Leukocyte- and Platelet Rich Fibrin (L-PRF) Application in Impacted Mandibular Third Molar Removal Surgery
Leukocyte- and Platelet Rich Fibrin (L-PRF) Application in Impacted Mandibular Third Molar Removal Surgery
The purpose of this study was to evaluate the influence of leukocyte- and platelet-rich
 fibrin (L-PRF) on impacted mandibular third molar (IMTM) extraction wound healing, patient
 postoperative discomfort, and incidence of alveolar osteitis (AO).

 Thirty patients (18 female, 12 male) who met the inclusion criteria for this split-mouth
 study underwent bilateral IMTM surgical extractions. Following extraction, randomization was
 done. One socket received L-PRF, and the other socket served as a regular blood clot control.
 Postoperatively, the soft tissue healing index (HI), pain according to visual analog scale
 (VAS), facial swelling using a horizontal and vertical guide, and incidence of AO were
 evaluated 1, 3, 7, and 14 days after surgery.
The study was designed as a prospective split-mouth clinical trial on patients who needed
 bilateral paired IMTM extractions. The study was conducted at the Maxillofacial Surgery
 Department of the Lithuanian University of Health Sciences. Approval by the local bioethics
 committee was granted (No. BEC-MF-01). All patients signed an informed consent.

 Patient selection Based on previously treated pilot cases, a power analysis was conducted for
 sample size estimation (Statistica 6.0, Dell Software, Round Rock, TX, USA). The calculations
 revealed a minimum sample size of n = 30, based on a power of 90% and an α of 5%.

 The inclusion criteria were as follows:

 1. Male and female patients with an age of 18 to 60 years;

 2. Healthy patients (American Society of Anesthesiologists Physical Status (ASA PS) index ≤
 2);

 3. Sole bilateral extractions of IMTMs during the same surgery;

 4. Complete root formation of tooth No. 38 and 48 at radiological examination;

 5. Absence of acute inflammation and/or infection in the IMTMs area;

 6. No history of nonsteroidal anti-inflammatory drug (NSAID) use in the past 4 weeks prior
 to surgery;

 7. Same-difficulty bilateral IMTM extractions, according to both Pederson and Juodzbalys &
 Daugela classifications;

 8. Signed informed consent.

 The following exclusion criteria were used:

 1. Smoking patients;

 2. IMTM surgery duration difference greater than 10 minutes for each side, seeking to avoid
 bias;

 3. Different surgical manipulations taken at each IMTM surgery site;

 4. Presence of any neoplastic lesion (benign or malignant), clinically or radiologically
 evaluable, at the site or close to the impacted tooth;

 5. Presence of any radiolucent lesion >1 cm at impacted tooth level;

 6. Absence of the adjacent tooth;

 7. Systemic conditions or pharmacological treatments altering oral microbiota or
 immunologic response;

 8. Alcohol or drug abuse;

 9. Pregnant or breastfeeding women;

 10. Lack of compliance by the patient or any other evidence suggesting that the patient was
 not likely to follow the study protocol.

 Preoperatively, two blind expert surgeons assessed patient Orthopantomograph (OPG) and/or
 cone beam computed tomography (CBCT), evaluating IMTM extraction difficulty and complexity
 according to Pederson and Juodzbalys & Daugela indexes for each IMTM bilaterally. The overall
 kappa index for inter-observers agreement was calculated by using weighted kappa. Only teeth
 having the same extraction difficulty scores according to both classifications were enrolled
 in the study.

 Pederson difficulty index assessment

 Pederson difficulty index was evaluated based on anatomical and radiographic features of the
 tooth to be extracted. Following scores were given according to OPG and/or CBCT findings:

 Spatial relationship

 -  Mesio angular 1

 -  Horizontal 2

 -  Vertical 3

 -  Distoangular 4

 Depth

 -  Level A 1

 -  Level B 2

 -  Level C 3

 Ramus relationship / space available

 -  Class I 1

 -  Class II 2

 -  Class III 3

 According to the difficulty score total score, the IMTMs were classified as:

 Minimally difficult (score 3 to 4) Moderately difficult (score 5 to 7) Very difficult (score
 7 to 10).

 Juodzbalys & Daugela classification Modification of the Juodzbalys & Daugela classification
 is related with the different interpretation of the final score, i.e. rating of surgical
 difficulty and the surgical time. Indexes and their evaluation remain unaltered. The impacted
 mandibular third molar is evaluated by considering its relationships with adjacent anatomical
 boundaries (second molar, mandibular ramus, alveolar crest, mandibular canal, corticals of
 the mandible) and its spatial position.

 The classification attributes a score from ranging from 0 to 3 (0=conventional; 1=simple;
 2=moderate; 3=complicated) to six items (M, R, A, C, B, S), according to the tooth
 positioning.

 -  in relation to the second molar (M): the wisdom tooth crown could be directed at, or
 above the equator of the second molar (conventional, score 0), directed below the
 equator, to the coronal third of the second molar root (simple, score 1), directed to
 the middle third of the second molar root (moderate, score 2), directed to the apical
 third of the second molar root (complicated, score 3)

 -  in relation to the mandibular ramus (R): the tooth could have sufficient space in the
 dental arch (conventional, score 0), could be partially impacted in the ramus (simple,
 score 1), completely impacted in the ramus (moderate, score 2), or completely impacted
 in the ramus in disto-angular or horizontal position (complicated, score 3).

 -  considering the apico-coronal position of the tooth in relationship with the alveolar
 crest (A): the tooth could be completely erupted (conventional, score 0), partially
 impacted but with the widest part of the crown (equator) above the bone (simple, score
 1), partially impacted but the widest part of the crown below the bone (moderate, score
 2), or completely encased into the bone (complicated, score 3).

 -  tooth relation with the mandibular canal is expressed as follows (C): ≥ 3 mm from the
 mandibular canal (conventional, score 0), contacting or penetrating the mandibular
 canal, with the wall of the mandibular canal which can be radiographically identified
 (simple, score 1), contacting or penetrating the mandibular canal, with the wall of the
 mandibular canal not radiographically identifiable (moderate, score 2), roots
 surrounding the mandibular canal (complicated, score 3).

 -  bucco-lingual position of the tooth in relation to mandibular lingual and buccal walls
 (B): closer to buccal wall (conventional, score 0), in the middle between lingual and
 buccal walls (simple, score 1), closer to lingual wall (moderate, score 2), closer to
 lingual wall, when the tooth is partially impacted or completely encased in the bone
 (complicated, score 3).

 -  the spatial tooth positioning (S) is defined as follows: vertical (conventional, score
 0), mesio-angular at ≤ 60◦ (simple, score 1), disto-angular at ≥ 120◦ (moderate, score
 2), horizontal or inverted (complicated, score 3).

 L-PRF preparation Prior to surgery, two 9 ml glass-coated tubes (Intra-Spin, Intra-Lock
 International, Boca Raton, USA) of patient venous blood were collected. Tubes were
 transferred to a centrifuge (Andreas Hettich GmbH & Co.KG, Tuttlingen, Germany) and processed
 for 12 minutes at 2800 rpm to prepare L-PRF, as described previously.

 Surgical procedure Surgical procedures were performed following a standardized surgical
 approach by one expert surgeon (PD). Surgical manipulations required for each tooth
 extraction were as follows: simple mobilization of the tooth, coronectomy, root separation,
 incision, osteotomy. After removal of IMTM and socket curettage, on the side randomly chosen
 by the opaque sealed envelope technique to be the study side, the extraction sockets were
 filled with two L-PRF clots, whereas on the other side (control), they were allowed to form a
 natural blood clot and undergo natural healing. Both sides were sutured with absorbable
 Polyglactin (Atramat, Internacional Farmacéutica S.A. de C.V., Mexico City, Mexico)
 interrupted 5-0 sutures. Patients were blinded to the study or control side selection.

 Duration of the surgical procedure was counted from the beginning of surgical manipulations
 to complete removal of mandibular wisdom tooth and wound closure for each side separately. If
 there was a time difference greater than 10 minutes or if a different surgical manipulation
 was used between each bilateral extraction, the patient was excluded from the study.

 Each patient was given systemic antibiotic prophylaxis (Clindamycin 600 mg per os) 1 hour
 before and 6 hours after surgery. Postoperatively oral Lornoxicam 8 mg was prescribed to use
 for pain control when needed. Patients were instructed to rinse with chlorhexidine 0.12%
 solution three times a day for 2 weeks and provided with both verbal and written
 postoperative instructions. The sutures were removed one week after surgery.

 Postoperative outcome evaluation Quality of soft tissue healing, incidence of AO, pain, and
 facial swelling were selected as postoperative outcome measures.

 Postextractional wound soft tissue healing evaluation Postextractional wound soft tissue
 healing was evaluated using a modified postextraction wound healing index (HI), which
 considered four parameters: bleeding, suppuration, tissue color, and consistency of the
 healing tissue. Each parameter involved 3 scoring levels; consequently, the cumulative
 scoring scale ranged from 4, corresponding to excellent healing, to 12, indicating severely
 impaired healing. HI was evaluated clinically by the same blinded examiner at the 1st, 3rd,
 7th, and 14th days after surgery.

 Alveolar osteitis evaluation Alveolar osteitis was characterized as postoperative continuous
 throbbing pain surrounding the alveolus that was not adequately relieved by analgesics and
 that increased in severity during a period of 1-3 days after tooth extraction. The pain was
 followed by partial or complete loss of the initial blood clot in the interior of the socket
 with or without halitosis.

 Pain evaluation Pain was assessed using a written questionnaire with a visual analog scale
 (VAS) every day by the patient during the first postoperative week. VAS consisted of 10 units
 in combination with a graphic rating scale, where the leftmost score 0 represented absence of
 pain and the rightmost score 10 indicated the worst possible, unbearable, excruciating pain.
 Patients were blind to the knowledge of L-PRF and control sides and were asked to evaluate a
 VAS pain score for each operated side every day separately for the first week after surgery.

 Facial swelling clinical assessment

 Facial swelling clinical assessments were performed by a single blinded examiner at baseline
 (before surgery) and at the 1st-, 3rd-, and 7th-day visits postoperatively at approximately
 the same time of day. Patients were seated in a relaxed position with the inferior border of
 the mandible parallel to the floor. Facial swelling in the operation side was evaluated using
 two facial measurements with nonexpandable tape:

 -  In the vertical dimension, measurement from the lateral canthus of the eye to the
 pogonion of the mandible was taken;

 -  In the horizontal dimension, the distance from the lower border of the tragus to the
 mouth commissure on both operated sides was measured.

 Facial swelling value was taken as the average value of these two measurements. The
 preoperative measurement was considered as the baseline value.
Inclusion Criteria:

 1. Male and female patients with an age of 18 to 60 years;

 2. Healthy patients (American Society of Anesthesiologists Physical Status (ASA PS) index
 ≤ 2);

 3. Sole bilateral extractions of IMTMs during the same surgery;

 4. Complete root formation of tooth No. 38 and 48 at radiological examination;

 5. Absence of acute inflammation and/or infection in the IMTMs area;

 6. No history of nonsteroidal anti-inflammatory drug (NSAID) use in the past 4 weeks
 prior to surgery;

 7. Same-difficulty bilateral IMTM extractions, according to both Pederson and Juodzbalys
 & Daugela classifications;

 8. Signed informed consent.

 Exclusion Criteria:

 1. Smoking patients;

 2. IMTM surgery duration difference greater than 10 minutes for each side, seeking to
 avoid bias;

 3. Different surgical manipulations taken at each IMTM surgery site;

 4. Presence of any neoplastic lesion (benign or malignant), clinically or radiologically
 evaluable, at the site or close to the impacted tooth;

 5. Presence of any radiolucent lesion >1 cm at impacted tooth level;

 6. Absence of the adjacent tooth;

 7. Systemic conditions or pharmacological treatments altering oral microbiota or
 immunologic response;

 8. Alcohol or drug abuse;

 9. Pregnant or breastfeeding women;

 10. Lack of compliance by the patient or any other evidence suggesting that the patient
 was not likely to follow the study protocol.