Bone mineral density (BMD) is the most important predictor of fracture risk. We performed the largest meta-analysis to date on lumbar spine and femoral neck BMD, including 17 genome-wide association studies and 32,961 individuals of European and East Asian ancestry. We tested the top-associated BMD markers for replication in 50,933 independent subjects and for risk of low-trauma fracture in 31,016 cases and 102,444 controls. We identified 56 loci (32 novel)associated with BMD atgenome-wide significant level (P<5×10−8). Several of these factors cluster within the RANK-RANKL-OPG, mesenchymal-stem-cell differentiation, endochondral ossification and the Wnt signalling pathways. However, we also discovered loci containing genes not known to play a role in bone biology. Fourteen BMD loci were also associated with fracture risk (P<5×10−4, Bonferroni corrected), of which six reached P<5×10−8 including: 18p11.21 (C18orf19), 7q21.3 (SLC25A13), 11q13.2 (LRP5), 4q22.1 (MEPE), 2p16.2 (SPTBN1) and 10q21.1 (DKK1). These findings shed light on the genetic architecture and pathophysiological mechanisms underlying BMD variation and fracture susceptibility.

Atypical femoral fractures (AFF) associated with long-term bisphosphonates (LTB) are a growing concern. Their etiology is unknown, but bone material properties might be deteriorated. In an AFF series, we analyzed the bone material properties by microindentation. Four groups of patients were included: 6 AFF, 38 typical osteoporotic fractures, 6 LTB, and 20 controls without fracture. Neither typical osteoporotic fractures nor controls have received any antiosteoporotic medication. A general laboratory workup, bone densitometry by dual-energy X-ray absorptiometry (DXA), and microindentation testing at the tibia were done in all patients. Total indentation distance (Total ID), indentation distance increase (IDI), and creep indentation distance (Creep ID) were measured (microns). Age-adjusted analysis of covariance (ANCOVA) was used for comparisons. Controls were significantly younger than fracture groups. Bisphosphonate exposure was on average 5.5 years (range 5 to 12 years) for the AFF and 5.4 years (range 5 to 8 years) for the LTB groups. Total ID (microns) showed better material properties (lower Total ID) for controls 36 (AE 6; mean AE SD) than for AFF 46 (AE 4) and for typical femoral fractures 47 (AE 13), respectively. Patients on LTB showed values between controls and fractures, 38 (AE 4), although not significantly different from any of the other three groups. IDI values showed a similar pattern 13 (AE 2), 16 (AE 6), 19 (AE 3), and 18 (AE 5). After adjusting by age, significant differences were seen between controls and typical (p < 0.001) and atypical fractures (p ¼ 0.03) for Total ID and for IDI (p < 0.001 and p < 0.05, respectively). There were no differences in Creep ID between groups. Our data suggest that patients with AFF have a deep deterioration in bone material properties at a tissue level similar to that for the osteoporotic fracture group. The LTB group shows levels that are in between controls and both type of fractures, although not statistically different. These results suggest that bisphosphonate therapy probably does not put the majority of patients at risk for AFF. ß

Single-nucleotide polymorphisms (SNPs) in regulatory regions of candidate genes may determine variability in bone mineral density (BMD) because they may be responsible for differences in levels of a gene product in response to external signals. Under this hypothesis, we scanned an 800-base pair (bp) region within the COL1A1 promoter, known to harbor cis elements important for in vivo expression, and we found two new polymorphisms: ؊1663indelT and ؊1997 G/T. The G to T transversion at ؊1997 was associated with lumbar spine BMD (p ‫؍‬ 0.015) when tested in a cohort of 256 postmenopausal women after adjusting by age, body weight, and years since menopause; a lower degree of association was detected also for femoral neck BMD in a subgroup of 146 women in univariate analysis and after adjusting by age (p ‫؍‬ 0.044). The polymorphism ؊1663indelT, which corresponds to a deletion of a T in a tract of eight T residues (؊1670 to ؊1663), did not show significant association with BMD. Interestingly, ؊1663indelT is in strong linkage disequilibrium (LD) with the previously described Sp1 polymorphism of intron 1, which in this study did not show association with BMD either. Significant interaction between ؊1997 G/T and ؊1663indelT (p ‫؍‬ 0.019), and between ؊1997 G/T and Sp1 (p ‫؍‬ 0.045) was observed also. Individuals heterozygous for the three polymorphisms showed the highest mean BMD value. Gel retardation assays showed that oligonucleotides containing either the ؊1663 or the ؊1997 polymorphic sites specifically bind primary osteoblast nuclear proteins. We named these binding sites as PCOL1 and PCOL2, respectively. In summary, this study describes two new SNPs in the COL1A1 promoter, which may affect bone mass determination. (J Bone Miner Res 2002;17:384 -393)

Context COVID-19 is a major health problem because of saturation of intensive care units (ICU) and mortality. Vitamin D has emerged as a potential treatment able to reduce the disease severity. Objective To elucidate the effect of calcifediol [25OHD3] treatment on COVID-19-related outcomes. Design Observational cohort study from March to May, 2020. Setting Patients admitted to COVID-19 wards of Hospital del Mar, Barcelona, Spain. Patients A total of 930 patients with COVID-19 were included. Ninety-two were excluded due to previous calcifediol intake. Intervention Of the remaining 838, a total of 447 received calcifediol (532ug on day one plus 266ug on day 3, 7, 15, and 30) whereas 391 were not treated at the time of hospital admission (Intention-to-Treat). Of the latter, 53 patients were treated later during ICU admission and were allocated in the treated group in a second analysis. In healthy subjects, calcifediol is about 3.2-fold more potent on a weight basis than cholecalciferol. Main Outcome Measures ICU admission and mortality. Results ICU assistance was required by 102 (12.2%) participants. Out of 447 patients treated with calcifediol at admission, 20 (4.5%) required ICU, compared to 82 (21%) out of 391 non-treated (p-value&lt;0.0001). Logistic regression of calcifediol treatment on ICU admission, adjusted by age, gender, linearized 25OHD levels at baseline, and comorbidities showed that treated patients had a reduced risk to require ICU (OR 0.13 [95% CI 0.07;0.23]). Overall mortality was 10%. In the Intention-to-Treat analysis, 21 (4.7%) out of 447 patients treated with calcifediol at admission died compared to 62 patients (15.9%) out of 391 non-treated (p=0.0001). Adjusted results showed a reduced mortality risk with an OR 0.21 [95% CI 0.10; 0.43]). In the second analysis, the obtained OR was 0.52 [95% CI 0.27;0.99]. Conclusions In patients hospitalized with COVID-19, calcifediol treatment significantly reduced ICU admission and mortality.

A technique for producing controlled interconnected porous structures for application as a tissue engineering scaffold is presented in this article. The technique is based on the fabrication of a template of interconnected poly(ethyl methacrylate) (PEMA) microspheres, the introduction of a biodegradable polymer, poly-epsilon-caprolactone (PCL), and the elimination of the template by a selective solvent. A series of PCL scaffolds with a porosity of 70% and pore sizes up to 200 microm were produced and characterized (both thermally and mechanically). Human chondrocytes were cultured in monolayer on bulk PCL disks or seeded into porous PCL scaffolds. Cell adhesion, viability, proliferation, and proteoglycan (PG) synthesis were tested and compared with monolayer cultures on tissue-treated polystyrene or pellet cultures as reference controls. Cells cultured on PCL disks showed an adhesion similar to that of the polystyrene control (which allowed high levels of proliferation). Stained scaffold sections showed round-shaped chondrocyte aggregates embedded into porous PCL. PG production was similar to that of the pellet cultures and higher than that obtained with monolayer postconfluence cultures. This shows that the cells are capable of attaching themselves to PCL. Furthermore, in porous PCL, cells maintain the same phenotype as the chondrocytes within the native cartilage. These results suggest that PCL scaffolds may be a suitable candidate for chondrocyte culture.

BackgroundMicroRNAs (miRNAs) are important regulators of gene expression, with documented roles in bone metabolism and osteoporosis, suggesting potential therapeutic targets. Our aim was to identify miRNAs differentially expressed in fractured vs nonfractured bones. Additionally, we performed a miRNA profiling of primary osteoblasts to assess the origin of these differentially expressed miRNAs.MethodsTotal RNA was extracted from (a) fresh femoral neck trabecular bone from women undergoing hip replacement due to either osteoporotic fracture (OP group, n = 6) or osteoarthritis in the absence of osteoporosis (Control group, n = 6), matching the two groups by age and body mass index, and (b) primary osteoblasts obtained from knee replacement due to osteoarthritis (n = 4). Samples were hybridized to a microRNA array containing more than 1900 miRNAs. Principal component analysis (PCA) plots and heat map hierarchical clustering were performed. For comparison of expression levels, the threshold was set at log fold change > 1.5 and a p-value < 0.05 (corrected for multiple testing).ResultsBoth PCA and heat map analyses showed that the samples clustered according to the presence or absence of fracture. Overall, 790 and 315 different miRNAs were detected in fresh bone samples and in primary osteoblasts, respectively, 293 of which were common to both groups. A subset of 82 miRNAs was differentially expressed (p < 0.05) between osteoporotic and control osteoarthritic samples.The eight miRNAs with the lowest p-values (and for which a validated miRNA qPCR assay was available) were assayed, and two were confirmed: miR-320a and miR-483-5p. Both were over-expressed in the osteoporotic samples and expressed in primary osteoblasts. miR-320a is known to target CTNNB1 and predicted to regulate RUNX2 and LEPR, while miR-483-5p down-regulates IGF2. We observed a reduction trend for this target gene in the osteoporotic bone.ConclusionsWe identified two osteoblast miRNAs over-expressed in osteoporotic fractures, which opens novel prospects for research and therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0149-2) contains supplementary material, which is available to authorized users.

Background: Zinc is an essential micronutrient that impacts host–pathogen interplay at infection. Zinc balances immune responses, and also has a proven direct antiviral action against some viruses. Importantly, zinc deficiency (ZD) is a common condition in elderly and individuals with chronic diseases, two groups with an increased risk for severe severe coronavirus disease 2019 (COVID-19) outcomes. We hypothesize that serum zinc content (SZC) influences COVID-19 disease progression, and thus might represent a useful biomarker. Methods: We ran an observational cohort study with 249 COVID-19 patients admitted in Hospital del Mar. We have studied COVID-19 severity and progression attending to SZC at admission. In parallel, we have studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) replication in the Vero E6 cell line modifying zinc concentrations. Findings: Our study demonstrates a correlation between serum zinc levels and COVID-19 outcome. Serum zinc levels lower than 50 µg/dL at admission correlated with worse clinical presentation, longer time to reach stability, and higher mortality. Our in vitro results indicate that low zinc levels favor viral expansion in SARS-CoV-2 infected cells. Interpretation: Low SZC is a risk factor that determines COVID-19 outcome. We encourage performing randomized clinical trials to study zinc supplementation as potential prophylaxis and treatment with people at risk of zinc deficiency.

Royal Society of ChemistryAreias, A.; Ribeiro, C.; Sencadas, V.; Garcia Giralt, N.; Diez Perez, A.; Gómez Ribelles, JL.; Lanceros Mendez, S. (2012). Influence of crystallinity and fiber orientation on hydrophobicity and biological response of poly(L-lactide) electrospun mats. Soft Matter. 8 (21) AbstractPoly(L-lactide) electrospun mates have been produced with random and aligned fiber orientation and degrees of crystallinity from 0 up to nearly 50%. These two factors, fiber alignment and degree of crystallinity strongly affect the hydrophobicity of the samples, being this larger for the aligned fiber mats and for the fibers with higher degree of crystallinity. Whereas the first effect can be associated to a decrease in the degree of porosity the second should be related to variations in the fiber roughness at nanometric scale and an increase in fiber stiffness. Proliferation of human chondrocytes cultured in monolayer on these substrates is similar in both aligned and non-aligned amorphous mats. Crystallization of the aligned mats, on the other hand, nearly suppresses proliferation and the cells produce higher amounts of aggrecan, characteristic of the extracellular matrix of hyaline cartilage.