Aberrant TGF-β activation in bone tendon insertion induces enthesopathy-like disease

Abstract

Enthesopathy is a disorder of bone, tendon, or ligament insertion. It represents one-fourth of all tendon-ligament diseases and is one of the most difficult tendon-ligament disorders to treat. Despite its high prevalence, the exact pathogenesis of this condition remains unknown. Here, we show that TGF-β was activated in both a semi-Achilles tendon transection (SMTS) mouse model and in a dorsiflexion immobilization (DI) mouse model of enthesopathy. High concentrations of active TGF-β recruited mesenchymal stromal stem cells (MSCs) and led to excessive vessel formation, bone deterioration, and fibrocartilage calcification. Transgenic expression of active TGF-β1 in bone also induced enthesopathy with a phenotype similar to that observed in SMTS and DI mice. Systemic inhibition of TGF-β activity by injection of 1D11, a TGF-β-neutralizing antibody, but not a vehicle antibody, attenuated the excessive vessel formation and restored uncoupled bone remodeling in SMTS mice. 1D11-treated SMTS fibrocartilage had increased proteoglycan and decreased collagen X and matrix metalloproteinase 13 expression relative to control antibody treatment. Notably, inducible knockout of the TGF-β type II receptor in mouse MSCs preserved the bone microarchitecture and fibrocartilage composition after SMTS relative to the WT littermate controls. Thus, elevated levels of active TGF-β in the enthesis bone marrow induce the initial pathological changes of enthesopathy, indicating that TGF-β inhibition could be a potential therapeutic strategy.

Keywords: Bone Biology; Bone disease.

Figure 1. Osteoclastic bone resorption in SMTS enthesopathy mice.

(A) μCT images of the PCT (sagittal view). Red arrowhead indicates altered morphology of the PCT. Red asterisk indicates bone marrow cavities. Scale bar: 500 μm. (B) Quantitative analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Tb.Pf in PCT determined by μCT analysis. (C) TRAP staining (magenta) in mouse PCT bone marrow. Scale bar: 200 μm. Red asterisks indicate bone marrow cavities. (D) Quantitative analysis of TRAP⁺ OCS/BS. (E) Quantitative analysis of active TGF-β1 in serum by ELISA. D0, prior to SMTS surgery; Sham, sham surgery; W1, 1 week after SMTS surgery. Data shown as mean ± SEM. n = 10. *P < 0.05 compared with the sham group.

Figure 2. Dysregulated TGF-β signaling is associated with trabecular bone changes in SMTS enthesopathy mice.

(A) Immunohistochemical analysis of pSmad2/3⁺ cells (brown) in mouse PCT bone marrow. The bottom panels show higher magnification of the boxed area in the top panels. Scale bar: 500 μm (top panels); 50 μm (bottom panels). (B) Quantitative analysis of the number of pSmad2/3⁺ cells per bone marrow area (mm²). (C) Immunostaining and (D) quantitative analysis of Ocn⁺ cells. Scale bar: 50 μm. (E) Quantitative analysis of osteocalcin in serum by ELISA. (F) Representative images of calcein double labeling of Achilles tendon enthesis 8 weeks after sham or SMTS operation with (G) quantification of MAR and BFR. Scale bar: 50 μm. Data shown as mean ± SEM. n = 10. *P < 0.05 compared with the sham group.

Figure 3. Fibrocartilage displays mineralization in Achilles tendon enthesis.

(A) SOFG staining of Achilles tendon enthesis compartment, proteoglycan (red), bone (green) and Achilles tendon (green). Black arrowheads indicate direct connection between bone marrow and CF. White arrowhead indicates blood vessel invasion from PCT bone to CF. Scale bar: 200 μm. (B) Quantitative analysis of area of CF and UF. (C and E) Immunohistochemical and (D and F) quantitative analysis of (C and D) COLX⁺ cells and (E and F) MMP13⁺ cells (bottom) in fibrocartilage of mouse Achilles tendon enthesis after SMTS surgery. Dotted lines separate CF and UF. Scale bars: 150 μm. Data shown as mean ± SEM. n = 10. *P < 0.05 compared with the D0 group or between groups. AT, Achilles tendon.

Figure 4. CED mice show an Achilles tendon enthesopathy phenotype.

(A) μCT images of the PCT (sagittal view). Red arrowhead shows bony projections. Scale bar: 500 μm. (B) Quantitative analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Tb.Pf in PCT determined by μCT analysis. (C) μCT-based microangiography of the calcaneus and (D) quantitative analysis of vessel number (VN) and vessel volume (VV). Scale bar: 100 μm. (E) Immunostaining and (F) quantitative analysis of Osx⁺ (brown) cells in the PCT bone marrow of WT and CED mice. Scale bar: 50 μm. (G) H&E staining of Achilles tendon enthesis. PCT bone, CF, and UF are separated by dotted lines. Scale bar: 200 μm. (H) Quantitative analysis of areas of CF and UF. (I) SOFG staining of Achilles tendon enthesis. Scale bar: 200 μm. Data shown as mean ± SEM. n = 10. *P < 0.05 compared between groups.

Figure 5. Nestin⁺ and LepR⁺ progenitor cells are increased in HO in CED mice.

(A and C) Immunostaining and (B and D) quantitative analysis of (A and B) Nestin⁺ (red) cells and (C and D) LepR⁺ cells in the PCT bone marrow 4 weeks after sham and SMTS operations. Scale bars: 30 μm. Data shown as mean ± SEM. n = 10. *P < 0.05 compared between groups.

Figure 6. Systemic injection of TGF-β1 antibody maintains Achilles tendon enthesis structure and reduces the unregulated TGF-β signaling.

(A) μCT images of the PCT (sagittal view) of mice treated with 5 mg per kg body weight of the TGF-β–neutralizing antibody 1D11 weekly for 30 days and analyzed 4 or 8 weeks after SMTS or sham surgery. Scale bar: 500 μm. (B) Quantitative analysis of BV/TV, Tb.Th, Tb.N, Tb.Sp, and Tb.Pf in PCT determined by μCT analysis. (C) Immunostaining and (D) quantitative analysis of Nestin⁺ cells (red) in the PCT bone marrow. Scale bar: 30 μm. (E) Immunostaining of CD31⁺ (red) vessels and the (F) quantification of the number of vessels positive for CD31 (per mm²). Scale bar: 100 μm. (G) Osx⁺ cells (brown) in the PCT and (H) quantifications of Osx⁺ cell number in PCT bone marrow and on PCT BS. Data shown as mean ± SEM. n = 10. *P < 0.05 compared between groups or to the sham group. Veh, vehicle.

Figure 7. Systemic injection of TGF-β antibody attenuates enthesis changes in enthesopathy mice.

(A) SOFG staining of Achilles tendon enthesis. PCT, CF, UF, and Achilles tendon (T) are separated by dotted lines. Scale bars: 200 μm. (B) Quantitative analysis of areas of CF and UF. (C and E) Immunohistochemical (staining) and (D and F) quantitative (bar chart, right) analysis of (C and D) COLX⁺ cells and (E and F) MMP13⁺ cells (bottom)in fibrocartilage (CF and UF are separated by dotted lines) of mouse Achilles tendon enthesis after SMTS surgery. Scale bars: 150 μm. Data shown as mean ± SEM. n = 10. *P < 0.05 compared between groups or to the sham group.

Figure 8. Nestin⁺ cells gives rise to vesselsin PCT bone marrow after SMTS.

(A, left) CD31⁺ cells (red), YFP⁺ cells (green), and (A, right) Emcn⁺ cells (red), YFP⁺ cells (green). (B) Quantitation in PCT bone marrow of Nestin-creERT2::R26R-EYFP mice 8 weeks after SMTS. Scale bar: 100 μm. n = 10 per group. Data shown as mean ± SEM.

Figure 9. Genetic knockout of Tgfbr2 in Nestin⁺ cells results in less changein Achilles tendon enthesis after SMTS.

(A) μCT images of the PCT (sagittal view) of Nestin-creERT2::Tgfbr2^fl/fl (Tgfbr2^–/–) mice after 2 months treatment with vehicle or tamoxifen after undergoing sham or SMTS surgery. Scale bar: 500 μm. (B) Quantitative analysis of structural parameters of PCT by μCT analysis. (C) Nestin⁺ (red) and (E) Osx⁺ (brown) cells in the PCT bone marrow treated with vehicle or tamoxifen 4 weeks after sham and SMTS operations. White dotted lines (C) indicate bone surface. B, bone. Scale bar: 50 μm. (D and F) Quantifications of the number of bone marrow cells positive for Nestin and Osx in PCT bone marrow and PCT BS (per mm²). (G) μCT-based microangiography of the calcaneus and (H) quantitative analysis of VN and VV. Red arrowhead indicates blood vessel invasion into the enthesis. Scale bar: 150 μm. Data shown as mean ± SEM. n = 10. *P < 0.05 compared between groups or to the sham group.

Figure 10. Deletion of Tgfbr2 in Nestin⁺ cells mitigates enthesis degradation in enthesopathy mice.

(A) SOFG staining of Achilles tendon enthesis. PCT, CF, UF, and Achilles tendon are separated by dotted lines. Scale bar: 200 μm. (B) Quantitative analysis of area of CF and UF. (C and E) Immunohistochemical and (D and F) quantitative analysis of (C and D) COLX⁺ and (E and F) MMP13⁺ cells in enthesis fibrocartilage. Scale bars: 150 μm. Data shown as mean ± SEM. n = 10. *P < 0.05 compared between or to the sham group.