Mechanically induced Ca^(2+) oscillations in osteocytes release extracellular vesicles and enhance bone formation

The vast osteocytic network is believed to orchestrate bone metabolic activity in response to mechanical stimuli through production of sclerostin, RANKL, and osteoprotegerin(OPG). However, the mechanisms of osteocyte mechanotransduction remain poorly understood. We've previously shown that osteocyte mechanosensitivity is encoded through unique intracellular calcium (Ca^(2+) ) dynamics. Here, by simultaneously monitoring Ca^(2+) and actin dynamics in single cells exposed to fluid shear flow, we detected actin network contractions immediately upon onset of flow-induced Ca^(2+) transients, which were facilitated by smooth muscle myosin and further confirmed in native osteocytes ex vivo. Actomyosin contractions have been linked to the secretion of extracellular vesicles(EVs), and our studies demonstrate that mechanical stimulation upregulates EV production in osteocytes through immunostaining for the secretory vesicle marker Lysosomal-associated membrane protein 1(LAMP1) and quantifying EV release in conditioned medium, both of which are blunted when Ca^(2+) signaling was inhibited by neomycin. Axial tibia compression was used to induce anabolic bone formation responses in mice, revealing upregulated LAMP1 and expected downregulation of sclerostin in vivo. This load-related increase in LAMP1 expression was inhibited in neomycin-injected mice compared to vehicle.Micro-computed tomography revealed significant load-related increases in both trabecular bone volume fraction and cortical thickness after two weeks of loading, which were blunted by neomycin treatment. In summary, we found mechanical stimulation of osteocytes activates Ca^(2+) -dependent contractions and enhances the production and release of EVs containing bone regulatory proteins. Further, blocking Ca^(2+) signaling significantly attenuates adaptation to mechanical loading in vivo, suggesting a critical role for Ca^(2+) -mediated signaling in bone adaptation.

Trabecular Plate Loss and Deteriorating Elastic Modulus of Femoral Trabecular Bone in Intertrochanteric Hip Fractures

Osteoporotic hip fracture is associated with significant trabecular bone loss, which is typically characterized as low bone density by dual-energy X-ray absorptiometry （DXA） and altered microstructure by micro-computed tomography （pCT）. Emerging morphological analysis techniques, e.g. individual trabecula segmentation （ITS）, can provide additional insights into changes in plate-like and rod-like trabeculae, two major micro- structural types serving different roles in determining bone strength. Using ITS, we evaluated trabecular microstructure of intertrochanteric bone cores obtained from 23 patients undergoing hip replacement surgery for intertrochanteric fracture and 22 cadaveric controls. Micro-finite element （~FE） analyses were performed to further understand how the abnormalities seen by ITS might translate into effects on bone strength. ITS analyses revealed that, near fracture site, plate-like trabeculae were seriously depleted in fracture patients, but trabecular rod volume was maintained. Besides, decreased plate area and rod length were observed in fracture patients. Fracture patients also showed decreased elastic moduli and shear moduli of trabecular bone. These results provided evidence that in intertrochanteric hip fracture, preferential loss of plate-like trabeculae led to more rod-like microstructure and deteriorated mechanical competence adjacent to the fracture site, which increased our understanding of the biomechanical pathogenesis of hip fracture in osteoporosis.

Abnormal subchondral bone remodeling and its association with articular cartilage degradation in knees of type 2 diabetes patients

Type 2 diabetes （T2D） is associated with systemic abnormal bone remodeling and bone loss. Meanwhile, abnormal subchondral bone remodeling induces cartilage degradation, resulting in osteoarthritis （OA）. Accordingly, we investigated alterations in subchondral bone remodeling, microstructure and strength in knees from T2D patients and their association with cartilage degradation. Tibial plateaus were collected from knee OA patients undergoing total knee arthroplasty and divided into non-diabetic （n---70） and diabetes （n = 51） groups. Tibial plateaus were also collected from cadaver donors （n = 20） and used as controls. Subchondral bone microstructure was assessed using micro-computed tomography. Bone strength was evaluated by micro-finite-element analysis. Cartilage degradation was estimated using histology. The expression of tartrate-resistant acidic phosphatase （TRAP）, osterix, and osteocalcin were calculated using immunohistochemistry. Osteoarthritis Research Society International （OARSI） scores of lateral tibial plateau did not differ between non-diabetic and diabetes groups, while higher OARSI scores on medial side were detected in diabetes group. Lower bone volume fraction and trabecular number and higher structure model index were found on both sides in diabetes group. These microstructural alterations translated into lower elastic modulus in diabetes group. Moreover, diabetes group had a larger number of TRAP~ osteoclasts and lower number of Osterix~ osteoprogenitors and Osteocalcin~ osteoblasts. T2D knees are characterized by abnormal subchondral bone remodeling and microstructural and mechanical impairments, which were associated with exacerbated cartilage degradation. In regions with intact cartilage the underlying bone still had abnormal remodeling in diabetes group, suggesting that abnormal bone remodeling may contribute to the early pathogenesis of T2D-associated knee OA.

How does Mechanical Loading Make Bone Microstructure: Modeling vs. Remodeling

Bone modeling and remodeling are governed by distinct biochemical processes that may hold unique opportunities for optimizing bone mass[1,2].Remodeling refers to the coupled resorption and subsequent formation on the bone surface,while modeling represents uncoupled formation or resorption.Mechanical loading is known to improve bone mass,though whether this occurs through modeling or remodeling(or by some combination)is unclear.Dynamic in vivo morphometry utilizing high resolution micro-CT and image registration has only recently become feasible and thus holds an untapped and expanding potential for understanding bone metabolism by quantifying and localizing formation/resorption and modeling/remodeling events.16-week-old mice were given 2 baseline weekly micro-CT scans of both tibiae prior to the initiation of daily unilateral loading(contralateral limb for nonloaded control).Weekly scanning and daily loading continued for 5 weeks.Registered images for each mouse in a global coordinate system revealed the time course of each voxel,and changes in bone mass were quantified as modeling or remodeling starting at the onset of loading.In cortical bone,after an initial response to loading in both regimes,modeling emerged as the dominant response.Loading effects were largest in areas of mechanical significance.For example,anabolic modeling on the periosteal surface of the half of the tibia in compression under axial load presented a strong effect of loading,whereas the same measure on the endosteal surface in the area in tension showed no difference.Similarly,in trabecular bone anabolic modeling was significantly increased with loading on trabecular plates but not rods(plates have been shown to be the major contributor to overall bone strength).The catabolic modeling response on the endosteal surface showed an interesting transition over time.Loading initially led to a significant suppression of catabolic modeling,but over time increased it to levels significantly beyond that of nonloaded controls.

Bone density, microarchitecture and stiffness in Caucasian and Caribbean Hispanic postmenopausal American women

Hispanic Americans of Caribbean origin are a fast-growing subset of the US population, but there are no studies on bone density, microstructure and biomechanical integrity in this minority group. In this study, we aimed to compare Caucasian and Caribbean Hispanic postmenopausal American women with respect to these characteristics. Thirty-three Caribbean Hispanics were age-matched to thirty-three Caucasian postmenopausal women. At the lumbar spine, the Hispanic women had significantly lower areal bone mineral density （aBMD）. At the radius by high-resolution peripheral quantitative computed tomography （HR-pQCT）, there were minimal differences between Hispanic and Caucasian women. At the tibia, Hispanic women had lower trabecular volumetric bone density and trabecular number, and higher trabecular separation. Individual trabecula segmentation （ITS） analyses indicated that at the tibia, Hispanic women not only had significantly lower bone volume fraction, but also had significantly lower rod bone volume fraction, plate trabecular number, rod trabecular number and lower plate-plate, plate-rod and rod-rod junction densities compared to Caucasian women. The differences in bone quantity and quality contributed to lower whole bone stiffness at the radius, and both whole bone and trabecular bone stiffness at the tibia in Hispanic women. In conclusion, Hispanic women had poorer bone mechanical and microarchitectural properties than Caucasian women, especially at the load-bearing distal tibia.

Intracellular calcium signaling in osteocytes:A mechano-transduction-mechano paradigm

Osteocytes in vivo are embedded in the mineralized extracellular bone matrix,where their cell bodies reside in the lacunae and are interconnected to neighboring osteocytes through numerous intercellular processes.The 3-dimensional（3D）osteocyte network positioning and ability to communicate with other bone cells make osteocytes ideal mechanosensors of bone.Thus the role of osteocyte network and intercellular communication between osteocytes in response to mechanical stimulation may clarify the mechanisms behind normal bone adaptation to mechanical loading.We have been using intracellular calcium([Ca2+]i)as a ubiquitous real-time signaling indicator for studying mechanotransduction in osteocytic network

Calcium signaling in osteocyte network

Introduction Osteocytes are interconnected through numerous intercellular processes,forming extensive cell networks throughout the bone tissue[1]. It has been shown that osteocyte density is an important physiological parameter,which decreases