Plasma rich in growth factors stimulates proliferation, migration, and gene expression associated with bone formation in human dental follicle cells

Abstract

Background/purpose: Plasma rich in growth factors (PRGFs), which is prepared from autologous blood from patients, has been reported with regards to bone regeneration for dental implants. Human dental follicle cells (hDFCs) have the capacity to commit to multiple cell types such as the osteoblastic lineage. The aim of this study is to evaluate the effects of PRGFs for mineralization in hDFCs.

Materials and methods: PRGFs was prepared from whole blood centrifuged at 460g for 8 minutes. hDFCs isolated from the dental follicle with collagenase/dispase were cultured with growth medium or osteogenic induction medium (OIM) containing PRGFs or fetal bovine serum. Concentrations of the growth factors were examined using an enzyme-linked immunosorbent assay kit. A cell migration assay was used for two-dimensional movement. Gene expressions were examined with real-time polymerase chain reaction using a DyNAmo SYBR Green quantitative polymerase chain reaction kit.

Results: The platelet concentration in PRGF Fraction 2 was 2.14-fold higher than in whole blood. White blood cells were not detected in PRGFs. Transforming growth factor-β levels were higher than insulin-like growth factor-1, platelet-derived growth factor-AB and -BB, and vascular endothelial growth factors in PRGF Fraction 2. Proliferation and migration by hDFCs increased in OIM supplemented with PRGFs in a dose-dependent manner and were higher in hDFCs cultured in OIM plus 10% PRGFs compared with OIM plus 10% fetal bovine serum. PRGFs upregulated the gene expression of type I collagen, osteomodulin, alkaline phosphatase, bone morphogenic protein-4, and transforming growth factor-β in hDFCs.

Conclusion: PRGFs may promote bone regeneration due to it including high levels of growth factors.

Keywords: PRGF; dental follicle cells; growth factors; osteogenic differentiation.

Figure 1

The levels of growth factors in serum and plasma rich in growth factor (PRGF) fractions. Serum and PRGFs were prepared from four healthy donors. Growth factor concentrations are expressed as the means ± standard deviation. F1 indicates PRGF fraction 1 and F2 indicates PRGF fraction 2. Bar indicates the mean value. IGF = insulin-like growth factor; PDGF = platelet-derived growth factor; TGF = transforming growth factor; VEGF = vascular endothelial growth factor.

Figure 2

Effect of plasma rich in growth factors (PRGFs) on human dental follicle cell proliferation. Human dental follicle cells were cultured in growth medium (GM), osteogenic induction medium (OIM) supplemented with 10% fetal bovine serum (FBS), or OIM supplemented with the indicated PRGF concentrations for 2 or 3 days. Values represent the means ± standard deviation. * P < 0.05. ** P < 0.01.

Figure 3

Effect of plasma rich in growth factors (PRGFs) on migration of human dental follicle cells (hDFCs). hDFCs were cultured in growth medium (GM), osteogenic induction medium (OIM) supplemented with 10% fetal bovine serum (FBS), or OIM supplemented with the indicated PRGF concentrations for 3 days. (A) Phase-contrast microscopy shows hDFC migration; (B) quantitation of cell migration using Image J software (National Institutes of Health, Bethesda, MD, USA). Values represent the means ± standard deviation.

Figure 4

Gene expression of type I collagen α1, osteomodulin (OMD), alkaline phosphatase (ALP), bone morphogenetc proteins (BMPs), and transforming growth factor (TGF)-β in human dental follicle cells. Human dental follicle cells were cultured in growth medium (GM), osteogenic induction medium (OIM) supplemented with 10% fetal bovine serum (FBS), or OIM supplemented with the indicated PRGFs concentrations for 3 or 7 days. The gene expression was examined with real-time polymerase chain reaction. Values represent the means ± standard deviation of the results from three independent experiments. * P < 0.05. ** P < 0.01. *** P < 0.05 compared with Culture Day 0.