L-PRF Secretome from Both Smokers/Nonsmokers Stimulates Angiogenesis and Osteoblast Differentiation In Vitro

Abstract

Leukocyte and Platelet-Rich Fibrin (L-PRF) is part of the second generation of platelet-concentrates. L-PRF derived from nonsmokers has been used in surgical procedures, with its beneficial effects in wound healing being proven to stimulate biological activities such as cell proliferation, angiogenesis, and differentiation. Cigarette smoking exerts detrimental effects on tissue healing and is associated with post-surgical complications; however, evidence about the biological effects of L-PRF derived from smokers is limited. This study evaluated the impact of L-PRF secretome (LPRFS) derived from smokers and nonsmokers on angiogenesis and osteoblast differentiation. LPRFS was obtained by submerging L-PRF membranes derived from smokers or nonsmokers in culture media and was used to treat endothelial cells (HUVEC) or SaOs-2 cells. Angiogenesis was evaluated by tubule formation assay, while osteoblast differentiation was observed by alkaline phosphatase and osterix protein levels, as well as in vitro mineralization. LPRFS treatments increased angiogenesis, alkaline phosphatase, and osterix levels. Treatment with 50% of LPRFS derived from smokers and nonsmokers in the presence of osteogenic factors stimulates in vitro mineralization significantly. Nevertheless, differences between LPRFS derived from smokers and nonsmokers were not found. Both LPRFS stimulated angiogenesis and osteoblast differentiation in vitro; however, clinical studies are required to determine the beneficial effect of LPRFS in smokers.

Keywords: angiogenesis; cell differentiation; cigarette smoking; leukocyte- and platelet-rich fibrin; osteoblast; secretome.

Figure 1

LPRFS from smokers and nonsmokers have similar PDGF-BB and FGF-2 growth factors and IL-6 levels. L-PRF membranes were obtained from S and NS after L-PRF Clot compression, and exudates released were recovered for cotinine quantification. L-PRF membranes were submerged in DMEM for 24 h to obtain LPRFS. (A) Cotinine was determined in L-PRF exudates derived from S, NS, and LPRFS-S. Quantification of (B) PDGF-BB, (C) FGF-2, and (D) IL-6 levels in LPRFS and conditioned medium of starved human gingival fibroblast as IL-6 control. ** p ≤ 0.0022; *** p ≤ 0.002; **** p ≤ 0.0001 Ex = L-PRF exudates, S = smokers, NS = nonsmokers, LPRFS = L-PRF secretome.

Figure 2

LPRFS from smokers and nonsmokers stimulates angiogenesis in vitro. HUVEC cells were treated for 6 h with 50% LPRFS-S or LPRFS-NS or positive (EBM + FGF) and negative (EBM + FGF + SUL or EBM) controls. Cells were stained with calcein and observed in an epifluorescence inverted microscope at 4×. Representative images of live stain HUVEC cells treated with (A) LPRFS or (B) Positive and (C) negative Controls. Quantification of (D) total tubes, (E) total tube length, (F) total loop area, (G) mean loop area, and (H) mean loop perimeter. * p ≤ 0.0140; ** p ≤ 0.085; *** p ≤ 0.005; **** p ≤ 0.001. S = smokers, NS = nonsmokers, LPRFS = L-PRF secretome, EBM = endothelial basal medium, F = FGF = fibroblast growth factor, SUL = sulphoraphane. Scale Bar = 50 microns.

Figure 3

LPRFS derived from smokers and nonsmokers modulates Alkaline phosphatase and Osterix protein levels. SaOs-2 cells were treated for 48 h with LPRFS-S or LPRFS-NS with or without osteogenic factors. DMEM supplemented with FBS 10% with or without osteogenic factors was used as a control. (A) Representative images of a Western blot for Alkaline phosphatase (Alp), Osterix (Osx), and Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in SaOs-2 cells treated in indicated conditions. Relative quantification of (B) Alp protein levels in cells treated with 100% LPRFS, (C) Alp protein levels in cells treated with 50% LPRFS, (D) Osx protein levels in cells treated with 100% LPRFS and (E) Osx protein levels in cells treated with 50% LPRFS. Representative images of Osx immunostaining in red and DAPI nuclear stain in blue, SaOs-2 cells were treated as indicated in (F) Controls, (G) 100% LPRFS-S (H) 50% LPRFS-S, (I) 100% LPRFS-NS, (J) 50% LPRFS-NS with or without osteogenic factors. * p ≤ 0.0455; ** p ≤ 0.0074; *** p ≤ 0.003; **** p ≤ 0.001. S = smokers, NS = nonsmokers, LPRFS = L-PRF secretome, Ost = osteogenic factors, RIU = relative intensity units. Scale Bar = 50 microns.

Figure 4

LPRFS derived from smokers and nonsmokers stimulates mineralization in vitro. SaOs-2 cells were treated for 11 days with LPRFS-S or LPRFS-NS with or without osteogenic factors. DMEM supplemented with FBS 10% with or without osteogenic factors was used as a control. Cells were stained with alizarin red stain. Representative images of cells treated with (A) LPRFS-S, (B) LPRFS-NS, or (C) 10% FBS. (D) Quantification of percentage area stained with alizarin red. * p ≤ 0.0247; ** p ≤ 0.061. S = smokers, NS = nonsmokers, LPRFS = L-PRF secretome, Ost = osteogenic factors. Scale Bar = 500 microns.