In Vivo Osteocyte Mechanotransduction: Recent Developments and Future Directions.

PURPOSE OF REVIEW: Mechanical loading is an essential stimulus for skeletal tissues. Osteocytes are primarily responsible for sensing mechanical stimuli in bone and for orchestrating subsequent responses. This is critical for maintaining homeostasis, and responding to injury/disease. The osteocyte mechanotransduction pathway, and the downstream effects it mediates, is highly complex. In vivo models have proved invaluable in understanding this process. This review summarizes the commonly used models, as well as more recently developed ones, and describes how they are used to address emerging questions in the field. RECENT FINDINGS: Minimally invasive animal models can be used to determine mechanisms of osteocyte mechanotransduction, at the cell and molecular level, while simultaneously reducing potentially confounding responses such as inflammation/wound-healing. The details of osteocyte mechanotransduction in bone are gradually becoming clearer. In vivo model systems are a key tool in pursing this question. Advances in this field are explored and discussed in this review.

Effects of estrogen deficiency and bisphosphonate therapy on osteocyte viability and microdamage accumulation in an ovine model of osteoporosis.

It has been proposed that osteocyte viability plays an important role in bone integrity, and that bone loss in osteoporosis may be partially due to osteocyte cell death following estrogen depletion. Osteoporosis treatments such as bisphosphonates can inhibit osteocyte apoptosis which in turn may also reduce remodeling. Consequently, microcracks in bone which are normally repaired by bone remodeling may accumulate. This study used an ovine model of osteoporosis to examine the effects of estrogen depletion and bisphosphonates on osteocyte apoptosis and microdamage accumulation. Skeletally mature ewes were randomly assigned into two equal groups; ovariectomy (OVX) and a non-treatment group (control). Half of these animals were sacrificed 12 months post-OVX. Twenty months post-OVX, a number of OVX animals were randomly selected and each received a supra-pharmacological dose of the bisphosphonate, zoledronic acid (Zol). This group and all the remaining animals were sacrificed 31 months post-OVX. A compact bone specimen was removed from the left metacarpal of each animal; half was used for osteocyte apoptosis detection and the remainder for microdamage analysis. Estrogen deficiency resulted in significant increases in the levels of osteocyte apoptosis while zoledronic acid significantly reduced the level of apoptosis in osteocytes. Zoledronic acid treatment resulted in the formation of more microcracks. However, these cracks were shorter than in control or OVX groups which may provide one explanation as to why increased damage levels following bisphosphonate treatment have not lead to increased fractures. This study also provides additional evidence of the importance of estrogen in preserving the osteocyte network.

Variation of trabecular microarchitectural parameters in cranial, caudal and mid-vertebral regions of the ovine L3 vertebra.

The lumbar vertebrae are major load-bearing structures within the spinal column. The current understanding of the microstructure of these bodies and their full role in load-bearing is incomplete. There is a need to develop our understanding of these issues to improve fracture prediction in musculoskeletal diseases such as osteoporosis. The lumbar vertebrae consist primarily of trabecular bone enclosed in a thin cortical shell, but little is known about how microstructural parameters vary within these structures, particularly in relation to the trabecular compartment. The specific aim of this study was to use micro-computed tomography to characterize the trabecular microarchitecture of the ovine L3 vertebra in cranial, mid-vertebra and caudal regions. The L3 vertebra was obtained from skeletally mature ewes (n = 18) more than 4 years old. Three-dimensional reconstructions of three pre-defined regions were obtained and microarchitectural parameters were calculated. Whereas there was no difference in bone volume fraction or structural model index between regions, trabecular number, thickness, spacing, connectivity density, degree of anisotropy and bone mineral density all displayed significant regional variations. The observed differences were consistent with the biomechanical hypothesis that in vivo loads are distributed differently at the endplates compared with the mid-vertebra. Thus, a more integrative approach combining biomechanical theory and anatomical features may improve fracture risk assessment in the future.

Effects of ovariectomy on bone turnover, porosity, and biomechanical properties in ovine compact bone 12 months postsurgery.

Compact bone makes up approximately 80% of the human skeleton by mass; but there are little data available on the effects of increased bone turnover on compact bone mechanical and material properties. This study addresses this question by measuring intracortical remodeling, resorption cavity number, and porosity in an ovariectomized (OVX) sheep model, and measures changes in biomechanical properties. Thirty-eight sheep were divided into two groups. Group 1 were controls (n = 19), and Group 2 were ovariectomized (OVX; n = 19). Fluorochrome dyes were administered intravenously to both groups at five time points over 12 months post-OVX to label sites of bone turnover. At 12 months post-OVX all animals were euthanized. Samples were harvested from the left metatarsal and were analyzed for intracortical bone turnover at five time points, the number of resorption cavities, and the level of intracortical porosity. The effects of these parameters on bone biomechanical properties were then measured. Bone turnover was increased in the OVX group at 6, 9, and 12 months (p < 0.05). Resorption was also higher in the OVX group at 12 months (p < 0.05). Furthermore, porosity was significantly increased in the OVX group at 12 months (p < 0.05). Stiffness and yield strength were reduced in the OVX group compared to controls (p = 0.05). Ultimate compressive strength and work to fracture did not differ between groups. These findings provide new insights into the mechanisms and effects of increased bone turnover on bone material and microstructural properties.

Effects of high bone turnover on the biomechanical properties of the L3 vertebra in an ovine model of early stage osteoporosis.

STUDY DESIGN: Investigations of the effects of high bone turnover on the L3 vertebra were carried out, using an ovariectomized (OVX) ovine model of early stage osteoporosis. OBJECTIVE: To assess the contribution of bone turnover to the biomechanics of L3. SUMMARY OF BACKGROUND DATA: Clinically, dual energy x-ray absorptiometry (DEXA) is used to measure bone mineral density (BMD). However, this can only predict 60% to 70% of bone strength; the remainder is due to bone quality. There is currently little information available on how strength is affected by changes in bone quality parameters, particularly bone turnover. Turnover can be assessed clinically using biochemical markers; however, this provides systemic values, whereas localized values are required to predict site-specific fracture risk. METHODS: Thirty-eight sheep were assigned to 2 groups (control, n = 19; OVX, n = 19). Both groups were intravenously administered a fluorochrome dye on the day of surgery and 3, 6, 9, and 12 months thereafter, to label sites of bone turnover. After 12 months, animals were killed and the spinal columns harvested. L3 vertebrae were scanned using DEXA. Bone turnover was quantified using epifluorescence microscopy, and microarchitecture was assessed by microCT scanning. Compressive testing was used to characterize the mechanical properties of the vertebrae. RESULTS: BMD and microarchitecture were unchanged in OVX compared with controls. However, bone turnover, as measured by fluorochrome labeled sites, was significantly increased in the OVX group in cortical and trabecular compartments. As a consequence, the biomechanical properties were significantly reduced in that group. CONCLUSION: These findings show that OVX resulted in changes in bone turnover, which reduced biomechanical properties in a model of early stage osteoporosis. These differences were present despite microarchitecture or BMD remaining unchanged. In the future, the ability to assess site-specific bone turnover would greatly enhance the accuracy with which fracture risk could be predicted.

The effects of increased intracortical remodeling on microcrack behaviour in compact bone.

The behaviour of microdamage in bone is related to its microstructural features and thus has an important role in tissue structural properties. However, it is not known how cracks behave in areas of increased intracortical remodeling. More remodeling creates wider variation in the properties of the primary microstructural features of cortical bone, namely osteons. This situation may occur after treatment involving parathyroid hormone or events such as menopause/ovariectomy. High turnover was modeled in this study by using ovariectomy (OVX) to induce surgical menopause in sheep. We hypothesized that osteon age would influence microcrack behaviour during propagation. Five fluorochrome dyes were administered intravenously at different time-points over 12 months post-OVX to label remodeling sites and all animals were then euthanized. Compact bone specimens (2x2x36 mm) were harvested from the right metatarsal. Samples were cyclically loaded to failure and then histological analyses were carried out. Cracks were categorized by length into three groups; short (<100 mum), intermediate (100-300 mum) and long (>300 mum). Numerical crack density (Cr.Dn) of long cracks was greater in controls compared with OVX. Controls also displayed a higher crack surface density (Cr.S.Dn) compared with OVX (p<0.05). The behaviour of short cracks did not differ between old and new osteons, but intermediate and long cracks preferentially stopped at newer osteons compared with older ones (p<0.05). This mechanism may have an important role in terms of prolonging fatigue life. We conclude that recently formed secondary osteons have a unique influence on propagating microcracks compared with older osteons. Therefore localized remodeling levels should be considered when studying microcrack behaviour in bone.

The behaviour of fatigue-induced microdamage in compact bone samples from control and ovariectomised sheep.

This study investigates the effect of microdamage on bone quality in osteoporosis using an ovariectomised (OVX) sheep model of osteoporosis. Thirty-four sheep were divided into an OVX group (n=16) and a control group (n=18). Fluorochromes were administered intravenously at 3 monthly intervals after surgery to label bone turnover. After sacrifice, beams were removed from the metatarsal and tested in three-point bending. Following failure, microcracks were identified and quantified in terms of region, location and interaction with osteons. Number of cycles to failure (Nf) was lower in the OVX group relative to controls by approximately 7%. Crack density (CrDn) was higher in the OVX group compared to controls. CrDn was 2.5 and 3.5 times greater in the compressive region compared to tensile in control and OVX bone respectively. Combined results from both groups showed that 91% of cracks remained in interstitial bone, approximately 8% of cracks penetrated unlabelled osteons and less than 1% penetrated into labelled osteons. All cases of labelled osteon penetration occurred in controls. Crack surface density (CrSDn), was 25% higher in the control group compared to OVX. It is known that crack behaviour on meeting microstructural features such as osteons will depend on crack length. We have shown that osteon age also affects crack propagation. Long cracks penetrated unlabelled osteons but not labelled ones. Some cracks in the control group did penetrate labelled osteons. This may be due the fact that control bone is more highly mineralized. CrSDn was increased by 25% in the control group compared to OVX. Further study of these fracture mechanisms will help determine the effect of microdamage on bone quality and how this contributes to bone fragility.