Scleraxis lineage cells contribute to organized bridging tissue during tendon healing and identify a subpopulation of resident tendon cells

Abstract

During tendon healing, it is postulated that tendon cells drive tissue regeneration, whereas extrinsic cells drive pathologic scar formation. Tendon cells are frequently described as a homogenous, fibroblast population that is positive for the marker Scleraxis (Scx). It is controversial whether tendon cells localize within the forming scar tissue during adult tendon healing. We have previously demonstrated that S100 calcium-binding protein A4 (S100a4) is a driver of tendon scar formation and marks a subset of tendon cells. The relationship between Scx and S100a4 has not been explored. In this study, we assessed the localization of Scx lineage cells (Scx^Lin) following adult murine flexor tendon repair and established the relationship between Scx and S100a4 throughout both homeostasis and healing. We showed that adult Scx^Lin localize within the scar tissue and organize into a cellular bridge during tendon healing. Additionally, we demonstrate that markers Scx and S100a4 label distinct populations in tendon during homeostasis and healing, with Scx found in the organized bridging tissue and S100a4 localized throughout the entire scar region. These studies define a heterogeneous tendon cell environment and demonstrate discrete contributions of subpopulations during healing. These data enhance our understanding and ability to target the cellular environment of the tendon.-Best, K. T., Loiselle, A. E. Scleraxis lineage cells contribute to organized bridging tissue during tendon healing and identify a subpopulation of resident tendon cells.

Keywords: S100a4; heterogeneity; myofibroblast; regeneration; tenocyte.

Figure 1

Scx^Lin localize within the forming scar tissue during flexor tendon healing. A) Scx-Cre^ERT2 mice were crossed to ROSA-Ai9 reporter (Scx^Ai9) to trace adult Scx^Lin at homeostasis and throughout healing (Scx^Lin). B) Mice were injected with TAM for 3 consecutive days followed by a 4-d WO prior to tendon injury and repair. C–F) Tendons were harvested uninjured (C) and at d 7 (D), 14 (E, E′), and 21 (F, F′) postrepair. Nuclear stain DAPI is blue. Tendon is outlined by white dotted lines and scar tissue by yellow dotted lines. White arrows indicate Scx^Lin with wavy morphology. N = 3–4 specimens per time point. Scale bars, 50 μm (E′, F′), 100 μm (C, D), 200 μm (E, F).

Figure 2

Alterations in Scx expression throughout early tendon healing. A) Scx^Ai9 mice were injected with TAM on d 0–2 postrepair to trace cells expressing Scx shortly after repair (Scx⁰⁻²). B–D) Scx⁰⁻² mice were harvested uninjured (B) and 14 d postrepair (C, C′). Scx^Ai9 mice were injected with TAM on d 5–7 postrepair to trace cells expressing Scx later in healing (Scx⁵⁻⁷) (D). E, F) Scx⁵⁻⁷ mice were harvested uninjured (E) and 14 d postrepair (F, F′). Nuclear stain DAPI is blue. Tendon is outlined by white dotted lines and scar tissue by yellow dotted lines. N = 3–4 specimens per induction scheme per time point. Scale bars, 50 μm (B, E), 100 μm (C′, F′), 200 μm (C, F).

Figure 3

Dual tracing of Scx^Lin and S100a4^GFP cells exhibits tendon cell heterogeneity. A) Scx^Ai9 mice were crossed to S100a4^GFP mice (Scx^Ai9;S100a4^GFP) to allow dual tracing of Scx lineage and S100a4⁺ cell populations. B) Scx^Ai9;S100a4^GFP mice were injected with TAM on 3 consecutive days and harvested 18 d following the final injection. C) Contralateral (opposite foot taken for d 14 repair) Scx^Ai9;S100a4^GFP tendons exhibit 4 distinct cell types with colored arrows indicating examples of each: Scx^Lin (red arrow), S100a4^GFP (green arrow), Scx^Lin; S100a4^GFP (yellow arrow), and dual-negative populations (blue arrow). Nuclear stain DAPI is blue. Scale bars, 20 μm. D, E) Quantification of individual and total Scx and S100a4 populations. N = 4.

Figure 4

Scx^Lin and S100a4^GFP populations differentially localize during tendon healing. A) Scx^Ai9; S100a4^GFP mice were injected with TAM on 3 consecutive days and allowed to WO for 4 d prior to repair. B, C) Scx^Lin; S100a4^GFP mice were harvested at 14 (B) and 21 (C) d postrepair. D, E) D 21 bridging (D) and peripheral scar tissue (E) exhibit differential cell contribution and morphology. The tendon is outlined by the white dotted lines and the scar tissue by yellow dotted lines. F, G) Quantification of Scx^Ai9, S100a4^GFP, and Scx^Ai9;S100a4^GFP % total area fluorescence at d 14 (B) and d 21 (C) for both bridging tissue (F) and total scar tissue (G) area. Scale bars, 200 μm (B, C), 20 μm (D, E). *P < 0.05, **P < 0.01, ****P < 0.0001.

Figure 5

Scx¹⁰⁻¹² and S100a4^GFP differentially localize during tendon healing. A, B) Scx^Ai9; S100a4^GFP mice were injected with TAM on d 10–12 postrepair (A) to generate Scx¹⁰⁻¹²; S100a4^GFP mice and were harvested at d 14 (B). C, D) Scx¹⁰⁻¹² cells were detected at the tendon stub (C) and bridging tissue (D). Tendon is outlined by white dotted lines and scar tissue by yellow dotted lines. E, F) Quantification of Scx^Ai9, S100a4^GFP, and Scx^Ai9;S100a4^GFP % total area fluorescence at d 14 for both bridging tissue (E) and total scar tissue (F) area. Scale bars, 100 μm (B), 20 μm (C, D). *P < 0.05.

Figure 6

Scx^Lin do not substantially differentiate into myofibroblasts. A) Scx^Ai9 mice were injected with TAM for 3 d and allowed to WO for 4 d (Scx^Lin). B) Coimmunofluorescence of Scx^Lin (RFP, labels tdTomato of ROSA-Ai9) and αSMA (myofibroblast marker) on Scx^Lin d 14 postrepair sections reveals minimal differentiation of Scx^Lin cells into myofibroblasts. Differentiation occurs primarily within the native tendon (B′) and not within the scar tissue (B″). C, C′) Nuclear stain DAPI is blue. Samples were then stained with Masson’s trichrome to analyze localization of organized collagen deposition at the repair site . Tendon is outlined by white dotted lines and scar tissue by yellow dotted lines. Demarcation of the organized-disorganized collagen boundary labeled with orange dotted lines. Scale bars, 200 μm (B, C), 50 μm (B′, B″), 20 μm (C′).