Nanotopographic cues and stiffness control of tendon-derived stem cells from diverse conditions

Abstract

Background

Tendon-derived stem cells (TDSCs) are key factors associated with regeneration and healing in tendinopathy. The aim of this study was to investigate the effects of mechanical stiffness and topographic signals on the differentiation of TDSCs depending on age and pathological conditions.

Materials and methods

We compared TDSCs extracted from normal tendon tissues with TDSCs from tendinopathic Achilles tendon tissues of Sprague Dawley rats in vitro and TDSCs cultured on nanotopographic cues and substrate stiffness to determine how to control the TDSCs. The tendinopathy model was created using a chemical induction method, and the tendon injury model was created via an injury-and-overuse method. Norland Optical Adhesive 86 (NOA86) substrate with 2.48 GPa stiffness with and without 800 nm-wide nanogrooves and a polyurethane substrate with 800 nm-wide nanogrooves were used.

Results

TDSCs from 5-week-old normal tendon showed high expression of type III collagen on the flat NOA86 substrate. In the 15-week normal tendon model, expression of type III collagen was high in TDSCs cultured on the 800 nm NOA86 substrates. However, in the 15-week tendon injury model, expression of type III collagen was similar irrespective of nanotopographic cues or substrate stiffness. The expression of type I collagen was also independent of nanotopographic cues and substrate stiffness in the 15-week normal and tendon injury models. Gene expression of scleraxis was increased in TDSCs cultured on the flat NOA86 substrate in the 5-week normal tendon model (P=0.001). In the 15-week normal tendon model, scleraxis was highly expressed in TDSCs cultured on the 800 nm and flat NOA86 substrate (P=0.043). However, this gene expression was not significantly different between the substrates in the 5-week tendinopathy and 15-week tendon injury models.

Conclusion

Development and maturation of tendon are enhanced when TDSCs from normal tendons were cultured on stiff surface, but not when the TDSCs came from pathologic models. Therapeutic applications of TDSCs need to be flexible based on tendon age and tendinopathy.

Keywords:

• gene expression
• differentiation
• animal models
• tendon-derived stem cells

Supplementary materials

Figure S1 H&E staining of the left Achilles tendon confirmed the pathological findings of tendinopathy and tendon injury models.

Note: Five-week-old tendinopathy model showed an irregular pattern of collagen fibers with multiple lipid vacuoles (A), and the 15-week-old tendon injury model showed a thickened irregular pattern of collagen fibers with abundant polymorphic nuclear cells (B).

Figure S2 Isolation of TDSCs was validated by identifying cells that positively stained for nucleostemin, OCT4, SSEA4, and tenomodulin. Each TDSC cell line was successfully differentiated into osteogenic, adipose, and chondrogenic cell lines, demonstrating their multipotent capacity.

Abbreviation: TDSCs, tendon-derived stem cells.

Acknowledgments

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HI16C1104) and the National Research Fund (2018R1D1A1B07047084).

Author contributions

Sun Jeong Kim, Philip D Tatman, Albert O Gee, Deok-Ho Kim, and Sang Jun Kim contributed to conception and design. Sun Jeong Kim, Da-Hyun Song, Deok-Ho Kim, and Sang Jun Kim contributed to acquisition of data. All authors contributed to interpretation of data, drafting of the article, gave final approval of the manuscript version to be published, and agreed to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.