Effect of 660-nm LED photobiomodulation on the proliferation and chondrogenesis of meniscus-derived stem cells (MeSCs)

Abstract

Meniscus-derived stem cells (MeSCs), a unique type of MSC, have outstanding advantages in meniscal cytotherapy and tissue engineering, but the effects and molecular mechanisms of PBM on MeSCs are still unclear. We used 660-nm LED light with different energy densities to irradiate six human MeSC samples and tested their proliferation rate via cell counting, chondrogenic differentiation capacity via the DMMB assay, mitochondrial activity via the MTT assay, and gene expression via qPCR. The proliferation ability, chondrogenic capacity and mitochondrial activity of the 18 J/cm² group were greater than those of the 4 J/cm² and control groups. The mRNA expression levels of Akt, PI3K, TGF-β3, Ki67 and Notch-1 in the 18 J/cm² group were greater than those in the other groups in most samples. After chondrogenic induction, the expression of Col2A1, Sox9 and Aggrecan in the 18 J/cm² group was significantly greater than that in the 4 J/cm² and control groups in most of the samples. The variation in the MTT values and Src, PI3K, Akt, mTOR and GSK3β levels decreased with time. The results showed that 660-nm LED red light promoted proliferation and chondrogenic differentiation and affected the gene expression of MeSCs, and the effects on gene expression and mitochondrial activity decreased with time.

Figure 1

The MeSCs were expanded in cell culture flasks until passage 3. The energy output in the area of light exposure was measured with a power meter.

Figure 2

MeSCs were subcultured every 10 days, and NCPD and CPDT measurements were performed after each passage. Compared with those in the 4 J/cm² and control groups, the proliferative capacity of the MeSCs in the 18 J/cm² group was greater at passage 3. n = 3; *p < 0.05, **p < 0.01.

Figure 3

MeSCs were subjected to the MTT assay at 24 h after irradiation, with the absorbance at 570/630 nm. n = 3; *p < 0.05, **p < 0.01.

Figure 4

MeSCs were subjected to the MTT assay at 4 and 24 h after irradiation, at which point the absorbance at 570/630 nm was measured. (a) Four hours after irradiation, the MTT values of the 18 and 30 J/cm² groups were significantly greater than those of the control group; (b) Twenty-four hours after irradiation, the MTT values of the 18 and 30 J/cm² groups were significantly greater than those of the control group; (c) The MTT value at 4 h after irradiation was greater than that at 24 h. n = 3; *p < 0.05, **p < 0.01.

Figure 5

Comparison of the effects of the PBM at different power densities. DMMB assays were used to determine the production of GAGs in differentiated MeSCs. (a) The values in the 18 and 4 J/cm² groups were greater than those in the control group for all samples, but there was no significant difference between the 18 and 4 J/cm² groups. n = 3; *p < 0.05, **p < 0.01. (b) Alcian blue staining; scale bars = 500 μm.

Figure 6

Gene expression results.

Figure 7

Gene expression results.