PRF vs EMD periodontal regenerative surgery : a double blind, randomized controlled trial on the treatment of furcation II involved molars

Periodontitis is an inflammatory disease of the supporting tissues of the
teeth, namely connective tissue attachment and alveolar bone. Severe forms of
periodontitis result in the loss of these tissues and eventually loss of the
teeth. Active treatment of periodontitis includes the non-surgical and the
surgical therapy. Ultimate aim of the periodontal therapy is to remove the
infection and inflammation and, if possible, reconstruct or regenerate the lost
periodontal tissues in order to retain the teeth as long as possible. In
periodontitis, molar teeth run the highest risk of being lost in comparison to
other teeth, because of their multi-rooted anatomy (furcation) which makes
proper oral hygiene and professional maintenance difficult.

In molar teeth. the degree of furcation involvement, as a measure of
inter-radicular bone loss, was demonstrated to be a prognostic indicator for
the risk of future attachment and tooth loss.Several studies have demonstrated
that, in multi-rooted teeth, sites presenting furcation defects respond less
favourably to non-surgical therapy, compared to sites that do not have
furcation involvement, and have higher risk for tooth-loss (Salvi 2014,
HIrschfield and Wasserman 1978). Therefore, improving the furcation involvement
of a molar could mean improving the prognosis of the tooth, and, as a
consequence, improving the masticatory function and the quality of life of the
patient.

Several surgical procedures have been proposed to treat molar teeth with
furcation involvement. The most commonly used is the open flap debridement
(OFD). In comparison to other surgical therapies, as respective of regenerative
surgeries, molars treated with OFD showed a higher rate of re-treatment need
(Dannewitz 2016).The periodontal resective surgery consists in bone resection
and either tunnelling of the furcation area or root resection or root
amputation. Drawbacks of these techniques are the need for endodontic
treatment, the sacrifice of alveolar bone and tooth substance and, as a
consequence, the high risk of complications.

In this perspective, the ultimate aim of the periodontal therapy is to
re-establish the supporting tissues that have been lost due to the periodontal
inflammation. To this aim, The Guided Tissue Regeneration (GTR) has been
attempted during the past decades to recreate a new functional attachment
apparatus in molar furcation areas. The need to focus future research on
regeneration was underlined by a consensus report from the American Academy of
Periodontology in 2014.

In the past 20 years, the use of enamel matrix derivate (EMD) in combination
with periodontal surgery, demonstrated to increase the chance of achieving
(partial) periodontal regeneration compared to OFD and Resection techniques
(Casarin et al. 2010). From the study of Casarin, it can be concluded that EMD
therapy promoted a reduction in the number of proximal furcations presenting a
diagnosis of class II after 24 months of treatment, compared with OFD therapy.
For these reasons, EMD is commonly used in periodontal regeneration.
Nevertheless, EMD is a xenogenic material of animal origin (porcine) and this
aspect, together with the high cost of the product itself, still can bring
problems in terms of patient acceptance (some religions don*t allow the use of
animal material in human body; some patients prefers not having any animal
derived material because of ethical reasons and/or beliefs).

More recently, an autologous material has been developed; Platelet Rich Fibrin
(PRF). PRF has demonstrated to be beneficial in wound healing, due to the
potential of slow Growth Factors release and the dense fibrin network. These
characteristics seem to be favourable especially in the early phase of the
wound healing process. In vitro studies and some clinical trials have reported
promising results also in periodontal regeneration. Its advantage is the
autologous origin and substantial reduced direct material cost for the patient
(Dohan Ehrenfest 2006, 2009, 2010). Up to date, no studies have been published
which compare PRF with EMD This is of importance to confirm PRF as a suitable
alternative to EMD in the surgical treatment of molar teeth with periodontal
destruction in the furcation area.

Compare PRF and EMD regenerative potential in the surgical treatment of molar
teeth with periodontal destruction in the furcation area.
Evaluate induced Healing potential of the two materials.
Correlate healing pattern with biomarkers in blood and gingival crevicular
fluid.

In this double blind, randomized controlled trial, 3 different treatment
modalities will be tested on the patient cohort.
The selected sites will be randomly allocated to receive A-PRF+ + OFD, EMD +
OFD or OFD .


The screening will be carried out on the patient that received periodontal
reevaluation at ACTA-Periodontology department e months after IPT . All the
patients will already have full periodontal clinical measurements including
Full Mouth Plaque Scores, Full Mouth Bleeding Scores, Pocket Depth, Attachment
Level and full radiographic examination before IPT (initial periodontal
treatment) since this is the standard protocol at the department of
Periodontology.
After being screened and selected, patients will be contacted telephonically to
ask for participation in this research. Patients will receive an information
brochure about the content and the aim of the research. They will have 2 weeks
time to think if they will agree to participate in this project. After
agreement patients will be asked to sign an inform consent to bring back the
day of data collection.
Standardized (same conventional- film holder) Digital single solo Radiograph
will be taken, using exposure of 0,1 s.
At the moment of the surgical intervention,GCF preoperative collections will be
performed. Blood will be sampled preoperatively to start the PRF preparation
protocol. Local anaesthesia is administered and bone sounding will be carried
out. intraoperative measurements will be also made by the surgeon.
Postoperative GCF samples, together with Early Wound Healing Assessment, will
also administer the postoperative Questionnaire to each patient.
Postoperative measurements and evaluations will be made at 6 months.

PRF preparation

The A-PRF+ will be prepared according the recently introduced spinning
protocol. Sterile plain glass-based vacuum tubes (A-PRF+ 10 mL tube) are used,
and the centrifugation protocol includes 1300 rpm for 8 minutes (Ghanaati et
al. 2014). After centrifugation, in the tubes is possible to retrieve 3 layers.
From bottom to the top, these are red blood cells and plasma, fibrin clot,
serum. In order to be removed, the PRF clot is gently taken from the tube and
separated from the red element phase at the base with pliers. The PRF clot is
then adapted in the proper PRF box (APRF, Nice, France), that allows a constant
compression due to the lid (cover of the PRF box) on top of the clot. The
compression lasts 5 minutes, after which it is possible to retrieve PRF
membranes, equal in size and thickness. The test material from the OFD + EMD
and OFD groups will be discarded according to the ACTA protocol for the
disposal of biohazard materials. The tubes (only component non-sterile) are
placed after centrifugation in a specific metal rack to keep them stable. At
that point, the top of the tube is removed with not sterile gloves, and the
fibrin clot is removed from the tube using sterile tweezers and sterile gloves.
The all procedure is performed in the surgical room, with sterile instruments
and in sterile setting. We highlight once more that PRF is an autologous
material and each clot is obtained for immediate use and use in the same
patient (only the OFD + PRF).



Surgical procedure

All surgical procedures are following standard principles and are performed by
one experienced clinician who is blinded for the treatment allocation until the
moment of placement of the material. Intraoral antisepsis will be performed
with 0,12 % Chlorhexidin rinse for 1 minute, and perioral tissues will be
disinfected with Chlorhexidin 0.12% as well. Local anesthesia on the buccal and
lingual aspects will be performed with xylocaine 1:80.000.
A microsurgical approach will be performed with modified or simplified papilla
preservation technique depending on the width of the inter-dental space
(Cortellini et al. 2001). If deep pockets are present in the distal side
(retromolar area), the access will be performed with a trap technique, leaving
as much tissue as possible in the middle of the ridge. This will allow a better
coverage and stabilization of the gingival flap. Full-thickness flaps will be
raised around the defects. The elevation of the flap will be performed in the
most atraumatic way possible, leaving interdental tissues intact. In addition,
the flaps will be extended apically to the defects and furcation entrances, to
ensure that the barrier at placement will rest on bone along its entire
peri-defect extension. Releasing incisions will be performed on the mid-buccal
and mid-lingual side of the most mesial tooth involved in the flap.
Degranulation will be thoroughly made with curettes, including removal of
granulation tissue from the furcation involved areas. After having access to
the bone anatomy, ultrasonic debridement of roots will be performed with saline
cooling, together with hand instruments. Clinical bone measurement will be at
this moment performed, including horizontal and vertical bone in the furcation
areas and, if present, measurements of infrabony defects (Tonetti et al. 2002).
No bone corrections will be performed in any case. If enamel projections or
enamel pearls are visible, they will be corrected and smoothened with diamond
coated burs, until reaching the dentin below.
At this point, the person in charge for the randomization will open a
randomized assigned envelope containing the treatment allocation and the
surgeon will be informed. about which of the three approaches will be used
(A-PRF or EMD or OFD only). If the patient is allocated to OFD alone group, the
area will be rinsed with physiological solution (NaCl 0,9%), followed by a
pause of 1 minute, and later copiously irrigated once again with the same
solution. This will resemble the application of regenerative materials. At the
end of the sham procedure, sutures are directly applied. In case of PRF
allocation, sterile prepared PRF membranes will be minced by sterile scissors
and compressed, as much as possible, into the furcation II defect of the
molars involved in the surgery. In order to guarantee clot stabilization, an
additional PRF membrane will be applied covering the graft material and
protecting the furcation entrance around the tooth profile. The eventual
application of the PRF will happen in less than 1 hour from the beginning of
the surgery. In case of EMD, the manufacturer protocol will be applied,
consisting of EDTA root conditioning for 1 min, rinsing with saline, and
immediately after by EMD application for 1 min and suturing. The papilla at
this point will be repositioned in the original buccal position. Horizontal 5-0
polypropylene tension-free sutures will be applied with the intention to
achieve primary closure. An additional horizontal external mattress suture with
a laurel loop is performed. This will allow the tissues to stay in a coronal
position. The duration of surgical session will be recorded for each procedure,
and it*s expected to be around 90 minutes.


Postoperative instructions

Every patient will be asked to avoid any form of brushing in the operated area
and to rinse first with hydrogen peroxide for 2 mins and later with
Chlorhexidine 0,12 % (PerioAid 0.12%, Dentaid, Barcelona, Spain) for 1 min
twice per day. These instructions will be continued for 4 weeks.
Painkillers use (Paracetamol 500 mg, max 6g per day) will be advised if
necessary and the patient will be requested to report the consumption of the
medication in the provided diary/ questionnaires.
Sutures will be removed after 14 days. A prophylaxis protocol via gentle
polishing with rubber cups and brushes will be performed on day 3, day 7, day
14, week 6 and month 3 after surgery.

This study also investigates whether there is a possible correlation between
the general blood count (numbers of white blood cells), a number of biomarkers
in the local gingival crevicular fluid (GCF) and early wound healing. The
hypothesis here is that the application of PRF or EMD positively influences
local wound healing.
 

Nature and extent of the burden and risks for patients due to their
participation:
Benefits for patients: the surgical procedures are standard periodontal
interventions to treat periodontal defects in molars with furcation problems.
Degranulation, cleaning of the affected root surfaces etc are standard. The
results of standard treatments for molars often result in deep recessions
(withdrawn gums) and it is difficult for patients to clean between the roots in
the new postoperative situation. So new methods to regenerate the affected bone
between the roots (furcation defects) and reduce the furcation defects from
class 2 to a class 1 are an important step forward. In addition, it is
important to avoid artificial or animal materials by means of autologous
regeneration.
The time for the procedure is standard 90 min, the application of the
experimental approaches (PRF or EMD) does not give an extension of the
estimated surgical time. Application of Enamel Matrix Proteins (EMD) is a
procedure that has been used in periodontal surgery for more than 20 years and
there are no reported risks. In the current study design, the application
applies EMD as a "positive control" for regeneration of the furcation defect.
The blood collection (venipuncture in the antecubital fossa), is a very low
risk for the patients; the sampling of the GCF is not invasive and does not
pose a risk for the patient: this procedure is generally accepted and executed
as a routine.
An extra effort is required from the patient by completing a questionnaire in
the form of a VAS scale every day during the first week after the procedure.
WIll be requested at day 14, 42 and 90. Filling out the questionnaire is
estimated to require a 5 minutes time (total 10 x 5 min = 50 min).
The assessment of wound healing on day 3 is the only extra appointment that is
not included in the regular protocol of the periodontology section at ACTA for
the surgical treatment of molars with furcation disorders. This extra visit
takes 20 minutes, but is essential to assess the early wound healing. All other
agreements are part of the regular clinical protocol for periodontal
regenerative surgery.
The extra time per patient is in total 135 min, divided as follows:
Questionnaire completion: total 10 x 5 min = 50 min.
Evaluation of wound healing : total 5 x 5 min = 25 min
Sampling GCF: total baseline plus 5 x 10 min = 60 min