Fig 1.
A healing model of the supraspinatus tendon enthesis.
(A) An enthesis injury was made in the left shoulder of ScxGFP transgenic mice. (B and C) The section stained with hematoxylin and eosin (HE) under 40-fold magnification (B) and 100-fold magnification (C) at 2 weeks after injury. Bar = 500 μm in B and 200 μm in C.
Fig 2.
Postnatal formation of the supraspinatus tendon enthesis in mice.
(A-H) Microscopic images of the supraspinatus tendon enthesis during postnatal development after staining with toluidine blue (A-D) and alkaline phosphatase (ALP)/alizarin red S (E-H). (I-L) Image of the second harmonic generation (SHG, white) of the tendon attachment sites. SSPT, supraspinatus tendon; hu, humerus; cFC, calcified fibrocartilage; ucFC, uncalcified fibrocartilage. Solid lines and dotted lines highlight the surface of humeral bone and the interface between cFC and ucFC, respectively. Scale bar = 100 μm.
Fig 3.
Scx+/Sox9+ cells participate in postnatal formation of fibrocartilaginous enthesis.
(A-D) Immunostaining of ScxGFP (green), Sox9 (red) and DAPI (blue). SSPT, supraspinatus tendon; hu, humerus; cFC, calcified fibrocartilage; ucFC, uncalcified fibrocartilage. Solid lines and dotted lines highlight the surface of humeral bone and interface between cFC and ucFC, respectively. Scale bar = 100 μm. (E-G) The number of cells positive for ScxGFP (E), Sox9 (F), and ScxGFP/Sox9 (G) was normalized to the total number of nuclei with DAPI staining in the supraspinatus tendon enthesis in neonates, and 3-, 6-, and 20-week-old mice. All values represent means ± SD. N = 6 for each time point. *p < 0.05.
Fig 4.
Healing process after supraspinatus tendon enthesis injury in 20-week-old mice.
(A-L) Microscopic images of the tendon-bone attachment sites at 3 days (d), and 1, 2, and 4 weeks (wks) after injury in 20-week-old (wo) mice, stained with toluidine blue (A-D), and alkaline phosphatase (ALP)/alizarin red S (E-H). Image of the second harmonic generation (SHG, white, I-L). hu, humerus. Solid lines highlight the surface of the humeral bone. Scale bar = 100 μm.
Fig 5.
Healing process after supraspinatus tendon enthesis injury in 3-week-old mice.
(A-L) Microscopic images of the tendon-bone attachment sites at 3 days (d), and 1, 2, and 4 weeks (wks) after injury in 3-week-old (wo) mice, stained with toluidine blue (A-D), and alkaline phosphatase (ALP)/alizarin red S (E-H). Image of the second harmonic generation (SHG, white, I-L). hu, humerus. Solid lines highlight the surface of humeral bone. Scale bar = 100 μm.
Fig 6.
Scx+/Sox9+ cells in healing after supraspinatus tendon enthesis injury in 20- and 3-week-old mice.
(A-J) Microscopic images of the injured enthesis at 3 days (d), and 1, 2, and 4 weeks (wks) and sham-operated control at 4 wks after immunostaining for ScxGFP (green) and Sox9 (red) along with DAPI (blue) in 20 (A-E)- and 3 (F-J)-week-old (wo) mice. Hu, humerus. White arrowheads indicate Scx+/Sox9+ cells. Solid lines highlight the surface of humeral bone. Scale bar = 100 μm. (K-M) The number of cells positive for Scx, Sox9, and Scx/Sox9 was normalized to the total number of nuclei with DAPI staining at the reparative tissue in 20 and 3 wo mice. All values represent means ± SD. N = 6 for each time point. *p < 0.05.
Fig 7.
Localization of Scx+/Sox9+ cells at the entheseal fibrocartilage in postnatal development and in healing.
(A-D) Microscopic images of the supraspinatus tendon enthesis at 4 weeks after enthesis injury in 3-week-old mice (A and B) and normal enthesis at 3 weeks of age in postnatal development (C and D). Toluidine blue staining (A and C) and immunostaining of ScxGFP (green) and Sox9 (red) along with DAPI (blue) (B and D) were performed. SSPT, supraspinatus tendon; FC, entheseal fibrocartilage; hu, humerus. White arrowheads indicate Scx+/Sox9+ cells. Scale bar = 100 μm.
Fig 8.
Cell proliferation in the healing site at 1 week after injury.
(A-H) Microscopic images of the tendon-bone attachment sites at 1 week after injury, immunostaining for ScxGFP (green, A and E), Ki-67 (red, B and F), DAPI (blue, C and G), and their merged image (D and H) in 20- and 3-week-old (wo) mice. (I) The number of cells positive for ScxGFP/Ki-67 was normalized to the total number of nuclei with DAPI staining at the reparative tissue in 20 and 3 wo mice. All values represent means ± SD. N = 6. *p < 0.05.