Reduction of mechanical loading in tendons induces heterotopic ossification and activation of the β-catenin signaling pathway

Abstract

Background: Tendons are the force transferring tissue that enable joint movement. Excessive mechanical loading is commonly considered as a primary factor causing tendinopathy, however, an increasing body of evidence supports the hypothesis that overloading creates microdamage of collagen fibers resulting in a localized decreased loading on the cell population within the damaged site. Heterotopic ossification is a complication of late stage tendinopathy, which can significantly affect the mechanical properties and homeostasis of the tendon. Here, we the examine the effect of mechanical underloading on tendon ossification and investigate its underlying molecular mechanism.

Method: Rabbit Achilles tendons were dissected and cultured in an underloading environment (3% cyclic tensile stain,0.25 ​Hz, 8 ​h/day) for either 10, 15 or 20 days. Using isolated tendon-derived stem cells (TDSCs) 3D constructs were generated, cultured and subjected to an underloading environment for 6 days. Histological assessments were performed to evaluate the structure of the 3D constructs; qPCR and immunohistochemistry were employed to study TDSC differentiation and the β-catenin signal pathway was investigated by Western blotting. Mechanical testing was used to determine ability of the tendon to withstand force generation.

Result: Tendons cultured for extended times in an environment of underloading showed progressive heterotopic ossification and a reduction in biomechanical strength. qPCR revealed that 3D TDSCs constructs cultured in an underloading environment exhibited increased expression of several osteogenic genes: these include RUNX2, ALP and osteocalcin in comparison to tenogenic differentiation markers (scleraxis and tenomodulin). Immunohistochemical analysis further confirmed high osteocalcin production in 3D TDSCs constructs subject to underloading. Western blotting of TDSC constructs revealed that β-catenin accumulation and translocation were associated with an increase in phosphorylation at Ser552 and decrease phosphorylation at Ser33.

Conclusion: These findings unveil a potential mechanism for heterotopic ossification in tendinopathy due to the underloading of TDSCs at the damage sites, and also that β-catenin could be a potential target for treating heterotopic ossification in tendons.

The translational potential: Tendon heterotopic ossification detrimentally affect quality of life especially for those who has atheletic career. This study reveals the possible mechanism of heterotpic ossification in tendon related to mechanical loading. This study provided the possible to develop a mechanical stimulation protocol for preventive and therapeutic purpose for tendon heterotopic ossification.

Keywords: Bioreactor; Heterotopic ossification; Mechanical loading; Mechanobiology; Tendon-derived stem cell.

Fig. 1

Flowchart of experimental plan. The rabbit Achilles tendons were allocated in four groups which are native tendon group for mechanical test, and NL (no loading), UL (underloading) and PL (physiological loading) groups in bioreactor.

Fig. 2

Histological assessment of rabbit Achilles tendons stained with either H&E or the von Kossa method. The tendons cultured without loading over a period of 10-20 days showed a pattern of progressive fiber disorientation and rupture (A); Normal morphology was shown in tendon subjected to 6% loading (B); Tendons subjected to 3% loading exhibited wavy collagen fibers (B) and progressive ossification confirmed by von Kossa staining (C, D). Average histological scores of tendon pathological changes (E), A score of 0 for each parameter represents normal and healthy structure, whereas 3 represents severe abnormality. Results are expressed as the mean±SEM. One-way ANOVA significance values were ∗p < 0.05 n = 5.

Fig. 3

Mechanical testing of rabbit Achilles tendons. (A) The Force versus displacement curves. (B) Mechanicl testing machine and loaded samples. (C) Maximum load and mean stiffness showed reduce mechanical properties of Achilles tendon tissue cultured ex vivo compared to native tendon tissue. Mechanical underloading and deprivation culture of tendon further reduced the mechanical properties compared to the one under physiological loading culture. Results are expressed as the mean ± SEM (n = 5). One-way ANOVA significance values were ∗p < 0.05.

Fig. 4

Tendon derived stem cells identification. (A) Formation of cell colonies TDSCs after culturing for 21 days at different seeding density of 100, 1000, 10000 cell/90mm petri dish. (B) Flow cytometry showing expression of mesenchymal stem cell marker (CD44, CD90), endothelial cell marker (CD34) and hematopoietic cell marker (CD45). (C) Differentiation potential of TDSCs after 21 days conditional medium stimulation. Calcium deposition was formed in osteogenic medium and stained by Alizarin Red. Lipid vacuoles were form in adipogenic medium and visualized by Oil red-O staining. Cell pellets were formed in chondrogenic medium and proteoglycan was stained by Alcian Blue.

Fig. 5

(A) Photographs of 3D TDSCs construct culture. The cell sheet was formed and detached from T75 culture flask, which was then rolled over the hooks forming tendon-like 3D construct. The tissues were either cultured without loading in 15ml tube for 6 days or subjected to 3% cyclic tensile strain at 0.25Hz, 8h/day for 6 days in the bioreactor system. (B) Histological and immunohistochemical assessment comparison of 3D constructs. The sample cultured with 3% loading exhibited aligned collagen fibers with higher positive staining of osteocalcin (∗∗p<0.01) than the tissue without loading. Staining of Tenomodulin showed no difference between two groups. The tissue formed without loading exhibited random extracellular matrix formation without specific differentiation pattern. Samples stained without primary antibodies acted as negative control group (N.C.). (C) Quantitation of immunohistochemical staining were performed as staining intensity using ImageJ software.

Fig. 6

(A) Comparison of differentiation-related markers gene expression on 3D TDSCs construct. 3% mechanical loading induced upregulation of COL1A1, ALP, RUNX2 and osteocalcin. The gene expression level is normalized against 36B4. Result are expressed as the mean±SEM. One-way ANOVA significance values were ∗p < 0.05 n=5; (B) Western blot analysis of protein phosphorylation and expression levels in 3D TDSC constructs cultured for 6 d, with or without 3% uniaxial loading. Total cell lysates were immunoblotted with the indicated antibodies. (C) Densitometric quantitation of bands (∗p<0.05, n = 3).

Fig. 7

Heatmap of extension distribution in tendon. In healthy tendon, extension is evenly distributed through the tendon. However, reduced local tension is formed in sub-ruptured site in the sub-failure tendon.

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