Effects and mechanisms of basic fibroblast growth factor on the proliferation and regenerative profiles of cryopreserved dental pulp stem cells

Abstract

Objectives: Various factors could interfere the biological performance of DPSCs during post-thawed process. Yet, little has been known about optimization of the recovery medium for DPSCs. Thus, our study aimed to explore the effects of adding recombinant bFGF on DPSCs after 3-month cryopreservation as well as the underlying mechanisms.

Materials and methods: DPSCs were extracted from impacted third molars and purified by MACS. The properties of CD146⁺ DPSCs (P3) were identified by CCK-8 and flow cytometry. After cryopreservation for 3 months, recovered DPSCs (P4) were immediately supplied with a series of bFGF and analysed cellular proliferation by CCK-8. Then, the optimal dosage of bFGF was determined to further identify apoptosis and TRPC1 channel through Western blot. The succeeding passage (P5) from bFGF pre-treated DPSCs was cultivated in bFGF-free culture medium, cellular proliferation and stemness were verified, and pluripotency was analysed by neurogenic, osteogenic and adipogenic differentiation.

Results: It is found that adding 20 ng/mL bFGF in culture medium could significantly promote the proliferation of freshly thawed DPSCs (P4) through suppressing apoptosis, activating ERK pathway and up-regulating TRPC1. Such proliferative superiority could be inherited to the succeeding passage (P5) from bFGF pre-stimulated DPSCs, meanwhile, stemness and pluripotency have not been compromised.

Conclusions: This study illustrated a safe and feasible cell culture technique to rapidly amplify post-thawed DPSCs with robust regenerative potency, which brightening the future of stem cells banking and tissue engineering.

Keywords: basic fibroblast growth factor; cell culture technique; cryopreservation; dental pulp stem cells; extracellular signal-regulated kinase pathway; transient receptor potential canonical 1 channel.

Figure 1

The cellular growth curve before and after cryopreservation, and cell proliferation of post‐thawed DPSCs and their succeeding passage. (A) The cell proliferation of non‐frozen DPSCs between the passage 3 (P3) and the passage 4 (P4) from day 1 to 7. No statistical differences were observed between the two passages at all timepoints. Data were represented as mean ± SD (n = 3) (B) The cellular viability of post‐thawed DPSCs (P4) was statistically lower compared to the before‐frozen cells (P3) from day 1 to 7. All data were represented as mean ± SD (n = 3), ^* P < .05. (C) Cell proliferation of post‐thawed DPSCs (P4) in the 20 ng/mL bFGF group was higher than other groups on day 5 and 7. (D) After supplemented with bFGF for 5 d, DPSCs (P4) were passaged to P5 and cultured with bFGF‐free medium. The succeeding passage (P5) also proliferated fastest in the 20 ng/mL bFGF group on day 5 and 7. All data were represented as mean ± SD (n = 3), ^* P < .05, ^** P < .01 and ^*** P < .01 vs the control (CM)

Figure 2

bFGF impacted on apoptosis of post‐thawed DPSCs and TRPC1 channel, and bFGF rescued cellular proliferation after TRPC1 inhibition. (A, C) Western blot analysis of Bcl‐2, Bax, p‐Erk1/2, Erk1/2 and TRPC1 expression in DPSCs with/ without bFGF treatment. (B, D) Quantification of these proteins in DPSCs with bFGF treatment indicated that down‐expression of Bax, up‐expression of Bcl‐2, p‐Erk1/2, Erk1/2 and TRPC1. All data were represented as mean ± SD (n = 3), ^** P < .01, ^*** P < .001 vs the control (CM). (E) TRPC1 inhibitor, SKF‐96365, effectively suppressed the proliferation of DPSCs (P4) at 10‐25 µmol/L. (F) DPSCs (P4) were pre‐treated with SKF‐96365 for 24 h and then cultured with/ without 20 ng/mL bFGF for another 24 h. Additional bFGF significantly increased the cell proliferation in a short‐term culture. ^* P < .05, ^** P < .01 and ^*** P < .001 vs the control group; ^# P < .05 vs the 1 μmol/L group. All data were represented as mean ± SD (n = 5)

Figure 3

Immunofluorescence labelled MSCs markers of the succeeding passage from bFGF pre‐treated DPSCs. (A) Post‐thawed DPSCs (P4) were firstly cultured with bFGF (20 ng/mL) for 5 d and passaged to P5, then stained with CD146 (green), STRO‐1 (red) and nucleus (blue). DPSCs (P5) in CM group were cultured in bFGF‐free medium. Scale bar: 200 μm. (B) Semi‐quantification of the fluorescence intensity of CD146 and STRO‐1. There is no statistical difference (NS) in the expression of these surface markers between bFGF pre‐treated and control DPSCs (P5). All data were represented as mean ± SD (n = 3)

Figure 4

Stemness of the succeeding passage from bFGF pre‐treated DPSCs. (A) Post‐thawed DPSCs (P4) were firstly cultured with bFGF (20 ng/mL) for 7 d and passaged to P5, then assessed the MSCs markers (CD73 and CD90) and haematopoietic stem cells (CD14 and HLA‐DR). The expression of these markers between bFGF pre‐treated and control DPSCs (P5) was similar. (B, C) Quantification of the proteins of CD146 and Nanog in bFGF pre‐treated and control DPSCs (P5) indicated no statistical difference (NS). All data were represented as mean ± SD (n = 3)

Figure 5

Neurogenic differentiation of the succeeding passage from bFGF pre‐treated DPSCs. In the Control group, DPSCs (P5) were cultured in bFGF‐free medium and not induced. In the Induction‐CM group, cells (P5) were cultured without bFGF and induced. In the Induction‐prime 20 ng/mL bFGF group, cells (P5) were passaged from bFGF pre‐treated DPSCs and induced. (A) Immunofluorescent staining showed neural markers of DPSCs (P5), including Nestin (green), NeuN (green), GFAP (red) and β‐tubulin III (red). Cell nuclei were stained with DAPI (blue). Scale bar: 100 μm. (B) Semi‐quantification of the fluorescent intensity of these neural markers. The induction enhanced these expressions, and the expression level of bFGF pre‐treated DPSCs (P5) was similar to the cells in bFGF‐free culture. All data were represented as mean ± SD (n = 3), ^* P < .05, ^** P < .01 and ^*** P < .001 vs the Control group

Figure 6

Osteogenic and adipogenic differentiation of the succeeding passage from bFGF pre‐treated DPSCs. In the Control group, DPSCs (P5) were cultured in bFGF‐free medium and not induced. In the Induction‐CM group, cells (P5) were cultured without bFGF and induced. In the Induction‐prime 20 ng/mL bFGF group, cells (P5) were passaged from bFGF pre‐treated DPSCs and induced. In the Control, cells did not display newly regenerated structure (top panel in A and C). At low and high magnification of induction groups (middle and right column in A and C), mineralized nodules were stained by Alizarin Red and lipid droplets were stained by Oil Red O Scale bar: 200 μm and 100 μm. (B, D) Semi‐quantification of the newly regenerated structure. Induction groups sustained similar potential to differentiate into osteoblast or adipocyte. All data were represented as mean ± SD (n = 3), ^*** P < .001 vs the Control group