The 532 nm Laser Treatment Promotes The Proliferation of Tendon-Derived Stem Cells and Up-Regulates Nr4a1 To Stimulate Tenogenic Differentiation
Abstract Background The combination of low-level laser therapy (LLLT) and stem cell transplantation with tendon-derived stem cells (TDSCs) as seed cells provides a new treatment strategy for tendon injury. Nevertheless, the effect of LLLT on the biological behavior of TDSCs and its internal mechanisms remain unclear. The purpose of this study was to verify the effect of LLLT with a wavelength of 532 nm on the proliferation and differentiation of TDSCs of Sprague-Dawley (SD) rats. Methods TDSCs were isolated from Achilles tendons of SD rats and identified by cell morphology and flow cytometric analysis. Energy density gradient experiment was performed to determine the ideal energy. Then TDSCs were treated with LLLT using a wavelength of 532 nm at a fluence of 15J/cm2. Cell response after irradiation was observed at 6, 12 and 24 hours to ascertain cell morphology and cell proliferation. RT-PCR was used to detect the RNA expression levels of the key genes of TDSCs differentiation including Scx, Tnmd, Mkx and Dcn, PPARγ, Sox9 and Runx2. Then gene chip microarray was used to detect the expression of differential genes after 532 nm laser intervention in TDSCs, and the target genes were screened out to verify the role of target genes in this process. Results When the 532 nm laser energy density was 15 J/cm2, the proliferation capacity of TDSCs was improved (2.73 ± 0.24 vs. 1.81 ± 0.71, P < 0.05), and the expression of genes related to tenogenic differentiation of TDSCs was significantly increased (P < 0.01), showing the potential of tenogenic differentiation. After RNA-seq and bioinformatics analyses, we speculated that Nr4a1 was involved in the tenogenic differentiation process of TDSCs regulated by 532 nm laser treatment. Subsequent experiments confirmed that Nr4a1 regulated the expression of the tenogenic differentiation genes scleraxis (Scx) and tenomodulin (Tnmd) in TDSCs, affecting the process. Conclusion A 532 nm laser with 15J/cm2 regulated the process of TDSC proliferation and tenogenic differentiation by up-regulating Nr4a, which could accelerate tendon healing.