Co-Treatment of Purified Annatto Oil (Bixa orellana L.) and Its Granules (Chronic®) Improves the Blood Lipid Profile and Bone Protective Effects of Testosterone in the Orchiectomy-Induced Osteoporosis in Wistar Rats.

This study aimed to evaluate and compare the effects of co-treatment with purified annatto oil (PAO) or its granules (GRA, Chronic®) with that of testosterone on the orchiectomy-induced osteoporosis in Wistar rats. After surgery, rats were treated from day 7 until day 45 with testosterone only (TES, 7 mg/kg, IM) or TES + PAO or GRA (200 mg/kg, p.o.). The following parameters were evaluated: food/water intake, weight, HDL, LDL, glucose, triglycerides (TG), total cholesterol (TC), alkaline phosphatase levels, blood phosphorus and calcium contents, femur weight, structure (through scanning electron microscopy), and calcium content (through atomic absorption spectrophotometry). Our results show that orchiectomy could significantly change the blood lipid profile and decrease bone integrity parameters. Testosterone reposition alone could improve some endpoints, including LDL, TC, bone weight, and bone calcium concentration. However, other parameters were not significantly improved. Co-treatment with PAO or GRA improved the blood lipid profile and bone integrity more significantly and improved some endpoints not affected by testosterone reposition alone (such as TG levels and trabeculae sizes). The results suggest that co-treatment with annatto products improved the blood lipid profile and the anti-osteoporosis effects of testosterone. Overall, GRA had better results than PAO.

Biocompatibility and Biological Performance Evaluation of Additive-Manufactured Bioabsorbable Iron-Based Porous Suture Anchor in a Rabbit Model.

This study evaluated the biocompatibility and biological performance of novel additive-manufactured bioabsorbable iron-based porous suture anchors (iron_SAs). Two types of bioabsorbable iron_SAs, with double- and triple-helical structures (iron_SA_2_helix and iron_SA_3_helix, respectively), were compared with the synthetic polymer-based bioabsorbable suture anchor (polymer_SAs). An in vitro mechanical test, MTT assay, and scanning electron microscope (SEM) analysis were performed. An in vivo animal study was also performed. The three types of suture anchors were randomly implanted in the outer cortex of the lateral femoral condyle. The ultimate in vitro pullout strength of the iron_SA_3_helix group was significantly higher than the iron_SA_2_helix and polymer_SA groups. The MTT assay findings demonstrated no significant cytotoxicity, and the SEM analysis showed cells attachment on implant surface. The ultimate failure load of the iron_SA_3_helix group was significantly higher than that of the polymer_SA group. The micro-CT analysis indicated the iron_SA_3_helix group showed a higher bone volume fraction (BV/TV) after surgery. Moreover, both iron SAs underwent degradation with time. Iron_SAs with triple-helical threads and a porous structure demonstrated better mechanical strength and high biocompatibility after short-term implantation. The combined advantages of the mechanical superiority of the iron metal and the possibility of absorption after implantation make the iron_SA a suitable candidate for further development.

Modulation of gut microbiota by chondroitin sulfate calcium complex during alleviation of osteoporosis in ovariectomized rats.

Although chondroitin sulfate calcium complex (CSCa) was claimed to have the bioactivity for bone care in vitro, its anti-osteoporosis bioactivity was little reported in vivo. Here, the effects of CSCa on osteoporosis rats were investigated. Results showed that, compared with the osteoporosis rats, CSCa could improve the bone mineral density and microstructure of femur, and change the bone turnover markers level in serum. 16S rRNA sequencing and metabolomics analysis indicated CSCa intervention altered the composition of gut microbiota along with metabolite profiles in ovariectomized rat faeces. The correlation analysis showed some gut microbiota taxa were significantly correlated with osteoporosis phenotypes and the enriched metabolites. Taken together, dietary CSCa intervention has the potential to alleviate the osteoporosis and related symptoms probably involving gut microbiota or the metabolite profiles as demonstrated in rats. This study provides some scientific evidence for the potential effects of CSCa as the food supplement on the osteoporosis.

The stereomicroscope and scanning electron microscope comparison of false starts bones produced by an oscillating autopsy saw.

In forensic anthropology, saw mark analysis plays an important role in cases of criminal dismemberment. Autopsy saw is not used by the perpetrator in cases of dismemberment, but the forensic pathologist may accidentally create false starts with this saw during an autopsy, especially while sampling bones for further analysis, and these autopsy false starts can be confused with false starts produced by the offender. In this study, the characteristics of 20 false starts were compared using stereomicroscopy (SM) and scanning electron microscopy (SEM). These bone false starts were selected at random from a previous study of 100 false starts created by an electrical oscillating autopsy saw on human femoral bones. That study had enabled the categorization of the lesions into two groups ("superficial group" and "deep group") with a 0.52 mm depth cut off, based on the dramatic differences in lesion characteristics between these two groups. In the current study, SEM confirmed the characteristics of the false starts (walls and profile shapes, striae, bone islands and bone debris were studied), and above all explained the mechanism whereby oval bone islands in deep lesions are formed. Bone islands are due to the horizontal and vertical movement of the oscillating autopsy blade.

Tuneable in-situ nanoCT workflow using FIB/SEM.

Inspired by the standard computed tomography, a new method of 3D X-ray imaging embedded in FIB-SEM microscope is proposed. The unique combination of TEM-like specimen stage enabling in lens STEM detection (referred to as CompuStage), nanomanipulator (referred to as EasyLift) facilitating in-situ sample transfer from bulk sample to TEM-like stage and pixelated in-situ Timepix X-ray detector in Helios G4 FX FIB-SEM system offers an unprecedented workflow. Motivated by common circular CT scan known from microCT world, the object under study is placed on CompuStage rod which enables two possible rotation (in TEM/SEM terminology called tilt) movements - α-tilt - rotation of the CompuStage rod around its axis, and ß-tilt - rotation around axis perpendicular to CompuStage rod. ß-tilt rotation enables a circular movement of the sample while α-tilt sets the correct position of sample with respect to target and detector. Thin metal lamella of suitable material welded to EasyLift manipulator needle is used as an X-ray target. The final target-sample geometry - position, distance - can be fine-tuned using position control of CompuStage and EasyLift and in-situ monitored by SEM. Both sample and target can also be easily prepared in-situ. Radiographs are recorded by Timepix detector with inherent noise-free operation and energy filtration. For the 3D reconstruction standard microCT reconstruction algorithm is used with the procedure adjusted for the format and quality of nanoCT images. The experiments were carried out on Helios G4 FX DualBeam using titanium and tungsten targets and various semiconductor samples. The ultimate resolution of the proposed method in orders of tens of nanometers was achieved both by the possibility of close target to sample positioning and of adjustment of primary beam energy down to low energies reducing the interaction volume in the target. Since the lower energy radiation is well suited for life-science, the method was also tested on several bio-samples using silver target. The silver target, thanks to its massive low energy Lα line, allowed to distinguish subtle structures in the resin embedded stained mouse brain and also to observe and reconstruct canaliculi in the mouse bone (earlier reported by Dierolf et al. 2010, Nature 467 436).

Liquid-phase ASEM imaging of cellular and structural details in cartilage and bone formed during endochondral ossification: Keap1-deficient osteomalacia.

Chondrogenesis and angiogenesis drive endochondral ossification. Using the atmospheric scanning electron microscopy (ASEM) without decalcification and dehydration, we directly imaged angiogenesis-driven ossification at different developmental stages shortly after aldehyde fixation, using aqueous radical scavenger glucose solution to preserve water-rich structures. An embryonic day 15.5 mouse femur was fixed and stained with phosphotungstic acid (PTA), and blood vessel penetration into the hypertrophic chondrocyte zone was visualised. We observed a novel envelope between the perichondrium and proliferating chondrocytes, which was lined with spindle-shaped cells that could be borderline chondrocytes. At postnatal day (P)1, trabecular and cortical bone mineralisation was imaged without staining. Additional PTA staining visualised surrounding soft tissues; filamentous connections between osteoblast-like cells and osteocytes in cortical bone were interpreted as the osteocytic lacunar-canalicular system. By P10, resorption pits had formed on the tibial trabecular bone surface. The applicability of ASEM for pathological analysis was addressed using knockout mice of Keap1, an oxidative-stress sensor. In Keap1-/- femurs, we observed impaired calcification and angiogenesis of epiphyseal cartilage, suggesting impaired bone development. Overall, the quick ASEM method we developed revealed mineralisation and new structures in wet bone tissue at EM resolution and can be used to study mineralisation-associated phenomena of any hydrated tissue.

Excessive salt consumption causes systemic calcium mishandling and worsens microarchitecture and strength of long bones in rats.

Excessive salt intake has been associated with the development of non-communicable diseases, including hypertension with several cardiovascular consequences. Although the detrimental effects of high salt on the skeleton have been reported, longitudinal assessment of calcium balance together with changes in bone microarchitecture and strength under salt loading has not been fully demonstrated. To address these unanswered issues, male Sprague-Dawley rats were fed normal salt diet (NSD; 0.8% NaCl) or high salt diet (HSD; 8% NaCl) for 5 months. Elevation of blood pressure, cardiac hypertrophy and glomerular deterioration were observed in HSD, thus validating the model. The balance studies were performed to monitor calcium input and output upon HSD challenge. The HSD-induced increase in calcium losses in urine and feces together with reduced fractional calcium absorption led to a decrease in calcium retention. With these calcium imbalances, we therefore examined microstructural changes of long bones of the hind limbs. Using the synchrotron radiation x-ray tomographic microscopy, we showed that trabecular structure of tibia and femur of HSD displayed a marked increase in porosity. Consistently, the volumetric micro-computed tomography also demonstrated a significant decrease in trabecular bone mineral density with expansion of endosteal perimeter in the tibia. Interestingly, bone histomorphometric analyses indicated that salt loading caused an increase in osteoclast number together with decreases in osteoblast number and osteoid volume. This uncoupling process of bone remodeling in HSD might underlie an accelerated bone loss and bone structural changes. In conclusion, long-term excessive salt consumption leads to impairment of skeletal mass and integrity possibly through negative calcium balance.

Effect of alendronate on the femoral metaphyseal defect under carbamazepine in ovariectomized rats.

BACKGROUND: The use of antiepileptic drugs and estrogen deficiency put forward higher requirements for bone defect regeneration. The present study investigated the effects of alendronate (ALN) on femoral bone defect in ovariectomized (OVX) rats under the influence of carbamazepine (CBZ). METHODS: One hundred female SD rats at 3 months of age were either sham-operated or OVX and divided into four groups: sham control (CON); OVX control (OVX); ovariectomized rats treated with CBZ via gavage (75 mg/kg/day; CBZ); ovariectomized rats treated with CBZ plus ALN (2 mg/kg/day; CBZ-ALN). A critical-sized femoral metaphyseal bone defect was established in all female SD rats. Animals from the CBZ and CBZ-ALN groups received drugs by gavage the day after bone defect surgery was performed. After the rats were sacrificed, the defected area located in the distal femur was harvested for evaluation by microcomputed tomography (micro-CT), hematoxylin and eosin (HE) staining, and Masson's trichrome staining. The samples were also analyzed by biomechanics and immunohistochemical evaluation (IHC). Besides, biochemical analysis evaluates all serum samples. RESULTS: The present study showed that ovariectomy changed the microstructural parameters of bone. The use of CBZ further decreased femur bone mass while treatment with ALN prevented bone loss. Compared to OVX and CBZ groups, CBZ-ALN group promoted bone neoformation and enhanced the ultimate load of the femur bone. However, the group of CBZ-ALN did not return to normal levels compared with the CON group. Besides, we noticed that CBZ-ALN group reduced tartrate-resistant acid phosphatase-5b (Tracp-5b) expression and had no significant effect on the expression of osteocalcin (OCN) and type I collagen (Col-I) in IHC compared with CBZ group. Biochemical analysis results presented that systemic delivery of CBZ showed pernicious effects on bone formation and resorption in ovariectomized rats, with the worse effects on C-terminal crosslinked telopeptide of type I collagen (CTX-1). Besides, a significant decrease in CTX-1 levels was observed in CBZ-ALN group as compared to the group of CBZ. CONCLUSION: These results demonstrated that ALN can effectively reverse the effects of CBZ on the microarchitectural properties of bone, and thus can have a positive effect on local bone neoformation in rats with osteoporosis. CLINICAL RELEVANCE: The dose of 2 mg/kg ALN improves the negative effect of prescription of CBZ at 75 mg/kg and promotes bone neoformation of femoral bony deficits.

Changes in the intra- and peri-cellular sclerostin distribution in lacuno-canalicular system induced by mechanical unloading.

INTRODUCTION: Mechanical stimuli regulate Sclerostin (Scl), a negative regulator of bone formation, expression in osteocytes. However, the detailed Scl distribution in osteocytes in response to mechanical unloading remains unclear. MATERIALS AND METHODS: Twelve-week-old male rats were used. The sciatic and femoral nerves on the right side were excised as mechanical unloading treatment. A sham operation was performed on the left side. One week after neurotrauma, the bone density of the femora was evaluated by peripheral quantitative computed tomography, and immunofluorescence was performed in coronal sections of the femoral diaphysis. The mean fluorescence intensity and fluorescent profile of Scl from the marrow to the periosteal side were analyzed to estimate the Scl expression and determine to which side (marrow or periosteal) the Scl prefers to distribute in response to mechanical unloading. The most sensitive region indicated by the immunofluorescence results was further investigated by transmission electron microscopy (TEM) with immunogold staining to show the Scl expression changes in different subcellular structures. RESULTS: In femur distal metaphysis, neurotrauma-induced mechanical unloading significantly decreased the bone density, made the distribution of Scl closer to the marrow on the anterior and medial side, and increased the Scl expression only on the lateral side. TEM findings showed that only the expression of Scl in canaliculi was increased by mechanical unloading. CONCLUSIONS: Our results showed that even short-term mechanical unloading is enough to decrease bone density, and mechanical unloading not only regulated the Scl expression but also changed the Scl distribution in both the osteocyte network and subcellular structures.

Investigation for GSK3ß expression in diabetic osteoporosis and negative osteogenic effects of GSK3ß on bone marrow mesenchymal stem cells under a high glucose microenvironment.

Osteoporosis is a common skeletal complication of diabetes mellitus (DM). The mechanisms underlying the pathophysiology of diabetic osteoporosis are complex. Glycogen synthase kinase-3ß (GSK-3ß) is a widely expressed serine/threonine kinase and associated with both DM and bone metabolism, which arouse our concern. In this study, we established the diabetic mouse model by high-fat diet combined with streptozotocin injection. Decreased bone mass and reduced osteogenesis were observed in femurs of the mice. Besides, we identified that there is an activated expression of GSK3ß in the bone marrow mesenchymal stem cells (BMSCs) of diabetic mice. To explore the link between GSK3ß and diabetic osteoporosis, we exposed BMSCs to a high glucose microenvironment in vitro and discovered that the glucose-induced GSK3ß activation has negative osteogenic effects on BMSCs by suppressing ß-catenin/Tcf7/Ccn4 signaling axis. Inhibition of GSK3ß by specific concentrations of LiCl could reverse the impaired osteogenesis of BMSCs and increase expression of ß-catenin, Tcf7 and Ccn4. Our research indicated that abnormal activation of GSK3ß plays a role in diabetic osteoporosis and might be a potential target to treat diabetic osteoporosis.

Kefir Peptides Prevent Estrogen Deficiency-Induced Bone Loss and Modulate the Structure of the Gut Microbiota in Ovariectomized Mice.

Osteoporosis is a major skeletal disease associated with estrogen deficiency in postmenopausal women. Kefir-fermented peptides (KPs) are bioactive peptides with health-promoting benefits that are produced from the degradation of dairy milk proteins by the probiotic microflora in kefir grains. This study aimed to evaluate the effects of KPs on osteoporosis prevention and the modulation of the composition of the gut microbiota in ovariectomized (OVX) mice. OVX mice receiving an 8-week oral gavage of 100 mg of KPs and 100 mg of KPs + 10 mg Ca exhibited lower trabecular separation (Tb. Sp), and higher bone mineral density (BMD), trabecular number (Tb. N) and bone volume (BV/TV), than OVX groups receiving Ca alone and untreated mice, and these effects were also reflected in bones with better mechanical properties of strength and fracture toughness. The gut microbiota of the cecal contents was examined by 16S rDNA amplicon sequencing. α-Diversity analysis indicated that the gut microbiota of OVX mice was enriched more than that of sham mice, but the diversity was not changed significantly. Treatment with KPs caused increased microbiota richness and diversity in OVX mice compared with those in sham mice. The microbiota composition changed markedly in OVX mice compared with that in sham mice. Following the oral administration of KPs for 8 weeks, the abundances of Alloprevotella, Anaerostipes, Parasutterella, Romboutsia, Ruminococcus_1 and Streptococcus genera were restored to levels close to those in the sham group. However, the correlation of these bacterial populations with bone metabolism needs further investigation. Taken together, KPs prevent menopausal osteoporosis and mildly modulate the structure of the gut microbiota in OVX mice.

Proanthocyanidin-rich grape seed extract improves bone loss, bone healing, and implant osseointegration in ovariectomized animals.

The purpose of the present study was to confirm if proanthocyanidin-rich grape seed extract (GSE) had the ability to improve bone health such as bone loss, bone healing, and implant osseointegration (defined as the direct connection between bone tissue and an implant) in ovariectomized (OVX) animals. We demonstrated that daily oral administration of GSE prevented bone loss in the lumbar vertebrae and femur in OVX mice. In addition, osteoclastogenesis in the lumbar spine bone of OVX mice, as assessed by histological and histomorphometric analyses, was accelerated but GSE prevented this dynamization, suggesting that GSE could counteract OVX-induced accelerated osteoclastogenic activity. In rats, OVX clearly impaired the healing of defects created on the calvaria, and GSE overcame this OVX-impaired healing. In the same way, osseointegration of a tibial implant in rats was retarded by OVX, and GSE counteracted the OVX-induced poor osseointegration, likely promoting bone healing by preventing imbalanced bone turnover. These results suggest that orally administered GSE improved implant osseointegration by mitigating the impaired bone health induced by OVX as a model of estrogen deficiency.

The structure of cortical bone as revealed by the application of methods for the calcified matrix study.

Calcination and decalcification are basic procedures useful to a morphological approach of a biological, composite material like cortical bone. The study was carried out on a whole human femur conserved in liquid (from an educational collection). Cortical fracturing and SEM observation of vascular canals surface collagen texture was used to study bone deproteination at scalar temperatures (400-1,200°C) and acid bone decalcification at crescent time intervals. Heating burned and vaporized the organic matrix with shrinkage of the bone specimens as documented by the weight loss and transverse surface morphometry. SEM showed a pattern of aligned spherulites at 400°C which maintained the collagen fibrils layout (like a mineral cast), followed by a spherulites fusion progression with the temperature increments. At 1200°C a crystalline-like structure of tightly-packed trapezohendron units. XRD analysis supported the SEM morphology displaying the complete Debey rings of hydroxyapatite and spotted Debey rings of withlockite. Surface Ca and P elution was documented after 12 hr of exposition to the acid solution by dissolution of spherulites and the whole canal surface decalcified in depth after 15 days by SEM-EDAX analysis. The periodic pattern of collagen fibrils was still evident up to 15 days of decalcification together with fine granular deposits of a not-collagenic proteic material, while after 30 days no period was observed in the decalcified fibrils. Collagen mineral cast at 400°C calcination. Complete crystalline transformation at 1200°C. Up to 15 days of decalcification fibrils period maintained.

Measuring Knee Bone Marrow Perfusion Using Arterial Spin Labeling at 3 T.

Bone perfusion is an essential physiological measure reflecting vasculature status and tissue viability of the skeletal system. Arterial spin labeling (ASL), as a non-invasive and non-contrast enhanced perfusion imaging method, is an attractive approach for human research studies. To evaluate the feasibility of ASL perfusion imaging of knee bone marrow in the distal femoral condyle at a 3 T MRI scanner, a study was performed with eight healthy volunteers (three males and five females, 26 ± 2 years old) and two patients (male, 15 and 11 years old) with diagnosed stage II juvenile osteochondritis dissecans (JOCD). ASL imaging utilized a flow-sensitive alternating inversion recovery method for labeling and a single-shot fast spin echo sequence for image readout. In addition to quantitative knee bone marrow ASL imaging, studies were also performed to evaluate the effects of prolonged post-bolus delay and varied labeling size. ASL imaging was successfully performed with all volunteers. Despite the benefits of hyper-intensive signal suppression within bone marrow, the use of a prolonged post-bolus delay caused excessive perfusion signal decay, resulting in low perfusion signal-to-noise ratio (SNR) and poor image quality. Bone marrow perfusion signal changed with the labeling size, suggesting that the measured bone marrow perfusion signal is flow-associated. The means and standard deviations of bone marrow blood flow, spatial SNR, and temporal SNR from the quantitative perfusion study were 38.3 ± 5.2 mL/100 g/min, 3.31 ± 0.48, and 1.33 ± 0.31, respectively. The imaging results from JOCD patients demonstrated the potential of ASL imaging to detect disease-associated bone marrow perfusion changes. This study demonstrates that it is feasible to perform ASL imaging of knee bone marrow in the distal femoral condyle at 3 T.

Sub-chronic exposure to Kalach 360 SL, Glyphosate-based Herbicide, induced bone rarefaction in female Wistar rats.

We investigated the effects of Kalach 360 SL (KL), Glyphosate (G)-based herbicide, on bone tissue in different groups of female Wistar rats. Group 1 (n = 6) received a standard diet and served as a control, groups 2 and 3 (n = 6 each) received 0.07 ml (D1: 126 mg/Kg) and 0.175 ml (D2: 315 mg/Kg) of KL dissolved in the water for 60 days. The plasma was used to examine the metabolic balance markers (calcium, phosphorus, phosphatase alkaline (PAL), and vitamin D (vit D) and hormonal status (oestrogen and thyroid hormones). As a result, sub-chronic exposure to KL induced a perturbation of bone metabolism (calcium and phosphorus) and hormonal status disturbance. The histological and immunohistochemical study of the thyroid gland revealed a disturbance in morphological structure and thyroid cells function. Moreover, the KL disrupting eff ;ect on thyroid function was investigated by measuring changes in plasma levels of thyroid hormones. Free triiodothyronine (FT3) and thyroxine (FT4) were decreased in female rats breast-fed from rats treated with D and D2 of KL. This eff ;ect was associated with an increase in the plasma level of thyroid-stimulating hormone (TSH). Thus, that KL leads to hypothyroidism. Decrease in levels of oestrogen and thyroid dysfunction led to a disruption in the skeletal bone. The histological study and SEM in bone results allowed us to observe, in rats exposed to KL, the thinning and discontinuity of bone trabecular with a significant decrease in the number of nodes (intertrabecular links).In conclusion, KL sub-chronic exposure caused an aspect of osteoporosis.

Assessment of the human bone lacuno-canalicular network at the nanoscale and impact of spatial resolution.

Recently, increasing attention has been given to the study of osteocytes, the cells that are thought to play an important role in bone remodeling and in the mechanisms of bone fragility. The interconnected osteocyte system is deeply embedded inside the mineralized bone matrix and lies within a closely fitted porosity known as the lacuno-canalicular network. However, quantitative data on human samples remain scarce, mostly measured in 2D, and there are gaps to be filled in terms of spatial resolution. In this work, we present data on femoral samples from female donors imaged with isotropic 3D spatial resolution by magnified X-ray phase nano computerized-tomography. We report quantitative results on the 3D structure of canaliculi in human femoral bone imaged with a voxel size of 30 nm. We found that the lacuno-canalicular porosity occupies on average 1.45% of the total tissue volume, the ratio of the canalicular versus lacunar porosity is about 37.7%, and the primary number of canaliculi stemming from each lacuna is 79 on average. The examination of this number at different distances from the surface of the lacunae demonstrates branching in the canaliculi network. We analyzed the impact of spatial resolution on quantification by comparing parameters extracted from the same samples imaged with 120 nm and 30 nm voxel sizes. To avoid any bias related to the analysis region, the volumes at 120 nm and 30 nm were registered and cropped to the same field of view. Our results show that the measurements at 120 and 30 nm are strongly correlated in our data set but that the highest spatial resolution provides more accurate information on the canaliculi network and its branching properties.

A Method to Interpolate Osteon Volume Designed for Histological Age Estimation Research.

Aging adult skeletal material is a crucial component of building the biological profile of unknown skeletal remains, but many macro- and microscopic methods have challenges regarding accuracy, precision, and replicability. This study developed a volumetric method to visualize and quantify histological remodeling events in three dimensions, using a two-dimensional serialized approach that applied circular polarizing microscopy and geographic information systems protocols. This approach was designed as a tool to extend current histological aging methodologies. Three serial transverse sections were obtained from a human femoral midshaft. A total sample size of 6847 complete osteons from the three sections was identified; 1229 osteons connected between all sections. The volume of all connected osteons was interpolated using ArcGIS area calculations and truncated cone geometric functions. Each section was divided into octants, and two random samples of 100 and of 30 connected osteons from each octant were generated. Osteon volume was compared between the octants for each random sample using ANOVA. Results indicated that the medial aspect had relative uniformity in osteon volume, whereas the lateral aspect showed high variability. The anterolateral-lateral octant had significantly smaller osteon volume, whereas the posterior-posterolateral octant had significantly larger osteon volume. Results also indicated that a minimum of 100 osteons is statistically more robust and more representative of normal osteon distribution and volume; the use of 30 osteons is insufficient. This research has demonstrated that osteon volume can be interpolated using spatial geometry and GIS applications and may be a tool to incorporate into adult age-at-death estimation techniques.

Growing up Tyrannosaurus rex: Osteohistology refutes the pygmy "Nanotyrannus" and supports ontogenetic niche partitioning in juvenile Tyrannosaurus.

Despite its iconic status as the king of dinosaurs, Tyrannosaurus rex biology is incompletely understood. Here, we examine femur and tibia bone microstructure from two half-grown T. rex specimens, permitting the assessments of age, growth rate, and maturity necessary for investigating the early life history of this giant theropod. Osteohistology reveals these were immature individuals 13 to 15 years of age, exhibiting growth rates similar to extant birds and mammals, and that annual growth was dependent on resource abundance. Together, our results support the synonomization of "Nanotyrannus" into Tyrannosaurus and fail to support the hypothesized presence of a sympatric tyrannosaurid species of markedly smaller adult body size. Our independent data contribute to mounting evidence for a rapid shift in body size associated with ontogenetic niche partitioning late in T. rex ontogeny and suggest that this species singularly exploited mid- to large-sized theropod niches at the end of the Cretaceous.

Acoustic diffusion constant of cortical bone: Numerical simulation study of the effect of pore size and pore density on multiple scattering.

While osteoporosis assessment has long focused on the characterization of trabecular bone, the cortical bone micro-structure also provides relevant information on bone strength. This numerical study takes advantage of ultrasound multiple scattering in cortical bone to investigate the effect of pore size and pore density on the acoustic diffusion constant. Finite-difference time-domain simulations were conducted in cortical microstructures that were derived from acoustic microscopy images of human proximal femur cross sections and modified by controlling the density (Ct.Po.Dn) ∈[5-25] pore/mm2 and size (Ct.Po.Dm) ∈[30-100] µm of the pores. Gaussian pulses were transmitted through the medium and the backscattered signals were recorded to obtain the backscattered intensity. The incoherent contribution of the backscattered intensity was extracted to give access to the diffusion constant D. At 8 MHz, significant differences in the diffusion constant were observed in media with different porous micro-architectures. The diffusion constant was monotonously influenced by either pore diameter or pore density. An increase in pore size and pore density resulted in a decrease in the diffusion constant (D =285.9Ct.Po.Dm-1.49, R2=0.989 , p=4.96×10-5,RMSE=0.06; D=6.91Ct.Po.Dn-1.01, R2=0.94, p=2.8×10-3 , RMSE=0.09), suggesting the potential of the proposed technique for the characterization of the cortical microarchitecture.