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issn:0171-967
1.  Reduced tissue-level stiffness and mineralization in osteoporotic cancellous bone 
Calcified tissue international  2014;95(2):125-131.
Osteoporosis alters bone mass and composition ultimately increasing the fragility of primarily cancellous skeletal sites; however, effects of osteoporosis on tissue-level mechanical properties of cancellous bone are unknown. Dual-energy x-ray absorptiometry (DXA) scans are the clinical standard for diagnosing osteoporosis though changes in cancellous bone mass and mineralization are difficult to separate using this method. The goal of this study was to investigate possible difference in tissue-level properties with osteoporosis as defined by donor T-scores. Spine segments from Caucasian female cadavers (58–92 yrs) were used. A T-score for each donor was calculated from DXA scans to determine osteoporotic status. Tissue level composition and mechanical properties of vertebrae adjacent to the scan region were measured using nanoindentation and Raman spectroscopy. Based on T-scores, six samples were in the Osteoporotic group (58–74 yrs) and four samples were in the Not Osteoporotic group (65–92 yrs). The indentation modulus and mineral to matrix ratio (mineral:matrix) were lower in the Osteoporotic group than the Not Osteoporotic group. Mineral:matrix ratio decreased with age (r2 = 0.35, p = 0.05), and the indentation modulus increased with a real bone mineral density (aBMD) (r2 = 0.41, p = 0.04).
This study is the first to examine cancellous bone composition and mechanical properties from a fracture prone location with osteoporosis. We found differences in tissue composition and mechanical properties with osteoporosis that could contribute to increased fragility in addition to changes in trabecular architecture and bone volume.
doi:10.1007/s00223-014-9873-4
PMCID: PMC4104238  PMID: 24888692
Nanoindentation; Raman spectroscopy; Osteoporosis; Human trabecular bone
2.  Deficiency in Perlecan/HSPG2 during Bone Development Enhances Osteogenesis and Decreases Quality of Adult Bone in Mice 
Calcified tissue international  2014;95(1):29-38.
Perlecan/Hspg2 (Pln) is a large heparan sulfate proteoglycan abundant in the extracellular matrix of cartilage and the lacuno-canalicular space of adult bones. While Pln function during cartilage development is critical, evidenced by deficiency disorders including Schwartz-Jampel Syndrome and dyssegmental dysplasia Silverman-Handmaker type, little is known about its function in development of bone shape and quality. The purpose of this study was to understand the contribution of Pln to bone geometric and mechanical properties. We used hypomorph mutant mice that secrete negligible amount of Pln into skeletal tissues and analyzed their adult bone properties using micro-computed tomography and three-point-bending tests. Bone shortening and widening in Pln mutants was observed and could be attributed to loss of growth plate organization and accelerated osteogenesis that was reflected by elevated cortical thickness at older ages. This effect was more pronounced in Pln mutant females indicating a gender-specific effect of Pln deficiency on bone geometry. Additionally, mutant females, and to a lesser extent mutant males, increased their elastic modulus and bone mineral densities to counteract changes in bone shape, but at the expense of increased brittleness. In summary, Pln deficiency alters cartilage matrix patterning and, as we now show, coordinately influences bone formation and calcification.
doi:10.1007/s00223-014-9859-2
PMCID: PMC4137566  PMID: 24798737
Perlecan; Heparan Sulfate; Proteoglycan; Bone Quality; Schwartz-Jampel Syndrome; dyssegmental dysplasia Silverman-Handmaker
3.  Measuring Differences in Compositional Properties of Bone Tissue by Confocal Raman Spectroscopy 
Calcified tissue international  2011;89(2):111-122.
The full range of fracture risk determinants arise from each hierarchical level comprising the organization of bone. Raman spectroscopy is one tool capable of characterizing the collagen and mineral phases at a near sub-micron length scale, but the ability of Raman spectra to distinguish compositional differences of bone is not well defined. Therefore, we analyzed multiple Raman peak intensities and peak ratios to characterize their ability to distinguish between the typically less mineralized osteonal tissue and the more mineralized interstitial tissue in intra-cortical human bone. To further assess origins of variance, we collected Raman spectra from embedded specimens and for 2 orientations of cut. Per specimen, Raman peak intensities or ratios were averaged among multiple sites within 5 osteons and 5 neighboring interstitial tissue. The peak ratios of ν1 phosphate (PO4) to Proline or Amide III detected the highest increases of 15.4% or 12.5%, respectively, in composition from osteonal to interstitial tissue. The coefficient of variance (COV) was less than 5% for each as opposed to a COV of ∼8% for the traditional ν1PO4/Amide I, a peak ratio that varied the most between transverse and longitudinal cuts for each tissue type. Although embedding affected Raman peaks, it did not obscure differences in most peak ratios related to mineralization between the 2 tissue types. In studies with limited sample size but sufficient number of Raman spectra per specimen for spatial averaging, ν1PO4/Amide III or ν1PO4/Proline is the Raman property that is most likely to detect a compositional difference between experimental groups.
doi:10.1007/s00223-011-9497-x
PMCID: PMC4471954  PMID: 21597909
Raman; Embedding; Cortical Bone; Collagen; Mineral
4.  Osteoporosis screening and treatment among Veterans with recent fracture after implementation of an electronic consult service 
Calcified tissue international  2014;94(6):659-664.
Purpose
Less than 24 percent of Veterans received appropriate evaluation and/or treatment for osteoporosis within 6 months of an index fracture. An electronic consult (E-consult) service was implemented at 3 Veterans Affairs Medical Centers to facilitate identification of and to recommend management for patients with recent fracture.
Method
The E-consult service used clinical encounter data based on ICD9 diagnosis codes to prospectively identify patients with potential osteoporotic fractures. Eligible patients' medical records were reviewed by a metabolic bone specialist and an E-consult note was sent to the patient's primary provider with specific recommendations for further management. Recommendations were initiated at the provider's discretion.
Results
Between 2011 and 2013, the E-consult service identified 444 eligible patients with a low-trauma fracture who were not already on treatment. One hundred twenty-nine (29.1%) consults recommended immediate bisphosphonate treatment and 258 (58.1%) recommended bone density assessments. Primary providers responded by prescribing bisphosphonates in 74 patients (57.4%) and by ordering bone density testing in 183 (70.9%) patients. At the facility level, prior to implementation of the E-consult service, the rate of osteoporosis treatment following a fracture was 4.8% for bisphosphonates and 21.3% for calcium/vitamin D. After implementation, the treatment rate increased to 7.3% for bisphosphonates (P = 0.02) and 35.2% for calcium/vitamin D (P < 0.01).
Conclusion
While feasible and relatively low cost, an E-consult service modestly improved the rate of osteoporosis treatment among patients with a recent fracture. These results suggest that a program with direct patient interaction is probably required to substantially improve treatment rates.
doi:10.1007/s00223-014-9849-4
PMCID: PMC4058771  PMID: 24699797
Osteoporosis; fracture; electronic medical record; bone density; health services research
5.  Type III collagen regulates osteoblastogenesis and the quantity of trabecular bone 
Calcified tissue international  2014;94(6):621-631.
Type III Collagen (Col3), a fibril-forming collagen, is a major extracellular matrix component in a variety of internal organs and skin. It is also expressed at high levels during embryonic skeletal development and is expressed by osteoblasts in mature bone. Loss of function mutations in the gene encoding Col3 (Col3a1) are associated with vascular Ehlers Danlos Syndrome (EDS). Although the most significant clinical consequences of this syndrome are associated with catastrophic failure and impaired healing of soft tissue structures, several studies have documented skeletal abnormalities in vascular EDS patients. However, there are no reports of the role of Col3 deficiency on the murine skeleton. We compared craniofacial and skeletal phenotypes in young (6-8 weeks) and middle-aged (>1 year) control (Col3+/+) and haploinsufficient (Col3+/−) mice, as well as young null (Col3−/−) mice using microcomputed tomography (μCT). Although Col3+/− mice did not have significant craniofacial abnormalities based upon cranial morphometrics, microCT analysis of distal femur trabecular bone demonstrated significant reductions in bone volume (BV), bone fraction volume (BV/TV), connectivity density (ConnD), structure model index (SMI) and trabecular thickness (TbTh) in young adult, female Col3+/− mice relative to wild-type littermates. The reduction in BV/TV persisted in female mice at one year of age. Next we evaluated the role of Col3 in vitro. Osteogenesis assays revealed that cultures of mesenchymal progenitors harvested from Col3−/− embryos display decreased alkaline phosphatase activity and reduced capacity to undergo mineralization. Consistent with this data, a reduction in osteogenic markers (type I collagen, osteocalcin and bone sialoprotein) correlates with reduced bone Col3 expression in Col+/− mice and with age in vivo. A small but significant reduction in osteoclast numbers was found in Col3+/− compared to Col3+/+ bones. Taken together, these findings indicate that Col3 plays a role in development of trabecular bone through its effects on osteoblast differentiation.
doi:10.1007/s00223-014-9843-x
PMCID: PMC4335719  PMID: 24626604
Type III collagen; Extracellular matrix; Bone formation; Osteogenesis; Osteoporosis; Mineralization
6.  Effects of 1,25-Dihydroxyvitamin D3 and Vitamin D3 on the Expression of the Vitamin D Receptor in Human Skeletal Muscle Cells 
Calcified tissue international  2014;96(3):256-263.
Vitamin D receptor (VDR) expression and action in non-human skeletal muscle have recently been reported in several studies, yet data on the activity and expression of VDR in human muscle cells are scarce. We conducted a series of studies to examine the (1) effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on VDR gene expression in human primary myoblasts, (2) effect of 16-week supplementation with vitamin D3 on intramuscular VDR gene expression in older women, and (3) association between serum 25-hydroxyvitamin D (25OHD) and intramuscular VDR protein concentration in older adults. Human primary myoblasts were treated with increasing concentrations of 1,25(OH)2D3 for 18 h. A dose-dependent treatment effect was noted with 1 nmol/L of 1,25OH2D3 increasing intramuscular VDR mRNA expression (mean fold change ± SD 1.36 ± 0.33; P = 0.05). Muscle biopsies were obtained at baseline and 16 weeks after vitamin D3 supplementation (4,000 IU/day) in older adults. Intramuscular VDR mRNA was significantly different from placebo after 16 weeks of vitamin D3 (1.2 ± 0.99; −3.2 ± 1.7, respectively; P = 0.04). Serum 25OHD and intramuscular VDR protein expression were examined by immunoblot. 25OHD was associated with intramuscular VDR protein concentration (R = 0.67; P = 0.0028). In summary, our study found VDR gene expression increases following treatment with 1,25OH2D3 in human myoblasts. 25OHD is associated with VDR protein and 16 weeks of supplementation with vitamin D3 resulted in a persistent increase in VDR gene expression of vitamin D3 in muscle tissue biopsies. These findings suggest treatment with vitamin D compounds results in sustained increases in VDR in human skeletal muscle.
doi:10.1007/s00223-014-9932-x
PMCID: PMC4429607  PMID: 25479835
Vitamin D; Skeletal muscle; Vitamin D receptor; Human; Primary myoblasts; Aging
7.  Mechanical Loading Reduces Inflammation-Induced Human Osteocyte-to-Osteoclast Communication 
Calcified Tissue International  2015;97(2):169-178.
Multiple factors contribute to bone loss in inflammatory diseases such as rheumatoid arthritis (RA), but circulating inflammatory factors and immobilization play a crucial role. Mechanical loading prevents bone loss in the general population, but the effects of mechanical loading in patients with RA are less clear. Therefore, we aimed to investigate whether mechanical stimuli reverse the stimulatory effect of RA serum on osteocyte-to-osteoclast communication. Human primary osteocytes were pretreated with 10 % RA serum or healthy control serum for 7 days, followed by 1 h ± mechanical loading by pulsating fluid flow (PFF). Nitric oxide (NO) and prostaglandin E2 were measured in the medium. Receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), interleukin-6 (IL-6), cyclooxygenase-2 (COX2), matrix-extracellular phosphoglycoprotein (MEPE), cysteine-rich protein 61 (CYR61), and SOST gene expression was quantified by qPCR. Osteoclast precursors were cultured with PFF-conditioned medium (PFF-CM) or static-conditioned medium (stat-CM), and osteoclast formation was assessed. RA serum alone did not affect IL-6, CYR61, COX2, MEPE, or SOST gene expression in osteocytes. However, RA serum enhanced the RANKL/OPG expression ratio by 3.4-fold, while PFF nullified this effect. PFF enhanced NO production to the same extent in control serum (2.6–3.5-fold) and RA serum-pretreated (2.7–3.6-fold) osteocytes. Stat-CM from RA serum-pretreated osteocytes enhanced osteoclastogenesis compared with stat-CM from control serum-pretreated osteocytes, while PFF nullified this effect. In conclusion, RA serum, containing inflammatory factors, did not alter the intrinsic capacity of osteocytes to sense mechanical stimuli, but upregulated osteocyte-to-osteoclast communication. Mechanical loading nullified this upregulation, suggesting that mechanical stimuli could contribute to the prevention of osteoporosis in inflammatory disease.
doi:10.1007/s00223-015-9999-z
PMCID: PMC4491366  PMID: 25967362
Rheumatoid arthritis; Generalized osteoporosis; Inflammatory cytokines; Pulsating fluid flow; Osteoclastogenesis
8.  A Dual Isotope Hybrid Whole Body Micro-PET/CT System Reveals Functional Heterogeneity and Early Local and Systemic Changes Following Targeted Radiation to the Murine Caudal Skeleton 
Calcified tissue international  2014;94(5):544-552.
The purpose of this study is to develop a longitudinal non-invasive functional imaging method using a dual isotope hybrid micro-PET/CT scanner in order to assess both the skeletal metabolic heterogeneity and the effect of localized radiation that models therapeutic cancer treatment on marrow and bone metabolism. Skeletally mature BALB/c female mice were given clinically relevant local radiation (16 Gy) to the hind limbs on day 0. Micro-PET/CT acquisition was performed serially for the same mice on days -5 and +2 with FDG and days -4 and +3 with NaF. Serum levels of pro-inflammatory cytokines were measured. Significant differences (p<0.0001) in marrow metabolism (measured by FDG) and bone metabolism (measured by NaF) were observed among bones before radiation which demonstrates functional heterogeneity in the marrow and mineralized bone throughout the skeleton. Radiation significantly (p<0.0001) decreased FDG uptake but increased NaF uptake (p=0.0314) in both irradiated and non-irradiated bones at early time points. An increase in IL-6 was observed with a significant abscopal (distant) effect on marrow and bone metabolic function. Radiation significantly decreased circulating IGF-1 (p<0.01). Non-invasive longitudinal imaging with dual isotope micro-PET/CT is feasible to investigate simultaneous changes in marrow and bone metabolic function in local and distant skeletal sites in response to focused radiation injury. Distinct local and remote changes may be affected by several cytokines activated early after local radiation exposure. This approach has the potential for longer term studies to clarify the effects of radiation on marrow and bone.
doi:10.1007/s00223-014-9839-6
PMCID: PMC3987955  PMID: 24562595
translational research; radiotherapy; micro-PET/CT; bone
9.  Three-Times-Weekly Administration of Teriparatide Improves Vertebral and Peripheral Bone Density, Microarchitecture, and Mechanical Properties Without Accelerating Bone Resorption in Ovariectomized Rats 
Calcified Tissue International  2015;97(2):156-168.
Daily and weekly administration of teriparatide (PTH1–34) reduces the risk of osteoporotic bone fractures. However, their effects on markers of bone formation and bone resorption differ. These results indicate that the dosing frequency of teriparatide may affect bone metabolism and bone structure, with different effects on bone strength. In the present study, to evaluate the dose-related effects of a low administration frequency of teriparatide on bone status, we investigated the effects of three-times-weekly administration of teriparatide (1.1, 5.6, or 28.2 µg/kg) for 12 months on bone parameters, including bone metabolism markers, bone mineral density (BMD), micro-computed tomography, and bone strength, using 6-month-old ovariectomized (OVX) rats. Three-times-weekly administration of teriparatide dose-dependently increased the BMD of the lumbar vertebra and femur in OVX rats, and increased serum osteocalcin (a marker of bone formation), but not type I collagen C-telopeptide (a marker of bone resorption). The trabecular number and thickness increased in the vertebrae and femur, as in prior reports of daily teriparatide administration in OVX rats. Cortical thickness increased only toward the endocortical side of the femur, unlike with daily administration. Bone strength of the vertebrae and proximal and shaft of the femur was correlated with the changes in BMD and bone structure. These results demonstrate the effects of low frequency, intermittent administration of teriparatide on the biomechanical, and microstructural properties of bone in OVX rats.
Electronic supplementary material
The online version of this article (doi:10.1007/s00223-015-9998-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s00223-015-9998-0
PMCID: PMC4491365  PMID: 25911187
Teriparatide; Three-times-weekly; Bone metabolism; Mechanical strength; Micro-computed tomography
10.  Application of Structural Rigidity Analysis to Assess Fidelity of Healed Fractures in Rat Femurs with Critical Defects 
Calcified tissue international  2010;86(5):397-403.
Approximately 6 million fractures occur each year in the United States, with an estimated medical and loss of productivity cost of $99 billion. As our population ages, it can only be expected that these numbers will continue to rise. While there have been recent advances in available treatments for fractures, assessment of the healing process remains a subjective process. This study aims to demonstrate the use of micro-computed tomography (μCT)-based structural rigidity analysis to accurately and quantitatively assess the progression of fracture healing over time in a rat model. The femora of rats with simulated lytic defects were injected with human BMP-2 cDNA at various time points postinjury (t = 0, 1, 5, 10 days) to accelerate fracture healing, harvested 56 days from time of injury, and subjected to μCT imaging to obtain cross-sectional data that were used to compute torsional rigidity. The specimens then underwent torsional testing to failure using a previously described pure torsional testing system. Strong correlations were found between measured torsional rigidity and computed torsional rigidity as calculated from both average (R2 = 0.63) and minimum (R2 = 0.81) structural rigidity data. While both methods were well correlated across the entire data range, minimum torsional rigidity was a better descriptor of bone strength, as seen by a higher Pearson coefficient and smaller y-intercept. These findings suggest considerable promise in the use of structural rigidity analysis of μCT data to accurately and quantitatively measure fracture-healing progression.
doi:10.1007/s00223-010-9353-4
PMCID: PMC4405879  PMID: 20354683
Fracture healing; Healing strength; Structural rigidity analysis; Segmental defect; Rat model
11.  Bone Fracture Risk is Not Associated with the Use of Glucagon-Like Peptide-1 Receptor Agonists: A Population-Based Cohort Analysis 
Calcified Tissue International  2015;97(2):104-112.
Glucagon-like Peptide-1 receptor agonists (GLP1-ra) are a relatively new class of anti-hyperglycemic drugs which may positively affect bone metabolism and thereby decrease (osteoporotic) bone fracture risk. Data on the effect of GLP1-ra on fracture risk are scarce and limited to clinical trial data only. The aim of this study was to investigate, in a population-based cohort, the association between the use of GLP1-ra and bone fracture risk. We conducted a population-based cohort study, with the use of data from the Clinical Practice Research Datalink (CPRD) database (2007–2012). The study population (N = 216,816) consisted of all individuals with type 2 diabetes patients with at least one prescription for a non-insulin anti-diabetic drug and were over 18 years of age. Cox proportional hazards models were used to estimate the hazard ratio of fracture in GLP1-ra users versus never-GLP1-ra users. Time-dependent adjustments were made for age, sex, lifestyle, comorbidity and the use of other drugs. There was no decreased risk of fracture with current use of GLP1-ra compared to never-GLP1-ra use (adjusted HR 0.99, 95 % CI 0.82–1.19). Osteoporotic fracture risk was also not decreased by current GLP1-ra use (adjusted HR 0.97; 95 % CI 0.72–1.32). In addition, stratification according to cumulative dose did not show a decreased bone fracture risk with increasing cumulative GLP1-ra dose. We showed in a population-based cohort study that GLP1-ra use is not associated with a decreased bone fracture risk compared to users of other anti-hyperglycemic drugs. Future research is needed to elucidate the potential working mechanisms of GLP1-ra on bone.
doi:10.1007/s00223-015-9993-5
PMCID: PMC4491344  PMID: 25894068
GLP1-ra; Fracture; Diabetes mellitus type 2; Cohort study; CPRD
12.  Combined effects of botulinum toxin injection and hindlimb unloading on bone and muscle 
Calcified tissue international  2013;94(3):327-337.
Bone receives mechanical stimulation from two primary sources, muscle contractions and external gravitational loading, but the relative contribution of each source to skeletal health is not fully understood. Understanding the most effective loading for maintaining bone health has important clinical implications for prescribing physical activity for the treatment or prevention of osteoporosis. Therefore, we investigated the relative effects of muscle paralysis and reduced gravitational loading on changes in muscle mass, bone mineral density and microarchitecture. Adult female C57Bl/6J mice (n=10/group) underwent one of the following: unilateral botulinum toxin (BTX) injection of the hindlimb, hindlimb unloading (HLU), both unilateral BTX injection and HLU, or no intervention. BTX and HLU each led to significant muscle and bone loss. The effect of BTX was diminished when combined with HLU, though generally the leg that received the combined intervention (HLU + BTX) had the most detrimental changes in bone and muscle. We found an indirect effect of BTX affecting the uninjected (contralateral) leg that led to significant decreases in bone mineral density and deficits in muscle mass and bone architecture relative to the untreated controls; the magnitude of this indirect BTX effect was comparable to the direct effect of BTX treatment and HLU. Thus, while it was difficult to definitively conclude whether muscle forces or external gravitational loading contribute more to bone maintenance, it appears that BTX-induced muscle paralysis is more detrimental to muscle and bone than hindlimb unloading.
doi:10.1007/s00223-013-9814-7
PMCID: PMC3921683  PMID: 24240478
disuse; botulinum toxin; mechanical loading; tail suspension; muscle-bone; interaction; hindlimb unloading; paralysis
13.  Effect of Vitamin B12 and Folic Acid Supplementation on Bone Mineral Density and Quantitative Ultrasound Parameters in Older People with an Elevated Plasma Homocysteine Level: B-PROOF, a Randomized Controlled Trial 
Calcified Tissue International  2015;96(5):401-409.
High plasma homocysteine (Hcy) levels are associated with increased osteoporotic fracture incidence. However, the mechanism remains unclear. We investigated the effect of Hcy-lowering vitamin B12 and folic acid treatment on bone mineral density (BMD) and calcaneal quantitative ultrasound (QUS) parameters. This randomized, double-blind, placebo-controlled trial included participants aged ≥65 years with plasma Hcy levels between 12 and 50 µmol/L. The intervention comprised 2-year supplementation with either a combination of 500 µg B12, 400 µg folic acid, and 600 IU vitamin D or placebo with 600 IU vitamin D only. In total, 1111 participants underwent repeated dual-energy X-ray assessment and 1165 participants underwent QUS. Femoral neck (FN) BMD, lumbar spine (LS) BMD, calcaneal broadband ultrasound attenuation (BUA), and calcaneal speed of sound (SOS) were assessed. After 2 years, FN-BMD and BUA had significantly decreased, while LS-BMD significantly increased (all p < 0.01) and SOS did not change in either treatment arm. No statistically significant differences between the intervention and placebo group were present for FN-BMD (p = 0.24), LS-BMD (p = 0.16), SOS (p = 0.67), and BUA (p = 0.96). However, exploratory subgroup analyses revealed a small positive effect of the intervention on BUA at follow-up among compliant persons >80 years (estimated marginal mean 64.4 dB/MHz for the intervention group and 61.0 dB/MHz for the placebo group, p = 0.04 for difference). In conclusion, this study showed no overall effect of treatment with vitamin B12 and folic acid on BMD or QUS parameters in elderly, mildly hyperhomocysteinemic persons, but suggests a small beneficial effect on BUA in persons >80 years who were compliant in taking the supplement.
doi:10.1007/s00223-015-9968-6
PMCID: PMC4415946  PMID: 25712255
DXA; QUS; Vitamin B12; Folic acid; Homocysteine
14.  [No title available] 
PMCID: PMC3949118  PMID: 24077875
15.  Cellular complexity of the bone marrow hematopoietic stem cell niche 
Calcified tissue international  2013;94(1):112-124.
The skeleton serves as the principal site for hematopoiesis in adult terrestrial vertebrates. The function of the hematopoietic system is to maintain homeostatic levels of all circulating blood cells, including myeloid cells, lymphoid cells, red blood cells, and platelets. This action requires the daily production of more than 500 billion blood cells every day. The vast majority of these cells are synthesized in the bone marrow, where they arise from a limited number of hematopoietic stem cells (HSCs) that are multipotent and capable of extensive self-renewal. These attributes of HSCs are best demonstrated by marrow transplantation, where even a single HSC can repopulate the entire hematopoietic system. HSCs are therefore adult stem cells capable of multilineage repopulation, poised between cell fate choices, which include quiescence, self-renewal, differentiation and apoptosis. While HSC fate choices are in part determined by multiple stochastic fluctuations of cell autonomous processes, according to the niche hypothesis, signals from the microenvironment are also likely to determine stem cell fate. While it had long been postulated that signals within the bone marrow could provide regulation of hematopoietic cells, it is only in the past decade that advances in flow cytometry and genetic models have allowed for a deeper understanding of microenvironmental regulation of HSCs. In this review, we will highlight the cellular regulatory components of the HSC niche.
doi:10.1007/s00223-013-9805-8
PMCID: PMC3936515  PMID: 24101231
16.  Molecular Mechanisms of Osteoblast/Osteocyte Regulation by Connexin43 
Calcified tissue international  2013;94(1):55-67.
Osteoblasts, osteocytes and osteoprogenitor cells are interconnected into a functional network by gap junctions formed primarily by connexin43 (Cx43). Over the past two decades, it has become clear that Cx43 is important for the function of osteoblasts and osteocytes. This connexin contributes to the acquisition of peak bone mass and it is a major modulator of cortical modeling. We review key data from human and mouse genetics on the skeletal consequences of ablation or mutation of the Cx43 gene (Gja1), and the molecular mechanisms by which Cx43 regulates the differentiation, function and survival of osteogenic lineage cells. We also discuss putative second messengers that are communicated by Cx43 gap junctions, the role of hemichannels, and the function of Cx43 as a scaffold for signaling molecules. Current knowledge demonstrates that Cx43 is more than a passive channel; rather, it actively participates in generation and modulation of cellular signals driving skeletal development and homeostasis.
doi:10.1007/s00223-013-9742-6
PMCID: PMC3815501  PMID: 23754488
Cx43; gap junction; bone; signal transduction; Runx2; Osterix
17.  Control of Bone Remodeling by the Peripheral Sympathetic Nervous System 
Calcified tissue international  2013;94(1):140-151.
The skeleton is no longer seen as a static, isolated, and mostly structural organ. Over the last two decades, a more complete picture of the multiple functions of the skeleton has emerged, and its interactions with a growing number of apparently unrelated organs have become evident. The skeleton not only reacts to mechanical loading and inflammatory, hormonal, and mineral challenges, but also acts of its own accord by secreting factors controlling the function of other tissues, including the kidney and possibly the pancreas and gonads. It is thus becoming widely recognized that it is by nature an endocrine organ, in addition to a structural organ and site of mineral storage and hematopoiesis. Consequently and by definition, bone homeostasis must be tightly regulated and integrated with the biology of other organs to maintain whole body homeostasis, and data uncovering the involvement of the central nervous system (CNS) in the control of bone remodeling support this concept. The sympathetic nervous system (SNS) represents one of the main links between the CNS and the skeleton, based on a number of anatomic, pharmacologic, and genetic studies focused on β-adrenergic receptor (βAR) signaling in bone cells. The goal of this report was to review the data supporting the role of the SNS and βAR signaling in the regulation of skeletal homeostasis.
doi:10.1007/s00223-013-9752-4
PMCID: PMC3883940  PMID: 23765388
Bone turnover; Remodeling; Mouse genetics/transgenetics; Neurotransmitters; Signal transduction
18.  Osteocytes: Master Orchestrators of Bone 
Osteocytes comprise the overwhelming majority of cells in bone and are its only true “permanent” resident cell population. In recent years, conceptual and technological advances on many fronts have helped to clarify the role osteocytes play in skeletal metabolism and the mechanisms they use to perform them. The osteocyte is now recognized as a major orchestrator of skeletal activity, capable of sensing and integrating mechanical and chemical signals from their environment to regulate both bone formation and resorption. Recent studies have established that the mechanisms osteocytes use to sense stimuli and regulate effector cells (e.g. osteoblasts and osteoclasts) are directly coupled to the environment they inhabit – entombed within the mineralized matrix of bone and connected to each other in multicellular networks. Communication within these networks is both direct (via cell-cell contacts at gap junctions) and indirect (via paracrine signaling by secreted signals). Moreover, the movement of paracrine signals is dependent on movement of both solutes and fluid through the space immediately surrounding the osteocytes (i.e. the Lacunar-Canalicular System, LCS). Finally, recent studies have also shown that the regulatory capabilities of osteocytes extend beyond bone to include a role in endocrine control of systemic phosphate metabolism. This review will discuss how a highly productive combination of experimental and theoretical approaches has managed to unearth these unique features of osteocytes and bring to light novel insights into the regulatory mechanisms operating in bone.
doi:10.1007/s00223-013-9790-y
PMCID: PMC3947191  PMID: 24042263
Osteocytes; Biomechanics; Mechanotransduction; Intercellular communication
19.  Notch signaling in Skeletal Stem Cells 
Calcified tissue international  2013;94(1):68-77.
The skeleton is originated from stem cells residing in the sclerotome and neural crest that undergo proliferation, migration and commitment. The development of the skeletal stem cells is influenced by many signaling pathways that govern cell fate determination, proliferation, differentiation and apoptosis. This review will focus on Notch signaling functions in regulating different cells types forming the skeletal system as well as the interplay between them to maintain homeostasis. Osteochondroprogenitors require Notch signaling to maintain the multipotency and to prevent from premature differentiation into osteoblast. Subsequently, over-activation of Notch signaling suppresses osteoblast maturation. Moreover, Notch signaling in osteochondroprogenitors is required for chondrocyte proliferation, hypertrophy and suppresses terminal differentiation. Translational studies demonstrated a crucial role of Notch signaling in osteosarcoma and osteoarthritis, where concepts derived from developmental pathways are often recapitulated. This brings hope of taking advantages of the molecular mechanisms learned from development to approach the pathological processes underlying abnormal bone/cartilage metabolism or tumorigenesis. Pharmacological agents that target Notch receptors or ligands in a tissue specific fashion would offer new opportunities for treating bone/cartilage diseases caused by dysregulation of Notch signaling.
doi:10.1007/s00223-013-9773-z
PMCID: PMC3947214  PMID: 23963632
Notch signaling; osteoblast differentiation; chondrogenesis; osteoclastogenesis
20.  Osteocyte-Driven Bone Remodeling 
Calcified tissue international  2013;94(1):25-34.
Osteocytes, the most abundant cells in bone, have been long postulated to detect and respond to mechanical and hormonal stimuli and to coordinate the function of osteoblasts and osteoclasts. The discovery that the inhibitor of bone formation sclerostin is primarily expressed in osteocytes in bone and it is downregulated by anabolic stimuli provided a mechanism by which osteocytes influence the activity of osteoblasts. Advances of the last few years provided experimental evidence demonstrating that osteocytes also participate in the recruitment of osteoclasts and the initiation of bone remodeling. Apoptotic osteocytes trigger yet to be identified signals that attract osteoclast precursors to specific areas of bone, which in turn differentiate to mature, bone resorbing osteoclasts. Osteocytes are also the source of molecules that regulate generation and activity of osteoclasts, such as OPG and RANKL; and genetic manipulations of the mouse genome leading to loss or gain of function, or to altered expression of either molecule in osteocytes, markedly affect bone resorption. This review highlights these investigations and discusses how the novel concept of osteocyte-driven bone resorption and formation impacts our understanding of the mechanisms by which current therapies control bone remodeling.
doi:10.1007/s00223-013-9774-y
PMCID: PMC3947228  PMID: 24002178
osteocyte; osteoclast; osteoblast; bone remodeling; RANKL; OPG; Sost
21.  Inflammation, telomere length and grip strength: a 10 year longitudinal study 
Calcified tissue international  2014;95(1):54-63.
Background
Telomere attrition has been associated with age related diseases although causality is unclear and controversial; low grade systemic inflammation (inflammaging) has also been implicated in age-related pathogenesis. Unpicking the relationship between ageing, telomere length (TL) and inflammaging is hence essential to the understanding of ageing and management of age-related diseases. This longitudinal study explores whether telomere attrition is a cause or consequence of ageing and whether inflammaging explains some of the associations between TL and one marker of ageing, grip strength.
Methods
We studied 253 Hertfordshire Ageing Study participants at baseline and 10 year follow up (mean age at baseline 67.1years). Participants completed a health questionnaire and had blood samples collected for immune-endocrine and telomere analysis at both time points. Physical ageing was characterised at follow-up using grip strength (GS).
Findings
Faster telomere attrition was associated with lower GS at follow-up (β=0.98, p=0.035). This association was completely attenuated when adjusted for inflammaging burden (p=0.86) over the same period. Similarly, greater inflammaging burden was associated with lower GS at follow-up (e.g. interleukin1β (IL-1β): β=−2.18, p=0.001), however, these associations were maintained when adjusted for telomere attrition (IL-1β, p=0.006).
Interpretation
We present evidence that inflammaging may be driving telomere attrition and in-part explains the associations which have previously been reported between TL and grip strength. Thus biomarkers of physical ageing, such as inflammaging, may require greater exploration. Further work is now indicated.
doi:10.1007/s00223-014-9862-7
PMCID: PMC4098723  PMID: 24858709
Telomere; epidemiology; sarcopenia; inflammation; ageing; osteoporosis; grip strength
22.  Cadherin-Mediated Cell-Cell Adhesion and Signaling in the Skeleton 
Calcified tissue international  2013;94(1):46-54.
Direct cell-to-cell interactions via cell adhesion molecules, in particular cadherins, are critical for morphogenesis, tissue architecture, and cell sorting and differentiation. Partially overlapping, yet distinct roles of N-cadherin (cadherin-2) and cadherin-11 in the skeletal system have emerged from mouse genetics and in vitro studies. Both cadherins are important for precursor commitment to the osteogenic lineage, and genetic ablation of Cdh2 and Cdh11 results in skeletal growth defects and impaired bone formation. While Cdh11 defines the osteogenic lineage, persistence of Cdh2 in osteoblasts in vivo actually inhibits their terminal differentiation and impairs bone formation. The action of cadherins involves both cell-cell adhesion and interference with intracellular signaling, and in particular the Wnt/β-catenin pathway. Both cadherin-2 and cadherin-11 bind to β-catenin, thus modulating its cytoplasmic pools and transcriptional activity. Recent data demonstrate that cadherin-2 also interferes with Lrp5/6 signaling by sequestering these receptors in inactive pools via axin binding. These data extend the biologic action of cadherins in bone forming cells, and provide novel mechanisms for development of therapeutic strategies aimed at enhancing bone formation.
doi:10.1007/s00223-013-9733-7
PMCID: PMC4272239  PMID: 23657489
Cadherins; cell-cell adhesion; osteoblast differentiation; Wnt/β-catenin signaling; bone formation
23.  1,25(OH)2D3 Induces a Mineralization Defect and Loss of Bone Mineral Density in Genetic Hypercalciuric Stone-Forming Rats 
Calcified tissue international  2014;94(5):531-543.
Genetic hypercalciuric stone-forming (GHS) rats, bred to maximize urine (u) calcium (Ca) excretion, demonstrate increased intestinal Ca absorption, increased bone Ca resorption and reduced renal Ca reabsorption, all leading to elevated uCa compared to the parental Sprague-Dawley (SD) rats. GHS rats have increased numbers of vitamin D receptors (VDR) at each site, with normal levels of 1,25(OH)2D3 (1,25D), suggesting their VDR is undersaturated with 1,25D. We have shown that 1,25D induces a greater increase in uCa in GHS than SD rats. To examine the effect of the increased VDR on the osseous response to 1,25D we fed GHS and SD rats an ample Ca diet and injected either 1,25D (12.5 (LD) or 25 (HD) ng/100 g body wt/d) or vehicle (veh) daily for 16d. Femoral areal bone mineral density (aBMD, by DEXA) was decreased in GHS+LD and GHS+HD relative to GHS+veh while there was no effect on SD. Vertebral aBMD was lower in GHS compared to SD and further decreased in GHS+HD. Both femoral and L6 vertebral volumetric BMD (by μCT) were lower in GHS and further reduced by HD. Histomorphometry indicated a decreased osteoclast number in GHS+HD compared to GHS+veh or SD+HD. In tibiae, GHS+HD trabecular thickness and number increased, with a 12-fold increase in osteoid volume but only a 3-fold increase in bone volume. Bone formation rate was decreased in GHS+HD relative to GHS+veh, confirming the mineralization defect. The loss of BMD and the mineralization defect in GHS rats contribute to increased hypercalciuria; if these effects persist they would result in decreased bone strength, making these bones more fracture prone. The enhanced effect of 1,25D in GHS indicates that the increased VDR are biologically active.
doi:10.1007/s00223-014-9838-7
PMCID: PMC4276134  PMID: 24481706
vitamin D; calcium; reabsorption; bone quality
24.  Increased Bone Mineral Density with Monthly Intravenous Ibandronate Contributes to Fracture Risk Reduction in Patients with Primary Osteoporosis: Three-Year Analysis of the MOVER Study 
Calcified Tissue International  2014;95(6):557-563.
The relationship between gains in bone mineral density (BMD) in the hip and the incidence of vertebral fractures in the MOVER study was examined. Japanese patients from the ibandronate and risedronate treatment groups whose hip BMD had increased during the 3-year treatment period were classified into those with or without vertebral fractures. In both the ibandronate group and the risedronate group, hip BMD gains in the patients who had developed no vertebral fractures during the treatment period were greater than in the patients who developed vertebral fractures. We categorized the gains in hip BMD at 6 months into 3 groups (≤0, >0 to ≤3, and >3 %), and used logistic regression analysis to estimate odds ratios and the probabilities of incidence of vertebral fractures at 12, 24, and 36 months. The current study demonstrated that greater gains in hip BMD during the first 6 months of treatment were associated with a reduction in the risk of subsequent vertebral fractures during the duration of treatment, and suggested that measurement of hip BMD gain at that time could lead to a prediction of the risk of the future vertebral fracture incidence.
doi:10.1007/s00223-014-9927-7
PMCID: PMC4239781  PMID: 25377907
Ibandronate; Vertebral fracture; Hip BMD change; Osteoporosis; MOVER study
25.  Efficacy of reversal of aortic calcification by chelating agents 
Calcified tissue international  2013;93(5):10.1007/s00223-013-9780-0.
Elastin specific medial vascular calcification, termed Monckeberg’s sclerosis has been recognized as a major risk factor for various cardiovascular events. We hypothesize that chelating agents, such as disodium ethylene diamine tetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA) and sodium thiosulfate (STS) might reverse elastin calcification by directly removing calcium (Ca) from calcified tissues into soluble calcium complexes. We assessed the chelating ability of EDTA, DTPA, and STS on removal of calcium from hydroxyapatite (HA) powder, calcified porcine aortic elastin, and calcified human aorta in vitro. We show that both EDTA and DTPA could effectively remove calcium from HA and calcified tissues, while STS was not effective. The tissue architecture was not altered during chelation. In the animal model of aortic elastin-specific calcification, we further show that local periadventitial delivery of EDTA loaded in to poly (lactic-co-glycolic acid) (PLGA) nanoparticles regressed elastin specific calcification in the aorta. Collectively, the data indicate that elastin-specific medial vascular calcification could be reversed by chelating agents.
doi:10.1007/s00223-013-9780-0
PMCID: PMC3809012  PMID: 23963635
Elastin; Demineralization; Calcium; Arteriosclerosis; Chelating complexes

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