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1.  Sauropod dinosaurs evolved moderately sized genomes unrelated to body size 
Sauropodomorph dinosaurs include the largest land animals to have ever lived, some reaching up to 10 times the mass of an African elephant. Despite their status defining the upper range for body size in land animals, it remains unknown whether sauropodomorphs evolved larger-sized genomes than non-avian theropods, their sister taxon, or whether a relationship exists between genome size and body size in dinosaurs, two questions critical for understanding broad patterns of genome evolution in dinosaurs. Here we report inferences of genome size for 10 sauropodomorph taxa. The estimates are derived from a Bayesian phylogenetic generalized least squares approach that generates posterior distributions of regression models relating genome size to osteocyte lacunae volume in extant tetrapods. We estimate that the average genome size of sauropodomorphs was 2.02 pg (range of species means: 1.77–2.21 pg), a value in the upper range of extant birds (mean = 1.42 pg, range: 0.97–2.16 pg) and near the average for extant non-avian reptiles (mean = 2.24 pg, range: 1.05–5.44 pg). The results suggest that the variation in size and architecture of genomes in extinct dinosaurs was lower than the variation found in mammals. A substantial difference in genome size separates the two major clades within dinosaurs, Ornithischia (large genomes) and Saurischia (moderate to small genomes). We find no relationship between body size and estimated genome size in extinct dinosaurs, which suggests that neutral forces did not dominate the evolution of genome size in this group.
doi:10.1098/rspb.2009.1343
PMCID: PMC2817110  PMID: 19793755
palaeogenomics; Dinosauria; genome size; genomics; Sauropodomorpha
2.  Microanatomical and Histological Features in the Long Bones of Mosasaurine Mosasaurs (Reptilia, Squamata) – Implications for Aquatic Adaptation and Growth Rates 
PLoS ONE  2013;8(10):e76741.
Background
During their evolution in the Late Cretaceous, mosasauroids attained a worldwide distribution, accompanied by a marked increase in body size and open ocean adaptations. This transition from land-dwellers to highly marine-adapted forms is readily apparent not only at the gross anatomic level but also in their inner bone architecture, which underwent profound modifications.
Methodology/Principal Findings
The present contribution describes, both qualitatively and quantitatively, the internal organization (microanatomy) and tissue types and characteristics (histology) of propodial and epipodial bones in one lineage of mosasauroids; i.e., the subfamily Mosasaurinae. By using microanatomical and histological data from limb bones in combination with recently acquired knowledge on the inner structure of ribs and vertebrae, and through comparisons with extant squamates and semi-aquatic to fully marine amniotes, we infer possible implications on mosasaurine evolution, aquatic adaptation, growth rates, and basal metabolic rates. Notably, we observe the occurrence of an unusual type of parallel-fibered bone, with large and randomly shaped osteocyte lacunae (otherwise typical of fibrous bone) and particular microanatomical features in Dallasaurus, which displays, rather than a spongious inner organization, bone mass increase in its humeri and a tubular organization in its femora and ribs.
Conclusions/Significance
The dominance of an unusual type of parallel-fibered bone suggests growth rates and, by extension, basal metabolic rates intermediate between that of the extant leatherback turtle, Dermochelys, and those suggested for plesiosaur and ichthyosaur reptiles. Moreover, the microanatomical features of the relatively primitive genus Dallasaurus differ from those of more derived mosasaurines, indicating an intermediate stage of adaptation for a marine existence. The more complete image of the various microanatomical trends observed in mosasaurine skeletal elements supports the evolutionary convergence between this lineage of secondarily aquatically adapted squamates and cetaceans in the ecological transition from a coastal to a pelagic lifestyle.
doi:10.1371/journal.pone.0076741
PMCID: PMC3797777  PMID: 24146919
3.  Evidence for the role of connexin 43-mediated intercellular communication in the process of intracortical bone resorption via osteocytic osteolysis 
Background
Connexin 43 (Cx43) is the predominant gap junction protein in bone. Mice with a bone-specific deletion of Cx43 (cKO) have an osteopenic cortical phenotype. In a recent study, we demonstrated that cKO mice are resistant to bone loss induced by hindlimb suspension (HLS), an animal model of skeletal unloading. This protective effect occurred primarily as a result of lower osteoclast-mediated bone resorption. Interestingly, we also documented a significant increase in cortical osteocyte apoptosis and reduced sclerostin production. In the present study, we investigated whether osteocytic osteolysis – bone resorption by osteocytes within lacunae – is induced by HLS and the potential effect of Cx43 deficiency on this process during unloading.
Methods
6-month-old male Cx43 cKO or wild-type (WT) mice were subjected to three weeks of HLS (Suspended) or normal loading conditions (Control) (n = 5/group). Lacunar morphology and tartrate-resistant acid phosphatase (TRACP) staining were assessed on sections of femur cortical bone. Experimental groups were compared via two-way ANOVA.
Results
Empty lacunae were 26% larger in cKO-Control vs. WT-Control (p < 0.05), while there was no difference in the size of empty lacunae between Control and Suspended within WT or cKO (p > 0.05). Similarly, there was a trend (p = 0.06) for 11% larger lacunae containing viable osteocytes for cKO-Control vs. WT-Control, with no apparent effect of loading condition. There was no difference in the proportion of TRACP + cells between WT-Control and cKO-Control (p > 0.05); however, WT-Suspended mice had 246% more TRACP + osteocytes compared WT-Control mice (p < 0.05). There was no difference in TRACP staining between cKO-Control and cKO-Suspended (p > 0.05).
Conclusions
Prior to undergoing apoptosis, osteocytes in cKO mice may be actively resorbing their respective lacunae via the process of osteocytic osteolysis. Interestingly, the proportion of TRACP + osteocytes increased dramatically following unloading of WT mice, an effect that was not observed in cKO mice subjected to HLS. The results of the present study provide initial evidence that osteocytic osteolysis is occurring in cortical bone in response to mechanical unloading. Furthermore, Cx43 deficiency appears to protect against osteocytic osteolysis in a manner similar to the inhibition of unloading-induced osteoclast activation that we have documented previously.
doi:10.1186/1471-2474-15-122
PMCID: PMC3984635  PMID: 24716486
Connexin 43; Osteocytic osteolysis; Hindlimb suspension; Unloading; Cortical bone; Porosity
4.  Mineralisation of collagen rich soft tissues and osteocyte lacunae in Enpp1−/− mice 
Bone  2014;69:139-147.
Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyse nucleotide triphosphates to the corresponding nucleotide monophosphates and the mineralisation inhibitor, pyrophosphate (PPi). This study examined the role of NPP1 in osteocytes, osteoclasts and cortical bone, using a mouse model lacking NPP1 (Enpp1−/−). We used microcomputed tomography (μCT) to investigate how NPP1 deletion affects cortical bone structure; excised humerus bones from 8, 15 and 22-week old mice were scanned at 0.9 μm. Although no changes were evident in the cortical bone of 8-week old Enpp1−/− mice, significant differences were observed in older animals. Cortical bone volume was decreased 28% in 22-week Enpp1−/− mice, whilst cortical porosity was reduced 30% and 60% at 15 and 22-weeks, respectively. This was accompanied by up to a 15% decrease in closed pore diameter and a 55% reduction in the number of pores. Cortical thickness was reduced up to 35% in 15 and 22-week Enpp1−/− animals and the endosteal diameter was increased up to 23%. Thus, the cortical bone from Enpp1−/− mice was thinner and less porous, with a larger marrow space. Scanning electron microscopy (SEM) revealed a decrease in the size and number of blood vessel channels in the cortical bone as well as a 40% reduction in the mean plan area of osteocyte lacunae. We noted that the number of viable osteocytes isolated from the long bones of Enpp1−/− mice was decreased ≤ 50%. In contrast, osteoclast formation and resorptive activity were unaffected by NPP1 deletion. μCT and histological analysis of Enpp1−/− mice also revealed calcification of the joints and vertebrae as well as soft tissues including the whisker follicles, ear pinna and trachea. This calcification worsened as the animals aged. Together, these data highlight the key role of NPP1 in regulating calcification of both soft and skeletal tissues.
Highlights
•We examine the role of NPP1 in osteocytes, osteoclasts and cortical bone.•Osteocyte lacunae are reduced in size and number in NPP1 knockout mice.•Sclerostin levels are increased in NPP1 knockout mice.•Osteoclast formation and resorptive activity are unaffected by NPP1 deletion.•Mice lacking NPP1 display widespread calcification of collagen rich soft tissues.
doi:10.1016/j.bone.2014.09.016
PMCID: PMC4228085  PMID: 25260930
NPP1; Osteocytes; Osteoclasts; Soft tissue mineralisation; Pyrophosphate
5.  Glucocorticoid-Treated Mice Have Localized Changes in Trabecular Bone Material Properties and Osteocyte Lacunar Size That Are Not Observed in Placebo-Treated or Estrogen-Deficient Mice 
This study compares changes in bone microstructure in 6-month-old male GC-treated and female ovariectomized mice to their respective controls. In addition to a reduction in trabecular bone volume, GC treatment reduced bone mineral and elastic modulus of bone adjacent to osteocytes that was not observed in control mice nor estrogen-deficient mice. These microstructural changes in combination with the macro-structural changes could amplify the bone fragility in this metabolic bone disease.
Introduction
Patients with glucocorticoid (GC)-induced secondary osteoporosis tend to fracture at higher bone mineral densities than patients with postmenopausal osteoporosis. This suggests that GCs may alter bone material properties in addition to BMD and bone macrostructure.
Materials and Methods
Changes in trabecular bone structure, elastic modulus, and mineral to matrix ratio of the fifth lumbar vertebrae was assessed in prednisolone-treated mice and placebo-treated controls for comparison with estrogen-deficient mice and sham-operated controls. Compression testing of the third lumbar vertebrae was performed to assess whole bone strength.
Results
Significant reductions in trabecular bone volume and whole bone strength occurred in both prednisolone-treated and estrogen-deficient mice compared with controls after 21 days (p < 0.05). The average elastic modulus over the entire surface of each trabecula was similar in all the experimental groups. However, localized changes within the trabeculae in areas surrounding the osteocyte lacunae were observed only in the prednisolone-treated mice. The size of the osteocyte lacunae was increased, reduced elastic modulus around the lacunae was observed, and a “halo” of hypomineralized bone surrounding the lacunae was observed. This was associated with reduced (nearly 40%) mineral to matrix ratio determined by Raman microspectroscopy. These localized changes in elastic modulus and bone mineral to matrix ratio were not observed in the other three experimental groups.
Conclusions
Based on these results, it seems that GCs may have direct effects on osteocytes, resulting in a modification of their microenvironment. These changes, including an enlargement of their lacunar space and the generation of a surrounding sphere of hypomineralized bone, seem to produce highly localized changes in bone material properties that may influence fracture risk.
doi:10.1359/JBMR.051103
PMCID: PMC1797152  PMID: 16491295
glucocorticoids; mice; elastic modulus; mineralization; bone strength
6.  Changes in Intracortical Microporosities Induced by Pharmaceutical Treatment of Osteoporosis as Detected by High Resolution Micro-CT 
Bone  2011;50(3):596-604.
Bone’s microporosities play important biologic and mechanical roles. Here, we quantified 3D changes in cortical osteocyte-lacunae and other small porosities induced by estrogen withdrawal and two different osteoporosis treatments. Unlike 2D measurements, these data collected via synchrotron radiation-based μCT describe the size and 3D spatial distribution of a large number of porous structures. Six-month old female Sprague-Dawley rats were separated into four groups of age-matched controls, untreated OVX, OVX treated with PTH, and OVX treated with Alendronate (ALN). Intracortical microporosity of the medial quadrant of the femoral diaphysis was quantified at endosteal, intracortical, and periosteal regions of the samples, allowing the quantification of osteocyte lacunae that were formed primarily before versus after the start of treatment. Across the overall thickness of the medial cortex, lacunar volume fraction (Lc.V/TV) was significantly lower in ALN treated rats compared to PTH. In the endosteal region, average osteocyte lacunar volume () of untreated OVX rats was significantly lower than in age-matched controls, indicating a decrease in osteocyte lacunar size in bone formed on the endosteal surface after estrogen withdrawal. The effect of treatment (OVX, ALN, PTH) on the number of lacunae per tissue volume (Lc.N/TV) was dependent on the specific location within the cortex (endosteal, intracortical, periosteal). In both the endosteal and intracortical regions, Lc.N/TV was significantly lower in ALN than in untreated OVX, suggesting a site-specific effect in osteocyte lacuna density with ALN treatment. There also were a significantly greater number of small pores (5–100 μm3 in volume) in the endosteal region for PTH compared to ALN. The mechanical impact of this altered microporosity structure is unknown, but might serve to enhance, rather than deteriorate bone strength with PTH treatment, as smaller osteocyte lacunae may be better able to absorb shear forces than larger lacunae. Together, these data demonstrate that current treatments of osteoporosis can alter the number, size, and distribution of microporosities in cortical rat lamellar bone.
doi:10.1016/j.bone.2011.12.012
PMCID: PMC3278519  PMID: 22226688
PTH; Alendronate; OVX; osteocyte lacunae; synchrotron micro-CT; cortical bone porosity
7.  Osteohistological variation in growth marks and osteocyte lacunar density in a theropod dinosaur (Coelurosauria: Ornithomimidae) 
BMC Evolutionary Biology  2014;14(1):231.
Background
Osteohistological examinations of fossil vertebrates have utilized a number of proxies, such as counts and spacing of lines of arrested growth (LAGs) and osteocyte lacunar densities (OLD), in order to make inferences related to skeletochronology and mass-specific growth rates. However, many of these studies rely on samplings of isolated bones from single individuals. These analyses do not take individual variation into account, and as a result may lead to misleading inferences of the physiology of extinct organisms. This study uses a multi-element, multi-individual sampling of ornithomimid dinosaurs to test the amount of individual variation in the aforementioned osteohistological indicators. Based on these results we also assess the conclusions of previous studies that tested paleohistological hypotheses using isolated elements.
Results
LAG number was found to be consistent within the hind limb bones of each individual, with the exception of the fibula, which preserves one additional LAG. Considerable differences in LAG spacing were found between elements of the sampled individuals, with larger variation found in elements of the foot compared with the femur, fibula, and tibia. Osteocyte lacunar density ranged between 29000 and 42000 osteocyte lacunae per mm3, and was found to vary more between hind limb bones of an individual and within bones, than between the average values of individuals.
Conclusions
The variation between hind limb elements in LAG number and LAG spacing suggests that direct comparisons of these elements may be misleading, and that LAG spacing is not a reliable proxy for mass-specific growth rates of an individual. Sampling of multiple bones should be performed as an internal check of model-based LAG retro-calculation and growth equations. The observation that osteocyte lacunar density varies more between individual bone elements than between average individual values suggests that the choice of sampled element can greatly influence the result, and care should be taken to not bias interpretations of the physiology of fossil tetrapods.
Electronic supplementary material
The online version of this article (doi:10.1186/s12862-014-0231-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12862-014-0231-y
PMCID: PMC4269922  PMID: 25421260
8.  Alterations in the Osteocyte Lacunar-Canalicular Microenvironment due to Estrogen Deficiency 
Bone  2012;51(3):488-497.
While reduced estrogen levels have been shown to increase bone turnover and induce bone loss, there has been little analysis of the effects of diminished estrogen levels on the lacunar-canalicular porosity that houses the osteocytes. Alterations in the osteocyte lacunar-canalicular microenvironment may affect the osteocyte’s ability to sense and translate mechanical signals, possibly contributing to bone degradation during osteoporosis. To investigate whether reduced estrogen levels affect the osteocyte microenvironment, this study used high-resolution microscopy techniques to assess the lacunar-canalicular microstructure in the rat ovariectomy (OVX) model of postmenopausal osteoporosis. Confocal microscopy analyses indicated that OVX rats had a larger effective lacunar-canalicular porosity surrounding osteocytes in both cortical and cancellous bone from the proximal tibial metaphysis, with little change in cortical bone from the diaphysis or cancellous bone from the epiphysis. The increase in the effective lacunar-canalicular porosity in the tibial metaphysis was not due to changes in osteocyte lacunar density, lacunar size, or the number of canaliculi per lacuna. Instead, the effective canalicular size measured using a small molecular weight tracer was larger in OVX rats compared to controls. Further analysis using scanning and transmission electron microscopy demonstrated that the larger effective canalicular size in the estrogen-deficient state was due to nanostructural matrix-mineral level differences like loose collagen surrounding osteocyte canaliculi. These matrix-mineral differences were also found in osteocyte lacunae in OVX, but the small surface changes did not significantly increase the effective lacunar size. The alterations in the lacunar-canalicular surface mineral or matrix environment appear to make OVX bone tissue more permeable to small molecules, potentially altering interstitial fluid flow around osteocytes during mechanical loading.
doi:10.1016/j.bone.2012.05.014
PMCID: PMC3412941  PMID: 22634177
osteoporosis; osteocyte; lacuna; canaliculi; periosteocytic remodeling
9.  Osteocytic osteolysis: time for a second look? 
BoneKEy Reports  2012;1:229.
Over 100 years ago it was suggested that osteocytes could remodel their surrounding environment by removing and replacing bone. In the 1960s and 1970s, many observations were made to suggest that osteocytes could resorb bone and increase the size of their lacunae. This concept became known as osteocytic osteolysis and studies suggested that it occurred in response to diverse stimuli such as parathyroid hormone, calcium restriction, hibernation and reproductive cycles. However, this concept fell out of favor in the late 1970s when it became clear that osteoclasts were the principal bone-resorbing cells in the skeleton. Over the past decade, we have increasingly appreciated that osteocytes are remarkably versatile cells and are involved in all aspects of skeletal biology, including the response to loading, the regulation of bone turnover and the control of mineral metabolism. Recent data have demonstrated that osteocytes remodel their perilacunar and canalicular matrix and participate in the liberation of skeletal calcium stores during lactation. In light of these new findings, it may be time to reassess the concept of osteocytic osteolysis and reconsider whether osteocyte lacunar and canalicular remodeling contributes more broadly to the maintenance of skeletal and mineral homeostasis.
doi:10.1038/bonekey.2012.229
PMCID: PMC3868715  PMID: 24363929
10.  Delineating bone's interstitial fluid pathway in vivo 
Bone  2004;34(3):499-509.
Although interstitial fluid flow has been suggested to play a role in bone adaptation and metabolism, the constituents and ultrastructure of this interstitial fluid pathway are not well understood. Bone's lacunar–canalicular porosity is generally believed to be a continuous interstitial fluid pathway through which osteocytes sense external mechanical loading as well as obtain nutrients and dispose of wastes. Recent electron microscopy studies have suggested that a fiber matrix surrounds the osteocytic cell processes and fills this pericellular fluid space. However, studies injecting tracer molecules into the bone vasculature have provided conflicting results about the pore size or the fiber spacing of the interstitial fluid pathway. In addition, whether the smaller collagen–apatite porosity in adult bone is also a continuous fluid pathway is still unclear. To delineate bone's interstitial fluid pathway, four tracers of various size were injected into rats: reactive red (approximately 1 nm), microperoxidase (MP, approximately 2 nm), horseradish peroxidase (HRP, approximately 6 nm), and ferritin (approximately 10 nm). Five minutes after injection, the tibiae were harvested and processed using histological protocols optimized to minimize processing time to reduce possible redistribution of tracer molecules. The number of blood vessels and osteocytic lacunae labeled with the tracers per unit bone area was then measured for mid-diaphysial cross-sections of the tibia. While none of the tracers was detected within the mineralized bone matrix (the collagen–apatite porosity) using light microscopy, all the tracers except ferritin were found to pass through the canaliculi and appear in the osteocytic lacunae. These results indicate that while small tracers (< 6 nm) readily pass through the lacunar–canalicular porosity in the absence of mechanical loading, there appears to be an upper limit or cutoff size between 6 and 10 nm for molecular movement from bone capillaries to osteocytic lacunae in rat long bone. This range of pore size contains the most likely fiber spacing (approximately 7 nm) that has been proposed for the lacunar–canalicular annular space based on the presence of a proteoglycan fiber matrix surrounding the osteocyte.
doi:10.1016/j.bone.2003.11.022
PMCID: PMC3929128  PMID: 15003797
Bone pore size; Bone permeability; Bone interstitial fluid; Tracer movement; Mass transport
11.  3D Assessment of Cortical Bone Porosity and Tissue Mineral Density Using High-Resolution Micro-CT: Effects of Resolution and Threshold Method 
Current micro-CT systems allow scanning bone at resolutions capable of three-dimensional characterization of intracortical vascular porosity and osteocyte lacunae. However, the scanning and reconstruction parameters along with the image segmentation method affect the accuracy of the measurements. In this study, the effects of scanning resolution and image threshold method in quantifying small features of cortical bone (vascular porosity, vascular canal diameter and separation, lacunar porosity and density, and tissue mineral density) were analyzed. Cortical bone from the tibia of Sprague-Dawley rats was scanned at 1-µm and 4-µm resolutions, reconstructions were density-calibrated, and volumes of interest were segmented using approaches based on edge-detection or histogram analysis. With 1-µm resolution scans, the osteocyte lacunar spaces could be visualized, and it was possible to separate the lacunar porosity from the vascular porosity. At 4-µm resolution, the vascular porosity and vascular canal diameter were underestimated, and osteocyte lacunae were not effectively detected, whereas the vascular canal separation and tissue mineral density were overestimated compared to 1-µm resolution. Resolution had a much greater effect on the measurements than did threshold method, with partial volume effects at resolutions coarser than 2 µm demonstrated in two separate analyses, one of which assessed the effect of resolution on an object of known size with similar architecture to a vascular pore. Although there was little difference when using the edge-detection versus histogram-based threshold approaches, edge-detection was somewhat more effective in delineating canal architecture at finer resolutions (1 – 2 µm). In addition, use of a high-resolution (1-µm) density-based threshold on lower resolution (4-µm) density-calibrated images was not effective in improving the lower-resolution measurements. In conclusion, if measuring cortical vascular microarchitecture, especially in small animals, a micro-CT resolution of 1 – 2 µm is appropriate, while a resolution of at least 1 µm is necessary when assessing osteocyte lacunar porosity.
doi:10.1002/jbmr.2012
PMCID: PMC3870034  PMID: 23775635
intracortical porosity; vascular porosity; micro-CT; resolution; partial volume effect
12.  Tissue strain amplification at the osteocyte lacuna: A microstructural finite element analysis 
Journal of biomechanics  2007;40(10):2199-2206.
A parametric finite element model of an osteocyte lacuna was developed to predict the microstructural response of the lacuna to imposed macroscopic strains. The model is composed of an osteocyte lacuna, a region of perilacunar tissue, canaliculi, and the surrounding bone tissue. A total of 45 different simulations were modeled with varying canalicular diameters, perilacunar tissue material moduli, and perilacunar tissue thicknesses. Maximum strain increased with a decrease in perilacunar tissue modulus and decreased with an increase in perilacunar tissue modulus, regardless of the thickness of the perilacunar region. An increase in the predicted maximum strain was observed with an increase in canalicular diameter from 0.362 to 0.421 μm. In response to the macroscopic application of strain, canalicular diameters increased 0.8% to over 1.0% depending on the perilacunar tissue modulus. Strain magnification factors of over 3 were predicted. However, varying the size of the perilacunar tissue region had no effect on the predicted perilacunar tissue strain. These results indicate that the application of average macroscopic strains similar to strain levels measured in vivo can result in significantly greater perilacunar tissue strains and canaliculi deformations. A decrease in the perilacunar tissue modulus amplifies the perilacunar tissue strain and canaliculi deformation while an increase in the local perilacunar tissue modulus attenuates this effect.
doi:10.1016/j.jbiomech.2006.10.040
PMCID: PMC2094105  PMID: 17196968
Bone; Osteocyte; Lacuna; Tissue strain; Finite element model
13.  Anatomy of the Distal Incus in Humans 
The anatomy of the distal incus, including the lenticular process, was examined in histological sections from 270 normal cadaveric human temporal bones aged between less than 1 month and 100 years. All but nine of these sectioned specimens showed signs of a bony connection between the long process of the incus and the flattened plate of the lenticular process, and in 108 specimens a complete bony attachment was observed in a single 20 μm section. In these 108 ears, the bony lenticular process consisted of a proximal narrow “pedicle” connected to a distal flattened “plate” that forms the incudal component of the incudo-stapedial joint. A fibrous joint capsule extended from the stapes head to the pedicle of the lenticular process on all sides, where it was considerably thickened. Three-dimensional reconstructions made from serial 20 μm sections of four bones provided views from all directions that easily convey the anatomical features of this region. Morphometric measurements of the bony architecture of the distal incus in 103 temporal bones were made, including lengths and cross-sectional areas, estimates of the percentage of lacunae containing osteocytes, and the degree of bone resorption. These measurements, analyzed as a function of age, provided an anatomic description over a large age range that can serve as a normal baseline against which structural pathology can be compared. Although none of the bony dimensions showed significant age dependence, the estimated percentage of bony lacunae that contain osteocytes decreased significantly with age. The results have implications for the roles of specific components on the coupling of ossicular motion across the incudo-stapedial joint, and provide insights regarding bone resorption at the level of the distal incus, which occurs clinically in some patients with chronic otitis media or after stapedectomy.
doi:10.1007/s10162-009-0179-6
PMCID: PMC2774412  PMID: 19685262
incus; lenticular process; incus necrosis; middle-ear mechanics; incudo-stapedial joint
14.  Osteocytes Remove and Replace Perilacunar Mineral during Reproductive Cycles 
Bone  2013;54(2):230-236.
Lactation is associated with an increased demand for calcium and is accompanied by a remarkable cycle of bone loss and recovery that helps to supply calcium and phosphorus for milk production. Bone loss is the result of increased bone resorption that is due, in part, to increased levels of PTHrP and decreased levels of estrogen. However, the regulation of bone turnover during this time is not fully understood. In the 1960’s and 1970’s many observations were made to suggest that osteocytes could resorb bone and increase the size of their lacunae. This concept became know as osteocytic osteolysis and studies suggested that it occurred in response to parathyroid hormone and/or an increased systemic demand for calcium. However, this concept fell out of favor in the late 1970’s when it was established that osteoclasts were the principal bone-resorbing cells. Given that lactation is associated with increased PTHrP levels and negative calcium balance, we recently examined whether osteocytes contribute to bone loss during this time. Our findings suggest that osteocytes can remodel their perilacunar and pericanalicular matrix and that they participate in the liberation of skeletal calcium stores during reproductive cycles. These findings raise new questions about the role of osteocytes in coordinating bone and mineral metabolism during lactation as well as the recovery of bone mass after weaning. It is also interesting to consider whether osteocyte lacunar and canalicular remodeling contributes more broadly to the maintenance of skeletal and mineral homeostasis.
doi:10.1016/j.bone.2013.01.025
PMCID: PMC3624069  PMID: 23352996
15.  Multiscale, Converging Defects of Macro-Porosity, Microstructure and Matrix Mineralization Impact Long Bone Fragility in NF1 
PLoS ONE  2014;9(1):e86115.
Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.
doi:10.1371/journal.pone.0086115
PMCID: PMC3897656  PMID: 24465906
16.  Osteocyte lacunae tissue strain in cortical bone 
Journal of biomechanics  2005;39(9):1735-1743.
Current theories suggest that bone modeling and remodeling are controlled at the cellular level through signals mediated by osteocytes. However, the specific signals to which bone cells respond are still unknown. Two primary theories are: (1) osteocytes are stimulated via the mechanical deformation of the perilacunar bone matrix and (2) osteocytes are stimulated via fluid flow generated shear stresses acting on osteocyte cell processes within canaliculi. Recently, much focus has been placed on fluid flow theories since in vitro experiments have shown that bone cells are more responsive to analytically estimated levels of fluid shear stress than to direct mechanical stretching using macroscopic strain levels measured on bone in vivo. However, due to the complex microstructural organization of bone, local perilacunar bone tissue strains potentially acting on osteocytes cannot be reliably estimated from macroscopic bone strain measurements. Thus, the objective of this study was to quantify local perilacunar bone matrix strains due to macroscopically applied bone strains similar in magnitude to those that occur in vivo. Using a digital image correlation strain measurement technique, experimentally measured bone matrix strains around osteocyte lacunae resulting from macroscopic strains of approximately 2000 microstrain are significantly greater than macroscopic strain on average and can reach peak levels of over 30,000 microstrain locally. Average strain concentration factors ranged from 1.1 to 3.8, which is consistent with analytical and numerical estimates. This information should lead to a better understanding of how bone cells are affected by whole bone functional loading.
doi:10.1016/j.jbiomech.2005.04.032
PMCID: PMC1866263  PMID: 15993413
Bone; Osteocyte lacuna; Micromechanics; Tissue strain
17.  Effect of Raloxifene Treatment on Osteocyte Apoptosis in Postmenopausal Women 
Calcified Tissue International  2007;81(3):183-190.
Increased osteocyte apoptosis, as the result of estrogen deficiency, could play a role in the decrease of bone mass and bone strength seen in postmenopausal osteoporosis. We investigated whether treatment with raloxifene of postmenopausal women with osteoporosis affects osteocyte apoptosis. Transiliac bone biopsies were obtained from 26 osteoporotic women at baseline and after 2 years of treatment with placebo or raloxifene. Immunohistochemical detection of cleaved caspase-3 was performed on sections from nondecalcified bone biopsies to visualize apoptosis. In the trabecular bone total osteocytes, positively stained osteocytes and empty lacunae were counted and percent positive cells and percent empty lacunae determined. Statistical evaluation was performed by Wilcoxon’s paired t-test and Spearman’s rank correlations. There was no significant difference in percentage positive osteocytes between baseline and follow-up biopsies in both the placebo and the raloxifene groups. The percentage empty lacunae increased significantly in the placebo group (11.20 ± 1.43 vs. 9.00 ± 2.25, P = 0.014) but not in the raloxifene group. At baseline in both groups combined, there was a negative correlation between indices of bone remodeling and the percentage positive osteocytes (bone formation rate/bone volume r = −0.67, P = 0.001). We found no direct evidence for an effect of raloxifene treatment on osteocyte apoptosis, but small effects of raloxifene treatment cannot be excluded. The percent of apoptotic osteocytes was dependent on the level of bone remodeling in an individual.
doi:10.1007/s00223-007-9050-0
PMCID: PMC2039811  PMID: 17676256
Apoptosis; Histomorphometry; Osteocyte; Postmenopausal osteoporosis; Raloxifene
18.  Constitutive protein kinase A activity in osteocytes and late osteoblasts produces an anabolic effect on bone 
Bone  2013;55(2):277-287.
Osteocytes have been implicated in the control of bone formation. However, the signal transduction pathways that regulate the biological function of osteocytes are poorly defined. Limited evidence suggests an important role for the Gs/cAMP pathway in osteocyte function. In the present study, we explored the hypothesis that cAMP-dependent kinase A (PKA) activation in osteocytes plays a key role in controlling skeletal homeostasis. To test this hypothesis, we mated mice harboring a Cre-conditional, mutated PKA catalytic subunit allele that encodes a constitutively active form of PKA (CαR) with mice expressing Cre under the control of the osteocyte-specific promoter, DMP1. This allowed us to direct the expression of CαR to osteocytes in double transgenic progeny. Examination of Cre expression indicated that CαR was also expressed in late osteoblasts. Cortical and trabecular bone parameters from 12-week old mice were determined by μCT. Expression of CαR in osteocytes and late osteoblasts altered the shape of cortical bone proximal to the tibia-fibular junction (TFJ) and produced a significant increase in its size. In trabecular bone of the distal femur, fractional bone volume, trabecular number, and trabecular thickness were increased. These increases were partially the results of increased bone formation rates (BFRs) on the endosteal surface of the cortical bone proximal to the TFJ as well as increased BFR on the trabecular bone surface of the distal femur. Mice expressing CαR displayed a marked increase in the expression of osteoblast markers such as osterix, runx2, collagen 1α1, and alkaline phosphatase (ALP). Interestingly, expression of osteocyte marker gene, DMP1, was significantly up-regulated but the osteocyte number per bone area was not altered. Expression of SOST, a presumed target for PKA signaling in osteocytes, was significantly down-regulated in females. Importantly, no changes in bone resorption were detected. In summary, constitutive PKA signaling in osteocytes and late osteoblasts led to a small expansion of the size of the cortical bone proximal to the TFJ and an increase in trabecular bone in female mice. This was associated with down-regulation of SOST and up-regulation of several osteoblast marker genes. Activation of the PKA pathway in osteocytes and late osteoblasts is sufficient for the initiation of an anabolic skeletal response.
doi:10.1016/j.bone.2013.04.001
PMCID: PMC3690773  PMID: 23583750
Osteocyte; Protein kinase A; Cyclic AMP; Dentin matrix protein 1
19.  Trabecular bone microstructure scales allometrically in the primate humerus and femur 
Most analyses of trabecular microarchitecture in mammals have focused on the functional significance of interspecific variation, but they have not effectively considered the influence of body size or phylogeny on bone architecture. The goals of this study were to determine the relationship between trabecular bone and body size in the humeral and femoral heads of extant primates, and to assess the influence of phylogeny on bone microstructure. Using a sample of 235 individuals from 34 primate species, ranging in body size from 0.06 to 130 kg, the relationships between trabecular bone structure and body size were assessed by using conventional and phylogenetic regression analyses. Bone volume fraction, trabecular thickness and trabecular spacing increase with body size, whereas bone surface-area-to-volume ratio decreases. Shape variables such as trabecular number, connectivity density and degree of anisotropy scale inversely with size. Most of these variables scale with significant negative allometry, except bone surface-area-to-volume ratio, which scales with slight positive allometry. Phylogenetic regressions indicate a relatively weak phylogenetic signal in some trabecular bone variables. These data demonstrate that, relative to body size, large primates have thinner and more tightly packed trabeculae than small primates. The relatively thin trabeculae in large primates and other mammals, coupled with constraints on trabecular thickness related to osteocyte function, suggest that increased skeletal loads in the postcranial joints of large mammals are probably mitigated not only through alterations in trabecular microarchitecture, but also through other mechanisms such as changes in cortical bone distribution, limb posture and gait speed.
doi:10.1098/rspb.2013.0172
PMCID: PMC3619467  PMID: 23486443
trabecular bone; primates; high-resolution computed tomography
20.  Preliminary Analysis of Osteocyte Lacunar Density in Long Bones of Tetrapods: All Measures Are Bigger in Sauropod Dinosaurs 
PLoS ONE  2013;8(10):e77109.
Osteocytes harbour much potential for paleobiological studies. Synchrotron radiation and spectroscopic analyses are providing fascinating data on osteocyte density, size and orientation in fossil taxa. However, such studies may be costly and time consuming. Here we describe an uncomplicated and inexpensive method to measure osteocyte lacunar densities in bone thin sections. We report on cell lacunar densities in the long bones of various extant and extinct tetrapods, with a focus on sauropodomorph dinosaurs, and how lacunar densities can help us understand bone formation rates in the iconic sauropod dinosaurs. Ordinary least square and phylogenetic generalized least square regressions suggest that sauropodomorphs have lacunar densities higher than scaled up or comparably sized mammals. We also found normal mammalian-like osteocyte densities for the extinct bovid Myotragus, questioning its crocodilian-like physiology. When accounting for body mass effects and phylogeny, growth rates are a main factor determining the density of the lacunocanalicular network. However, functional aspects most likely play an important role as well. Observed differences in cell strategies between mammals and dinosaurs likely illustrate the convergent nature of fast growing bone tissues in these groups.
doi:10.1371/journal.pone.0077109
PMCID: PMC3812986  PMID: 24204748
21.  Cartilaginous Epiphyses in Extant Archosaurs and Their Implications for Reconstructing Limb Function in Dinosaurs 
PLoS ONE  2010;5(9):e13120.
Extinct archosaurs, including many non-avian dinosaurs, exhibit relatively simply shaped condylar regions in their appendicular bones, suggesting potentially large amounts of unpreserved epiphyseal (articular) cartilage. This “lost anatomy” is often underappreciated such that the ends of bones are typically considered to be the joint surfaces, potentially having a major impact on functional interpretation. Extant alligators and birds were used to establish an objective basis for inferences about cartilaginous articular structures in such extinct archosaur clades as non-avian dinosaurs. Limb elements of alligators, ostriches, and other birds were dissected, disarticulated, and defleshed. Lengths and condylar shapes of elements with intact epiphyses were measured. Limbs were subsequently completely skeletonized and the measurements repeated. Removal of cartilaginous condylar regions resulted in statistically significant changes in element length and condylar breadth. Moreover, there was marked loss of those cartilaginous structures responsible for joint architecture and congruence. Compared to alligators, birds showed less dramatic, but still significant changes. Condylar morphologies of dinosaur limb bones suggest that most non-coelurosaurian clades possessed large cartilaginous epiphyses that relied on the maintenance of vascular channels that are otherwise eliminated early in ontogeny in smaller-bodied tetrapods. A sensitivity analysis using cartilage correction factors (CCFs) obtained from extant taxa indicates that whereas the presence of cartilaginous epiphyses only moderately increases estimates of dinosaur height and speed, it has important implications for our ability to infer joint morphology, posture, and the complicated functional movements in the limbs of many extinct archosaurs. Evidence suggests that the sizes of sauropod epiphyseal cartilages surpassed those of alligators, which account for at least 10% of hindlimb length. These data suggest that large cartilaginous epiphyses were widely distributed among non-avian archosaurs and must be considered when making inferences about locomotor functional morphology in fossil taxa.
doi:10.1371/journal.pone.0013120
PMCID: PMC2948032  PMID: 20927347
22.  The enigmas of bone without osteocytes 
BoneKEy Reports  2013;2:343.
One of the hallmarks of tetrapod bone is the presence of numerous cells (osteocytes) within the matrix. Osteocytes are vital components of tetrapod bone, orchestrating the processes of bone building, reshaping and repairing (modeling and remodeling), and probably also participating in calcium-phosphorus homeostasis via both the local process of osteocytic osteolysis, and systemic effect on the kidneys. Given these critical roles of osteocytes, it is thought-provoking that the entire skeleton of many fishes consists of bone material that does not contain osteocytes. This raises the intriguing question of how the skeleton of these animals accomplishes the various essential functions attributed to osteocytes in other vertebrates, and raises the possibility that in acellular bone some of these functions are either accomplished by non-osteocytic routes or not necessary at all. In this review, we outline evidence for and against the fact that primary functions normally ascribed to osteocytes, such as mechanosensation, regulation of osteoblast/clast activity and mineral metabolism, also occur in fish bone devoid of these cells, and therefore must be carried out through alternative and perhaps ancient pathways. To enable meaningful comparisons with mammalian bone, we suggest thorough, phylogenetic examinations of regulatory pathways, studies of structure and mechanical properties and surveys of the presence/absence of bone cells in fishes. Insights gained into the micro-/nanolevel structure and architecture of fish bone, its mechanical properties and its physiology in health and disease will contribute to the discipline of fish skeletal biology, but may also help answer questions of basic bone biology.
doi:10.1038/bonekey.2013.77
PMCID: PMC3692265  PMID: 24422081
23.  Dose-Response Effect of Human Equivalent Radiation in the Murine Mandible. Part I. A Histomorphometric Assessment 
Plastic and reconstructive surgery  2011;128(1):114-121.
Background
Our lab previously demonstrated that radiation significantly alters new bone formation in the murine mandible impeding the use of distraction osteogenesis (DO) as a viable reconstructive option after radiatiotherapy in Head and Neck Cancer (HNC). We hypothesize that the deleterious effects of radiation on regenerate formation results from a dose response (DR) depletion of essential osteogenic cells. Our specific aim is to use quantitative histomorphometry (QHM) to objectively measure the human equivalent DR effects of radiation on the integrity of the mandible’s cellular and tissue composition.
Methods
20 Sprague-Dawley rats were randomIzed into three radiation dosage groups: low (5.91Gy), middle (7Gy) and high (8.89Gy) delivered in 5 daily fractions. These dosages approximated 75%, 100%, and 150% respectively of the bioequivalent dose the mandible experiences in the clinical regimen of HNC patients. Hemimandibles were harvested 56 days post-radiation, and stained with Gomori Trichrome. QHM was performed using Bioquant software and analysis with a one-way ANOVA Kruskal-Wallis test.
Results
Our data revealed a statistically significant diminution in the mean number of osteocytes . We also demonstrated a corresponding significant increase in the mean values of empty lacunae. Both of these QHM changes demonstrated a DR relationship.
Conclusion
Our study supports our hypothesis that radiation induces a DR depletion in osteocytes and an increase in empty lacunae. These reliable and reproducible metrics can now be utilized to determine the efficacy of therapies aimed at safeguarding the cells essential for optimal bone regeneration and potentially enhance the use of DO in HNC patients.
doi:10.1097/PRS.0b013e31821741d4
PMCID: PMC3132535  PMID: 21701328
24.  Osteocyte Wnt/β-Catenin Signaling Is Required for Normal Bone Homeostasis▿  
Molecular and Cellular Biology  2010;30(12):3071-3085.
β-Catenin-dependent canonical Wnt signaling plays an important role in bone metabolism by controlling differentiation of bone-forming osteoblasts and bone-resorbing osteoclasts. To investigate its function in osteocytes, the cell type constituting the majority of bone cells, we generated osteocyte-specific β-catenin-deficient mice (Ctnnb1loxP/loxP; Dmp1-Cre). Homozygous mutants were born at normal Mendelian frequency with no obvious morphological abnormalities or detectable differences in size or body weight, but bone mass accrual was strongly impaired due to early-onset, progressive bone loss in the appendicular and axial skeleton with mild growth retardation and premature lethality. Cancellous bone mass was almost completely absent, and cortical bone thickness was dramatically reduced. The low-bone-mass phenotype was associated with increased osteoclast number and activity, whereas osteoblast function and osteocyte density were normal. Cortical bone Wnt/β-catenin target gene expression was reduced, and of the known regulators of osteoclast differentiation, osteoprotegerin (OPG) expression was significantly downregulated in osteocyte bone fractions of mutant mice. Moreover, the OPG levels expressed by osteocytes were higher than or comparable to the levels expressed by osteoblasts during skeletal growth and at maturity, suggesting that the reduction in osteocytic OPG and the concomitant increase in osteocytic RANKL/OPG ratio contribute to the increased number of osteoclasts and resorption in osteocyte-specific β-catenin mutants. Together, these results reveal a crucial novel function for osteocyte β-catenin signaling in controlling bone homeostasis.
doi:10.1128/MCB.01428-09
PMCID: PMC2876685  PMID: 20404086
25.  Drilling k-wires, what about the osteocytes? An experimental study in rabbits 
Introduction
The function of osteocytes regarding osteonecrosis has been underestimated for a long time. Recently it has been suggested that apoptosis of osteocytes results in strong osteoclastic bone resorption. Death of osteocytes due to drilling may therefore increase the risk of K-wire loosening. The purposes of our in vivo study were to assess the minimal drill time needed to notice disappearance of osteocytes and to measure the distance of the empty osteocyte lacunae surrounding the drill tract in relation with the insertion time, directly and 4 weeks after drilling Kirschner (K-) wires into the femur and tibia of rabbits.
Materials and methods
Trocar tipped K-wires (70 mm length and 0.6 mm thickness) were drilled into the femur and tibia of 14 New Zealand white rabbits [mean body weight 2.81 kg (2.66–3.09 kg)]. Six rabbits were terminated following surgery (t = 0) and eight rabbits were terminated 4 weeks (t = 4) after surgery. Following termination, hematoxylin and eosin stained sections were cut from femur and tibia until the drill hole was visible. The sections were evaluated under a light microscope for the presence or absence of osteocytes in osteocyte lacunae surrounding the drill holes.
Results
All osteocyte lacunae were empty around the K-wires in 50 and 87% of the cases, directly and 4 weeks after the surgery, respectively. The osteocytes disappeared especially beyond a drilling time of 37 s (P = 0.011) and 27 s (P = 0.008) at t = 0 and t = 4, respectively. Furthermore, a significantly positive correlation was seen between the distances of the empty osteocyte lacunae surrounding the drill holes in relation with time at t = 0 (P = 0.008) and t = 4 (P = 0.000).
Conclusion
Although only drilling without cooling was studied, short drilling times may prevent the disappearance of osteocytes in case cooling is not used in clinical practice as is the case in percutaneous K-wire insertion.
doi:10.1007/s00402-007-0382-z
PMCID: PMC2092411  PMID: 17598116
Animal; Kirschner wire; Drilling; Histology; Insertion time

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