Advanced Platelet-Rich Fibrin: A New Concept for Cell-Based Tissue Engineering by Means of Inflammatory Cells
Choukroun's platelet-rich fibrin (PRF) is obtained from blood without adding anticoagulants. In this study, protocols for standard platelet-rich fibrin (S-PRF) (2700 rpm, 12 minutes) and advanced platelet-rich fibrin (A-PRF) (1500 rpm, 14 minutes) were compared to establish by histological cell detection and histomorphometrical measurement of cell distribution the effects of the centrifugal force (speed and time) on the distribution of cells relevant for wound healing and tissue regeneration. Immunohistochemistry for monocytes, T and B -lymphocytes, neutrophilic granulocytes, CD34-positive stem cells, and platelets was performed on clots produced from four different human donors. Platelets were detected throughout the clot in both groups, although in the A-PRF group, more platelets were found in the distal part, away from the buffy coat (BC). T- and B-lymphocytes, stem cells, and monocytes were detected in the surroundings of the BC in both groups. Decreasing the rpm while increasing the centrifugation time in the A-PRF group gave an enhanced presence of neutrophilic granulocytes in the distal part of the clot. In the S-PRF group, neutrophils were found mostly at the red blood cell (RBC)-BC interface. Neutrophilic granulocytes contribute to monocyte differentiation into macrophages. Accordingly, a higher presence of these cells might be able to influence the differentiation of host macrophages and macrophages within the clot after implantation. Thus, A-PRF might influence bone and soft tissue regeneration, especially through the presence of monocytes/macrophages and their growth factors. The relevance and feasibility of this tissue-engineering concept have to be proven through in vivo studies.
Clots after centrifugation: (a) depicts a platelet-rich fibrin (PRF) clot removed from the centrifugation tube with forceps and placed on sterile gauze. (b) highlights four PRF clots after careful removal of the red blood cell (RBC) fraction count with scissors. The PRF clots are placed in Choukroun's PRF Box. The box is especially designed for the processing of the PRF. It is composed of an outer lid, an inner lid used for compressing the clots, a perforated plate as seen in the picture, and a bottom used for conserving the exudated fluid. As seen in the image on the right-hand side, the two sockets are used to compress the clots, if needed.
Total scan of a fibrin clot along its longitudinal axis (Masson-Goldner staining). RBC represents the red blood cell fraction. The buffy coat (BC) is the transformation zone between the RBC fraction and fibrin clot, and FC represents the fibrin clot. The three bars within the scan and the arrows show close-ups of the respective areas. The red arrows mark cells that are entrapped within the fibrin network.
Immunohistochemical reaction of the cells detected within the clots of the two experimental groups. Images S1–S6 show close-ups of the transformation zone between red blood cell (RBC), buffy coat (BC), and fibrin clot (FC) with different immunohistochemical staining for CD 3-, CD15-, CD20-, CD34-, CD61-, and CD68-positive cells in the respective picture for platelet-rich fibrin (PRF) clots produced with the standard PRF protocol. As can be seen in the different images, the stained cells are mainly located within the border between the RBC count and the BC. Depending on the immunohistochemical marker, the cells are more or less prominent in the PRF clot. The red arrows mark the cells stained cells (positive) within the respective picture. The same applies for the Figure A1 through A6. These images are taken from PRF clots produced with the advanced PRF protocol.
Two total scans of the standard platelet-rich fibrin (PRF): left, hematoxylin and eosin (H&E) staining, total scan, 100 × magnification, and the advanced PRF; right, Masson-Goldner staining, total scan, 100 × magnification. Between both total scans, each immunohistochemical marker is portrayed by a colored bar (CD34–CD61). Within each of the total scans, the distribution of each cell type is depicted by separately colored bars. By looking at the total scans of both the standard and advanced PRF, it becomes apparent that CD61-positive cells (platelets) are distributed evenly throughout the clot, although the amount of platelets appears to decrease toward the peripheral parts of the clot in the S-PRF group. Additionally, the modification of the protocol resulted in an increased migration of CD15-positive cells into the A-PRF clot.
Comparative penetration of the different cell types into the clot for both the standard platelet-rich fibrin (S-PRF) and advanced platelet-rich fibrin (A-PRF). As can be seen in this diagram, a highly significant difference (**P < .01) in distribution was established for neutrophilic granulocytes (CD15) between both groups.
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