Elsevier

Journal of Biomechanics

Volume 78, 10 September 2018, Pages 94-101
Journal of Biomechanics

Ex-vivo observation of calcification process in chick tibia slice: Augmented calcification along collagen fiber orientation in specimens subjected to static stretch

https://doi.org/10.1016/j.jbiomech.2018.07.024Get rights and content

Abstract

Bone formation through matrix synthesis and calcification in response to mechanical loading is an essential process of the maturation in immature animals, although how mechanical loading applied to the tissue increases the calcification and improves mechanical properties, and which directions the calcification progresses within the tissue are largely unknown. To address these issues, we investigated the calcification of immature chick bone under static tensile stretch using a newly developed real-time observation bioreactor system. Bone slices perpendicular to the longitudinal axis obtained from the tibia in 2- to 4-day-old chick legs were cultured in the system mounted on a microscope, and their calcification was observed up to 24 h while they were stretched in the direction parallel to the slice. Increase in the calcified area, traveling distance and the direction of the calcification and collagen fiber orientation in the newly calcified region were analyzed. There was a significant increase in calcified area in the bone explant subjected to tensile strain over ∼3%, which corresponds to the threshold strain for collagen fibers showing alignment in the direction of stretch, indicating that the fiber alignment may enhance tissue calcification. The calcification progressed to a greater distance to the stretching direction in the presence of the loading. Moreover, collagen fiber orientation in the calcified area in the loaded samples was coincided with the progression angle of the calcification. These results clearly show that the application of static tensile strain enhanced tissue calcification, which progresses along collagen fibers aligned to the loading direction.

Introduction

Bone tissue is subjected to both tensile and compressive loadings during body movements in vivo. The loadings applied at the tissue level induced hydrostatic and fluid shear stress at the cellular level (Curry, 2002). These stimuli are known to maintain a balance of activities of osteocytes, osteoblasts and osteoclasts, contributing to tissue homeostasis when the loading is within a physiological range (Robling et al., 2006). In the absence of the loading, however, bone resorption by osteoclasts become more evident than bone synthesis (Jaworski et al., 1980). Thus, mechanical loading plays crucial roles in bone tissue homeostasis as well as bone remodeling.

It is also exhibited that the role of mechanical loading in bone tissue homeostasis is different between mature and immature animals. As mentioned above, in mature animals the absence of mechanical loading results in porous bone matrix due to shifting of the cellular activities to bone resorption. In contrast, in immature animals, the absence of the loading results in the inhibition of bone growth (Amprino, 1985, Uhthoff and Jaworski, 1978), possibly due to the lack of stimuli to upregulate bone matrix synthesis. These differences suggest that cells in immature bones possess the sensitivities and the responsiveness to mechanical loading different from those in mature bones. Similar differences in mechanical responses to mechanical loading between immature and mature animals have also been reported in other load-bearing tissues such as tendon (Fujie et al., 2000). Because bone cells in immature tissue are more active in producing bone matrix components, it is important to understand how the cells in immature bones respond to mechanical loading and how these responses contribute to the maturation of the bone tissue. Such information would also be useful to the development of strategies of bone tissue engineering and regenerative medicine.

We have previously developed an ex vivo tissue loading system and utilized it to examine responses of bone tissue to mechanical loading in an explant model obtained from 0-day-old chick tibia (Maeda et al., 2017). It was demonstrated that an application of cyclic compression with an amplitude of 0.3 N to the explant with a thickness of 3 mm elevated elastic moduli significantly at the end of a 3-day loading culture period from the level measured before loading culture. This was associated with an increase in the area of newly calcified tissue. In the absence of loading during the culture, such elevation of the moduli nor increase in the calcified area was evident. However, it is still unknown how mechanical loading applied to the explant increases the calcification and the elastic moduli, and which directions the calcification progresses in response to the loading. Meanwhile, we noticed in the histological sections of 0-day-old and 3-day-old chick tibias that the progression of the calcification was at several micrometers per hour on average. This indicates that the progress of bone calcification in situ could be observed using tissue culture technique in conjunctions with a microscopic observation during a period of several hours. Therefore, the present study was performed to establish a bioreactor system on a microscope whereby a real-time monitoring of bone calcification is enabled, and using the system and the newborn chick tibia model we investigated the calcification of immature bone tissue under static tensile stretch.

Section snippets

Specimens

All animal experiments were approved by the institutional review board for animal care at Nagoya Institute of Technology (Approval Nos. 18003 and 19003) and were performed in accordance with the Guide for Animal Experimentation, Nagoya Institute of Technology. Bone explant was obtained from the tibia in 2- to 4-day-old chick legs (Fig. 1). The animal was sacrificed using CO2 gas. Both left and right legs were sterilized with 70% ethanol, and the tibia was excised within a clean bench. A bone

Results

Fig. 4(a) presents the relationship between the calcification ratio obtained at 24 h and the level of tensile strain applied to the cartilage region. It was exhibited that the calcification ratio in stretched samples remained at the levels of unstretched control samples when the strain level applied was low, whereas the ratio in stretched samples subjected to higher levels of strain was markedly higher than those of unstretched samples. However, the trend was opposite in the strain level over

Discussion

We have successfully established the experimental system to conduct the real-time observation of the calcification of immature bone tissue subjected to static tensile stretch. Results obtained showed that the application of static tensile strain to a slice of the tibia from immature chick induced significant enhancement of tissue calcification when tensile strain exceeded 3%. The progression of enhanced tissue calcification was in the direction same with the applied strain. It was also

Acknowledgements

This work was supported in part by KAKENHI from JSPS (Nos. 15H02209, 15H05860, and 16K01346) and AMED-CREST from Japan Agency for Medical Research and Development (JP17gm0810005).

Conflict of interest

The authors have neither financial nor personal relationships with other people or organizations that could inappropriately influence the present work.

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