Cortical Bone Area Research Articles

The role of Zfp467 in mediating the pro-osteogenic and anti-adipogenic effects on bone and bone marrow niche.

Conditional deletion of the PTH receptor (Pth1r) in mesenchymal progenitors reduces osteoblast differentiation and bone mass while enhancing adipogenesis and bone marrow adipose tissue. Mechanistically, PTH suppresses the expression of Zfp467, a pro-adipogenic zinc finger transcription factor. Consequently, Pth1r deficiency in mesenchymal progenitors leads to increased Zfp467 expression. Based on these observations, we hypothesized that genetic loss of Zfp467 would lead to a shift in marrow progenitor cell fate towards osteogenesis and increased bone mass. To test this hypothesis, we generated Zfp467-/- mice. Zfp467-/- mice (-/-) were significantly smaller than Zfp467+/+ mice (+/+). μCT showed significantly higher trabecular bone and cortical bone area in -/- vs. +/+, and histomorphometry showed higher structural and dynamic formation parameters in -/- mice vs. +/+. Femoral gene expression including Alpl, Sp7, and Acp5 were increased in -/-mice, whereas Adiponectin, Cebpa, Lepr, and Ppraγ mRNA were lower in -/- mice. Similarly, Fabp4 and Lep in the inguinal depot were also decreased in -/- mice. Moreover, marrow adipocyte numbers were reduced in -/- vs +/+ mice (p<0.007). In vitro, COBs and BMSCs-/- showed more positive ALP and Alizarin Red staining and a decrease in ORO droplets. Pth1r mRNA and protein levels were increased in COBs and BMSCs from -/- mice vs +/+ (p<0.02 for each parameter, -/- vs. +/+). -/- cells also exhibited enhanced endogenous levels of cAMP vs. control cells. Moreover, in an ovariectomy (OVX) mouse model, Zfp467-/- mice had significantly lower fat mass but similar bone mass compared to OVX +/+ mice. In contrast, in a high fat diet (HFD) mouse model, in addition to reduced adipocyte volume and adipogenesis related gene expression in both peripheral and bone marrow fat tissue, greater osteoblast number and higher osteogenesis related gene expression were also observed in -/- HFD mice vs. +/+ HFD mice. Taken together, these results demonstrate that ZFP467 negatively influences skeletal homeostasis and favors adipogenesis. Global deletion of Zfp467 increases PTHR1, cAMP and bone turnover, hence its repression is a component of PTH signaling and its regulation. These data support a critical role for Zfp467 in early lineage allocation and provide a novel potential mechanism by which PTH acts in an anabolic manner on the bone remodeling unit.

Quantitative Computed Tomography Assessment of Bone Deficits in Ambulatory Children and Adolescents with Spina Bifida: Importance of Puberty.

ABSTRACTPathologic fractures of the femur and tibia are common in youth with spina bifida (SB). These fractures may be associated with deficient bone accrual due to decreased ambulation and skeletal loading. This prospective cohort study used quantitative computed tomography (QCT) to assess three‐dimensional (3D) bone properties in children and adolescents with SB. Eighty‐three ambulatory youth with SB underwent QCT imaging of the tibia at up to four annual visits between ages 6 to 16 years (294 total visits averaging 3.5 visits/patient). A total of 177 controls without disability and 10 non‐ambulatory youth with SB underwent imaging once. Bone geometric properties (cortical bone area, cross‐sectional area, cortical thickness, cortical density, and moments of inertia) were measured at the mid‐diaphysis (50% of bone length); cross‐sectional area, cancellous density, and density‐weighted area were measured in the proximal (13% of bone length) and distal (90% of bone length) metaphyses. Bone properties were compared between the ambulatory SB and control participants, among SB neurosegmental subgroups (sacral, low lumbar, mid lumbar and above) as a function of pubertal stage (prepubertal, pubertal, postpubertal), and considering SB type (myelomeningocele, lipomyelomeningocele) using linear mixed effects models adjusted for sex, age, height percentile, and body mass index (BMI) percentile. Only cancellous density of both metaphyses and weighted area of the proximal metaphysis differed between ambulatory children with SB and controls before puberty. However, significant deficits in all bone properties manifested during and after puberty as moderate bone growth in the SB group failed to keep pace with the large increases normally observed during puberty. The bone deficits primarily affected patients with myelomeningocele, and similar deficits were observed at all neurosegmental levels except that cancellous density was closer to normal in the sacral group. Descriptive analysis of the 10 non‐ambulatory youth with SB showed greater bone deficits than ambulatory children, particularly for cancellous density in the distal metaphysis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Combinations of trabecular and cortical bone properties distinguish various loading modalities between athletes and controls.

Variation in trabecular and cortical bone properties is often used to infer habitual behavior in the past. However, the structures of both types of bone are rarely considered together and may even contradict each other in functional interpretations. We examine trabecular and cortical bone properties in various athletes and sedentary controls to clarify the associations between combinations of cortical and trabecular bone properties and various loading modalities. We compare trabecular and cortical bone properties using peripheral quantitative computed tomography scans of the tibia between groups of 83 male athletes (running, hockey, swimming, cricket) and sedentary controls using Bayesian multilevel models. We quantify midshaft cortical bone rigidity and area (J, CA), midshaft shape index (Imax/Imin), and mean trabecular bone mineral density (BMD) in the distal tibia. All groups show unique combinations of biomechanical properties. Cortical bone rigidity is high in sports that involve impact loading (cricket, running, hockey) and low in nonimpact loaded swimmers and controls. Runners have more anteroposteriorly elliptical midshafts compared to other groups. Interestingly, all athletes have greater trabecular BMD compared to controls, but do not differ credibly among each other. Results suggest that cortical midshaft hypertrophy is associated with impact loading while trabecular BMD is positively associated with both impact and nonimpact loading. Midshaft shape is associated with directionality of loading. Individuals from the different categories overlap substantially, but group means differ credibly, suggesting that nuanced group-level inferences of habitual behavior are possible when combinations of trabecular and cortical bone are analyzed.

MicroCT for Scanning and Analysis of Mouse Bones.

The purpose of this Chapter is to present a detailed description of methods for performing bone Micro-Computed Tomography (microCT) scanning and analysis. MicroCT is an x-ray imaging method capable of visualizing bone at the micro-structural scale, that is, 1-100 µm resolution. MicroCT is the gold-standard method for assessment of 3D bone morphology in studies of small animals. As applied to the small bones of mice or rats, microCT can efficiently and accurately assess bone structure (e.g., cortical bone area [Ct.Ar]) and micro-structure (e.g., trabecular bone volume fraction [Tb.BV/TV]). The particular application described herein is for post mortem mouse femur specimens. The material presented should be generally applicable to many commercially available laboratory microCT systems, although some details are specific to the system used in our lab (Scanco mCT 40; SCANCO Medical AG, Bruttisellen, Switzerland).

FN1 promotes chondrocyte differentiation and collagen production via TGF-β/PI3K/Akt pathway in mice with femoral fracture

Fibronectin (FN) functions as a potent stimulator of osteogenic differentiation, and bone fracture healing. In FN family, FN1 acts as an interactive protein gene product to mediate chondrocyte adhesion. However, its effect on fracture healing remains elusive. Therefore, we aimed to investigate the involvement of FN1 in fracture healing. Hard callus formations were found at fracture site with thicker periosteum in lateral cortical bone area outside the fracture site in model mice. The decreased number of osteogenic cells in the middle of the callus region and increased extracellular matrix were suggestive of successful induction. Immunoblotting and RT-qPCR revealed that expression of FN1 was increased in tissues of fracture mice. As displayed by Safranin-fast green staining hematoxylin-eosin staining, the overexpression of FN1 at fracture site promoted osteoid formation and chondrocyte differentiation. The stimulating role of FN1 in collagen production was evidenced by increased levels of Col2, Col1, ColX, Osteonectin, and Osteocalcin and enhanced BMD, BV, BV/TV and Tb.Th values verified by immunoblotting and immunohistochemical staining. Additionally, the upregulation of FN1 contributed to promoted TGF-β, c-Caspase-9/t-Caspase-9 ratio and NF-κB p65 protein expression as well as lowered p-PI3K/PI3K and p-AKT/AKT ratios, implying the positive correlation between FN1 and the TGF-β/PI3K/Akt signaling pathway. The key findings of the present study provided evidence indicating that overexpression of FN1 contributes to fracture healing by activation of the TGF-β/PI3K/Akt signaling pathway.

Osteoprotective effect of green tea polyphenols and annatto-extracted tocotrienol in obese mice is associated with enhanced microbiome vitamin K2 biosynthetic pathways

The role of the gut microbiome in bone health has received significant attention in the past decade. We investigated the effects of green tea polyphenols (GTP) and annatto-extracted tocotrienols (AT) on bone properties and gut microbiome in obese mice. Male mice were assigned to a two (no AT vs. 400 mg/kg diet AT) × two (no GTP vs. 0.5% w/v GTP) factorial design, namely control, G, T, and G+T group respectively, for 14 weeks. The 4th lumbar vertebra (LV-4) and femur were harvested for bone microstructural analysis using μ-CT. Microbiome analysis using 16S rRNA gene sequencing of cecal feces was performed. AT increased bone volume at distal femur. GTP increased serum procollagen type 1 N-terminal propeptide concentration, bone volume at the distal femur and the LV-4, and trabecular number at distal femur; whereas GTP decreased trabecular separation at distal femur. Interactions between GTP and AT were observed in serum C-terminal telopeptide of type I collagen level (control>G=T=G+T) as well as the cortical bone area (control<G=T=G+T) and thickness (T≥G+T≥G≥control) at femur mid-diaphysis. Redundancy analysis showed a significant difference in the gut microbiome profile among different groups and the relative abundance of Akkermansia muciniphila, Clostridum saccharogumia, and Subdoligranulum variabile was increased in the GTP- and AT-supplemented groups. Functional profiling of the gut microbiome showed the combination of GTP and AT induced biosynthetic pathways for vitamin K2. Our results suggest that GTP and AT supplementation benefits bone properties in obese mice through modifying gut microbiome composition and function.

Abstract P140: Myeloid Activation in Hypertension and Effects on Bone: A New End Organ?

Hypertension affects approximately one-half of the adult population. Another common disease is osteoporosis, which affects 53 million people in the United States. Clinical studies have shown an association between hypertension and bone loss. We hypothesized that experimental hypertension would be associated with osteoporosis. In Angiotensin (Ang) II-induced hypertension, there is an increase in hematopoietic stem cells (HSCs) expressing the master myeloid transcription factor PU.1 (2.99 ± 0.92) vs. sham (0.17 ± 0.04,p=0.007). Here we reported an increase an osteoclasts number per mm 2 by Tartrate-resistant acid phosphatase staining in DOCA-salt hypertension (27.4 ± 2.3) vs. sham (12.09 ± 1.7, p=0.0002). Using microCT analysis of femoral bone, we found that Ang II-induced hypertension caused striking bone loss as reflected by decreases in cortical bone area, cortical thickness and trabecular number (see Table). We also examined bone strength using a three-point biomechanical testing. In keeping with the micro-CT data, both forms of hypertension were associated with increased bone fragility, reflected by several parameters including maximum force to failure and rigidity (see Table). Recent studies have implicated T cells in the genesis of osteoporosis, and in keeping with this, we found almost two-fold increase in both CD4 + and CD8 + T cells in the marrow of mice with DOCA-salt hypertension. Thus, in two models of experimental hypertension there is marked bone loss and the development of bone fragility, potentially related to T cell accumulation and cytokine release. These findings might explain the association between osteoporosis and hypertension.

Total urinary polyphenols and longitudinal changes of bone properties. The InCHIANTI study

SummaryThe aim of this study was to evaluate the association of levels of urinary total polyphenols considered as a proxy measure of polyphenol intake, with longitudinal changes of bone properties, in the InCHIANTI study. Dietary intake of polyphenols appears to be associated with future accelerated deterioration of bone health.IntroductionPolyphenols, micronutrients ingested through plant-based foods, have antioxidant and anti-inflammatory properties and may contribute to osteoporosis prevention. We evaluated associations of high levels of urinary total polyphenols (UTP), a proxy measure of polyphenol intake, with longitudinal changes of bone properties in a representative cohort of free-living participants of the InCHIANTI study.MethodsThe InCHIANTI study enrolled representative samples from the registry list of two towns in Tuscany, Italy. Baseline data were collected in 1998 and follow-up visits in 2001 and 2004. Of the 1453 participants enrolled, 956 consented to donate a 24-h urine sample used to assess UTP, had dietary assessment, a physical examination, and underwent a quantitative computerized tomography (pQCT) of the tibia. From pQCT images, we estimated markers of bone mass (BM), diaphyseal design (DD), and material quality (MQ). Mixed models were used to study the relationship between baseline tertiles of UTP with changes of the bone characteristics over the follow-up.ResultsAt baseline, higher levels of UTP were positively correlated with markers of BM, DD, and MQ. Compared with lower tertile of UTP, participants in the intermediate and highest tertiles had higher cortical bone area, cortical mineral content, and cortical thickness. However, participants in the intermediate and highest UTP tertiles experienced accelerated deterioration of these same parameters over the follow-up compared with those in the lowest UTP tertile.ConclusionsDietary intake of polyphenols estimated by UTP and dietary questionnaire was associated with long-term accelerated deterioration of bone health. Our study does not support the recommendation of increasing polyphenol intake for osteoporosis prevention.

IMPACT OF MATERNAL EXERCISE ON CORTICAL GEOMETRY AND TRABECULAR MICROARCHITECTURE IN MOUSE OFFSPRING

Exercise during gestation is safe and has many positive effects on the offspring, such as improved cardiovascular health and nervous system development. However, whether gestational exercise positively impacts skeletal development is unknown. PURPOSE: To determine whether maternal exercise throughout gestation and lactation positively impacts cortical geometry and trabecular microarchitecture of the femur in mouse male and female offspring. METHODS: In this longitudinal study, sexually mature C57BL/6 female mice were given ad lib access to a standard rodent chow. Two weeks before mating, females were randomized into two groups: voluntary wheel running treatment (EX) or sedentary control (SED). Females were mated to C57BL/6 males and continued treatment or control through both gestation and lactation. One male and one female offspring from each dam (n=7/group/sex) were selected and given ad lib access to a high-fat diet until 16 weeks of age, when final body weight was measured and femora collected. Cortical geometry of the mid-diaphysis and trabecular microarchitecture of the distal right femur were assessed via micro-computed tomography (μCT) with a voxel size of 12 μm. ANOVA or ANCOVA with final body weight (BW) as a covariate was used to determine the effects of maternal exercise on trabecular and cortical bone outcomes, respectively. RESULTS: There were no differences in BW between SED and EX male offspring. EX male offspring had smaller total area (Tt.Ar, p=0.075) and marrow area (Ma.Ar, p=0.025), and a higher cortical-to-total area ratio (Ct.Ar/Tt.Ar, p=0.017) compared to SED males. EX males had lesser trabecular thickness (p=0.049) but higher connectivity density (p=0.029) compared to SED. EX female offspring had greater BW than SED offspring (p=0.064). EX females had smaller Tt.Ar (p=0.017), Ma.Ar (p=0.033), and Ct.Ar (p=0.063) versus SED. EX female offspring had greater trabecular spacing (p=0.046) and degree of anisotropy (p=0.040), and lower trabecular number (p=0.034) compared to SED. CONCLUSION: Maternal exercise during gestation and lactation decreased cortical bone area independent of body weight and negatively impacted trabecular microarchitecture in both male and female offspring.

Interscholastic Athletics and Bone Strength: The Iowa Bone Development Study.

Pashkova, A, Hartman, JM, Letuchy, EM, and Janz, KF. Interscholastic athletics and bone strength: the Iowa bone development study. J Strength Cond Res 36(5): 1271-1276, 2022-The objective of this study was to determine the relationship between adolescents' participation in various interscholastic sports and differences in bone strength outcomes. Subjects (N = 380) were recruited from the Iowa Bone Development Study and categorized based on sport participation into 3 power groups: no-power, low-power, and high-power. Sports such as basketball, cheerleading/poms, gymnastics, volleyball, track, football, tennis, and soccer were considered high-power. Peripheral quantitative computed tomography (pQCT) was used to determine bone measures of polar stress-strain index (measure of torsion strength), cortical content (measure of cortical bone size and area at the 66% tibia site), and bone strength index (measure of compression strength based on total bone density and area at the 4% tibia site). Adjusted pairwise comparison for group least squares means high-power sport participation compared with no-power sport participation showed significant differences in all bone strength outcomes for both men and women (p value < 0.01). There was a significant difference in all bone strength measures between low-power and no-power groups for men (p value < 0.05), but not women. Because of decreasing levels of physical activity in late adolescence, the promotion of high-power sports may be particularly important for optimal bone development in the final years before peak bone mass.

E-vapor aerosols do not compromise bone integrity relative to cigarette smoke after 6-month inhalation in an ApoE\u2013/\u2013 mouse model

Cigarette smoke (CS) exposure is one of the leading risk factors for human health. Nicotine-containing inhalable products, such as e-cigarettes, can effectively support tobacco harm reduction approaches. However, there are limited comparative data on the effects of the aerosols generated from electronic vapor products (e-vapor) and CS on bone. Here, we report the effects of e-vapor aerosols and CS on bone morphology, structure, and strength in a 6-month inhalation study. Eight-week-old ApoE–/– mice were exposed to aerosols from three different e-vapor formulations—CARRIER (propylene glycol and vegetable glycerol), BASE (CARRIER and nicotine), TEST (BASE and flavor)—to CS from 3R4F reference cigarettes at matched nicotine concentrations (35 µg/L) or to fresh air (Sham) (N = 10 per group). Tibiae were analyzed for bone morphology by µCT imaging, biomechanics by three-point bending, and by histological analysis. CS inhalation caused a significant decrease in cortical and total bone volume fraction and bone density relative to e-vapor aerosols. Additionally, CS exposure caused a decrease in ultimate load and stiffness. In contrast, bone structural and biomechanical parameters were not significantly affected by e-vapor aerosol or Sham exposure. At the dissection time point, there was no significant difference in body weight or tibia bone weight or length among the groups. Histological findings revealed microcracks in cortical bone areas among all exposed groups compared to Sham control. In conclusion, because of the bone-preserving effect of e-vapor aerosols relative to CS exposure, e-vapor products could potentially constitute less harmful alternatives to cigarettes in situations in which bone health is of importance.

Oxytocin and bone quality in the femoral neck of rats in periestropause

The objective of this study is to identify whether oxytocin (OT) contributes to the reduction of osteopenia in the femoral neck of rats in periestropause. Animals in irregular estrous cycles received two NaCl injections (0.15 mol/L) or OT (134 μg/kg) over a 12-h interval, and after thirty-five days without treatments, the biological sample collection was performed. The oxytocin group (Ot) demonstrated the highest enzymatic activity of alkaline phosphatase (p = 0.0138), lowest enzymatic activity of tartrate-resistant acid phosphatase (p = 0.0045), higher percentage of compact bone (p = 0.0359), cortical expression of runt-related transcription factor 2 (p = 0.0101), osterix (p = 0.0101), bone morphogenetic protein-2/4 (p = 0.0101) and periostin (p = 0.0455). Furthermore, the mineral-to-matrix ratio (ν1PO4/Proline) was higher and type-B carbonate substitution (CO3/ν1PO4) was lower (p = 0.0008 and 0.0303) in Ot group. The Ot showed higher areal bone mineral density (p = 0.0050), cortical bone area (p = 0.0416), polar moment of inertia, maximum, minimum (p = 0.0480, 0.0480, 0.0035), bone volume fraction (p = 0.0166), connectivity density (p < 0.0001), maximal load (p = 0.0003) and bone stiffness (p = 0.0145). In Ot percentage of cortical pores (p = 0.0102) and trabecular number (p = 0.0088) was lower. The results evidence action of OT in the reduction of osteopenia, suggesting that it is a promising anabolic strategy for the prevention of primary osteoporosis during the periestropause period.

Age-related and sex-specific effects on architectural properties and biomechanical response of the C57BL/6N mouse femur, tibia and ulna

Aging is known to reduce bone quality and bone strength. We sought to determine how aging affects the biomechanical and architectural properties of various long bones, and if sex influences age related differences/changes. While researchers have extensively studied these changes in individual bones of mice, there is no comprehensive study of the changes in the bones from the same mice to study the changes with aging. We performed three point bending tests and microcomputed tomography (microCT) analysis on femurs, tibiae and ulnae. Three point bending tests were utilized to calculate biomechanical parameters and imaging was also performed using high resolution microCT to reveal both cortical and trabecular microarchitecture C57BL/6N mice were divided into three age groups: 6, 12 and 22 months. Each age and sex group consisted of 6–7 mice. The ultimate load to failure (UL), elastic stiffness (ES), modulus of elasticity (E) and the moment of inertia about bending axis (MOI) for each bone was calculated using three point bending test. MicroCT scans of all the bones were analyzed to determine cortical bone volume per tissue volume (C.BV/TV), trabecular bone volume per tissue volume (Tb.BV/TV), cortical bone area (B.Ar) using CTAn's microCT analysis and tested for correlation with the biomechanical parameters. Mean (standard error) values of UL in femur decreased from 19.8(0.6) N to 12.8(1.1) N (p < .01) and 17.9(0.6) N to 14.6(1.0) N (p = .02) from 6 to 22 months groups in males and females respectively. Similarly, UL in tibia decreased from 19.8(0.5) N to 14.3(0.2) N (p < .01) and 14.4(0.6) N to 9.5(1.0) N (p < .01) from 6 to 22 months group in males and females respectively. ES in femur decreased from 113.2(7) N/mm to 69.6(6.7) N/mm (p < .01) from 6 to 22 months in males only. ES in tibia decreased from 78.6(3.2) N/mm to 65.0(2.3) N/mm (p = .01) and 53.1(2.9) N/mm to 44.0(1.7) N/mm (p = .02) from 6 to 22 months in males and females respectively. Interestingly, ES in ulna increased from 8.2(0.8) N/mm to 10.9(1.0) N/mm (p = .051) from 6 to 22 months of age in females only. E in femur decreased from 4.0(0.4) GPa to 2.8(0.2) GPa (p = .01) and 6.7(0.5) GPa to 4.5(0.4) GPa (p = .01) from 6 to 22 months of age in males and females respectively while tibia showed no change. However, E in ulna increased from 7.0(0.8) GPa to 11.0(1.1) GPa (p = .01) from 6 to 22 months of age in females only. Changes in age and sex-related bone properties were more pronounced in the femur and tibia, while the ulna showed fewer overall differences. Most of the changes were observed in biomechanical compared to architectural properties and female bones are more severely affected by aging. In conclusion, our data demonstrate that care must be taken to describe bone site and sex-specific, rather than making broad generalizations when describing age-related changes on the biomechanical and architectural properties of the skeleton.

Chronic Ethanol Feeding in Mice Decreases Expression of Genes for Major Structural Bone Proteins in a Nox4-Independent Manner.

Bone loss in response to alcohol intake has previously been hypothesized to be mediated by excessive production of reactive oxygen species via NADPH oxidase (Nox) enzymes. Nox4 is one of several Nox enzymes expressed in bone. We investigated the role of Nox4 in the chondro-osteoblastic lineage of the long bones in mice during normal chow feeding and during chronic ethanol feeding for 90 days. We generated mice with a genotype (PrxCre +/- Nox4 fl/fl) allowing conditional knockout of Nox4 in the limb bud mesenchyme. Adult mice had 95% knockdown of Nox4 expression in the femoral shafts. For mice on regular chow, only whole-body Nox4 knockout mice had clearly increased cortical thickness and bone mineral density in the tibiae. When chronically fed a liquid diet with and without ethanol, conditional Nox4 knockout mice had slightly reduced dimensions of the cortical and trabecular regions of the tibiae (P < 0.1). The ethanol diet caused a significant reduction in cortical bone area and cortical thickness relative to a control diet without ethanol (P < 0.05). The ethanol diet further reduced gene expression of Frizzled related protein (Frzb), myosin heavy chain 3, and several genes encoding collagen and other major structural bone proteins (P < 0.05), whereas the Nox4 genotype had no effects on these genes. In conclusion, Nox4 expression from both mesenchymal and nonmesenchymal cell lineages appears to exert subtle effects on bone. However, chronic ethanol feeding reduces cortical bone mass and cortical gene expression of major structural bone proteins in a Nox4-independent manner. SIGNIFICANCE STATEMENT: Excessive alcohol intake contributes to osteopenia and osteoporosis, with oxidative stress caused by the activity of NADPH oxidases hypothesized to be a mediator. We tested the role of NADPH oxidase (Nox) 4 in osteoblast precursors in the long bones of mice with a conditional Nox4 knockout model. We found that Nox4 exerted effects independent of alcohol intake, and ethanol effects on bone were Nox4-independent.

Effects of Rambutan Peel (Nepheliumlappaceum) PhenolicExtract on RANKL-Induced Differentiation of RAW264.7 Cells into Osteoclasts and Retinoic Acid-Induced Osteoporosis in Rats

Previous studies have shown that rambutan peel phenolic (RPP) extract has excellent biological activities due to its abundant phenolic content and profile. In this study, the potential anti-osteoporosis (OP) effects of RPP were evaluated by suppressing receptor activator nuclear factor-kappa B ligand (RANKL)-induced differentiation of RAW264.7 cells into osteoclasts and amelioratingretinoic acid-induced OP in rats. Our results showed that RPP efficiently decreased the formation of tartrate-resistant acid phosphatase (TRAP)-positive cells and reduced total TRAP activity in RAW264.7 cells under RANKL stimulation. RPP treatment significantlyameliorated retinoid acid-induced calcium loss in rats (p < 0.05). The serum phosphorus level of osteoporotic rats was increased by RPP treatment, and the serum levels of total alkaline phosphatase and osteocalcin in osteoporotic rats were further reduced. RPP treatment improved the qualities of the femur and tibia, such asbone mineral density, bone length, bone maximum load, cortical bone area ratio, and trabecularelative bone density in osteoporotic rats to some extent. Furthermore, histological analysis showed that RPP effectively improved the bone microstructure of osteoporotic rats by regulating the cortical bone thickness and trabecular bone separation. These results indicate that RPP could have potential applications as a newnutraceutical and functional food in the prevention of OP.

Finite-Element analysis of a lateral femoro-tibial impact on the total knee arthroplasty

Background and ObjectiveTotal knee arthroplasty (TKA) is a routine surgery performed to treat patients with severe knee osteoarthritis. The success of a TKA depends strongly on the initial stability of the prosthetic components and its long-term osseointegration due to the optimal distribution of mechanical stresses in the surrounding bones under the effect of the different biomechanical loads applied to the Femur-TKA-Tibia system. The purpose of this study is to analyze the level and the distribution of the induced stresses in a Femur-TKA-Tibia system subjected to combined triaxial forces, which mimic a femoral mechanical shock. MethodsIn this study, complex TKA system implanted in both femoral and tibial bones has been analyzed numerically with a three-dimensional finite-element method. A virtual model is designed to examine in silico the effect of the combined triaxial forces acting on this prosthesis in femoral region. Anatomical three-dimensional finite-element models of both femoral and tibial bones were constructed to calculate the interfacial stresses around the TKA components. The 3D finite-element processing program ABAQUS was used to perform the analysis. ResultsThe stresses propagated in the bone regions adjacent to the TKA osseointegrated components, and the decreased in their magnitude to the outer region. These stresses reached the highest level in the cortical bone areas that are right next to the proximal upper attachment portions of the TKA osseointegrated components. The magnitude of the stresses in the tibial component is higher than that in the femoral component. Finally, it is very important to emphasize the role of the polyethylene articulating spacer in the shock absorption of bone support sections. Thus, this component should be preserved mechanically from the impact of high shocks in order to maintain healthy TKA systems. ConclusionsOptimizing TKA model by controlling the biomechanical stresses distributed within its both components and supporting bones is a valid approach to achieving favorable long-term outcomes. The 3D finite-element analysis provides an effective pre-operative method for planning patient-specific TKA prostheses, and for designing future models that preserves the biomechanical function of the Femur-TKA-Tibia system.

The effects of estradiol are modulated in a tissue-specific manner in mice with inducible inactivation of ERα after sexual maturation.

Mouse models with lifelong inactivation of estrogen receptor-α (ERα) show that ERα is the main mediator of estrogenic effects in bone, thymus, uterus, and fat. However, ERα inactivation early in life may cause developmental effects that confound the adult phenotypes. To address the specific role of adult ERα expression for estrogenic effects in bone and other nonskeletal tissues, we established a tamoxifen-inducible ERα-inactivated model by crossing CAGG-Cre-ER and ERαflox/flox mice. Tamoxifen-induced ERα inactivation after sexual maturation substantially reduced ERα mRNA levels in cortical bone, trabecular bone, thymus, uterus, gonadal fat, and hypothalamus, in CAGG-Cre-ERαflox/flox (inducible ERαKO) compared with ERαflox/flox (control) mice. 17β-estradiol (E2) treatment increased trabecular bone volume fraction (BV/TV), cortical bone area, and uterine weight, while it reduced thymus weight and fat mass in ovariectomized control mice. The estrogenic responses were substantially reduced in inducible ERαKO mice compared with control mice on BV/TV (-67%), uterine weight (-94%), thymus weight (-70%), and gonadal fat mass (-94%). In contrast, the estrogenic response on cortical bone area was unaffected in inducible ERαKO compared with control mice. In conclusion, using an inducible ERαKO model, not confounded by lack of ERα during development, we demonstrate that ERα expression in sexually mature female mice is required for normal E2 responses in most, but not all, tissues. The finding that cortical, but not trabecular bone, responds normally to E2 treatment in inducible ERαKO mice strengthens the idea of cortical and trabecular bone being regulated by estrogen via different mechanisms.

Calcium Deficiency in Diet Decreases the Magnesium Content in Bone and Affects Femur Physicochemical Properties in Growing Rats.

This study evaluates the effect of three calcium levels in the diet (normal, moderate, and severe calcium depletion) on bone metabolism of male Wistar rats during their growth period. Bone mineral density (BMD) and femur length were determined in vivo during the growth stage using a single X-ray transmission system. The apparent calcium absorption was calculated in the rat adolescent and adulthood stages. At the end of the experiment, calcium concentrations in serum and urine were analyzed. The bones were evaluated postmortem to corroborate in vivo analyses. Microstructural properties of cortical and trabecular tissues of femurs bones were assessed using scanning electron microscopy. Bone mineral contents (Mg, Ca, P, and K) were quantified by inductively coupled plasma. Severe calcium depletion in the diets in the development stage affects the bone quality parameters such as bone mineral density and mineral content. Moreover, it was found thinner cortical and trabecular bone areas. Additionally, it was found that severe calcium depletion increased the apparent absorption of calcium as a defense mechanism, but with the decrease of the BMD peak, and the thickness of cortical bone as well as trabecular bone porosity. The severe calcium depletion increased the efficiency of apparent absorption calcium as a defense mechanism, but, even so, decreases the BMD peak as well as the thickness of cortical bone and trabecular bone porosity.

Higher fracture prevalence and smaller bone size in patients with hEDS/HSD-a prospective cohort study.

The Ehlers-Danlos syndromes (EDS) comprise a group of heritable connective tissue disorders caused by defects in the biosynthesis, secretion, and/or organization of fibrillar collagens which might impair bone strength. Our aim was to compare fracture prevalence, volumetric and areal bone mineral density (BMD), bone geometry, muscle size and the muscle-bone interaction, body composition and longitudinal changes therein between patients with hypermobile EDS (hEDS) or hypermobility spectrum disorder (HSD), and healthy control subjects. Cross-sectional data comprised 39 female hEDS/HSD patients (age 41 ± 11years) and 43 age-matched controls. After 8years, 27 hEDS/HSD and 17 control subjects were re-evaluated. Tibial trabecular and cortical volumetric BMD, bone mineral content (BMC), cortical bone geometry, and lower leg muscle cross-sectional area (CSA) were measured using pQCT. Body composition, areal BMD, and BMC were determined by DXA. At baseline, patients with hEDS/HSD presented with a smaller cortical bone area, smaller cortical thickness and muscle CSA, and a higher fracture prevalence than control subjects (all p < 0.05). No differences in areal or volumetric BMD were found. Longitudinally, muscle CSA decreased in both groups and muscle density decreased in the hEDS/HSD group (p < 0.001) whereas all bone parameters remained unchanged. hEDS/HSD patients have a cortical bone size deficit compared with controls, possibly contributing to their increased fracture risk. They presented with decreased muscle CSA but normal bone/muscle area ratio, suggesting that this bone size deficit is likely secondary to decreased mechanical loading. Further, there were no arguments for accelerated bone loss in hEDS/HSD subjects.

\u03b1SMA Osteoprogenitor Cells Contribute to the Increase in Osteoblast Numbers in Response to Mechanical Loading

Bone is a dynamic tissue that site-specifically adapts to the load that it experiences. In response to increasing load, the cortical bone area is increased, mainly through enhanced periosteal bone formation. This increase in area is associated with an increase in the number of bone-forming osteoblasts; however, the origin of the cells involved remains unclear. Alpha-smooth muscle actin (αSMA) is a marker of early osteoprogenitor cells in the periosteum, and we hypothesized that the new osteoblasts that are activated by loading could originate from αSMA-expressing cells. Therefore, we used an in vivo fate-mapping approach in an established axial loading model to investigate the role of αSMA-expressing cells in the load-induced increase in osteoblasts. Histomorphometric analysis was applied to measure the number of cells of different origin on the periosteal surface in the most load-responsive region of the mouse tibia. A single loading session failed to increase the number of periosteal αSMA-expressing cells and osteoblasts. However, in response to multiple episodes of loading, the caudal, but not the cranial, periosteal surface was lined with an increased number of osteoblasts originating from αSMA-expressing cells 5 days after the initial loading session. The proportion of osteoblasts derived from αSMA-labeled progenitors increased by 70% (p < 0.05), and the proportion of αSMA-labeled cells that had differentiated into osteoblasts was doubled. We conclude that αSMA-expressing osteoprogenitors can differentiate and contribute to the increase in periosteal osteoblasts induced by mechanical loading in a site-specific manner.