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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. [ABSTRACT FROM AUTHOR]

Background: Risperidone is one of the most reliable and effective antipsychotics for schizophrenia treatment. However, the mechanism of action of risperidone is not yet fully understood. Traf2 and Nck-interacting protein kinase (TNIK), a schizophrenia susceptibility gene, is associated with risperidone treatment response. Our previous in vitro experiments confirmed that downregulated TNIK affected the effect of risperidone on downstream targets. However, the effect of downregulated TNIK on risperidone-induced molecular expression remains to be further explored. Methods: Transcriptome analysis was performed on U251 cells subjected to risperidone, TNIK siRNA, and no treatment, respectively. Compared to the notreatment group, two groups of DEGs were screened out and then intersected with the schizophrenia-related genes to screen the cross-talk genes. Those DEGs were analyzed using GO and KEGG. STRING and Cytoscape were used to construct a protein-protein interaction (PPI) network for the cross-talk gene. Results: The results showed that the parathyroid hormone synthesis, secretion, and action were significantly enriched after risperidone treatment. Downregulated TNIK could have an impact on the collagen-containing extracellular matrix, signaling receptor activator activity, and PI3K-Akt signaling pathway. Interestingly, bone mineralization function and calcium signaling pathway were enriched in the cross-talk genes. Additionally, FGFR2, FGF1, and FGFR might be the potential targets for TNIK affecting the effects of risperidone. Conclusion: The study indicated that risperidone primarily influences functions and/or pathways associated with bone metabolism, potentially contributing to the adverse effect of osteoporosis. Our study may offer a novel perspective on investigating the mechanisms underlying the adverse effects of risperidone. [ABSTRACT FROM AUTHOR]

Different biological models can be evaluated using mathematical models in both qualitative and quantitative ways. A fractional bone mineralization model involving Caputo’s fractional derivative is presented in this work. The fractional mathematical model is beneficial because of its memory carrying property. An appropriate fractional order of the derivative can be chosen that is more closely related to experimental or actual data. The dynamical system of equations for the process of bone mineralization is examined qualitatively and quantitatively in this article. A numerical simulation has been performed for the model. The model’s parameters have undergone sensitivity analysis and their effects on the model variables have been explored. By studying the mineralization patterns in bone, different diseases can be cured, and it can also be examined how the deviations from healthy mineral distributions lead to specific bone diseases. [ABSTRACT FROM AUTHOR]

Craniometaphyseal Dysplasia (CMD) is a rare skeletal disorder that can result from mutations in the ANKH gene. This gene encodes progressive anksylosis (ANK), which is responsible for transporting inorganic pyrophosphate (PPi) and ATP from the intracellular to the extracellular environment, where PPi inhibits bone mineralization. When ANK is dysfunctional, as in patients with CMD, the passage of PPi to the extracellular environment is reduced, leading to excess mineralization, particularly in bones of the skull. Zebrafish may serve as a promising model to study the mechanistic basis of CMD. Here, we provide a detailed analysis of the zebrafish Ankh paralogs, Ankha and Ankhb, in terms of their phylogenic relationship with ANK in other vertebrates as well as their spatiotemporal expression patterns during zebrafish development. We found that a closer evolutionary relationship exists between the zebrafish Ankhb protein and its human and other vertebrate counterparts, and stronger promoter activity was predicted for ankhb compared to ankha. Furthermore, we noted distinct temporal expression patterns, with ankha more prominently expressed in early development stages, and both paralogs also being expressed at larval growth stages. Whole-mount in situ hybridization was used to compare the spatial expression patterns of each paralog during bone development, and both showed strong expression in the craniofacial region as well as the notochord and somites. Given the substantial overlap in spatiotemporal expression but only subtle patterning differences, the exact roles of these genes remain speculative. In silico analyses predicted that Ankha and Ankhb have the same function in transporting PPi across the membrane. Nevertheless, this study lays the groundwork for functional analyses of each ankh paralog and highlights the potential of using zebrafish to find possible targeted therapies for CMD. [ABSTRACT FROM AUTHOR]

Physiological processes within the human body are regulated in approximately 24‐h cycles known as circadian rhythms, serving to adapt to environmental changes. Bone rhythms play pivotal roles in bone development, metabolism, mineralization, and remodeling processes. Bone rhythms exhibit cell specificity, and different cells in bone display various expressions of clock genes. Multiple environmental factors, including light, feeding, exercise, and temperature, affect bone diurnal rhythms through the sympathetic nervous system and various hormones. Disruptions in bone diurnal rhythms contribute to the onset of skeletal disorders such as osteoporosis, osteoarthritis and skeletal hypoplasia. Conversely, these bone diseases can be effectively treated when aimed at the circadian clock in bone cells, including the rhythmic expressions of clock genes and drug targets. In this review, we describe the unique circadian rhythms in physiological activities of various bone cells. Then we summarize the factors synchronizing the diurnal rhythms of bone with the underlying mechanisms. Based on the review, we aim to build an overall understanding of the diurnal rhythms in bone and summarize the new preventive and therapeutic strategies for bone disorders. [ABSTRACT FROM AUTHOR]

Epimedium is thought to enhance the integrity of tendons and bones, ease joint discomfort and rigidity and enhance kidney function. Although glucocorticoids are commonly used in clinical practice, the mechanism by which the active compound Epimedin C (EC) alleviates glucocorticoid‐induced osteoporosis (GIOP) is not well understood. The therapeutic potential of EC in treating GIOP was evaluated using alizarin red S staining, calcein immersion and fluorescence imaging, and bone mineralization, bone mass accumulation and bone density in zebrafish larvae were determined. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the key signalling pathways related to bone development were identified. A protein–protein interaction network (PPIN) was constructed to identify osteoclast characteristic genes and the findings were verified using real‐time quantitative PCR (RT‐qPCR). The bone tissue damage caused by prednisolone was reduced by EC. It also altered physiological processes, improved bone density, boosted mineralization and increased bone mass and activity. Subsequent empirical investigations showed that EC impacted the major signalling pathways involved in bone development, such as osteoclast differentiation, oestrogen, MAPK, insulin resistance, PPAR and AMPK signalling pathways. It also decreased the expression of genes typical of osteoclasts. The results of our study uncover a previously unknown function of EC in controlling bone formation and emphasize the potential of EC as a therapeutic target. The osteoprotective effect of EC indicates its potential as a cost‐effective strategy for treating GIOP. [ABSTRACT FROM AUTHOR]

Simple Summary: Supplementing piglets and growing–finishing pigs' diets with a new biosynthetic 6-phytase can be an effective replacement for inorganic phosphorus to improve growth performance, feed efficiency, bone mineralization, and availability of nutrients in weaned piglets and growing–finishing pigs. Phytase addition improved the availability of nutrients, especially of phosphorus and calcium and, therefore, growth performance and bone mineralization in weaning and growing–finishing pigs fed diets reduced in digestible phosphorus and calcium. In addition, the present study highlights that the main pathways (fecal and urinary) of phosphorus and calcium excretion in weaned piglets can be influenced not only by dietary levels of these minerals but also by phytase supplementation. Two experiments were performed to evaluate the effect of a biosynthetic 6-phytase added at 500 phytase unit (FTU)/kg diet on growth performance, bone mineralization, and nutrient digestibility and retention in weaned piglets and growing–finishing pigs. Experiments were performed on 90 weaned male and female piglets with an average initial body weight (BW) at 7.7 ± 0.73 kg, 26 days of age) and 300 male and female growing pigs (initial BW: 21.0 ± 3.44 kg) for 43 and 98 days in experiments 1 and 2, respectively. In each experiment, the animals were assigned to one of three treatments according to a randomized complete block design. The treatments consisted of a positive-control (PC) diet formulated to meet nutrient requirements; a negative-control (NC) diet reduced similarly in calcium (Ca) and digestible P by 0.15 and 0.12% points in phases 1 and 2, respectively, in piglets and by 0.14, 0.11, and 0.10% points, respectively, in phases 1, 2, and 3 in growing–finishing pigs, compared with PC diet; and a NC diet supplemented with the new 6-phytase at 500 FTU/kg diet (PHY). The dietary P and Ca depletion reduced (p < 0.05) the final BW (−11.9%; −7.8%,), average daily gain (ADG, −17.8%; −10.1%), average daily feed intake (ADFI, −9.9%; −6.0%), gain-to-feed (G:F) ratio (−8.9%; −4.6%), and apparent total tract digestibility (ATTD) of P (−7.7% points; −6.7% points) in nursery piglets and growing pigs, respectively. It also decreased (p < 0.001) P and Ca retention by 6.1 and 9.4% points, respectively, in nursery pigs and ash, P, and Ca contents in metacarpal bones by 18.4, 18.4, and 16.8%, respectively, in growing pigs. Compared to animals fed the NC diet, phytase supplementation improved (p < 0.001) the final BW (+7.7%; +11.3%), ADG (+12.5%; +15.0%), G:F ratio (+8.4%; +5.8%), ATTD of Ca (+10.8% points; +7.2% points), and ATTD of P (+18.7% points; +16.6% points) in weaned piglets and growing pigs, respectively. In addition, phytase also increased (p < 0.001) P and Ca retention by 6.1 and 9.4% points, respectively, in nursery pigs and ash, P, and Ca contents in metacarpal bones by 17.7, 15.0, and 15.2%, respectively, in growing pigs. The final BW, ADG, G:F ratio, and bone traits in animals fed the NC diet supplemented with phytase were comparable to animals fed the PC diet. This finding indicates the ability of this novel biosynthetic phytase to restore performance and bone mineralization by improving the availability of P and Ca in piglets and growing pigs fed P- and Ca-deficient diets. [ABSTRACT FROM AUTHOR]

ABSTRACT Sunflower cake is an alternative feed with nutritional potential to be used in poultry feeding. However, the high fiber content and the presence of antinutritional factors can compromise the availability of nutrients in the diet and influence the bone development of pullets, which can be mitigated with the dietary inclusion of exogenous enzymes. In this context, the objective of this study was to evaluate the effect of multi-enzyme supplementation in rations containing sunflower cake to pullets on mineral digestibility and quality and bone mineral composition. A total of 1020 pullets 7-weeks-age from the commercial strain Hy-Line, 540 white pullets and 480 brown pullets, were distributed in a randomized experimental design in a 2×2+1 factorial arrangement, with two sunflower cake levels (10 and 20% for white pullets and 12 and 24 % for brown pullets); two ways of enzymatic supplementation (absence and presence of multi-enzyme complex), and one control diet based on corn and soybean meal, totaling five treatments with six replicates of 18 white pullets and 16 brown pullets. Between week 14 and week 15, a metabolic trial was performed to determine the digestibility coefficient of ration minerals. To assess bone quality of the tibia and femur, one pullet of the 12th and 17th week of age was selected and killed. There was no significant interaction between the tested factors (sunflower cake x multi-enzyme complexes) for any of the variables studied. The inclusion level of sunflower cake did not influence the growth and bone quality parameters of the pullets during the growing phase. However, a presence of the multi-enzyme complex provided significant improvements in Ca, P and Mg digestibility coefficient. It is concluded that the multi-enzyme supplementation in the diets increases the digestibility of the mineral in the rations, contributing to the maintenance of bone quality of pullets fed with up to 24% sunflower cake.

The objective of the present study was to improve our understanding of the complex biological process of bone mineralization by performing mathematical modeling with the Caputo-Fabrizio fractional operator. To obtain a better understanding of Komarova's bone mineralization process, we have thoroughly examined the boundedness, existence, and uniqueness of solutions and stability analysis within this framework. To determine how model parameters affect the behavior of the system, sensitivity analysis was carried out. Furthermore, the fractional Adams-Bashforth method has been used to carry out numerical and graphical simulations. Our work is significant owing to its comparison of fractional- and integer-order models, which provides novel insight into the effectiveness of fractional operators in representing the complex dynamics of bone mineralization.

Background: In standard fortification of human milk (HM), the HM macronutrient content is assumed, and a fixed amount of a multinutrient fortifier is added to achieve recommended nutrient intakes. In target fortification, the HM macronutrient content is regularly measured, guiding the addition of modular macronutrient supplements to the fortified HM, to achieve the nutritional targets more precisely. Objective: The study aimed to investigate whether this addition of modular supplements, unaccompanied by mineral supplementation, predispose to metabolic bone disease (MBD). Methods: This is a secondary analysis of a larger study of infants born with <33 weeks gestational age. Fortifications based on the assumed (Group 1) or measured (Group 2) of the HM macronutrient content were compared, using low serum phosphate levels as an indicator of MBD, and length growth as a surrogate of bone growth. Results: Eighty‑four infants were included, 35 in Group 1 and 49 in Group 2. During the exposure period, infants of Group 2 received higher mean fat (6.1 vs. 5.3 g/kg/day, P < 0.001) and carbohydrate (13.0 vs. 11.7 g/kg/day, P < 0.001) intakes; in addition, they exhibited lower mean serum phosphate (5.5 vs. 6.0 mg/dL, P = 0.022) and faster mean length velocity (1.06 vs. 0.89 cm/week, P = 0.003). Conclusions: These findings suggest that feeding fortified HM with extra fat and carbohydrate content, unaccompanied by mineral supplementation, promotes increased bone growth, as indicated by accelerated length growth, but with insufficiently mineralized osteoid, indicated by low serum phosphate levels. Intervention studies using direct biomarkers of bone mass content and mineral density are necessary to corroborate our findings. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bone loss is a well-known phenomenon in the older population leading to increased bone fracture risk, morbidity, and mortality. Supplementation of eggshell membrane (ESM) is evaluated due to its possible application to prevent bone loss and usage in osteoporosis therapy. The similar organic chemical composition of ESM and human bone is described in detail as both mainly consist of collagen type I, chondroitin sulfate, dermatan sulfate, hyaluronic acid and elastan. ESM and its components are reported to improve mineralization in bone tissue. In many studies ESM intake reduced pain in patients with joint disorders and reduced inflammatory processes. Additionally, ESM improved calcium uptake in human cells. These findings in comparison with a clinical pilot study reporting pain reduction in osteoporotic patients and increased osteoblast activity in in vitro assays support ESM to be a beneficial supplement for bone health. In this systematic review we combined chemical structure analysis with clinical studies to give a more comprehensive picture with novel explanations.

Despite advances in neonatal care, metabolic bone disease of prematurity (MBDP) remains a common problem in preterm infants. The development of non-invasive and affordable diagnostic approaches can be highly beneficial in the diagnosis and management of preterm infants at risk of MBDP. In this study, we present an ultrasound method called pulsed vibro-acoustic analysis to investigate the progression of bone mineralization in infants over time versus weight and postmenstrual age. The proposed pulsed vibro-acoustic analysis method is used to evaluate the vibrational characteristics of the bone. This method uses the acoustic radiation force of ultrasound to vibrate the bone. The generated acoustic waves are detected using a hydrophone placed on the skin over the tibia. The frequency of vibration and the speeds of received acoustic waves have information regarding the material property of the bone. We examined the feasibility of this method through an in vivo study consisting of 25 preterm and 10 full term infants. The pulsed vibro-acoustic data were acquired longitudinally in preterm infants with multiple visits and at a single visit in full term infants. Speed of sound and mean peak frequency of slow and fast sound waves recorded by hydrophone were used to analyze bone mineralization progress. Linear mixed model was used for statistical analysis in characterizing the mineralization progress in preterm infants compared to data from full term subjects. Significance changes in wave parameters (speed of sound and mean peak frequency) with respect to the postmenstrual age and weight in preterm infants were observed with p-values less than 0.05. Statistical significances in speed of sound measurement for both fast and slow waves were observed between preterm and full term infants, with p-values of

Study aim: This cross-sectional study examined the relationship between biological factors, physical activity (PA), bone turnover markers (BTMs) and bone mineral density (BMD) in women with subclinical hypothyroidism (SCH) and healthy.

Craniometaphyseal Dysplasia (CMD) is a rare skeletal disorder that can result from mutations in the ANKH gene. This gene encodes progressive anksylosis (ANK), which is responsible for transporting inorganic pyrophosphate (PPi) and ATP from the intracellular to the extracellular environment, where PPi inhibits bone mineralization. When ANK is dysfunctional, as in patients with CMD, the passage of PPi to the extracellular environment is reduced, leading to excess mineralization, particularly in bones of the skull. Zebrafish may serve as a promising model to study the mechanistic basis of CMD. Here, we provide a detailed analysis of the zebrafish Ankh paralogs, Ankha and Ankhb, in terms of their phylogenic relationship with ANK in other vertebrates as well as their spatiotemporal expression patterns during zebrafish development. We found that a closer evolutionary relationship exists between the zebrafish Ankhb protein and its human and other vertebrate counterparts, and stronger promoter activity was predicted for ankhb compared to ankha. Furthermore, we noted distinct temporal expression patterns, with ankha more prominently expressed in early development stages, and both paralogs also being expressed at larval growth stages. Whole-mount in situ hybridization was used to compare the spatial expression patterns of each paralog during bone development, and both showed strong expression in the craniofacial region as well as the notochord and somites. Given the substantial overlap in spatiotemporal expression but only subtle patterning differences, the exact roles of these genes remain speculative. In silico analyses predicted that Ankha and Ankhb have the same function in transporting PPi across the membrane. Nevertheless, this study lays the groundwork for functional analyses of each ankh paralog and highlights the potential of using zebrafish to find possible targeted therapies for CMD.

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.

Two experiments were performed to evaluate the effect of a biosynthetic 6-phytase added at 500 phytase unit (FTU)/kg diet on growth performance, bone mineralization, and nutrient digestibility and retention in weaned piglets and growing–finishing pigs. Experiments were performed on 90 weaned male and female piglets with an average initial body weight (BW) at 7.7 ± 0.73 kg, 26 days of age) and 300 male and female growing pigs (initial BW: 21.0 ± 3.44 kg) for 43 and 98 days in experiments 1 and 2, respectively. In each experiment, the animals were assigned to one of three treatments according to a randomized complete block design. The treatments consisted of a positive-control (PC) diet formulated to meet nutrient requirements; a negative-control (NC) diet reduced similarly in calcium (Ca) and digestible P by 0.15 and 0.12% points in phases 1 and 2, respectively, in piglets and by 0.14, 0.11, and 0.10% points, respectively, in phases 1, 2, and 3 in growing–finishing pigs, compared with PC diet; and a NC diet supplemented with the new 6-phytase at 500 FTU/kg diet (PHY). The dietary P and Ca depletion reduced (p < 0.05) the final BW (−11.9%; −7.8%,), average daily gain (ADG, −17.8%; −10.1%), average daily feed intake (ADFI, −9.9%; −6.0%), gain-to-feed (G:F) ratio (−8.9%; −4.6%), and apparent total tract digestibility (ATTD) of P (−7.7% points; −6.7% points) in nursery piglets and growing pigs, respectively. It also decreased (p < 0.001) P and Ca retention by 6.1 and 9.4% points, respectively, in nursery pigs and ash, P, and Ca contents in metacarpal bones by 18.4, 18.4, and 16.8%, respectively, in growing pigs. Compared to animals fed the NC diet, phytase supplementation improved (p < 0.001) the final BW (+7.7%; +11.3%), ADG (+12.5%; +15.0%), G:F ratio (+8.4%; +5.8%), ATTD of Ca (+10.8% points; +7.2% points), and ATTD of P (+18.7% points; +16.6% points) in weaned piglets and growing pigs, respectively. In addition, phytase also increased (p < 0.001) P and Ca retention by 6.1 and 9.4% points, respectively, in nursery pigs and ash, P, and Ca contents in metacarpal bones by 17.7, 15.0, and 15.2%, respectively, in growing pigs. The final BW, ADG, G:F ratio, and bone traits in animals fed the NC diet supplemented with phytase were comparable to animals fed the PC diet. This finding indicates the ability of this novel biosynthetic phytase to restore performance and bone mineralization by improving the availability of P and Ca in piglets and growing pigs fed P- and Ca-deficient diets.

Bone has several crucial functions. It is essential for locomotion and allows our body to stand erect against gravity. A mismatch between the mechanical stresses applied to it and its mechanical resistance leads to fractures. Bone also has numerous endocrine functions. It acts as a reservoir for minerals such as calcium and phosphorus, making it the target of calciotropic hormones that mobilize these minerals, particularly calcium, according to the body's needs. Additionally, bone secretes hormones, notably fibroblast growth factor 23 (FGF23), which regulates urinary excretion of phosphate and the bioavailability of active vitamin D. Bone mineralization is the process that facilitates the organized deposition of minerals in the bone matrix, providing rigidity and appropriate mechanical resistance. This process is compromised in genetically related bone mineralization disorders, such as those causing hypophosphatemia or hypophosphatasia. Conversely, calcification can be pathological, affecting soft tissues like the blood vessels, as seen in generalized arterial calcification of infancy (GACI) or arterial calcification due to CD73 deficiency (ACDC). The aim of this article is to first present the composition and structure of the mineralized bone matrix, to review the current understanding of the molecular mechanisms of mineralization, and finally to discuss the conditions associated with ectopic calcification and the underlying mechanisms. [ABSTRACT FROM AUTHOR]

This study investigated the impact of calcium (Ca) and vitamin D supplements on bone metabolism, bone measurement, lameness, and selection rate in gilts fed five dietary treatments. Two Ca levels (6.85/6.42 [adequate; ACa] or 8.99/8.56 [high; HCa] g/kg) were combined with either 856 IU/kg vitamin D3 (Danish feeding standards; adequate; AD3) or 50 μg/kg 25-hydroxyvitamin D3 (high; HHyD) to create ACaAD3, HCaAD3, ACaHHyD, and HCaHHyD diets. The values 6.85/6.42 and 8.99/8.56 g/kg correspond to adequate and high Ca supply for gilts weighing 32 to 100 and 100 to 180 kg body weight (BW), respectively. The fifth diet was a combination of HCa and 2,000 IU/kg vitamin D3 (high; HD3) to create HCaHD3. Two hundred gilts were phase fed the dietary treatments from 32 to 100 and 100 to 180 kg BW until they were slaughtered, either at 100 or 180 kg BW. The gilts were weighed fortnightly, and plasma and urine samples were collected at 100 and 180 kg BW. At slaughter, the 2nd and 3rd metacarpal bones were collected for bone parameters measurements. Lameness and selection rate were assessed within the last 7 d at 100 and 180 kg BW. Dietary treatments did not affect gilts' growth performance and plasma concentration of Ca, but urinary concentration of Ca was greater in HCa-supplemented gilts at both 100 (P = 0.003) and 180 (P = 0.05) kg BW. Plasma concentration of vitamin D3 (P < 0.001) and 25-hydroxyvitamin D3 (P < 0.001) showed dose-dependent responses at both 100 and 180 kg BW. Bone-specific alkaline phosphatase was greater (P = 0.02) in the plasma sample collected at 180 kg BW in gilts fed the HCaHD3 diet and tended to be greater in gilts fed the ACaAD3 diet (P = 0.06). The bone ash content (P = 0.02) was greater in gilts fed the HCaAD3 diet and slaughtered at 100 kg BW compared with gilts fed the ACaAD3 and ACaHHyD diets. However, bone weight, length, thickness, dry matter, and mineral content did not differ among the dietary treatments at both 100 and 180 kg BW (P > 0.05). Neither lameness nor selection rate was affected by the dietary treatments. The average daily gain of gilts weighing 32 to 100 and 100 to 180 kg BW showed a positive correlation with bone strength (r = 0.37; P < 0.001) and bone ash content (r = 0.24; P = 0.02), respectively. In conclusion, higher Ca and vitamin D3 supplementation slightly increased bone ash content but had no effect on lameness or selection rate of the gilts compared to those fed according to the Danish nutrient standards., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Abamectin (ABM) is a widely used pesticide in agriculture and veterinary medicine, which primarily acts by disrupting the neurological physiology of pests, leading to their paralysis and death. Its extensive application has resulted in contamination of many natural water bodies. While the adverse effects of ABM on the growth and development of non-target organisms are well documented, its impact on bone development remains inadequately studied. The present study aimed to investigate the effects of environmentally relevant concentrations of ABM (1, 5, 25 μg/L) on early bone development in zebrafish. Our results indicated that ABM significantly affected both cartilage and bone development of zebrafish larvae, accompanied by dose-dependent increase in deformity and mortality rates, as well as exacerbated apoptosis. ABM exposure led to deformities in the ceratobranchial (cb) and hyosymplectic (hs), accompanied by significant increases in the length of the palatoquadrate (pq). Furthermore, significant decreases in the CH-CH angle, Meckel's-Meckel's angle, and Meckel's-PQ angle were noted. Even at the safe concentration of 5 μg/L (1/10 of the 96 h LC 50 ), ABM delayed the process of bone mineralization in zebrafish larvae. Real-time fluorescent quantitative PCR results demonstrated that ABM induced differential gene expression associated with cartilage and bone development in zebrafish. Thus, this study provides preliminary insights into the effects and molecular mechanisms underlying ABM's impact on the bone development of zebrafish larvae and offers new evidence for a better understanding of its toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ammonia is a major pollutant of freshwater environments. Previous studies have indicated that ammonia exposure adversely affects the physiology of freshwater fish. However, its effect on bone mineralization in freshwater fish larvae remains unclear. In this study, zebrafish larvae were used as a model to investigate the effects of different ammonia levels (0, 2.5, 5, and 10 mM NH 4 Cl) on the survival rate, body length, and bone mineralization of fish. The survival rate of zebrafish embryos exposed to different NH 4 Cl concentrations for 8 days was not affected. In contrast, the body length and bone mineralization of zebrafish larvae at 8 days post fertilization (dpf) were significantly reduced at 5 and 10 mM NH 4 Cl exposure. Further investigations revealed that ammonia exposure decreased the mRNA expression of osteoblast-related genes and increased that of osteoclast-related genes. Additionally, exposure to 5 mM and 10 mM NH4Cl induced the production of reactive oxygen species (ROS). 10 mM-but not 5 mM-NH 4 Cl exposure reduced the calcium and phosphorus content in 8 dpf zebrafish larvae. In conclusion, ammonia exposure induces bone resorption, while decreasing the calcium and phosphorus content of the whole body and bone formation, resulting in impaired bone mineralization in fish larvae., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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-17β (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. [Display omitted] • Regulatory-relevant concentration of E2 has negative effects on mineralization. • BPA treatment inhibited mineralization but with less severe effects than E2. • E2 and BPA act differently on the expression of gene markers of bone cells. • Elevated concentrations of E2 and BPA exert opposite effects on growth. [ABSTRACT FROM AUTHOR]

Exposure to plastic-derived estrogen-mimicking endocrine-disrupting bisphenols can have a long-lasting effect on bone health. However, gestational exposure to bisphenol A (BPA) and its analogue, bisphenol S (BPS), on offspring's bone mineralization is unclear. The effects of in-utero bisphenol exposure were examined on the offspring's bone parameters. BPA and BPS (0.0, 0.4 μg/kg bw) were administered to pregnant Wistar rats via oral gavage from gestational day 4–21. Maternal exposure to BPA and BPS increased bone mineral content and density in the offspring aged 30 and 90 days (P < 0.05). Plasma analysis revealed that alkaline phosphatase, and Gla-type osteocalcin were significantly elevated in the BPS-exposed offspring (P < 0.05). The expression of BMP1 , BMP4 , and their signaling mediators SMAD1 mRNAs were decreased in BPS-exposed osteoblast SaOS-2 cells (P < 0.05). The expression of extracellular matrix proteins such as ALPL, COL1A1, DMP1, and FN1 were downregulated (P < 0.05). Bisphenol co-incubation with noggin decreased TGF-β1 expression, indicating its involvement in bone mineralization. Altered mineralization could be due to dysregulated expression of bone morphogenetic proteins and signalling mediators in the osteoblast cells. Thus, bisphenol exposure during gestation altered growth and bone mineralization in the offspring, possibly by modulating the expression of Smad-dependent BMP/TGF-β1 signalling mediators. [Display omitted] • Fetal exposure to BPS increases the offspring's whole-body bone mineral content and density, similar to BPA. • In-utero BPS exposure decreases plasma calcium and increases ALP and Gla-type osteocalcin in the offspring. • Bisphenol exposure modulates Smad-dependent BMP/TGF-β1 signaling in the SaOS-2 cells. [ABSTRACT FROM AUTHOR]

Background: Risperidone is one of the most reliable and effective antipsychotics for schizophrenia treatment. However, the mechanism of action of risperidone is not yet fully understood. Traf2 and Nck-interacting protein kinase ( TNIK ), a schizophrenia susceptibility gene, is associated with risperidone treatment response. Our previous in vitro experiments confirmed that downregulated TNIK affected the effect of risperidone on downstream targets. However, the effect of downregulated TNIK on risperidone-induced molecular expression remains to be further explored., Methods: Transcriptome analysis was performed on U251 cells subjected to risperidone, TNIK siRNA, and no treatment, respectively. Compared to the no-treatment group, two groups of DEGs were screened out and then intersected with the schizophrenia-related genes to screen the cross-talk genes. Those DEGs were analyzed using GO and KEGG. STRING and Cytoscape were used to construct a protein-protein interaction (PPI) network for the cross-talk gene., Results: The results showed that the parathyroid hormone synthesis, secretion, and action were significantly enriched after risperidone treatment. Downregulated TNIK could have an impact on the collagen-containing extracellular matrix, signaling receptor activator activity, and PI3K-Akt signaling pathway. Interestingly, bone mineralization function and calcium signaling pathway were enriched in the cross-talk genes. Additionally, FGFR2, FGF1, and FGFR might be the potential targets for TNIK affecting the effects of risperidone., Conclusion: The study indicated that risperidone primarily influences functions and/or pathways associated with bone metabolism, potentially contributing to the adverse effect of osteoporosis. Our study may offer a novel perspective on investigating the mechanisms underlying the adverse effects of risperidone., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yuan, Li, Li, Fu, Ning, Wang, Zhang, Yu and Xu.)

The Forkhead box transcription factors FOXC1 and FOXC2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, whereas the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed, with fewer Indian hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocytes and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that FOXC1 and FOXC2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, FOXC1 and FOXC2 regulate function in hypertrophic chondrocyte remodeling to allow primary ossification center formation and osteoblast recruitment., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

We report the genome sequence of the human fecal isolate Lacticaseibacillus paracasei LPC100 from the NORDBIOTIC collection, comprising a 3.075 Mb chromosome and three plasmids (61 kb, 12 kb, and 7 kb). Genetic content reveals the strain's beneficial features-complete lactose metabolic pathway, potential production of bacteriocins, and short-chain fatty acids., Competing Interests: The authors declare no conflict of interest.

The tunable nature of two-dimensional nanostructured Layered Double Hydroxides [LDH] and their effective intercalation ability with biomolecules make them inevitable functional hybrid materials for versatile biomedical applications. In an attempt to meet the limitations in existing bone reconstruction practice, the present work reports the synthesis of zinc and iron-based LDH [Zn-Fe LDH] and its subsequent intercalation with thymoquinone [TQ-Zn-Fe LDH] to augment osteoblastic differentiation and proliferation for accelerated bone mineralization. Structural studies depicted the polycrystalline and amorphous nature of Zn Fe LDH and TQ-Zn-Fe LDH respectively. The biocompatible nature of LDHs was proven by negative cytotoxicity, cell adhesion, and proliferation assays studied in MG-63 cell lines. Gene expression studies of the nanocomposites showed a fold change of 5–10 in osteoblastic differentiation and proliferation. However, TQ-Zn-Fe LDH treated cells showed significantly higher protein translation levels of osteogenesis markers and elevated alkaline phosphatase secretion and calcium deposition on day 21, confirming their osteogenic efficiency. Thus, acquired results speculate the osteogenic potential of thymoquinone intercalated TQ-Zn-Fe-LDH to meet the limitations in bone reconstruction practice. [Display omitted] • Thymoquinone intercalated Zn Fe LDH provides a multidirectional effect on osteogenesis. • LDH helps in the augmentation of osteoblastogenesis and inhibition of osteoclastogenesis. • Sequential expression of alkaline Phosphatase aids in maintaining the bone homeostasis. • TQ-Zn-Fe LDH aids in accelerated mineralization by enhanced calcium deposition. • The synergistic effect of Zn, Fe, and TQ makes it a promising candidate for bone regeneration. [ABSTRACT FROM AUTHOR]

Bone loss is a well-known phenomenon in the older population leading to increased bone fracture risk, morbidity, and mortality. Supplementation of eggshell membrane (ESM) is evaluated due to its possible application to prevent bone loss and usage in osteoporosis therapy. The similar organic chemical composition of ESM and human bone is described in detail as both mainly consist of collagen type I, chondroitin sulfate, dermatan sulfate, hyaluronic acid and elastan. ESM and its components are reported to improve mineralization in bone tissue. In many studies ESM intake reduced pain in patients with joint disorders and reduced inflammatory processes. Additionally, ESM improved calcium uptake in human cells. These findings in comparison with a clinical pilot study reporting pain reduction in osteoporotic patients and increased osteoblast activity in in vitro assays support ESM to be a beneficial supplement for bone health. In this systematic review we combined chemical structure analysis with clinical studies to give a more comprehensive picture with novel explanations., Competing Interests: JPF reported receiving personal fees from Apomedica Pharmazeutische Produkte GmbH as employee.The authors have no conflict of interest to declare., (© 2024 The Authors.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Majhi et al.)

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., (© 2024 The Authors. Pediatric Pulmonology published by Wiley Periodicals LLC.)

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 (ZnSO 4 ·7H 2 O), 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 ., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Md. Amzad Hossain reports financial support was provided by University Grants Commission of Bangladesh. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

The objective of the present study was to improve our understanding of the complex biological process of bone mineralization by performing mathematical modeling with the Caputo-Fabrizio fractional operator. To obtain a better understanding of Komarova's bone mineralization process, we have thoroughly examined the boundedness, existence, and uniqueness of solutions and stability analysis within this framework. To determine how model parameters affect the behavior of the system, sensitivity analysis was carried out. Furthermore, the fractional Adams-Bashforth method has been used to carry out numerical and graphical simulations. Our work is significant owing to its comparison of fractional- and integer-order models, which provides novel insight into the effectiveness of fractional operators in representing the complex dynamics of bone mineralization.

[Display omitted] • Biomimicking PCL-Gelatin nanofibrous matrix membrane as periosteum substitute. • Sustained delivery of osteo-inductive bioactive molecule by nanofibrous matrix. • Osteo-inductive and osteo-conductive nanofibrous matrix to promote bone regeneration. • Enhanced calcium deposition by the biomimetic nanofibrous matrix. The resemblance of nanofibers to the extracellular matrix (ECM) makes it suitable for various tissue engineering application. In this study, electro-spun polycaprolactone (PCL)-gelatin nanofibers had been fabricated with the incorporation thymoquinone (TQ) biomolecule as a periosteum substitute to promote regeneration of bone. The fabricated nanofibrous matrix possess osteo-conductive and osteo-inductive property. It is biocompatible and also acts as an excellent drug delivery vehicle. The significant expression of the bone matrix proteins, alkaline phosphatase secretion and calcium deposition studied in the MG-63 osteoblast like cells seeded on the nanofibrous matrix proves that it makes a suitable periosteum-substitute by enhancing bone mineralization. [ABSTRACT FROM AUTHOR]

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

A total of 882 pigs (PIC TR4 × [Fast LW × PIC L02]; initially 33.2 ± 0.31 kg) were used in a 112-d study to evaluate the effects of different bones and analytical methods on the assessment of bone mineralization response to changes in dietary P, phytase, and vitamin D in growing pigs. Pens of pigs (20 pigs per pen) were randomized to one of five dietary treatments with nine pens per treatment. Dietary treatments were designed to create differences in bone mineralization and included: 1) P at 80% of NRC (2012) standardized total tract digestible (STTD) P requirement, 2) NRC STTD P with no phytase, 3) NRC STTD P with phytase providing an assumed release of 0.14% STTD P from 2,000 FYT/kg, 4) high STTD P (128% of the NRC P) using monocalcium phosphate and phytase, and 5) diet 4 with additional vitamin D3 from 25(OH)D3. On day 112, one pig per pen was euthanized for bone, blood, and urine analysis. Additionally, 11 pigs identified as having poor body condition which indicated a history of low feed intake (unhealthy) were sampled. There were no differences between treatments for final body weight, average daily gain, average daily feed intake, gain to feed, or bone ash measurements (treatment × bone interaction) regardless of bone ash method. The response to treatment for bone density and bone mineral content was dependent upon the bone sampled (density interaction, P = 0.053; mineral interaction, P = 0.078). For 10th rib bone density, pigs fed high levels of P had increased (P < 0.05) bone density compared with pigs fed NRC levels with phytase, with pigs fed deficient P, NRC levels of P with no phytase, and high STTD P with extra 25(OH)D3 intermediate, with no differences for metacarpals, fibulas, or 2nd ribs. Pigs fed extra vitamin D from 25(OH)D3 had increased (P < 0.05) 10th rib bone mineral content compared with pigs fed deficient P and NRC levels of P with phytase, with pigs fed industry P and vitamin D, and NRC P with monocalcium intermediate. Healthy pigs had greater (P < 0.05) serum Ca, P, vitamin D concentrations, and defatted bone ash than those unhealthy, with no difference between the two health statuses for non-defatted bone ash. In summary, differences between bone ash procedures were more apparent than differences between diets. Differences in bone density and mineral content in response to dietary P and vitamin D were most apparent with 10th ribs., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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 ± 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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Despite advances in neonatal care, metabolic bone disease of prematurity (MBDP) remains a common problem in preterm infants. The development of non-invasive and affordable diagnostic approaches can be highly beneficial in the diagnosis and management of preterm infants at risk of MBDP. In this study, we present an ultrasound method called pulsed vibro-acoustic analysis to investigate the progression of bone mineralization in infants over time versus weight and postmenstrual age. The proposed pulsed vibro-acoustic analysis method is used to evaluate the vibrational characteristics of the bone. This method uses the acoustic radiation force of ultrasound to vibrate the bone. The generated acoustic waves are detected using a hydrophone placed on the skin over the tibia. The frequency of vibration and the speeds of received acoustic waves have information regarding the material property of the bone. We examined the feasibility of this method through an in vivo study consisting of 25 preterm and 10 full term infants. The pulsed vibro-acoustic data were acquired longitudinally in preterm infants with multiple visits and at a single visit in full term infants. Speed of sound and mean peak frequency of slow and fast sound waves recorded by hydrophone were used to analyze bone mineralization progress. Linear mixed model was used for statistical analysis in characterizing the mineralization progress in preterm infants compared to data from full term subjects. Significance changes in wave parameters (speed of sound and mean peak frequency) with respect to the postmenstrual age and weight in preterm infants were observed with p-values less than 0.05. Statistical significances in speed of sound measurement for both fast and slow waves were observed between preterm and full term infants, with p-values of <0.01 and 0.02, respectively. The results of this pilot study indicate the potential use of vibro-acoustic analysis for monitoring the progression of bone mineralization in preterm infants.