Elsevier

Tissue and Cell

Volume 65, August 2020, 101362
Tissue and Cell

A new insight of Platelet-Rich Fibrin clots morphology and their elemental composition

https://doi.org/10.1016/j.tice.2020.101362Get rights and content

Highlights

  • Platelet-Rich Fibrin clot was composed mainly by C and O.

  • PRF’s clot also reveals little amounts of N, Na, P, Cl and S.

  • Removal of extracellular elements enriched the morphology of PRF’s zones.

  • PRF’s fibrin density and organization increases from proximal to distal portion.

Abstract

This study analyzed the architecture of Platelet-Rich Fibrin (PRF) clots and assessed their elemental composition in order to provide new insight into this biomaterial. Five surplus PRF clots (2,700 RPM, 12 min.) donated by patients (63.6 ± 12.3 years old) were prepared for use in dental clinical procedures. The internal three-dimensional morphology of the red zones and the thirds of the yellow zones of the clots were analyzed by Variable Pressure Scanning Electron Microscope (VPSEM) after sample preparation by two methods: 1. Fixation (2.5% gluataraldehyde); and 2. Fixation with subsequent partial removal of extracellular elements (8 N, HCl). Semi-quantitative elemental analysis was performed by energy-dispersive X-ray spectrometry (EDX). VPSEM analysis showed erythrocytes in both the red zone and the yellow zone, which consisted mainly of fibrin. Removal of extracellular elements enriched the morphology of both zones; the organization of the fibrin was observed to differ in the thirds of the yellow zone, with increasing density and organization to distal. The elements that compose organic substances (C-Carbon, N-Nitrogen, O-Oxygen, Na-Sodium and P-Phosphorus) and halogens (Cl-Chloride and S-Sulfur) were detected; the highest concentrations were of C, followed by O (p < 0.05), in the proximal region of the fibrin. The results of the present study suggest organization of fibrin in the PRF clot, and also reveal the distribution of the elements present in the different regions of the clot. Improved understanding of these characteristics may favor the use of this biomaterial by increasing its efficiency and functionality.

Introduction

Platelet-Rich Fibrin (PRF) is a second generation platelet concentrate obtained by an immediate blood centrifugation process requiring anticoagulants and gelling agents. It is considered autologous and biocompatible, and is economic and easy to obtain (Choukroun et al., 2006; Singh et al., 2013; Canellas et al., 2017; Al-Hamed et al., 2017; Isobe et al., 2017).

PRF has been shown to be able to shorten tissue regeneration and healing periods; it can act as a biodegradable scaffold and favors the development of micro-vascularization and epithelial cell migration (Montanari et al., 2013; Cortese et al., 2017).

PRF is characterized by a dense fibrin network with a high content of platelets, macrophages, neutrophils and leukocytes, which play a central role in phagocytosis of debris, microorganisms and necrotic tissues. It also presents sustained release of anti-inflammatory cytokines (Choukroun et al., 2006). The PRF clot fulfills the three important criteria for the regeneration of biological tissues: I. it serves as a three-dimensional fibrin pathway; II. it contains autologous cells; and III. it is considered a reservoir of natural growth factors, which may be released gradually over a period of time and which play a crucial role in the repair of hard and soft tissues (Choukroun et al., 2006; Panda et al., 2016).

Among the identified growth factors contained in PRF are: Insulin-like Growth Factor-1(IGF-1), Epidermal Growth Factor (EGF), Transforming Growth Factor-β (TGF-β), Vascular Endothelial Growth Factor (VEGF) and Platelet-Derived Growth Factors (PDGFs) (Arunachalam et al., 2016).

The use of PRF was first introduced in medicine for the treatment of ulcers and wounds that are difficult to cure (Choukroun et al., 2006). Currently, PRF is used in different biomedical areas including dentistry, where it has already been used in: regenerative treatment of grade II furcal lesions (Sharma and Pradeep, 2011); maxillary sinus lift procedures with simultaneous implant placement, improving natural bone regeneration around the implants (Mazor et al., 2009); surgical treatment of periapical lesions, where it has assisted early wound closure, bone maturation and the final aesthetic result of the periodontal soft tissues (Singh et al., 2013). It has proven to be a safe and predictable method for increasing deficient alveolar ridges in preparation for endo-osseous implant placement (Montanari et al., 2013), and for improving stability in the posterior area of the maxilla in dental implant insertion procedures (Tabrizi et al., 2018).

The cellular content and three-dimensional architecture of PRF clots were described by Dohan Ehrenfest et al. (2010); these authors mainly evaluate the characteristics of the two main regions of the clot, the red erythrocytes zone and the yellow fibrin zone. Other studies have analyzed different portions of these zones by assessing their cell concentrations. (Kobayashi et al., 2012; Bootkrajang et al., 2020; Miron et al., 2020).

The aim of the present study was to analyze the three-dimensional architecture of the red and yellow zones of PRF clots, assessing the morphological characteristics of the thirds which make up the yellow fibrin zone, and including an analysis of the elemental composition of each of the regions in order to provide new insight into this biomaterial.

Section snippets

Obtaining PRF clots

Five adult patients, 3 women and 2 men (63.6 ± 12.3 years old) treated at a private dental clinic in Temuco (Chile) underwent dental procedures in which the use of PRF clots was indicated. None of the participants reported systemic diseases or consumption of anticoagulants. Normally, an excessive number of clots is usually prepared in these clinical procedures as a means of ensuring sufficient material in case of unforeseen events. The clots used in the present study were found to be surplus to

Morphological analysis of three-dimensional architecture - SEM

PRF clots that were fixed without extracellular element removal (Fig. 2A) revealed clear morphological differences between the red zone, which contained erythrocytes, and the fibrin zone, which appeared as a dense amorphous mass. The interface between the zones showed tangled erythrocytes with thin fibrin strands in the red zone (Fig. 2B), in contrast to a dense mass of fibrin containing cells with morphology suggestive of erythrocytes interspersed in the fibrin network in the yellow zone (Fig.

Discussion

The present study analyzed the three-dimensional architecture and elemental composition of different regions of PRF clot samples obtained from patients for use in dental clinical procedures, in order to provide new insight into this biomaterial used in clinical procedures in various biomedical areas (Choukroun et al., 2006; Singh et al., 2013; Montanari et al., 2013; Sharma and Pradeep, 2011; Mazor et al., 2009; Tabrizi et al., 2018).

The three-dimensional architecture of the PRF clot was

Conclusions

The results obtained in the present study reveal differences in the morphology/three-dimensional architecture of the red zone (erythrocytes) and the various thirds of the yellow zone (fibrin), in which the density and organization of the fibrin network increases distally. The elements detected in the samples were those present in organic and halogen compounds, with a greater number of chemical elements in the proximal third at the interface between the red and yellow zones.

Improved

Funding

Financed (partially, English editing) by Dirección de Investigación, Universidad de La Frontera.

CRediT authorship contribution statement

Fernando José Dias: Conceptualization, Investigation, Formal analysis, Writing - original draft, Writing - review & editing. Camila Venegas: Investigation, Writing - original draft, Data curation. Eduardo Borie: Investigation, Formal analysis, Writing - review & editing. Alain Arias: Investigation, Data curation. Ii-sei Watanabe: Methodology, Resources, Validation. Ramón Fuentes: Conceptualization, Resources, Project administration, Supervision, Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors would like to thank Scientific and Technological Bioresource Nucleus (BIOREN—Universidad de La Frontera), especially the Biochemist Karina Godoy, for the support in the analyses with the VP-scanning electron microscope and EDX.

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