Behavior of Gingival Fibroblasts on Titanium Implant Surfaces in Combination with either Injectable-PRF or PRP

Xuzhu Wang, Yufeng Zhang, Joseph Choukroun, Shahram Ghanaati, Richard J Miron, Xuzhu Wang, Yufeng Zhang, Joseph Choukroun, Shahram Ghanaati, Richard J Miron

Abstract

Various strategies have been employed to speed tissue regeneration using bioactive molecules. Interestingly, platelet concentrates derived from a patient's own blood have been utilized as a regenerative strategy in recent years. In the present study, a novel liquid platelet formulation prepared without the use of anti-coagulants (injectable-platelet-rich fibrin, i-PRF) was compared to standard platelet-rich plasma (PRP) with gingival fibroblasts cultured on smooth and roughened titanium implant surfaces. Standard PRP and i-PRF (centrifuged at 700 rpm (60× g) for 3 min) were compared by assays for fibroblast biocompatibility, migration, adhesion, proliferation, as well as expression of platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), collagen1 (COL1) and fibronectin (FN). The results demonstrate that i-PRF induced significantly higher cell migration, as well as higher messenger RNA (mRNA) levels of PDGF, TGF-β, collagen1 and fibronectin when compared to PRP. Furthermore, collagen1 synthesis was highest in the i-PRF group. These findings demonstrate that liquid platelet concentrates can be formulated without the use of anticoagulants and present much translational potential for future research. Future animal and clinical trials are now necessary to further investigate the potential of utilizing i-PRF for soft tissue regenerative protocols in combination with various biomaterials.

Keywords: blood; fibrin; fibroblasts; platelet-rich fibrin; platelets; regeneration; wound healing.

Conflict of interest statement

Joseph Choukroun is the founder of Process of Platelet-Rich Fibrin (PRF), which supplied the PRF machine utilized in this study. All other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The percentage of human gingival fibroblasts quantified with a Live/Dead assay at 24 h on tissue culture plastic (TCP), Pickled titanium (PT) and Sand-blasted with Large grit particles followed by Acid-etching (SLA) with platelet rich plasma (PRP) or injectable platelet-rich fibrin (i-PRF) (* denotes difference between control TCP and experimental group, p < 0.05).
Figure 2
Figure 2
Effects of surface topography and PRP/i-PRF on the migration of human gingival fibroblasts (A) Cell migration was assessed after 24 h. (Scale bars = 100 µm); (B) Cell migration was quantified by normalizing to the control TCP group. (* denotes significant difference between control and experimental group, p < 0.05; ** denotes significantly higher than all other groups, p < 0.05).
Figure 3
Figure 3
Effect of surface topography in combination with PRP and i-PRF on the adhesion and proliferation of human gingival fibroblasts. (A) Cell adhesion at 2, 4 and 8 h and (B) Cell proliferation at 1, 3 and 5 days. (* denotes significant difference between control and experimental group, p < 0.05; ** denotes significantly higher than all other groups, p < 0.05; # denotes significant difference between PT and SLA surfaces p < 0.05).
Figure 4
Figure 4
Effects of surface topography in combination with PRP and i-PRF on the morphology of human gingival fibroblasts (A) Human gingival fibroblasts cultured with and without PRP or i-PRF at 8 h were stained for F-actin (green) and nuclei (blue) (Scale bars = 100 µm); (B) Average surface planar area of cells. (* denotes significant difference between control and experimental group, p < 0.05; ** denotes significantly higher than all other groups, p < 0.05; # denotes significant difference between PT and SLA surfaces, p < 0.05).
Figure 5
Figure 5
Real-time PCR of human gingival fibroblasts cultured on TCP, PT and SLA, with and without PRP or i-PRF for mRNA levels of (A) PDGF; (B) TGF-β; (C) COL1 and (D) FN1. (* denotes significant difference between control and experimental group, p < 0.05; ** denotes significantly higher than all other groups, p < 0.05; # denotes significant difference between PT and SLA surfaces, p < 0.05).
Figure 6
Figure 6
Immunofluorescent staining of collagen type 1 for human gingival fibroblasts seeded on TCP, PT and SLA surfaces with and without PRP and i-PRF at 7 days. (A) The merged fluorescent images of collagen type 1 staining (green) with DAPI staining (blue). (Scale bars = 100 µm) (B) Quantified values of collagen type 1 staining in comparison to control TCP sample (* denotes significant difference between control and experimental group, p < 0.05, ** denotes significantly higher than all other groups, p < 0.05, # denotes significant difference between PT and SLA surfaces, p < 0.05).

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