Table 1.
Mouse primer sequences for qRT-PCR.
Fig 1.
The properties of TSCs differ from that of TNCs isolated from GFP mouse patellar tendons.
TSCs exhibit cobble stone shape (A) and TNCs are a mix of elongated and spindle-like cells (yellow and red arrows, B). Immunostaining shows that nucleostemin (NS) is highly expressed in TSCs (C, E), but NS expression is much lower in TNCs (D, F). The images of E and F are enlarged images of the boxes in C and D, respectively. Semi-quantification of positively stained cells shows that 89% of TSCs express NS and about 12% of TNCs express NS (G). Graph bars represent the mean ± SD. *p < 0.05 compared to TSCs. White bars: 100 μm; yellow bars: 25 μm.
Fig 2.
Dead cells are predominant in irradiated mouse patellar tendons.
(A) A normal, untreated mouse has black hair. (B, C) Live/dead cell viability assay results show that normal mouse patellar tendon sections have more than 98% live cells as evidenced by green stained cell numbers and a few cells are dead cells which were stained by red fluorescence. (D) An irradiated (6 Gy) mouse exhibits loss of hair and change in hair color to brown or grey. (E, F) Live/dead cell viability assay results show that irradiation has killed most of the cells as evidenced by red fluorescence. n = 3 wild type untreated mice (A-C), and n = 3 irradiated wild type mice (D-F). Red bars: 50 μm; white bars: 100 μm.
Fig 3.
Cells are viable after TSCs and TNCs are injected into irradiated patellar tendons.
A, B: Cell viability tested by live/dead assay kit. C-D: GFP expression from injected patellar tendons due to either GFP-TSCs (C, blue: DAPI staining) or GFP-TNCs (D, blue: DAPI staining). E-F: GFP-cell expression tested by immunostaining (blue: DAPI staining). G-H: Merged images of the C, D with E, F (only green with red for clarity). After one week, many cells are alive in the tendons injected with TSCs (green fluorescent cells in A) and TNCs (green fluorescent cells in B). GFP is highly expressed in tendon tissues injected either with TSCs (C) or TNCs (D) (white arrows in C and D). Immunostaining further demonstrated that these green fluorescent cells are GFP positive cells (yellow arrows in E, F) as evidenced by merged images expressing both green and red fluorescence (yellow fluorescence in G, H). For mice, n = 3 for both groups, meaning 3 irradiated GFP-TSC mice (top row) and 3 irradiated GFP-TNC mice (bottom row) were used in these experiments. Yellow bars: 50 μm; white bars: 100 μm.
Fig 4.
Cell morphological changes in tendon tissue sections after irradiation, injection, and treadmill running.
The H&E staining results show that normal cells (A, B) maintain the elongated shape (blue arrows in B) and irradiated cells (C, D) increase in density (red arrows in D). The cells either in TSCs injected tissues (E, F) or in TNCs injected tissues (G, H) do not change the shape in cage control condition (black arrows in F, H). Also, the cells either in TSCs injected tissues (I, J) or in TNCs injected tissues (K, L) do not change the shape after MTR (green arrows in J, L). However, the cells in tendon tissues injected with GFP-TSCs (M, N) assume a round shape (yellow arrows in N) after ITR. In contrast, the cells in TNCs-injected tissues (O, P) do not change shape (white arrows in P) after ITR. The images in the second and fourth columns are enlarged images of the boxes in the first and third columns, respectively. Red bars: 500 μm; white bars: 25 μm. MTR: 13 m/min, 50 min/day, 5 days a week for 3 weeks after a week’s training; ITR: 13 m/min, 3 hrs, 4 hrs, and 5 hrs/day in the 2nd, 3rd, and 4th week respectively after a week’s training. N = 6 untreated control mice were utilized in A, B, and n = 3 irradiation mice were utilized in C, D. For irradiated cage, MTR, and ITR mice, n = 3 mice (6 tendons) with GFP-TSCs, and n = 3 mice (6 tendons) with GFP-TNCs for each group were used.
Fig 5.
ITR induces chondrogenic differentiation of TSCs in irradiated tendons.
Histochemical staining by Alcian blue and nuclear fast red shows that the cells in cage control tendons injected either with TSCs (A, B) or with TNCs (C, D) are still in elongated shape (blue arrows in A, C), and very few cells in cage control groups are positive for Alcian blue staining (A-D). Furthermore, both TSCs and TNCs in MTR tendons are still elongated shape (black arrows in F, H) and a few TSCs are positively stained by Alcian blue after MTR (E, F). However, some cells in ITR tendons are round shaped (white arrows in I, J), and many TSCs are positively stained by Alcian blue (I, J) after ITR. The TNCs are still in elongated shape (red arrows in K, L) and very little positive staining by Alcian blue (K, L). Semi-quantification of the chondrocyte staining shows that 66% of TSCs are positively stained with Alcian blue, and only 5% TNCs after ITR, and very few cells in cage control tendons and TSCs in MTR tendons are positively stained by Alcian blue (M). Graph bars represent the mean ± SD. *p < 0.05 compared to normal tendon. For each irradiated cage, MTR, and ITR group, n = 6 mice as described in Fig 5. Red bars: 50 μm; black bars: 12.5 μm.
Fig 6.
ITR induces significant non-tenocyte related gene expression in TSCs of irradiated tendons.
Both MTR and ITR induce significant expression of tenocyte related genes, collagen I and tenomodulin in TSCs (A), however, only ITR induces non-tenocyte related gene expression of LPL, Runx-2, and SOX-9 in TSCs (B). MTR and ITR also significantly induce collagen I and tenomodulin (C) without the induction of non-tenocyte related gene expression in TNCs (D). For each group sample, n = 6 tendons were used for analysis. Graph bars represent the mean ± SD. *p < 0.05 compared to each cage control group.
Fig 7.
ITR induces non-tenocyte differentiation of TSCs in irradiated tendons determined by immunostaining.
Top panel (A): A-C: TSCs-transplanted, irradiated tendons without running; D-F: TSCs-transplanted, irradiated tendon after MTR; G-I: TSCs-transplanted, irradiated tendon after ITR; Bottom panel (B): J-L: TNCs-transplanted, irradiated tendon without running; M-O: TNCs-transplanted, irradiated tendon after MTR; P-R: TNCs-transplanted, irradiated tendons after ITR. There is no positive staining in TSCs-transplanted, irradiated tendons (A-C) and TNCs-transplanted (J-L) without running for all three non-tenocyte markers including SOX-9 (A, J), Runx-2 (B, K), and PPARγ (C, L). A few positively stained cells are found in TNCs-transplanted, irradiated tendons after ITR (P-R), and in TSCs-transplanted, irradiated tendons after MTR (D-F). However, a high percentage TSCs (G-I) are positively stained after ITR indicating the abundant presence of chondrocytes (G), osteocytes (H) and adipocytes (I) in mouse tendon tissues injected with TSCs that have undergone ITR. Three colors–green, red, and blue (DAPI) are merged in each of the images. Also, for each group sample, n = 6 tendons were used for analysis. Bars: 100 μm.