Table 1.
A list of primers used for gene expression analysis.
Figure 1.
The gross appearance of mouse tendons after treadmill running.
A–C. Patellar tendons. D–F. Achilles tendons. It is evident that without treadmill running (or cage control), both mouse patellar tendon (A) and Achilles tendon (D) were shiny and avascular (arrows). After moderate treadmill running (MTR), the normal appearance of both patellar and Achilles tendons (B, E) apparently remained unchanged (arrows). After intensive treadmill running (ITR), the areas in and around the patellar and Achilles tendons appeared more vascularized, instead of a normal glistening and white appearance, suggesting neo-vascularization (C, F; arrows) had occurred.
Figure 2.
MGF expression in mouse patellar (A) and Achilles (B) tendons in response to moderate treadmill running (MTR) and intensive treadmill running (ITR).
Compared to non-running controls, MTR upregulated MGF gene expression in patellar tendons by more than 5-fold, whereas ITR increased the same gene expression by over 17-fold. Similarly, MGF increased 5 times in the Achilles tendons of the MTR group and 7 times in the ITR group (Data are shown as mean ± SD, n = 6. (*p<0.05 with respect to control; #p<0.05 with respect to MTR).
Figure 3.
The morphology and colony formation of TSCs isolated from mouse tendons with and without treadmill running.
A–C. Patellar tendon stem cells (PTSCs); and D–F. Achilles tendon stem cells (ATSCs). The stem cells were isolated from the same amounts of patellar and Achilles tendon samples of mice subjected to treadmill running and cage controls. With MTR, and more so with ITR, both PTSCs and ATSCs grew more quickly and formed larger colonies than cage controls. Bars: 100 µm.
Figure 4.
The population doubling time (PDT) of TSCs isolated from mouse tendons.
TSCs were seeded in a 6-well plate and cultured to determine PDT. Mouse treadmill running enhanced proliferation of TSCs from patellar tendons (A) and Achilles tendons (B), as indicated by decreased PDT compared to cage controls. In fact, ITR stimulated cell growth more quickly than MTR (A, B) (*p<0.05 with respect to cage control; #p<0.05 with respect to MTR).
Table 2.
Number of TSCs cultured from patellar tendons of mice subjected to treadmill running.
Table 3.
Number of TSCs cultured from Achilles tendons of mice subjected to treadmill running.
Figure 5.
The expression of stem cell markers by patellar tendon stem cells (PTSCs) in response to mouse treadmill running.
TSCs were seeded in a 6-well plate, cultured and incubated with goat anti-mouse nucleostemin/Cy3-conjugated donkey anti-goat IgG antibodies (A, D, G), rabbit anti-mouse Oct-4/Cy3-conjugated donkey anti-rabbit IgG antibodies (B, E, H) or FITC-conjugated mouse anti-SSEA-1 (C, F, I). Compared to PTSCs from cage control mice (A–C), more cells in MTR (D–F) and ITR (G–I) groups had increased expression of stem cell markers nucleostemin (NS), Oct-4, and SSEA-1. Note that the extent of the immunostaining on these stem cell markers is apparently running-intensity-dependent. Positively stained cells were also counted to calculate percentage staining (J). (*p<0.05 with respect to control; #p<0.05 with respect to MTR). Bar: 100 µm.
Figure 6.
The expression of stem cell markers by Achilles tendon stem cells (ATSCs) in response to mouse treadmill running.
TSCs were seeded in a 6-well plate, cultured and incubated with goat anti-mouse nucleostemin/Cy3-conjugated donkey anti-goat IgG antibodies (A, D, G), rabbit anti-mouse Oct-4/Cy3-conjugated donkey anti-rabbit IgG antibodies (B, E, H) or FITC-conjugated mouse anti-SSEA-1 (C, F, I). Compared to ATSCs from cage control mice (A–C), more cells in MTR (D–F) and ITR (G–I) groups increased the expression of stem cell markers nucleostemin (NS), Oct-4, and SSEA-1. It is apparent that the extent of the immunostaining on these stem cell markers is running-intensity-dependent. Positively stained cells were also counted to calculate percentage staining (J), which shows no significant difference between the groups. Bar: 100 µm.
Figure 7.
The expression of tenocyte and non-tenocyte related genes in patellar (A) and Achilles (B) mouse tendons in response to treadmill running.
Total RNA collected from the tendons of controls and mice in the MTR and ITR groups were subjected to qRT-PCR. As shown, MTR only increased the expression of tenocyte related genes in the two types of tendons (Coll. I, or collagen type I; Tenom or tenomodulin), whereas ITR increased the expression of both tenocyte and non-tenocyte related genes (LPL: a marker for adipocyte; Sox9: a marker for chondrocyte; Runx2 and Osterix: two markers for osteoblasts). (*p<0.05 with respect to the corresponding controls; #p<0.05 with respect to MTR. In A, however, # in Coll. I represents p<0.05 with respect to ITR).
Figure 8.
The expression of tenocyte and non-tenocyte related genes in patellar (A) and Achilles (B) TSCs in response to mechanical loading in vitro.
Total RNA were collected from TSCs stretched to 4% or 8% for qRT-PCR analysis. In PTSCs under low mechanical loading (green, 4% stretching), only those genes related to tenocytes (Coll. I, or collagen type I; Tenom or tenomodulin) were highly expressed, but under high mechanical loading (red, 8% stretching), both tenocyte and non-tenocyte related genes increased their expression. Similar results were obtained for ATSCs in response to low (4%) and high (8%) mechanical loading. Note the different scale in gene expression by PTSCs and ATSCs between the two loading conditions (*p<0.05, with respect to non-loaded cells; #p<0.05 with respect to 4% stretching).
Figure 9.
The effects of mechanical loading on gene expression in the mouse patellar tenocytes in vitro.
Total RNA were collected from tenocytes stretched to 4% or 8% for qRT-PCR analysis. Both low (4%) and high (8%) mechanical stretching caused the expression of tenocyte-related genes (Coll. I, or Collagen type I; and Tenom or tenomodulin). However, regardless of the magnitude of the mechanical loading condition, the expression of non-tenocyte related genes LPL, Sox9, and Runx2 was not induced (*p<0.05, with respect to non-loaded cells; #p<0.05 with respect to 8% stretching).