Effects of Different Centrifuged Platelet Concentrates on Bone Remodelling Around Dental Implants
Condition: Edentulous Jaw; Bone Loss, Alveolar; Resorption, Bone
MeSH terms: Jaw, Edentulous; Alveolar Bone Loss; Bone Resorption
Study type: Interventional

Summary: 
      Platelet concentrates obtained from blood have been used as regenerative biomaterials in
      periodontal surgery. Along with the migration and proliferation of osteogenic cells,
      platelets accelerate bone regeneration by increasing the formation of blood vessels and
      inducing inflammatory reactions. Experimental studies revealed that growth factors released
      from platelets enhance osteoblastic differentiation on the implant surface, and enlarge the
      contact surface of the bone and implant. Platelet-rich fibrin (PRF), a platelet concentrate,
      was introduced by Choukroun in 2001, and it contains a significant amount of cytokines.
      Advanced-platelet rich fibrin (A-PRF), discovered in 2014, is a PRF derivative with a denser
      leukocyte concentration and a softer consistency. Concentrated growth factor (CGF), another
      platelet derivative, differs from A-PRF since it contains many concentrated growth factors
      trapped in a more rigid fibrin structure. It was reported that both A-PRF and CGF, obtained
      with variable centrifuge speeds, accelerated the proliferation and differentiation of bone
      cells.

      Stimulated osteoblasts and osteocytes initiate the remodelling process by producing
      macrophage colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand
      (RANKL).Previous studies reported that TNF-α initiated bone resorption independently of
      RANKL.Osteoprotegerin (OPG) is a soluble cytokine receptor of the TNF family and is produced
      by osteoblasts, fibroblasts, and a number of host cells. OPG binds to RANKL and prevents the
      RANKL-RANK interaction Therefore, it inhibits osteoclastic activity. The RANKL/OPG ratio is
      used as an indicator for estimating bone remodelling, osteoclastic activity, or osteogenesis.

      The interactions among cytokines, growth factors, chemokines, and chemical mediators during
      blood clot formation result in a complex signalling process. High concentrations of cytokines
      and growth factors in the wound promote the migration of macrophages, neutrophils, and
      lymphocytes. Therefore, it was reported that the cytokines released from the fibrin matrix
      might affect those signaling pathways. In this study, investigators hypothesised that the
      application of CGF or A-PRF in dental implantation would contribute to inflammation,
      proliferation and the remodeling process. Therefore, the aim of this study was to investigate
      the effects of CGF and A-PRF on the osseointegration of dental implants in clinical,
      radiographic, and biochemical aspects.
    

Detailed description: 
      This study was conducted in Periodontology clinics. The study protocol was approved by the
      Hatay Mustafa Kemal University Human Ethics Committee before the initiation of the study
      (approval number:). The patients who met the inclusion criteria and who volunteered to
      participate in the study were given detailed information about the study and provided
      informed consent before the procedure.

      Patient selection:

      The inclusion criteria of the individuals found to be healthy from their medical histories
      and who had received clinical examination and had radiological imaging were as follows:

        -  Above 18 years of age

        -  Symmetrical edentulous areas in the mandible

        -  Sufficient bone width and height permitting ideal dental implant placement

        -  Individuals who do not smoke

        -  At least 2 mm of keratinized gingiva width and 3 mm soft tissue thickness at the implant
           site

      Individuals meeting any of the criteria listed below were excluded from the study:

        -  Patients with systemic disorders, including diabetes mellitus, metabolic bone disease,
           rheumatoid arthritis, mucocutaneous disorders, immunological disorders, hepatitis, or
           HIV

        -  Individuals who were on antibiotics, anti-inflammatory agents, corticosteroids,
           immunosuppressants, anticoagulants, or hormonal contraceptives for any reason, at least
           6 months prior to the procedure, and those who were on bisphosphonates at that time or
           previously.

        -  Individuals with any pathology or defects at the implant site.

        -  History augmentation at the implant site.

        -  Individuals with severe periodontal disease and poor oral hygiene.

        -  Severe caries or endodontic lesions in teeth adjacent to the implant.

        -  Those who use intraoral orthodontic or prosthetic appliances that make plaque control
           difficult.

        -  Pregnant and lactating individuals. Randomization and Blinding Both the study groups and
           the surgical site were determined by drawing a closed envelope immediately before
           surgery. Clinical and radiological records of the study were obtained by an experienced
           periodontist who was unaware of the allocation

      Surgical Procedure

      Infiltrative anaesthesia was provided with an anaesthetic solution containing 1:100,000
      epinephrine. A mid-crestal incision was made using a 15c scalpel, and a full-thickness flap
      was elevated. The drilling procedure was performed (600 rpm and 25 Ncm) according to the
      implant diameter and the need for an edentulous area in the mandible. A countersink drill was
      used for all implants to minimise the crestal bone stress. In case of the presence of a
      natural tooth adjacent to the implant site, a distance of at least 1.5 mm between the tooth
      and the implant cavity was left, and the presence of at least 1 mm bone plates in the buccal
      and lingual sides of the implant. CGF or A-PRF autologous obtained from centrifuged blood was
      placed in a PRF box. In this way, the liquid rich in growth factor and platelets leaking from
      the fibrin network was allowed to flow into the box. The liquid was collected with a syringe
      and applied over the implant surface until no dry area was left. In addition, the implant
      site was filled with this liquid, and 0.5 mm subcrestal placement of the implant was provided
      at 40 rpm and 25 Ncm torque. The membrane obtained from the CGF or A-PRF was placed over the
      implant, enough to cover the crestal bone, and a healing cap was placed. Implant placement
      without CGF or A-PRF was performed on the opposite side of the jaw in the control group. The
      stability of the implant was determined using resonance frequency analysis. The surgical site
      was sutured using a 5/0 silk. All patients were advised not to take any medications, unless
      necessary. The sutures were removed at the 10 days after surgery. All surgeries were
      performed by a physician with five years of experience Preparation Procedure of CGF and A-PRF
      Before starting the implant surgery, 9 mL of blood was withdrawn from the cubital vein of the
      patient's forearm, transferred into a tube without anticoagulant, and centrifuged. CGF was
      obtained using an automatically adjusted centrifuge device, fired at alternating and
      controlled speeds (2 min at 2700 rpm, 4 min at 2400 rpm, 4 min at 2700 rpm, and 3 min at 3000
      rpm) (MEDIFUGE, Silfradentsrl, S. Sofia, Italy). In the A-PRF group, blood was centrifuged at
      1400 rpm for 14 min (Duo Centrifuge Process for PRF, France). Centrifugation resulted in
      three layers in the tube: a red blood cell layer at the bottom, a platelet-poor serum layer
      at the top, and a fibrin gel layer containing growth factors and platelets in the middle. The
      serum in the upper layer was removed with a sterile syringe, and the fibrin layer was removed
      from the tube using a clamp. The red blood cells in the lower layer were removed from the
      fibrin structure using a scissors.

      Clinical Measurements and Resonance Frequency Analysis Four sites of each healing cap,
      mesial, distal, buccal, and lingual surfaces, were analysed for plaque index (PI), gingival
      index (GI), pocket depth (PD), and gingival bleeding index (GBI). All clinical measurements
      were repeated at 2, 4, and 12 weeks after the procedure. Resonance frequency analysis was
      performed intraoperatively and postoperatively at the fourth and twelfth month.

      Measurement of Marginal Bone Levels:

      Calibrated panoramic radiographs obtained on the day of surgery and 3 months after were used
      to determine the marginal bone level. The implant-fixture platform was considered as the
      threshold for measuring changes in the marginal bone level. When the marginal bone around the
      dental implant decreased below the platform, two horizontal lines were drawn on the
      radiograph: the first plane was located on the platform of the implant fixture, and the
      second was on the alveolar crest or at the bottom of the bone defect. The average of the
      marginal bone loss was obtained by measuring the distance between the two points left by the
      lines drawn from the mesial and distal surfaces of the cylindrical part of the implant on the
      horizontal lines. All radiological measurements were performed by two examiners experienced
      in their fields.

      Collection of Peri-implant Crevicular Fluid (PICF):

      First, the implant site was isolated from saliva and dried with air. An absorbent filter
      paper strip was placed 1 mm into the pocket in the mesial and distal surfaces of the healing
      cap, and was left there for 30 s to allow absorption of a sufficient amount of peri-implant
      crevicular fluid. The volume of the fluid was measured using a calibrated electronic device
      (Periotron 8010, Oraflow, Amityville, NY, USA). Paper strips contaminated with blood or
      plaque were excluded from the study. The paper strips were transferred into an empty
      Eppendorf tube covered with paraffin wax and stored at 80 °C until analysis.

      Biochemical Analysis:

      Initially, fresh phosphate buffered saline (PBS) was prepared saline (pH: 7.00 137 mM NaCl,
      10 mM Na2HPO4, and 2.7 mM KCl]. PBS of 300 µL) was added to each Eppendorf tube with two
      paper strips inside, left at room temperature for 30 min, and then centrifuged at 12,000 × g
      at 4°C for 15 min. Following centrifugation, the strips in the Eppendorf tube were removed,
      and the supernatant obtained was used for ELISA. Tumour necrosis factor alpha (TNF-α)
      (Boster), sRANKL (Soluble Receptor Activator of Nuclear Factor-Kb Ligand) (Elabscience) and
      Osteoprotegerin (OPG) (Boster) levels were measured by ELISA at 450 nm wavelength using
      commercial kits and Thermo Fisher Scientific Multiscan Go- Finland ELISA Reader. All analyses
      were performed in accordance with the manufacturer's instructions.

      Statistical Analysis:

      The data were analysed at 95% confidence using the SPSS 21 software. Continuous variables
      were expressed as mean, standard deviation, median, minimum, and maximum. Categorical
      variables are expressed as frequencies and percentages. The normality of the distribution of
      the data was analysed using the Shapiro-Wilk test, and Mann Whitney U and Student t tests
      were used to compare independent groups. The Wilcoxon signed rank test and Friedman test were
      used to compare dependent variables. The comparisons of two measurement points were analysed
      using the Wilcoxon test with Bonferroni correction (p <0.016) if a significant difference was
      determined using the Friedman test. The p value was determined as 0.05, for all analyses.
    

Intervention name(s): Application of concentrated growth factor with dental implants; Application of advanced platelet rich fibrin with dental implants; Control side of concentrated growth factor study group; Control side of advanced platelet rich fibrin study group
Intervention type(s): Procedure; Procedure; Procedure; Procedure
Intervention description(s): Concentrated growth factor(CGF) liquid applied to the implant cavity also CGF membrane covered socket and implant; Advanced platelet rich fibrin (A-PRF) liquid applied to the implant cavity also A-PRF membrane covered socket and implant; Traditional implant methods applied; Traditional implant methods applied
Intervention MeSH terms: Mitogens
Allocation: Randomized
Intervention model: Parallel Assignment
Intervention model description: This is a split-mouth design study. Accordingly, our study was conducted on 40 patients, including 20 patients in the CGF(Concentrated Growth Factor) study group and 20 patients in the A-PRF(Advanced Platelet Rich Fibrin) study group. While the implant combined with any regenerative material (CGF or A-PRF) was applied to the test site randomly assigned in the mouth, dental implants were placed according to traditional methods without applying any additional material to the contralateral site. Implants were placed symmetrically in the region of incisors, premolars or molar teeth in the mandible.
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Treatment
Keywords: Concentrated Growth Factor; Advanced Platelet Rich Fibrin; Inflammatory Cytokines; Implant Osseointegration; Peri-implant Crevicular Fluid