Effect of simultaneous and sequential use of TGF-β1 and TGF-β3 with FGF-2 on teno/ligamentogenic differentiation of periodontal ligament stem cells

IF 2.2 4区医学 Q2 DENTISTRY, ORAL SURGERY & MEDICINE Archives of oral biology Pub Date : 2024-03-13 DOI:10.1016/j.archoralbio.2024.105956

Fazele Atarbashi-Moghadam , Ali Azadi , Hanieh Nokhbatolfoghahaei , Niloofar Taghipour

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Abstract

Objective

The periodontal ligament is a crucial part of the periodontium, and its regeneration is challenging. This study compares the effect of simultaneous and sequential use of FGF-2 and TGF- $β$ 1 with FGF-2 and TGF- $β$ 3 on the periodontal ligament stem cells (PDLSCs) teno/ligamentogenic differentiation.

Design

This study comprises ten different groups. A control group with only PDLSCs; FGF-2 group containing PDLSCs with a medium culture supplemented with FGF-2 (50 ng/mL). In other experimental groups, different concentrations (5 ng/mL or 10 ng/mL) of TGF- $β$ 1&- $β$ 3 simultaneously or sequentially were combined with FGF-2 on the cultured PDLSCs. TGF- $β$ was added to the medium after day 3 in the sequential groups. Methyl Thiazolyl Tetrazolium (MTT) assay on days 3, 5, and 7 and Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) analysis after day 7 were conducted to investigate PLAP1, SCX, and COL3A1, RUNX2 genes. All experiments were conducted in a triplicate. The One-way and Two-way ANOVA with Tukey post hoc were utilized to analyze the results of the MTT and RT-qPCR tests, respectively. A p-value less than 0.05 is considered significant.

Results

The proliferation of cells on days 3, 5, and 7 was not significantly different among different experimental groups (P > 0.05). A higher expression of the PLAP1, SCX, and COL3A1 have been seen in groups with sequential use of growth factors; among these groups, the group using 5 ng/mL of TGF- $β 3$ led other groups with the most amount of significant upregulation in PLAP1(17.69 ± 1.11 fold; P < 0.0001), SCX (5.71 ± 0.38 fold; P < 0.0001), and COL1A3 (6.35 ± 0.39 fold; P < 0.0001) expression, compared to the control group. The expression of the RUNX2 decreased in all groups compared to the control group; this reduction was more in groups with sequential use of growth factors.

Conclusion

The sequential use of growth factors can be more effective than simultaneous use in teno/ligamentogenic differentiation of PDLSCs. Moreover, treatment with 5 ng/mL TGF-β3 after FGF-2 was more effective than TGF- $β$ 1.

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同时和依次使用 TGF-β1 和 TGF-β3 与 FGF-2 对牙周韧带干细胞韧带分化的影响

目的牙周韧带是牙周的重要组成部分，其再生具有挑战性。本研究比较了同时和先后使用 FGF-2 和 TGF-β1 与 FGF-2 和 TGF-β3 对牙周韧带干细胞（PDLSCs）韧带分化的影响。对照组只含 PDLSCs；FGF-2 组含 PDLSCs，培养基中添加 FGF-2（50 ng/mL）。在其他实验组中，不同浓度（5 ng/mL 或 10 ng/mL）的 TGF-β1&-β3 同时或依次与 FGF-2 结合作用于培养的 PDLSCs。连续组在第 3 天后在培养基中加入 TGF-β。第 3、5 和 7 天进行甲基噻唑基四氮唑（MTT）检测，第 7 天后进行定量实时聚合酶链反应（RT-qPCR）分析，以研究 PLAP1、SCX 和 COL3A1、RUNX2 基因。所有实验均一式三份。MTT 和 RT-qPCR 试验结果分别采用单因素方差分析和双因素方差分析，并进行 Tukey post hoc 分析。结果第 3、5 和 7 天细胞的增殖在不同实验组之间无明显差异（P > 0.05）。在连续使用生长因子的实验组中，PLAP1、SCX 和 COL3A1 的表达量较高；在这些实验组中，使用 5 ng/mL TGF-β3 的实验组的 PLAP1 表达量明显高于其他实验组（17.69±1.11倍；P< 0.0001）、SCX（5.71±0.38倍；P< 0.0001）和COL1A3（6.35±0.39倍；P< 0.0001）的表达量均高于对照组。与对照组相比，所有组中 RUNX2 的表达都有所下降；在连续使用生长因子的组中，这种下降幅度更大。此外，在 FGF-2 之后使用 5 ng/mL TGF-β3 比 TGF-β1 更有效。

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来源期刊

Archives of oral biology 医学-牙科与口腔外科

CiteScore

5.10

自引率

3.30%

发文量

177

审稿时长

26 days

期刊介绍： Archives of Oral Biology is an international journal which aims to publish papers of the highest scientific quality in the oral and craniofacial sciences. The journal is particularly interested in research which advances knowledge in the mechanisms of craniofacial development and disease, including: Cell and molecular biology Molecular genetics Immunology Pathogenesis Cellular microbiology Embryology Syndromology Forensic dentistry