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INTRODUCTION
The orthodontic profession has witnessed a growing interest in accelerated orthodontic tooth movement (OTM), driven by patient preferences and the desire to mitigate the risks associated with prolonged treatment durations, including root resorption, decalcification, and periodontal problems.1 OTM occurs because of remodeling processes within the alveolar bone and periodontal ligament (PDL). This process is characterized by bone resorption on the compression side of the tooth and bone deposition on the tension side.
Orthodontic forces trigger disturbances in the microcirculation of the PDL, leading to an initial inflammatory response characterized by increased vascular permeability and leukocyte migration. Subsequently, the migrated cells release inflammatory cytokines and pro-inflammatory lymphocytic growth and chemotactic factors.
Numerous methods have been suggested to expedite tooth movement, ranging from invasive surgical techniques, such as osteotomies and corticotomy, to noninvasive approaches, such as pulsed electromagnetic fields, mechanical vibration, and low-level laser therapy.4,5 Among the noninvasive methods, biomaterials, such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF), have been recommended as promising alternatives for expediting OTM while minimizing the risk of bone and periodontal loss.6,7 The platelet concentration is a source of autologous growth factors and secretory cytokines, which play a significant role in angiogenesis, wound healing, and bone regeneration.8
In recent years, leukocytes have been included in PRP compositions, known as leukocyte–PRP (L-PRP), and are gaining increased attention for their therapeutic potential in tissue regeneration. L-PRP can enhance bone regeneration by upregulating cellular proliferation, viability, migration, angiogenesis, and osteogenesis.9,10 Furthermore, the presence of leukocytes in PRP correlates with elevated levels of catabolic cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha, leading to the production of catabolic metalloproteinases.11,12 Moreover, the bone turnover efficiency of PRF is higher than that of PRP because of its extended period of activity.13 Overall, leukocyte–PRF (L-PRF) has demonstrated better persistent release of pro-inflammatory factors from its thin and flexible fibrin matrix than PRP and PRF.14
Although existing literature has addressed the effects of L-PRF on OTM in extraction sockets, its influence on soft tissue healing and pain remains unexplored. This randomized controlled trial aimed to fill this knowledge gap by investigating the effects of L-PRF on maxillary canine retraction and rotation and its role in soft tissue healing and pain in individuals with Class I bimax protrusion and Class II div I malocclusion by comparing these effects to conventional solutions.
MATERIALS AND METHODS
Trial design
This was a single-center, randomized split-mouth clinical trial employing a 1:1 allocation ratio. The methodology used remained unchanged after trial completion.
Participants, eligibility, and study setting
This study spanned a period of 3 months, from October to December 2022, and was conducted within the Orthodontics and Dentofacial Orthopaedics Department at SCB Dental College and Hospital, Cuttack, India. This study was approved by the Institutional Ethics Committee of SCB Dental College and Hospital, Cuttack, India (IES/SCBDCH/092/2021) and registered in the Clinical Trial Registry of India (CTRI) (registration number: CTRI/2023/01/048932). Although the registration application was submitted before the trial commenced, registration confirmation was received after the trial concluded in January 2023. The protocol is accessible on the CTRI website (https://ctri.nic.in/clinicaltrials/login.php). Participants of both sexes, aged between 18 and 25 years, were included based on predefined inclusion and exclusion criteria. All owners of eligible participants provided informed consent after receiving a comprehensive explanation of the study procedures.
Inclusion criteria:
Class I bimaxillary protrusion requiring first premolar extraction
Class II div 1 malocclusion requiring maxillary first premolar extraction
Moderate crowding or spacing (2–5 mm) in the maxillary and mandibular arches
Exclusion criteria:
Sample size derivation
The methods of Pandis15 (standard for split-mouth design) were used for sample size estimation:
where σ pertains to the standard deviation, (µ1–µ2) intra-person difference, and ƒ (α, β) a function of significance level and power. With a difference between sides of 0.5 mm and canine distalization movement of 1 mm per month and a standard deviation of the difference of 0.7 mm at a 5% significance level (95% confidence interval) and 80% power, the computed sample was rounded up to 16 cases. Power analysis was performed for all outcomes. The distribution of malocclusions is shown in Figure 1.
Interim analyses and stopping guidelines
This study did not involve any interim analyses, and no specific stopping guidelines were established.
Randomization
Random allocation was performed using a computer-assisted function that generated random numbers (Microsoft Office 2022, Microsoft, Random, Redmond, WA, USA). The Consolidated Standards of Reporting Trials statement was used as a guide (Figure 2).16 One extraction side was randomly assigned for L-PRF insertion, whereas the contralateral extraction side of the same patient was allocated for the conventional method. Sequentially numbered opaque envelopes were prepared, each containing information about the corresponding group based on the generated order. These envelopes remain sealed in a secure location until they were opened by the main researcher (SKS). The randomization process was overseen by an individual who was not directly involved in the study, thus ensuring impartial management.
Allocation concealment
The evaluator obtained each randomized allocation via a centralized, secure, web-based interface (RandoWeb, Assistance Publique-Hopitaux de Paris [AP-HP], Paris, France). This sequence was masked until the intervention was assigned.
Blinding
Immediately following the extraction, the researcher responsible for applying L-PRF opened one envelope and performed the specified procedure (L-PRF or conventional treatment). Only the main researcher (SKS) was aware of the intervention used for each patient. All other investigators, including SKD, involved in recruitment, and AKB, handling data collection/outcomes, were blinded to the intervention. The allocation details were not disclosed to the participants during the study. SKS ensured the confidentiality of personal data for potential and enrolled participants through the secure collection, sharing, and maintenance of protocols. This involved using a computer without Internet access, implementing password protection, and encrypting data stored on a USB drive.
Interventions
Pre-surgical orthodontic preparation
All 16 participants underwent orthodontic treatment performed by a single orthodontist. Pre-adjusted edgewise Mclaughlin–Bennett–Trevisi brackets of 0.018 inch (in) slot with –70 torque for maxillary canines (Leone SpA, Firenze, Italy) were bonded to all teeth. In contrast, triple-slot molar tubes were connected or banded to the molars. Bonding was performed using Transbond XT (3M Unitek, Monrovia, CA, USA) and cured using a light-emitting diode curing light (Coltene/Whaledent Inc., Cuyahoga Falls, OH, USA). The bands were luted with a glass ionomer luting cement (GC Fuji I; GC Corporation, Tokyo, Japan). Alignment and leveling were initiated using light wires, that is, 0.012-in nickel-titanium (NiTi), 0.014-in NiTi, and 0.016-in round (NiTi) arch wire, and eventually progressed to stiffer wires, such as 0.016 × 0.022-in rectangular NiTi, 0.016 × 0.022-in rectangle stainless steel (SS), and 0.017 × 0.025-in rectangular SS arch wires. Complete alignment of the dentition was performed before the initiation of canine retraction.
Micro-implant placement
After alignment and leveling, a guiding template fabricated from a 0.017 × 0.025-in rectangular SS wire was used to correct the positioning of the micro-implant during its placement. After placing one of the free arms in the molar tube slot, intraoral periapical radiographs were obtained to determine the implant placement site. Micro-implants of 1.5 mm width and 8 mm length (FavAnchor® microimplant color code: pink, S. H. Pitkar Orthotools Ltd., Pune, India) were inserted under 2% local infiltration of lignocaine between the first maxillary first molar and second premolar on either side at a height of 5 mm from the alveolar crest (Figure 3). After leveling and alignment, atraumatic extraction of the maxillary premolars was performed under local anesthesia with 2% lignocaine diluted to 1:200,000.
Preparation and placement of leukocyte–platelet-rich fibrin plug
Using specific guidelines17 Choukroun’s PRF was used for preparing LPRF were used to prepare and place L-PRF on the experimental side before extracting the first premolars. Approximately 10 mL of venous blood (Figure 4A) was centrifuged without anticoagulants for 12 minutes at 2,700 rpm. At the apex of the tube, a layer of plasma devoid of platelets was observed, and an RBC clog was formed at the base (Figure 4B). Using sterile tweezers, the middle portion of the L-PRF plug was extracted and dissected using a BP No. 15 blade, approximately 2 mm below the intersection between the middle and lower layers (Figure 4C). After carefully inserting the L-PRF plug into the socket (Figure 4D), a 3-0 silk suture was used to close the socket. Similarly, the contralateral first premolar was gently extracted, and the socket was covered with a sterile gauze pack. All participants received post-extraction instructions and appropriate medications. The interventions were concurrently provided to each patient.
Orthodontic intervention for individual canine retraction
Individual canine retraction after extraction was accomplished using sliding mechanics of a 0.016 × 0.022-in SS arch wire with the help of a microimplant placed between the first molar and second premolar and a closed NiTi coil spring to exert a constant force of 150 g, which was measured through a Dontrix gauge at the sites.
Study records and outcome measure assessment
Pre-treatment records, such as lateral cephalograms, orthopantomogram, plaster models, and intra- and extra-oral photographs, were obtained.
Outcome measures
Primary outcome
Canine distalization or retraction, which was intraorally measured using a digital Vernier caliper (Absolute Digimatic Caliper, Mitutoyo, Kawasaki, Japan) as the distance between the distal aspect of the maxillary canine bracket and the mesial aspect of the molar tube slot (Figure 5A and 5B), as the primary outcome. The measurements were recorded every 2 weeks (six time points, after placement of L-PRF): on the day of L-PRF placement (T0), 15th day (T1), 30th day (T2), 45th day (T3), 60th day (T4), 75th day (T5), and 90th day (T6) for 3 months between L-PRF and control sites.
Secondary outcomes
Canine rotation: Occlusograms were used to measure the rotation of canines using the method described by Ziegler and Inglervall.18 The angle formed by the line running through the mid-palatal raphe and the line connecting the canine’s mesial and distal contact sites was measured. The change in angle indicated the amount of rotation that occurred after the completion of canine retraction. The recordings were performed at the start (T0) and end of the study period (T6) (Figure 6).
Soft tissue healing and pain: The Healing Index by Landry et al.19 and the numerical pain rating scale by McCaffery and Beebe20 were used to evaluate soft tissue response and pain in the extraction sockets of L-PRF and control sites on the 3rd, 7th, and 15th days (Figure 7). No changes in trial outcomes were made after the trial commenced.
Statistical analyses
Statistical Package for the Social Sciences (SPSS version 21; IBM Corp., Armonk, NY, USA) was used for statistical analyses. Data normality was assessed using the Shapiro–Wilk test. For intragroup comparisons among different study visits, repeated measures analysis of variance was used. The intraclass correlation coefficient was used to evaluate examiner reliability. Unpaired Student’s t test was used to compare the L-PRF and control groups. A paired t test was used after summing the data at the cluster level to account for the clustering effects. The effect estimates were calculated by dividing the difference between the two groups (treatment group mean minus control group mean) by the standard deviation of one of the groups. Pearson’s correlation coefficients were also calculated, and the chi-squared test was used to determine whether there was a significant difference between categorical variables. For all statistical inferences, P < 0.05 was considered significant.
RESULTS
Participant flow
This study included 16 participants and 32 sides, with 16 participants each randomly assigned to the L-PRF and the control groups. All participants were tracked until the end of the study period (Figures 1 and 2). The intraclass correlation coefficient was r = 0.84, demonstrating high evaluator reliability.
Baseline data
Age was categorized into two subgroups: ≤ 20 and > 20 years. Most of the study participants (62.5%) belonged to the age group of > 20 years. The mean age of the participants was 22.25 ± 2.26 years. Most study participants (75%) were female (Figure 1).
Primary outcome
Canine distalization
Table 1 shows the degree of canine retraction or distalization on the L-PRF and control sides at various time intervals.
The mean changes in canine distalization between the L-PRF and the control groups were compared from T0 to T6 at monthly intervals and overall (Table 2). At each of the three time points, T0–T2 (1.24 ± 0.13 mm vs. 0.68 ± 0.12 mm, P < 0.001), T2–T4 (1.55 ± 0.10 mm vs. 0.77 ± 0.08 mm, P < 0.001), and T4–T6 (0.83 ± 0.06 mm vs. 0.77 ± 0.07 mm, P = 0.002), the difference between the L-PRF and the control side was statistically significant, indicating higher canine movement on the L-PRF side compared with the control side.
Overall, from T0 to T6, the distalization of the canine at the L-PRF side was higher (3.66 ± 0.24 mm) compared with that of the control side (2.18 ± 0.13 mm), the difference of which was statistically significant (P < 0.001) (Table 2). This indicates that L-PRF has the potential to induce rapid tooth movement when applied to an extraction socket. Figure 7 shows intraoral images of canine retraction 30 days post-treatment.
Secondary outcomes
Canine rotation
Inter-group comparison (Table 3) showed that the difference between the L-PRF and control sides at both T0 (33.99 ± 9.31° vs. 35.01 ± 5.30°, P = 0.705) and T6 (27.07 ± 9.20° vs. 30.12 ± 5.30°, P = 0.259) was nonsignificant, which signifies that L-PRF has little role in canine rotational movement.
Intra-group comparisons at T0 and T6 (Table 3) showed a significant difference in the mean for both the L-PRF (33.99 ± 9.31° vs. 27.07 ± 9.20°, P < 0.0001) and control (35.01 ± 5.30° vs. 30.12 ± 5.30°, P < 0.0001) sides. The fact that SS ligatures were used to tie the canines on both sides during retraction might have contributed to the nonsignificant differences between the two sides.
Healing and pain index
Inter-group comparison (Table 4) of both healing and pain index scores showed a significant difference in the mean for both the L-PRF and control sides on the 3rd, 7th, and 15th days, indicating that L-PRF aids in faster healing and pain modulation.
DISCUSSION
Numerous surgical and nonsurgical approaches have been suggested to hasten tooth movement and yield diverse outcomes. Surgical techniques, such as tooth extraction and periodontally accelerated osteogenic orthodontics, induce a regional acceleratory phenomenon (RAP),21,22 reducing the duration of orthodontic treatment. Despite their apparent success, these methods require comprehensive long-term evaluations.23,24
Minimally invasive techniques have come to the forefront in the pursuit of less invasive approaches to expedite OTM. PRP has emerged as a recent addition to noninvasive methods aimed at accelerating tooth movement,7 offering the dual benefit of preventing alveolar bone loss while triggering a RAP-like effect and fostering alveolar bone regeneration.25
Animal studies have indicated that L-PRF results in increased tooth mobility, with a 1.7-fold increase observed in rat models and a 2.13-fold increase in dog models.6,7 A recently published systematic review suggested that platelet-rich concentrates might play a role in accelerating OTM in animal models.26
Effects of L-PRF on canine distalization
Except at T6, the current study found a statistically significant retraction on the L-PRF side compared with the control side at all time points (T1, T2, T3, T4, and T5). Biweekly canine retraction changes on the L-PRF side were 1.5 times more at T1, 2.5 times more at T2 and T3, almost twice at T4, and nearly similar at T5 and T6 compared with that on the control side.
Studies have presented divergent perspectives regarding the use of L-PRF in canine retraction. These conflicting findings may stem from variations in the intervention procedures, including distribution methods (membrane, injection, or plugs), dosage, concentrate presentation (L-PRF, PRF, or PRP), and observation periods.27-30
Studies investigating the placement of the L-PRF clot in extraction sockets have yielded mixed results. In some instances, canine retraction was less pronounced on the control side compared with that on the L-PRF side when canine distalization commenced immediately after the extraction of the first premolar.27,28
Retraction rates of 0.32 (control side) and 0.52 (L-PRF side) mm/month were reported by Nemtoi et al.28 (P = 0.006). Tehranchi et al.27 observed that throughout all subsequent visits (biweekly for 4 months), the teeth migrated faster on the L-PRF side than on the control side (P = 0.006). In contrast to the above, a study by Reyes Pacheco et al.31 found a considerably superior canine distalization rate (0.23 mm/month) on the control side, although they began canine retraction 15 days post-initial premolar extraction (L-PRF side: 0.67 mm/month, P = 0.004; control side: 0.90 mm/month). An 8-week study recently published by Barhate et al.32 demonstrated that when L-PRF was inserted into the extraction socket, canine retraction was significantly higher on the L-PRF side for the first 4 weeks and comparable for the remaining 4 weeks. Thus, canine distalization after L-PRF insertion may be predominantly caused by normal alveolar bone remodeling in response to a constant and optimal force magnitude. When injectable PRF was used, other studies22,30,33-35 revealed an increase in the canine retraction rate.
It is not desirable to draw conclusions by merely synthesizing the final output of each incorporated study, owing to their different trial times. Nevertheless, we may acquire a deeper understanding of the PRF effect by performing a time-dependent evaluation of each study’s data. For the first 3 months, PRF succeeded in accelerating OTM, with the most significant rate of acceleration occurring around the interval during the second month. The current study showed an accelerating effect of L-PRF during the first 3 months of placement, which is consistent with other studies reported in the literature.27,30,33,34 However, three of them27,30,34 showed a maximal effect during the second month after L-PRF application, implying that L-PRF exerts the most significant effect on OTM acceleration after the first month.
Although the causal mechanism remains unknown, we can presume that (1) immediately following L-PRF application, there is increased production of cytokines, which produce bone remodeling and then accelerate OTM,30 and (2) after a defined period, as L-PRF dissociates and there is a concomitant decrease in growth factors and cytokines, there may be some decrease in OTM via a negative feedback mechanism.29 However, the natural effect and mechanism of L-PRF and PRP must be elucidated by additional studies using yardstick protocols and PRP/L-PRF parameters.
Rotational effect of L-PRF
The mean change in canine rotation was not statistically significant between the L-PRF and control sides at T0 and T6. This contradicts the findings of Reyes Pacheco et al.,31 who demonstrated that the mean canine rotations were 5.80° on the test side and 8.50° on the control side (P = 0.001). The L-PRF side showed higher retroclination at the end of the distalization period. In contrast to the study by Reyes Pacheco et al.,31 three studies in the literature29,33,34 found statistically nonsignificant variations in canine rotation between the control and L-PRF, which is consistent with the result of the current study.
Study generalizability and limitations
This study was a split-mouth trial. Compared with parallel-group trials, this design requires fewer patients, which may result in non-normal distributions and affect the results. Although the sample size was determined using standard methods, a larger sample size would have resulted in a more foreseeable outcome and provided more value to the current study. The second limitation is derived from the fact that OTM is multifactorial and involves multiple cytokines and growth factors. Because such factors have not been considered, the effect cannot be fully generalized. In addition, as the measurement was performed using standard instruments, some degree of inaccuracy could creep in, as it was performed on the chair side in the patient’s mouth. Further studies considering other OTM-associated biomarkers and growth factors can provide a broader perspective and improve our understanding of tooth movement.
CONCLUSIONS
Cumulatively, the L-PRF group demonstrated a significantly greater tooth movement as compared to conventional treatment group (P < 0.001); however, there was no significant difference thereafter. There was also a significant improvement in healing and pain reduction on the L-PRF side compared with that on the control side.
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