Advanced Platelet- Rich Fibrin (A-PRF): A Gender Based Study on Cell Population

Platelet Rich Fibrin (PRF) is a natural fibrin-based biomaterial prepared from an anticoagulant-free blood harvest without any artificial biochemical modification that allows obtaining fibrin membranes enriched with platelets and growth factors. PRF is superior to other platelet concentrates like Platelet Rich Plasma (PRP) due to its ease and inexpensive method of preparation and also it does not need any addition of exogenous compounds like bovine thrombin and calcium chloride. A blood sample of 10 individuals with healthy periodontium and no systemic diseases was collected and centrifuged to get platelet rich fibrin. Platelet rich fibrin is studied histologically to compare the ability of tissue regeneration and wound healing capacity between males and females. Neutrophils and lymphocytes are increased in A-PRF compared to S-PRF. Neutrophils and lymphocytes are proportionately more in males than females in both A-PRF and S-PRF. Monocytes are similar in APRF & S-PRF. Cells are more concentrated in S-PRF. Cells are more widely distributed in A-PRF. Original Research Article Kumaran et al.; JPRI, 32(28): 1-7, 2020; Article no.JPRI.59774 2 Fibrin is more dense in A-PRF compared to S-PRF. Hence A-PRF especially in males could have a greater potential of tissue regeneration and wound healing capacity. Although it is not conclusive due to limited sample size, further increase in sample size can throw more light on the clinical implications. The difference in regenerative potential of platelet concentrates can pave the way for specialised and focussed treatment strategies resulting in more favourable patient outcomes.


INTRODUCTION
In clinical research, the development of biomaterial that are used to regulate the inflammation and increase the speed of healing process is one of the great challenges [1]. Healing is a complex process, which is initiated by clot formation, followed by proliferative stage which comprises of epithelialization, angiogenesis, granulation tissue formation, collagen deposition and finally collagen maturation and contraction [2,3]. But understanding this entire process is still incomplete; however, it is known that platelets play a crucial role not only in hemostasis, but also in the wound healing process [4]. Materials like hydroxyapatite, freeze dried bone graft, tricalcium phosphate, bioactive glass etc. have been widely used and tested for their contribution in healing and regeneration of soft and hard tissues [5]. Recent research on growth factors has recognized that the best tissue regenerative stimulus is present amongst the autologous growth factors, which have clinically proven to induce regeneration and tissue healing [6]. It led to the introduction of Platelet rich plasma (PRP). PRP is the autogenous material that is obtained by mixing of bovine in platelet rich concentrate.It is known as the first generation of platelet concentrate [7]. Currently, the studies have been focussed on the use of an autogenous material called Platelet Rich Fibrin (second generation platelet concentrate) that provides an osteoconductive scaffold along with growth factors to stimulate patient's own cells towards a regenerative response. It was first described by Dr. Joseph Choukroun in France to promote wound healing in implants [5]. PRF is a natural fibrin-based biomaterial prepared from an anticoagulant-free blood harvest without any artificial biochemical modification that allows obtaining fibrin membranes enriched with platelets and growth factors. PRF is superior to other platelet concentrates like PRP due to its ease and inexpensive method of preparation and also it does not need any addition of exogenous compounds like bovine thrombin and calcium chloride. It is advantageous than autogenous graft also because an autograft requires a second surgical site and procedure [8].
Applications of PRF has been found in various branches of dentistry such as endodontics, oral and maxillofacial surgery, periodontics, tissue engineering. In the branch of endodontics, it is used in the treatment of open apex for regeneration of pulp-dentin complex, in combination with Mineral trioxide aggregate (MTA) to create root end barriers in apexification procedures to prevent extrusion of material, in regenerative pulpotomy and to fill in bony defect [5]. It also has a advantage of faster healing of cavitated periapical lesions when treated with PRF, graft stabilisation, wound sealing [9,10]. In the branch of oral and maxillofacial surgery, it is used as filling material in avulsion sockets, bone augmentation in sinus lifts for posterior maxilla augmentation for implants, bony defects etc, ridge preservation, guided bone regeneration [5]. In Periodontics, it is used as a treatment of intrabony defects [5,[11][12][13], gingival recession, guided tissue regeneration, periapical lesions. In tissue engineering, it is used in vitro cultivation of human periosteal cells for bone [5]. Choukroun et al. reported that low speed centrifugation concept (LSCC) enhances the regeneration potential of fluid PRF-based matrices [14]. Richard et al. reported that females and old age produce larger PRF membranes due to the lower red blood cell count in their peripheral blood [15]. Hence a study was planned to know the efficacy of PRF between males and females and between two different centrifugation protocols standard platelet rich fibrin (S-PRF), (9 mL; 2700 rpm for 12 minutes) and (2) advanced platelet rich fibrin (A-PRF) 10 mL; 1500 rpm for 14 minutes) [16]. Previously we have worked on plenty of topics in Periodontology [17][18][19][20][21][22][23][24][25][26][27][28][29]. In this study, we assess the cells in platelet rich fibrin and compare the standard platelet rich fibrin (S-PRF) protocol and advanced platelet rich fibrin (A-PRF) protocol between males and females.

Production of PRF
The classical technique for PRF preparation was invented by Dr. Joseph Choukroun in 2000. It is the current PRF technique authorized by the French Health Ministry in which PRF is prepared without using an anticoagulant during blood harvesting or bovine thrombin during gelling [8].
For preparation of PRF, blood sample without anticoagulant was collected from 10 individuals (5 males,5 females) who were healthy volunteers with no periodontal problems or systemic disorders, in the age range between 18 to 22 years For each individual, 2 tubes of peripheral blood were collected and immediately placed in a pre-programmed centrifuge (PC-O2, PROCESS for PRF, Nice, France). Centrifugation was performed according to the following two protocols: (1) Standard PRF, sterile glass coated plastic tube (9 mL; 2700 rpm for 12 minutes) and (2) Advanced PRF, sterile plain glass-based vacuum tubes (A-PRF10 tube) (10 mL; 1500 rpm for 14 minutes) [16].
After centrifugation, 3 layers are obtained in the test tube (Fig. 1). The topmost layer consisting of acellular PPP (platelet poor plasma), PRF clot in the middle and red blood cells (RBCs)at the bottom of the test tube. The middle layer of PRF clot is then removed with sterile tweezers (Fig. 2) and separated from the underlying RBC layer using scissors and then transferred on a sterile dish and subsequently fixed with 4% paraformaldehyde for 24hrs. It is supposed that the junction of PRF to the RBC layer is rich in growth factors and therefore this region is preserved [30].

Microscopic Analysis of the Platelet Rich Fibrin (A-PRF & S-PRF)
For microscopic analysis, the samples were chemically fixed and processed in an alcohol series and xylene. Subsequently, paraffin embedding was performed, and 10 sections were cut with a semi-automated microtome and affixed on albumin coated glass slides. Before staining, samples underwent a deparaffinization and rehydratation process by sequential immersion in xylene followed by descending concentrations of ethanol. Samples were histologically stained with standard protocols for hematoxylin and eosin (H&E). Microscopic analysis was done using Binocular research microscope (Lieca, 10X and 40 X magnification). The slides were subjected to robust screening to check for the increase or decrease in the various cells. In this study, it is found that Neutrophils and lymphocytes are proportionately more in males than females in both A-PRF and S-PRF.
Neutrophil contribution to tissue repair is that neutrophils convert monocytes into macrophages which initiates release of multiple cytokines and growth factors. The neutrophils become apoptotic and are cleared by macrophages. It is also characterised by the release of the tissuerepairing cytokines transforming growth factor-β (TGFβ) and interleukin-10 (IL-10) [31,32].
Robertson in his study reported that drugs that promote neutrophil apoptosis have a therapeutic potential to accelerate tissue repair [33]. Hence the higher the neutrophils the higher was the regenerative capacity of tissues. We can find that males have more neutrophils than females which suggest that S-PRF and A-PRF from males may have the more regenerative potential than females.
In this study, we found that neutrophils and lymphocytes are more in A-PRF than S-PRF.J. Choukroun, S.
Ghanaati, in their study demonstrated that reducing the relative centrifugation force (RCF) from a high range to low range with in PRF based matrices lead to a significant increase of leucocyte and platelet number. It also resulted in the increase of growth factor concentration such as vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGF-β1).They concluded that the low speed centrifugation concept (LSCC) enhances the regeneration potential of fluid PRF-based matrices. Consequently, the reduction of relative centrifugation force by application of low speed centrifugation concept opens up new avenues for advanced PRF-matrices, in which the cell-cell communication between platelets and leukocytes and that of these cells within the recipient tissue might result in improved wound healing and enhanced tissue regeneration [14].
Bagdadi in his study, concluded that A-PRF+, prepared with a reduced relative centrifugation force, displayed significantly higher vascular endothelial growth factor (VEGF) concentration over the study period of 10 days than A-PRF and PRF Epidermal growth factor (EGF) and transforming growth factor-β (TGF-β1) were comparable in A-PRF and A-PRF+, which were significantly higher than PRF [34]. Wend in his study found that total growth factor release of Platelet derived growth factor (PDGF-BB), transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) in low and medium relative centrifugation force (RCF) were both significantly higher than those in the high RCF group. Vascular endothelial growth factor (VEGF) and MMP-9 were significantly higher in the low RCF group compared to high RCF. These findings support the LSCC (low speed centrifugation concept), which confirms that improved PRF-based matrices may be generated through RCF reduction [35]. In our study, neutrophils and lymphocytes are more in A-PRF than S-PRF. Hence it is clear that A-PRF which had a reduced centrifugation speed have improved PRF matrices which in turn have a greater potential of tissue regeneration.
Miron et al. in his study reported that the size of the membranes produced from females was 17% larger than those produced in males. As the role of centrifugation is to separate blood layers transitionally over time, these differences were thought to be observed due to females generally containing lower hematocrits levels within their peripheral blood compared to males [15].

CONCLUSION
There is an increase in neutrophils in A-PRF and S-PRF of males compared to females. This indirectly implies a greater potential for tissue regeneration and wound healing capacity in the A-PRF from males. As this study is limited in sample size, definitive conclusions cannot be made. This can be overcome by more detailed studies with histomorphometrical analysis of the various cells with larger sample sizes. The difference in regenerative potential of platelet concentrates can pave the way for specialised and focussed treatment strategies resulting in more favorable patient outcomes.

CONSENT
As per international standard or university standard, patients' written consent has been collected and preserved by the author(s).

ETHICAL APPROVAL
As per international standard or university standard written ethical approval has been collected and preserved by the author(s).