(M-500) Application of tenocytes-based injectable putty for tendon regeneration in Achilles defect rat model

Introduction:: The tendon is a connective tissue that acts like a bridge between bone and muscle tissue to transmit mechanical force. The Achilles tendon, called by calcaneal tendon, is the largest and strongest tendon in the human body. Still, excessive movements, gradual wear, injuries, inflammations, and aging can induce tendon damage. Currently, the treatment methods are divided into injecting corticosteroid drugs and suturing part of the torn tendon tissues. However, the revision surgery cases are increased because of randomly produced collagen fibers that lead to reduced mechanical properties.

Cell therapy emerged in tissue engineering fields to regenerate damaged tissues, but the method showed low cell survival and poor cell localization after injecting cells. To overcome the limitation of single-cell injection treatment, the authors concentrated on cell sheet engineering technology to use a high-purity extracellular matrix (ECM) produced by cells. Moreover, we have manipulated the cell sheets as an injectable type to ease controlling it and named Teno-putty in this study. Besides, the authors have found that the cell sheet formation was faster under cultured in low oxygen tension than in normal conditions due to accelerating cell proliferation.

Thus, we hypothesized that Teno-putty containing accumulated tendon ECM cultured under hypoxic conditions would enhance the mechanical properties and regeneration of the Achilles tendon more than the single-cell injected groups in the tendon defects rat model.


Materials and Methods:: Tenocytes, tendon-derived fibroblasts, were isolated from the Achilles tissues and characterized with tendon-related protein markers like Collagen type I/III, Tenomodulin, Tenascin-C, and Decorin using immunocytochemistry (ICC)/western blot analysis. After that, the hypoxia condition during the culture period was confirmed by the expression of hypoxia-inducible factor-1 alpha (HIF-1ɑ) with ICC analysis. We fabricated injectable Teno-putty with manipulation procedures using the cell sheet made by tenocytes. Furthermore, the cell proliferation rate was examined after manipulation to check the maintenance of cellular function and the application possibility for in vivo study.

Manipulated Teno-putty was implanted in the Achilles tendon defects rat model for 2, 4, and 8 weeks. We compared it with the positive control (healthy state), negative control (any materials were not treated to the defects), and single-cell injected groups. In addition, we verified tendon regeneration by scoring tendon tissues after sacrifice, observing collagen fiber stained with a picrosirius red, and measuring mechanical properties with the universal testing machine (UTM).


Results, Conclusions, and Discussions:: In the hypoxic condition analysis, we observed the expression of HIF-1ɑ from the three days of culture. Remarkedly, we found that hypoxic conditions increase the proliferation and sheet formation of the tendon-derived cell. Thus, the authors have regarded that the culture method under hypoxia supported the accumulation of tendon ECM components. This condensed ECM contributed to the structural stability of the sheets. When we checked the cellular function after the manipulation steps, Teno-putty showed a similar level of proliferation rate to the un-manipulated group. We applied it to rats' Achilles tendon defects model to verify tissue regeneration.

In the mechanical testing results, the maximum load and stiffness of negative control and single-cell injection groups were more excessive than healthy tendons. In particular, the mechanical properties of the negative control group were rapidly increased than others. Only Teno-putty implanted group showed a similar to positive control at eight weeks. Therefore, we regarded those excessive mechanical properties involved severe concerns, which healing delay, pain, and even re-injury. This tendency is almost the same as visible scoring results. After animal experiments, we scored the tendon tissues extracted from each group. Teno-putty implanted groups were shown similar values to the positive control. Likewise, the uniaxial collagen alignment was observed in positive control and Teno-putty groups in histological observation. In contrast, the negative control group was not presented with any alignments.


In this study, we conducted a new approach to manipulating cell sheets as the injectable type and a comparative analysis with other treatment methods to verify its effectiveness in tendon regeneration.

The concept of tissue therapy products in the tissue engineering field has yet to be established. The Teno-putty showed potentially expanding the tissue engineering field and will be a novel approach beyond cell therapy.

Keywords: Tendon regeneration, Achilles tendon, Tenocytes, Hypoxia


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