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Effect of Periodontal Ligament Stem Cells-Derived Conditioned Medium on Gene Expression and Differentiation of Tumor Necrosis Factor-α-Challenged Osteoblasts
Objectives Tumor necrosis factor-α (TNF-α) causes bone resorption in periodontitis. It induces the production of receptor activator of NF-κB ligand (RANKL) from osteoblasts, leading to the disturbance of bone homeostasis through RANKL, RANK, and osteoprotegerin (OPG) axis. This study aimed to explore the effect of periodontal ligament stem cells-derived conditioned medium (PDLSCs-CM) on gene expression related to bone homeostasis and the differentiation of TNF-α-challenged osteoblasts.
Materials and Methods Human osteoblasts were cultured with 50 ng/mL of TNF-α and 0, 1, 10, and 100 µg/ mL of PDLSCs-CM. Osteoblasts cultured without TNF-α and PDLSCs-CM were served as control. Gene expression of RANKL, OPG, and interleukin-1β (IL-1β) was evaluated by reverse transcription quantitative polymerase chain reaction at 48 hours. The early-stage and late-stage differentiation of TNF-α-challenged osteoblasts without or with PDLSCs-CM was explored by alkaline phosphatase (ALP) activity and alizarin red staining, respectively, at day 1, 3, 6, 9, and 12.
Statistical Analysis Mann–Whitney U test was used to analyze the differences in gene expression of TNF-α-challenged osteoblasts at 24 and 48 hours, and Kruskal–Wallis test was used to analyze the effect of PDLSCs-CM on gene expression and ALP activity among all experimental groups using SPSS software version 21.0. Statistical significance was considered with p-value less than 0.05.
Results Expression of RANKL, OPG and IL-1β was significantly upregulated in TNF-α-challenged osteoblasts compared to the untreated control. The PDLSCs-CM at 1 and 10 μg/mL downregulated gene expression of TNF-α-challenged osteoblasts compared to the group without PDLSCs-CM, but the difference did not reach statistical significance. The ALP activity was decreased in TNF-α-challenged osteoblasts. The addition of PDLSCs-CM did not alter ALP activity of TNF-α-challenged osteoblasts. Alizarin red staining was comparable in the TNF-α-challenged osteoblasts cultured without or with PDLSCs-CM.
Conclusions The PDLSCs-CM did not alter gene expression involved in bone homeostasis and differentiation of TNF-α-challenged osteoblasts.
Keywordsconditioned medium - mesenchymal stem cells - osteoblasts - osteoprotegerin - RANKL - tumor necrosis factor-alpha
All authors have made substantial contributions to conception and design of the study. P.V. and S.S.S. have been involved in data collection and data analysis. P.V., S.S.S., S.R., and H.S. have been involved in data interpretation, drafting the manuscript and revisiting it critically and have given final approval of the version to be published.
Article published online:
10 August 2023
© 2023. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)
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