
==== Front
Braz Oral Res
Braz Oral Res
bor
Brazilian Oral Research
1806-8324
1807-3107
Sociedade Brasileira de Pesquisa Odontológica - SBPqO

01900
10.1590/1807-3107bor-2024.vol38.0089
Original research/Mouth and Jaw Surgery
Impact of L-PRF on pain and healing outcomes in lower third molar surgery: a randomized split-mouth trial
https://orcid.org/0000-0002-5893-4047
MORAES Raissa Pinheiro (a)
https://orcid.org/0000-0002-3262-3347
COSTA Fábio Wildson Gurgel (a)
https://orcid.org/0000-0002-1513-9027
SILVA Paulo Goberlânio de Barros (a)
https://orcid.org/0000-0002-3142-1268
CARVALHO Francisco Samuel Rodrigues (a)
https://orcid.org/0000-0002-8090-1540
PAZ Jéssica Emanuella Rocha Moura (b)
https://orcid.org/0009-0005-8628-575X
MATOS Gabriel Carvalho (c)
https://orcid.org/0000-0001-9978-0542
GURGEL Marcela Lima (d)
https://orcid.org/0000-0002-5985-8955
CETIRA FILHO Edson Luiz (a)
https://orcid.org/0000-0002-7188-9308
SOARES Eduardo Costa Studart (a)
(a) Universidade Federal do Ceará – UFCE, Postgraduate Program in Dentistry, Fortaleza, CE, Brazil.
(b) Universidade de São Paulo – USP, School of Dentistry of Ribeirao Preto, Ribeirão Preto, SP, Brazil.
(c) Private Dental Office, Fortaleza, CE, Brazil.
(d) Universidade Federal do Ceará – UFCE, Postgraduate Program in Dentistry, Fortaleza, CE, Brazil.
Corresponding Author : Fábio Wildson Gurgel Costa E-mail: fwildson@yahoo.com.br
Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.

13 9 2024
2024
38 e08912 10 2023
16 4 2024
13 5 2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

This study explored the effects of L-PRF on pain, soft tissue healing, periodontal condition, and post-extraction bone repair of mandibular third molars (3Ms). A randomized, prospective, triple-blind, split-mouth clinical trial was conducted with 34 volunteers. Eligible patients were randomly allocated into two treatments: G1 (without L-PRF), G2 (alveoli filled with L-PRF), in which the removal of bilateral 3Ms was performed at the same surgical time. Outcomes were assessed according to a visual analogue scale (pain), soft tissue scoring system (wound healing), periodontal probing of mandibular second molar. Bone repair was determined by volumetric analysis (ITK-SNAP software) and fractal analysis (ImageJ software). An intention-to-treat approach to Statistical analysis was used. L-PRF reduced pain in the 7-day postoperative follow-up (p = 0.019) and not only improved soft tissue healing after 1 month of follow-up (p = 0.021), but also probing depth (distal face) in 3 months postoperatively (p = 0.011). Significant alveolar reduction occurred in 3 months after surgery in both treatments (p < 0.05), however, this was more significant in G1 (p = 0.016). The fractal dimension showed no statistical differences. L-PRF improved postoperative clinical parameters of pain, soft tissue healing, and periodontal condition, suggesting that it has a beneficial effect on preserving the alveolar ridge and accelerating the initial repair process.

Biological Products
Pain
Wound Healing
Oral Surgical Procedures
Tooth Socket
CNPq315479/2021-3 Fundação Cearense de Apoio ao Desenvolvimento Científico e TecnológicoFPD-0213-00294.01.00/23 The authors thank the Brazilian Society of Dental Research (SBPqO) for the opportunity to present this study at 40th Annual Meeting of the SBPqO, where it was awarded first place. Furthermore, this work was partially supported by the Brazilian National Council for Scientific and Technological Development (CNPq) which provided a PQ fellowship in category 2 to Dr. Fábio Costa (process number 315479/2021-3). Dr. Marcela Gurgel is supported by the Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico post-doctoral fellowship (process number FPD-0213-00294.01.00/23).
==== Body
pmcIntroduction

Mandibular third molar surgery is a procedure frequently associated with postoperative discomfort, primarily due to the inflammatory events that naturally occur because of the surgical trauma. 1 Therefore, it is essential to closely monitor the healing process and bone remodeling to prevent unfavorable outcomes. 2

Numerous studies have been conducted with a focus on strategies performed with the aim of not only reducing postoperative symptoms but also enhancing and accelerating the healing process. For this purpose, biomaterials such as platelet aggregates have been developed. These aggregates have the potential to regulate inflammatory events and promote tissue regeneration, thereby contributing to the treatment of bone defects. 3

Platelet aggregates can be categorized into various types, with two notable variants: Platelet-Rich Plasma (PRP) and Leukocyte-Platelet-Rich Fibrin (L-PRF). Distinct protocols are employed to obtain each type of platelet, involving differences in centrifugation protocol, impact on injured tissue, and cellular composition, among other factors. 4

L-PRF, a natural alternative to PRP, is gaining popularity in maxillofacial surgery because of its biological advantages. These benefits arise from its denser fibrin network structure, gradual release of growth factors and cytokines, presence of mononuclear leukocytes, integration of circulating microparticles and adhesive glycoproteins, and its easy, quick, and cost-effective processing. 5 Moreover, another possible benefit of L-PRF on third molar alveoli is related to the periodontal health of second molars. Surgical removal of fully impacted third molars has been found to lead to loss of attachment on the distal portion of the second molar, irrespective of the flap design used or the initial height of the alveolar bone. 6 A longitudinal study has shown that third molar extraction leads to periodontal breakdown on the distal surface of the second molar, leading to increased probing depth, and immunological-microbiological changes. 7

The widespread adoption of L-PRF for alveolar filling after mandibular third molar extraction is due to its numerous advantages. However, its impact on postoperative morbidity remains unclear. A recent meta-analysis had the aim of assessing the effects of novel PRF centrifugation methods on postoperative outcomes and soft tissue healing after mandibular third molar removal. This meta-analysis took into account that surgery of mandibular teeth involves more invasive procedures, such as larger flaps, ostectomies, and odontosections. These types of procedures generally generate greater postoperative morbidity The aforementioned analysis found a limited number of studies that specifically examined the benefits of L-PRF using a split-mouth design, with only four trials meeting the inclusion criteria. 6 Moreover, only one study in the meta-analysis addressed bone healing as an outcome. 7 This emphasizes a significant gap in the current literature relative to the application of L-PRF in mandibular third molar surgery.

The lack of studies on this specific topic highlights the need for additional research to fully comprehend the potential benefits and limitations of L-PRF in this context of promoting favorable outcomes and enhancing bone healing. In view of the foregoing, the aim of our present study was to evaluate whether L-PRF has positive effects on pain, soft tissue healing, periodontal condition, and, most importantly, the quantity and quality of bone repair after mandibular third molar extraction.

Methods

Trial design

A triple-blind, crossover, clinical trial was conducted at the Walter Cantídio University Hospital, Federal University of Ceará, from November 2019 to December 2020. Patients referred for bilateral mandibular third molar extraction participate in the studs, with approval from the Research Ethics Committee (CAAE 19487419.0.0000.5054). A term of Informed consent was obtained in accordance with the Declaration of Helsinki, and the study followed the CONSORT guidelines for quality and transparency. 8

Sample size and power

Two sample size calculations were conducted, one for bone repair and another for clinical variables, with the larger value chosen. Following the research of Ritto et al., 9 which showed a significant increase in bone mineral density with L-PRF (954.100±500.768 vs. 522.514±352.281), 23 patients were initially estimated to meet the study requirements relative to statistical power and confidence level (90% power, 95% confidence). To account for potential dropouts, an additional 20% was added, resulting in a total of 28 patients. As the study involved bilateral mandibular third molar extraction (split-mouth) from each patient, a total of 56 extractions were performed in all 28 participants.

With a grade 5 healing score of 96.4% for the L-PRF group compared with 67.9% for the non-L-PRF group, the present sample size of 28 patients per treatment yielded a statistical power of 81.29% to reject the null hypothesis. In a new sample size calculation, a total of 28 patients was necessary for a power of 80%.

Eligibility criteria

This study sample consisted of individuals aged 18 to 35 years, classified as healthy according to the American Society of Anesthesiologists (ASA I). Participants included both males and females with bilaterally located mandibular second and third molars. Furthermore, in the thorough clinical examination, they showed no signs of inflammation in the periodontal tissues and were devoid of pain, local inflammation, or associated pathologies. The criteria for inclusion of third molars entailed consistent bilateral patterns of root formation, positioning, and level of impaction, in accordance with the classification established by Pell, Gregory, and Winter. Patients included in the study were required to have previously been recommended for the extraction of their mandibular third molars, and their willingness to participate was confirmed by their informed consent, a term that was read and signed prior to their involvement.

Exclusion criteria referred to pregnant women, smokers, individuals with systemic conditions that could affect inflammation and healing reactions, and those with a platelet count below 150,000 ml/mm3. Furthermore, participants who missed postoperative assessments, did not follow recommendations, experienced intraoperative complications, or had surgical procedures exceeding a 15-minute interval between each side (measured from starting the incision through to removal of tooth) were also excluded from the study.

Treatment characterization and randomization

The patients initially underwent evaluation by the lead researcher. This consisted of a combination of medical history and clinical examination. The data collected were recorded on a standardized patient chart specifically designed for the study. This information included details about the chief complaint, dental and medical history, degree of tooth impaction (classified radiographically by using Pell and Gregory [1933] and Winter [1929] criteria), and stages of development of the tooth and root (Nolla stages). Data from the Pernambuco Index (PI) 11 were also included. These were used to assess surgical complexity based on factors such as occlusal plane level, retromolar space, tooth angulation, curvature and root count, relationship with the second molar, patient’s age, and Body Mass Index (BMI). During postoperative follow-up time intervals, a calibrated examiner measured all the variables being analyzed.

Participants were randomly allocated to two treatment groups: G1 (control), which involved extraction without L-PRF insertion, and G2 (experimental intervention), where L-PRF was inserted into the alveolar socket during extraction. To maintain patient blinding relative to the alveolus receiving L-PRF treatment, surgical drapes were used to cover patients’ eyes, and the platelet aggregate was discreetly introduced Into the alveolar socket

Each participant had both mandibular third molars extracted during a single surgical session. The allocation of treatments to specific alveolar sockets was randomized using the “randbetween” function in Microsoft Excel® software, version 2010. Random numbers were generated and sealed in opaque envelopes, which were held by a collaborator not involved in surgical procedures or outcome assessments. The envelopes were opened only after the removal of both mandibular teeth. The side for starting the surgery was also randomly determined.

Surgical procedure

To ensure the safety of both patients and researchers, stringent biosafety protocols were followed. This included the use of sterile instruments, hydrogen peroxide mouthwash, and the wearing of appropriate personal protective equipment. As a proactive measure to mitigate the potential spread of the COVID-19 (SARS-CoV-2) pandemic, scheduling was adjusted to accommodate only one patient per shift, and the clinic was exclusively reserved for the designated surgical procedure. Furthermore, strict measures were implemented to prevent individuals with COVID-19 symptoms from visiting the clinic.

All surgical procedures were performed by a single surgeon in the same outpatient clinic, in accordance with standardized techniques for third molar extraction. The procedure began with the administration of local anesthesia, using 4% articaine hydrochloride with 1:200,000 adrenaline (Articaine®, DFL, Rio de Janeiro, Brazil). Subsequent to anesthesia, a triangular flap was created, and any bone obstructing tooth extraction was removed using a high-speed handpiece with a proficient carbide bur (#702, FGXL, Brazil), continually irrigated with a 0.9% saline solution. Tooth sectioning with the #702 bur and subsequent removal were performed, if necessary, using either a straight or Seldin elevator. All operative steps were performed consistently on both sides.

Platelet preparation

From each patient, 20 ml of venous blood was collected and immediately transferred to sterile glass tubes with no anticoagulants or interfering substances. These tubes were then centrifuged using a Montserrat centrifuge (Oxford, England) in accordance with Choukroun’s protocol (3,000 rpm for 10 minutes), a method widely used in the literature. 12 This process resulted in a product with acellular plasma at the top, L-PRF gel in the middle, and red blood cells at the bottom. The L-PRF gel was meticulously extracted from the glass tube using sterile instruments, separating it from other residual blood components.

After tooth extraction, the operating surgeon (researcher) left the operating room. Then, a second surgeon and an assistant entered the room to insert the L-PRF into the surgical flap on one side and suture both surgical sites. This approach ensured that the researcher remained unaware of the insertion procedure, maintaining blindness relative to assignment of the treatments to each side. Suturing was performed using 4-0 nylon threads. After suturing, the patient was instructed to bite down on a piece of gauze for 30 minutes and to follow the postoperative guidelines provided.

Drug protocol

Patients were instructed to adhere to a medication regimen that included taking amoxicillin 500 mg every eight hours for seven days. They were also advised to take ibuprofen 600 mg every twelve hours for three days. Moreover, dipyrone was prescribed as an additional analgesic option, with a recommended dosage of 500 mg every six hours for three days.

Pain and soft tissue assessments

Postoperative pain was assessed at twelve-hour intervals and one week after the procedure using the Visual Analog Scale (VAS), with separate scales for each side. 9 The healing was evaluated on the seventh day. and one month after the operation. For this assessment, an adapted version of the healing index (HI) scores, developed by Landry and colleagues, was used. 13 This score provides a comprehensive evaluation of the tissue, considering factors such as incision margin epithelialization, tissue color, response to palpation (presence or absence of bleeding), presence of granulation tissue, and incidence of suppuration. 14

Periodontal condition

Moreover, before surgery and three months after surgery, the probing depth on the distal (D), distobuccal (DB), and distolingual (DL) sites of the mandibular second molar was measured using a North Carolina probe (no. 15). The probe was inserted into the gingival sulcus along the longitudinal axis of the tooth until slight resistance was encountered.

Imaging evaluation

Cone-beam computed tomography (CBCT) scans were performed using a Kodak K 9000 3D machine (Kodak Dental Systems, Carestream Health, Toronto, Canada). The scans were performed with a kilovoltage range of 65 to 75 Kvp, using 8 to 12 mA, and an exposure time of 23 seconds. These CBCT images were obtained at time intervals immediately after the surgical procedure (T0), as well as at one month (T1) and three months (T2) after the surgery. Importantly, the images were stored in Digital Imaging and Communications in Medicine (DICOM) format, enabling subsequent assessment of alveolar bone volume and quality.

The alveolar bone volume and quality were evaluated by an examiner who was unaware of the particular surgical protocol used, ensuring a blinded evaluation process.

Volumetric analysis

Alveolar volumes were calculated at two specific time points by an experienced evaluator (M.L.G.) who was skilled in performing three-dimensional segmentation and image processing: immediately after the surgical procedure (T0) and three months post-surgery (T2). This was accomplished using ITK-SNAP software, version 3.8 (http://www.itksnap.org/), which allowed for the processing of tomographic images from both the right and left alveoli at both stages.

The process started with conversion of the DICOM images into the Neuroimaging Informatics Technology Initiative (NIfTI) format. After this, regions of interest (ROIs) were segmented to cover the entire length of the alveoli. The software then automatically calculated the alveolar volumes for these segmented regions, provided measurements in cubic millimeters (cm3) (Figure 1).

Figure 1 Alveolar reconstruction using the ITK-SNAP software. A. Sagittal slices; B. Axial slice; C. Coronal slice; D. Three-dimensional reconstruction of alveoli for volume calculation.

Fractal analysis

The bone quality was also assessed in a blinded manner by a calibrated researcher (R.M.P.), This involved fractal analysis (FA) at three time intervals: immediately after the surgical procedure (T0), one-month post-surgery (T1), and three months post-surgery (T2). The process began by selecting coronal sections from the original tomographic images. These sections were chosen at various thicknesses: PR1 (panoramic radiograph with a 1 mm thickness), PR5 (panoramic radiograph with a 5 mm thickness), and PR10 (panoramic radiograph with a 10 mm thickness). The mean values of these sections were then calculated and represented as the mean PR value. Furthermore, a sagittal section labeled as SR1 (sagittal radiograph with a thickness of 1 mm) was chosen.

The scans chosen were exported as JPEG (Joint Photographic Experts Group) images to the Image J software (https://imagej.nih.gov/). The process of calculating the fractal dimension occurred after defining a region of interest (ROI) that covered the extent of surgical alveolus, while excluding the crestal lamina dura. To assess the images, the box-counting method introduced by White and Rudolph 15 was used. These images were saved with acquisition of grayscale images with a bit depth of 8.

For each CT scan (T0, T1, and T2) of each patient, an internal control measurement of 5x5 mm was defined, located consistently in an area shared across all scans. This region was typically situated between the mandibular foramen and the base of the mandible. This internal control was used to compare the FA values in the sagittal slices. The ROI for this control was a spherical measurement of 5x5 mm.

The subsequent steps in the FA calculation included duplicating the chosen ROI image (excluding the crestal lamina dura), applying a Gaussian filter (sigma-35), subtracting it from the original image, adding a gray value of 128, binarizing the image, and then subjecting it to erosion, dilation, and skeletonization. These processes were performed to obtain the fractal dimension value (Figure 2).

Figure 2 Step-by-step analysis of the fractal dimension of the dental alveoli. A. Coronal slice (5-mm thick) of a CBCT scan; B. Delimitation of the region of interest (ROI) C. ROI (dental socket); D. Blurred image with Gaussian filter; E. Image subtraction result; F. – Addition of a gray value threshold of 128; G. Binary image; H. Eroded image; I. Dilated image; J. Skeletonized image.

Statistical analysis

Quantitative data were presented in terms of mean and standard deviation values and subjected to the Kolmogorov-Smirnov normality test. Subsequently, pairwise comparisons were made using either the Wilcoxon or Friedman/Dunn tests. Whereas categorical data were represented as absolute and percentage frequency values, and they were compared by using the McNemar test.

All analyses were performed by adhering to a 95% confidence interval, the difference limit defined as significant between groups was p < 0.05, and the procedures were performed within the SPSS® (Statistical Package for the Social Sciences) version 20.0 software designed for Windows®. An intention-to-treat (ITT) approach was used, incorporating all randomized patients who were included in the study. Moreover, a treatment received analysis was performed, focusing on patients who actually underwent the intervention, thereby assessing the effectiveness and outcomes of the treatment within the intended population.

RESULTS

Sample characterization

A total of forty patients were initially assessed, but six were excluded because they did not meet the inclusion criteria (one declined to participate, and five had unilateral third molars). Consequently, 34 patients were enrolled in the study for an intention-to-treat (ITT) analysis. However, six patients were lost to follow-up as they did not return for the postoperative tomographic examination. Therefore, the final sample consisted of 28 patients for a treatment received (TR) analysis. This final sample included a total of 56 tooth extractions, involving 16 female and 12 male patients (Figure 3).

Figure 3 Flow Diagram Following the CONSORT Statement.

The participants’ ages ranged from 18 to 35 years (mean age of 22.36 ± 4.14 years). There were no statistically significant differences among the study variables in both the ITT and TR analyses. These variables included factors such as sample size, gender distribution, age, degree of impaction, surgical difficulty index, and procedure time. The absence of significant variation may reflect the careful standardization of the teeth and the consistent uniformity of the surgical procedures performed. Moreover it is worth noting that each patient served as his/her own reference and this may have contributed to this outcome (Table 1).

Table 1 Sample characterization.

Variable	TR analyses	ITT analyses	
Control	Experimental	Control	Experimental	
n (%)	n (%)	n (%)	n (%)	
Pernambuco index	
Low	19 (67.9%)	19 (67.9%)	24 (70.6%)	24 (70.6%)	
Moderate	9 (32.1%)	9 (32.1%)	10 (29.4%)	10 (29.4%)	
Pell & Gregory’s classification	
IA	5 (17.9%)	5 (17.9%)	6 (17.6%)	6 (17.6%)	
IB	3 (10.7%)	3 (10.7%)	3 (8.8%)	3 (8.8%)	
IIA	10 (35.7%)	10 (35.7%)	11 (32.4%)	11 (32.4%)	
IIB	8 (28.6%)	8 (28.6%)	12 (35.3%)	12 (35.3%)	
IIC	2 (7.1%)	2 (7.1%)	2 (5.9%)	2 (5.9%)	
Winter’s classification	
Mesioangular	7 (25.0%)	7 (25.0%)	10 (29.4%)	10 (29.4%)	
Vertical	19 (67.9%)	19 (67.9%)	22 (64.7%)	22 (64.7%)	
Distoangular	1 (3.6%)	1 (3.6%)	1 (2.9%)	1 (2.9%)	
Horizontal	1 (3.6%)	1 (3.6%)	1 (2.9%)	1 (2.9%)	
Surgical time (min)*	8.07 ± 5.06	8.39 ± 6.94	7.26 ± 4.96	7.61 ± 6.57	
*mean †standard deviation; TR, treatment received; ITT, intention to treat; min, minutes.

Pain and soft tissue assessments

The use of L-PRF in the alveoli resulted in a reduction in postoperative pain at both of the time intervals examined. A statistically significant difference in means was observed over the seven-day period (p = 0.019), while this difference was not apparent in the 12-hour follow-up (ITT analysis, p = 0.301; TR analysis, p = 0.307) (Table 2).

Table 2 Pain and probing depth assessment.

Variable	TR analyses	ITT analyses	
Control	Experimental	p-value	Δ	Control	Experimental	p-value	Δ	
VAS (cm)	
12h	3.36 ± 2.06	3.11 ± 1.85	0.301a	-0.25 ± 1.21	3.23 ± 2.06	3.00±1.79	0.307a	-0.23±1.18	
7d	1.32 ± 1.81	0.64 ± 1.06	0.019a	-0.68 ± 1.39	1.12 ± 1.70	0.56±0.99	0.018a	-0.56±1.31	
p-value	0.001a	<0.001a	-	-	0.001a	< 0.001a	-	-	
DB (MM)	
PO	3.46 ± 0.64	3.46 ± 0.88	-	0.00 ± 0.77	-	-	-	-	
3 mth	2.96 ± 0.69	2.89 ± 0.74	0.626a	-0.07 ± 0.77	-	-	-	-	
p-value	0.003a	0.025a	-	-	-	-	-	-	
D (mm)	
PO	3.66 ± 0.82	3.33 ± 0.51	-	-0.33 ± 1.03	-	-	-	-	
3 mth	3.43 ± 0.74	3.14 ± 0.80	0.011a	-0.29 ± 0.53	-	-	-	-	
p-value	1.000a	0.157a	-	-	-	-	-	-	
DL (mm)	
PO	3.39 ± 0.63	3.50 ± 0.69	-	0.11 ± 0.74	-	-	-	-	
3 mth	3.11 ± 0.63	3.04 ± 0.64	0.678a	-0.07 ± 0.90	-	-	-	-	
p-value	0.087a	0.015a	-	-	-	-	-	-	
*p < 0.05; Wilcoxon atest. TR: treatment received; ITT: intention to treat; VAS, Visual Analog Scale; DB: distobuccal; D: distal; DL, distolingual; PO: preoperative assessment; mth = month.

Periodontal condition

The assessment of the Healing Index (HI) and probing depth showed statistically significant differences at the one-month postoperative evaluation (p = 0.021) and the three-month follow-up for the distal (D) aspect (p =0.011), respectively (Tables 2 and 3). Furthermore, considering the probing depth at the distal sites of the second molar three months postoperatively, we observed no increase in these measurements from baseline to the 3-month postoperative follow-up time interval among any of the patients included in the trial. Consequently, based on this assessment, and within this context. we did not observe any instances of distal bone loss at the second molar

Table 3 Soft tissue healing and alveolar volume values.

Variable	Control	Experimental	p-value	Δ	
n (%)	n (%)	n (%)	
Soft tissue healing scores (7 d)	
1	0 (0.0%)	0 (0.0%)	0.446	-	
2	10 (35.7%)	5 (17.9%)	 	-	
3	16 (57.1%)	19 (67.9%)	 	-	
4	2 (7.1%)	4 (14.3%)	 	-	
5	0 (0.0%)	0 (0.0%)	 	-	
Soft tissue healing scores (1 mth)	
1	0 (0.0%)	0 (0.0%)	0.021	-	
2	0 (0.0%)	0 (0.0%)	 	-	
3	0 (0.0%)	0 (0.0%)	 	-	
4	9 (32.1%)*	1 (3.6%)	 	-	
5	19 (67.9%)	27 (96.4%)*	 	-	
p-value	< 0.001	< 0.001	 	 	
Alveolar volume (PR 1)	
T0	622.86 ± 285.79mm3	590.10 ± 291.09mm3	0.127a	32.76 ± 181.25	
T3	186.96 ± 131.97mm3	229.06 ± 146.09mm3	0.116a	-42.10 ± 150.55	
p-value	<0.001a	<0.001a	-	 	
Variation (absolute number)	-435.90 ± 239.06mm3	-361.04 ± 224.76mm3	0.016a	-74.86 ± 184.19	
Variation (%)	-68.96 ± 17.54mm3	-59.55 ± 20.62mm3	0.084a	-9.41 ± 22.54	
*p < 0.05; McNemar’s test (n. %); aWilcoxon test; mth, month; PR 1: panoramic radiograph with a thickness of 1 mm.

Bone repair imaging evaluation

In the volumetric analysis, there were no statistically significant differences observed between the control and experimental treatments at both T0 (p = 0.127) and T3 (p = 0.116) time intervals. However, both treatments showed a significant reduction in alveolar volume three months post-surgery (p < 0.01). Furthermore, the difference between T3 and T0 (Δ) exhibited a significant distinction (p = 0.016), with the control group demonstrating a more pronounced reduction in alveolar volume (Δ = -435.90 ± 239.06) compared with the experimental intervention group (Δ = -361.04 ± 224.76) (Table 3).

Relative to fractal analysis (FA), in the experimental intervention group, PR5 and SR1 values showed a consistent increase from T1 onwards. In contrast, in the control group, this increase became apparent only after T2. Nevertheless, it is important to note that the differences in trabecular complexity between the two treatments were not statistically significant during the one and three-month postoperative follow-up time intervals (p > 0.05) (Table 4).

Table 4 Fractal analysis for bone quality assessment.

Variable	Control	Experimental	p-value	Δ	
PR 1	
T0	1.104 ± 0.075	1.097 ± 0.060	0.687a	-0.01 ± 0.09	
T1	1.149 ± 0.070*	1.158 ± 0.054*	0.589a	0.01 ± 0.09	
T3	1.068 ± 0.065*†	1.051 ± 0.059*†	0.225a	-0.02 ± 0.07	
p-value	< 0.001b	< 0.001b	-	 	
PR 5	
T0	1.117 ± 0.066	1.114 ± 0.072	0.842a	0.00 ± 0.09	
T1	1.142 ± 0.061	1.156 ± 0.060*	0.285a	0.01 ± 0.07	
T3	1.077 ± 0.064*	1.067 ± 0.058*†	0.586a	-0.01 ± 0.09	
p-value	< 0.001b	< 0.001b	-	 	
PR 10	
T0	1.126 ± 0.084	1.124 ± 0.064	0.881a	0.00 ± 0.08	
T1	1.123 ± 0.079	1.144 ± 0.064	0.121a	0.02 ± 0.07	
T3	1.103 ± 0.075	1.111 ± 0.057	0.666a	0.01 ± 0.10	
p-value	0.248b	0.215b	-	 	
Mean PR	
T0	1.116 ± 0.059	1.112 ± 0.046	0.748a	0.00 ± 0.07	
T1	1.138 ± 0.057*	1.152 ± 0.043*	0.139a	0.01 ± 0.05	
T3	1.082 ± 0.052*†	1.077 ± 0.039*†	0.644a	-0.01 ± 0.06	
p-value	0.001b	< 0.001b	-	 	
SR 1	 	 	 	 	
T0	1.165 ± 0.072	1.132 ± 0.066	0.056a	-0.03 ± 0.09	
T1	1.195 ± 0.068	1.206 ± 0.037*	0.441a	0.01 ± 0.07	
T3	1.103 ± 0.069*†	1.088 ± 0.068*†	0.335a	-0.01 ± 0.08	
p-value	0.005b	< 0.001b	-	 	
Sagittal (internal)	
T0	1.106 ± 0.115	1.083 ± 0.114	0.350a	-0.02 ± 0.13	
T1	1.123 ± 0.114	1.128 ± 0.075	0.796a	0.01 ± 0.12	
T3	1.100 ± 0.114	1.108 ± 0.075	0.712a	0.01 ± 0.11	
p-value	0.105b	0.409b	-	 	
aWilcoxon test; bFriedman/Dunn test. *p < 0.05 vs. T0; †p < 0.05 vs. T1; PR 1: panoramic radiograph with a thickness of 1 mm; PR 5: panoramic radiograph with a thickness of 5 mm; PR 10: panoramic radiograph with a thickness of 10 mm.

Discussion

In the present split-mouth study, in which each patient served as both the experimental and control individuals, no significant differences were observed between the treatments in terms of degree of impaction and procedure time. These findings suggested a potential reduction in bias, thereby enhancing the precision of assessing the influence of L-PRF on alveolar healing after mandibular third molar surgery.

Despite the extensive body of existent(?) research, a lack of appropriate standardization and methodological design has been observed, contributing to conflicting outcomes and controversies within the literature. 16 Consequently, this investigation adhered to a methodological framework similar to that of studies conducted by Ritto et al., 9 Kapse et al., 17 and Ozgul et al. 18 These studies, classified in a systematic review with meta-analysis by Zhu et al. 19 as exhibiting a low risk of methodological bias, were used to guide the methodology of this study.

One of the most extensively scrutinized clinical parameters is pain, a highly subjective experience that varies from patient to patient. This underscores the significance of using a split-mouth clinical trial design. Whereas some researchers opt to use the number of analgesics consumed as an indicator of pain intensity, 20 the present study focused solely on the Visual Analog Scale (VAS) as the assessment tool. This choice was motivated because the VAS is the measurement most widely used and frequently referenced in the literature. 9,17,18

Relative to the timing of pain analysis, we conducted evaluations at two specific time intervals. First, a 12-hour post-surgery assessment was performed, as this time interval typically represents the peak of pain. In addition, a follow-up assessment occurred one week after surgery, as this time interval is commonly used to conclude postoperative evaluations for surgical procedures. We observed an improvement in pain levels among patients who received L-PRF at both evaluation time intervals. However, statistically significant improvement was only evident during the one-week follow-up. One possible explanation for this outcome could be that since tooth extractions occurred on the same day, patients may have encountered challenges when distinguishing and individually evaluating each surgical site immediately after the procedure. This perspective aligns with the findings presented by Ozgul et al., 18 which could be considered a limitation of our study.

When evaluating split-mouth studies, variations in outcomes have appeared Some investigations have pointed out the potential of L-PRF to mitigate postoperative pain. 17 Conversely, other studies have proposed that L-PRF might not significantly influence this outcome, 9,19 raising questions about its effectiveness. Similarly, while certain authors have reported that L-PRP could enhance soft tissue healing, others have found no significant distinctions. 9,17 In the present study, patients exhibited notable enhancements in both pain severity and soft tissue healing.

The periodontal condition of the distal surface of the second molar may have had a direct influence on the healing process in the region adjacent to and near the third molar. The application of L-PRF to fill the alveolus could promote effective healing by potentially reducing the infiltration of microorganisms, thus positively affecting the periodontal health of the mandibular second molar. This, in turn, would reduce the likelihood of periodontal pocket formation, resulting in a decrease in probing depth, as demonstrated in our study.

Analysis of prior research may contribute to understanding the mechanisms underlying the benefits of L-PRF in pain alleviation, by improving soft tissue healing, and improving periodontal diseases.

In post-extraction sockets, L-PRF has been demonstrated to enhance epithelialization and reduce postoperative discomfort, as demonstrated by higher levels of healing and decreased levels of pain and analgesic consumption reported in L-PRF-treated patients in comparison with controls. 21 This suggested that L-PRF may exert its effects by promoting tissue regeneration and modulation of pain perception.

According to a study evaluating the impact of L-PRF on soft tissue healing and its relationship with local growth factor and cytokine concentrations, tissue healing scores were higher, and there was less postoperative pain at L-PRF sites. This was correlated with increased levels of growth factors such as platelet-derived growth factors (PDGFs) and basic fibroblast growth factor (bFGF), which are known to play roles in tissue repair and regeneration. 22

The regenerative potential of L-PRF in periodontal therapy is related to its capacity to enhance both hard and soft tissue regeneration. A fibrin network formed by the gradual polymerization that occurs during PRF preparation promotes effective cell migration and proliferation, which enhances cicatrization. 9 The therapeutic benefits of L-PRF in treating periodontal hard and soft tissue abnormalities are probably due in part to this mechanism. Moreover, it was observed that the use of L-PRF increased bone density after extraction of the mandibular third tooth, suggesting that it had a favorable impact on bone healing. 23 This might be because the L-PRF matrix releases growth factors that could promote osteogenesis.

In terms of complications, only one single case of alveolar osteitis and one instance of postsurgical bleeding were observed, both occurring on the control side. Alveolar osteitis, though relatively infrequent, can generate significant discomfort for patients after the procedure. According to Zhu et al., 19 while L-PRF may not entirely prevent this complication, it might contribute to reducing its incidence. Relative to bleeding, the use of L-PRF tends to lower the risk of post-surgical bleeding due to the platelet aggregation facilitated by the higher platelet concentration. 17

This trial focused on L-PRF and its impact on clinical and imaging outcomes after third molar surgery. Conversely, some evidence has been shown to suggest that different types of PRF can have varying effects on pain and healing outcomes after third molar surgeries. A study comparing advanced PRF (A-PRF) and standard PRF (S-PRF) found that A-PRF resulted in significantly improved pain, swelling, and mouth opening outcomes in comparison with S-PRF. 24 This suggested that there may be differences between types of PRF in terms of their efficacy. Thus, the extent of these benefits and the differences between types of PRF require further investigation to fully understand their clinical implications.

In terms of cost-benefit, while the studies did not provide explicit economic analyses, the use of L-PRF is derived from the patient’s blood, which may reduce the costs associated with synthetic or donor materials and the risk of disease transmission. Furthermore, the simplicity of the technique suggested that it may not require significant additional resources or specialized equipment, potentially offering a cost-effective treatment option. 25 Despite these benefits, we believe that this intervention should not be generalized. It is important to thoroughly assess each patient’s medical history, overall health status, and specific surgical needs to determine whether L-PRF therapy would be appropriate for them. In the majority of cases, L-PRF can be a valuable adjunctive treatment in promoting wound healing, reducing inflammation, and enhancing tissue regeneration in oral surgical procedures as observed in the present trial. However, there is a lack of evidence-based practice regarding the use of L-PRF in patients with blood disorders.

Conclusion

In summary, the finding os this clinical trial’s emphasize the positive impact of L-PRF on various postoperative clinical parameters, including pain, soft tissue healing, and periodontal condition. These results suggest that L-PRF has the potential to enhance alveolar ridge preservation and expedite the initial healing process after mandibular third molar extraction. This means that L-PRF may be a useful alternative, with a cost-benefit ratio, in extraction cases in which there is a higher risk of postoperative morbidity and in other areas where implant rehabilitation is required in the future. The present study maintains strong internal validity due to its rigorous methodology, although its external validity is somewhat limited by specific sample characteristics. In view of the critical importance of this topic in the field of oral sciences, there is a compelling need for future well-designed trials to investigate the use of L-PRF in third molar surgeries across diverse populations. This type of research should prioritize patient-centered outcomes to provide more comprehensive insights into the clinical advantages of L-PRF.

Acknowledgments

The authors thank the Brazilian Society of Dental Research (SBPqO) for the opportunity to present this study at 40th Annual Meeting of the SBPqO, where it was awarded first place. Furthermore, this work was partially supported by the Brazilian National Council for Scientific and Technological Development (CNPq) which provided a PQ fellowship in category 2 to Dr. Fábio Costa (process number 315479/2021-3). Dr. Marcela Gurgel is supported by the Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico post-doctoral fellowship (process number FPD-0213-00294.01.00/23).
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References

1 Santos BF Costa FO Pinto AA Júnior Araújo AV Cyrino RM Cota LO Postoperative pain and edema control following different protocols of preemptive analgesia in the surgical removal of impacted third molars: a triple-blind parallel randomized placebo-controlled clinical trial J Craniomaxillofac Surg 2021 08 49 8 694 704 10.1016/j.jcms.2021.01.002 33994294
2 Tatullo M Marrelli M Cassetta M Pacifici A Stefanelli LV Scacco S et al Platelet Rich Fibrin (P.R.F.) in reconstructive surgery of atrophied maxillary bones: clinical and histological evaluations Int J Med Sci 2012 9 10 872 880 10.7150/ijms.5119 23155361
3 He Y Chen J Huang Y Pan Q Nie M Local application of platelet-rich fibrin during lower third molar extraction improves treatment outcomes J Oral Maxillofac Surg 2017 12 75 12 2497 2506 10.1016/j.joms.2017.05.034 28672135
4 Dohan Ehrenfest DM Rasmusson L Albrektsson T Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leucocyte- and platelet-rich fibrin (L-PRF) Trends Biotechnol 2009 03 27 3 158 167 10.1016/j.tibtech.2008.11.009 19187989
5 Dohan DM Choukroun J Diss A Dohan SL Dohan AJ Mouhyi J et al Platelet-rich fibrin (PRF): a second-generation platelet concentrate. Part I: technological concepts and evolution Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006 03 101 3 e37 e44 10.1016/j.tripleo.2005.07.008 16504849
6 Li ZB Qu HL Zhou LN Tian BM Chen FM Influence of non-impacted third molars on pathologies of adjacent second molars: a retrospective study J Periodontol 2017 05 88 5 450 456 10.1902/jop.2016.160453 27976596
7 Tian Y Sun L Qu H Yang Y Chen F Removal of nonimpacted third molars alters the periodontal condition of their neighbors clinically, immunologically, and microbiologically Int J Oral Sci 2021 02 13 1 5 10.1038/s41368-020-00108-y 33550328
8 Ramos EU Bizelli VF Pereira Baggio AM Ferriolli SC Silva Prado GA Farnezi Bassi AP Do the new protocols of platelet-rich fibrin centrifugation allow better control of postoperative complications and healing after surgery of impacted lower third molar? A systematic review and meta-analysis J Oral Maxillofac Surg 2022 07 80 7 1238 1253 10.1016/j.joms.2022.03.011 35439437
9 Ritto FG Pimentel T Canellas JV Junger B Cruz M Medeiros PJ Randomized double-blind clinical trial evaluation of bone healing after third molar surgery with the use of leukocyte- and platelet-rich fibrin Int J Oral Maxillofac Implants 2019 08 48 8 1088 1093 10.1016/j.ijom.2019.01.020 30910410
10 Schulz KF Altman DG Moher D CONSORT Group CONSORT 2010 statement: updated guidelines for reporting parallel group randomised trials PLoS Med 2010 03 7 3 e1000251 10.1371/journal.pmed.1000251 20352064
11 Carvalho RW Vasconcelos BC Pernambuco index: predictability of the complexity of surgery for impacted lower third molars Int J Oral Maxillofac Implants 2018 02 47 2 234 240 10.1016/j.ijom.2017.07.013
12 Ehrenfest DMD Del Corso M Diss A Mouhyi J Charrier JB Three-dimensional architecture and cell composition of a Choukroun's platelet-rich fibrin clot and membrane J Periodontol 2010 04 81 4 546 555 10.1902/jop.2009.090531 20373539
13 Lingamaneni S Mandadi LR Pathakota KR Assessment of healing following low-level laser irradiation after gingivectomy operations using a novel soft tissue healing index: a randomized, double-blind, split-mouth clinical pilot study J Indian Soc Periodontol 2019 23 1 53 57 10.4103/jisp.jisp_226_18 30692744
14 Varghese MP Manuel S Kumar L K S Potential for osseous regeneration of platelet-rich fibrina: a comparative study in mandibular third molar impaction sockets J Oral Maxillofac Surg 2017 07 75 7 1322 1329 10.1016/j.joms.2017.01.035 28249808
15 White SC Rudolph DJ Alterations of the trabecular pattern of the jaws in patients with osteoporosis Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999 11 88 5 628 635 10.1016/S1079-2104(99)70097-1 10556761
16 Baslarli O Tumer C Ugur O Vatankulu B Evaluation of osteoblastic activity in extraction sockets treated with platelet-rich fibrin Med Oral Patol Oral Cir Bucal 2015 01 20 1 e111 e116 10.4317/medoral.19999 25475771
17 Kapse S Surana S Satish M Hussain SE Vyas S Thakur D Autologous platelet-rich fibrin: can it secure a better healing? Oral Surg Oral Med Oral Pathol Oral Radiol 2019 01 127 1 8 18 10.1016/j.oooo.2018.08.010 30287202
18 Ozgul O Senses F Er N Tekin U Tuz HH Alkan A et al Efficacy of platelet rich fibrin in the reduction of the pain and swelling after impacted third molar surgery: randomized multicenter split-mouth clinical trial Head Face Med 2015 11 11 1 37 10.1186/s13005-015-0094-5
19 Zhu J Zhang S Yuan X He T Liu H Wang J et al Effect of platelet-rich fibrin on the control of alveolar osteitis, pain, trismus, soft tissue healing, and swelling following mandibular third molar surgery: an updated systematic review and meta-analysis Int J Oral Maxillofac Implants 2021 03 50 3 398 406 10.1016/j.ijom.2020.08.014
20 Medeiros-Albuquerque AF Roriz-Fonteles CS Nascimento-Costa JJ Viana-Silva JR Barros-Silva PG Studart-Soares EC et al RT-qPCR study of COX-1 and -2 genes in oral surgical model comparing single-dose preemptive ibuprofen and etoricoxib: a randomized clinical trialy J Clin Exp Dent 2020 04 12 4 e371 e380 10.4317/jced.56447 32382387
21 Castro AB Meschi N Temmerman A Pinto N Lambrechts P Teughels W et al Regenerative potential of leucocyte- and platelet-rich fibrin. Part A: intra-bony defects, furcation defects and periodontal plastic surgery: a systematic review and meta-analysis J Clin Periodontol 2017 01 44 1 67 82 10.1111/jcpe.12643 27783851
22 Daugela P Grimuta V Sakavicius D Jonaitis J Juodzbalys G Influence of leukocyte- and platelet-rich fibrin (L-PRF) on the outcomes of impacted mandibular third molar removal surgery: a split-mouth randomized clinical trial Quintessence Int 2018 49 5 377 388 10.3290/j.qi.a40113 29629438
23 Munoz F Jiménez C Espinoza D Vervelle A Beugnet J Haidar Z Use of leukocyte and platelet-rich fibrin (L-PRF) in periodontally accelerated osteogenic orthodontics (PAOO): clinical effects on edema and pain J Clin Exp Dent 2016 04 8 2 e119 e124 10.4317/jced.52760 27034749
24 Shruthi TM Shetty AD Akash KS Ahmed F Shetty N Singarapu R Evaluation of effects of platelet-rich fibrin on treatment outcomes after impacted mandibular third molar surgery: a randomized controlled clinical study Natl J Maxillofac Surg 2022 08 13 4 Suppl 1 S46 S51 10.4103/njms.NJMS_16_20 36393932
25 Eren G Atilla G Platelet-rich fibrin in the treatment of localized gingival recessions: a split-mouth randomized clinical trial Clin Oral Investig 2014
