Low-intensity pulsed ultrasound enhances immunomodulation and facilitates osteogenesis of human periodontal ligament stem cells by inhibiting the NF-κB pathway

Abstract

Human periodontal ligament stem cells (hPDLSCs) are vital in cellular regeneration and tissue repair due to their multilineage differentiation potential. Low intensity pulsed ultrasound (LIPUS) has been applied for treating bone and cartilage defects. This study explored the role of LIPUS in the immunomodulation and osteogenesis of hPDLSCs. hPDLSCs were cultured in vitro, and the effect of different intensities of LIPUS (30, 60, and 90 mW/cm²) on hPDLSC viability was measured. hPDLSCs irradiated by LIPUS and stimulated by lipopolysaccharide (LPS) and LIPUS (90 mW/cm2) were co-cultured with peripheral blood mononuclear cells (PBMCs). Levels of immunomodulatory factors in hPDLSCs and inflammatory factors in PBMCs were estimated, along with determination of osteogenesis-related gene expression in LIPUS-irradiated hPDLSCs. The mineralized nodules and alkaline phosphatase (ALP) activity of hPDLSCs and levels of IκBα, p-IκBα, and p65 subunits of NF-κB were determined. hPDLSC viability was increased as LIPUS intensity increased. Immunomodulatory factors were elevated in LIPUS-irradiated hPDLSCs, and inflammatory factors were reduced in PBMCs. Osteogenesis-related genes, mineralized nodules, and ALP activity were promoted in LIPUS-irradiated hPDLSCs. The cytoplasm of hPDLSCs showed increased IκBα and p65 and decreased p-IκBα at increased LIPUS intensity. After LPS and LIPUS treatment, the inhibitory effect of LIPUS irradiation on the NF-κB pathway was partially reversed, and the immunoregulation and osteogenic differentiation of hPDLSCs were decreased. LIPUS irradiation enhanced immunomodulation and osteogenic differentiation abilities of hPDLSCs by inhibiting the NF-κB pathway, and the effect is dose-dependent. This study may offer novel insights relevant to periodontal tissue engineering.

Keywords: Human periodontal ligament stem cells; Immunomodulation; Low intensity pulsed ultrasound; NF-κB pathway; Osteogenic differentiation.

Fig. 1

LIPUS treatment increased hPDLSC viability. A expression of surface markers of hPDLSCs were detected using flow cytometry; B hPDLSCs were stained with alizarin red; C hPDLSCs were stained with oil red O; D viability of hPDLSCs measured on the 1st, 3rd, 5th, 7th, and 9th day after LIPUS irradiation using the CCK-8 assay; E viability of hPDLSCs irradiated by different intensities of LIPUS (30, 60, and 90 mW/cm²) on the 9th day measured using the CCK-8 assay. Cellular experiments were repeated three times. Data are presented as the mean ± standard deviation and were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test, **p < 0.01 vs. control group

Fig. 2

LIPUS promoted immunoregulation of hPDLSCs. A mRNA expression of immunoregulatory factors (TGF-β, IDO, and IL-10) in hPDLSCs detected using RT-qPCR; B mRNA expression of inflammatory factors (TNF-α, IFN-γ, and IL-lα) in PBMCs measured using RT-qPCR; C, D proteins level of immunoregulatory factors (TGF-β, IDO, and IL-10) in the supernatant of hPDLSCs and inflammatory cytokines (TNF-A, IFN-y, IL-1α) in the supernatant of PBMCs was detected using ELISA; Cell experiments were repeated three times. Data are presented as mean ± standard deviation and were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test, ** p < 0.01, * p < 0.05

Fig. 3

LIPUS facilitated osteogenic differentiation of hPDLSCs. A mRNA expression of osteogenic differentiation-related genes (Runx2, OPN, OSX, and OCN) detected using RT-qPCR; B number of mineralized nodules in hPDLSCs measured using alizarin red staining; C ALP activity of hPDLSCs measured using ALP staining. Cell experiments were repeated three times. Data are presented as mean ± standard deviation and were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test, **p < 0.01, * p < 0.05

Fig. 4

LIPUS promoted immunoregulation and osteogenic differentiation of hPDLSCs by inhibiting the NF-κB signaling pathway. A: protein levels of IκBα, p-IκBα, p65, and p-p65 examined using western blotting; B entry of p65 into nuclei detected using immunofluorescence; C proteins level of immunoregulatory factors (TGF-β, IDO, and IL-10) in hPDLSCs and inflammatory cytokines (TNF-α, IFN-γ, IL-1α) in PBMCs was detected using ELISA; D mRNA levels of immunoregulatory factors (TGF-β, IDO, and IL-10) in hPDLSCs and inflammatory factors (TNF-α, IFN-γ, and IL-lα) in PBMCs in co-culture system were measured using RT-qPCR; E mRNA levels of osteogenic differentiation-related genes (Runx2, OPN, OSX, and OCN) in hPDLSCs in co-culture system were detected using RT-qPCR; F protein levels of osteogenic differentiation-related genes (Runx2, OPN, OSX, and OCN) in hPDLSCs in co-culture system were detected using WB. G number of mineralized nodules in hPDLSCs in co-culture system counted using alizarin red staining; H ALP activity of hPDLSCs in co-culture system was measured using ALP staining. Cellular experiments were repeated three times. Data are presented as mean ± standard deviation and were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test, **p < 0.01, * p < 0.05