move the tooth into the new bone area. Although goats are gentle and easy to operate, the roots of goats are thick and tight and it is difficult to realize the experimental stage of tooth movement, while rabbits and rats are rodents with small teeth, which is inconvenient to observe and operate. The Beagle dog chosen for this experiment has a docile character and is able to complete the whole process of the experiment with its own cooperation during mandibular retraction and dentition movement, which on the one hand reduces the number of anesthesia on the animal, and on the other hand does not pose a danger to the operator because the observation position is inside the mouth, and does not adversely affect the experiment due to animal agitation. In addition, the canine dentition is not significantly different from that of humans, and its tolerance is better than that of other animals. 3.2 Retractors A good fixation of the retractor during retraction osteogenesis is an important condition to ensure the osteogenesis effect. Therefore, the length, size and shape of the retractor, whether it is made to fit, whether it is firmly placed, and whether the direction and magnitude of the adjustment force are appropriate and accurate all affect the stability of the retractor, which becomes a critical part of the test. The retractor used in this test is designed and made by professional senior technician according to the test requirements, in line with the characteristics of the experiment itself. However, the retractor should be more miniaturized, not only to reduce the feeling of foreign body in the mouth, but also not to cause damage to the periodontium and its soft tissues. 3.3 Preparation of specimens In this experiment, 10% neutral formalin was used to soak the specimens, because neutral formalin is added with phosphate buffer, which is not easily oxidized and can maintain pH 7.0 for a long time, which is very beneficial to the fixation (especially long-term fixation) and staining effect of the tissue specimens, making the normal tissue cells morphology, hierarchy, bright colors, and normal tissue cells basically without atrophy and deformation [4]. The selection of an appropriate decalcifying solution is crucial for the preparation of high-quality conventional sections of decalcified bone tissue, and there are various components of decalcifying solutions that should be selected according to the tissue to be decalcified [5]. Given the need for simultaneous observation of different tissues such as tooth-periodontal membrane-alveolar bone-jaw bone, mixed acid decalcification means that the decalcification rate is maintained and the different components of the mixed acid will play their respective different roles to complement each other without causing damage to the tissue. 3.4 Selection of force and duration of tooth movement The changes in the root and periodontal tissues under different force values of traction may be influenced by many factors, such as age, gender, nutrition, and treatment course. Most scholars believe that the value of traction force is not easy to be too large, otherwise serious root resorption will occur and lead to local inflammatory reaction of the root, Schwartz [6] showed that the most suitable orthodontic force does not exceed the capillary pressure, i.e., 20-26 g/cm2, such a mild and long-lasting orthodontic force allows the teeth to produce the desired movement without causing damage to the root and periodontal tissues and is considered as “Classical mechanics”, therefore, scholars recommend that the size of the orthodontic force applied must take into account the surface area of the tooth root. However, it is clinically difficult to determine the optimal orthodontic force for each tooth due to the difficulty of accurately measuring the root surface area and the different force required for different tooth movement patterns. Therefore, combining clinical experience and literature, two force values of 50g and 100g were chosen to move the experimental teeth into the new bone zone of the distraction osteogenesis in this experiment. Foreign studies [7, 8] found that the duration of the orthodontic force also causes root resorption, which is as important as the effect of the value of the traction force on root resorption. If orthodontic treatment is performed with intermittent forces, the interval between them not only restores the resorbed bone, but also prevents further root resorption [9]. However, the results of the study by Ruping Jiang [10] showed that although the intermittent application of external force may be more consistent with the physiological state of cells in the organism, subjecting tissue cells to the same force for a long period of time may not be conducive to the physiological function of the cells, but may increase root resorption. Therefore, in this experiment, NiTi helical tension springs were used to apply the force. The NiTi helical spring stress fatigue force decayed slowly, and the force was applied once every 2 weeks, so the loading force could be considered as a continuous force. In this study, the experimental tooth was moved directly through the ligature wire fixed tension spring, and it was found that there was instability in the movement, and the traction direction was not always consistent, so the tooth did not always move in the pre-determined direction as a whole. When tilted movement occurred, the stress was concentrated on the top of the alveolar ridge, which led to horizontal resorption on both the traction and tension sides of the alveolar ridge, followed by a loss of alveolar bone height. In addition, the tension spring tends to collect food debris, which makes it difficult to self-clean, thus aggravating gingivitis, and some potential factors may also lead to the loss of alveolar bone height. Therefore, improvements in tooth movement and material selection should be made accordingly. However, it has also been reported that there is a small loss of alveolar bone height in orthodontic teeth during orthodontic treatment, but excessive resorption never occurs after completion of orthodontic treatment[11 ] . The animal model chosen for this experiment is representative, and a scientific, simple and reliable animal model can be established with a homemade retraction device, using dogs as the experimental subjects, and the speed and frequency of retraction can be precisely controlled. It provides the basis of animal models for future experiments of moving teeth at different times, but this experiment is only a small sample of animal experiments, and the conclusions obtained still need further research and demonstration.