Development of a novel bioreactor for the mechanbiological study of the tendon-bone interface

Corluka, Slavko; Croft, Andreas Shaun; Garnier, Manuel; Wangler, Sebastian; Moser, Helen Laura; Künzler, Michael; Gantenbein, Benjamin; Schär, Michael (2023). Development of a novel bioreactor for the mechanbiological study of the tendon-bone interface. In: Annual Meeting of the Swiss Society of Biomedical Engineering. 13 September.

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Introduction

Tendons are crucial in transmitting mechanical force from muscles to bones. However, this task is challenging due to the significant difference in tissue resistance between the elastic tendon and the stiff bone. Consequently, stress concentrations occur at the interface between the tendon and bone, known as the enthesis.

The enthesis possesses a specialized structure comprising four zones with increasing calcification of the extracellular matrix from the tendon to the bone. [1] Excessive loading of the enthesis can lead to degeneration, resulting in the loss of the four-zone structure, tendon rupture, and the need for surgical refixation. Unfortunately, the current surgical refixation techniques have a high re-rupture rate, primarily due to the formation of scar tissue instead of the original fourzone structure during healing. [2] To address this issue and reduce re-rupture rates, it is essential to understand the healing process of the enthesis after tendon refixation, particularly the role of mechanical loading.

Therefore, we aimed to develop an innovative bioreactor capable of precise mechanical loading in ex-vivo models, surpassing existing devices. In this study, we present the development of our new bioreactor and report our initial tests on entheses obtained from freshly slaughtered sheep using this state-of-the-art device.

Material and Methods The bioreactor consisted of a linear stage (Igus, Köln,

Germany), a step motor (NEMA 24, Igus, Köln, Germany), a load cell (Pushton, Zhengzhou, China), and an Arduino microcontroller (Mega 2560, Arduino). The linear stage provided precise positioning control, driven by the step motor. The Arduino microcontroller programmed the bioreactor's functions, controlling motion, speed, direction, and force application using the load cell. It included a safety mechanism to prevent motor damage in case of resistance or sample rupture. Sheep enthesis samples from a local abattoir were harvested and securely fastened inside a stainless-steel chamber using custom-made clamps. The chamber was sealed with a plastic beaker containing 5% fetal calf serum in high-glucose Dulbecco's Modified Eagle Medium (HG-DMEM). The fully assembled bioreactor was placed in a normoxic incubator at 37°C with 5% CO2.

Cultivation and mechanical loading of the sheep enthesis in the bioreactor were conducted for four days. On the fourth day, the cell viability of the mechanically loaded enthesis was assessed using the LIVE/DEAD Viability kit (ThermoFisher, MA, USA) and compared to a free-floating enthesis without mechanical loading.

Results

We successfully developed a bioreactor capable of controlling ex vivo enthesis loading based on predefined strain or force values. The bioreactor's output includes force measurement within the range of 15 to 200 N, with an accuracy of ±10% of the measured value and a repeatability of ±0.2 N. The absolute position of the linear displacement demonstrates a precision of ±0.1 mm, with a repeatability of ±0.05 mm. All position, force, and time data are recorded for subsequent evaluation.

In our preliminary tests using cultivated entheses obtained from freshly slaughtered sheep, we observed a high initial cell viability of 97.6% ± 0.044% immediately after harvesting. However, by day 4 in the free-floating condition, the cell viability decreased to

63.9% ± 0.073%. In contrast, the mechanically stimulated sample exhibited a higher cell viability of

74.4% ± 0.029% on day 4, indicating a positive effect of mechanical loading on cell viability compared to the free-floating condition.

Discussion

The newly developed bioreactor combines user-friendly operation, adaptability, and flexible sample loading, greatly expanding its range of applications. Initial test outcomes indicate a favorable effect of mechanical loading on cell viability in ex vivo bioreactor cultures of entheses extracted from freshly slaughtered sheep. To establish the validity of these observations, further assessment using larger sample sizes is imperative. Moreover, a comprehensive understanding of the exact implications of mechanical loading on enthesis requires diligent exploration of culture conditions and mechanical loading patterns.

References

1. Loukopoulou et al, Eur Cell Mater. 2022 May 5;43:162-

178.

2. Vinestock et al, Am J Pathol. 2022 Aug;192(8):1122-1135

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