Red and Yellow Injectable Platelet-Rich Fibrin Demonstrated Differential Effects on Periodontal Ligament Stem Cell Proliferation, Migration, and Osteogenic Differentiation

Prakan Thanasrisuebwong, Sirichai Kiattavorncharoen, Rudee Surarit, Chareerut Phruksaniyom, Nisarat Ruangsawasdi, Prakan Thanasrisuebwong, Sirichai Kiattavorncharoen, Rudee Surarit, Chareerut Phruksaniyom, Nisarat Ruangsawasdi

Abstract

The biological benefits of using two fractions derived from injectable platelet-rich fibrin (i-PRF) in bone regeneration remain unclear. Thus, the current study examined two fractionation protocols producing yellow i-PRF and red i-PRF on periodontal ligament stem cells (PDLSCs). The i-PRF samples from five donors were harvested from two different levels, with and without a buffy coat layer, to obtain red and yellow i-PRF, respectively. The PDLSCs were isolated and characterized before their experimental use. The culture medium in each assay was loaded with 20% of the conditioned medium containing the factors released from the red and yellow i-PRF. Cell proliferation and cell migration were determined with an MTT and trans-well assay, respectively. Osteogenic differentiation was investigated using alkaline phosphatase and Alizarin red staining. The efficiency of both i-PRFs was statistically compared. We found that the factors released from the red i-PRF had a greater effect on cell proliferation and cell migration. Moreover, the factors released from the yellow i-PRF stimulated PDLSC osteogenic differentiation earlier compared with the red i-PRF. These data suggest that the red i-PRF might be suitable for using in bone regeneration because it induced the mobilization and growth of bone regenerative cells without inducing premature mineralization.

Keywords: bone regeneration; cell migration; cell proliferation; injectable platelet-rich fibrin; osteogenic differentiation; periodontal ligament stem cells.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Periodontal ligament stem cell characterization. (a) The isolated cells were evaluated for single colony formation (scale bar = 5 mm), (b) and multi-lineage differentiation potency; adipogenic differentiation, neurogenic differentiation, and osteogenic differentiation, (scale bars = 100 µm). (c) Analysis of the stem cell markers showed 70‒99% CD105+, CD90+, CD73+, CD146+, and CD34− cells.
Figure 2
Figure 2
Proliferative effects on periodontal ligament stem cells. (a) Cell culture medium containing the release from the red i-PRF enhanced cell proliferation significantly greater than the medium consisting of the release from the yellow i-PRF at day 5. (b) Live cell images of cell number in the different culture mediums (scale bars = 200 µm). Significance was assessed using one-way ANOVA followed by post hoc least-significant difference tests to compare the differences between groups. The data are shown as individual donor pairs. * p < 0.05.
Figure 3
Figure 3
Migration of periodontal ligament stem cells. Conditioned medium obtained from the release from red or yellow i-PRFs was loaded into the growth medium supplemented with fetal bovine serum as the positive control. The medium without any supplement was the negative control. The number of migrated cells was significantly higher in the red i-PRF group than the yellow i-PRF group as shown (a) quantitatively and (b) microscopically (scale bar = 200 µm). Significance was assessed using one-way ANOVA followed by post hoc least-significant difference tests to compare the differences between groups. The data are shown as individual donor pairs. * p < 0.05.
Figure 4
Figure 4
Osteogenic differentiation of periodontal ligament stem cells. Effects of the release from the i-PRF types on osteogenic differentiation were tested in parallel with osteogenic medium as the positive control and growth medium as the negative control. The release from the yellow i-PRF improved osteogenic differentiation more than the release from the red i-PRF demonstrated a significantly higher (a) alkaline phosphatase activity, (b) percent of calcification and (c) alizarin red staining (scale bar = 100 µm). Significance was assessed using one-way ANOVA followed by post hoc least-significant difference tests to compare the differences between groups. The data are shown as individual donor pairs. * p < 0.05 and *** p < 0.001.

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