Mechanical stimulation of growth plate chondrocytes: Previous approaches and future directions

Abstract

Growth plate cartilage resides near the ends of long bones and is the primary driver of skeletal growth. During growth, both intrinsically and extrinsically generated mechanical stresses act on chondrocytes in the growth plate. Although the role of mechanical stresses in promoting tissue growth and homeostasis has been strongly demonstrated in articular cartilage of the major skeletal joints, effects of stresses on growth plate cartilage and bone growth are not as well established. Here, we review the literature on mechanobiology in growth plate cartilage at macroscopic and microscopic scales, with particular emphasis on comparison of results obtained using different methodological approaches, as well as from whole animal and in vitro experiments. To answer these questions, macroscopic mechanical stimulators have been developed and applied to study mechanobiology of growth plate cartilage and chondrocytes. However, the previous approaches have tested a limited number of stress conditions, and the mechanobiology of a single chondrocyte has not been well studied due to limitations of the macroscopic mechanical stimulators. We explore how microfluidics devices can overcome these limitations and improve current understanding of growth plate chondrocyte mechanobiology. In particular, microfluidic devices can generate multiple stress conditions in a single platform and enable real-time monitoring of metabolism and cellular behavior using optical microscopy. Systematic characterization of the chondrocytes using microfluidics will enhance our understanding of how to use mechanical stresses to control the bone growth and the properties of tissue-engineered growth plate cartilage.

Keywords: Growth plate chondrocyte; bone growth; mechanobiology; microfluidics.

Fig. 1. Endochondral bone generation (reproduced from [1] with permission).

(a) Condensation of mesenchymal cells (blue). (b) Condensed cells developed into chondrocytes. (c) Hypertrophic chondrocytes are generated at the center of condensation. (d) Bone collar is formed, and hypertrophic chondrocytes induce mineralized extracellular matrix (ECM) and invasion of blood vessels. (e) Primary spongiosa is generated by osteoblasts. (f) Osteoblasts of bone collar and primary spongiosa become cortical bone and trabecular bone, respectively. (g) Secondary ossification center is formed at the end of bone and columns of chondrocytes are generated in the proliferation zone of growth plate. Haematopoietic marrow is generated in bone marrow space. c: chondrocytes, h: hypertrophic chondrocytes, bc: bone collar, ps: primary spongiosa, soc: secondary ossification center, col: columns of proliferating chondrocytes, hm: Haematopoietic marrow.

Fig. 2. Growth plate structure.

(a) An image of the rat tibial growth plate shows reserve zone (or resting zone), proliferative zone, and hypertrophic zone (reproduced from [8] with permission). (b) A schematic of growth plate structure (reproduced from [7] with permission). The function of growth plate is regulated by molecular signals of parathyroid hormone-related protein (PTHrP) and Indian Hedgehog (IHH). (c) Process of chondrocyte column formation (reproduced from [6] with permission).

Fig. 3. External fixators.

(a) An external fixator actuated by a calibrated spring. The magnitude of the applied force is proportional to the change in the spring length (reproduced from [16] with permission). (b-c) An external fixator actuated by pneumatic pressure (reproduced from [17] from permission). (b) The bladder operated by pressurized air pushes the one of the fixation to generate compression on the sample.

Fig. 4. Macroscopic compression device to compress hydrogel-chondrocyte constructs or cartilage samples.

(a) A sample is compressed by two plungers actuated by a motorized actuator (reproduced from [19] with permission). (b) Mechanical testing equipment for unconfined compression test of a sample (reproduced from [23] with permission). (c) Bioreactor (FX-4000C™ Flexcell® Compression Plus™ System) actuated by air pressure to compress multiple samples (reproduced from [26]).

Fig. 5. Two-dimensional (2D) cell stretching device.

(a) 2D cell stretcher actuated by vacuum pressure. The cells are cultured on thin elastic membrane, and the tension is applied to the cells by pulling the thin elastic membrane with vacuum pressure (modified from Liu et al. [28]). (b) The elastic membrane bending system with four points. Depending on the relative height between inner and outer supporting points, the strain on the cell seeded elastic membrane can be manipulated. The strain (ε) can be calculated with the equation shown above. t is the thickness of elastic membrane, d is the displacement of loading part of the device, a is the distance between the inner and outer contact points, and L is the distance between outer contact points (modified from Sun et al. [27]).

Fig. 6.. Examples of microfluidic devices used in cell biology studies.

(a) Microfluidic device generating multiple tensions in a single platform. (reproduced from [39] with permission). (b) Singe axon injury model generated in a microfluidic device. (reproduced from [40] with permission). (c) Multi channel shear stress generator. (reproduced from [41] with permission). (d) Stiffness gradient generator on a hydrogel (reproduced from [42] with permission). (e) Lung-on-a-chip device which mimics the physiology of alveolar-capillary interface of lung (reproduced from [44] with permission).