| Abstract|| |
Context: The platelet rich fibrin (PRF) is second generation platelet concentrate that has been widely used and researched for stimulation and acceleration of soft tissue and osseous healing. Its continuous delivery of growth factors and proteins mimic the need of physiological wound healing and regenerative tissue processes.
Aims and Objectives: The aim of this study was to evaluate the efficacy of PRF in osseous regeneration after enucleation of cystic lesions. The objectives of this study were: (1) To evaluate osseous regeneration radiographically with the use of PRF in intrabony defects after cystic enucleation. (2) To evaluate the degree of bone density in intrabony defects with the use of PRF postoperatively after 1 st , 3 rd , and 6 th months.
Subjects and Methods: 10 cases of cystic lesions were treated using PRF after cystic enucleation. Follow-up radiographs (orthopantomogram) were taken 1 st , 3 rd , and 6 th months postoperatively. Bone density was measured with grayscale histogram using Adobe Photoshop 7.0 software.
Results: The subsequent follow-up examinations revealed progressive, predictable, and significant radiographic osseous regeneration.
Conclusion: The use of PRF in management of cystic lesions seems to be a novel therapeutic approach promoting faster osseous regeneration within 6 months postoperatively however further study is required with larger sample size and with a control group.
Keywords: Cystic lesions, histogram, osseous regeneration, platelet rich fibrin
|How to cite this article:|
Meshram VS, Lambade PN, Meshram PV, Kadu A, Tiwari MS. The autologous platelet rich fibrin: A novel approach in osseous regeneration after cystic enucleation: A pilot study. Indian J Dent Res 2015;26:560-4
The primary goal of any surgical treatment is maintenance of optimum health, comfort, function and esthetics of the patient. Repair and regeneration are two aspects of healing after any uncompromised surgery. Repair is defined as the healing of wound by tissue that has not fully restored the architecture or function of the part. Regeneration is defined as the reproduction and reconstitution of a host or injured part to restore the architecture and function of the tissue.  Bone grafts and bone regenerative materials are commonly used today for treating intrabony defects in periapical surgery with varying degrees of success. However, the key to tissue regeneration is to stimulate a coordinated cascade of healing events that results in integrated tissue formation. This is possible only with the use of growth factors, extracellular matrix, and bone morphogenetic proteins (BMPs) instead of routinely used synthetic bone grafts; as the latter induce bone regeneration by osteoconduction, while former stimulate regeneration by osteoinduction. 
|How to cite this URL:|
Meshram VS, Lambade PN, Meshram PV, Kadu A, Tiwari MS. The autologous platelet rich fibrin: A novel approach in osseous regeneration after cystic enucleation: A pilot study. Indian J Dent Res [serial online] 2015 [cited 2019 Jul 18];26:560-4. Available from: http://www.ijdr.in/text.asp?2015/26/6/560/176915
The enhancement of regenerative processes of human body by utilizing the patient's own blood is a unique concept. Platelet rich fibrin (PRF) was first introduced by Choukroun et al. in 2001. 
PRF protocols require neither the use of anticoagulants nor bovine thrombin. It is simply the centrifuged blood that contains fibrin clots; where growth factors and cytokines are abundantly trapped in a fibrin meshwork. The growth factors present in it are biologically active substances that are involved in tissue repair mechanisms such as chemotaxis, cell proliferation, angiogenesis, extracellular matrix deposition, and remodeling.  PRF releases (through degranulation) at least seven different growth factors as well as cytokines that stimulates bone and soft tissue healing. Thus, PRF is an easy and cost-effective way to obtain high concentrations of growth factors for soft and hard tissue regeneration in wound healing.
| Subjects and Methods|| |
This pilot study was conducted in the Department of Oral and Maxillofacial Surgery, 10 patients (6 male and 4 female) diagnosed with cystic lesions based on clinical and radiographic findings with age groups ranging from 23 years to 45 years were selected randomly for the study.
Exclusion criteria involved patients having any systemic disease that contraindicated the surgical intervention, or with insufficient platelet count for PRF preparation and those having history of coagulation defect or under anticoagulant treatment. Informed consent was taken from all the patients. According to standard surgical protocol, all cystic lesions were enucleated and PRF was placed in the bony defects.
Armamentarium and techniques
The armamentarium required for production of PRF: Tabletop centrifugal machine with the maximum speed of 3500 rpm with glass test tubes of maximum 15 ml capacity; collection kit: Tweezers, scissors, tourniquet, stopwatch [Figure 1].
Platelet rich fibrin protocol
The PRF preparation protocol is very simple and easy. Around 10 ml of whole venous blood is collected in Vacutainer tubes of 15 ml capacity without adding anticoagulant. The Vacutainer tubes are then placed in tabletop centrifugal machine at 3000 rpm for 10 min [Figure 2].
|Figure 2: Use of platelet rich fibrin in cystic lesion; (a) preoperative radiograph (b) osseous defect (c) placement of PRF (d) surgical closure|
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The resultant product consists of the following three layers:
Topmost layer consisting of acellular plasma or platelet poor plasma PRF clot in the middle. Red blood cells (RBCs) at the base.
Due to the absence of the anticoagulant, blood begins to coagulate as soon as it comes in contact with the glass surface. Therefore for successful preparation of PRF, speedy blood collection and immediate centrifugation before the clotting cascade is initiated, is absolutely essential.  Next, the middle layer, i.e., PRF clot is removed and separated from RBC base. The serum which is present along with PRF clot is exudated and fine PRF membrane thus achieved is placed in the intrabony defects [Figure 2].
Periapical radiographs and orthopantomograms (OPG) were taken preoperatively and on 1 st , 3 rd , and 6 th months postoperatively to access radiographic bone density at the site of PRF placement. Bone density was measured with the help of grayscale histogram study of OPG images of bony defects obtained through Adobe Photoshop 7.0 software. (By Adobe systems incorporated in United States. copyright 1990-2002).
| Results|| |
This pilot study evaluated the clinical efficacy of PRF in the treatment of cystic lesions. PRF consists of high concentration of growth factors lodged in a fibrin mesh, which optimizes wound healing.
In our study, none of the patients complained of any severe or unusual pain. There were no signs of infection or rejection, untoward reaction, wound dehiscence or extrusion of material in any of the patients, and an uneventful soft tissue healing was achieved. The histopathological diagnosis of the cystic lesions is given in [Table 1]. Patients were followed up on 1 st , 3 rd and 6 th months postoperatively. Follow-up radiographic examination by grayscale histogram study revealed progressive, predictable and significant radiographic osseous regeneration and an increase in bone density. Radiographically, all patients showed that PRF promotes faster osseous regeneration within the 3 rd postoperative month and within 6 th postoperative month complete bone regeneration was seen [Figure 3] and [Figure 4]. This indicates that PRF may be considered as a novel surgical additive which can be used to accelerate osseous regeneration in intra bony defects.
|Figure 3: Histogram study of orthopantomograms images preoperatively and on 1st, 3rd, 6th months|
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|Figure 4: Bar diagram showing bone density observed by histogram study at periodic intervals|
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| Discussion|| |
Four critical factors that influence bone regeneration after the periapical surgery are primary wound closure, angiogenesis as a blood supply and source of undifferentiated mesenchymal cells, space maintenance, and stability of the wound (PASS principle). 
Our pilot study was aimed to initiate and accelerate bone regeneration to its maximum predictability with the use of PRF. This article will serve to introduce a second generation platelet concentrate, i.e., PRF as "regenerative matrix" in promoting osseous regeneration in intrabony defect after cystic enucleation.
Osteoinductive growth factors that are involved in bone formation are transforming growth factor-β (TGF-β), BMPs, insulin like growth factors and fibroblasts growth factors. The vasculature is formed by two mechanisms: Vasculogenesis and angiogenesis. During wound healing and repair of ischemic tissues, the endothelial cells and their precursors actively participate in the healing process. The major angiogenic factors includes fibroblasts growth factors, platelet-derived growth factors (PDGFs), and vascular endothelial growth factors (VEGF). 
Surgeons have long sought of a product that could act both as a tissue regenerator as well as a hemostatic agent and PRF satisfactory fulfills this purpose. The scientific reason behind use of platelet concentrates lies in the fact that its abundance in growth factors plays a crucial role in hard and soft tissue repair mechanisms. ,
PRF production protocol attempts to concentrate platelets and releases cytokines in a fibrin clot. Activation and degranulation of platelets is important to initiate and support aggregation at the healing site and release cytokines (interleukin [IL]-1 β and IL-6, tumor necrosis factor-α) and growth factors (TGF-β, PDGF, VEGF) that stimulate cell migration and proliferation within the fibrin matrix, which begins the first stage of healing. 
PRF is in the form of a platelet concentrate which offers several advantages including promoting wound healing, bone growth and maturation, graft stabilization, wound sealing, hemostasis, and improving the handling properties of graft materials. PRF is the concentrated suspension of growth factors that are involved in wound healing and are postulated as promoters of tissue regenerations. 
The use of these platelets and immune concentrates during bone grafting procedures is well-known in literature. According to Simonpieri et al.,  the following four advantages are achieved, First, the fibrin clot plays an important mechanical role, with the PRF membrane maintaining and protecting the grafted biomaterial and the PRF fragments serving as biological connectors between the bone particles. Second, when these fibrin networks are integrated into the regenerative sites, they facilitate cellular migration, particularly for endothelial cells, necessary for neo-angiogenesis, vascularization, and survival of graft. Third, there is a gradual release of cytokines as the fibrin matrix resorbs, thus creating a perpetual process of healing. Finally, the presences of leucocytes and cytokines in the fibrin network can play a significant role in self-regulation of inflammatory and infectious phenomena within the grafted material.
The literature shows successful use of autologous PRF as an adjunct to promote healing and osseous regeneration in human mandibular 3 rd molar extraction site. , The improvement in wound healing, decrease in pain, and increase in bone density signifies and highlights the use of PRF as a valid method in promoting and accelerating soft and hard tissue regeneration. 
The use of PRP and PRF is widely used in various situations such as tooth avulsion or extraction, mucogingival surgery, guided tissue regeneration or bone filling of periodontal infrabony defects as well as ridge augmentation.  The use of PRF in combination with freeze dried bone allograft (FDBA) to enhance bone regeneration in sinus floor elevation is also reported. It has been found that there are histological similarities observed in both the group (FDBA alone and FDBA + PRF) which makes it possible to consider sinus floor augmentation surgery with a shorter healing period before implant placement (4 months instead of 8 months). 
Parikh et al.  evaluated the healing of bilateral periapical lesions with and without using platelet rich plasma with the help of computed tomography, they concluded that a comparison between PRP aided healing and normal healing process has been made on the same individual and suggested that PRP thus initiates bone regeneration within 8 weeks postoperatively. Some studies also suggest addition of hydroxyapatite crystals which further helps to induce the rapid rate of bone formation. 
Initially with the development of technologies to obtain platelet concentrates lead to the formation of fibrin adhesive concentrated platelet rich plasma, the use of PRP for various procedures is well-known. The drawbacks of PRP are biochemical blood handling with addition of anticoagulants, some studies also shows that PRP has limited potential to stimulate bone regeneration. The toxic effects of bovine thrombin on body cells is also been demonstrated. 
The PRF is a second generation platelet concentrates which has various added advantages over the traditionally used PRP. The production of PRF is very economical plus it does not require biochemical handling of blood, thus eliminating the redundant step of adding bovine thrombin which is necessary in PRP for conversion of fibrinogen to fibrin. The slow polymerization process of PRF is another advantage over the PRP as it resembles natural healing process and the network of fibrin generated is very similar to natural one and this leads to a more efficient cell migration and proliferation.
PRF is efficacious clinically and radiologically in treatment of intrabony defects after enucleation of various periapical lesions, where complete bone regeneration is seen within 6 th months postoperatively. 
In our study, 10 cases of cystic lesions were treated using PRF after cystic enucleation to evaluate the efficacy of PRF in osseous regeneration of defects left after enucleation. Though the results of our study are preliminary, it shows promising, progressive, predictable, and significant radiographic osseous regeneration in all the cases. Satisfactory osseous regeneration was observed on 3 rd postoperative month and within 6 th postoperative month, complete bone regeneration was seen and good bone density was noticed. The grayscale histogram study revealed a rapid increase in bone density in the bony defect. The use of PRF in intraoral bony defects after cystic enucleation promotes faster regeneration because of gradual release of growth factors lodged in the fibrin matrix.
| Conclusion|| |
The preliminary results of our study are promising and reveal progressive, predictable, and significant radiographic osseous regeneration in all the cases. As PRF is entirely autologous in nature, it would prove to be useful for defecting resolution in patients who are unable to afford expensive regenerative therapies, rather than leaving the defect to heal on its own. As an initial study, we recommend the use of PRF in treatment of cystic lesions for promoting faster osseous regeneration. However, long term comparative studies with control group and adequate sample sizes comparing PRF with other regenerative treatment modalities are essential for better perspectives on this promising biomaterial.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Vikas Sukhadeo Meshram
Department of Oral and Maxillofacial Surgery, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]