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Background: Vitamin D and dairy protein may stimulate bone mineralization and linear growth in children, but previous studies show inconsistent results and have not examined their combined effects. Objectives: To investigate combined and separate effects of vitamin D supplementation and high-protein (HP) compared with normal-protein (NP) yogurt intake on children's bone mineralization and linear growth. Methods: In a 2 × 2-factorial trial, 200 healthy, 6-to 8-year-old, Danish, children with light skin (55°N) were randomized to 20 μg/d vitamin D3 or placebo and to substitute 260 g/d dairy with HP (10 g protein/100 g) or NP (3.5 g protein/100 g) yogurt for 24 weeks during an extended winter. Outcomes were total body less head (TBLH) and lumbar spine bone mineral density (BMD), bone mineral content (BMC), and bone area (BA) by dual-energy X-ray absorptiometry, height, and biomarkers of bone turnover and growth. The primary outcome was TBLH BMD. Results: In total, 184 children (92%) completed the study. The baseline serum 25-hydroxyvitamin D was 80.8 ± 17.2 nmol/L, which increased by 7.2 ± 14.1 nmol/L and decreased by 32.3 ± 17.5 nmol/L with vitamin D and placebo, respectively. The baseline protein intake was 15.4 ± 2.4 energy percentage (E%), which increased to 18.3 ± 3.4 E% with HP. There were no vitamin D-yogurt interactions and no main effects of either intervention on TBLH BMD. However, vitamin D supplementation increased lumbar spine BMD and TBLH BMC compared to placebo, whereas HP groups showed lower increments in lumbar spine BMD, TBLH BMC and BA, and plasma osteocalcin compared to NP groups. Height, growth factors, and parathyroid hormone levels were unaffected. Conclusions: Although there were no effects on whole-body BMD, vitamin D increased bone mass and spinal BMD, whereas high compared with normal dairy protein intake had smaller incremental effects on these outcomes. This supports a recommended vitamin D intake of around 20 μg/d during winter but not use of HP dairy products for improved bone mineralization among healthy, well-nourished children. This trial was registered at clinicaltrials.gov as NCT03956732.

The aim of this study was to determine how different doses of phytase in diets with extruded soybean seeds and rapeseed meal affected pigs’ growth performance, meat quality, bone mineralization, and fatty acid profiles. Sixty pigs were divided into three treatments by sex and body mass. Pigs were divided into starter (25 days), grower (36 days), and finisher (33 days) periods and fed with mash diets. No phytase was used in the control group diet, whereas in Phy1 and Phy2, 100 g and 400 g of phytase per ton of mixture were used, respectively. The feed conversion ratio and meat color were significantly correlated with phytase. Phytase supplementation had no effect on the growth of pigs, but total phosphorus was significantly increased in the bones and meat of the pigs. The enzyme additive reduced the C22:4 n-6 acid content in the meat, whereas other results were not significantly affected. The data suggest that the addition of phytase at a dosage of 100 g/ton to diets with extruded full-fat soya seeds and rapeseed meal can be valuable, as it reduces the FCR and increases the P content in the meat and bones.

This study aims to determine how different doses of phytase in diets including extruded soya and lupine seeds affect fatteners’ performance, meat quality, bone mineralization, and fatty acid profile. Sixty pigs were divided into three treatment groups. The control group was offered a diet without phytase, whereas the Phy100 and Phy400 groups were provided with 100 g and 400 g of phytase per ton of their diet, respectively. The animals from both experimental groups were characterized by a significantly (p < 0.05) higher body weight gain and lower feed efficiency in the starter period than the control group. Unfortunately, their meat had lower (p < 0.05) fat content, gluteal muscle thickness, and water-holding capacity. In the meat, a higher phosphorus content (p < 0.05) was found, and in the bones, a higher calcium (for Phy400) content was found when phytase was added to the pigs’ diet. The pigs from the Phy100 group tended to have higher mean backfat thickness and C18:2 n-6 content in their fat, but lower C22:5 n-3 content, than the other groups. A higher dosage of phytase is not necessary for the diets of fatteners with extruded full-fat soya and lupin seeds.

Sturgeons and paddlefishes (Acipenseriformes) occupy the basal position of ray-finned fishes, although they have cartilaginous skeletons as in Chondrichthyes. This evolutionary status and their morphological specializations make them a research focus, but their complex genomes (polyploidy and the presence of microchromosomes) bring obstacles and challenges to molecular studies. Here, we generated the first high-quality genome assembly of the American paddlefish (Polyodon spathula) at a chromosome level. Comparative genomic analyses revealed a recent species-specific whole-genome duplication event, and extensive chromosomal changes, including head-to-head fusions of pairs of intact, large ancestral chromosomes within the paddlefish. We also provide an overview of the paddlefish SCPP (secretory calcium-binding phosphoprotein) repertoire that is responsible for tissue mineralization, demonstrating that the earliest flourishing of SCPP members occurred at least before the split between Acipenseriformes and teleosts. In summary, this genome assembly provides a genetic resource for understanding chromosomal evolution in polyploid nonteleost fishes and bone mineralization in early vertebrates.

Bone loss is among the most frequent changes seen in astronauts during space missions. Although weightlessness is known to cause high bone resorption and a rapid decrease in bone minerals and calcium, the underlying mechanisms are not yet fully understood. In our work, we investigated the influence of random positioning machine (RPM) exposure on the mineralization process in the SAOS-2 cell line, in osteogenic and non-osteogenic conditions, by examining changes in their mineralizing capacity and in the expression of PTX3, a positive regulator of bone mineralization. We analyzed cell viability by MTS assay and the mineralization process after staining with Toluidine Blue and Alizarin Red, while PTX3 expression was investigated by immunocytochemistry and western blotting analysis. Our results showed that RPM exposure increased cells’ viability and improved their mineralizing competence when not treated with osteogenic cocktail. In contrast, in osteogenic conditions, cells exposed to RPM showed a reduction in the presence of calcification-like structures, mineral deposits and PTX3 expression, suggesting that the effects of RPM exposure on mineralizing matrix deposition depend on the presence of osteogenic factors in the culture medium. Further studies will be needed to clarify the role of potential mineralization markers in the cellular response to the simulated biological effects of microgravity, paving the way for a new approach to treating osteoporosis in astronauts exposed to spaceflight.

The formation of the hard tissues that provide support and mobility to organisms is achieved through the interplay of inorganic crystals and an organic framework composed of collagen and a small percentage of non-collagenous proteins. Despite their clinical relevance, the mechanisms governing mineralization of the extracellular matrix are still poorly understood. By using 3D electron tomography and high-resolution electron microscopy imaging and spectroscopy, it has been demonstrated that mineralization proceeds through a spherulitic-like crystal growth process. First, aggregates of disordered crystals form in the interfibrillar spaces, which lead to the mineralization of adjacent fibrils. Mineral propagates steadily through the inter- and intrafibrillar spaces of the collagen structure forming layered spherulites that grow to confluence. The structure of the collagen fibrils serves as a protein scaffold to guide the formation of a myriad of platelet-shaped crystallites that make up each of these spherulites. At their periphery, nanosized unmineralized areas remain, leading to the formation of the characteristic lacy pattern observed in the transversal cross-section of mature calcified tissues. This study provides fundamental insights into the bone formation process and represents a potential strategy for complex materials design, Projekt DEAL

The effects of Ca:P total ratio and particle size of oyster shell meal (OSM) were evaluated in broiler diets. In Experiment 1, 800 broilers (22-42 days old) were distributed in a 2×2 factorial design, with two Ca:P ratios (1.7 and 2.0:1) and two OSM particle sizes (coarse = 1,354 µm and fine = 428 µm), totaling four treatments with 10 repetitions with 20 broilers. Feed intake, weight gain, and feed conversion ratio were calculated. In Experiment 2, 1,280 broilers were distributed in a 2×2×2 factorial design (1.7 and 2.0:1 Ca:P ratios; coarse and fine OSM; male and female broilers), with eight treatments and 16 repetitions with 10 broilers. Apparent metabolizability of dry matter, Ca, P, and apparent metabolizable energy (AME), as well as bone resistance, bone weight, ash, Ca, and P content in the tibia were assessed. Growth performance was not affected (P > 0.05). Coarse OSM increased tibia Ca content in male broilers (P < 0.001), and higher Ca:P ratio improved bone ash and bone resistance in both sexes (P < 0.001), but reduced P content in male broilers (P < 0.05); male broilers displayed heavier bones with higher ash content than females (P < 0.05). Metabolizability of Ca was improved with coarse OSM (P < 0.05); whereas metabolizability of DM, P, and AME was not affected (P > 0.05). In conclusion, diets with a Ca:P total ratio of 2.0:1 containing coarser OSM improved bone mineral composition, particularly in male broilers, and coarse OSM improved the metabolizability of Ca in broilers regardless of the Ca:P total ratio or broiler sex. RESUMO: Dois experimentos foram conduzidos para avaliar os efeitos do tamanho de partícula da farinha de ostras (FO) e relação Ca:P total em dietas para frangos de corte. No primeiro experimento, 800 frangos (22 a 42 dias) foram distribuídos em um delineamento fatorial 2x2: 2 relações Ca:P (1,7 e 2,0:1) e dois tamanhos de partícula da FO (grossa = 1354 µm e fina = 428 µm), totalizando quatro tratamentos com 10 repetições de 20 aves. O consumo de ração, o ganho de peso e a conversão alimentar foram calculados. No segundo experimento, 1.280 frangos foram distribuídos em um fatorial 2x2x2 (relações Ca:P 1,7 e 2,0:1; FO grossa e fina; aves machos e fêmeas) com oito tratamentos e 16 repetições de 10 aves. Foram avaliados: metabolizabilidade aparente da matéria seca, Ca e P, energia metabolizável aparente (EMA), peso e resistência óssea, conteúdo de cinzas, Ca e P na tíbia. As variáveis de desempenho não foram afetadas (P > 0,05). O uso de FO grossa aumentou o conteúdo de Ca na tíbia de frangos machos (P < 0,001), e a relação Ca:P de 2,0:1 aumentou o conteúdo de cinzas e aprimorou resistência óssea em ambos os sexos (P < 0,001), porém reduziu P na tíbia dos machos (P < 0,05); frangos machos também tiveram ossos mais pesados e maior conteúdo de cinzas do que fêmeas (P < 0,05). A metabolizabilidade de Ca foi melhorada com FO grossa, enquanto a metabolizabilidade da matéria seca, P, e EMA não foram afetadas (P > 0,05). Conclui-se que as dietas com relação Ca:P de 2,0:1 e com FO grossa resultaram em melhor composição mineral óssea - particularmente em frangos machos - e a FO grossa melhorou a metabolizabilidade de Ca independentemente da relação Ca:P ou do gênero das aves.

Background: Preterm infants have a low level of bone mineralization compared to those born at term, since 80% of calcium incorporation occurs at the end of pregnancy. The purpose of the present study was to investigate the effect of reflex locomotion therapy on bone modeling and growth in preterm infants and to compare its effect with those of other Physiotherapy modalities. Methods: A multicentre randomized controlled clinical trial was conducted (02/2016 - 07/2020). 106 preterm infants born at the Virgen de la Arrixaca University Clinical Hospital, the General University Hospital of Elche and the Tor-recardenas University Hospital of Almeria, between 26 and 34 weeks with hemodynamic stability, complete enteral nutrition and without any metabolic, congenital, genetic, neurological or respiratory disorders were evaluated for inclusion. Infants were randomly assigned to three groups: one group received reflex locomotion therapy (EGrIt); another group received passive mobilizations with gentle joint compression (EGpmc); and the control group received massage (CG). All treatments were carried out in the neonatal units lasting one month. The main outcome measure was bone formation and resorption measured with bone biomarkers. A mixed ANOVA was used to compare the results of bone biomarkers, and anthropometric measurements. Results: Infants were randomized to EGrIt (n = 38) , EGpmc (n = 32) , and CG (n = 36). All groups were similar in terms of gender (p = 0.891 female 47.2%), gestational age (M=30.753, SD= 1.878, p = 0.39) and birth weight (M=1413.45, SD =347.36, p= 0.157). At the end of the study, significant differences were found between the groups in their interaction in bone formation, measured with osteocalcin [F (2,35)=4.92, p = 0.013, eta(2)(p) = 0.043], in benefit of the EGrIt. Conclusions: Reflex locomotion therapy has been effective in improving bone formation, more so than other Physiotherapy modalities. Therefore, reflex locomotion therapy could be considered one of the most effective physiotherapeutic modalities for the prevention and treatment of osteopenia of prematurity.

Summary: The objective was to evaluate the effects of different levels of phytase in diets with varying amounts of phytate on live performance and bone characteristics of broiler chickens at 21 D. The treatments consisted of a 3 × 5 factorial arrangement, with 3 levels of phytate, high (HP), medium (MP), and low (LP), and 5 diets consisting of a positive control (PC); negative control (NC, with a reduction of 0.15% of calcium [Ca] and 0.15% of phosphorus [P]), and NC diet plus 0, 500, 1,000, or 1,500 FTU kg−1 of phytase. Feed intake peaked with supplementation of 1,051 FTU kg−1 phytase to the LP diets. Bone ash of broilers receiving LP showed a maximum response at 1,101 FTU kg−1. Birds receiving the NC diet had a larger hypertrophic cartilage zone (P < 0.05) than those receiving the PC diet. Serum Ca and P of birds receiving the NC treatment and LP diet were lower than those in broilers fed with the MP and HP diets. Broilers in the NC plus 500 FYT kg−1 treatments had lower tibia P levels than those in the PC treatment; also, broilers receiving HP diets had a higher tibia Ca content than those receiving LP diets (P < 0.05). In general, bone P of birds fed with diets containing HP was higher than that of birds on a LP or MP diet (P < 0.05). The use of 1,101 FTU kg−1 resulted in better bone characteristics when fed with the lowest phytate level, and this level does not negatively affect the other parameters evaluated.

This study investigated the influence of meat and bone meal (MBM), phytase, and antibiotics (AB) on the performance, intestinal pH, ileal digestibility, cecal microbiota, and tibial mineralization in Ross 308 broilers challenged with necrotic enteritis (NE). A total of 672-day-old male Ross 308 chicks were allocated to 8 treatments with 6 replicate pens, with 14 birds each. The study employed a 2 × 2 × 2 factorial arrangement of treatments: MBM (no or yes), AB (no or yes, zinc bacitracin + salinomycin), and phytase level (500 or 1,500 FTU/kg; both using 500 matrix recommendations). Diets were based on wheat–soybean meal–canola meal. All birds were challenged with Eimeria spp on day 9 and Clostridium perfringens (C. perfringens) strain EHE-NE18 on day 14 and day 15. On day 21 (postchallenge), birds fed MBM had reduced weight gain (WG; P

In recent years, much progress has been made in understanding the mechanisms of bone growth and development over a lifespan, including the crosstalk between muscle and bone, to achieve optimal structure and function. While there have been significant advances in understanding how to help improve and maintain bone health in normal individuals, there is limited knowledge on whether these mechanisms apply or are compromised in pathological states. X-linked hypophosphatemia (XLH) (ORPHA:89936) is a rare, heritable, renal phosphate-wasting disorder. The resultant chronic hypophosphatemia leads to progressive deterioration in musculoskeletal function, including impaired growth, rickets, and limb deformities in children, as well as lifelong osteomalacia with reduced bone quality and impaired muscle structure and function. The clinical manifestations of the disease vary both in presentation and severity in affected individuals, and many of the consequences of childhood defects persist into adulthood, causing significant morbidity that impacts physical function and quality of life. Intervention to restore phosphate levels early in life during the critical stages of skeletal development in children with XLH could optimize growth and may prevent or reduce bone deformities in childhood. A healthier bone structure, together with improved muscle function, can lead to physical activity enhancing musculoskeletal health throughout life. In adults, continued management may help to maintain the positive effects acquired from childhood treatment, thereby slowing or halting disease progression. In this review, we summarize the opinions from members of a working group with expertise in pediatrics, epidemiology, and bone, joint and muscle biology, on potential outcomes for people with XLH, who have been optimally treated from an early age and continue treatment throughout life.

The objective of this study was to evaluate the influence of the titanium nanotube diameter and the effect of silicon carbide (SiC) coating on the proliferation and mineralization of pre-osteoblasts on titanium nanostructured surfaces. Anodized titanium sheets with nanotube diameters of 50 and 100 nm were used. The following four groups were tested in the study: (1) non-coated 50 nm nanotubes; (2) SiC-coated 50 nm titanium nanotubes; (3) non-coated 100 nm nanotubes and (4) SiC-coated 100 nm nanotubes. The biocompatibility and cytotoxicity of pre-osteoblasts were evaluated using a CellTiter-BlueCell Viability assay after 1, 2, and 3 days. After 3 days, cells attached to the surface were observed by SEM. Pre-osteoblast mineralization was determined using Alizarin-Red staining solution after 21 days of cultivation. Data were analyzed by a Kruskal–Wallis test at a p-value of 0.05. The results evidenced biocompatibility and non-cytotoxicity of both 50 and 100 nm diameter coated and non-coated surfaces after 1, 2 and 3 days. The statistical analysis indicates a statistically significant higher cell growth at 3 days (p < 0.05). SEM images after 3 days demonstrated flattened-shaped cells without any noticeable difference in the phenotypes between different diameters or surface treatments. After 21 days of induced osteogenic differentiation, the statistical analysis indicates significantly higher osteoblast calcification on coated groups of both diameters when compared with non-coated groups (p < 0.05). Based on these results, we can conclude that the titanium nanotube diameter did not play any role on cell viability or mineralization of pre-osteoblasts on SiC-coated or non-coated titanium nanotube sheets. The SiC coating demonstrated biocompatibility and non-cytotoxicity and contributed to an increase in osteoblast mineralization on titanium nanostructured surfaces when compared to non-coated groups.

Bone physiology is regulated by osteoblast and osteoclast activities, both involved in the bone remodeling process, through deposition and resorption mechanisms, respectively. The imbalance between these two phenomena contributes to the onset of bone diseases. Among these, osteoporosis is the most common metabolic bone disorder. The therapies currently used for its treatment include antiresorptive and anabolic agents associated with side effects. Therefore, alternative therapeutic approaches, including natural molecules such as coumarin and their derivatives, have recently shown positive results. Thus, our proposal was to investigate the effect of the coumarin derivative umbelliferon (UF) using an interesting model of human osteoblasts (hOBs) isolated from osteoporotic patients. UF significantly improved the activity of osteoporotic-patient-derived hOBs via estrogen receptor 1 (ESR1) and the downstream activation of β-catenin pathway. Additionally, hOBs were co-cultured in microgravity with human osteoclasts (hOCs) using a 3D system bioreactor, able to reproduce the bone remodeling unit in bone loss conditions in vitro. Notably, UF exerted its anabolic role by reducing the multinucleated cells. Overall, our study confirms the potential efficacy of UF in bone health, and identified, for the first time, a prospective alternative natural compound useful to prevent/treat bone loss diseases such as osteoporosis.

Despite the significant advancement in bone tissue engineering, it is still challenging to find a desired scaffold with suitable mechanical and biological properties, efficient bone formation in the defect area, and antibacterial resistivity. In this study, the zeolite (ZSM-5) scaffold was developed using the space holder method, and a novel vancomycin-loaded alginate coating was developed on it to promote their characteristics. Our results demonstrated the importance of alginate coating on the microstructure, mechanical, and cellular properties of the ZSM-5 scaffold. For instance, a three-fold increase in the compressive strength of coated scaffolds was observed compared to the uncoated ZSM-5. After the incorporation of vancomycin into the alginate coating, the scaffold revealed significant antibacterial activity against Staphylococcus aureus (S. aureus). The inhibition zone increased to 35 mm. Resets also demonstrated 74 ± 2.5% porosity, 4.3 ± 0.07 MPa strength in compressive conditions, acceptable cellular properties (72.3 ± 0.2 (%control) cell viability) after 7 days, good cell attachment, and calcium deposition. Overall, the results revealed that this scaffold could be a great candidate for bone tissue engineering.

The present trial was designed to assess the effect of phytase, multi-strain probiotic, Saccharomyces cerevisiae, and fumaric acid on performance, nutrient digestibility, bone physical parameters and mineralization, blood constituents, bone and gut histomorphology, and duodenal phosphorus transporter genes of broiler chickens fed a decreased non-phytate phosphorus (nPP) diet for 5 weeks. A total of 480 broiler chickens were allotted to six dietary groups and eight replicates each: (1) positive control diet with recommended levels of nPP (PC; 0.48, 0.44, and 0.41% in the three feeding phases); (2) negative control diet with a decreased dietary nPP (NC; 0.28, 0.24, and 0.21% in the three feeding phases); (3) NC + 600 FTU/kg phytase (PHY); (4) NC + 0.05% multi-strain probiotic (PRO); (5) NC + 0.2% Saccharomyces cerevisiae (SC); and (6) NC + 0.2% fumaric acid. Growth performance data were recorded weekly, and blood sampling was performed at days 21 and 35 of age. Bone quality traits, gut and tibia histology, nutrient digestibility, and intestinal gene expression analyses were conducted at the end of the trial (35 days of age). Final body weight and total gain at day 35 of age of the broiler chickens fed with the PHY, PRO, and SC diets were greater (p < 0.01) than in NC, where broilers fed with the PRO and PHY diets had higher values and were similar to that of PC. There was a non-significant variation in the cumulative feed intake among the treatment groups. The PHY and PRO groups had better FCR than the PC group (p < 0.05), and FA and SC had an FCR equivalent to that of PC. The PHY and PRO broilers had greater dressing % than the NC group (p < 0.05) and even better than PC. The PHY, PRO, SC, and FA broilers had higher relative weights of spleen and bursa of Fabricius (p < 0.01) than NC. In comparison to NC, the PHY, PRO, and SC groups improved (p < 0.05) CP, CF, Ca, and P digestibility. Greater tibia breaking strength of the low nPP-supplemented groups was shown to be associated with higher tibia ash, Ca, and P concentrations (p < 0.01) and increased (p < 0.001) tibia cortical area thickness. At days 21 and 35 of age, the dietary supplements to low nPP diets reduced (p < 0.05) serum total cholesterol, triglyceride, triiodothyronine, thyroxine, glucose, and alkaline phosphatase levels, while serum Ca and P concentrations were improved (p < 0.05) compared to NC. All supplements led to enhancement (p < 0.01) in villi height and width and villi absorptive surface area when compared with NC and were even comparable to that of PC. The mRNA expression of NaP-IIb was up-regulated (p < 0.001) in the duodenum of PRO and FA broilers at day 35 of age compared with NC, and their expression levels were similar to that of PC, indicating greater P availability. It is concluded that dietary supplementation of PHY, PRO, SC, and FA to a low nPP diet was advantageous and mitigated the negative impacts of P reduction on the growth performance, health, nutrient digestibility, and bone quality of broilers.

Paeonia suffruticosa is a magnificent and long-lived woody plant that has traditionally been used to treat various diseases including inflammatory, neurological, cancer, and cardiovascular diseases. In the present study, we demonstrated the biological mechanisms of paeonoside (PASI) isolated from the dried roots of P. suffruticosa in pre-osteoblasts. Herein, we found that PASI has no cytotoxic effects on pre-osteoblasts. Migration assay showed that PASI promoted wound healing and transmigration in osteoblast differentiation. PASI increased early osteoblast differentiation and mineralized nodule formation. In addition, PASI enhanced the expression of Wnt3a and bone morphogenetic protein 2 (BMP2) and activated their downstream molecules, Smad1/5/8 and β-catenin, leading to increases in runt-related transcription factor 2 (RUNX2) expression during osteoblast differentiation. Furthermore, PASI-mediated osteoblast differentiation was attenuated by inhibiting the BMP2 and Wnt3a pathways, which was accompanied by reduction in the expression of RUNX2 in the nucleus. Taken together, our findings provide evidence that PASI enhances osteoblast differentiation and mineralized nodules by regulating RUNX2 expression through the BMP2 and Wnt3a pathways, suggesting a potential role for PASI targeting osteoblasts to treat bone diseases including osteoporosis and periodontitis.

There is limited information on the effects of enteric pathogen on bone quality in rapidly growing broiler chicks. We examined tibia and femur attributes (length, diameter, relative weight of ash content [AC] to the BW, ash concentration [AP]) and serum bone-turnover markers including receptor activator of nuclear factor kappa-B ligand (RANKL) for resorption, alkaline phosphatase (ALP) for mineralization, and selected serum metabolites in 14-day-old broilers challenged with Eimeria. A total of 160 (80 males and 80 females) 1-day-old Ross × Ross 708 chicks were used. Based on BW, birds were placed within sex in cages (5 birds per cage) and fed chick starter diets to day 9 of age. On day 9, half of the cages were orally gavaged with 1 mL of Eimeria culture (100,000 oocysts of E. acervulina and 25,000 oocysts of E. maxima) and the other half (unchallenged control) received 1 mL 0.9% saline in distilled water. On day 14, 2 birds were randomly selected and necropsied for intestinal lesion score, blood, tibia, and femur samples. Data were analyzed in a 2 (challenged vs. unchallenged) × 2 (males vs. females) factorial arrangement. There was no interaction (P > 0.05) between Eimeria and sex on any measurement. Whereas there were no intestinal lesions in unchallenged birds, Eimeria resulted in lesion score (0 to 4) of 3.35, 2.59 and 0.11 in duodenum, jejunum and ileum, respectively. Eimeria challenge decreased (P < 0.05) tibia AC and AP by 10 and 8.2%, respectively but had no (P > 0.10) effect on femur attributes. Generally, males showed (P < 0.05) longer and wider bones with more AC compared with the female. Circulating serum RANKL concentration increased (P = 0.017) in response to Eimeria challenge and was negatively correlated with tibia AC (–0.731; P = 0.021). Our findings showed that Eimeria damage to the intestinal physiology had adverse effects on long bone attributes linked to increased resorption.

ObjectivesLong bone defects often require surgical intervention for functional restoration. The ‘gold standard’ treatment is autologous bone graft (ABG), usually from the patient’s iliac crest. However, autograft is plagued by complications including limited supply, donor site morbidity, and the need for an additional surgery. Thus, alternative therapies are being actively investigated. Autologous bone marrow (BM) is considered as a candidate due to the presence of both endogenous reparative cells and growth factors. We aimed to compare the therapeutic potentials of autologous bone marrow aspirate (BMA) and ABG, which has not previously been done.MethodsWe compared the efficacy of coagulated autologous BMA and ABG for the repair of ulnar defects in New Zealand White rabbits. Segmental defects (14 mm) were filled with autologous clotted BM or morcellized autograft, and healing was assessed four and 12 weeks postoperatively. Harvested ulnas were subjected to radiological, micro-CT, histological, and mechanical analyses.ResultsComparable results were obtained with autologous BMA clot and ABG, except for the quantification of new bone by micro-CT. Significantly more bone was found in the ABG-treated ulnar defects than in those treated with autologous BMA clot. This is possibly due to the remnants of necrotic autograft fragments that persisted within the healing defects at week 12 post-surgery.ConclusionAs similar treatment outcomes were achieved by the two strategies, the preferred treatment would be one that is associated with a lower risk of complications. Hence, these results demonstrate that coagulated BMA can be considered as an alternative autogenous therapy for long bone healing. Cite this article: Z. X. H. Lim, B. Rai, T. C. Tan, A. K. Ramruttun, J. H. Hui, V. Nurcombe, S. H. Teoh, S. M. Cool. Autologous bone marrow clot as an alternative to autograft for bone defect healing. Bone Joint Res 2019;8:107–117. DOI: 10.1302/2046-3758.83.BJR-2018-0096.R1.

Bone is a complex organ maintained by three main cell types: osteoblasts, osteoclasts, and osteocytes. During bone formation, osteoblasts deposit a mineralized organic matrix. Evidence shows that bone cells release extracellular vesicles (EVs): nano-sized bilayer vesicles, which are involved in intercellular communication by delivering their cargoes through protein–ligand interactions or fusion to the plasma membrane of the recipient cell. Osteoblasts shed a subset of EVs known as matrix vesicles (MtVs), which contain phosphatases, calcium, and inorganic phosphate. These vesicles are believed to have a major role in matrix mineralization, and they feature bone-targeting and osteo-inductive properties. Understanding their contribution in bone formation and mineralization could help to target bone pathologies or bone regeneration using novel approaches such as stimulating MtV secretion in vivo, or the administration of in vitro or biomimetically produced MtVs. This review attempts to discuss the role of MtVs in biomineralization and their potential application for bone pathologies and bone regeneration.

Introducción La osteopenia es frecuente en los prematuros, y cursa con deficiencia de la disponibilidad mineral del hueso. Si bien el tratamiento de Fisioterapia con movilizaciones pasivas con presión articular (MPPA) ha mostrado beneficios, se desconoce el efecto de la terapia de locomoción refleja (TLR) Objetivos Determinar el efecto de la TLR en la mineralización, modelado óseo, y el crecimiento en bebés prematuros, y su comparación con las MPPA. Metodología Se realizaron dos ensayos clínicos aleatorizados con tres grupos de tratamiento y una muestra de 106 bebés prematuros. El primer estudio, multicéntrico, con pacientes procedentes del Hospital Clínico Universitario Virgen de la Arrixaca (HCUVA), el Hospital Torrecárdenas de Almería y el Hospital General Universitario de Elche; y el segundo estudio, con pacientes prematuros de HCUVA. Se comparó el efecto de la TLR frente a las MPPA y el masaje en la mineralización, el remodelado óseo y el crecimiento de los prematuros. Resultados Se encuentran diferencias significativas a favor del grupo tratado con TLR en la mineralización (p=0.049), la formación ósea (p=0.013), y la talla (p=0.018), respecto a los demás grupos. No se observaron diferencias en las variables antropométricas de peso y perímetro cefálico. Conclusiones La TLR ha mostrado ser más efectiva en la mejora de la mineralización ósea que las MMPA, la formación ósea y el crecimiento en bebés prematuros. Este hecho convierte a la TLR como una de las modalidades de Fisioterapia de elección para el tratamiento y la prevención de la osteopenia del prematuro. Introduction Osteopenia is common in preterm infants, and it is produced by a deficiency in bone mineral availability. Although Physiotherapy treatment with passive mobilizations with gentle joint compression (PMC) has shown benefits, the effect of reflex locomotion therapy (RLT) is unknown. Objectives To determine the effect of RLT on mineralization, bone remodelling, and growth in preterm infants, and its comparison with PMC. Methods Two randomized clinical trials were conducted with three treatment groups and a sample of 106 premature babies. The first multicentre study with patients from the Virgen de la Arrixaca University Clinical Hospital (HCUVA), the Torrecárdenas Hospital in Almería and the General University Hospital of Elche; and the second study, with preterm infants from HCUVA. The effect of RLT versus PMC and massage on mineralization, bone remodeling and growth of preterm infants was compared. Results There are significant differences in favour of the group treated with RLT in mineralization (p = 0.049), bone formation (p = 0.013), and height (p = 0.018), compared to the other groups. No differences were observed in the anthropometric variables of weight and head circumference. Conclusions RLT has been shown to be more effective in improving bone mineralization, bone formation, and growth than PMC in preterm infants. This fact makes RLT one of the physiotherapy modalities of choice for the treatment and prevention of osteopenia of

Scope: Osteoporosis poses a health challenge especially for postmenopausal women. This study aims to explore nutritional strategies to counteract bone demineralization in ovarierectomized (OVX) rats. Methods and Results: OVX rats (n = 49) are fed with one of six different diets, where two different calcium sources (dairy calcium or calcium carbonate) are provided alone or in combination with either inulin (5%) or lactose (0.5%). In addition, a calcium-deficient diet is included. Calcium supplementation increases intestinal concentrations of short-chain fatty acids (SCFAs) and the abundance of fecal Acinetobacter and Propionibacterium. Accompanied with these effects, rats fed with calcium-fortified diets have higher bone mineral density, bone mineral content and femur mechanical strength, lower serum levels of bone markers, and lower expression of calcium absorption-related genes (transient receptor potential vanilloid type 6 (TRPV6), calcium-binding protein (CaBP) compared with control. Inulin supplementation results in a markedly increased production of intestinal SCFAs, a decreased intestinal pH, an increased abundance of Allobaculum and Bifidobacterium, and an increased expression of Trpv6. Inulin and lactose show beneficial effects on spine bone. Conclusion: Calcium modulates gut microbiome composition and function. A pronounced effect of inulin on metabolic activity in the gastrointestinal tract is evident, and lactose supplementation decreases jejunal pH that might be associated with slightly enhanced bone mineralization.

Phosvitin (PV) from egg yolk is an excellent substrate for the production of phosphopeptides, which have a strong calcium chelating capacity and promoting calcium absorption and bone mineralization. This study investigated the effect of PV hydrolysates produced using a effective preparation method (high temperature (121°C) and mild pressure (0.1 MPa), HTMP) or HTMP pretreatment and trypsin hydrolysis combination (HTMP-PV18) on the physiology of an osteoblast MC3T3-E1 cells line. The proliferation, apoptosis, and differentiation of MC3T3-E1 cells were analyzed using the CCK-8, flow cytometry, and RT-PCR reactions, respectively. Both the HTMP-PV and HTMP-PV18 increased the proliferation, and inhibited the apoptosis of MC3T3-E1 cells significantly. The HTMP-PV increased the proliferation of MC3T3-E1 cells by 147.12 ± 2.11% and the HTMP-PV18 by 136.43 ± 4.51%. In addition, the HTMP-PV and HTMP-PV18 effectively promoted the expression of genes related to the OPG/RANKL signaling channel during cell differentiation. This indicated that both the HTMP-PV and HTMP-PV18 have the potential to promote bone mineralization by improving the proliferation and differentiation of osteoblastic cells.

Dioxins (including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are highly toxic and persistent chemicals widely distributed in the environment in trace amounts, and are side products of industrial and chemical processes. Exposure to dioxins leads to multiorgan morphological and functional abnormalities, including within the bone tissue, disrupting its microarchitecture and mechanical properties. Xanthohumol (XN) is a chemical compound classified as a prenylated flavonoid, distinguished by multidirectional biological action. The aim of the study is to assess whether xanthohumol, as a substance with strong antioxidant and anti-inflammatory properties, has the ability to eliminate the negative effects of TCDD on bone tissue. The experiment was conducted on adult Japanese quails. Two different doses of TCDD and xanthohumol were administered to birds. After euthanasia of animals, the research material in the form of cranial vault and hind limb bone was collected, and their mineral compositions of calcium, phosphorus, magnesium, zinc, and iron concentrations were determined using atomic emission spectrometry in an acetylene-air flame method. Our results indicate that the administration of TCDD at a low dose causes more dynamic changes in the concentration of elements in bone, in comparison to a higher dose of dioxin. Results show also that higher doses of the XN cause the linear increase in the concentration of phosphorus and iron in the bone of the hind limb, and calcium in the bones of the cranial vault. In conclusion, our experiment shows that the use of TCDD and XN in Japanese quails together in various doses influences the content of phosphorus, magnesium, zinc, and iron in the research material.

Bone mineralization is an orchestrated process by which mineral crystals are deposited by osteoblasts; however, the detailed mechanisms remain to be elucidated. The presence of P2X7 receptor (P2X7R) in immature and mature bone cells is well established, but contrasting evidence on its role in osteogenic differentiation and deposition of calcified bone matrix remains. To clarify these controversies in the present study, we investigated P2X7R participation in bone maturation. We demonstrated that the P2X7R is expressed and functional in human primary osteoblasts, and identified in the P2RX7 promoter several binding sites for transcription factors involved in bone mineralization. Of particular interest was the finding that P2X7R expression is enhanced by nuclear factor of activated T cells cytoplasmic 1 (NFATc1) overexpression, and accordingly, NFATc1 is recruited at the P2RX7 gene promoter in SaOS2 osteoblastic-like cells. In conclusion, our data provide further insights into the regulation of P2X7R expression and support the development of drugs targeting this receptor for the therapy of bone diseases.

Background: The prevalence of vertebral fractures (VF) and their association with clinical risk factors and outcomes are poorly documented in chronic kidney disease (CKD) cohorts. The aim of the study was to evaluate the prevalence of VF in patients with non-dialysis dependent CKD (NDD-CKD), their value in predicting mortality and its correlation with parameters of bone mineral metabolism and vascular calci?cation. Materials and Methods: 612 NDD 3-5 stage CKD patients participating in the OSERCE-2 study, a prospective, multicenter, cohort study, were prospectively evaluated and categorized into two groups according to presence or absence of VF at enrollment. VF were assessed with lateral radiographs and Genant semi-quantitative method was applied. Three radiologists specialized in musculoskeletal radiology performed consensual reading of individual images obtained using a Raim DICOM Viewer and a Canon EOS 350 camera to measure with Java Image software in those who had traditional acetate X-ray. Factors related to VF were assessed by logistic regression analysis. Association between VF and death over a 3-year follow-up was assessed by Kaplan-Meier survival curves and Cox-proportional hazard models. Results: VF were detected in 110patients(18%). Serumphosphatelevels(OR0.719,95%CI0.532to0.972,p = 0.032),ankle-brachial index < 0.9 (OR 1.694, 95% CI 1.056-2.717, p = 0.029) and treatment with bisphosphonates (OR 5.636, 95% CI 1.876-16.930, p = 0.002) were independently related to the presence of VF. After a median follow-up of 35 months (IQR: 17-37 months), 62 patients (10%) died. The causes of death were cardiovascular (n = 21, 34%) and infectious (n = 11, 18%). In the crude analysis, fractured patients group had poorer survival (log-rank test, p = 0.02). After multivariate adjustment for age, MDRD, albumin, diabetes mellitus, comorbidity, Adragao Score > 3 and serum phosphate, the presence of VF (HR 1.983, 95% CI 1.009-3.898, p = 0.047) were an independent predictor of all-cause mortality. Conclusions: In our study 18% of patients with NDD-CKD have VF. Factors associated with VF were age, low serum phosphate levels and peripheral vascular disease. The presence of VF was an independent risk factor for mortality in stages 3-5 NDD-CKD patients. Clinical trials are needed to con?rm whether this relationship is causal and reversible with treatment for osteoporosis. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

A calcium deficiency is detected in more than 80% of children. This is the result of inadequate consumption of milk and dairy products which are the main food sources of calcium. There is a correlation between deficiency of calcium intake with food in childhood and the risk of osteopenia and osteoporosis in subsequent life periods. With insufficient exogenous intake of calcium, its concentration in the blood decreases which stimulates bone resorption. The factors that further limit the consumption of dairy products are lactase deficiency and cow's milk protein allergy. In order to ensure the intake of the necessary amount of calcium, it is advisable to use vitaminmineral complexes in children that contain not only a sufficient amount of calcium and vitamin D but also other micronutrients required for bone formation.

The aim of this study was to investigate the effects of olive leaf and marigold extracts on the apparent total tract digestibility (ATTD) of the principal nutrients and energy, as well as on mineral utilization (Ca, P, Mg, Mn, Fe, Cu and Zn) in relation to bone characteristics in broilers fed walnut- or linseed oil-supplemented diets. Thirty-six 12-day-old commercial broilers Ross 308 were reared in metabolic cages, assigned to one of the six dietary treatments (3 × 2 factorial design): three supplements (not supplemented, olive leaf extract, or marigold extract), and two oils (walnut or linseed oil). The results showed that the marigold extract reduced Zn and P balances and tended to lower the balance of ash and Mg, and the ATTD of Zn and Mg. Diets with linseed oil increased the ATTD of acid detergent fiber and reduced the ATTD of the organic residue and Cu. No differences in the bone characteristics of tibia were observed between treatments. These results indicated that the inclusion of marigold extract had a negative effect on the Zn and P balance, and that neither extract had any major effect on the digestion and utilization of energy and other investigated nutrients, or on bone mineralization, irrespective of the oil source included in the diet.

Larsen, MN, Nielsen, CM, Ørntoft, CØ, Randers, M, Manniche, V, Hansen, L, Hansen, PR, Bangsbo, J, and Krustrup, P. Physical fitness and body composition in 8–10-year-old Danish children are associated with sports club participation. J Strength Cond Res 31(12): 3425–3434, 2017—We investigated whether physical fitness and body composition in 8–10-year-old Danish children are associated with sports club participation. The study included 423 schoolchildren, comprising 209 girls and 214 boys, of whom 67 and 74%, respectively, were active in sports clubs. Yo-Yo intermittent recovery level 1 for children (YYIR1C), balance, jump and coordination tests, together with dual-energy X-ray absorptiometry, were used to determine exercise capacity and body composition. Children active in sports clubs had better (p < 0.05) YYIR1C (33%, 767 ± 26 vs. 575 ± 29 m), 20-m sprint (3%, 4.33 ± 0.03 vs. 4.48 ± 0.04 seconds), coordination (6%, 68 ± 1 vs. 72 ± 1 second), and balance test performances (9%, 19.3 ± 0.5 vs. 21.2 ± 0.7 falls·min−1) and lower fat mass index (16%, 3.8 ± 0.1 vs. 4.5 ± 0.2 kg[fat]·m−2) than children not active in sports clubs. Ball game players had better (p < 0.05) YYIR1C (38%, 925 ± 39 vs. 671 ± 28 m), 20-m sprint (4%, 4.25 ± 0.03 vs. 4.42 ± 0.04 seconds), and coordination test performances (5%, 65 ± 1 vs. 69 ± 1 second), along with higher (p < 0.05) lean body mass (5%, 24.00 ± 0.22 vs. 22.83 ± 0.25 kg) and whole-body BMD (2%, 0.90 ± < 0.01 vs. 0.88 ±

Two trials were conducted to determine the non-phytate phosphorus (nPP) requirement for broiler under heat stress. In both trials, birds were distributed in a completely randomized 4 × 2 factorial design with four nPP concentrations: 0.25, 0.35, 0.45, and 0.55%, and two Ca supply techniques: Ca fixed at 0.899% (CaF) or varying along with nPP aiming a 2:1 Ca to nPP ratio (CaV). Both trials had eight pens/treatment, with nine and five birds/pen for exp. 1 and exp. 2, respectively. nPP concentration had no effect on feed intake (FI), body weight gain (BWG), feed conversion ratio (FCR), nor fat deposition ratio (FDR). nPP levels showed a linear effect on protein deposition ratio (PDR) only for CaF diets. The nPP levels had a significant effect, regardless the technique adopted, on tibia phosphorus (TibP), which varied quadratically, on tibia calcium (TibCa) that increased quadratically and linearly, respectively, on CaF and CaV diets, and on tibia ash (TibAsh) that showed a quadratic effect for both. No effect was observed on Ca to P ratio in the tibia (TibCa:TibP). The nPP levels showed a linear increase effect over phosphorus intake (PI), phosphorus excreted (PE), and phosphorus retained (PR), and a linear decrease effect on phosphorus retention coefficient (PRC). Therefore, the nPP requirement for broilers from 8 to 21 days of age that provided better performance and bone variables were 0.250 and 0.484%, respectively, for CaF diets and 0.250 and 0.511%, respectively, for CaV diets.

Summary Aluminum doped magnetite (Fe3O4) magnetic nanoparticle (MNP) has been used as an effective sorbent to remove phosphorus (P) species in poultry processing wastewaters. It was found that spent MNP contains approximately 13% of P by weight. A study was conducted to evaluate the potential of using spent MNP as a P source. A total of 240 one-day-old male broilers were randomly allocated to four dietary treatments and six replicate each for 18 days. Treatments consisted of 1) positive control (PC) with 0.45% nonphytate-P (nPP); 2) negative control (NC) with a reduction of 0.15% nPP; 3) NC with additional 0.075% of P from magnetic nanoparticle (NC + 0.075% P-MNP); and 4) NC with additional 0.15% of P from magnetic nanoparticle (NC + 0.15% P-MNP). Body weight and feed intake were recorded at day 7, 14, and 18. Three birds were randomly selected from each cage for Dual-energy X-ray Absorptiometry (DXA) scanning at d18. The liver, heart, and spleen were collected and analyzed for mineral content. Ileal digesta were collected from the rest of the birds for P digestibility analysis. Data were subjected to SAS using one-way ANOVA model with GLM procedure. The birds fed with additions of 0.075% and 0.15% P from MNP had an increased (P = 0.017) body weight gain at d8 to d14 compared to those with NC diet. Significant lower (P ≤ 0.008) body mineral density and body mineral content were observed in the birds with NC diet compared to those with PC, NC + 0.075% P- magnetic nanoparticle, and NC + 0.15% P- magnetic nanoparticle diets. In addition, the birds with NC + 0.075% P- magnetic nanoparticle and NC + 0.15% P- magnetic nanoparticle had similar body mineral contents and body mineral densities compared to the PC-fed birds, indicating P from spent magnetic nanoparticle has incorporated into the bone mineralization. No significant differences were found between the treatments for mineral residues (Al and Fe) in liver, heart and spleen samples. The ileal P digestibility of dry matter and P was not influenced by the presence of magnetic nanoparticle in diets. In conclusion, 0.15% P from spent magnetic nanoparticle could be efficiently and safely used to replace the same amount P from dicalcium phosphate in the diets for broilers.

The objective of the present study was to investigate the effects of chronic lead exposure on the mineral properties of alveolar bone. For this purpose, female Wistar rats (n = 8) were exposed to 1000 ppm lead acetate in drinking water for 90 days, while the control group (n = 5) was treated with sodium acetate. The alveolar bone structure and chemical composition of the dissected man‐ dibles were examined using micro‐computed tomography (micro‐CT), scanning electron micros‐ copy (SEM), inductively coupled plasma optical emission spectrometry (ICP‐OES), attenuated total reflection Fourier transform infrared spectroscopy (ATR‐FTIR), and X‐ray diffraction (XRD) tech‐ niques to determine possible alterations in alveolar bone due to lead exposure. In addition, changes in bone mechanical properties were analysed using a three‐point bending test. Exposure to lead induced notable changes in bone mineralization and properties, specifically a reduction of the tra‐ becular thickness and bone mineral density. Furthermore, there was a reduction in carbonate con‐ tent and an increase in bone mineral crystallinity. These changes in bone mineralization could be explained by an alteration in bone turnover due to lead exposure. Three‐point bending showed a trend of decreased displacement at failure in the mandibles of lead‐exposed rats, which could com‐ promise the mechanical stability and normal development of the dentition., MICINN‐CGL2011‐25906 (Spain), UBACYT‐ 20020150100006BA (Argentina)

Summary: An experiment was conducted to evaluate the effects of phytase and multicarbohydrase (MC) on growth performance, bone ash, and nutrient digestibility in broilers fed a nutritionally reduced diet. A total of 480 Cobb 500 male broilers were randomly allocated to 8 treatments with 6 replicates of 10 birds each. Experimental diets consisted of a positive control (PC); a negative control (NC) with the reduction of ME, CP, Ca, and nonphytate phosphorus by 75 kcal/kg, 1.5, 0.15, and 0.15%, respectively; NC plus phytase at 500, 1,000, or 1,500 FTU/kg as T3–T5; and further plus MC at 500 mg/kg as T6–T8. Results showed that during 0–18 d of age, feed conversion ratio (FCR) was increased (P

Summary An experiment was conducted to evaluate high doses of phytase associated with multicarbohydrase complex in diets with a nutritional reduction on feed cost, performance, intestinal morphometry, digesta viscosity, blood and bone parameters, and carcass yield of broilers. For this purpose, 1,540 1-day-old male broiler chicks were distributed in a completely randomized design with 5 treatments, with 14 replicates and 22 birds each. The treatments consisted of the following: positive control (PC), a diet meeting the nutritional requirements without enzymes; negative control (NC), a PC diet with a reduction of 100 kcal/kg AME, 0.15% available P, 0.12% Ca, 4% digestible lysine, and 5% Na; NC diet + multicarbohydrase complex (MCC) + phytase at 3 levels (1,000, 1,500, and 2,000 FTU/kg). The results demonstrated that the use of phytase associated with MCC in nutrient-restricted diets maintained broiler performance in the prestarter phase. From 1 to 42 d, broilers fed enzyme-supplemented diets had a better performance than those receiving nutrient-restricted diets without enzymes. The different levels of phytase associated with MCC affected the serum biochemical parameters of broilers; however, these modifications did not influence bone characteristics. The mode of action of enzymes is substrate-dependent, which may have limited enzyme activity. As a result, no improvements in carcass yield or intestinal and bone parameters were observed. It is concluded that diets with nutritional reduction and addition of 1,000 FTU/kg phytase in association with MCC maintain the performance and is a feasible strategy to reduce feed cost.

Paeonia suffruticosa has been extensively used as a traditional medicine with various beneficial effects, paeonolide (PALI) was isolated from its dried roots. This study aimed to investigate the novel effects and mechanisms of PALI in pre-osteoblasts. Here, cell viability was evaluated using an MTT assay. Early and late osteoblast differentiation was examined by analyzing the activity of alkaline phosphatase (ALP) and by staining it with Alizarin red S (ARS). Cell migration was assessed using wound healing and Boyden chamber assays. Western blot and immunofluorescence analyses were used to examine the intracellular signaling pathways and differentiation proteins. PALI (0.1, 1, 10, 30, and 100 μM) showed no cytotoxic or proliferative effects in pre-osteoblasts. In the absence of cytotoxicity, PALI (1, 10, and 30 μM) promoted wound healing and transmigration during osteoblast differentiation. ALP staining demonstrated that PALI (1, 10, and 30 μM) promoted early osteoblast differentiation in a dose-dependent manner, and ARS staining showed an enhanced mineralized nodule formation, a key indicator of late osteoblast differentiation. Additionally, low concentrations of PALI (1 and 10 μM) increased the bone morphogenetic protein (BMP)–Smad1/5/8 and Wnt–β-catenin pathways in osteoblast differentiation. Particularly, PALI (1 and 10 μM) increased the phosphorylation of ERK1/2 compared with BMP2 treatment, an FDA-approved drug for bone diseases. Furthermore, PALI-mediated early and late osteoblast differentiation was abolished in the presence of the ERK1/2 inhibitor U0126. PALI-induced RUNX2 (Cbfa1) expression and nuclear localization were also attenuated by blocking the ERK1/2 pathway during osteoblast differentiation. We suggest that PALI has biologically novel activities, such as enhanced osteoblast differentiation and bone mineralization mainly through the intracellular ERK1/2-RUNX2 signaling pathway, suggesting that PALI might have therapeutic action and aid the treatment and prevention of bone diseases, such as osteoporosis and periodontitis.

Hypophosphatasia (HPP) is a rare autosomal dominant or recessive metabolic disorder caused by mutations in the tissue nonspecific alkaline phosphatase gene (ALPL). To date, over 300 different mutations in ALPL have been identified. Disease severity is widely variable with severe forms usually manifesting during perinatal and/or infantile periods while mild forms are sometimes only diagnosed in adulthood or remain undiagnosed. Common clinical features of HPP are defects in bone and tooth mineralization along with the biochemical hallmark of decreased serum alkaline phosphatase activity. The incidence of severe HPP is approximately 1 in 300,000 in Europe and 1 in 100,000 in Canada. We present the clinical and molecular findings of 83 probands and 28 family members, referred for genetic analysis due to a clinical and biochemical suspicion of HPP. Patient referrals included those with isolated low alkaline phosphatase levels and without any additional clinical features, to those with a severe skeletal dysplasia. Thirty-six (43.3%) probands were found to have pathogenic ALPL mutations. Eleven previously unreported mutations were identified, thus adding to the ever increasing list of ALPL mutations. Seven of these eleven were inherited in an autosomal dominant manner while the remaining four were observed in the homozygous state. Thus, this study includes a large number of well-characterized patients with hypophosphatasemia which has permitted us to study the genotype:phenotype correlation. Accurate diagnosis of patients with a clinical suspicion of HPP is crucial as not only is the disease life-threatening but the patients may be offered bone targeted enzymatic replacement therapy. © 2017 Wiley Periodicals, Inc.

Alternations of collagen and mineral at the molecular level may have a significant impact on the strength and toughness of bone. In this study, scanning transmission electron microscopy (STEM) and electron energy-loss spectroscopy (EELS) were employed to study structural and compositional changes in bone pathology at nanometer spatial resolution. Tail tendon and femoral bone of osteogenesis imperfecta murine (oim, brittle bone disease) and wild type (WT) mice were compared to reveal defects in the architecture and chemistry of the collagen and collagen-mineral composite in the oim tissue at the molecular level. There were marked differences in the substructure and organization of the collagen fibrils in the oim tail tendon; some regions have clear fibril banding and organization, while in other regions fibrils are disorganized. Malformed collagen fibrils were loosely packed, often bent and devoid of banding pattern. In bone, differences were detected in the chemical composition of mineral in oim and WT. While mineral present in WT and oim bone exhibited the major characteristics of apatite, examination in EELS of the fine structure of the carbon K ionization edge revealed a significant variation in the presence of carbonate in different regions of bone. Variations have been also observed in the fine structure and peak intensities of the nitrogen K-edge. These alterations are suggestive of differences in the maturation of collagen nucleation sites or cross-links. Future studies will aim to establish the scale and impact of the modifications observed in oim tissues. The compositional and structural alterations at the molecular level cause deficiencies at larger length scales. Understanding the effect of molecular alterations to pathologic bone is critical to the design of effective therapeutics.

Background Nondairy beverages, produced from soy, rice, oat, almond, or coconut, are increasingly being used as alternatives to dairy milk, with the perception that they are healthier and/or more sustainable products than dairy products. Objective The aim of this study was to compare the effects of supplementing either bovine milk, soy, or almond-based beverages to young, growing rats fed an intact-protein diet or a diet that had protein substituted with amino acids (AA-diet). Methods Three-week-old male Sprague-Dawley rats were randomly assigned to 5 groups (n = 10/group) and fed ad libitum for 4 wk. Two control groups were fed either standard AIN-93G food [20% casein (CN) protein] or AIN-93G with amino acids (AAs) equivalent to CN protein, and water to drink. Three treatment groups were fed AIN-93G AA and supplemented with either bovine ultra-heat treatment (UHT) milk or soy or almond UHT beverages. Rat weight gain and food intakes were recorded. During week 4, body composition was assessed using DEXA to determine lean soft tissue, fat, and bone mass. At trial end, bone biomechanical properties and blood plasma mineral concentrations were measured. Results At the end of the trial, animals supplemented with almond beverage were lightest (P > 0.05), with higher plasma calcium concentrations (P > 0.05) and lower bone mineral content (BMC) and bone density (P > 0.05) than animals supplemented with milk or soy beverage. Soy-supplemented animals had similar BMC and bone density compared with milk-supplemented animals, although the soy group gained most weight (P > 0.05) and had the highest fat:lean ratio (P > 0.05) compared with other groups. Conclusions In the model tested, supplementing rats with bovine UHT milk and soy UHT beverage provided favorable bone health outcomes. Conversely, almond UHT beverage was not an effective supplement and could be detrimental to bone mineralization and strength outcomes.

Deoxynivalenol (DON) is one of the most abundant and important trichothecene mycotoxins produced by Fusarium species. In chickens, DON intake causes feed refusal, impairs performance, gut barrier function, and immunity, and raises oxidative stress. To determine the effect of DON on bone mineralization and serum calcium and phosphorus, 80 newly-hatched chickens were fed 4 diets with 0, 2.5, 5, and 10 mg DON/kg feed in this pilot study. In week 5, chickens were euthanized, femur and tibiotarsus bones were separated from the meat, and after incineration ash composition, as well as serum calcium and phosphorus, were determined using clinical biochemistry. Dietary DON reduced chicken dry matter, calcium, and phosphorus intake, and subsequently body and leg weight. DON affected bone density and composition of the tibiotarsus more drastically than of the femur. However, lower mineral intake did not solely explain our observations of the quadratically lower tibiotarsus density and ash content, as well as linearly decreased Ca content in the femur and tibiotarsus with increasing DON levels. Linearly decreasing serum phosphorus concentrations with increasing DON levels further supported impaired mineral homeostasis due to DON. In conclusion, already low dietary DON contamination of 2.5 mg/kg feed can compromise bone mineralization in chickens.

Hypophosphatasia (HPP) is a multi-systemic metabolic disorder caused by loss-of-function mutations in the ALPL gene that encodes the mineralization-associated enzyme, tissue-nonspecific alkaline phosphatase (TNSALP). HPP is characterized by defective bone and dental mineralization, leading to skeletal abnormalities with complications resulting in significant morbidity and mortality. Management of HPP has been limited to supportive care until the introduction of a recently approved enzyme replacement therapy employing bone-targeted recombinant human TNSALP, asfotase alfa (AA). This new therapy has been transformative as it improves survival in severely affected infants, and overall quality of life in children and adults with HPP. This review provides an overview of HPP, focusing on important steps in the development of AA enzyme replacement therapy, including the drug design, preclinical studies in the HPP mouse model, and outcomes from clinical trials and case report publications to date, with special attention given to response to therapy of skeletal manifestations, biochemical features, and other clinical manifestations. The limitations, adverse effects, and outcomes of AA are outlined and the place in therapy for individuals with HPP is discussed.

Summary: An experiment was conducted to evaluate the effects of dietary nonphytate phosphorus level and a novel corn-expressed phytase (GraINzyme, Agrivida Inc., Woburn, MA) supplementation on broiler growth performance and bone mineralization. Broilers were fed graded levels of nonphytate phosphorus from 0.20 to 0.44% increased by 0.06% and 4 levels of corn-expressed phytase (500; 1,500; 3,000; and 4,500 FTU/kg). Dicalcium phosphate was used as the reference for inorganic phosphorous. Increasing dietary nonphytate phosphorus level or supplementation of phytase linearly (P < 0.01) increased the bodyweight gain, feed intake, tibia fat–free dry weight, ash weight, ash percentage, and ash concentration. Feed conversion ratio was linearly (P < 0.01) improved as the dietary nonphytate phosphorus level was gradually increased. Graded nonphytate phosphorus levels were log-transformed and regressed against the bodyweight gain, tibia ash weight, ash percentage, and ash concentration for calculating the nonphytate phosphorus relative to the phytase efficacy. Both raw and standardized responses were regressed against dietary nonphytate phosphorus levels. Bodyweight gain was less sensitive than bone mineralization results. From the regression estimates using standardized responses, bodyweight gain was about 92% of tibia ash concentration in terms of sensitivity. The calculated nonphytate phosphorus relative to the equivalence for bodyweight gain, tibia ash weight, ash percentage, and ash concentration were 0.166, 0.158, 0.163, and 0.183%, respectively. Results suggest that the corn-expressed phytase is effectively released in the P form phytate and improves broiler growth performance and bone mineralization. Bone mineralization is a better indicator for the broiler response to dietary nonphytate phosphorus levels than bodyweight gain.

Palm oil/olein (PO/POL) is used in infant formulas to imitate the fatty acid profile of human milk (HM) and achieve similar levels of palmitic acid (PA). However, the positions of fatty acids on the triacylglyceride differ between PO/POL and HM, which affect fat absorption and produce unintended physiological consequences. Recent papers have reviewed evidence for physiological benefits of PO/POL and beta-palmitate (sn-2-palmitate) in infant formulas. The aim of the present review is to supplement the assessment of available clinical evidence on the physiological effects of PO/POL formulas in healthy infants. We intend to focus on PO/POL and not on sn-2-palmitate, since the latter was recently extensively reviewed. Clinical evidence supports that PO/POL in infant formulas leads to a lower fat, DHA, palmitate and calcium absorption, and bone mineralization; soft stools; and growth (weight accretion) compared to formulas without PO/POL. Consequently, it seems prudent to be considerate and cautious when adding PO/POL to infant formulas. While HM is the gold standard for infant nutrition, the development of infant formula should be based on achieving positive physiological outcomes, rather than just replicating HM nutrient composition.

The macro- and micro-features of bone can be assessed by using imaging methods. However, nano- and molecular features require more detailed characterization, such as use of e.g., vibrational spectroscopy and X-ray scattering. Nano- and molecular features also affect the mechanical competence of bone tissue. The aim of the present study was to reveal the effects of mineralization and its alterations on the mineral crystal scale, by investigating the spatial variation of molecular composition and mineral crystal structure across the cross-section of femur diaphyses in young rats, and healthy and osteoporotic mature rats (N=5). Fourier transform infrared spectroscopy and scanning small- and wide-angle X-ray scattering (SAXS/WAXS) techniques with high spatial resolution were used at identical locations over the whole cross-section. This allowed quantification of point-by-point information about the spatial distribution of mineral crystal volume. All measured parameters (crystal dimensions, degree of orientation and predominant orientation) varied across the cortex. Specifically, the crystal dimensions were lower in the central cortex than in the endosteal and periosteal regions. Mineral crystal orientation followed the cortical circumference in the periosteal and endosteal regions, but was less well-oriented in the central regions. Central cortex is formed rapidly during development through endochondral ossification. Since rats possess no osteonal remodeling, this bone remains (until old age). Significant linear correlations were observed between the dimensional and organizational parameters, e.g., between crystal length and degree of orientation (R2=0.83, p

Purpose: The prevalence of lifestyle diseases has escalated, and effective exercise training programmes are warranted. This study tested the hypothesis that regular participation in small-sided team handball training could provide beneficial health effects on cardiovascular, skeletal, and muscular parameters in young adult untrained men. Method: Twenty-six untrained 20–30-year-old men were randomly allocated to either a team handball training group (HG; n = 14), which completed 1.9 ± 0.3 training sessions per week over 12 weeks, or an inactive control group (CG; n = 12). Physiological training adaptations were assessed pre- and post interventions by DXA scans, blood samples, muscle biopsies, and physical tests. Results: The average heart rate during training was equivalent to 84 ± 4% of maximal heart rate. Compared to CG, HG displayed significant increases in VO2max (11 ± 6%), proximal femur bone mineral density (2 ± 1%), whole-body bone mineral content (2 ± 1%), intermittent endurance performance (32 ± 16%), incremental treadmill test performance (16 ± 7%) and muscle citrate synthase activity (22 ± 28%) as well as decreases in total fat mass (7 ± 7%) and total fat percentage (6 ± 7%) (all p < 0.05). There were no significant changes in muscle mass, blood pressure, resting heart rate, muscle hydroxyl-acyl-dehydrogenase activity, or blood lipids (all p > 0.05). Conclusion: Participation in regular recreational team handball training was associated with positive cardiovascular, skeletal, and muscular adaptations, including increased maximal oxygen uptake, increased muscle enzymatic activity, and improved bone mineralization as well as lower fat percentage. These findings suggest that recreational team handball training may be an effective health-promoting activity for young adult men.

Fibroblast growth factor-23 (FGF23) is a bone-derived hormone suppressing phosphate reabsorption and vitamin D hormone synthesis in the kidney. At physiological concentrations of the hormone, the endocrine actions of FGF23 in the kidney are αKlotho-dependent, because high-affinity binding of FGF23 to FGF receptors requires the presence of the co-receptor αKlotho on target cells. It is well established that excessive concentrations of intact FGF23 in the blood lead to phosphate wasting in patients with normal kidney function. Based on the importance of diseases associated with gain of FGF23 function such as phosphate-wasting diseases and chronic kidney disease, a large body of literature has focused on the pathophysiological consequences of FGF23 excess. Less emphasis has been put on the role of FGF23 in normal physiology. Nevertheless, during recent years, lessons we have learned from loss-of-function models have shown that besides the paramount physiological roles of FGF23 in the control of 1α-hydroxylase expression and of apical membrane expression of sodium-phosphate co-transporters in proximal renal tubules, FGF23 also is an important stimulator of calcium and sodium reabsorption in distal renal tubules. In addition, there is an emerging role of FGF23 as an auto-/paracrine regulator of alkaline phosphatase expression and mineralization in bone. In contrast to the renal actions of FGF23, the FGF23-mediated suppression of alkaline phosphatase in bone is αKlotho-independent. Moreover, FGF23 may be a physiological suppressor of differentiation of hematopoietic stem cells into the erythroid lineage in the bone microenvironment. At present, there is little evidence for a physiological role of FGF23 in organs other than kidney and bone. The purpose of this mini-review is to highlight the current knowledge about the complex physiological functions of FGF23.

Sclerostin has emerged as an important regulator of bone mass. We have shown that sclerostin can act by targeting late osteoblasts/osteocytes to inhibit bone mineralization and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into individual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared with unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared with unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionized calcium concentration in the perfusate and the release of beta-CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis, and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released beta-CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the periosteocyte matrix. Refereed/Peer-reviewed

Objectives: To longitudinally assess bone mineral content (BMC), bone mineral density (BMD), and whole-body lean mass obtained through bone densitometry by dual-energy X-ray absorptiometry (DXA) in preterm newborns (PTNs) and compare them with full-term newborns (FTNs) from birth to 6 months of corrected postnatal age. Methods: A total of 28 adequate for gestational age (AGA) newborns were studied: 14 preterm and 14 full-term newborns. DXA was used to determine BMC, BMD, and lean mass in three moments: 40 weeks corrected post-conceptual age, as well as 3 and 6 months of corrected postnatal age. PTNs had gestational age ≤ 32 weeks at birth and were fed their mother's own milk or milk from the human milk bank. Results: All infants had an increase in BMC, BMD, and lean body mass values during the study. PTNs had lower BMC, BMD, and lean mass at 40 weeks of corrected post-conceptual age in relation to FTNs (p

Background: Fibroblast growth factor-23 (FGF-23) is a hormone principally produced by osteocytes/osteoblasts. In patients with chronic kidney disease (CKD), FGF-23 levels are usually elevated and can reach up to 300 – 400 times the normal range. FGF-23 is regulated by local bone-related and systemic factors, but the relationship between circulating FGF-23 concentrations and bone remodeling and mineralization in CKD has not been well characterized. In the current study, we examined the relationship between FGF-23 levels and bone histomorphometry parameters in adult patients with renal osteodystrophy. Material and methods: 36 patients on dialysis (CKD-5D) underwent bone biopsies after tetracycline double labeling. Blood drawings were done at time of biopsy to determine serum levels of markers of bone and mineral metabolism. Results: Patients with high bone turnover had higher values of serum FGF-23 than patients with low bone turnover. FGF-23 levels correlated with activation frequency (ρ = 0.60, p 100 days) was mainly seen in patients with FGF-23 levels less than 2,000 pg/mL, while very high levels of FGF-23 are associated with normal mineralization lag time.

ObjectivesTo longitudinally assess bone mineral content (BMC), bone mineral density (BMD), and whole‐body lean mass obtained through bone densitometry by dual‐energy X‐ray absorptiometry (DXA) in preterm newborns (PTNs) and compare them with full‐term newborns (FTNs) from birth to 6 months of corrected postnatal age.MethodsA total of 28 adequate for gestational age (AGA) newborns were studied: 14 preterm and 14 full‐term newborns. DXA was used to determine BMC, BMD, and lean mass in three moments: 40 weeks corrected post‐conceptual age, as well as 3 and 6 months of corrected postnatal age. PTNs had gestational age ≤ 32 weeks at birth and were fed their mother's own milk or milk from the human milk bank.ResultsAll infants had an increase in BMC, BMD, and lean body mass values during the study. PTNs had lower BMC, BMD, and lean mass at 40 weeks of corrected post‐conceptual age in relation to FTNs (p