Fig. 3: PI3K-Akt signalling is involved in tenocyte proliferation and migration in the early regenerative phase, and tendon thickening and chondrometaplasia in the late regenerative phase after tendon injury in vivo.

a Experimental scheme for evaluating tendon regeneration in vivo. Scx-CreER^T2; Rosa26-tdTomato mice were injected with 0.075 mg of tamoxifen three times. Tamoxifen was washed out for 72 h, and tenotomy was performed (P 10-day). Vehicle (control; Ctrl) or ZSTK474 (ZSTK) was administered and evaluated 2 h, 3 d, and 28 d later. b Haematoxylin-eosin staining (H&E) and TUNEL staining of the Achilles tendon stub 2 h after tenotomy. Scx-lineage cells [tdTomato-positive (tdTomato⁺) cells] were stained with an RFP antibody. White dotted lines indicate the distal tendon stub. c Quantification of TUNEL-positive (TUNEL⁺) cells shown in (b). Data are shown as the mean ± SEM of three independent biological samples (three independent sections per mouse). A two-tailed Student’s t-test was used for statistical analyses. There was no significant difference in the number of apoptotic cells in tendon stubs between the control and ZSTK groups. d H&E and immunofluorescence staining using RFP and Ki67 antibodies for the Achilles tendon stub was performed 3 days post-tenotomy. The distal tendon stub is indicated by white dotted lines, while the tdTomato⁺ tendon area is indicated by yellow dotted lines. e Quantification of the percentage of Ki67 positive (Ki67⁺) cells shown in (d). Data are shown as the mean ± SEM of three independent biological samples (three independent sections per mouse). The percentage of Ki67⁺ cells significantly decreased in the ZSTK group (two-tailed Student’s t-test; p = 0.0439). f Quantification of the area of the tendon with tdTomato-positive (tdTomato⁺) cells shown in (d). Data are shown as the mean ± SEM of three independent biological samples (three independent sections per mouse). The ZSTK group showed a significantly smaller tdTomato⁺ area at the tendon stub than the control group (two-tailed Student’s t-test; p = 0.0003), indicating that the migration of tdTomato⁺ cells decreased. g Haematoxylin-eosin staining (H&E), Safranin O and fast green staining (SaO+FG), and Picrosirius Red (PSR) staining of Achilles tendons 28 days after tenotomy. The regenerated tendons and intact plantaris tendons are indicated by white dotted lines and black asterisks, respectively. h Magnified images of the black dotted squares in (g). i Quantification of neotendon thickness shown in (g). Data are shown as the mean ± SEM of four independent biological samples (two independent sections per mouse). The transverse diameter of the thickest part of the regenerated tendon, excluding distal and proximal tendon stubs, was measured as neotendon thickness. The ZSTK group showed a significant decrease in neotendon thickness compared with the control group (two-tailed Student’s t-test; p = 0.0232). j Percentage of thick fibres in the neotendon. The thick fibre area was measured in PSR staining sections. Data are shown as the mean ± SEM of four independent biological samples (two independent sections per mouse). The ZSTK group had a significantly higher percentage of thick fibres than the control group (two-tailed Student’s t-test; p = 0.0064). k Immunofluorescence staining was performed using an RFP antibody at the boundary between the regenerated tendon and the remaining distal tendon stub at 28 days after tenotomy. Lower migration of tdTomato⁺ cells into the regenerated tendons was observed in the ZSTK group than in the control group. CA; calcaneus. l Quantification of the distance of proximally migrating tdTomato-positive (tdTomato⁺) cells in neotendons shown in (k). The distance between the most proximal tdTomato+ cell and the calcaneus was measured. Data are shown as the mean ± SEM of four independent biological samples (three independent sections per mouse). The distance in the ZSTK group was significantly smaller than that in the control group (two-tailed Student’s t-test; p = 0.0311). m Quantification of the area with tdTomato⁺ cells shown in (k). Data are shown as the mean ± SEM of four independent biological samples (three independent sections per mouse). The area of tdTomato⁺ cells in the ZSTK group was significantly smaller than that in the control group (two-tailed Student’s t-test; p = 0.0309). n Immunofluorescence staining using the Col2 antibody. Yellow dotted circles indicate Col2 positive (Col2⁺) chondrometaplastic regions in regenerated tendons. o Quantification of the number of Col2⁺ cells shown in (n). Data are shown as the mean ± SEM of four independent biological samples (three independent sections per mouse). The number of Col2⁺ cells significantly decreased in the ZSTK group compared to the control groups (two-tailed Mann Whiteny-U test, p = 0.0286). p Immunofluorescence staining using the S100b antibody. Yellow dotted circles indicate S100b positive (S100b⁺) chondrometaplastic regions in regenerated tendons. q Quantification of the number of S100b⁺ cells shown in (p). Data are shown as the mean ± SEM of four independent biological samples (three independent sections per mouse). The number of S100⁺ cells significantly decreased in the ZSTK group compared to the control groups (two-tailed Student’s t-test, p = 0.0461). r Transmission electron microscope (TEM) pictures showing collagen fibrils in the neotendon at 28 days after tenotomy. s Distribution of the collagen fibril diameter in the neotendon. The diameter of 1000 collagen fibrils was measured in TEM pictures shown in (r) (three independent biological samples in each group). Collagen fibril diameter in the ZSTK group tends to be larger than that in the Ctrl group. * Scale bars, 100 nm (s), 50 μm (n, p), 100 μm (d, h), 200 μm (b, k), 400 μm (g).