Figure 1.
SMA9 model labels 4 distinct cell populations in and around the tendon midsubstance.
Following tamoxifen injection, SMA9+ cells (Red) are found in 1) smooth muscle cells in large blood vessels outside of the tendon (A), 2) perivascular cells in smaller vessels (A,B,G), 3) paratenon/sheath/retinacular cells on the tendon surface (B,D,E,G), and 4) tendon fibroblasts within the midsubstance (C,F,H) of the patellar (PT), flexor carpi ulnaris (FCU) tendon, Achilles (AT), and superficial digital flexor tendons (SDF) on 21 days (A–C,G) and 42 days (D–F,H) following injection. Images and 3D reconstructions (G) were created using two photon microscopy in SMA9 (A–C,G) and SMA9-ScxGFP mice (D–F,H). SMA9+ cells within the tendon body are ScxGFP+ (green) (F,H), unlike SMA9+ in the paratenon and tendon sheath (D–E). B&C are optical slices at the tendon surface and 50 µm deep in the PT, respectively. D–F are optical slices at the surface of the sheath, middle of the sheath, and 130 µm deep, respectively. G displays SMA9+ cells in the retinaculum covering the SDF and AT. Blue – second harmonic generation (SHG) for collagen. Scale bars = 100 µm. The authors encourage the readers to view figure S1 for the anatomical location of each panel in this figure.
Figure 2.
SMA9+ cells are found in the tendon body, perimysium, and muscle fibers at the myotendinous junction.
A–D) SMA9+ (red) cells in the supraspinatus tendon (ST) span from the anterior surface near the enthesis (ENT) to the myotendinous junction (MTJ) at 42 days post-injection. E–G) SMA9+ and SMA9/ScxGFP+ cells are found at the muscle-tendon interface and in the perimysium (PM) of the extensor digiti minimi (EDM) tendon at 21 days. Dotted lines in E–G refer to orthogonal slices in the x–y, x–z, and y–z planes. SMA9/ScxGFP+ tendon fibroblasts and SMA9+ paratenon cells and muscle fibers are found at the MTJ of the Achilles tendon (H–J) 42 days. H&I are optical slices at the tendon surface and 30 µm deep in the AT, respectively (J is 3D reconstruction of H and I). All images were taken using two photon microscopy. Blue – second harmonic generation (SHG) for collagen. Scale bars = 100 µm. Anatomical locations of images can be found in figure S1.
Figure 3.
SMA9 model transiently labels an amplifying resident progenitor population in the tendon midsubstance during growth.
A pulse chase experiment where two tamoxifen injections were given on consecutive days to 3–4 week old mice revealed that the initial number (3.1±1.5% of total) of resident SMA9+ cells (red, arrows) in the tendon midsubstance at 2 days (A,D,G) following injection expanded over 4-fold to 13.5±4.4% at 21 days (B,E,G) and then reduced to day 2 levels by 70 days (C,F,G). Trabecular bone in the tibia shows a similar trend, while muscle fibers in the quadriceps were labeled at later time points (C). No SMA9+ cells were found in the fibrocartilage of the enthesis or ligaments in the knee at any time point. In addition, the number of expanded cells on day 21 was significantly higher in the patellar (PT) and supraspinatus (ST) tendons compared to the achilles (AT) tendon (H, p<0.05). Blue – dapi counterstained nuclei. PCL – posterior cruciate ligament. Scale bars = 250 µm. *significantly different than other time points, ∧significantly different than other tendons. Error bars denote ± SD.
Figure 4.
Unlike the SMA9 model, GDF5-9 cells are found within the fibrocartilage of the tendon enthesis and throughout the length of ligaments.
GDF5-9+ cells are expressed in cells within the cruciate ligaments (ACL, PCL) from bone to bone (A,B,D,E). GDF5-9+ cells are concentrated near the enthesis of the supraspinatus tendon (ST) and do not extend along the length of the midsubstance (C,F). However, GDF5-9+ cells are found along the posterior half of the PT (A,D). GDF5-9 cells are also found within articular cartilage, menisci, collateral ligaments, synovium, and epiphyseal bone in the knee. These expression patterns are similar between the ages of P0 (A–C) and P56 (D–F) except the epiphyseal bone is not labeled at P0, which is before secondary ossification. Blue – dapi counterstained nuclei. LCL – lateral collateral ligament, MCL – medial collateral ligament. Scale bars = 250 µm.
Figure 5.
SMA9+ progenitors form a collagenous bridge over the anterior surface of the PT defect.
Two photon imaging shows that SMA9+ cells within the thickened paratenon synthesize collagen (SHG signal in blue and grey) as they form the paratenon bridge. These cells originate from the medial and lateral tendon struts (G,N) and also from the anterior aspect of the tibia (O,P) and patella (data not shown). While these cells originate from the paratenon and vasculature, they infiltrate the adjacent struts over time (J). The collagen in the bridge matures from 1 week (A–G) to 5 weeks (H–U). G,N,U) 3D reconstructions in the axial view depicting the 3 levels in the anterior view (A–F, H–M, O–T). Scale bars = 100 µm.
Figure 6.
SMA9+ progenitors in the expanded paratenon differentiate into ScxGFP+ cells at 2 weeks post-injury.
SMA9+ cells (white arrows, D–F) at one week post-injury (A–J) are predominately negative for ScxGFP as seen in two photon image stacks in SMA9-ScxGFP mice. However, these cells differentiate into ScxGFP+ cells (yellow arrows, N–P,V) at 2 weeks (K–T) within the collagenous regions of the paratenon bridge while SMA9+ only cells still remain on the anterior surface (white arrows, K–M). By 5 weeks, there is reduced overlap in SMA9 and ScxGFP expression (V). J,T) Axial views depicting the 3 levels in the anterior views (A–J, K–T) of the defects at 1 and 2 weeks, respectively. U) Total area of SMA9 and ScxGFP positive signal at 1, 2, and 5 weeks. V) SMA9-ScxGFP double positive area normalized to either total SMA9 or ScxGFP area, respectively. Scale bars = 100 µm. *significantly different than 1 and 5 weeks, ∧significantly different than 1 week (p<0.05). Error bars denote ± SD. Full z-stacks can be found in Video S1.
Figure 7.
SMA9+ cells from the paratenon/periosteum of the adjacent tendon and bone are the main contributors to PT healing.
Schematic depicts the origin and directional expansion of cells that contribute to PT defect healing. These cells originate within the paratenon in the tendon and retinaculum of the medial/lateral aspects as well as paratenon/periosteum over the proximal and distal bones. As the cells form the anterior bridge over the defect space, they begin to align along the tendon axis. MR – medial retinaculum, LR – lateral retinaculum.