Rom J Morphol Embryol 2014, 55(3):961–964

RJME
CASE REPORTS Romanian Journal of
Morphology & Embryology
http://www.rjme.ro/

Clinical and histopathological studies using fibrin-rich

plasma in the treatment of bisphosphonate-related
osteonecrosis of the jaw
OCTAVIAN DINCĂ1), SABINA ZURAC2), FLORICA STĂNICEANU2), MIHAI BOGDAN BUCUR3),
DANA CRISTINA BODNAR4), CRISTIAN VLĂDAN1), ALEXANDRU BUCUR1)
1)
“Prof. Dr. Dan Theodorescu” Clinical Hospital for Oro-Maxillo-Facial Surgery, Bucharest, Romania; Department of Oral and
Maxillofacial Surgery, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
2)
Department of Pathology, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania
3)
Private practice Dentimplant, Bucharest, Romania
4)
Department of Restorative Odontotherapy, Faculty of Dental Medicine, “Carol Davila” University of Medicine and Pharmacy,
Bucharest, Romania

Abstract
The authors report their experience using platelet-rich fibrin (PRF) therapy for the treatment of ten patients presenting bisphosphonate-
related osteonecrosis of the jaw (BRONJ). The aim of our study was to evaluate the effect of this therapy on recurrent BRONJ and to
describe the clinical and histopathological/immunohistochemical staining features of PRF treatment. As such, we describe the method we
used and report the results observed in the areas treated as well as side effects. The reported results recommend the safety and efficacy
of PRF in treatment of BRONJ.
Keywords: platelet-rich fibrin (PRF), bisphosphonate-related osteonecrosis of the jaw (BRONJ), maxillofacial.

 Introduction BRONJ was dental extraction in all cases. The diagnosis

was performed basing on clinical and radiographic features.
Bisphosphonate-related osteonecrosis of the jaw After the BRONJ diagnosis, all patients discontinued
(BRONJ) is a challenging complication resulting from the the use of bisphosphonate.
long-term therapy with bisphosphonates [1]. Currently, not All patients included in study were treated with i.v.
only the pathogenesis is still not complete understood, bisphosphonates for oncological reasons and meet the
but the management of BRONJ remains challenging following criteria: absence of diagnosed malignancies,
because there is no definitive treatment other than palliative lack of maxillofacial radiotherapy medical history and
methods [2]. Current recommendations contraindicate recurrent BRONJ after application of the protocol proposed
aggressive surgery because its results are unpredictable
by American Association of Oral and Maxillofacial
and may trigger disease progression [3]. The application
Surgeons (AAOMS): Chlorhexidine 0.12% mouth rinse,
of platelet-rich fibrin (PRF) in the surgical site may
local irrigation with Povidone-Iodine and oral antibiotics
constitute a new alternative method of BRONJ treatment
– Amoxicillin/Clavulanic Acid (4 g/day) for 10 days.
[4]. Dohan et al. (2006) developed the production protocol
Patients with immunologic diseases, diabetes mellitus,
of PRF attempts to accumulate platelets and released
low blood concentration of thrombocytes, chemotherapy
cytokines in a fibrin clot. The platelets and leukocyte
or radiotherapy were excluded.
cytokines are important part in role-play of this bio-
Presurgical standard blood analyses showed normal
material, but the fibrin matrix supporting them is very
variables, particularly platelet and leukocyte concentra-
helpful in constituting the determining elements responsible
tions. With the patient’s consent, treatment consisted in
for therapeutic potential of PRF [5].
superficial removal of bone sequestra/superficial curettage
The aims of our study were to describe the clinical
and Choukroun’s fibrin-rich plasma (PRF) therapy. A
and histopathological/immunohistological staining features
single team (the authors) performed all the surgeries and
and to evaluate the PRF treatment outcomes of patients
applied the PRF in these patients. Every patient was
with BRONJ.
operated with local anesthesia and conscious sedation.
PRF clots were prepared as described by Dohan et al.
 Patients, Methods and Results (2006) [5]. Blood was obtained several minutes before
We conducted a retrospective analysis of 10 consecu- starting surgery and prior to anesthesia administration.
tive patients who presented with bisphosphonate-related Whole blood was drawn into four to eight A-PRF tubes
osteonecrosis of the jaw (BRONJ) stage II (Marx classi- without anticoagulant and was immediately centrifuged at
fication), diagnosed and treated between August 2013 1300 rpm for 14 minutes using a centrifuge specifically
and February 2014. The precipitating event leading to designed for this application.

ISSN (print) 1220–0522 ISSN (on-line) 2066–8279

962 Octavian Dincă et al.
Post-surgical antibiotherapy was prescribed to all patients were examined by detecting the clinical signs of
patients with oral Clindamycin 0.9 g daily in divided BRONJ: pain, swelling, non-healing/exposed necrotic
doses, for 10 days. The sutures were removed 10 days bone, and/or fistulas with connection to the bone. The
postoperatively. maintenance of mucosal closure without clinical signs
Samples of PRF masses were fixed in 10% buffered of residual infection or exposed bone at 30 days of
formalin and routinely processed using Thermo Scientific evaluation was determined to indicate successful treatment.
STP 420D Tissue Processor. After embedding the tissue As the literature does not contraindicate this therapy
fragments, the resulting paraffin blocks were cut into 3 µm in BRONJ [6], no ethical problems were raised. The
thick sections with a semi-automated Rotary Microtome patients were informed about the aim and design of the
Leica RM2245. The slides were routinely stained with study, and written consent was obtained.
Hematoxylin–Eosin (HE); immunohistochemical stains Written informed consents were obtained from the
were performed for CD61 (Novocastra-Leica Biosystems, patients for publication of this case series and accom-
Newcastle Upon Tyne, U.K., 1:100, clone 2F2, heat panying images.
induced epitope retrieval pH 6). As detection system, we Ten consecutive patients (six women and four men),
used Novolink Polymer (Leica/Novocastra) and DAB mean age 59±15 years at the time of diagnosis had been
(3,3’-diaminobenzidine) chromogen. Slides were counter- treated with i.v. bisphophonates. After a BRONJ diagnosis,
stained with Mayer’s Hematoxylin, rehydrated and mounted drug holiday has been determined by the oncologist in
with glycerol gelatin. Immunohistochemical stains were all the cases. Of the 10 treated sites, three were located
analyzed using a microscope Nikon 80i. in the maxilla and seven in the mandible.
Monitoring of mucosal healing was carried out The main characteristics of the 10 consecutive patients
postoperatively at 3, 5 and 10 days and at 30 days. The belonging to the study are presented in Table 1.
Table 1 – A summary of findings for each case
Age Bisphosphonate Wound healing
No. Gender Medical condition Localization
[years] and dosage (30 days)
1. F 42 Zoledronic Acid breast cancer mandible healed
2. F 68 Zoledronic Acid breast cancer mandible healed
3. M 69 Zoledronic Acid prostate cancer maxilla healed
4. M 52 Ibandronate bowel cancer mandible healed
5. F 59 Zoledronic Acid breast cancer maxilla healed
6. F 58 Ibandronate kidney cancer mandible healed
7. M 63 Zoledronic Acid prostate cancer mandible healed
8. F 30 Ibandronate multiple myeloma mandible healed
9. M 79 Zoledronic Acid prostate cancer maxilla healed
10. F 75 Zoledronic Acid multiple myeloma mandible healed
F – Female; M – Male.

In all cases, we performed removal of the bone maxillofacial surgery has been implicated in different
sequestrations and curettage in the bone tissue until procedures such as socket preservation, sinus lift and bone
clear bleeding appeared from the subjacent bone. After augmentation, root coverage procedures, and healing in
that, bone cavities were filled with PRF clots. PRF donor site with good results [7].
membranes were used to protect the filled bone defect To date, no universally accepted management protocol
from muco-invagination. Mobilization of a mucoperi- is available to treat BRONJ, because the etiopathogenic
osteal flap was made to obtain a hermetic closure at the mechanism remains unclear [8]. The response to surgical
wound margins. No postoperative complications were debridement is unpredictable and there is a risk of
observed and all 10 patients were treated successfully. worsening bone exposure [9].
All the patients in our case series improved after PRF Bisphosphonate toxicity to epithelial cells has been
treatment, with mucosal healing. These patients continued well documented [10, 11]. Bisphosphonates inhibits cell
with follow-up visits, without evidence of exposed bone proliferation and the capacity for wound healing in
after 30 days (Figure 1). murine oral keratinocytes [12]. However, the reports of
HE staining was used for histological examination autologous PRF use for BRONJ treatment are limited [13].
by light microscopy. The results showed in all cases Therefore, what the benefit brought about by PRF should
necrotic lamellar bone fragments with acute and chronic be studied, which is the purpose of the present study.
inflammatory cells well bacterial colonies (Figure 2). PRF Additional biological effects of PRF may contribute to
clots consisted of a matrix of fibrin embedding numerous the improvement of clinical outcome in recurrent BRONJ.
platelets (Figure 3A). Platelets, CD61-positive, were To our knowledge, PRF works via the degranulation of the
diffusely distributed in PRF clot (Figure 3B). alpha granules in platelets, which contain the synthesized
and prepackaged growth factors [14]. The secreted growth
 Discussion factors bind to their transmembrane receptors on adult
mesenchymal stem cells, osteoblasts and endothelial cells
Some perspective is needed regarding what we are and then cause matrix formation and osteoid production
showing with our case series. Use of PRF in oral and trough cellular message transforming [15].
 Clinical and histopathological studies using fibrin-rich plasma in the treatment of bisphosphonate… 963

Figure 1 – (A) Initial clinical aspect of the BRONJ Figure 2 – Tissue specimen from patient No. 3 (of 10)
lesion (white arrow); (B) Surgical removal of bone with BRONJ, demonstrating a bone resorption lacuna
sequestration (white arrow); (C) PRF clot (white arrow) (black arrows) and rare multinucleated osteoclasts
placed between the bone tissue and the mucoperiosteal (white arrows). HE staining, ×100. Scale bar = 200 µm.
flap, to promote healing processes; (D) Clinical aspects
of the BRONJ lesion after surgical debridement and
application of PRF showing a mucosa with normal
aspect without signals of inflammatory process or bone
exposure (white arrow).

Figure 3 – (A) PRF clot – fibrin (white arrows) and platelets (black arrows). HE staining, ×400. Scale bar = 25 µm.
(B) Platelets (CD61-positive) aggregated areas of maximum positivity (maximal density of platelets) (black arrows)
within the fibrin network. CD61 immunostaining, ×1000. Scale bar = 20 μm.
Besides, PRF also contains three proteins in blood are heterogeneous in terms of localization of lesions and
known to act as cell adhesion molecules for osteo- age of patients. Further studies are necessary to assess
conduction and as a matrix for bone and connective tissue the long-term effectiveness of PRF, and a larger sample
[16]. PRF creates the conditions for the development of size is recommended.
microvascularization, guides epithelial cell migration to
its surface [17, 18] and facilitates cellular migration,  Conclusions
particularly for endothelial cells necessary for the neo-
Within the limits of this study, PRF led to favorable
angiogenesis [19]. Thus, PRF acts not just like a fibrin
clinical improvement in recurrent BRONJ. However,
matrix too accelerate the healing of wound [20], but also
the long-term outcome needs better assessments, and
by its high concentration of high amounts of growth further researches should address this issue.
factors such as TGBβ or PDGF (platelet-derived growth
factor) released by the platelets [19]. Author contribution
Although the sample size was small, results from the All authors have equally contributed to this study.
present investigation showed that PRF was effective in
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Corresponding author
Cristian Vlădan, Teaching assistant, MD, DDS, PhD, Department of Oral and Maxillofacial Surgery, “Carol Davila”
University of Medicine and Pharmacy, 17–21 Plevnei Avenue, 010221 Bucharest, Romania; Phone +4021–
314 41 31, Fax +4021–315 80 01, e-mail: [email protected]

Received: February 6, 2014

Accepted: August 26, 2014