自组装肽 P-4 影响根尖乳头干细胞 (SCAP) 的活力和成骨分化。

The Self-assembling peptide P-4 influences viability and osteogenic differentiation of stem cells of the apical papilla (SCAP).

机构信息

Department of Restorative Dentistry, Dental Materials Division, University of Campinas, Av Limeira, 901. CEP 13.414-903, Piracicaba, São Paulo, Brazil.

Department of Biosciences, Physiological Sciences Division, University of Campinas, Av Limeira, 901. CEP 13.414-903, Piracicaba, São Paulo, Brazil.

出版信息

J Dent. 2023 Jul;134:104551. doi: 10.1016/j.jdent.2023.104551. Epub 2023 May 16.

DOI:10.1016/j.jdent.2023.104551

PMID:37201776

Abstract

OBJECTIVE

To analyze the effect of P-4 self-assembly peptide on cell viability and osteogenic capacity of SCAPs through mineral deposition and gene expression of osteogenic markers.

METHODS

SCAPs were seeded in contact with P-4 (10 µg/ml, 100 µg/ml and 1 mg/ml) solution. Cell viability was evaluated using a colorimetric assay MTT: 3-(4,5-dimethyl-thiazolyl-2)-2,5- diphenyltetrazolium bromide) in an experimental time of 24, 48 and 72 h (n = 7). Mineral deposition and quantification provided by the cells was tested using the Alizarin Red staining and Cetylpyridinium Chloride (CPC), respectively, after 30 days (n = 4). Gene expression of Runt-related transcription factor 2 (RUNX2), Alkaline phosphatase (ALP) and Osteocalcin (OCN) was quantified using quantitative polymerase chain reaction (RT-qPCR), at 3 and 7 days with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene, and relative gene expression was measured using the ΔΔCq method. Data were analyzed using Kruskall-Wallis followed by multiple comparisons, and T-test for gene expression with α=0.05.

RESULTS

All tested concentrations (10 µg/ml, 100 µg/ml and 1 mg/ml) were not cytotoxic at time 24 and 48 h. After 72 h, a slight decrease in cell viability was observed for the lowest concentration (10 µg/ml). The concentration of 100 µg/ml P-4 showed the highest mineral deposition. However, qPCR analysis of P-4 (10 µg/ml) showed upregulation of RUNX2 and OCN at 3 days, with downregulation of ALP at 3 and 7d CONCLUSION: P-4 did not affect cell viability, induced mineral deposition in SCAPs, and upregulated the expression of RUNX2 and OCN genes at 3 days, while downregulating ALP expression at 3 and 7 days.

CLINICAL SIGNIFICANCE

Based on the results obtained in this study it can be stated that self-assembling peptide P-4 is a potential candidate to induce mineralization on dental stem cells for regenerative purposes and also for a clinical use as a capping agent without compromising the cells health.

摘要

目的

通过矿化沉积和骨形成标志物基因表达来分析 P-4 自组装肽对 SCAPs 细胞活力和成骨能力的影响。

方法

将 SCAPs 与 P-4（10μg/ml、100μg/ml 和 1mg/ml）溶液接触。通过 24、48 和 72 小时的 MTT（3-(4,5-二甲基噻唑基-2)-2,5-二苯基四氮唑溴化物）比色测定法评估细胞活力（n=7）。使用茜素红染色和十六烷基吡啶氯（CPC）分别测试细胞矿化和定量，在第 30 天（n=4）时。使用定量聚合酶链反应（RT-qPCR）定量检测 runt 相关转录因子 2（RUNX2）、碱性磷酸酶（ALP）和骨钙素（OCN）的基因表达，以甘油醛-3-磷酸脱氢酶（GAPDH）为管家基因，在第 3 天和第 7 天进行测量，并使用 ΔΔCq 法测量相对基因表达。使用 Kruskal-Wallis 进行数据分析，随后进行多重比较，并用 T 检验进行基因表达分析，α=0.05。

结果

在 24 和 48 小时时，所有测试浓度（10μg/ml、100μg/ml 和 1mg/ml）均无细胞毒性。在 72 小时时，最低浓度（10μg/ml）观察到细胞活力略有下降。100μg/ml P-4 浓度显示出最高的矿化沉积。然而，P-4（10μg/ml）的 qPCR 分析显示，RUNX2 和 OCN 在第 3 天上调，而 ALP 在第 3 天和第 7 天下调。