The goal of orthodontic treatment is effective movement with minimal damage to the teeth and supporting tissues (1). Examining the factors affecting the amount of tooth movement and bone remodeling and root resorption during the application of orthodontic forces is one of the important challenges in orthodontic treatment. During orthodontic tooth movement, extensive remodeling of bone, periodontal ligament, cementum, and sutures occurs when orthodontic forces are applied to the tooth and bone. These changes show the adaptability of the periodontal ligament and the bone tissue around it (2). Most orthodontists are concerned about root resorption as an undesirable side effect of orthodontic treatment, and in the past few decades, several studies have been conducted on the etiology of root resorption because in permanent teeth it is considered a pathological condition. It should be noted that the main cause of root resorption is the activation of osteoclast-like cells that attack the roots of teeth (3).

Cementum forms a protective layer on the dentin, which is mainly mineralized collagen. Repeated bending of the tooth root may lead to cracks and loss of the entire cementum layer. Local inflammation and exposure of the underlying mineralized dentin may lead to root osteoclasticity resorption (2). Inflammatory root resorption during orthodontic treatment is a pathological process that occurs as a result of the loss of mineralized cementum and dentin at the same time during the removal of hyalinized tissue (4). This process is dependent on several factors, among which the external ones can be divided into two general categories: biological factors and mechanical factors. The effective mechanical factors include the amount of dental movements, the type of movement, the amount of orthodontic force, and the duration of force application (5). The effective biological factors include genetic predisposition and systemic factors such as hormonal imbalance, root morphology, tooth agenesis, and medications that the patient receives (6). In general, root resorption is known as a multifactorial phenomenon. During the movement of teeth, forces are transferred to the periodontal ligament through the teeth, the areas under pressure of the ligament cause the production and penetration of osteoclasts effective in bone resorption, and the areas under tension cause the differentiation of osteoblasts and bone deposition, and an imbalance between resorption and deposition along with the loss of the protective role of cementum may affect resorptive cementoclasts and osteoclasts of the root (7).

Although the amount of orthodontic force and the duration of force application have been stated as vital factors of root resorption, it is possible that pharmacological factors affecting the speed of tooth movement also affect the rate of root resorption (8). Molecules produced from different damaged tissues or drugs and nutrients that are regularly consumed by the patient can reach the tissues around the tooth that are under mechanical stress through blood circulation and communication with the target cell (9). Platelets rich in plasma are an autologous concentration of platelets in a small volume of plasma, and the number of platelets in blood varies between 150,000-350,000. In contrast, the number of platelets in plasma-rich platelet is about 1,000,000 microliters of platelets, which are rich in growth factors and cytokines, including granulocyte, macrophage colony stimulating factor, Tumor necrosis factor alpha (TNF-α), Interleukin-1β (IL-1β), IL-6, IL-8, endothelial growth factors (EFG), basic fibroblast growth factor (BFGF), granulocyte colony stimulating factor, vascular endothelial growth factor (VEGF), transforming growth factor (TGF-β2), growth factor PDGF, PDGFab, PDGFbb, and insulin-like factor (IGF) (10).

High concentration of cytokines such as IL-1, IL-2, IL-3, IL-8 and TNF-α played an important role in bone regeneration. In addition, IL-1 through its receptor on osteoclasts, stimulates osteoclast function. It was also found that mechanical stress caused by orthodontic treatment increases the production of prostaglandin (PGE) and IL-β1 in the periodontal ligament (11, 12). Many growth factors in plasma-rich platelets to stimulate osteoblastic and osteoclastic activities together have the potential to play an important role with their rich content of cytokines during the indirect activity of differentiation and activation of all remaining bone cells, supporting the idea that PRP can have a significant effect on orthodontic movements.

The platelets in this autologous plasma concentrate release their alpha granule locally at the wound site after the coagulation process. This alpha granule contains a group of growth factors that cause the proliferation and differentiation of cells that are essential for osteogenesis. Therefore, in addition to procoagulant effects, PRP is a source of growth factors in creating and maintaining wound healing by accelerating bone healing, promoting fibroblast proliferation, and increasing tissue concentration. A platelet-rich plasma gel is formed by mixing PRP from centrifuged autologous whole blood (with thrombin and calcium chloride). The addition of thrombin and calcium chloride to PRP automatically activates alpha-granules to release the following biological growth factors: platelet growth factor (PDGF), transforming growth factor beta (TGF-b), vascular endothelial growth factor (VEGF), insulin-like growth factor I, epidermal growth factor (EGF), and epithelial cell growth factor (13).

According to the collection of above materials and the increasing tendency towards orthodontic treatments, especially in adults, which causes root resorption to be one of the most undesirable results of orthodontic treatment, and also by considering the use of PRP in preserving bone and cementum during orthodontic movements, this study examines the amount of root resorption and the number of cementoblasts and cementoclasts.

The sample in this research included 6 adult male dogs of mixed breed with an age between 10-12 months and an approximate weight of 15-20 kg. For each dog, after consultation with a veterinarian, all research processes, including surgical and medical tests, were performed under general anesthesia and using ketamine hydrochloride as an anesthetic and xylazine HCI as an agonist until the end of the experiment (day 63), and the animals were euthanized with an intravenous injection of 50 mg per kilogram of phenobarbital.

Preparation of High Platelet Plasma

Preparation Method of PRP

After obtaining blood samples from the cephalic vein of the dogs, the blood samples were centrifuged in 5 cc test tubes containing sodium citrate in a PRGF centrifuge machine manufactured by BTI Biotechnology Company in Spain at a speed of 460 g for 8 minutes. The test tubes were placed opposite each other to balance the device. After centrifugation, plasma was separated from red blood cells. In the centrifuged test tube, red blood cells were placed at the bottom and a thin layer of white blood cells was seen on it. Above it, the plasma was divided from bottom to top into three layers: PRGF (Plasma rich in growth factors), PGF (0.5 cc) (Plasma with growth factors), and PPGF (0.5 cc) (Plasma poor in growth factors). A pipette was used to separate the layers. Removing the layers by pipette was done slowly and carefully so as not to mix the layers. Three sterile and empty white test tubes were numbered and used to separate the mentioned three layers. First, with a pipette, 500 microliters (0.5 cc) of the upper part, i.e. PPGF, was removed and poured into tube number 1. The volume of the first layer is different depending on the hematocrit of the animal, and the number of platelets per unit volume is similar to the number of platelets in peripheral blood. To separate the second part (PGF) by a 500-microliter pipette, the removed plasma was emptied into tube number 2. The second layer has a higher concentration of platelets than the first layer. The third part, namely PRGF, was mostly separated with a 100 microliter pipette because of its proximity to red blood cells and needing more preciseness. Because of having the highest concentration of platelets, this layer is considered the most important layer. If the obtained plasma is cloudy after centrifugation, it indicates hemolysis and the tubes were not used. After separating the PRGF layer, it was placed on a heater until use to be kept at the same temperature of 36 Celsius degrees. Then 0.5 cc of PRP mixture was mixed with 0.5 thrombin and calcium chloride, and around the second premolar at 8 points (midbuccal, midlingual, distobuccal, distolingual, a point in the middle of the distal surface, mesiolingual, mesiobuccal, and a point in the middle of the mesial surface), interligament injection was done. On the control side, a mixture of 0.5 cc of calcium chloride and 0.5 cc of placebo was injected.

Orthodontic Trends

In this experiment, the maxillary first premolar tooth was pulled due to having a thin and short root on the left and right side, and then a 9 mm titanium fennernickel with an average force of 200 grams was tied from the canine tooth as an anchorage unit to the maxillary second premolar as a movement unit. Besides, to fix the spring, ligature wire 0.014 inch (0.0014, LIGATURE TIES, ORTHOTRCHNOLOGY, USA) was placed around the second premolar teeth and canine tooth in two rings and was etched for 30 seconds around the canine tooth and second premolar completely with 37% phosphoric acid. Then the surface of the tooth was smeared with bonding and cured by light cure for 20 seconds. In the next step, the composite was completely placed around the teeth and the ligature and was cured for 40 seconds.

Using Plasma with High Platelets

The experimental group and the control group were randomly selected and placed on the right or left side. In each dog, 50 units of mixed syringe of thrombin-CACL2 and 50 units of PRP were randomly injected on one side and were considered as the experimental group. On the opposite side as the control group, only thrombin-CACL2 and placebo were injected in the amount of 50 units. 0.5 cc of PRP mixture with 0.5 cc of thrombin-CACLI2 was injected into the PDL around the second premolar in 8 points (midbuccal, midlingual, distobuccal, distolingual, a point in the middle of the distal surface, mesiobuccal, mesiolingual, and a point in the middle of the mesial surface.

The duration of the study was 63 days and the injection was done on the 1^st, 21^st, and 42^nd days, and after 63 days the dogs were euthanized.

Tissue Surgeries

After the end of the experiment (9 weeks), the dogs were treated and the wires and the device were removed. The dogs were coded to avoid bias.

RESULTS

Table 4-1. Descriptive analysis of research variables

The variable average of tooth root resorption ratio in the histological sections of dog alveolar bone during the application of orthodontic force with PRP injection in the experimental group (0.315) is less than that in the control group (0.345, without PRP injection). Also, according to standard deviation values, the dispersion of the root resorption ratio in the experimental group (0.0266) is less than that in the control group (0.0446).

The average number of cementoblasts in the histological sections of dog alveolar bone during the application of orthodontic force with PRP injection in the experimental group (12.83) is more than that in the control group (7.50, without PRP injection). Also, according to standard deviation values, the dispersion of the number of cementoblasts in the experimental group (1.329) is less than that in the control group (1.643).

The average number of cementoclasts in the histological sections of dog alveolar bone during the application of orthodontic force with PRP injection in the experimental group (5.17) is less than that in the control group (6.00, without PRP injection). Also, according to standard deviation values, the dispersion of the number of cementoclasts in the experimental group (0.753) is less than that in the control group (1.095).

- Comparison of Tooth Root Resorption Ratio

The average amount of tooth root resorption in histological sections during the application of orthodontic force is higher than the average amount of tooth root resorption in histological sections during the application of orthodontic force along with local injection of PRP.

To investigate hypothesis 1, non-parametric Wilcoxon test was used and the rank difference between the experimental group and the control group was considered. The results are given in Table 2-4.

Table 2. Comparison of tooth root resorption ratio in the experimental group with the control group

Since the significance level of the test is greater than 0.05 (p-value = 0.104), there is no significant difference in the ratio of tooth root resorption in the experimental group and the control group, and hypothesis 1 is not confirmed. Although the average positive rank is higher than the negative rank, which means that the ratio of tooth root resorption in the sample of the control group is higher than that of the experimental group, this difference is not significant in general and can be caused by an error.

- Examining the Number of Cementoblasts

The average number of cementoblasts in histological sections during orthodontic force application is less than the average number of cementoblasts in histological sections during orthodontic force application along with local injection of PRP.

To investigate hypothesis 2, non-parametric Wilcoxon test was used and the rank difference between the experimental group and the control group was considered. The results are given in Table 3-4.

Table 3. Comparison of the number of cementoblasts in the experimental group with the control group

Since the significance level of the test is less than 0.05 (p-value = 0.027), there is a significant difference in the number of cementoblasts formed between the experimental group and the control group. In addition, because the average of negative ranks is higher than the average of positive ranks, the rank values ​​of the experimental group are higher than those of the control group. In other words, it can be concluded that the number of cementoblasts in the histological sections during the application of orthodontic force (control group) is less than the number of cementoblasts in the histological sections during the application of orthodontic force with PRP injection (experimental group). In other words, hypothesis 2 is confirmed.

  - Comparing the Number of Cementoclasts

The average number of cementoclasts in histological sections during the application of orthodontic force is higher than the average number of cementoclasts in histological sections during the application of orthodontic force along with local injection of PRP.

To investigate hypothesis 3, non-parametric Wilcoxon test was used and the rank difference between the experimental group and the control group was taken into account. The results are given in Table 4-4

Table 4. Comparison of the number of cementoclasts in the experimental group with the control group

Since the significance level of the test is greater than 0.05 (p-value = 0.157), there is no significant difference in the number of cementoclasts formed between the experimental group and the control group, and so hypothesis 3 is not confirmed. Although the average of positive rank is higher than that of the negative rank, that is, the average number of cementoclasts in the sample of the control group is more than that of the experimental group, this difference is generally not significant and can be caused by an error.

In this study, the effect of local injection of platelet-rich plasma (PRP) on the amount of tooth root resorption and the number of cementoblasts and cementoclasts during the application of orthodontic force in dogs' teeth was investigated.

Acceleration of orthodontic tooth movement through the injection of biological materials into the periodontal ligament has recently been investigated. Tooth root resorption during orthodontic treatments is one of the major problems that can cause irreparable defects on teeth (14).

So far, no definitive reason has been provided to determine the cause of root resorption (15). The occurrence of more resorption has a significant relationship with the use of heavier and uncontrolled forces (16). Of course, there have been reports that hormonal changes and genetic factors can also cause root resorption (14).

 A large number of previous studies conducted on animals have investigated the effect of local injection of specific medicinal substances on dental movements through submucosal injection. However, in this study, the injection in periodontal fibers was done with the aim of improving the tissue reaction under the influence of the growth factors in PRP. The difference in the speed of tooth movement in this study after the first week was clearly observed and the markers related to root resorption in the gingival fluid were also tested. In the platelet-rich plasma method, the large number of platelets accelerates the process of tooth movement in one area.

Periodontal regeneration includes the formation of new alveolar bone, new cementum, and functional periodontal ligaments. New methods of periodontal treatment to improve regeneration include the use of Mesenchymal Stem Cells 1 (MSCs) and platelet-rich plasma (PRP). There is also great interest in using PRP to treat periodontal defects. Platelets are a rich source of growth factors (GF), such as platelet-derived growth factor (PDGF), transforming growth factor (TGF-b), and vascular endothelial growth factor, which modulate the wound healing response in hard and soft tissues. Therefore, PRP after destruction of platelets may promote wound healing by increasing the level of growth factors (GF) in the wound site. Recent in vitro studies have shown that PRP has positive effects on gingival fibroblasts, oral osteoblasts, and periodontal ligament fibroblasts. A preliminary in vivo study recently published by a group of authors shows that PRP promotes the formation of new cementum along with periodontal fibers in fenestration defects in rats. However, the exact role of platelets in periodontal tissue regeneration still needs to be further examined (17).

In hypothesis 1 in this study, we stated that the average amount of tooth root resorption in histological sections during the application of orthodontic force is higher than the average amount of tooth root resorption in histological sections during the application of orthodontic force along with local injection of PRP. Since the significance level of the test is greater than 0.05 (p-value = 0.104), there is no significant statistical difference in the ratio of tooth root resorption in the experimental group with the control group, and so hypothesis 1 is not confirmed. After the experiment, Zhang observed that the periapical radiolucency has disappeared and the walls of the canals have thickened and new tissues have been formed. In addition, Yang stated that the use of PRP reduces enclosis and increases the formation of cementum and PDL-like tissue. Rashidi observed the greater amount of cementum formation and remodeling activity as well as a decrease in root resorption in the experimental group. Kobayashi stated that the main effect of PRP is soft tissue repair and has little effect on the differentiation of osteoblasts, so it cannot affect the resorptive and restorative activities of hard tissue. In a 12-month clinical study for pulp regeneration with PRP, Herini observed external and internal root resorption with periapical radiolucency after 6 months of follow-up in the experimental group. Javardna stated in a report of 3 cases of patients during five years that PRP has shown high clinical results in the field of periapical healing, apical closure, and increasing the thickness of the tooth root wall. Histological results from animal and human species show the formation of cementoid and osteoid tissues on the walls of the treated canals with increased thickness, and no resorption was observed. Besides, Morari stated that with the use of PRP in 100% of cases, he observed an increase in root length and apical obstruction, and so our study is consistent with the studies of Zhang, Rashidi, Yang, Morari, and Javardna, and it is in contradiction with the study of Kobayashi and Herini. The reason for this in the human investigation by Herini was in the difference in the experiment process, the injection dose, and the samples preparation conditions, and the reason for the difference with Kobayashi's study is in the manner of conducting the experiment in the cell culture. In hypothesis 2, we stated that the average number of cementoblasts in histological sections during the application of orthodontic force is less than the average number of cementoblasts in histological sections during the application of orthodontic force along with local injection of PRP. Since the significance level of the test is less than 0.05 (p-value=0.027), there is a significant difference in the number of cementoblasts formed in the experimental group and the control group. In other words, it can be concluded that the number of cementoblasts in the histological sections during the application of orthodontic force (control group) is less than the number of cementoblasts in the histological sections during the application of orthodontic force along with injection (PRP of the experimental group), and so hypothesis 2 is confirmed. Yang mentioned that the use of PRP on root surfaces during tooth placement in dogs can reduce dental ankylosis and increase PDL-like and cementum-like tissue formation.

Rashidi stated that in the tissue morphometric analysis, there are much more osteoblasts, cementoblasts, and osteoclasts in the experimental group compared to the control group. In addition, Ngata's results stated that in the experimental group, new cementum along with collagen fibers enter diagonally or perpendicularly on the root surface, but in the control group, no new cementum was observed, which indicates cementoblastic activity and an increase in the number of cementoblasts in the experimental group. Through morphometric analysis, Zhang observed that the cementum-like tissues have increased in growth, the canal walls have thickened, and the apex is closed. Besides, by examining the histometric factors of the connective tissue of the new cementum of the new bone, Ferariasevada observed the increase in the length of the new cementum area in the experimental groups. These results are in line with our study, but are in contrast with the study of Kobayashi and Herini, which is the reason for conducting the experiment in the cell culture environment, human society, the difference in the working method, the preparation method of PRP, and the dose used in the experiment. In the third hypothesis, we stated that the number of cementoclasts in histological sections during orthodontic force application is higher than the average of cementoclasts in histological sections during orthodontic force application along with local PRP injection. Since the significance level of the test is greater than 0.05 (p-value = 0.157), there is no significant difference in the number of cementoclasts formed between the experimental group and the control group, and so hypothesis 3 is not confirmed, which is in contrast with the findings of the studies of Morari, Ngata, Zhang, Kobayashi, and Sawada, but is in line with the studies of Rashidi, Gulak, and Herini. The reason for this difference in the examined samples is the dose of PRP and the way PRP is used in the experiment.

The findings of the present study show that PRP injection increases the number of cementoblasts and increases the amount of cementum in the teeth during orthodontic movements, which in this case can help to reduce complications caused by root resorption during orthodontic movements.

1. Owman-Moll, Petra. "Orthodontic tooth movement and root resorption with special reference to force magnitude and duration. A clinical and histological investigation in adolescents." Swedish Dental Journal Supplement 1995, no. 105, pp. 1-45.
2. Graber, Thomas M. Orthodontics: Current Principles and Techniques. 4th ed., Elsevier Mosby, 2005, pp. 145-149.
3. Verna, Carl, Davide Zaffe, and Guiseppe Siciliani. "Histomorphometric study of bone reactions during orthodontic tooth movement in rats." Bone, vol. 24, no. 4, Apr. 1999, pp. 371-379. doi:10.1016/S8756-3282(98)00196-3.
4. Gonzales, Carlos, et al. "Force magnitude and duration effects on amount of tooth movement and root resorption in the rat molar." Angle Orthodontist, vol. 78, no. 3, May 2008, pp. 502-509. doi:10.2319/033007-145.1.
5. Abuabara, Alan. "Biomechanical aspects of external root resorption in orthodontic therapy." Medical Oral Pathology Oral Cirugia Bucal, vol. 12, no. 8, Dec. 2007, pp. E610-3.
6. Proffit, William R. Contemporary Orthodontics. 4th ed., Elsevier Mosby, 2007, pp. 338-363.
7. Acar, Ali, et al. "Continuous vs. discontinuous force application and root resorption." Angle Orthodontist, vol. 69, no. 2, Apr. 1999, pp. 159-163; discussion 163-4.
8. Weltman, Bede, et al. "Root resorption associated with orthodontic tooth movement: a systematic review." American Journal of Orthodontics and Dentofacial Orthopedics, vol. 137, no. 4, Apr. 2010, pp. 462-476; discussion 12A. doi:10.1016/j.ajodo.2009.06.019.
9. Krishnan, Vinod, and Ze'ev Davidovitch. "The effect of drugs on orthodontic tooth movement." Orthodontics & Craniofacial Research, vol. 9, no. 4, 2006, pp. 163-171. doi:10.1111/j.1601-6343.2006.00370.x.
10. Rashid, Abeer, et al. "Effect of platelet-rich plasma on orthodontic tooth movement in dogs." Orthodontics & Craniofacial Research, vol. 20, no. 2, May 2017, pp. 102-110. doi:10.1111/ocr.12184.
11. Davidovitch, Ze'ev, et al. "Neurotransmitters, cytokines, and the control of alveolar bone remodeling in orthodontics." Dental Clinics of North America, vol. 32, no. 3, 1988, pp. 411-35.
12. Saito, Mitsuo, et al. "Interleukin 1 beta and prostaglandin E are involved in the response of periodontal cells to mechanical stress in vivo and in vitro." American Journal of Orthodontics and Dentofacial Orthopedics, vol. 99, no. 3, 1991, pp. 226-40.
13. Albanese, Alfredo, et al. "Platelet-rich plasma (PRP) in dental and oral surgery: from the wound healing to bone regeneration." Immunity & Ageing, vol. 10, no. 1, Jun. 2013, p. 23. doi:10.1186/1742-4933-10-23.
14. Liu, Lin, et al. "Effects of local administration of clodronate on orthodontic tooth movement and root resorption in rats." European Journal of Orthodontics, vol. 26, 2004, pp. 469-473. doi:10.1093/ejo/26.5.469.
15. Nanda, Ravindra. Biomechanics and Esthetics Strategies in Clinical Orthodontics. Elsevier Saunders, 2005, Chap. 2, pp. 28.
16. Rahgozar, S., G. Pakravan, and K. Ghaedi. "Cellular adhesions and signaling pathways in platelets." Scientific Journal of Iran Blood Transfusion Organization, vol. 8, no. 1, 2011, pp. 60-73.
17. Zhang, D.-D., et al. "Histologic Comparison between Platelet-rich Plasma and Blood Clot in Regenerative Endodontic Treatment: An Animal Study." Journal of Endodontics, vol. 40, no. 9, 2014, pp. 1388-1393. doi:10.1016/j.joen.2014.02.019.