Relative Bone Volume Research Articles

MiR-375-3p negatively regulates osteogenesis by targeting and decreasing the expression levels of LRP5 and β-catenin.

Wnt signaling pathways are essential for bone formation. Previous studies showed that Wnt signaling pathways were regulated by miR-375. Thus, we aim to explore whether miR-375 could affect osteogenesis. In the present study, we investigated the roles of miR-375 and its downstream targets. Firstly, we revealed that miR-375-3p negatively modulated osteogenesis by suppressing positive regulators of osteogenesis and promoting negative regulators of osteogenesis. In addition, the results of TUNEL cell apoptosis assay showed that miR-375-3p induced MC3T3-E1 cell apoptosis. Secondly, miR-375-3p targeted low-density lipoprotein receptor-related protein 5 (LRP5), a co-receptor of the Wnt signaling pathways, and β-catenin as determined by luciferase activity assay, and it decreased the expression levels of LRP5 and β-catenin. Thirdly, the decline of protein levels of β-catenin was determined by immunocytochemistry and immunofluorescence. Finally, silence of LRP5 in osteoblast precursor cells resulted in diminished cell viability and cell proliferation as detected by WST-1-based colorimetric assay. Additionally, all the parameters including the relative bone volume from μCT measurement suggested that LRP5 knockout in mice resulted in a looser and worse-connected trabeculae. The mRNA levels of important negative modulators relating to osteogenesis increased after the functions of LRP5 were blocked in mice. Last but not least, the expression levels of LRP5 increased during the osteogenesis of MC3T3-E1, while the levels of β-catenin decreased in bone tissues from osteoporotic patients with vertebral compression fractures. In conclusion, we revealed miR-375-3p negatively regulated osteogenesis by targeting LRP5 and β-catenin. In addition, loss of functions of LRP5 damaged bone formation in vivo. Clinically, miR-375-3p and its targets might be used as diagnostic biomarkers for osteoporosis and might be also as novel therapeutic agents in osteoporosis treatment. The relevant products of miR-375-3p might be developed into molecular drugs in the future. These molecules could be used in translational medicine.

Analog Method for Radiographic Assessment of Heterotopic Bone in Fibrodysplasia Ossificans Progressiva

Severe progressive multifocal heterotopic ossification (HO) is a rare occurrence seen predominantly in patients who have fibrodysplasia ossificans progressiva (FOP) and is difficult to quantitate owing to patient-, disease-, logistical-, and radiation-related issues. The purpose of this study was to develop and validate a scoring system based on plain radiographs for quantitative assessment of HO lesions in patients with FOP. Institutional review board approval was obtained from the University of Pennsylvania, and all data comply with Health Insurance Portability and Accountability Act regulations. The University of Pennsylvania Institutional Animal Care and Use Committee approved the use of mice in this study. First, we used a mouse model of FOP-like HO to validate a semiquantitative analog scale for estimating relative heterotopic bone volume. Second, we used this validated scale to estimate the relative amount of HO from a retrospective analysis of plain radiographs from 63 patients with classic FOP. Finally, the scale was applied to a retrospective analysis of computed tomographic images from three patients with FOP. In the FOP-mouse model, the observed rating on the analog scale is highly correlated to heterotopic bone volumes measured by microcomputed tomography (R2 = 0.89). The scoring system that was applied to radiographs of patients with FOP captured the clinical range of HO typically present at all axial and appendicular sites. Analysis of computed tomographic scans of patients with FOP found that observed radiograph ratings were highly correlated with HO volume (R2 = 0.80). The scoring system described here could enable practical, quantitative assessment of HO in clinical trials to evaluate new treatment modalities, especially for FOP. The development of the six-point analog scale described here provides and validates a much-needed, reproducible, and quantifiable method for describing and assessing HO in patients with FOP. This scale has the potential to be a key descriptor that can inform patients with FOP and clinicians about disease progression and response of HO lesions to interventions and treatments.

Blockade of NF-κB and MAPK pathways by ulinastatin attenuates wear particle-stimulated osteoclast differentiation invitro and invivo.

Ulinastatin, a urinary trypsin inhibitor (UTI), is widely used to clinically treat lipopolysaccharide (LPS)-related inflammatory disorders recently. Adherent pathogen-associated molecular patterns (PAMPs), of which LPS is the best-studied and classical endotoxin produced by Gram-negative bacteria, act to increase the biological activity of osteopedic wear particles such as polymethyl-methacrylate (PMMA) and titanium particles in cell culture and animal models of implant loosening. The present study was designed to explore the inhibitory effect of UTI on osteoclastogenesis and inflammatory osteolysis in LPS/PMMA-mediated Raw264.7 cells and murine osteolysis models, and investigate the potential mechanism. The in vitro study was divided into the control group, LPS-induced group, PMMA-stimulated group and UTI-pretreated group. UTI (500 or 5000 units/ml) pretreatment was followed by PMMA (0.5 mg/ml) with adherent LPS. The levels of inflammatory mediators including tumour necrosis factor-α (TNF-α), matrixmetallo-proteinases-9 (MMP-9) and interleukin-6 (IL-6), receptor activation of nuclear factor NF-κB (RANK), and cathepsin K were examined and the amounts of phosphorylated I-κB, MEK, JNK and p38 were measured. In vivo study, murine osteolysis models were divided into the control group, PMMA-induced group and UTI-treated group. UTI (500 or 5000 units/kg per day) was injected intraperitoneally followed by PMMA suspension with adherent LPS (2×108 particles/25 μl) in the UTI-treated group. The thickness of interfacial membrane and the number of infiltrated inflammatory cells around the implants were assessed, and bone mineral density (BMD), trabecular number (Tb.N.), trabecular thickness (Tb.Th.), trabecular separation (Tb.Sp.), relative bone volume over total volume (BV/TV) of distal femur around the implants were calculated. Our results showed that UTI pretreatment suppressed the secretion of proinflammatory cytokines including MMP-9, IL-6, TNF-α, RANK and cathepsin K through down-regulating the activity of nuclear factor kappa B (NF-κB) and MAPKs partly in LPS/PMMA-mediated Raw264.7 cells. Finally, UTI treatment decreased the inflammatory osteolysis reaction in PMMA-induced murine osteolysis models. In conclusion, these results confirm the anti-inflammatory potential of UTI in the prevention of particle disease.

Effects of combined ovariectomy with dexamethasone on rat lumbar vertebrae.

This study investigated the effects of combined ovariectomy with dexamethasone treatment on rat lumbar vertebrae in comparison with osteoporosis induced via ovariectomy or dexamethasone alone, and analysis of the associated molecular mechanism. Sixty-two female Sprague-Dawley rats (3 months' old) were randomly divided into five treatment groups: an untreated baseline (BL) group; those receiving a sham operation (SHAM); those receiving a dexamethasone injection alone (DEXA); those undergoing bilateral ovariectomy (OVX); and those subjected to both ovariectomy and dexamethasone injection (OVX-DEXA). Animals in the BL group were euthanized at the beginning of the experiment, whereas animals in the remaining groups were euthanized at the end of the first month (M1), second month (M2), or third month (M3). Bone mineral density, bone microarchitecture, biomechanical properties of vertebrae, and serum levels of estrogen, amino-terminal propeptide of type I collagen (PINP), and β-C-telopeptide of type I collagen (β-CTX) were measured. In addition, we examined biglycan, runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), lipoprotein receptor-related protein-5 (LRP-5), cathepsin K (CTSK), and sclerostin mRNA expression. Bone mineral content and bone mineral density were markedly lower in the OVX-DEXA group compared with the OVX group at all time points examined. The relative bone surface (BS/TV, mm(-1), relative bone volume (BV/TV,%), and trabecular number (Tb.N, 1/mm) were markedly lower in the OVX-DEXA group compared with the remaining groups, whereas trabecular separation (Tb.Sp, mm) was markedly higher in the OVX-DEXA group compared with the remaining groups at M2 or M3. The OVX-DEXA group showed lower compressive strength and lower stiffness compared with the other groups at M2 and M3. Compressive displacement and energy absorption capacity were also markedly lower in the OVX-DEXA group compared with the OVX group at M3. Estradiol levels were markedly lower in the OVX-DEXA group compared with the other groups. Biglycan, runt-related transcription factor 2, osteoprotegerin, and lipoprotein receptor-related protein-5 were down-regulated in the DEXA, OVX, and OVX-DEXA groups compared with the BL and SHAM groups, whereas cathepsin K and sclerostin were up-regulated in the OVX-DEXA group compared with the DEXA and OVX groups. Ovariectomy combined with dexamethasone induced more serious osteoporosis in the rat lumbar spine than either ovariectomy or dexamethasone alone. The combined effect may be due to a combination of suppressed bone formation and increased bone resorption related to an estradiol deficit.

The effect of oxytocin on osseointegration of titanium implant in ovariectomized rats

ABSTRACTIntroduction: Oxytocin (OT) was reported to control differentiation of human mesenchymal stem cells and reverse osteoporosis (OP). This study investigated the effect of systematical treatment of OT on implant osseointegration in ovariectomized (OVX) rats. Material and methods: Twenty female rats received bilateral ovariectomy. Twelve weeks later, all animals were randomly assigned to control or experimental group. Each rat received two implants at the distal femoral metaphysis. From the first postoperative day, rats in experiment group received subcutaneous injection of OT (1 mg/kg · d), while animals in control group received vehicle. Twelve weeks after implantation, specimens containing implants were harvested and evaluated by histology, micro-CT, and push-out test. Tibiae were also harvested to evaluate the effect of OT on intact bone tissue of OVX rats. Results: Compared with control, OT treatment increased the relative bone volume surrounding the implant by 2.2 times, the percent implant osseointegration by 0.62 times, and the maximum push-out force by 2.25 times. Increased bone mass was also observed in histological sections of distal femur with implant and intact bone tissue of the proximal tibiae. Conclusion: Systemic administration of OT promoted peri-implant bone healing and osseointegration of titanium implant and recovered the negative effects of OP in undisturbed bone tissue partially.

Effects of Sclerostin Antibody on the Healing of Femoral Fractures in Ovariectomised Rats.

The inhibition of sclerostin by the systemic administration of a monoclonal antibody (Scl-Ab) significantly increased bone mass and strength in fractured bones in animal models and non-fractured bones in ovariectomised (OVX) rats. In this study, the effects of Scl-Ab on healing were examined in a closed fracture model in OVX rats. Sixty Sprague-Dawley rats underwent an ovariectomy or a sham operation at 4months of age, and a closed fracture of the right femur was performed 3months later. Subcutaneous injections with Scl-Ab (25mg/kg) or saline were then administered on day 1 after the fracture and twice a week for 8weeks (n=20 per group), at which time the fractured femurs were harvested for micro-computed tomography analysis, four-point bending mechanical testing and histomorphometric analysis to examine bone mass, bone strength and dynamic bone formation at the fracture site. The angiogenesis at the fracture site was also examined. Bone marrow stem cells were also isolated from the fractured bone to perform a colony-forming unit (CFU) assay and an alkaline phosphatase-positive (ALP(+)) CFU assay. OVX rats treated with Scl-Ab for 8weeks had significantly increased bone mineral density and relative bone volume compared with OVX rats treated with saline. Similarly, maximum loading, energy to maximum load and stiffness in Scl-Ab-treated OVX rats were significantly higher than those in saline controls. The mineral apposition rate (MAR), mineralising surface (MS/BS) and bone formation rate (BFR/BS) were also significantly increased in Scl-Ab-treated group compared with the saline-treated group in OVX rats. Furthermore, the Scl-Ab-treated group had more CFUs and ALP(+) CFUs than the saline-treated group in OVX rats. No significant difference in angiogenesis at the fracture site was found between the groups. Our study demonstrated that Scl-Ab helped to increase bone mass, bone strength and bone formation at the fracture site in a closed femoral fracture model in OVX rats. Bone marrow stem cells in OVX rats injected with Scl-Ab also had increased CFUs and ALP(+) CFUs.

Small-Diameter Awls Improve Articular Cartilage Repair After Microfracture Treatment in a Translational Animal Model

Background: Microfracture is the most commonly applied arthroscopic marrow stimulation procedure. Hypothesis: Articular cartilage repair is improved when the subchondral bone is perforated by small-diameter microfracture awls compared with larger awls. Study Design: Controlled laboratory study. Methods: Standardized rectangular (4 × 8 mm) full-thickness chondral defects (N = 24) were created in the medial femoral condyle of 16 adult sheep and debrided down to the subchondral bone plate. Three treatment groups (n = 8 defects each) were tested: 6 microfracture perforations using small-diameter awls (1.0 mm; group 1), large-diameter awls (1.2 mm; group 2), or without perforations (debridement control; group 3). Osteochondral repair was assessed at 6 months in vivo using established macroscopic, histological, immunohistochemical, biochemical, and micro–computed tomography analyses. Results: Compared with control defects, histological cartilage repair was always improved after both microfracture techniques (P < .023). Application of 1.0-mm microfracture awls led to a significantly improved histological overall repair tissue quality (7.02 ± 0.70 vs 9.03 ± 0.69; P = .008) and surface grading (1.05 ± 0.28 vs 2.10 ± 0.19; P = .001) compared with larger awls. The small-diameter awl decreased relative bone volume of the subarticular spongiosa (bone volume/tissue volume ratio: 23.81% ± 3.37% vs 30.58% ± 2.46%; P = .011). Subchondral bone cysts and intralesional osteophytes were frequently observed after either microfracture treatment. Macroscopic grading, DNA, proteoglycan, and type I and type II collagen contents as well as degenerative changes within the adjacent cartilage remained unaffected by the awl diameter. Conclusion: Small-diameter microfracture awls improve articular cartilage repair in the translational sheep model more effectively than do larger awls. Clinical Relevance: These data support the use of small microfracture instruments for the surgical treatment of cartilage defects and warrant prolonged clinical investigations.

Micro-computed tomography and histomorphometric analysis of human alveolar bone repair induced by laser phototherapy: a pilot study

Immediate dental implant placement in the molar region is critical, because of the high amount of bone loss and the discrepancy between alveolar crest thickness and the implant platform. Laser phototherapy (LPT) improves bone repair. The aim of this study was to evaluate the human alveolar bone repair 40 days after molar extraction in patients submitted to LPT. Twenty patients were selected for this randomized controlled clinical trial; 10 underwent LPT (laser group) with a GaAlAs diode laser (808nm, 100mW, 0.04cm2, 75J/cm2, 30s per point, 3J per point, at five points). The control group patients (n=10) were not irradiated. Forty days later, the tissue formed inside the sockets was analyzed by micro-computed tomography and histomorphometry. Data from the two groups were compared with Student's t-test and Pearson's correlation test. The relative bone volume was significantly higher in the laser group (P<0.0001). The control group showed negative correlations (P<0.01) between number and thickness, and between number and separation of trabeculae, and a positive correlation between thickness and separation of trabeculae. The laser group showed a significant negative correlation between the number and thickness of trabeculae (P<0.01). The results suggest that LPT is able to accelerate alveolar bone repair after molar extraction, leading to a more homogeneous trabecular configuration represented by thin and close trabeculae.

Characterization of bone turnover and energy metabolism in a rat model of primary and secondary osteoporosis

BackgroundAn experimental rat model served for evaluation of bone- and energy metabolism in early and late stages of osteoporosis. For the early stage, we hypothesized that bilateral ovariectomy (OVX)+multi-deficiency diet (OVXD; depletion of vitamin D, calcium, vitamin K, phosphorus) would induce increased bone turnover while the late stage would be characterized by enhanced bone catabolism. Obesity, insulin resistance and hyperleptinemia would be seen during the whole course of disease.Healthy female Sprague Dawley rats (n=41) aged 10 weeks were randomly assigned to sham and treatment groups and sacrificed at 3, 12, and 14 months after the study began. ResultsIn the early phase, OVXD was associated with an increase in body weight, but not, however, in later stages. There was a decrease in bone mineral density and relative bone volume (BV/TV) as assessed by Dual Energy X-ray Absorptiometry and micro computed tomography that was most severe in the later stages of disease, indicating bone catabolism.Osteocalcin limiting bone formation was increased initially, whereas later stages (14 months) were characterized by elevated osteopontin, suggesting bone remodeling. Severe hyperparathyroidism was present during all stages of disease. Only the early phases of disease were characterized by hyperinsulinemia and increased adrenocorticotrophic stimulating hormone, whereas in the late stage hypoleptinemia rather than hyperleptinemia was seen. ConclusionMarkers of bone and energy metabolism reflected both an increased bone turn over and ongoing bone remodeling associated with initial hyperinsulinemia. Osteopontin and osteocalcin can be used to differentiate early and late stages of osteoporosis.

The effect of granulocyte-colony stimulating factor on rotator cuff healing after injury and repair.

The failure rate of tendon-bone healing after repair of rotator cuff tears remains high. A variety of biologic- and cell-based therapies aimed at improving rotator cuff healing have been investigated, and stem cell-based techniques have become increasingly more common. However, most studies have focused on the implantation of exogenous cells, which introduces higher risk and cost. We aimed to improve rotator cuff healing by inducing endogenous stem cell mobilization with systemic administration of granulocyte-colony stimulating factor (G-CSF). We asked: (1) Does G-CSF administration increase local cellularity after acute rotator cuff repair? (2) Is there histologic evidence that G-CSF improved organization at the healing enthesis? (3) Does G-CSF administration improve biomechanical properties of the healing supraspinatus tendon-bone complex? (4) Are there micro-MRI-based observations indicating G-CSF-augmented tendon-bone healing? After creation of full-thickness supraspinatus tendon defects with immediate repair, 52 rats were randomized to control or G-CSF-treated groups. G-CSF was administered for 5 days after repair and rats were euthanized at 12 or 19 postoperative days. Shoulders were subjected to micro-MR imaging, stress relaxation, and load-to-failure as well as blinded histologic and histomorphometric analyses. G-CSF-treated animals had significantly higher cellularity composite scores at 12 and 19 days compared with both control (12 days: 7.40 ± 1.14 [confidence interval {CI}, 5.98-8.81] versus 4.50 ± 0.57 [CI, 3.58-5.41], p = 0.038; 19 days: 8.00 ± 1.00 [CI, 6.75-9.24] versus 5.40 ± 0.89 [CI, 4.28-6.51], p = 0.023) and normal animals (12 days: p = 0.029; 19 days: p = 0.019). There was no significant difference between G-CSF-treated animals or control animals in ultimate stress (MPa) and strain, modulus (MPa), or yield stress (MPa) and strain at either 12 days (p = 1.000, p = 0.104, p = 1.000, p = 0.909, and p = 0.483, respectively) or 19 days (p = 0.999, p = 0.964, p = 1.000, p = 0.988, and p = 0.904, respectively). There was no difference in MRI score between G-CSF and control animals at either 12 days (2.7 ± 1.8 [CI, 1.08-4.24] versus 2.3 ± 1.8 [CI, 0.49-4.17], p = 0.623) or 19 days (2.5 ± 1.4 [CI, 1.05-3.94] versus 2.3 ± 1.5 [CI, 0.75-3.91], p = 0.737). G-CSF-treated animals exhibited significantly lower relative bone volume compared with normal animals in the entire humeral head (24.89 ± 3.80 [CI, 20.17-29.60) versus 32.50 ± 2.38 [CI, 29.99-35.01], p = 0.009) and at the supraspinatus insertion (25.67 ± 5.33 [CI, 19.04-32.29] versus 33.36 ± 1.69 [CI, 31.58-35.14], p = 0.027) at 12 days. Further analysis did not reveal any additional significant relationships with respect to regional bone volume or trabecular thickness between groups and time points (p > 0.05). Postoperative stem cell mobilization agents may be an effective way to enhance rotator cuff repair. Future studies regarding the kinetics of mobilization, the homing capacity of mobilized cells to injured tissues, and the ability of homing cells to participate in regenerative pathways are necessary.

Deteriorating effect on bone metabolism and microstructure by passive cigarette smoking through dual actions on osteoblast and osteoclast.

There is no clear evidence to show the direct causal relationship between passive cigarette smoking and osteoporosis. Furthermore, the underlying mechanism is unknown. The objective of this study is to demonstrate the effects of long-term passive cigarette smoking on bone metabolism and microstructure by a mouse model and cell culture systems. BALB/c mice were exposed to 2 or 4 % cigarette smoke for 14 weeks. The bone turnover biochemical markers in urine and serum and also the bone micro-architecture by micro-CT were compared with the control group exposed to normal ambient air. In the cell culture experiments, mouse MC3T3-E1 and RAW264.7 cell lines to be employed as osteoblast and osteoclast, respectively, were treated with the sera obtained from 4 % smoking or control mice. Their actions on cell viability, differentiation, and function on these bone cells were assessed. The urinary mineral and deoxypyridinoline (DPD) levels, and also the serum alkaline phosphatase activity, were significantly higher in the 4 % smoking group when compared with the control group, indicating an elevated bone metabolism after cigarette smoking. In addition, femoral osteopenic condition was observed in the 4 % smoking group, as shown by the decrease of relative bone volume and trabecular thickness. In isolated cell studies, osteoblast differentiation and bone formation were inhibited while osteoclast differentiation was increased. The current mouse smoking model and the isolated cell studies demonstrate that passive cigarette smoke could induce osteopenia by exerting a direct detrimental effect on bone cells differentiation and further on bone remodeling process.

Hyperbaric oxygen therapy as a prevention modality for radiation damage in the mandibles of mice

BackgroundRadiation therapy (RT) as part head and neck cancer treatment often leads to irradiation of surrounding normal tissue, such as mandibular bone. A reduced reparative capacity of the bone can lead to osteoradionecrosis (ORN). Hyperbaric oxygen therapy (HBOT) is used to treat ORN, based on its potential to raise the oxygen tension in tissues. However, prevention of radiation-induced damage is of great interest. Our purpose was to investigate whether HBOT could prevent radiation-induced damage to murine mandibles. MethodsTwenty-eight mice were irradiated in the head and neck region with a single dose (15 Gy) and half of them were subsequently subjected to HBOT. Another 14 mice did not receive any treatment and served as controls. Ten and 24 weeks after RT, mandibles were harvested and analysed histologically and by microcomputed tomography (micro-CT). ResultsMicro-CT analysis showed a reduction in relative bone volume by RT, which was partly recovered by HBOT. Trabecular thickness and separation were also positively influenced by HBOT. Morphologically, HBOT suppressed the osteoclast number, indicating decreased resorption, and decreased the amount of lacunae devoid of osteocytes, indicating increased bone viability. ConclusionsHBOT was able to partly reduce radiation-induced effects on microarchitectural parameters, resorption, and bone viability in mouse mandibles. HBOT could therefore potentially play a role in the prevention of radiation-induced damage to human mandibular bone.

Gender- and region-specific alterations in bone metabolism in Scarb1-null female mice.

A positive correlation between plasma levels of HDL and bone mass has been reported by epidemiological studies. As scavenger receptor class B, type I (SR-BI), the gene product of Scarb1, is known to regulate HDL metabolism, we recently characterized bone metabolism in Scarb1-null mice. These mice display high femoral bone mass associated with enhanced bone formation. As gender differences have been reported in HDL metabolism and SR-BI function, we investigated gender-specific bone alterations in Scarb1-null mice by microtomography and histology. We found 16% greater relative bone volume and 39% higher bone formation rate in the vertebrae from 2-month-old Scarb1-null females. No such alteration was seen in males, indicating gender- and region-specific differences in skeletal phenotype. Total and HDL-associated cholesterol levels, as well as ACTH plasma levels, were increased in both Scarb1-null genders, the latter being concurrent to impaired corticosterone response to fasting. Plasma levels of estradiol did not differ between null and WT females, suggesting that the estrogen metabolism alteration is not relevant to the higher vertebral bone mass in female Scarb1-null mice. Constitutively, high plasma levels of leptin along with 2.5-fold increase in its expression in white adipose tissue were measured in female Scarb1-null mice only. In vitro exposure of bone marrow stromal cells to ACTH and leptin promoted osteoblast differentiation as evidenced by increased gene expression of osterix and collagen type I alpha. Our results suggest that hyperleptinemia may account for the gender-specific high bone mass seen in the vertebrae of female Scarb1-null mice.

Osseous wound repair under inhibition of the axis of advanced glycation end-products and the advanced glycation end-products receptor

Blockade of advanced glycation end-products (AGE) is able to reduce diabetic complications and control periodontitis. This study aimed to determine whether the application of aminoguanidine (AG), an AGE inhibitor, or N-phenacylthiazolium bromide (PTB), an AGE breaker, facilitates the healing of an osseous wound in non-diabetic animals. 2.6 mm diameter full-thickness osseous wounds were created bilaterally in 54 healthy Sprague-Dawley rats. Rats received daily normal saline, AG, or PTB injections respectively and were euthanized after 7 days, 14 days, or 28 days (n=6). The wound healing pattern was assessed by micro-computed tomography, histology, histochemistry for the fiber arrangement, and the gene expression levels of AGE receptor, tumor necrosis factor-α, type I collagen, and fibronectin. Under the AG and PTB administration, osteogenesis was apparently promoted in the early stages of healing, but the union of the osseous wound and the fibril re-arrangement was apparently retarded. No significant difference was found in any of the micro-computed tomography parameters as compared to the control in the first 14 days, whereas the relative bone volume was significantly higher in the control at Day 28. AGE receptor and tumor necrosis factor-α were depressed in the PTB group, but only temporarily at Day 14 in the AG group. Therefore, at Day 14, type I collagen was significantly upregulated in the PTB group, and fibronectin was significantly increased in the AG group. Anti-AGE agents reduced inflammation but did not apparently facilitate osteogenesis during the osseous wound repair.

Trabecular bone structure in the primate wrist

Trabecular (or cancellous) bone has been shown to respond to mechanical loading throughout ontogeny and thus can provide unique insight into skeletal function and locomotion in comparative studies of living and fossil mammalian morphology. Trabecular bone of the hand may be particularly functionally informative because the hand has more direct contact with the substrate compared with the remainder of the forelimb during locomotion in quadrupedal mammals. This study investigates the trabecular structure within the wrist across a sample of haplorhine primates that vary in locomotor behaviour (and thus hand use) and body size. High-resolution microtomographic scans were collected of the lunate, scaphoid, and capitate in 41 individuals and eight genera (Homo, Gorilla, Pan, Papio, Pongo, Symphalangus, Hylobates, and Ateles). We predicted that particular trabecular parameters would 1) vary across suspensory, quadrupedal, and bipedal primates based on differences in hand use and load, and 2) scale with carpal size following similar allometric patterns found previously in other skeletal elements across a larger sample of mammals and primates. Analyses of variance (trabecular parameters analysed separately) and principal component analyses (trabecular parameters analysed together) revealed no clear functional signal in the trabecular structure of any of the three wrist bones. Instead, there was a large degree of variation within suspensory and quadrupedal locomotor groups, as well as high intrageneric variation within some taxa, particularly Pongo and Gorilla. However, as predicted, Homo sapiens, which rarely use their hands for locomotion and weight support, were unique in showing lower relative bone volume (BV/TV) compared with all other taxa. Furthermore, parameters used to quantify trabecular structure within the wrist scale with size generally following similar allometric patterns found in trabeculae of other mammalian skeletal elements. We discuss the challenges associated with quantifying and interpreting trabecular bone within the wrist.

Dual gate ultrasonic backscatter technique compared to x-ray microtomography parameters

Over 52 million Americans suffer low bone mass and at least 10 million suffer from osteoporosis. This study seeks to develop a dual gate ultrasonic technique for predicting bone quality as well as bone quantity. Ultrasonic pulses from a 5 MHz transducer were propagated into regions of porous bone in 18 bone specimens from one bovine and four human donors. The dual gate technique considered the normalized mean of the backscatter difference (nMBD), which is the power difference between two gated regions of 2 μs each placed 1 μs apart over the returned signal. This ultrasonic parameter was compared to eight X-Ray MicroCT parameters describing bone quality and quantity. Among these are the Structural Model Index (SMI) and Relative Bone Volume (BV/TV). SMI grades the structure of a specimen based on its plate and rod characteristics, making it a bone quality characteristic. SMI produced an R value of 0.982 with nMBD. BV/TV, a bone quantity indicator, finds the ratio of bone volume in the total specimen volume, and showed an R value of 0.993 with nMBD.

Deterioration of mouse femur microstructure by heavy cigarette smoking and the effect of smoking cessation

Smoking is a risk factor of osteoporosis. To investigate the dose effect of smoking, mice were subjected to ambient air, 2% or 4% cigarette smoke exposure for 14 weeks. To evaluate the effect of smoking cessation, ambient air or 4% cigarette smoke were given to mice for 20 weeks, while the smoking cessation group stopped smoking in the last 6 weeks. Urine and serum bone turnover markers; as well as femora and tibias mineral content, histology and microstructure were assessed.Deoxypyridinoline (DPD), minerals excretion in urine and serum alkaline phosphatase (ALP) activity increased after heavy smoking. Bone weight and mineral contents had no difference in all groups. Relative bone volume and trabecular thickness were lower with elevation in structure model index after 4% smoking. Smoking cessation returned body weight to control level. Serum ALP activity decreased after smoking cessation. Urine DPD level in smoking groups were significantly higher. Less intact trabecular structure in distal femur and reduced femur growth plate thickness were found in smoking groups.To conclude, chronic heavy smoking increases bone turnover and deteriorates bone microstructure in mice. Smoking cessation does not reverse the ill effects of chronic heavy smoking. The study was supported by research funds from the Jockey Club Centre for Care and Control and the Direct Grant, the Chinese University of Hong Kong.

Effect of long-term voluntary exercise on the induction of osteoarthritis by a very high-fat diet in aged mice

Purpose: Short-term voluntary exercise protects against cartilage proteoglycan loss in young mice fed a very high-fat diet. Exercise also reduces the clustering of metabolic inflammatory markers and improves glucose metabolism without weight loss, suggesting a role for systemic metabolic factors in mediating obesity-associated knee OA. We hypothesized that despite an increase in joint loading, long-term exercise would protect against knee OA in aged mice fed a very high-fat diet by reducing age and diet-associated increases in weight and glucose intolerance. Methods: Male C57BL/6J mice were fed either a control fat (CF; 10% kcal fat; n=21) or a very high fat (HF; 60% kcal fat; n=22) diet starting at 6 wks of age. At 26 wks, mice from each diet (n=9) were single-housed in cages with a running wheel until the end of the study at 52 wks of age. We compared joint loading between CF and HF fed mice using a custom force-instrumented running wheel. Body composition was quantified by DEXA and dissection of fat pads. Glucose tolerance testing was conducted at 24 and 48 wks. We evaluated OA pathology by MicroCT and histomorphometry. MicroCT was used to evaluate subchondral bone thickness and relative trabecular bone volume (BV/TV) in the medial and lateral tibial compartments. Stained sagittal knee sections were graded for cartilage OA severity using a modified Mankin scale (0-24), and anterior and posterior medial tibial osteophytes were graded using a semi-quantitative scale (0-3) by two blinded graders. Results: Prior to exercise, body weight and body fat were increased 71% and 63%, respectively, with HF feeding. Voluntary running distance (7.1 vs. 2.7 km/day), speed (59 vs. 33 cm/s) and peak limb force (115 vs. 92 %bodyweight) were lower in HF fed animals (p<0.05). Exercise did not significantly decrease body weight or epididymal fat pad mass in HF animals, although weight and fat mass were reduced with exercise in CF animals (Fig. 1A, B). Glucose tolerance area under the curve (AUC) was greater in exercised but not sedentary HF fed animals compared to activity-matched dietary controls (Fig 1C). A HF diet increased cartilage OA scores in both activity groups (Fig. 2A, p=0.007), although there was no effect of exercise. There was, however, a trend for decreased osteophyte formation in exercised animals regardless of diet (Fig. 2B, p=0.05). Conclusions: Contrary to our hypothesis, long-term voluntary wheel running exercise did not protect against HF diet-induced obesity, glucose intolerance, or OA severity in aged mice. Unlike short-term exercise in young HF-fed mice, mice that have already developed substantial diet-induced obesity show a decreased propensity for long-term voluntary wheel running exercise, which may contribute to the minimal activity-dependent changes with HF feeding in this study. Increased OA severity with long-term HF feeding was primarily due to increased cartilage degradation and proteoglycan loss and not due to increased osteophyte formation. Future work is needed to determine if systemic inflammatory markers are reduced with exercise in CF or HF fed animals to better understand the relationship between systemic metabolic inflammation and OA pathology.Figure 2Effect of a HF diet (filled bars) and long-term exercise on OA severity in 52-wk old mice. Bars sharing the same letters are not significantly different from each other (p>0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Computational identification and quantification of trabecular microarchitecture classes by 3-D texture analysis-based clustering

High resolution peripheral quantitative computed tomography (HR-pQCT) permits the non-invasive assessment of cortical and trabecular bone density, geometry, and microarchitecture. Although researchers have developed various post-processing algorithms to quantify HR-pQCT image properties, few of these techniques capture image features beyond global structure-based metrics. While 3D-texture analysis is a key approach in computer vision, it has been utilized only infrequently in HR-pQCT research. Motivated by high isotropic spatial resolution and the information density provided by HR-pQCT scans, we have developed and evaluated a post-processing algorithm that quantifies microarchitecture characteristics via texture features in HR-pQCT scans. During a training phase in which clustering was applied to texture features extracted from each voxel of trabecular bone, three distinct clusters, or trabecular microarchitecture classes (TMACs) were identified. These TMACs represent trabecular bone regions with common texture characteristics. The TMACs were then used to automatically segment the voxels of new data into three regions corresponding to the trained cluster features. Regional trabecular bone texture was described by the histogram of relative trabecular bone volume covered by each cluster. We evaluated the intra-scanner and inter-scanner reproducibility by assessing the precision errors (PE), intra class correlation coefficients (ICC) and Dice coefficients (DC) of the method on 14 ultradistal radius samples scanned on two HR-pQCT systems. DC showed good reproducibility in intra-scanner set-up with a mean of 0.870±0.027 (no unit). Even in the inter-scanner set-up the ICC showed high reproducibility, ranging from 0.814 to 0.964. In a preliminary clinical test application, the TMAC histograms appear to be a good indicator, when differentiating between postmenopausal women with (n=18) and without (n=18) prevalent fragility fractures. In conclusion, we could demonstrate that 3D-texture analysis and feature clustering seems to be a promising new HR-pQCT post-processing tool with good reproducibility, even between two different scanners.

Diabetes-related impairment in bone strength is established early in the life course

To evaluate properties of bone quantity/quality using young non-obese Type 1 (T1D)-diabetic (NOD) prone and syngenic non-diabetic (NOD.scid) mice. Quantitative bone assessment of tibia was conducted using dual-energy X-ray absorptiometry (DXA) for the evaluation of body mass, bone mineral content, body fat mass and lean mass. Qualitative assessment was accomplished by three-point breakage for assessment of force to failure and micro-computed tomography for evaluation of trabecular and cortical properties of bone. In addition, fasting blood was evaluated prior to sacrifice at week eleven and fifteen to evaluate and compare glucose homeostasis between the strains of mice. Our findings support a perturbation in the relationship between bone quantity, quality, and subsequently, the association between structure and strength. There were no differences in DXA-assessed body composition (body fat, % fat mass and lean mass) and bone composition (bone mineral content and bone mineral density) between strains. However, relative to NOD.scid, NOD mice had lower trabecular bone volume, relative trabecular bone volume, trabecular number and trabecular total material density (P < 0.05). Conversely, NOD mice had greater cortical total mean volume (P < 0.05). General linear models analysis adjusted for body weight revealed a significant contribution of T1D to bone health as early as 5 wk. It is well-established that diabetes is a significant risk factor for increased fractures, although the underlying mechanisms are not fully understood. Investigation of bone parameters encompassing strength and structure early in the life course will facilitate the elucidation of the pathogenesis of impaired bone integrity.