Moderate treadmill running exercise prior to tendon injury enhances wound healing in aging rats

Abstract

The effect of exercise on wound healing in aging tendon was tested using a rat moderate treadmill running (MTR) model. The rats were divided into an MTR group that ran on a treadmill for 4 weeks and a control group that remained in cages. After MTR, a window defect was created in the patellar tendons of all rats and wound healing was analyzed. We found that MTR accelerated wound healing by promoting quicker closure of wounds, improving the organization of collagen fibers, and decreasing senescent cells in the wounded tendons when compared to the cage control. MTR also lowered vascularization, increased the numbers of tendon stem/progenitor cells (TSCs) and TSC proliferation than the control. Besides, MTR significantly increased the expression of stem cell markers, OCT-4 and Nanog, and tenocyte genes, Collagen I, Collagen III and tenomodulin, and down-regulated PPAR-γ, Collagen II and Runx-2 (non-tenocyte genes). These findings indicated that moderate exercise enhances healing of injuries in aging tendons through TSC based mechanisms, through which exercise regulates beneficial effects in tendons. This study reveals that appropriate exercise may be used in clinics to enhance tendon healing in aging patients.

Keywords: Gerotarget; aging rat; proliferation; tendon stem cell; treadmill running; wound healing.

Figure 1. The effect of MTR on gross morphology of aging rat patellar tendons

A. Young tendon (3 months); B. Aging tendon (20 months); C. Aging tendon after MTR. Compared to young tendon A. aging tendon B. appears vascular and less intact. After MTR C., aging tendon has regained its intact structure and has decreased number of blood vessels.

Figure 2. The effect of MTR on the structure and cellularity of aging rat patellar tendon

A. Tendon section from cage control aging rat. B. Tendon section from aging rat after MTR. It is evident that MTR increases the number of typical tendon cells that are more elongate (green arrows) B. and decreases the number of round shaped cells presumably non-tenocytes (red arrows) A.. Insets represent magnified sections. Bars: 50 μm.

Figure 3. The effect of MTR on wound healing in aging rat patellar tendon

A., C. Control aging tendons. B., D. Tendons from aging rats after MTR. At 4 weeks, the window defect in control aging rats A. appears bruised and vascular. But MTR accelerated healing of the wound B., which is evidently smaller and seems to be closing (blue arrow). At 8 weeks, the wound in the control group still looks bruised and vascular (red arrow) C., whereas in the MTR group D. the wound for the most part appears to be healed.

Figure 4. Histological analysis of wound healing in aging rat patellar tendons

A., C. Control aging tendon; B., D. Aging tendon from rats after MTR. At 4 weeks, the control aging tendons have large numbers of lipid-like structures in the wound site (green arrow) A.. But these structures are reduced in aging tendons after the MTR regimen (yellow arrow) B.. At 8 weeks, the control aging tendon appears to be healing but the lipid-like structures still remain C. while in the MTR group, the normal-like tendon structure is regained in aging tendons, which have well organized matrix and evenly distributed cells D.. Bars: 200 μm.

Figure 5. β-gal staining of healing wound in aging rat patellar tendon

At 4 weeks, over 90% of wound area in the control aging tendons stained positive for the senescence marker, β-gal A.-E.; however, less than 30% of wound area in MTR group was positive for β-gal B.-F.. At 8 weeks, senescent marker expressing cells were still found in control aging tendons C.-G., while only a few cells in MTR group were positive for β-gal D.-H.. Red bars: 500 μm; Black bars: 200 μm.

Figure 6. MTR increases the colony number and colony size of TSCs from aging rat patellar tendons

A., B., C. - TSCs from control aging rats; D., E., F. - TSCs from aging rats after MTR. The number of colonies in control aging rats A. is much smaller than those in aging rats after MTR regimen D.. The number of cells per colony was also lower in the control group B., C. than MTR group E., F.. Note that the cells in MTR group F. are cobblestone shaped, the typical shape of “authentic” TSCs, while many cells in cage control group C. are spindle shaped. Bars: 100 μm.

Figure 7. MTR enhances the proliferative potential of aging rat TSCs

TSCs were isolated both from MTR and control groups. It is shown that TSCs in MTR group grew much quicker than control group because the former had a reduced PDT compared to control group (*P < 0.05).

Figure 8. MTR enhances stem cell marker expression in TSCs from aging rats

Expression of the three stem cell markers, OCT-4, Nanog and nucleostemin (NS) is minimal in the control aging rat TSCs A., C., E. but their expression level is increased in aging rats on an MTR regimen B., D., F.. Semi-quantification G. of these stem cell markers by counting the positively stained cells showed that MTR increased OCT-4 and Nanog expression but not NS. Bars: 100 μm, *P < 0.05 MTR vs. the respective control.

Figure 9. MTR enhance both stem cell and tenocyte related marker expression but decreases non-tenocyte related gene expression in aging rats

A. Stem cell marker genes, OCT-4 and Nanog; B. Tenocyte related genes, Collagen I, Collagen III, and tenomodulin; C. Non-tenocyte related genes, PPAR-γ, Collagen II, and Runx-2. Compared to the control, MTR markedly increased the expression of both stem cell marker genes as well as tenocyte related genes. In contrast, all three non-tenocyte related genes decreased > 50% when aging rats were subjected to MTR. *P < 0.05 MTR compared to the respective control.