Tendon-Derived Stem Cell Differentiation in the Degenerative Tendon Microenvironment

Abstract

Tendinopathy is prevalent in athletic and many occupational populations; nevertheless, the pathogenesis of tendinopathy remains unclear. Tendon-derived stem cells (TDSCs) were regarded as the key culprit for the development of tendinopathy. However, it is uncertain how TDSCs differentiate into adipocytes, chondrocytes, or osteocytes in the degenerative microenvironment of tendinopathy. So in this study, the regulating effects of the degenerative tendon microenvironment on differentiation of TDSCs were investigated. TDSCs were isolated from rat Achilles tendons and were grown on normal and degenerative (prepared by stress-deprived culture) decellularized tendon slices (DTSs). Immunofluorescence staining, H&E staining, real-time PCR, and Western blot were used to delineate the morphology, proliferation, and differentiation of TDSCs in the degenerative microenvironment. It was found that TDSCs were much more spread on the degenerative DTSs than those on normal DTSs. The tenocyte-related markers, COL1 and TNMD, were highly expressed on normal DTSs than the degenerative DTSs. The expression of chondrogenic and osteogenic markers, COL2, SOX9, Runx2, and ALP, was higher on the degenerative DTSs compared with TDSCs on normal DTSs. Furthermore, phosphorylated FAK and ERK1/2 were reduced on degenerative DTSs. In conclusion, this study found that the degenerative tendon microenvironment induced TDSCs to differentiate into chondrogenic and osteogenic lineages. It could be attributed to the cell morphology changes and reduced FAK and ERK1/2 activation in the degenerative microenvironment of tendinopathy.

Figure 1

The morphology and stemness characteristics of TDSCs: (a) morphology and (b) colon-formation ability of TDSCs and flow cytometric analysis of (c) CD44, (d) CD90, (e) CD45, and (f) CD106 expression of TDSCs.

Figure 2

Multidifferentiation potential of TDSCs: (a-b) Oil Red O staining, (c-d) Safranin O staining, and (e-f) Alizarin Red S staining.

Figure 3

Live/dead, H&E staining, and MMP expressions of normal and degenerative tendons. Live/dead staining of (a) normal and (b) degenerative tissues. H&E staining of (c-d) normal and (e-f) degenerative tendons. (g) Comparison of expressions of MMP 1 and MMP 2 in normal and degenerative tendons (n = 8). ^∗ P < 0.05, compared with normal tendons.

Figure 4

Decellularization of normal and degenerative tendons. DAPI staining of (a-b) normal and (c-d) degenerative DTSs before and after decellularization, respectively. (e) DNA content of normal and degenerative DTSs before and after decellularization. ^∗ P < 0.05, compared with the after decellularization group. The error bars represent the standard deviation of measurements for 5 separated samples (n = 5). (f) Elastic modulus of normal and degenerative DTSs. ^∗ P < 0.05, compared with normal DTSs. The error bars represent the standard deviation of measurements for 6 points in 3 separated samples (n = 18).

Figure 5

Live/dead staining and proliferative ability of TDSCs on normal and degenerative DTSs. Live/dead staining of TDSCs on normal DTSs at (a) day 1, (b) day 7, and (c) day 14. And live/dead staining of TDSCs on degenerative DTSs at (d) day 1, (e) day 7, and (f) day 14. (g) Proliferative ability assessment of TDSCs on normal and degenerative DTSs. ^∗ P < 0.05, compared with normal DTSs. The error bars represent the standard deviation of measurements for 3 replicates of 3 separated samples (n = 9).

Figure 6

F-actin (labeled with Alexa488-phalloidin) and nucleus (labeled with DAPI) staining and merged images of TDSCs. TDSCs on (a–f) normal and (g–l) degenerative DTSs magnified 100 and 200 times, respectively.

Figure 7

The differentiation of TDSCs toward tenogenic, chondrogenic, and osteogenic lineages. Relative gene expression of tenocyte markers COL1, SCX, and TNMD, chondrocyte markers COL2 and SOX9, and osteocyte markers Runx2 and ALP. The results were represented as the calculated comparative expression ratios of normal and degenerative DTS groups to the culture dish group. ^∗ P < 0.05, compared with normal DTSs. The error bars represent the standard deviation of measurements for 2 replicates of 8 separated samples (n = 16).

Figure 8

Gene and protein expression of FAK and ERK: (a) Relative gene expressions of integrin β1, FAK, and ERK and (b) Western blot from whole-cell lysates showing expressions of phosphorylated FAK^Tyr397 and phosphorylated ERK1/2^{Thr202/Tyr204} in TDSCs on a culture dish (as the control) and normal and degenerative DTSs. ^∗ P < 0.05, compared with normal DTSs. The error bars represent the standard deviation of measurements for 2 replicates of 8 separated samples (n = 16).