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. 2020 Mar;10(3):396-406.
doi: 10.1002/2211-5463.12792. Epub 2020 Feb 11.

All-trans Retinoic Acid and Human Salivary histatin-1 Promote the Spreading and Osteogenic Activities of Pre-Osteoblasts in Vitro

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Free PMC article

All-trans Retinoic Acid and Human Salivary histatin-1 Promote the Spreading and Osteogenic Activities of Pre-Osteoblasts in Vitro

Wei Sun et al. FEBS Open Bio. .
Free PMC article

Abstract

Cell-based bone tissue engineering techniques utilize both osteogenic cells and biomedical materials, and have emerged as a promising approach for large-volume bone repair. The success of such techniques is highly dependent on cell adhesion, spreading, and osteogenic activities. In this study, we investigated the effect of co-administration of all-trans retinoic acid (ATRA) and human salivary peptide histatin-1 (Hst1) on the spreading and osteogenic activities of pre-osteoblasts on bio-inert glass surfaces. Pre-osteoblasts (MC3T3-E1 cell line) were seeded onto bio-inert glass slides in the presence and absence of ATRA and Hst1. Cell spreading was scored by measuring surface areas of cellular filopodia and lamellipodia using a point-counting method. The distribution of fluorogenic Hst1 within osteogenic cells was also analyzed. Furthermore, specific inhibitors of retinoic acid receptors α, β, and γ, such as ER-50891, LE-135, and MM-11253, were added to identify the involvement of these receptors. Cell metabolic activity, DNA content, and alkaline phosphatase (ALP) activity were assessed to monitor their effects on osteogenic activities. Short-term (2 h) co-administration of 10 μm ATRA and Hst1 to pre-osteoblasts resulted in significantly higher spreading of pre-osteoblasts compared to ATRA or Hst1 alone. ER-50891 and LE-135 both nullified these effects of ATRA. Co-administration of ATRA and Hst1 was associated with significantly higher metabolic activity, DNA content, and ALP activity than either ATRA or Hst1 alone. In conclusion, co-administration of Hst1 with ATRA additively stimulated the spreading and osteogenicity of pre-osteoblasts on bio-inert glass surfaces in vitro.

Keywords: all-trans retinoic acid; cell spreading; histatin-1; osteogenic cells; pre-osteoblasts.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Light micrographs depicting the spreading of pre‐osteoblasts in the presence or absence of 10 µm ATRA. Bar = 50μm. (B) Folds of cell spreading surface area in the presence or absence of 1, 10, and 20 µm ATRA. Data are shown as mean ± SD (n = 6). (C) Light micrographs of the cell spreading in the presence or absence of 10 µm Hst1. Bar = 50μm. (D) Folds of cell spreading surface area in the presence or absence of 1, 10, and 20 µm Hst1. Data were shown as mean ± SD (n = 6). Data were plotted using graphpad prism (GraphPad Software version 6.0) and analyzed by one‐way ANOVA with Bonferroni's post hoc test for multiple comparisons. **P < 0.01 comparing with control group; n.s., no statistically significant difference.
Figure 2
Figure 2
(A) Light micrographs depicting the spreading of pre‐osteoblasts in the presence or absence of 10 µm Hst1 or 10 µm ATRA. Bar = 50μm. (B) Folds of spreading surface area of pre‐osteoblasts in the presence or absence of 10 µm Hst1 or 10 µm ATRA. Data are shown as mean ± SD (n = 6). Data were plotted using graphpad prism (GraphPad Software version 6.0) and analyzed by one‐way ANOVA with Bonferroni's post hoc test for multiple comparisons. *P < 0.05; ***P < 0.001. (C) Time‐dependent cell spreading surface area [expressed in folds with the value of the control group (no Hst1 and no ATRA) at first time point as 1] in the presence or absence of 10 µm Hst1 or 10 µm ATRA. Data were shown as mean ± SD (n = 6). Data were analyzed by two‐way ANOVA with Tukey test for multiple comparisons. § P < 0.05 indicating a significant difference compared with the values in the groups of Hst1 or ATRA; & P < 0.05 indicating a significant difference compared with the value in the control group at the same time point; # P < 0.05 indicating a significant difference compared with the value in the same treatment group at the earlier time point.
Figure 3
Figure 3
(A) Fluorescent micrographs depicting the spreading of pre‐osteoblasts (stained with FITC‐Phalloidin) in the presence or absence of 10 µm Hst1 and 10 µm ATRA on bio‐inert glass surface. Bar = 50μm. (B) The surface area per pre‐osteoblast in the presence or absence of 10 µm Hst1 and 10 µm ATRA on bio‐inert glass surface. Data were plotted using graphpad prism (GraphPad Software version 6.0) and analyzed by one‐way ANOVA with Bonferroni's post hoc test for multiple comparisons. Data were shown as mean ± SD (n > 20). *P < 0.05; ***P < 0.001; ****P < 0.0001.
Figure 4
Figure 4
Folds of spreading surface area of pre‐osteoblasts that were treated with (A) co‐administered 10 µm ATRA and Hst1 with or without the pretreatment with MM [10 µm MM‐11253, the antagonist of retinoic acid receptor (RAR)γ], ER (10 µm ER‐50891, the antagonist of RARα), and LE (10 µm LE‐135, the antagonist of RARβ); (B) 10 µm Hst1 in the presence or absence of 10 µm MM or 10 µm ATRA; (C) co‐administered 10 µm Hst1 in the presence or absence of 10 µm LE135 or 10 µm ATRA. Data were plotted using graphpad prism (GraphPad Software version 6.0) and analyzed by one‐way ANOVA with Bonferroni's post hoc test for multiple comparisons. Data were shown as mean ± SD (n = 6). *P < 0.05; ***P < 0.001.
Figure 5
Figure 5
(A) Folds of the metabolic activities of pre‐osteoblasts within 5 days after a short (2 h) treatment with either no Hst1, no ATRA (control), or 10 µm Hst1, or 10 µm ATRA, or co‐administered 10 µm ATRA and 10 µm Hst1 with α‐MEM containing 10% FBS during seeding (n = 6). (B) Folds of DNA content at 0 day and 5 days after response graph of DNA content after a short (2 h) treatment with either no Hst1, no ATRA (control), or 10 µm Hst1, or 10 µm ATRA, or co‐administered 10 µm ATRA and 10 µm Hst1 with α‐MEM containing 10% FBS during seeding (n = 6). (C) Folds of ALP activity at 3 days after a short (2 h) treatment either without Hst1 or ATRA (control), or with 10 µm Hst1, or 10 µm ATRA, or co‐administered 10 µm ATRA and 10 µm Hst1 with α‐MEM containing 2% FBS during seeding (n = 8). Data were plotted using graphpad prism (GraphPad Software version 6.0) analyzed by one‐way ANOVA with Bonferroni's post hoc test for multiple comparisons. Data were shown as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

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