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Role of Transforming Growth Factor Beta 1 in Reducing Orthodontic Relapse: Rabbit Model Study

Saba H. Al Zubaidi1,*, Niam R. Alsaleem1, Muhammad A. Alkataan2
1Department of Pedodontics Orthodontics Preventive Dentistry, College of Dentistry, University of Mosul, Mosul, Iraq.
2Department of Biochemistry, College of Medicine, Ninevah University, Mosul, Iraq.

ABSTRACT

Background: This study attempted to evaluate clinically and histologically the effect of local injection of transforming growth factor beta 1 (TGFβ1) on the rate of orthodontic relapse. 

Methods: 24 male albino rabbits were used, that grouped randomly into two groups (12 rabbits in each): positive control and TGFβ1 groups. Each group was further divided into 3 subgroups with 4 rabbits in each. Modified orthodontic appliances were cemented to the mandibular central incisors and tooth movement was carried out for 2 weeks, followed by a retention period for 3 weeks where positive control and TGFβ1 groups received local injections of phosphate buffer saline and TGFβ1 solutions, respectively, then the teeth allowed to relapse after removing the appliances for 20 days. The relapse was estimated clinically. Histological and immunohistochemical analyses were carried out following the completion of the experiment. 

Result: The TGFβ1 group showed a significantly lower rate of relapse than the positive control group. Histologically, during the relapse period, a significant increase in the number of osteoblasts, a decrease in the number of osteoclast, which was also confirmed by immunohistochemical analyses and a lesser PDL width was seen in TGFβ1 group when compared to the positive control group. local injection of TGFβ1 can significantly reduce the orthodontic relapse in the rabbit model.

INTRODUCTION

Orthodontic tooth movement is a procedure that includes mild, reversible periodontium damage along with physiologic adaptation of the alveolar bone to mechanical strains (Wise and King, 2008). Orthodontic tooth movement can take place by simultaneous activation of bone resorption by osteoclasts and bone synthesis by osteoblasts (Nakai et al., 2023).
       
Teeth tend to “relapse,” or return to their original position, if there is no retention phase following successful orthodontic treatment (Martin et al., 2023). Relapse is thought to be a complex issue with multiple potential causes. It might be due to the time needed for gingival and periodontal tissue reorganization, unstable teeth position after orthodontic treatment and changes caused by growth (Abdulraheem et al., 2020; Giancotti et al., 2020).
       
The transforming growth factor beta (TGFβ) is a pleiotropic cytokine that regulates various biological functions like regulating inflammatory and cell cycle responses (Pokharel et al., 2016). TGFβ is a part of the superfamily. It has three isoforms in mammalian cells: TGFβ1, which is the most abundant and TGFβ2 and TGFβ3, which are rare (Katz et al., 2016).
       
Managing the remodeling of alveolar bone following orthodontic tooth movement is an essential approach in preventing relapse (Lin et al., 2023). Previous studies confirm the ability of the TGFβ1 to enhance proliferation and differentiation of osteoblast cells and their progenitor and subsequently enhance bone formation (Zhang et al., 2019 and Xu et al., 2020). Therefore, this study aimed to utilize the osteogenic properties of TGFβ1 to promote the formation of osteoblasts and subsequently generate more new bone on the relapse side, with the goal of reducing the rate of orthodontic relapse. To the best of our knowledge, this work represents the first attempt in the field of orthodontics to utilize TGFβ1 with the aim of minimizing relapse following orthodontic tooth movement.

MATERIALS AND METHODS

The samples of the study consisted of (24) healthy male Albino rabbits, weighing 1000-2800 g, age 6-12 months. The rabbits were housed under standard animal care of food, water access, temperature and humidity (Yildiz-Gulay and Gulay, 2024). The male rabbits were chosen to ensure a hormonal balance for validating the study results and to exclude any hormonal fluctuations associated with the estrous cycle in females, which could potentially impact bone metabolism and tooth movement (Alaa et al., 2023). The animals were divided randomly into two main groups (12 rabbits in each), positive control and TGFβ1 groups. Each group was further subdivided into three subgroups (4 rabbits in each) as follow (Fig 1).
 

Fig 1: Timeline for establishing the rabbit relapse movement model.


 
Positive control day 0 (PCD0), day 10 (PCD10) and day 20 (PCD20) groups
 
 All rabbits  received 200 ml local  injection of  phosphate- buffered saline solution (PBS) (Himedia, India) every 3 days, during retention period. The animals in PCD0, PCD10 and PCD20 groups were euthanized on day 0, 10 and 20, respectively, after debanding of orthodontic appliance.
 
Transforming growth factor beta 1 day 0 (TGFβ1D0), day 10 (TGFβ1D10) and day 20 (TGFβ1D20) groups
 
All rabbits received local injection of TGFβ1 (1ng/ml) (Elabscience biotechnology Inc., USA) (200 ml) every 3 days (Zhang et al., 2019), during retention period. The animals in TGFβ1D0, TGFβ1D10 and TGFβ1D20 groups were euthanized on day 0, 10 and 20 receptively after debanding of orthodontic appliance.
 
Experimental design
 
Rabbits were anesthetized with an intramuscular injection of the mixture of 35 mg/kg body weight ketamine hydrochloride and 10 mg/kg body weight xylazine. Modified orthodontic appliance consisting of two bands (stainless steel, LA 00, Dentarum, Germany) into which bracket with 0.022 * 0.030" slot dimension is attached. A nickel titanium open coil spring, of about 5 circles length, was inserted along the sectional stainless steel wire (0.016*0.022") to exert a reciprocal lateral force of about 50g on both mandibular incisors (Vani et al., 2022). Ligature wire (0.01 inch) was used to ligate sectional wire into the bracket’s slot and the wire was bended on both ends to avoid trauma to the rabbits (Al-Fakhry and Al-Sayagh, 2022; Vieira et al., 2019). Then the appliance cemented to the mandibular incisors and actived by removing the ligature wire that squeezed the open coil spring and allow the teeth to move for (2 weeks), (Fig 2).
 

Fig 2: Steps of orthodontic appliance insertion.


       
After that, the appliance was retained in position for 3 weeks. Latter, the appliance was removed and the teeth were allowed to relapse. The amount of relapse was measured using electronic digital vernia (accurate to 0.01mm) (Generic, China), at the level of the mesial tip of the two mandibular incisors at 0,2,4,7,10,13,17 and 20 days. During retention, TGFβ1 and PC groups received local injections of TGFβ1 and PBS solution respectively. The syringe tip inserted near the mesial side of the left mandibular incisor, into the mucogingival junction, in the vestibule (buccaly and lingually).
 
Histological assessment
 
The lower jaw was fixed in 10% neutral buffered formalin for at least 48 h. After fixation, the specimens were passed through the following process: Decalcification, dehydration, clearing, infiltration and embedding (Yildiz-Gulay and Gulay, 2019). Longitudinal serial sections were cut for each specimen of 5ìm thickness in a labiolingual direction parallel to the long axis of the root of the mandibular incisor. The specimens were prepared for haematoxylin and eosin staining according to Suvana et al., (2019) and for immunohistochemistry analysis. The analysis was carried out in two areas and were determined by orienting the long axes of the tooth vertically. Then, two horizontal lines were drawn perpendicular to the long axes of the tooth. The first line (cervical) was drawn at level of alveolar crest and the other line (apical) was drawn at a distance of (9960 μm) from the first horizontal line, the distance between the two lines which represent the straight part of the mandibular incisor’s root. The distance between the two lines was divided equally into two halves. The target area was the mesial aspect of the mandibular left incisor tooth.
 
Histological evaluation includes
 
Number of osteoblasts, numbers of osteoclasts: that are counted on the alveolar wall next to the mesial side of the root of the left mandibular incisor. PDL width which represents the distance between the mesial surface of the mandibular left central incisor’s root and new bone surface at 2 regions (at the level of cervical and apical line).
 
Immunohistochemistry evaluation
 
Then the expression of bone alkaline phosphatase (bALP) and tartrate-resistant acid phosphatase (TRAP 5b) were evaluated on semi-quantitive scale measuring the intensity of immunoreaction. The score ranks usually lie in a range from “negative” (0) to “strong” (3) (Agustina et al., 2018).        
       
The clinical and histological parameters were analyzed using IBM SPSS Statistics, Version 20. Nonparametric analysis including, descriptive statistics (mean±SD) and the Mann-Whitney test was used to compare the means of all parameters. Differences at p<0.05 were considered statistically significant.

RESULTS AND DISCUSSION

Clinical observations
 
Relapse occurred in both groups and the relapse distance increased over time to reach its maximum value at day (20). However, the greater RD occurred in the PC group (Table 1).
 

Table 1: Amount of space gained, remaining space, relapse distance and percentage of relapse for positive control and transforming Growth Factor Beta 1 groups.


 
Histological analysis
 
The number of osteoblasts
 
on the day (0), highest counts of osteoblasts were seen in both groups with a gradual reduction in their number until reached the lowest number on the day (20). The TGFβ1 group was statistically significant the highest number of osteoblasts, Table 2, Fig 3.
 

Table 2: Comparison of osteoblasts and osteoclasts number among positive control and transforming growth factor beta 1 groups for three time intervals in two regions.


 

Fig 3: Hematoxylin and eosin stained section photomicrograph for the TGFb1 group.


 
The number of osteoclast
 
 The lowest number of osteoclasts was recorded on day (0), with a highest number was reached on the day (10) and then there was a reduction in the number of osteoclasts on the day (20). In both upper and lower areas, TGFβ1 group was significant the lowest number of osteoclasts over the relapse period, Table 2, Fig 3.
 
Periodontal ligament width
 
On day (0), the widest PDL width was recorded for both groups, with a gradual reduction in the width over time to record the narrowest width on day (20). In both regions, the PDL width was significantly the narrowest in TGFβ1 group during the relapse period, Table 3, Fig 3.
 

Table 3: Comparison of the periodontal ligament width among positive control and transforming growth factor beta 1 groups for three time intervals in two regions.


 
Bone ALP and TRAP 5b immunoreaction
 
For BALP, on day (0), strong immunoreaction was detected in the TGFβ1 group when compared to the PC group and then the reaction decreased over the relapse period to record the weakest reaction on day (20), with the TGFβ1 group was the highest level of BALP during the experimental period with a significant differences on day 0 and 10. While for TRAP5b, on day 0, a low level of this marker can be seen in both groups, to reach its maximum value, with a significant elevated immunoreaction in PC group on day 10 in the upper half and on day 10 and 20 in the lower half. Then the immunoreaction slows down until reached day (20), Table 4, Fig 4.
 

Table 4: Comparison of ALP and TRAP5b levels for positive control and transforming growth factor beta 1 at three experimental periods in the upper and lower halves.


 

Fig 4: Immunohistochemical expression of: A,B) BALP for the TGFβ1 group on day 0 and 20 respectively, C,D) TRAP for the TGFβ1 group on day 0 and 10 , respectively.


       
Higher rates of post-orthodontic relapse happened at the initial time after appliance removal. This result was in line with the findings of Reitan (1967), who observed that a higher initial relapse happens when the tooth experiences a rebound shift in the socket. More than half of the relapse distance occurred by 10th day, then the relapse rate decreased until the end of the experiment. This result is congruent with the findings of Al-Fakhry (2022).
       
The greater number of osteoblast in TGFβ1group on day 0 was due to the effect of TGFβ1 on bone cells. TGFβ1 stimulates bone cell proliferation and osteogenic differentiation (Zhao et al., 2018), displays chemotactic attraction of osteoblasts (Karst et al., 2004), prevents osteoblast apoptosis (Indriasari et al., 2019) and activates differentiation of MSCs to into osteoblasts (Li et al., 2022). The gradual reduction in the number of osteoblasts on days 10 and 20 because during the relapse, the previous tension site became pressure site and alveolar bone resorption occurred according to pressure-tension theory (Viazis et al., 2023).
       
The presence and activity of ALP during osteogenic differentiation signal that MSCs are undergoing differentiation into osteoblasts (Capulli et al., 2014). ALP is an essential biomarker of osteoblastic activity (Sanchez et al., 2020). The present study demonstrated a direct correlation between the number of osteoclasts and orthodontic relapse, underscoring the significance of bone resorption during the relapse period. Therefore, the control group showed a higher rates of relapse and osteoclast counts, while the TGFβ1 group displayed the opposite profile because of its impact on osteoblasts, as concluded by Takai et al., (1998) in that, high concentrations of TGFβ1 (1-10 ng/ml) increase the expression and secretion of OPG and decreased that of RANKL by osteoblasts.
       
TRAP functions to break down osteopontin, a protein that attaches to integrins on the surface of osteoclasts and enhances their adherence, thus, enhancing osteoclast migration by inhibiting this adhesion (Janckila and Yam, 2009). The level of TRAP5b comes in line with the number of osteoclast, since TRAP 5b is normally secreted by osteoclasts during bone resorption and can be measured as biochemical markers of bone resorption (Schini et al., 2023). TRAP reflect osteoclasts cell number (Park et al., 2024) and activity (Jiang et al., 2020).
       
TGFβ1 group showed the narrowest PDL width, which is due to the greater anabolic effect of TGFβ1. With time, there was a reduction in the width of the PDL fibers because it became the compression side during relapse. This implies that tension of periodontal ligament fibers plays a role in relapse. This result comes in agreement with Aoki et al., (2023).
       
Limitation of the study: The number of animals is limited, so to achieve more accurate and reliable results, the number of animals should be increased. In addition, there was difficulty in sample selection as well as difficulty in determination the exact age of the animals.

CONCLUSION

Local injection of TGFβ1 during the retention period is associated clinically with a significant reduction of orthodontic relapse as compared to PC group. Histological finding support the clinical result which manifested by significant increase in the number of osteoblast and reduction in the number of osteoclast. The immunohistochemical reaction comes in line with the histological parameters and characterized by the highest level of BALP and the lowest level of TRAP 5b throughout the relapse period as compared to PC group.  Also, TGFβ1 group had reduced PDL width as compared to PC group which confirm the anabolic effect of TGFβ1.

ACKNOWLEDGEMENT

The authors are grateful to the University of Mosul, College of Dentistry, for continuous support.
 
Ethical approval
 
The protocol of study was approved by the research ethics committee of University of Mosul / College of Dentistry, REC reference no. UoM.Dent/ DM.12/ 23.

CONFLICT OF INTEREST

All authors declare that they have no conflict of interest.

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