Seeing the Unseen: Cell Strain and Mechanosensing

Main Text

Each year there are 1.5 million osteoporotic fractures in the United States with an annual direct cost of $12–18 billion, but these data underrepresent the full burden (1). While osteoporotic fractures seldom result in death, they can lead to a precipitous decline in health. Hip fracture, a common type of osteoporotic fracture, leaves 26% of patients disabled and requires nearly 20% of patients to enter a nursing home. Startlingly, ∼20% of hip fracture patients die within this first year (1). Maintenance of healthy bone is a result of a complex and not fully understood process; mechanical loading is a critical signal in bone metabolism, but a clear understanding of its influence remains elusive, particularly because visualizing cells in their native environment during loading is challenging.

In this issue of the Biophysical Journal, Verbruggen et al. (2) quantify living osteoblast and osteocyte strain in situ while investigating the effects of early and long-term estrogen deficiency in a rat model of postmenopausal osteoporosis. Using confocal microscopy, femur specimens were cut to visualize viable cells under tissue-level, strain-controlled loading in their natural environment. We believe that this novel technique is the closest researchers have come to observing osteocytes and osteoblasts in vivo. Although it is unknown how small tissue-level strains are sensed by osteocytes within a stiff mineral matrix, they are the accepted orchestrator of bone adaptation and remodeling. The osteocyte strain characterization presented by Verbruggen et al. (2) will drastically advance the field of bone mechanosensing.

The mystery of osteocyte mechanosensation stems from in vitro substrate deformation studies showing osteogenic responses at a very high threshold, which Verbruggen et al. (2) define as 10,000 με, compared to physiological macrolevel strain in the range of 400–3000 με (3). One microstrain (με) is the strain that produces a deformation of one part per million. To transduce physiological strains, it is hypothesized that amplification in local matrix strain or fluid flow stimulates osteocytes (4). Bone mechanical loading results in fluid flow through the extensive lacunar-canalicular environment where osteocytes reside. The results of Verbruggen et al. (2) explicitly demonstrate a strain amplification mechanism where tissue-level strain of 3000 με results in an average maximum osteocyte strain of 31,028 με, which is much higher than the assumed osteogenic threshold. Regardless of whether this strain is due primarily to local matrix deformation or is influenced by fluid flow, their results provide direct validation of strain amplification.

Many models of osteocyte mechanosensing exist, but they can be grouped into three flavors: (Fig. 1 A): process adhesions, (Fig. 1 B) cell body adhesions, or (Fig. 1 C) direct flow sensing. The results of Verbruggen et al. (2) show maximum strain in the dendritic process, which support the first theory of a mechanosensing role at cell-matrix adhesions of the osteocyte process. Schaffler et al. (3) observed that osteocyte processes are tethered through flexible proteoglycans to the canalicular cell wall at regular intervals and are connected rigidly through an undetermined mechanism at other discrete locations. Mathematical models demonstrate that these anatomical constructs produce strain amplification with the help of fluid flow, and several in vitro models indicate the mechanosensitivity of the osteocyte process to strain, although there is, to date, little supporting in vivo data.

Figure 1.

Osteocyte mechanosensing theories can be divided into three main groups: (A) dendritic-process focal-adhesion sensing where strain and fluid flow initiate a cellular response; (B) cell body focal-adhesion sensing where lacunar matrix strain is detected; and (C) direct fluid flow sensing, such as where the primary cilium detects lacunar fluid flow. To see this figure in color, go online.

Focal adhesions of the cell body are the second potential locus for osteocyte mechanosensing. This area has at times been dismissed because it was assumed that local matrix strain was too low. Furthermore, compared to the canalicular space of the cell processes, additional stimulus from fluid shear stress is reduced in the more spacious lacuna. However, in this space, Nicolella et al. (5) showed that gross strains of 2000 με can lead to an amplified lacunar tissue strain of 30,000 με, which is well above the osteogenic threshold; and while Verbruggen et al. (2) highlight the strain amplification in the cell process, their results show a similarly high strain amplification at the cell membrane within the lacuna. Several cell body adhesions could contribute to the mechanosensing in the lacuna. For example, Bidwell and Pavalko (6) propose that a mechanical stimulus at the membrane is shuttled from focal adhesions to the nucleus via a mechanosome leading to a change in gene activity.

Finally, osteocyte mechanosensing can occur from direct flow sensing at the cell membrane. Qin et al. (7) demonstrated that fluid flow alone was sufficient to stimulate an osteogenic response; they isolated the ulnae from ambulatory loading in adult turkeys and dynamically pressurized the intramedullary canal to induce fluid flow, which resulted in periosteal and endosteal bone formation. A large number of signaling systems have been shown to be involved in the response to fluid flow (8). The primary cilium, an immotile antenna-like organelle, is one promising flow sensor and is associated with the release of prostaglandin E₂, a regulator of bone metabolism, as a response to fluid shear stress of 1 Pa (9). Validated computational studies show that physiological loading results in lacunar-canalicular system shear stresses in the range of 0.8–3 Pa (4). Interestingly, these poorly understood sensory cilia are extremely sensitive to small flow rates and deflect under shear stresses as little as 0.03 Pa (9). This high level of sensitivity could be another explanation for the paradox of how low-level physiological tissue strain produces an osteogenic response and why the lower fluid shear stresses in the lacunar space are still significant.

Verbruggen et al. (2) provide critical data for the discussion of bone cell mechanotransduction and shed light on the fact that some adaptive response is happening at the cellular level during skeletal disease. Comparing early to late-stage specimens, they found that osteocytes from the ovariectomized specimens—a model for postmenopausal osteoporosis—had higher strains at the earlier time point than the control and that this difference disappeared at a later time point. Presumably, some adaptation took place between the two time points and this indicates that there is an equilibrium state for osteocytes (2). While bone disease is sometimes in the shadows of the attention garnered by cancer and heart disease, it contributes significantly to the burden of healthcare (1); the five-year survival rate after osteoporotic hip fracture is comparable to that of breast cancer (10). Given the aging population in the United States, it is expected that by 2020, half of the people over the age of 50 will be at risk for developing osteoporosis of the hip and even more for developing osteoporosis elsewhere (1). Bone mechanobiology research is imperative to ameliorate this burden and the crucial work of Verbruggen et al. (2) will move the field forward by delineating in vivo cell strain.