This study aimed to verify the ability of TGF-β3 and mechanical strain to stimulate the differentiation of TMSCs into tenocytes. We hypothesized that TGF-β3 and mechanical strain induce higher expression of tenocyte-related genes and collagen as end products. TGF-β3 and mechanical strain stimulated TMSC differentiation into tenocytes, although the combination of the two did not induce significantly higher TMSC differentiation.
TMSCs have some advantages over MSCs obtained from other sources. TMSCs are waste tissues from tonsillectomies; thus, they can be obtained via less invasive procedures, as additional surgery is not necessary . Moreover, TMSCs can differentiate more stably than other MSCs, making them suitable for cell banking . Therefore, TMSCs can be easily applied to autografts or allografts . TMSCs have the potential to differentiate into a wide range of tissues. According to previous studies, TMSCs can be osteogenic, adipogenic, chondrogenic, myogenic, and tenogenic . Additionally, TMSCs can be applied in the field of tissue engineering, as the control of the inter- and extracellular environments is possible.
We previously reported that a low concentration of TGF-β3 can induce tenogenic differentiation of TMSCs and increase the expression of SCX, TNMD, and TNC . Similar results were obtained in this study when comparing the group treated with and without TGF-β3 in the static condition. However, in this study, an increase in the expression of SCX mRNA could not be observed when only TGF-β3 was used to treat TMSCs. Given that the protocol for TMSC stimulation using TGF-β3 was the same for this study and the previous one , we thought that the variations might have resulted from the differences in MSC sources. Regarding the expression of SCX, we believe that additional experiments will be required.
We measured the simultaneous effect of TGF-β3 and mechanical strain on TMSCs differentiation into tenocytes. In previous studies, TMSCs were used for chondrogenesis and adipogenesis using a modified 3D scaffold [19,20,21]. Park et al. reported that a 2D/3D hybrid cell culture system with TGF-β3 increases the expression of chondrogenic genes, such as SRY-Box transcription factor 9 (SOX9), COL II, COL II A1, and COL VII. Additionally, Patel et al. reported that a composite system of graphene oxide/polypeptide thermogel stimulates the expression of adipogenic genes, such as PPAR-γ, CEBP-α, LPL, AP2, ELOVL3, and HSL. However, there are insufficient studies regarding the tenogenic expression of TMSCs upon mechanical strain and TGF-β3 treatment, although Yu et al. reported that TGF-β3 induces tenogenesis of TMSCs.
As shown in Fig. 1, the mRNA expression of tenogenic genes, such as SCX, was significantly higher when the mechanical strain was applied than under static conditions. SCX is a transcription factor that leads to tenocyte differentiation and suppresses non-tenogenic capacity . These results are similar to those of previous studies in that mechanical strain stimulates the differentiation of MSCs into tenocytes. As shown in Fig. 3, the mRNA expression levels of osteogenic and chondrogenic genes were similar among the static control, 2, and 5% groups. Similarly, Zhang et al. reported that the expression of non-tenocyte-related genes was not significantly altered when mechanical loading was applied to tenocytes . Thus, we believe that mechanical strain can stimulate the differentiation of TMSCs, particularly to tenocytes.
In cells without TGF-β3, dsDNA concentration decreased, while the amount of normalized collagen increased as the intensity of mechanical strain increased. The concentration of dsDNA indicates the quantity of cell proliferation, whereas the amount of collagen as an end product indicates the extent of cell differentiation [24, 25]. The extent of cell proliferation and differentiation is inversely proportional . Therefore, mechanical strain without TGF-β3 stimulates cell differentiation rather than proliferation.
Even though SCX was increased with loading and TGF-β3 combinations at days 7 and 14, the two treatments did not have a significant synergistic effect on the expression of other genes. For example, in cells treated with TGF-β3, the mRNA expression level of COL1, COL3, and COL1/3 among mechanical strain groups except COL3 at day1 had no significant difference. Moreover, under TGF-β3 treatment, mRNA expression was lesser with mechanical strain than without mechanical strain for TNMD at day 14, TNC at day 14, and COL3 at day 1. The mRNA expression of TNC at day 14 and COL3 at day 1 decreased when mechanical strain was applied with TGF-β3 treatment, and the mRNA expression of TNMD at day 1 and DCN without TGF-β3 treatment was higher than that obtained under the combination of TGF-β3 with mechanical strain. Furthermore, in the combination case, the concentration of collagen as an end-product did not significantly change. Although TGF-β3 stimulates TMSC differentiation into tenocytes, the combination of TGF-β3 and mechanical strain does not appear to significantly increase the mRNA expression of tendon-related genes. This result concurs with those of previous studies, which showed that mechanical loading inhibits the differentiation of MSCs with TGF-β3 supplementation . Thorpe et al. reported that continuous dynamic compression from 0 to 42 days in the presence of TGF-β3 inhibits chondrogenesis, whereas delayed dynamic compression after TGF-β3 treatment from 0 to 21 days stimulates chondrogenesis . Therefore, the effect of mechanical loading on MSC differentiation can be manipulated according to the MSC stage.
Furthermore, previous studies have shown that mechanical strain activates the TGF-β3 pathway, which stimulates TMSC differentiation into tenocytes . In this study, TGF-β3 and mechanical strain were treated from the outside of the cell rather than measuring the amount of TGF-β3 expression inside the cell. Hence, this study offers a unique perspective in that the combination of TGF-β3 and mechanical strain did not significantly affect TMSC differentiation. However, the mRNA expression of tenocyte-related genes increased in cells treated with TGF-β3 compared to that in those lacking TGF-β3 treatment. Hence, this study had similar results to previous studies in that TGF-β3 stimulated the differentiation of TMSCs into tenocytes.
This study had some limitations. First, only TGF-β3 was used as a chemical stimulant for differentiation. Other chemicals, such as TGF-β1 or vascular endothelial growth factor, induce TMSC differentiation as well. A combination of TGF-β1 and TGF-β3 treatment can stimulate TMSC differentiation into tenocytes . Second, the effect of TGF-β3 and mechanical strain was only measured for 7 days. Long-term measurements are required to investigate the lasting effect of TGF-β3 and mechanical strain and any possible changes that could occur after a longer duration. Lastly, an immunocytochemistry assay was not conducted. Through immunocytochemistry, the presence of tenogenic proteins as a result of TMSC differentiation was verified . However, in this study, only collagen was measured as the end-product.