Compressed vertebrae in Atlantic salmon Salmo salar: evidence for metaplastic chondrogenesis as a skeletogenic response late in ontogeny

Anterior/posterior (a/p) compression of the vertebral column, referred to as 'short tails', is a recurring event in farmed Atlantic salmon. Like other skeletal deformities, the problem usually becomes evident in a late life phase, too late for preventive measures, making it difficult to understand the aetiology of the disease. We use structural, radiological, histological, and mineral analyses to study 'short tail' adult salmon and to demonstrate that the study of adult fish can provide important insights into earlier developmental processes. 'Short tails' display a/p compressed vertebrae throughout the spine, except for the first post-cranial vertebrae. The vertebral number is unaltered, but the intervertebral space is reduced and the vertebrae are shorter. Compressed vertebrae are characterized by an unchanged central part, altered vertebral end plates (straight instead of funnel-shaped), an atypical inward bending of the vertebral edges, and structural alterations in the intervertebral tissue. The spongiosa is unaffected. The growth zones of adjacent vertebrae fuse and blend towards the intervertebral space into chondrogenic tissue. This tissue produces different types of cartilage, replacing the notochord. The correspondence in location of intervertebral cartilage and deformed vertebral end plates, and the clearly delimited, unaltered, central vertebral parts suggest that the a/p compression of vertebral bodies is a late developmental disorder that may be related to a metaplastic shift of osteogenic tissue into chondrogenic tissue in the vertebral growth zone. Given the lack of evidence for infections, metabolic disorders and/or genetic disorders, we propose that an altered mechanical load could have caused the transformation of the bone growth zones and the concomitant replacement of the intervertebral (notochord) tissue by cartilaginous tissues in the 'short tails' studied here. This hypothesis is supported by the role that notochord cells are known to play in spine development and in maintaining the structure of the intervertebral disk.     

Effects of tissue preservation on murine bone mechanical properties

Murine bone specimens are used extensively in skeletal research to assess the effects of environmental, physiologic and pathologic factors on their mechanical properties. Given the destructive nature of mechanical testing, it is normally performed as a terminal procedure, where specimens must be preserved without affecting their mechanical properties. To this end, we aimed to study the effects of tissue preservation (freezing and formalin fixation) on the elastic and viscoelastic mechanical properties of murine femur and vertebrae. A total of 120 femurs and 180 vertebral bodies (L3–L5) underwent non-destructive cyclic loading to assess their viscoelastic properties followed by mono-cyclic loading to failure to assess their elastic properties. All specimens underwent re-hydration in 0.9% saline for 30 min prior to mechanical testing. Analysis indicated that stiffness, modulus of elasticity, yield load, yield strength, ultimate load and ultimate strength of frozen and formalin-fixed femurs and vertebrae were not different from fresh specimens. Cyclic loading of both femurs and vertebrae indicated that loss, storage and dynamic moduli were not affected by freezing. However, formalin fixation altered their viscoelastic properties. Our findings suggest that freezing and formalin fixation over a 2-week period do not alter the elastic mechanical properties of murine femurs and vertebrae, provided that specimens are re-hydrated for at least half an hour prior to testing. However, formalin fixation weakened the viscoelastic properties of murine bone by reducing its ability to dissipate viscous energy. Future studies should address the long-term effects of both formalin fixation and freezing on the mechanical properties of murine bone.     

Rib and vertebral micro‐anatomical characteristics of hydropelvic mosasauroids

Houssaye, A. & Bardet, N. 2011: Rib and vertebral micro‐anatomical characteristics of hydropelvic mosasauroids. Lethaia, Vol. 45, pp. 200–209. Mosasauroids are squamates secondarily adapted to an aquatic life that dominated the sea during the Late Cretaceous. Mosasauroids display distinct types of morphologies illustrating steps in the adaptation of this lineage to increasing obligatory habits. Hydropelvic mosasauroids (sensu Caldwell & Palci) were the most highly adapted to an open‐sea environment. Contrary to plesiopelvic forms, they are considered to have relied on a hydrodynamic, rather than hydrostatic buoyancy and body trim control strategies. This led previous authors to consider that these taxa would favour bone lightening (osteoporotic‐like condition) rather than bone mass increase. Although an osteoporotic‐like state was indeed described in Clidastes and Tylosaurus, bone mass increase was reported in Platecarpus. As a matter of fact, the new analysis of vertebral thin sections of various taxa combined with the reanalysis of the rib sections available in Sheldon’s PhD thesis in a micro‐anatomical perspective reveals the absence of both bone mass increase and bone lightening in these organisms. These taxa in fact display a vertebral micro‐anatomy much peculiar within squamates. It characteristically corresponds to a true network of thin trabeculae whose tightness varies between taxa, probably as a result of both species and individual size differences, particularly the latter. In addition, analysis of the pattern of vascularization as observed in hydropelvic mosasauroids, which is unique amongst squamates, suggests that large size in hydropelvic mosasauroids would mainly rely on protracted rather than faster growth rates. □Histology, hydropelvic mosasauroids, micro‐anatomy, ribs, vascularization, vertebrae.     

Mechanical implications of pneumatic neck vertebrae in sauropod dinosaurs

The pre-sacral vertebrae of most sauropod dinosaurs were surrounded by interconnected, air-filled diverticula, penetrating into the bones and creating an intricate internal cavity system within the vertebrae. Computational finite-element models of two sauropod cervical vertebrae now demonstrate the mechanical reason for vertebral pneumaticity. The analyses show that the structure of the cervical vertebrae leads to an even distribution of all occurring stress fields along the vertebrae, concentrated mainly on their external surface and the vertebral laminae. The regions between vertebral laminae and the interior part of the vertebral body including thin bony struts and septa are mostly unloaded and pneumatic structures are positioned in these regions of minimal stress. The morphology of sauropod cervical vertebrae was influenced by strongly segmented axial neck muscles, which require only small attachment areas on each vertebra, and pneumatic epithelia that are able to resorb bone that is not mechanically loaded. The interaction of these soft tissues with the bony tissue of the vertebrae produced lightweight, air-filled vertebrae in which most stresses were borne by the external cortical bone. Cervical pneumaticity was therefore an important prerequisite for neck enlargement in sauropods. Thus, we expect that vertebral pneumaticity in other parts of the body to have a similar role in enabling gigantism.     

MMP‐13 (Matrix MetalloProteinase 13) expression might be an indicator for increased ECM remodeling and early signs of vertebral compression in farmed Atlantic salmon (Salmo salar L.)

Vertebral body compression is a common problem in commercial farming of Atlantic salmon. Although risk factors, such as vaccination and malnutrition, have been identified, the etiology is largely unknown. Histological studies of Atlantic salmon have shown that in a compressed deformity (platyspondyly) the length of the compact bone is reduced while the notochord start to form atypical chrondrogenic structures. In mammals, similar remodeling activities have been linked to inflammatory processes in the tissue. Hence, we wanted to investigate whether the compressed vertebrae in Atlantic salmon showed presence of local (IL‐1β, TNF‐α1), systemic (IgM) and chronic (MMP‐13, MMP‐9) immune responses (measured with quantitative PCR). Unvaccinated groups of Atlantic salmon that would later develop high or low prevalence of vertebral compression during on‐growth in seawater were sampled at seawater transfer, and 3 and 6 weeks after seawater transfer. In addition, compressed and normal vertebrae from the high deformity prevalence group were sampled 44 weeks after transfer to seawater. MMP‐13 was significantly up‐regulated in the group that developed a high prevalence of deformity, and also significantly up‐regulated in compressed vertebrae, 44 weeks after seawater transfer. In compressed vertebrae, MMP‐13 was equally up‐regulated in the notochord, compact bone and trabecular bone. The results of the present study suggest that MMP‐13 may serve as an early indicator for bone remodeling which may lead to vertebral compression, and that there is a relationship between the development of vertebral compression and increased remodeling activities in farmed Atlantic salmon.     

The use of preserved tissue in finite element modelling of fresh ovine vertebral behaviour

The aim of this study was to investigate whether the predicted finite element (FE) stiffness of vertebral bone is altered when using images of preserved rather than fresh tissue to generate specimen-specific FE models. Fresh ovine vertebrae were used to represent embalmed (n = 3) and macerated dry-bone (n = 3) specimens and treated accordingly. Specimens were scanned pre- and post-treatment using micro-computed tomography. A constant threshold level derived from these images was used to calculate the respective bone volume fraction (BV/TV) from which the conversion factor validated for fresh tissue was used to determine material properties that were assigned to corresponding FE models. Results showed a definite change in the BV/TV between the fresh and the preserved bone. However, the changes in the predicted FE stiffness were not generally greater than the variations expected from assignment of loading and boundary conditions. In conclusion, images of preserved tissue can be used to generate FE models that are representative of fresh tissue with a tolerable level of error.     

Quantification of a rat tail vertebra model for trabecular bone adaptation studies

A feedback controlled loading apparatus for the rat tail vertebra was developed to deliver precise mechanical loads to the eighth caudal vertebra (C8) via pins inserted into adjacent vertebrae. Cortical bone strains were recorded using strain gages while subjecting the C8 in four cadaveric rats to mechanical loads ranging from 25 to 100 N at 1 Hz with a sinusoidal waveform. Finite element (FE) models, based on micro computed tomography, were constructed for all four C8 for calculations of cortical and trabecular bone tissue strains. The cortical bone strains predicted by FE models agreed with strain gage measurements, thus validating the FE models. The average measured cortical bone strain during 25-100 N loading was between 298 +/- 105 and 1210 +/- 297 microstrain (muepsilon). The models predicted average trabecular bone tissue strains ranging between 135 +/- 35 and 538 +/- 138 mu epsilon in the proximal region, 77 +/- 23-307 +/- 91 muepsilon in the central region, and 155 +/- 36-621 +/- 143 muepsilon in the distal region for 25-100 N loading range. Although these average strains were compressive, it is also interesting that the trabecular bone tissue strain can range from compressive to tensile strains (-1994 to 380 mu epsilon for a 100 N load). With this novel approach that combines an animal model with computational techniques, it could be possible to establish a quantitative relationship between the microscopic stress/strain environment in trabecular bone tissue, and the biosynthetic response and gene expression of bone cells, thereby study bone adaptation.     

Phenotypic plasticity and mechano‐transduction in the teleost skeleton

The proper formation, growth and maintenance of many bones depends on the mechanical loads generated by gravity and muscles. Mechanical loading by muscle forces does not only affect bone growth and maintenance in adult and juvenile vertebrates, but also affects larval and embryonic bone development. We have reviewed the current understanding of mechanotransduction in birds and mammals and compared it to teleosts. The major mechanosensing cells in the adult mammalian and avian skeleton are osteocytes. They are interconnected via cell processes and are contained within a canalicular network. Basal teleosts have osteocytes but their connectivity is questionable and the presence of a functional canalicular network is unlikely. Advanced teleosts have acellular bone and therefore lack osteocytes. Yet the skeleton of teleosts does show adaptive responses to changes in mechanical load. In these animals it is likely that osteoblasts, bone surface cells and chondrocytes act as mechanosensors. The factors expressed by osteocytes upon mechanical stimulation have been extensively investigated in vitro and in vivo in adult mammals and birds. Less is, however, known about the mechanotransduction pathway during embryonic bone development. The zebrafish presents new opportunities to analyze the mechanotransduction pathway during early (larval) bone formation due to the ex utero development and genetic analyses.     

Trabecular shear stress in human vertebral cancellous bone: intra- and inter-individual variations

Correlation of the mean and standard deviation of trabecular stresses has been proposed as a mechanism by which a strong relationship between the apparent strength and stiffness of cancellous bone can be achieved. The current study examined whether the relationship between the mean and standard deviation of trabecular von Mises stresses can be generalized for any group of cancellous bone. Cylindrical human vertebral cancellous bone specimens were cut in the infero-superior direction from T12 of 23 individuals (inter-individual group). Thirty nine additional specimens were prepared similarly from the T4-T12 and L2-L5 vertebrae of a 63 year old male (intra-individual group). The specimens were scanned by micro-computed tomography (microCT) and trabecular von Mises stresses were calculated using finite element modeling. The expected value, standard deviation and coefficient of variation of the von Mises stress were calculated form a three-parameter Weibull function fitted to von Mises stress data from each specimen. It was found that the average and standard deviation of trabecular von Mises shear stress were: (i) correlated with each other, supporting the idea that high correlation between the apparent strength and stiffness of cancellous bone can be achieved through controlling the trabecular level shear stress variations, (ii) dependent on anatomical site and sample group, suggesting that the variation of stresses are correlated to the mean stress to different degrees between vertebrae and individuals, and (iii) dependent on bone volume fraction, consistent with the idea that shear stress is less well controlled in bones with low BV/TV. The conversion of infero-superior loading into trabecular von Mises stresses was maximum for the tissue at the junction of the thoracic and lumbar spine (T12-L1) consistent with this junction being a common site of vertebral fracture.     

Effects of vegetable feed ingredients on bone health in Atlantic salmon

The aim of the present study was to examine if dietary inclusion of vegetable lipids (VL) and proteins (VP) influenced markers of bone health in Atlantic salmon. Triplicate groups were fed one of four different diets; 100% fish protein (FP) and fish lipids (FL) (FPFL), 80% VP and 35% VL (80VP35VL), 40% VP and 70% VL (40VP70VL), or 80% VP and 70% VL (80VP70VL) for 12 months on‐growth in sea water. Fish were analyzed for vertebral bone mineralization (mineral content, as % of bone dry weight), vertebral deformities (radiology), vertebral bone mRNA expression of factors involved in mineralization (bone gla protein, bgp) and growth regulation (igf‐I and growth hormone receptor), as well as plasma vitamin D metabolites. The fish grew from 0.35 to 4 kg during the experimental period. At the end of the experiment, significantly lower prevalence of fish with one or more deformed vertebrae was observed in the 80VP70VL group (11%) compared to the other groups (33–43%). There was a significant higher relative expression of igf‐I mRNA in vertebral bone of fish fed the 80VP70VL diet compared to control fish (FPFL), while the other genes studied were unaffected. Elevated plasma 25‐hydroxyvitamin D₃ recorded in the marine feed group is discussed as a predictor for later development of bone deformities. In conclusion, the present study shows that high inclusion levels of vegetable lipids and proteins may have a positive effect on bone health in Atlantic salmon postsmolts.     

Natural variability and effects of cleaning and storage procedures on vertebral chemistry of the blacktip shark Carcharhinus limbatus

Key methodological assumptions regarding the degree of natural variability and influence of sample handling and storage of elasmobranch vertebral chemistry were assessed using laser‐ablation inductively coupled plasma mass spectrometry. Vertebral chemistry of juvenile blacktip sharks Carcharhinus limbatus was examined to identify whether differences existed among different regions of the vertebral column, between thoracic vertebrae of individual fish or within individual vertebrae. Additionally, the effects of bleach exposure and storage in ethanol on vertebral chemistry were compared. No significant variation in vertebral chemistry was found among different regions of the vertebral column or between thoracic vertebrae, but significant differences among transect locations within individual vertebrae were observed. The variation at all three levels appears comparable with published data on sagittal otoliths of bilaterally symmetrical teleost fishes. The experimental assessment of potential treatment effects indicated vertebral chemistry was not significantly affected by bleach or exposure to ethanol. Taken together, these results support the idea that vertebrae taken from the same region of the vertebral column can be treated as equivalent and at least certain elements remain robust to exposure to bleach and ethanol.     

A comparison between porcine,ovine, and bovine intervertebral disc anatomy and single lamella annulus fibrosus tensile properties

This project aimed to compare gross anatomical measures and biomechanical properties of single lamellae from the annulus fibrosus of ovine and porcine lumbar vertebrae, and bovine tail vertebrae. The morphology of the vertebrae of these species differ significantly both from each other and from human, yet how these differences alter biomechanical properties is unknown. Geometric parameters measured in this study included: 1) absolute and relative intervertebral (IVD) and vertebral body height and 2) absolute and relative intervertebral disc (IVD) anterior‐posterior (AP) and medial‐lateral (ML) widths. Single lamella tensile properties included toe‐region stress and stretch ratio, stiffness, and tensile strength. As expected, the bovine tail IVD revealed a more circular shape compared with both the ovine and porcine lumbar IVD. The bovine tail also had the largest IVD to vertebral body height ratio (due to having the highest absolute IVD height). Bovine tail lamellae were also found to be strongest and stiffest (in tension) while ovine lumbar lamellae were weakest and most compliant. Histological analysis revealed the greatest proportion of collagen in the bovine corroborating findings of increased strength and stiffness. The observed differences in anatomical shape, connective tissue composition, and tensile properties need to be considered when choosing an appropriate model for IVD research. J. Morphol. 277:244–251, 2016. © 2015 Wiley Periodicals, Inc.     

A primary phosphorus‐deficient skeletal phenotype in juvenile Atlantic salmon Salmo salar: the uncoupling of bone formation and mineralization

To understand the effect of low dietary phosphorus (P) intake on the vertebral column of Atlantic salmon Salmo salar, a primary P deficiency was induced in post‐smolts. The dietary P provision was reduced by 50% for a period of 10 weeks under controlled conditions. The animal's skeleton was subsequently analysed by radiology, histological examination, histochemical detection of minerals in bones and scales and chemical mineral analysis. This is the first account of how a primary P deficiency affects the skeleton in S. salar at the cellular and at the micro‐anatomical level. Animals that received the P‐deficient diet displayed known signs of P deficiency including reduced growth and soft, pliable opercula. Bone and scale mineral content decreased by c. 50%. On radiographs, vertebral bodies appear small, undersized and with enlarged intervertebral spaces. Contrary to the X‐ray‐based diagnosis, the histological examination revealed that vertebral bodies had a regular size and regular internal bone structures; intervertebral spaces were not enlarged. Bone matrix formation was continuous and uninterrupted, albeit without traces of mineralization. Likewise, scale growth continues with regular annuli formation, but new scale matrix remains without minerals. The 10 week long experiment generated a homogeneous osteomalacia of vertebral bodies without apparent induction of skeletal malformations. The experiment shows that bone formation and bone mineralization are, to a large degree, independent processes in the fish examined. Therefore, a deficit in mineralization must not be the only cause of the alterations of the vertebral bone structure observed in farmed S. salar. It is discussed how the observed uncoupling of bone formation and mineralization helps to better diagnose, understand and prevent P deficiency‐related malformations in farmed S. salar.     

Regional variation in morphology of vertebral centra and intervertebral joints in striped bass,Morone saxatilis

The vertebral column of fishes has traditionally been divided into just two distinct regions, abdominal and caudal. Recently, however, developmental, morphological, and mechanical investigations have brought this traditional regionalization scheme into question. Alternative regionalization schema advocate the division of the abdominal vertebrae into cervical, abdominal, and in some cases, transitional regions. Here, we investigate regional variation at the level of the vertebrae and intervertebral joint (IVJ) tissues in the striped bass, Morone saxatilis. We use gross dissection, histology, and polarized light imaging to quantify vertebral height, width, length, IVJ length, IVJ tissue volume and cross‐sectional area, and vertical septum fiber populations, and angles of insertion. Our results reveal regional differences between the first four (most rostral) abdominal vertebrae and IVJs and the next six abdominal vertebrae and IVJs, supporting the recognition of a distinct cervical region. We found significant variation in vertebral length, width, and height from cranial to caudal. In addition, we see a significant decline in the volume of notochordal cells and the cross‐sectional area of the fibrous sheath from cranial to caudal. Further, polarized light imaging revealed four distinct fiber populations within the vertical septum in the cervical and abdominal regions in contrast with just one fiber population found in the caudal region. Measurement of the insertion angles of these fiber populations revealed significant differences between the cervical and abdominal regions. Differences in vertebral, IVJ, and vertical septum morphology all predict greater range of motion and decreased stiffness in the caudal region of the fish compared with the cervical and abdominal regions. J. Morphol., 2012. © 2011 Wiley Periodicals, Inc.     

Lordotic vertebrae in sea bass (Dicentrarchus labrax L.) are adapted to increased loads

Lordosis in fish is an abnormal ventral curvature of the vertebral column, accompanied by abnormal calcification of the afflicted vertebrae. Incidences of lordosis are a major problem in aquaculture and often correlate with increased swimming activity. To understand the biomechanical causes and consequences of lordosis, we mapped the morphological changes that occur in the vertebrae of European sea bass during their development from larva to juvenile. Our micro-CT analysis of lordotic and non-lordotic vertebrae revealed significant differences in their micro-architecture. Lordotic vertebrae have a larger bone volume, flattened dorsal zygapophyses and extra lateral ridges. They also have a larger second moment of area (both lateral and dorso-ventral) than non-lordotic vertebrae. This morphology suggests lordotic vertebrae to be adapted to an increased bending moment, caused by the axial musculature during increased swimming activity. We hypothesize the increase in swimming activity to have a two-fold effect in animals that become lordotic. The first effect is buckling failure of the axial skeleton due to an increased compressive load. The second effect is extra bone deposition as an adaptive response of the vertebrae at the cellular level, caused by an increased strain and strain rate in these vertebrae. Lordosis thus comprises both a buckling failure of the vertebral column and a molecular response that adapts the lordotic vertebrae to a new loading regime.     

Effect of saddleback syndrome and vertebral deformity on the body shape and size in hatchery-reared juvenile red spotted grouper, Epinephelus akaara (Perciformes: Serranidae): a geometric morphometric approach

This study examined how saddleback syndrome (SBS) and vertebral deformity affect the body shape and size of juvenile stage red spotted grouper, Epinephelus akaara, using the landmark‐based geometric morphometrics method. According to the criterion of skeletal conditions, three groups, i.e. vertebral deformity, SBS, and normal groups, were identified. The results revealed significant differences in body shape among the three groups, in which the vertebral‐deformed group had the deepest mid‐body, the broadest anterior part, and a shortened caudal peduncle, while the SBS group showed the shallowest mid‐body and the narrowest anterior part. The normal group had a body shape intermediate between the vertebral and SBS groups. A comparison of body size among the three groups revealed significant differences in centroid size, with the vertebral‐deformed and SBS groups showing smallest and largest centroid size, respectively. This study illuminates that not all skeletal deformities lead to smaller body size. We suggest that rearing conditions might have caused the deformities reported herein.     

Fluid shear stress induces Runx‐2 expression via upregulation of PIEZO1 in MC3T3‐E1 cells

Mechanically induced biological responses in bone cells involve a complex biophysical process. Although various mechanosensors have been identified, the precise mechanotransduction pathway remains poorly understood. PIEZO1 is a newly discovered mechanically activated ion channel in bone cells. This study aimed to explore the involvement of PIEZO1 in mechanical loading (fluid shear stress)‐induced signaling cascades that control osteogenesis. The results showed that fluid shear stress increased PIEZO1 expression in MC3T3‐E1 cells. The fluid shear stress elicited the key osteoblastic gene Runx‐2 expression; however, PIEZO1 silencing using small interference RNA blocked these effects. The AKT/GSK‐3β/β‐catenin pathway was activated in this process. PIEZO1 silencing impaired mechanically induced activation of the AKT/GSK‐3β/β‐catenin pathway. Therefore, the results demonstrated that MC3T3‐E1 osteoblasts required PIEZO1 to adapt to the external mechanical fluid shear stress, thereby inducing osteoblastic Runx‐2 gene expression, partly through the AKT/GSK‐3β/β‐catenin pathway.     

Bone resorption and bone remodelling in juvenile carp, Cyprinus carpio L.

The present study considers the important role of bone resorption for bone growth in general, and aims to clarify if and how bone resorption contributes to the skeletal development of carp, Cyprinus carpio L., a teleost species with ‘normal’ osteocyte‐containing (cellular) bone. To ensure the identification of osteoclasts and sites of bone resorption independently from the morphology of the bony cells, bones were studied by histological procedures, and by demonstration of the enzymes which serve as osteoclast markers, viz. tartrate resistant acid phosphatase (TRAP), ATPase and a vacuolar proton pump. Two types of bone‐resorbing cells were observed in juvenile carp: (1) multinucleated giant cells displaying morphological and biochemical attributes which are known from mammalian osteoclasts; and (b) flat cells which lack a visible ruffled border and for which identification requires the performance of enzyme histochemical procedures. Bone resorption performed by osteoclasts mainly occurs at endosteal bone surfaces. To a lesser extent, bone resorption also takes place at periosteal bone surfaces, but without an apparent connection to bone growth. The latter observation, and the occurrence of bone remodelling, suggest that the endoskeleton of juvenile carp might be involved in mineral metabolism. Morphological differences and biochemical similarities to bone resorption in teleosts with acellular bone are discussed.     

Low magnitude and high frequency mechanical loading prevents decreased bone formation responses of 2T3 preosteoblasts

Bone loss due to osteoporosis or disuse such as in paraplegia or microgravity is a significant health problem. As a treatment for osteoporosis, brief exposure of intact animals or humans to low magnitude and high frequency (LMHF) mechanical loading has been shown to normalize and prevent bone loss. However, the underlying molecular changes and the target cells by which LMHF mechanical loading alleviate bone loss are not known. Here, we hypothesized that direct application of LMHF mechanical loading to osteoblasts alters their cell responses, preventing decreased bone formation induced by disuse or microgravity conditions. To test our hypothesis, preosteoblast 2T3 cells were exposed to a disuse condition using the random positioning machine (RPM) and intervened with an LMHF mechanical load (0.1–0.4 g at 30 Hz for 10–60 min/day). Exposure of 2T3 cells to the RPM decreased bone formation responses as determined by alkaline phosphatase (ALP) activity and mineralization even in the presence of a submaximal dose of BMP4 (20 ng/ml). However, LMHF mechanical loading prevented the RPM‐induced decrease in ALP activity and mineralization. Mineralization induced by LMHF mechanical loading was enhanced by treatment with bone morphogenic protein 4 (BMP4) and blocked by the BMP antagonist noggin, suggesting a role for BMPs in this response. In addition, LMHF mechanical loading rescued the RPM‐induced decrease in gene expression of ALP, runx2, osteomodulin, parathyroid hormone receptor 1, and osteoglycin. These findings suggest that preosteoblasts may directly respond to LMHF mechanical loading to induce differentiation responses. The mechanosensitive genes identified here provide potential targets for pharmaceutical treatments that may be used in combination with low level mechanical loading to better treat osteoporosis or disuse‐induced bone loss. J. Cell. Biochem. 106: 306–316, 2009. © 2009 Wiley‐Liss, Inc.