Lower microhardness along with less heterogeneous mineralization in the femoral neck of individuals with type 2 diabetes mellitus indicates higher fracture risk.

There is still limited understanding of the microstructural reasons for the higher susceptibility to fractures in individuals with type 2 diabetes mellitus (T2DM). In this study, we examined bone mineralization, osteocyte lacunar parameters, and microhardness of the femoral neck trabeculae in 18 individuals with T2DM who sustained low-energy fracture (T2DMFx: 78 ± 7 years, 15 women and 3 men) and 20 controls (74 ± 7 years, 16 women and 4 men). Femoral necks of the T2DMFx subjects were obtained at a tertiary orthopedic hospital, while those of the controls were collected at autopsy. T2DMFx individuals had lower trabecular microhardness (P = .023) and mineralization heterogeneity (P = .001), and a tendency to a lower bone area with mineralization above 95th percentile (P = .058) than the controls. There were no significant intergroup differences in the numbers of osteocyte lacunae per bone area, mineralized lacunae per bone area, and total lacunae per bone area (each P > .05). After dividing the T2DMFx group based on the presence of vascular complications (VD) to T2DMFxVD (VD present) and T2DMFxNVD (VD absent), we observed that microhardness was particularly reduced in the T2DMFxVD group (vs. control group, P = .02), while mineralization heterogeneity was significantly reduced in both T2DMFx subgroups (T2DMFxNVD vs. control, P = .002; T2DMFxVD vs. control, P = .038). The observed changes in mineralization and microhardness may contribute to the increased hip fracture susceptibility in individuals with T2DM.

Evaluation of Osteogenic Potential of a Polysaccharide-Based Hydrogel Coating on Titanium.

INTRODUCTION: Reducing the healing period after surgical placement of dental implants can facilitate the loading of dental prostheses. AIM: The aim is to compare the osteogenic potential of unmodified titanium disks with titanium disks that were surface-modified or hydrogel-coated. MATERIALS AND METHODOLOGY: One hundred eight titanium disks (Ø6 × 2-mm) were divided into three groups: (1) unmodified titanium as control (Ti-C); (2) sandblasted and acid-etched (Ti-SLA), and (3) coated with tamarind kernel polysaccharide hydrogel grafted with acrylic acid (Ti-TKP-AA). The osteogenic potential and cytotoxic effect of various groups of titanium were compared using human osteoblasts Saos-2. The surface topography of the titanium disks and morphology of osteoblasts grown on disks were investigated by scanning electron microscopy (n = 3). Cell attachment to the disks and actin expression intensity were investigated by confocal imaging (n = 3). Cytotoxicity was quantified by cell viability assay (n = 9). Osteoblast maturation was determined by alkaline phosphatase assay (n = 9). Cell mineralization was quantified by Alizarin red staining (n = 9). One-way analysis of variance followed by Tukey's multiple comparisons test was used for intergroup comparisons (α= 0.05). RESULTS: The surface modifications on Ti-SLA and Ti-TKP-AA support better morphology and proliferation of osteoblasts than Ti-C (P< 0.001) and significantly higher levels of actin cytoskeleton accumulation (P< 0.0001). Ti-TKP-AA showed a significantly higher maturation rate than Ti-C (P< 0.001). Ti-TKP-AA showed > twofold increased mineralization than Ti-C and Ti-SLA (P< 0.001). CONCLUSIONS: TKP-AA hydrogel-coated titanium promotes faster osteoblast proliferation, maturation, and mineralization than SLA-treated or untreated titanium. These advantages can be explored for achieving early osseointegration and prosthetic loading of titanium dental implants.

Estradiol-17ß and bisphenol A affect growth and mineralization in early life stages of seabass.

Natural and synthetic estrogens are contaminants present in aquatic ecosystems. They can have significant consequences on the estrogen-sensitive functions of organisms, including skeletal development and growth of vertebrate larvae. Synthetic polyphenols represent a group of environmental xenoestrogens capable of binding the receptors for the natural hormone estradiol-17ß (E2). To better understand how (xeno-)estrogens can affect the skeleton in fish species with high ecological and commercial interest, 16 days post-hatch larvae of the seabass were experimentally exposed for 7 days to E2 and Bisphenol A (BPA), both used at the regulatory concentration of surface water quality (E2: 0.4 ng.L-1, BPA: 1.6 µg.L-1) or at a concentration 100 times higher. Skeletal mineralization levels were evaluated using Alizarin red staining, and expression of several genes playing key roles in growth, skeletogenesis and estrogen signaling pathways was assessed by qPCR. Our results show that E2 exerts an overall negative effect on skeletal mineralization at the environmental concentration of 0.4 ng.L-1, correlated with an increase in the expression of genes associated only with osteoblast bone cells. Both BPA exposures inhibited mineralization with less severe effects and modified bone homeostasis by regulating the expression of gene encoding osteoblasts and osteoclasts markers. Our results demonstrate that environmental E2 exposure inhibits larval growth and has an additional inhibitory effect on skeleton mineralization while both BPA exposures have marginal inhibitory effect on skeletal mineralization. All exposures have significant effects on transcriptional levels of genes involved in the skeletal development of seabass larvae.

Dietary zinc requirement of juvenile stinging catfish Heteropneustes fossilis based on growth performance, haematology, and tissue mineral composition.

This investigation was done to determine how much zinc (Zn) the stinging catfish, Heteropneustes fossilis, needs in its diet. Five isonitrogenous (34.5% protein) and isolipidic (6.0% lipid) diets were prepared to contain graded levels of Zn (0, 10, 20, 30, and 40 mg kg-1), supplied as zinc sulfate (ZnSO4·7H2O), and referred to as Zn0, Zn10, Zn20, Zn30, and Zn40, respectively. A total of 600 fish (initial body weight: 1.41 ± 0.02 g) were stocked in 15 glass aquaria (40 fish/aquarium), each with 180 L water capacity. For ten weeks, each diet was hand fed to three groups of fish twice daily until they appeared satisfied. The highest weight gain and specific growth rate, and lowest feed conversion ratio were recorded in fish fed with a 30 mg Zn kg-1 diet. Zn contents in bone and muscle linearly increased up to 30 mg kg-1 Zn and then remained stable, while iron (Fe) and copper (Cu) contents in bone and muscle had an inverse pattern with the inclusion level of dietary Zn. Increasing dietary Zn levels up to 30 mg kg-1 was found to improve values of hematological parameters such as red blood cell (RBC), white blood cell (WBC), haemoglobin (Hb), and haematocrit (HCT). These values, however, decreased when the dietary Zn level was further increased. The serum alkaline phosphatase level was the highest in fish fed a diet containing 30 mg kg-1 of Zn. Regression analyses based on weight gain, specific growth rate, and bone and muscle Zn concentrations indicated that the optimum dietary Zn requirement for stinging catfish was in a range of 27.4-36.5 mg kg-1.

Diagnostic survey of analytical methods used to determine bone mineralization in pigs.

Pigs from 64 commercial sites across 14 production systems in the Midwest United States were evaluated for baseline biological measurements used to determine bone mineralization. There were three pigs selected from each commercial site representing: 1) a clinically normal pig (healthy), 2) a pig with evidence of clinical lameness (lame), and 3) a pig from a hospital pen that was assumed to have recent low feed intake (unhealthy). Pigs ranged in age from nursery to market weight, with the three pigs sampled from each site representing the same age or phase of production. Blood, urine, metacarpal, fibula, 2nd rib, and 10th rib were collected and analyzed. Each bone was measured for density and ash (defatted and non-defatted technique). A bone × pig type interaction (P < 0.001) was observed for defatted and non-defatted bone ash and density. For defatted bone ash, there were no differences among pig types for the fibulas, 2nd rib, and 10th rib (P > 0.10), but metacarpals from healthy pigs had greater (P < 0.05) percentage bone ash compared to unhealthy pigs, with the lame pigs intermediate. For non-defatted bone ash, there were no differences among pig types for metacarpals and fibulas (P > 0.10), but unhealthy pigs had greater (P < 0.05) non-defatted percentage bone ash for 2nd and 10th ribs compared to healthy pigs, with lame pigs intermediate. Healthy and lame pigs had greater (P < 0.05) bone density than unhealthy pigs for metacarpals and fibulas, with no difference observed for ribs (P > 0.10). Healthy pigs had greater (P < 0.05) serum Ca and 25(OH)D3 compared to unhealthy pigs, with lame pigs intermediate. Healthy pigs had greater (P < 0.05) serum P compared to unhealthy and lame pigs, with no differences between the unhealthy and lame pigs. Unhealthy pigs excreted significantly more (P < 0.05) P and creatinine in the urine compared to healthy pigs with lame pigs intermediate. In summary, there are differences in serum Ca, P, and vitamin D among healthy, lame, and unhealthy pigs. Differences in bone mineralization among pig types varied depending on the analytical procedure and bone, with a considerable range in values within pig type across the 14 production systems sampled.

There is little literature or data comparing bone diagnostic results for healthy, lame, and unhealthy pigs. Typically, diagnosticians assessing clinical lameness cases in pigs will measure bone mineralization along with histopathological evaluation to diagnose and assess the severity of metabolic bone disease. Bone ash is the primary method to determine bone mineralization, with the removal of the lipid in the bone (defatting) before the bone is ashed, compared to not removing the lipid before the ashing (non-defatted). Defatting the bone reduces the amount of variation across the bones compared to non-defatting. In this diagnostic survey, there was no difference among the healthy, lame, or unhealthy pigs when comparing defatted bone ash, however, unhealthy pigs had an increased bone ash percentage compared to the healthy and lame pigs when the bones were assessed using the non-defatted procedure. There was variation across production systems and pig types for serum vitamin D. When comparing the pig types, healthy pigs had increased serum Ca, P, and vitamin D [25(OH)D3] compared to the unhealthy pigs, with the lame pigs intermediate.

Impact of deoxynivalenol in a calcium depletion and repletion nutritional strategy in piglets.

This study evaluated the effect of dietary calcium (Ca) levels and deoxynivalenol (DON) contamination on Ca and phosphorus (P) utilization and bone mineralization in piglets. During an initial 13-d depletion phase, 64 piglets (15.7 ±â€…0.7 kg) received a control (DON-) or DON-contaminated treatment (DON+, 2.7 mg DON/kg) with either a low Ca (Ca-, 0.39%) or normal Ca level (Ca+, 0.65%) with a constant digestible P level (0.40%). A second group of 16 piglets received DON- or DON+ treatments for 9 d for gene expression analysis. During the subsequent 14-d repletion phase, all piglets were fed a Ca+ DON- diet containing 0.65% Ca and 0.35% digestible P without DON. After 5 d of the depletion phase, the absorption of P (DON × Ca; P < 0.05) and Ca was increased by the Ca- (P < 0.01) and DON+ (P < 0.01) diet. After 13 d, feed conversion ratio (P < 0.01) and average daily feed intake (P = 0.06) tended to decrease with the Ca- diet. The bone mineral content (BMC) gain was decreased by Ca, especially with Ca- DON + (DON × Ca, P < 0.05). The P absorption was increased by Ca- DON + (DON × Ca, P < 0.01), although the P retention efficiency was only increased by Ca+ DON + (DON × Ca, P < 0.001). The absorption of Ca was increased by DON+ (P < 0.001), and the Ca efficiency was increased by Ca- DON- (DON × Ca, P < 0.01). After 9 d, the gene expression of intestinal claudin 12 (P < 0.01) and CYP24A1 (P < 0.05), femur cortical RANKL (P < 0.05) and OPG (P = 0.06), and renal calbindin D9K (P < 0.05) and Klotho (P = 0.07) were decreased by DON+. The Ca (P = 0.06) and magnesium (P < 0.01) concentrations were decreased by DON+, and the Ca (P = 0.06) and P digestibility (P < 0.01) were increased. After the repletion phase, Ca- piglets recovered their BMC deficit, but not those receiving DON+ (DON × Ca; P = 0.06). The Ca (P < 0.05) and P (P = 0.06) retention efficiency tended to increase with Ca-. The absorption of Ca and P was increased by Ca- and DON+ (DON × Ca, P < 0.05). The results show that piglets increased their Ca and P utilization efficiency, allowing them to recover the BMC deficit caused by Ca-, but not when the piglets were exposed to DON. Pigs previously receiving Ca-deficient diet with DON still have lower body Ca and P, leading to elevated calcitriol concentrations and enhanced Ca and P intestinal absorption. The fact that DON decreased the expression of genes implicated in Ca intestinal and renal transport and P excretion after 9 d can potentially explain the reduced plasma Ca concentration.

Calcium (Ca) deficiency can increase how efficiently pigs use Ca and phosphorus (P), but exposure to the mycotoxin deoxynivalenol (DON), often found in pig feed ingredients, can impact the digestibility and excretion of Ca and P. In our study, piglets received a diet with or without DON-contamination and either low Ca (0.39%) or normal Ca levels (0.65%) during a 13-d depletion phase, followed by a 14-d repletion phase where all piglets were fed a normal Ca diet without DON. The short Ca-depletion phase is known to improve the utilization efficiency of Ca and P in piglets by increasing the retention of these nutrients through both depletion and repletion phases and the Ca and P digestibility through the repletion phase, which allows recovery of the bone mineralization deficit that occurred during Ca deficiency. However, the diet contaminated with DON prevented pigs from recovering from their bone mineralization deficit observed during the Ca-depletion phase, even though they were better able to absorb and digest Ca and P during both phases. This was supported by the reduced expression of genes involved in Ca intestinal absorption, renal transport, osteoclastogenesis, and P excretion.

Impact of Trace Mineral Source and Phytase Supplementation on Prececal Phytate Degradation and Mineral Digestibility, Bone Mineralization, and Tissue Gene Expression in Broiler Chickens.

The objective of this study was to determine how different sources of Zn, Mn, and Cu in the feed without and with phytase affect prececal myo-inositol hexakisphosphate (InsP6) breakdown to myo-inositol (MI), prececal P digestibility, bone mineralization, and expression of mineral transporters in the jejunum of broiler chickens. A total of 896 male broiler chicks (Cobb 500) were distributed to 7 diets with 8 replicate pens (16 birds per floor pen). Experimental diets were fed from day 0 to 28. Diets were without or with phytase supplementation (0 or 750 FTU/kg) and were supplemented with three different trace mineral sources (TMS: sulfates, oxides, or chelates) containing 100 mg/kg Zn, 100 mg/kg Mn, and 125 mg/kg Cu. Prececal InsP6 disappearance and P digestibility were affected by interaction (phytase × TMS: P ≤ 0.010). In diets without phytase supplementation, prececal InsP6 disappearance and P digestibility were greater (P ≤ 0.001) in birds fed chelated minerals than in birds fed sulfates or oxides. However, no differences were observed between TMS in diets with phytase supplementation. Ileal MI concentration was increased by exogenous phytase but differed depending on TMS (phytase × TMS: P ≤ 0.050). Tibia ash concentration as well as Zn and Mn concentration in tibia ash were increased by phytase supplementation (P < 0.010), but the Cu concentration in tibia ash was not (P > 0.050). Gene expression of the assayed mineral transporters in the jejunum was not affected by diet (P > 0.050), except for Zn transporter 5 (phytase × TMS: P = 0.024). In conclusion, the tested TMS had minor effects on endogenous phytate degradation in the digestive tract of broiler chickens. However, in phytase-supplemented diets, the choice of TMS was not relevant to phytate degradation under the conditions of this study.

Long-term effects of high-dose systemic corticosteroids on growth and bone mineral density in patients treated for childhood interstitial lung disease (chILD).

BACKGROUND: Children's interstitial lung disease (chILD) is a rare and potentially life-threatening condition. For many chILD conditions, systemic corticosteroids (sCCS) are considered the primary treatment despite a broad spectrum of potential side effects. AIM: We aimed to determine the long-term effects of sCCS treatment on growth, bone mineral density (BMD), and body composition after chILD. MATERIALS AND METHODS: This descriptive cross-sectional single-center study included patients diagnosed with chILD before the age of 18 years treated with sCCS in the period 1998-2020. Dual-energy X-ray absorptiometry, anthropometric measurements, bone age determination, and blood tests were performed in 53 (55% males) of 89 eligible patients. RESULTS: Median (range) age was 19.3 (6.4;30.7 years). Participants received a median (range) cumulative sCCS dose of 1144 (135; 6178) mg over a 2.0 (0.1; 13.8) years period and latest dose was administered 11.7 (1.2; 19.6) years before follow-up. Mean delta height (height standard deviation scores [SDS] - target height SDS) was reduced at sCCS treatment initiation (mean: -0.55, 95% confidence interval [CI]: -0.91; -0.20, p < .005) and at sCCS treatment cessation (mean: -0.86, 95% CI:-1.22; -0.51, p < .001), but normalized in the majority at follow-up (mean: -0.29, 95% CI:-0.61; 0.03, p = .07). Mean (SD) BMD z-score for the spine and whole body was -0.34 (1.06) and 0.52 (1.13), with no significant correlation to sCCS dose. Excess body fat (>30% in females, >25% in males) was found in 58% of patients. CONCLUSION: Long-term treatment with sCCS did not cause significant long-term reduction of height but showed subtle effects on fat mass percentage and BMD. Given the severity of chILD, the observed long-term effects of sCCS on growth and BMD appear acceptable.

The ecto-nucleotide pyrophosphatase/phosphodiesterase 2 promotes early osteoblast differentiation and mineralization in stromal stem cells.

The phosphodiesterase enzymes mediate calcium-phosphate deposition in various tissues, although which enzymes are active in bone mineralization is unclear. Using gene array analysis, we found that a member of ecto-nucleotide pyrophosphatase/phosphodiesterase family, ENPP2, was strongly down-regulated with age in stromal stem cells that produce osteoblasts and make bone. This is in keeping with reduced bone formation in older animals. Thus, we hypothesized that ENPP2 is, at least in part, an early mediator of bone formation and thus may reflect reduced bone formation with age. Since ENPP2 has not previously been shown to have a role in osteoblast differentiation, we studied its effect on bone differentiation from stromal stem cells, verified by flow cytometry for stem cell antigens. In these remarkably uniform osteoblast precursors, we did transfection with ENPP2 DsiRNA, scrambled DsiRNA, or no transfection to make cells with normal or greatly reduced ENPP2 and analyzed osteoblast differentiation and mineralization. Osteoblast differentiation down-regulation was shown by alizarin red binding, silver staining, and alkaline phosphatase activity. Differences were confirmed by real-time PCR for alkaline phosphatase (ALPL), osteocalcin (BGLAP), and ENPP2 and by Western Blot for Enpp2. These were decreased, ∼50%, in osteoblasts transfected with ENPP2 DsiRNA compared with cells transfected with a scrambled DsiRNA or not transfected (control) cells. This finding is the first evidence for the role of ENPP2 in osteoblast differentiation and mineralization.NEW & NOTEWORTHY We report the discovery that the ecto-nucleotide pyrophosphatase/phosphodiesterase, ENPP2, is an important regulator of early differentiation of bone-forming osteoblasts.

Spheroids as a 3D in vitro model to study bone and bone mineralization.

Traumas, fractures, and diseases can severely influence bone tissue. Insight into bone mineralization is essential for the development of therapies and new strategies to enhance bone regeneration. 3D cell culture systems, in particular cellular spheroids, have gained a lot of interest as they can recapitulate crucial aspects of the in vivo tissue microenvironment, such as the extensive cell-cell and cell-extracellular matrix (ECM) interactions found in tissue. The potential of combining spheroids and various classes of biomaterials opens also new opportunities for research within bone tissue engineering. Characterizing cellular organization, ECM structure, and ECM mineralization is a fundamental step for understanding the biological processes involved in bone tissue formation in a spheroid-based model system. Still, many experimental techniques used in this field of research are optimized for use with monolayer cell cultures. There is thus a need to develop new and improving existing experimental techniques, for applications in 3D cell culture systems. In this review, bone composition and spheroids properties are described. This is followed by an insight into the techniques that are currently used in bone spheroids research and how these can be used to study bone mineralization. We discuss the application of staining techniques used with optical and confocal fluorescence microscopy, molecular biology techniques, second harmonic imaging microscopy, Raman spectroscopy and microscopy, as well as electron microscopy-based techniques, to evaluate osteogenic differentiation, collagen production and mineral deposition. Challenges in the applications of these methods in bone regeneration and bone tissue engineering are described. STATEMENT OF SIGNIFICANCE: 3D cell cultures have gained a lot of interest in the last decades as a possible technique that can be used to recreate in vitro in vivo biological process. The importance of 3D environment during bone mineralization led scientists to use this cell culture to study this biological process, to obtain a better understanding of the events involved. New and improved techniques are also required for a proper analysis of this cell model and the process under investigation. This review summarizes the state of the art of the techniques used to study bone mineralization and how 3D cell cultures, in particular spheroids, are tested and analysed to obtain better resolved results related to this complex biological process.

The Interplay between Muscular Grip Strength and Bone Mineral Density with Consideration of Metabolic and Endocrine Parameters in Individuals with Turner Syndrome.

INTRODUCTION: Patients with Turner syndrome (TS) often face skeletal and muscular challenges, including reduced bone mineral density (BMD) and muscle weakness. This comprehensive study sheds light on the complex interplay between muscle strength, BMD, and metabolic and endocrine parameters in TS and healthy subjects. METHODS: A cross-sectional study involving 42 TS patients and 70 healthy women was conducted. All patients had their BMD determined in the L1-L4 lumbar spine section and in the whole skeleton as well as the parameters of body fat mass (BF), and visceral fat mass (VF) were also determined. The maximum gripping force was measured with a hydraulic manual dynamometer. In addition, a number of blood hormonal and metabolic parameters were determined. RESULTS: In the TS group, hand grip strength correlated positively with triglyceride levels but not with BMD. Healthy individuals had a positive link between hand grip strength and BMD, while patients with TS did not show a significant association between the two. A trend suggested that longer recombinant human growth hormone (rhGH) therapy might improve BMD in the L1-L4 region. Multiple linear regression analysis revealed that muscle strength assessment may be a potential exponent of reduced BMD, and also used clinically in young adult women but not in individuals with TS. CONCLUSIONS: The relationship between BMD variables and hand grip might differ between the two groups, potentially indicating distinct musculoskeletal characteristics in TS patients. Longer rhGH therapy in TS patients may have a positive effect on BMD in the L1-L4 region. Understanding the intricate relationships between these factors is important for optimizing clinical management strategies and improving the quality of life for TS patients.

Diagnostic and New Therapeutic Approaches to Two Challenging Pediatric Metabolic Bone Disorders: Hypophosphatasia and X-linked Hypophosphatemic Rickets.

The diagnosis and management of metabolic bone disease among children can be challenging. This difficulty could be due to many factors, including limited awareness of these rare conditions, the complex pathophysiology of calcium and phosphate homeostasis, the overlapping phenotype with more common disorders (such as rickets), and the lack of specific treatments for these rare disorders. As a result, affected individuals could experience delayed diagnosis or misdiagnosis, leading to improper management. In this review, we describe the challenges facing diagnostic and therapeutic approaches to two metabolic bone disorders (MBD) among children: hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH). We focus on explaining the pathophysiological processes that conceptually underpin novel therapeutic approaches, as well as these conditions' clinical or radiological similarity to nutritional rickets. Particularly in areas with limited sun exposure and among patients not supplementing vitamin D, nutritional rickets are still more common than HPP and XLH, and pediatricians and primary physicians frequently encounter this disorder in their practices. More recently, our understanding of these disorders has significantly improved, leading to the development of novel therapies. Asfotas alfa, a recombinant, human-tissue, nonspecific alkaline phosphatase, improved the survival of patients with HPP. Burosumab, a human monoclonal anti-FGF23 antibody, was recently approved as a specific therapy for XLH. We also highlight the current evidence on these two specific therapies' safety and effectiveness, though long-term data are still needed. Both HPP and XLH are multisystemic disorders that should be managed by multidisciplinary teams. Finally, recognizing these conditions in early stages will enable affected children and young adults to benefit from newly introduced, specific therapies.

Effect of dietary zinc source, zinc concentration, and exogenous phytase on intestinal phytate degradation products, bone mineralization, and zinc status of broiler chickens.

This study aimed to determine the effect of Zn source and dietary level on intestinal myo-inositol hexakisphosphate (InsP6) disappearance, intestinal accumulation of lower InsP and myo-inositol (MI), prececal mineral digestibility, bone mineralization, and Zn status of broilers without and with exogenous phytase in the feed. Male Ross 308 broilers were allocated in groups of 10 to 8 treatments with 8 pens each. Experimental diets were fed from d 7 to d 28 and contained 33 mg/kg dry matter plant-intrinsic Zn. Experimental factors were phytase supplementation (0 or 750 FTU/kg) and Zn source (none [0 mg/kg Zn], Zn-sulfate [30 mg/kg Zn], Zn-oxide [30 mg/kg Zn]). Additional treatments with 90 mg/kg Zn as Zn-sulfate or Zn-oxide and phytase were included to test the effect of Zn level. No Zn source or Zn level effects were observed for ADG, feed conversion ratio, prececal P digestibility, intestinal InsP6 disappearance, and bone ash concentration. However, those measurements were increased by exogenous phytase (P < 0.001), except the feed conversion ratio, which was decreased (P < 0.001). Ileal MI concentrations were affected by phytase × Zn source interaction (P < 0.030). Birds receiving exogenous phytase and Zn supplementation had the highest MI concentrations regardless of exogenous Zn source, whereas MI concentrations were intermediate for birds receiving exogenous phytase only. Exogenous phytase and exogenous Zn source increased the Zn concentration in bone and blood of broilers (P < 0.001). In conclusion, measures of exogenous phytase efficacy were not affected by phytase × Zn source interaction. Further studies are needed to rule out an effect from Zn sources other than those tested in this study and to investigate the effect of Zn supplementation on endogenous phosphatases. The missing effect of increasing Zn supplementation from 30 to 90 mg/kg in phytase-supplemented diets gives reason to reconsider the Zn supplementation level used by the industry.

Advancing our understanding of the influence of drug induced changes in the gut microbiome on bone health.

The gut microbiome has been implicated in a multitude of human diseases, with emerging evidence linking its microbial diversity to osteoporosis. This review article will explore the molecular mechanisms underlying perturbations in the gut microbiome and their influence on osteoporosis incidence in individuals with chronic diseases. The relationship between gut microbiome diversity and bone density is primarily mediated by microbiome-derived metabolites and signaling molecules. Perturbations in the gut microbiome, induced by chronic diseases can alter bacterial diversity and metabolic profiles, leading to changes in gut permeability and systemic release of metabolites. This cascade of events impacts bone mineralization and consequently bone mineral density through immune cell activation. In addition, we will discuss how orally administered medications, including antimicrobial and non-antimicrobial drugs, can exacerbate or, in some cases, treat osteoporosis. Specifically, we will review the mechanisms by which non-antimicrobial drugs disrupt the gut microbiome's diversity, physiology, and signaling, and how these events influence bone density and osteoporosis incidence. This review aims to provide a comprehensive understanding of the complex interplay between orally administered drugs, the gut microbiome, and osteoporosis, offering new insights into potential therapeutic strategies for preserving bone health.

The effect of bone and analytical methods on the assessment of bone mineralization response to dietary phosphorus, phytase, and vitamin D in nursery pigs.

A total of 360 pigs (DNA 600 × 241, DNA; initially 11.9 ±â€…0.56 kg) were used in a 28-d trial to evaluate the effects of different bones and analytical methods on the assessment of bone mineralization response to dietary P, vitamin D, and phytase in nursery pigs. Pens of pigs (six pigs per pen) were randomized to six dietary treatments in a randomized complete block design with 10 pens per treatment. Dietary treatments were designed to create differences in bone mineralization and included: (1) 0.19% standardized total tract digestibility (STTD) P (deficient), (2) 0.33% STTD P (NRC [2012] requirement) using monocalcium phosphate, (3) 0.33% STTD P including 0.14% release from phytase (Ronozyme HiPhos 2700, DSM Nutritional Products, Parsippany, NJ), (4) 0.44% STTD P using monocalcium phosphate, phytase, and no vitamin D, (5) diet 4 with vitamin D (1,653 IU/kg), and (6) diet 5 with an additional 50 µg/kg of 25(OH)D3 (HyD, DSM Nutritional Products, Parsippany, NJ) estimated to provide an additional 2,000 IU/kg of vitamin D3. After 28 d on feed, eight pigs per treatment were euthanized for bone (metacarpal, 2nd rib, 10th rib, and fibula), blood, and urine analysis. The response to treatment for bone density and ash was dependent upon the bone analyzed (treatment × bone interaction for bone density, P = 0.044; non-defatted bone ash, P = 0.060; defatted bone ash, P = 0.068). Thus, the response related to dietary treatment differed depending on which bone (metacarpal, fibula, 2nd rib, or 10th rib) was measured. Pigs fed 0.19% STTD P had decreased (P < 0.05) bone density and ash (non-defatted and defatted) for all bones compared to 0.44% STTD P, with 0.33% STTD P generally intermediate or similar to 0.44% STTD P. Pigs fed 0.44% STTD P with no vitamin D had greater (P < 0.05) non-defatted fibula ash compared to all treatments other than 0.44% STTD P with added 25(OH)D3. Pigs fed diets with 0.44% STTD P had greater (P < 0.05) defatted second rib ash compared to pigs fed 0.19% STTD P or 0.33% STTD P with no phytase. In summary, bone density and ash responses varied depending on bone analyzed. Differences in bone density and ash in response to P and vitamin D were most apparent with fibulas and second ribs. There were apparent differences in the bone ash percentage between defatted and non-defatted bone. However, differences between the treatments remain consistent regardless of the analytic procedure. For histopathology, 10th ribs were more sensitive than 2nd ribs or fibulas for the detection of lesions.

Lameness is defined as impaired movement or deviation from normal gait. There are many factors that can contribute to lameness, including but not limited to: infectious disease, genetic and conformational anomaly, and toxicity that affects the bone, muscle, and nervous systems. Metabolic bone disease is another cause of lameness in swine production and can be caused by inappropriate levels of essential vitamins or minerals. To understand and evaluate bone mineralization, it is important to understand the differences in diagnostic results between different bones and analytical techniques. Historically, percentage bone ash has been used as one of the procedures to assess metabolic bone disease as it measures the level of bone mineralization; however, procedures and results vary depending on the methodology and type of bone measured. Differences in bone density and ash in response to dietary P and vitamin D were most apparent in the fibulas and second ribs. There were apparent differences in the percentage of bone ash between defatted and non-defatted bone; however, the differences between the treatments remain consistent regardless of the analytic procedure. For histopathology, 10th ribs were more sensitive than 2nd ribs or fibulas for detection of lesions associated with metabolic bone disease.

Association of bone mineralization markers with dietary nutrient intake in adolescents with and without biochemical osteomalacia.

Background: Dietary intake is widely known to play a crucial role in achieving peak bone mass among children and adolescents. Unfortunately, this information is lacking among Arab adolescents, an understudied demographic that has recently been observed to have a high prevalence of abnormal mineralization markers [low serum 25(OH)D, high serum alkaline phosphatase (ALP), low calcium (Ca) and/or inorganic phosphate (Pi)] suggestive of biochemical osteomalacia (OM, defined as any 2 of the 4 parameters). In order to fill this gap, we aimed to evaluate the associations of serum markers of biochemical OM with dietary intake of macronutrients, vitamins and trace minerals. Methods: Saudi adolescents (N = 2,938, 57.8% girls), aged 12-17 years from 60 different schools in Riyadh, Saudi Arabia were included. Dietary intake of nutrients was calculated following a semi-quantitative 24 h dietary recall over 3 weekdays and 1 weekend-day using a validated food frequency questionnaire. Compliance to reference daily intake (RDI) of macronutrients, vitamins and trace minerals were calculated. Fasting blood samples were collected and circulating levels of 25(OH)D, ALP, Ca, and Pi were analyzed. Results: A total of 1819 (1,083 girls and 736 boys) adolescents provided the dietary recall data. Biochemical OM was identified in 175 (9.6%) participants (13.5% in girls, 3.9% in boys, p < 0.01) while the rest served as controls (N = 1,644). All participants had serum 25(OH)D levels <50 nmoL/L. Most participants had very low dietary intakes of Ca (median ~ 290 mg) and vitamin D (median ~ 4 µg) which are far below the RDI of 1,300 mg/day and 20 µg/day, respectively. In contrast, excess dietary intakes of Pi, Na, K, and Fe were observed in all participants. In the biochemical OM group, thiamine and protein intake were significant predictors of serum 25(OH)D, explaining 4.3% of the variance perceived (r = 0.23, adjusted r2 = 4.3%, p = 0.01). Among controls, dietary vitamin C and vitamin D explained 0.6% of the total variation in serum 25(OH)D (r = 0.09, adjusted r2 = 0.6%, p = 0.004). Conclusion: Arab adolescents do not meet the RDI for dietary Ca and vitamin D, and none have sufficient vitamin D status (25(OH)D levels >50 nmol/L) but they exceed the RDI for dietary Pi. Interpreting these data in the light of the increased prevalence of rickets in Arab countries, food fortification to optimise vitamin D and Ca intake in Saudi adolescents should be considered.

Enhancing the effectiveness of Alkaline Phosphatase and bone matrix proteins by tunable metal-organic composite for accelerated mineralization.

The irregular expression of bone matrix proteins occurring during the mineralization of bone regeneration results in various deformities which poses a major concern of orthopedic reconstruction. The limitations of the existing reconstruction practice paved a way for the development of a metal-organic composite [TQ-Sr-Fe] with Metal ions strontium [Sr] and iron [Fe] and a biomolecule Thymoquinone [TQ] in an attempt to enhance the bone mineralization due to their positive significance in osteoblast differentiation, proliferation and maturation. TQ-Sr-Fe was synthesized by in-situ coprecipitation and subjected to various characterization to determine their nature, compatibility and osteogenic efficiency. The crystallographic and electron microscopy analysis reveals sheet like structure of the composite. The negative cytotoxicity of TQ-Sr-Fe in the MG 63 cell line signified their biocompatibility. Cell adhesion and proliferation rate affirmed osteoconductive and osteoinductive nature of the composites and it was further supported by the gene expression of osteoblastic differentiation. The sequential expression of bone matrix proteins such as OCN, SPARC, COL 1, and Alkaline Phosphatase elevate the calcium deposition of MG-63 osteoblast like cells and initiates mineralization compared to control. Thus, the metal-organic composite TQ-Sr-Fe would make a suitable composite for accelerating mineralization process which would leads to faster bone regeneration.

Avermectin reduces bone mineralization via the TGF-ß signaling pathway in zebrafish.

Avermectin, a widely used insecticide, is primarily effective against animal parasites and insects. Given its extensive application in agriculture, a large amount of avermectin accumulates in natural water bodies. Studies have shown that avermectin has significant toxic effects on various organisms and on the nervous system, spine, and several other organs in humans. However, the effects of avermectin on bone development have not been reported yet. In this study, zebrafish embryos were treated with different concentrations of avermectin to explore the effects of avermectin on early bone development. The results showed that avermectin disturbed early bone development in zebrafish, caused abnormal craniofacial chondrogenesis, and reduced bone mineralization. Avermectin treatment significantly reduced mineralization in zebrafish scales and increased osteoclast activity. Real-time quantitative PCR results showed that avermectin decreased the expression of genes related to osteogenesis and transforming growth factor-ß (TGF-ß) and bone morphogenetic protein (BMP) signaling pathways. The TGF-ß inhibitor SB431542 rescued avermectin-induced bone mineralization and osteogenesis related gene expression in zebrafish during early development. Thus, this study provides insight into the mechanism of damage caused by avermectin on bone development, thus helping demonstrate its toxicity.

Age-related decline in bone mineral transport and bone matrix proteins in osteoblasts from stromal stem cells.

We studied osteoblast bone mineral transport and matrix proteins as a function of age. In isolated bone marrow cells from long bones of young (3 or 4 mo) and old (18 or 19 mo) mice, age correlated with reduced mRNA of mineral transport proteins: alkaline phosphatase (ALP), ankylosis (ANK), the Cl-/H+ exchanger ClC3, and matrix proteins collagen 1 (Col1) and osteocalcin (BGLAP). Some proteins, including the neutral phosphate transporter2 (NPT2), were not reduced. These are predominately osteoblast proteins, but in mixed cell populations. Remarkably, in osteoblasts differentiated from preparations of stromal stem cells (SSCs) made from bone marrow cells in young and old mice, differentiated in vitro on perforated polyethylene terephthalate membranes, mRNA confirmed decreased expression with age for most transport-related and bone matrix proteins. Additional mRNAs in osteoblasts in vitro included ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), unchanged, and ENPP2, reduced with age. Decrease with age in ALP activity and protein by Western blot was also significant. Transport protein findings correlated with micro-computed tomography of lumbar vertebra, showing that trabecular bone of old mice is osteopenic relative to young mice, consistent with other studies. Pathway analysis of osteoblasts differentiated in vitro showed that cells from old animals had reduced Erk1/2 phosphorylation and decreased suppressor of mothers against decapentaplegic 2 (Smad2) mRNA, consistent with TGFß pathway, and reduced ß-catenin mRNA, consistent with WNT pathway regulation. Our results show that decline in bone density with age reflects selective changes, resulting effectively in a phenotype modification. Reduction of matrix and mineral transport protein expression with age is regulated by multiple signaling pathways.NEW & NOTEWORTHY This work for the first time showed that specific enzymes in bone mineral transport, and matrix synthesis proteins, in the epithelial-like bone-forming cell layer are downregulated with aging. Results were compared using cells extracted from long bones of young and old mice, or in essentially uniform osteoblasts differentiated from stromal stem cells in vitro. The age effect showed memory in the stromal stem cells, a remarkable finding.

Analysis of Morphological Traits, Mineralization, and Mechanical Properties of Femoral Bones in Young and Adult European Hares (Lepus europaeus).

Lagomorphs, which include hares, rabbits, and pikas, are herbivorous animals renowned for their exceptional running abilities. The femur, the largest and strongest bone in their bodies, plays a crucial role in supporting their weight and facilitating movement. This study aimed to investigate the structural and functional changes in the femora of hares during their development in a sex-dependent manner, and the influence of aging on femur structure and function. Various mechanical properties, including stiffness and strength, as well as densitometry and morphology, were evaluated. The study was conducted on n = 24 hares collected from a hunting district in the Lublin region of Poland and divided into four groups: young females, adult females, young males and adult males (n = 6 animals each). Dual-energy X-ray absorptiometry (DXA) was used to measure bone mineral content (BMC) and bone mineral density (BMD), and a three-point bending test was performed to assess mechanical properties. The findings revealed age-related differences in bone properties, with adult males exhibiting increased BMC, and BMD compared to young males. Geometrical properties of the femora mid-diaphysis, such as cortical index and cross-sectional area, remained relatively unchanged during maturation. Regarding mechanical properties, the femora of young males exhibited higher elastic work compared to those of young females, while the femora of adult males exhibited higher elastic and breaking work than those of adult females. The stiffness of femora was higher in adult females compared to young females. The results provide insights into the development and aging of hare femora and contribute to our understanding of the relationship between bone mechanical properties, musculoskeletal system, and aging in the wild. This knowledge can inform animal husbandry practices in captivity and enhance our broader understanding of the ecological functions of lagomorphs.