Alkaline Phosphatase Activity Research Articles

Alginate-gelatin based nanocomposite hydrogel scaffold incorporated with bioactive glass nanoparticles and fragmented nanofibers promote osteogenesis: From design to in vitro studies

The current study proposes fragmented nanofibers of polycaprolactone (FNF) with bioactive glass nanoparticles (nBG) incorporated into a polymeric matrix of alginate-gelatin for the creation of a hydrogel scaffold. Four groups were prepared: control, bioactive glass containing scaffold (BG), fragmented nanofibers with bioactive glass scaffold (FNF(PCL) + BG), and fragmented composite nanofibers scaffold (FNF (PCL + BG)). FNF (PCL + BG) scaffolds revealed a more controlled degradation rate, with approximately 20 % degradation occurring after 28 compared. The FNF(PCL) + BG scaffolds had the highest compressive strength in both dry and wet states. Following 14 days of incubation in simulated body fluid, hydroxyapatite formation had occurred on the surface of scaffolds containing nBG, and after 28 days on other groups tested. Cell studies revealed that the FNF(PCL) + BG scaffolds had superior cell viability without inhibiting cell proliferation. The FNF(PCL) + BG and FNF(PCL + BG) scaffolds had the highest alkaline phosphatase (ALP) activity and FNF(PCL) + BG scaffolds showed to support osteogenic differentiation.

Age-dependent haemogram and sex-dependent serum biochemistry values in semi-feral Konik horses.

Semi-feral, free-roaming Konik polski horses are used in some European countries for preserving semi-open pasture landscapes. The estimation of their health status is still limited by insufficient data on various blood parameters. Therefore, our study aimed at the sex- and age-dependent analysis of haemogram and selected biochemistry parameters in healthy, semi-feral Koniks. In order to reach this aim, we took blood samples from 53 female and 18 male (8 uncastrated, 10 castrated) Koniks living in two Middle German nature reserves. They were of different age (9-266 months) and without signs of illness. Blood samples were analysed by an accredited laboratory. We identified age- but not sex-dependent changes in the white blood cell count (WBC). Higher age mainly caused a decrease in lymphocytes. Therefore, WBC correlated negatively and granulocyte-to-lymphocyte ratio positively with increasing age. Serum values of selected biochemical parameters did not depend on age but showed some sex-related differences. In this regard, serum total protein, triglyceride and the enzymatic activities of alkaline phosphatase, aspartate transaminase and g-glutamyltransferase were higher in males than females. However, the sex dependency of these enzymatic activities was restricted to uncastrated males. They also showed higher serum values for calcium and selenium than castrated males or all females. As far as the respective group sizes permitted, we then calculated age- or sex-dependent reference interval values for all parameters analysed. These values improve now the estimation of the health status of semi-feral, free-roaming Konik horses and provide a stable basis for future studies.

Straw return can increase maize yield by regulating soil bacteria and improving soil properties in arid and semi-arid areas

Straw return has been found to benefit soil fertility and crop yield, however, by which it affects microbial communities to mediate soil factors driving crop yields under maize continuous cropping systems in dryland areas is still unclear. To fill this gap, a 6-year field experiment was established with five straw return amounts (T0, T1, T2, T3, and T4, representing 0, 3,000, 6,000, 9,000, and 12,000kgha-1 of straw, respectively), and investigated the effects of on soil properties, enzymes, bacterial community composition and diversity, and crop yields. Our analysis showed that soil organic carbon (SOC), total nitrogen (TN), and total phosphorus (TP) contents significantly increased by 1-8%, 5-25%, and 2-9% under straw return treatments, respectively, compared to the T0, and soil catalase, urease, and alkaline phosphatase activities increased by at least 34.00%. Additionally, crop yield significantly increased by 4.23-12.00% under T1-T4 treatments, and showed highly significant relationships with SOC, TN, and TP. Importantly, we found straw return significantly altered the community of bacteria involved in the carbon and nitrogen cycle, and their abundance of strong responses depending on the amounts of straw return. For example, straw input increased the abundance of Proteobacteria (+2.64–5.57%), Acidobacteria (+3.82–13.83%), and Bacteroidetes (+15.37–30.49%). Similarly, the amount of straw application increased the bacterial diversity indexes (Shannon, 2.65–10.93%; Chao1, 13.47–18.50%), and had significant positive correlations with SOC, TN, and TP contents. Structural equation models (SEM) revealed that straw return management practice had positive and indirect effects on crop yields by influencing soil properties or the bacteria community. In conclusion, our findings revealed common associations and variations of bacterial community diversity with soil factors and crop yields at different straw return rates, and these findings provide insights and options for the development of better straw return strategies and sustainable agriculture in semi-arid regions.

Determinants of rhizospheric organic carbon fractions and accumulation in four different vegetations of coastal saline-alkali soils

Rhizosphere processes are key pathways through which plants influence the carbon cycling of diverse ecosystems and are closely associated with vegetation types. However, the environmental responses of rhizospheric soil organic carbon (SOC) mediated by the interaction between rhizospheric effect and vegetation type remain uncertain, severely limiting our assessment of the carbon sequestration potential of different coastal vegetation. Here, we identified the core factors driving changes in various soil carbon fractions in the rhizosphere of four coastal vegetation types by testing the contents of the reactive, dissolved, particulate, mineral-associated, heavy and light fractions of SOC and their relationships with physicochemical properties and enzyme activities. It was found that the rhizosphere effect significantly influenced SOC, with rhizospheric SOC contents varying significantly among vegetation types, ranging from 2.12 to 9.04 g/kg. Besides, rhizospheric soil pH was found to contribute significantly to SOC fractions (except for light organic carbon) and exert significant inhibitory effects on SOC, reactive organic carbon, mineral-associated organic carbon and heavy organic carbon. Despite the different responses of various SOC fractions to physicochemical properties and enzyme activities, soil available phosphorus and alkaline phosphatase activity emerged as core environmental factors influencing rhizospheric SOC accumulation across different coastal vegetation types. This study presents a promising framework for understanding the determinants of SOC fractions in rhizospheric soil, and their accumulation in the vegetation of coastal saline-alkali soils. In addition to alkalinity, phosphorus acquisition capacity can be a key predictor of coastal SOC fractions and their accumulation.

The Benefits of Whole-Exome Sequencing in the Differential Diagnosis of Hypophosphatasia.

Hypophosphatasia (HPP) is a rare inherited disorder characterized by the decreased activity of tissue-nonspecific alkaline phosphatase (TNSALP), caused by mutations in the ALPL gene. The aim of this study was to conduct differential diagnostics in HPP patients using whole-exome sequencing (WES). The medical records of HPP patients and the genetic testing of the ALPL gene were reviewed. Seven patients were recruited and underwent WES using the Illumina or MGI sequencing platforms. All of the exome samples were matched onto a GRCh38.p13 reference genome assembly by using the Genome Analysis ToolKit (GATK) and the BWA MEM read aligner. We present the clinical and molecular findings of the seven patients referred for genetic analyses due to a clinical and biochemical suspicion of HPP. In two patients out of three (with identified heterozygous variants in the ALPL gene), we also identified c.682T>A in exon 3 of the WNT10A gene and c.3470del in exon 23 of the SMC1A gene variants for the first time. In four patients, variants in the ALPL gene were not detected, but WES allowed us to identify for the first time rare variants (c.5651A>C in exon 36 of the TRIO gene, c.880T>G in exon 6 of the TRPV4 gene, c.32078-1G>T in intron 159 of the TTN gene, c.47720_47721del in exon 235 of the TTN gene, and c.1946G>A in exon 15 of the SLC5A1 gene) and to conduct differential diagnostics with HPP. Using WES, for the first time, we demonstrate the possibility of early differential diagnostics in HPP patients with other rare genetic diseases.

The association of vitamin D insufficiency with the prevalence of obesity in children: implications for serum calcium levels, alkaline phosphatase activity, and bone maturation.

Vitamin D deficiency has been identified as a potential risk factor for various adverse health outcomes. However, its specific role in metabolic regulation and skeletal development in school-aged children is not fully understood. This study aimed to explore the correlation between vitamin D deficiency and childhood obesity rates, and its impact on serum calcium, alkaline phosphatase, and bone age in children. The study analyzed clinical data from 159 school-aged children who underwent medical examinations. Participants were divided into the 25-hydroxyvitamin D3 (25(OH)D3) deficiency group and the 25(OH)D3 normal group based on their serum levels. We compared body mass index (BMI), total cholesterol (TC), triglycerides (TG), Ca, ALP, bone age, fasting blood glucose (FBG), and hemoglobin A1c (HbA1c) between the two groups. Logistic regression and Spearman correlation analyses were performed to further investigate relationships between 25(OH)D3 levels and metabolic and bone-related markers. This study showed that the 25(OH)D3 deficiency cohort exhibited significantly higher BMI, TC, TG, and ALP levels, with lower Ca levels and delayed bone age compared to the normal group. Logistic regression analysis identified Ca, ALP, and bone age as significant predictors of 25(OH)D3 deficiency. Subgroup analysis showed that in the 25(OH)D3 deficient group, children with higher BMI had elevated TC, ALP levels, and delayed bone age, while Ca levels were lower. Correlation analysis confirmed the predictive value of these markers for 25(OH)D3 deficiency. Our findings demonstrate that 25(OH)D3 deficiency is strongly associated with obesity in school-aged children and may negatively affect normal skeletal development. Regular monitoring of 25(OH)D3 levels in school-aged children is essential for ensuring proper growth and development, especially in those at risk for obesity.

The uremic toxin indoxyl sulfate decreases osteocyte RANKL/OPG and increases wnt inhibitor RNA expression that is reversed by PTH

Abstract Renal osteodystrophy (ROD) leads to increased fractures, potentially due to underlying low bone turnover in chronic kidney disease (CKD). We hypothesized that indoxyl sulfate (IS), a circulating toxin elevated in CKD and ligand for the aryl hydrocarbon receptor (AhR), may target the osteocytes leading to bone cell uncoupling in ROD. The IDG-SW3 osteocytes were cultured for 14 days (early) and 35 days (mature osteocytes) and incubated with 500 μM of indoxyl sulfate after dose finding studies to confirm AhR activation. Long-term incubation of IS for 14 days led to decreased expression of Tnfsf11/Tnfrsf11b ratio (RANKL/OPG), which would increase osteoclast activity, and increased expression of Wnt inhibitors Sost and Dkk1, which would decrease bone formation in addition to decreased mineralization and alkaline phosphatase (ALP) activity. When osteocytes were incubated with IS and the AhR translocation inhibitor CH223191, mineralization and ALP activity were restored. However, the Tnfsf11/Tnfrsf11b ratio and Sost, Dkk1 expression were not altered compared to IS alone, suggesting more complex signaling. In both early and mature osteocytes, co-culture with PTH and IS reversed the IS-induced upregulation of Sost and Dkk1, and IS enhanced the PTH-induced increase of the Tnfsf11/Tnfrsf11b ratio. Co-culture of IS with PTH additively enhanced the AhR activity assessed by Cyp1a1 and Cyp1b1 expression. In summary, IS in the absence of PTH, increased osteocyte mRNA Wnt inhibitor expression in both early and mature osteocytes and decreased mRNA expression ofTnfsf11/Tnfrsf11b ratio and mineralization in early osteocytes. These changes would lead to decreased resorption and formation resulting in low bone remodeling. These data suggest IS may be important in the underlying low turnover bone disease observed in CKD when PTH is not elevated. In addition, when elevated PTH, IS interacts to further increase Tnfsf11/Tnfrsf11b ratio for osteoclast activity in both early and mature osteocytes which would worsen bone resorption.

Effect of rhizosphere soil microbial communities and environmental factors on growth and the active ingredients of Angelica sinensis in Gansu Province, China.

The growth and accumulation of active ingredients of Angelica sinensis were affected by rhizosphere soil microbial communities and soil environmental factors. However, the correlationship between growth and active ingredients and soil biotic and abiotic factors is still unclear. This study explored rhizosphere soil microbial community structures, soil physicochemical properties, enzyme activities, and their effects on the growth and active ingredient contents of A. sinensis in three principal cropping areas. Results indicated that the growth indices, ligustilide, ferulic acid contents, and soil environmental factors varied in cropping areas. Pearson correlation analysis revealed that the growth of A. sinensis was affected by organic matter, total nitrogen, total phosphorus, and available phosphorus; ferulic acid and ligustilide accumulation were related to soil catalase and alkaline phosphatase activities, respectively. Illumina MiSeq sequencing showed that the genera Mortierella and Conocybe were the dominant fungal communities, and Sphingomonas, Pseudomonas, Bryobacter, and Lysobacter were the main bacterial communities associated with the rhizosphere soil. Kruskal-Wallis one-way ANOVA and Spearman correlation conjoint analysis demonstrated a significant positive correlation (p < 0.001) among the composition of the rhizosphere microbial communities at all three sampling sites. The growth and active ingredient accumulation of A. sinensis not only was significantly susceptible to the bacterial communities of Sphingomonas, Epicoccum, Marivita,Muribaculum, and Gemmatimonas but also were significantly influenced by the fungal communities of Inocybe, Septoria, Tetracladium, and Mortierella (p < 0.05). Our findings provide a scientific basis for understanding the relationship between the growth and active ingredients in A. sinensis and their corresponding rhizosphere soil microbial communities, soil physicochemical properties, and enzyme activities.

Effect of P Reduction on phoD-Harboring Bacteria Community in Solar Greenhouse Soil

Phosphorus (P) enrichment frequently occurs in the soil used in greenhouse vegetable production (GVP). Minimizing the application of P fertilizer represents a crucial approach to mitigating the accumulation of P in the soil and enhancing its utilization efficiency. However, the changes in bacterial communities and the turnover mechanism of soil P fractions related to soil P cycling after P fertilizer reduction are still unclear. To unravel these complexities, we devised three experimental treatments: conventional nitrogen (N), P, and potassium (K) fertilizer (N1P1K1); conventional N and K fertilizer without P (N1P0K1); and no fertilizer (N0P0K0). These experiments were conducted to elucidate the effects of P reduction on cucumber plant growth, soil P fractions, and the phoD-harboring bacterial community in the P-rich greenhouse soil. The results showed that there were no significant differences between the N1P1K1 and N1P0K1 treatments in terms of plant growth, yield, and P uptake, and the values for the N0P0K0 treatment were significantly lower than those for the N1P1K1 treatment. In a state of P depletion (N0P0K0, N1P0K1), the main P sources were Resin-Pi, NaHCO3-Pi, NaHCO3-Po, and NaOH-Pi. The contents of NaOH-Po and CHCl-Po in the N1P0K1 treatment increased significantly. Without P fertilizer, alkaline phosphatase (ALP) activity, phoD gene abundance, and bacterial community diversity were significantly increased. The abundance of Ensifer in the N0P0K0 and N1P0K1 treatments was 8 and 10.58 times that in the N1P1K1 treatment, respectively. Additionally, total phosphorus (TP) and available nitrogen (AN) were key factors affecting changes in the phoD bacterial community, while Shinella, Ensifer and Bradyrhizobium were the main factors driving the change in soil P fractions, and NaHCO3-Pi and NaOH-Pi were key factors affecting crop yield. Therefore, reducing the application of P fertilizer will increases the diversity of phoD-gene-harboring bacterial communities and promote organic P mineralization, thus maintaining the optimal crop yield.

Antioxidant 1,2,3,4,6-Penta-O-galloyl-β-D-glucose Alleviating Apoptosis and Promoting Bone Formation Is Associated with Estrogen Receptors.

1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG) is the main phenolic active ingredient in Paeoniae Radix Alba, which is commonly used for the treatment of osteoporosis (OP). PGG is a potent natural antioxidant, and its effects on OP remain unknown. This study aimed to investigate the effects of PGG on promoting bone formation and explore its estrogen receptor (ER)-related mechanisms. A hydrogen peroxide-induced osteoblast apoptosis model was established in MC3T3-E1 cells. The effects of PGG were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, alkaline phosphatase (ALP) staining, RT-qPCR, and Western blot methods. Furthermore, a prednisolone-induced zebrafish OP model was employed to study the effects in vivo. ER inhibitors and molecular docking methods were used further to investigate the interactions between PGG and ERs. The results showed that PGG significantly enhanced cell viability and decreased cell apoptosis by restoring mitochondrial function, attenuating reactive oxygen species levels, decreasing the mitochondrial membrane potential, and enhancing ATP production. PGG enhanced ALP expression and activity and elevated osteogenic differentiation. PGG also promoted bone formation in the zebrafish model, and these effects were reversed by ICI182780. These results provide evidence that the effects of PGG in alleviating apoptosis and promoting bone formation may depend on ERs. As such, PGG is considered a valuable candidate for treating OP.

Optimizing Irrigation Scheduling to Enhance Nutrient Uptake and Soil Microbial Activity in Linseed Cultivation (Linum usitatissimum L.)

Linseed crop cultivated under residual moisture and water scarcity conditions in rabi season. To improve the productivity of linseed irrigation water is applied at different stages of crop. So, to optimize the irrigation scheduling and enhance the nutrient uptake and soil microbial activity, this experiment was conducted at Main Agriculture Research Station, University of Agricultural Sciences, Raichur (which comes under North Eastern Dry zone of Karnataka) (India) during rabi 2023-24. The experiment was carried out with Randomized Complete Block Design (RCBD) with five treatments and four replications. The results revealed that, pre sowing irrigation fb three irrigations at vegetative stage (30-35 DAS), flowering stage (40-45 DAS) and capsule development stage (60-65 DAS) (T4) recorded significantly higher nutrient uptake by linseed crop (46.07, 19.87 and 39.83 NPK kg ha-1). The rainfed condition treatment (T5) recorded significantly lower nutrient uptake by the crop (19.35, 8.15 and 18.40 NPK kg ha-1). Among the different treatments, pre sowing irrigation fb three irrigations at vegetative stage (30-35 DAS), flowering stage (40-45 DAS) and capsule development stage (60-65 DAS) recorded significantly higher soil microbial population of bacteria (16.1 X 106 cfu g-1 of soil), fungi (13.8 X 104 cfu g-1 of soil) and actinomycetes (6.8 X 103 cfu g-1 of soil) as compared to other treatments. The soil enzymatic activity was significantly influenced by scheduling of irrigation at different growth stages in linseed. At harvest among the different treatments, pre sowing irrigation fb three irrigations at vegetative stage (30-35 DAS), flowering stage (40-45 DAS) and capsule development stage (60-65 DAS) recorded significantly higher dehydrogenase, urease and alkaline phosphatase activity (18.63 μg TPF g-1 soil day-1, 22.5 μg NH4-N g-1 soil hr-1 and 14.4 µg PNP g-1 soil hr-1, respectively).

Ultraviolet laser induced periodic surface structures positively influence osteogenic activity on titanium alloys.

Endoprostheses might fail due to complications such as implant loosening or periprosthetic infections. The surface topography of implant materials is known to influence osseointegration and attachment of pathogenic bacteria. Laser-Induced Periodic Surface Structures (LIPSS) can improve the surface topography of orthopedic implant materials. In this preclinical in vitro study, laser pulses with a wavelength in the ultraviolet (UV) spectrum were applied for the generation of LIPSS to positively influence formation of extracellular matrix by primary human Osteoblasts (hOBs) and to reduce microbial biofilm formation in vitro. Laser machining was employed for generating UV-LIPSS on sample disks made of Ti6Al4V and Ti6Al7Nb alloys. Sample disks with polished surfaces were used as controls. Scanning electron microscopy was used for visualization of surface topography and adherent cells. Metal ion release and cellular metal levels were investigated by inductively coupled plasma mass spectrometry. Cell culture of hOBs on sample disks with and without UV-LIPSS surface treatments was performed. Cells were investigated for their viability, proliferation, osteogenic function and cytokine release. Biofilm formation was facilitated by seeding Staphylococcus aureus on sample disks and quantified by wheat germ agglutinin (WGA) staining. UV-LIPSS modification results in topographies with a periodicity of 223nm ≤ λ ≤ 278nm. The release of metal ions was found increased for UV-LIPSS on Ti6Al4V and decreased for UV-LIPSS on Ti6Al7Nb, while cellular metal levels remain unaffected. Cellular adherence was decreased for hOBs on UV-LIPSS Ti6Al4V when compared to controls while proliferation rate was unaffected. Metabolic activity was lower on UV-LIPSS Ti6Al7Nb when compared to the control. Alkaline phosphatase activity was upregulated for hOBs grown on UV-LIPSS on both alloys. Less pro-inflammatory cytokines were released for cells grown on UV-LIPSS Ti6Al7Nb when compared to polished surfaces. WGA signals were significantly lower on UV-LIPSS Ti6Al7Nb indicating reduced formation of a S. aureus biofilm. Our results suggest that UV-LIPSS texturing of Ti6Al7Nb positively influence bone forming function and cytokine secretion profile of hOBs in vitro. In addition, our results indicate diminished biofilm formation on UV-LIPSS treated Ti6Al7Nb surfaces. These effects might prove beneficial in the context of long-term arthroplasty outcomes.

A photo-triggered bioactive nanohydrogel loaded with niobium carbide for stem cell osteogenic differentiation

Photo-Cross-Linkable hydrogel has attracted immense interest in the regeneration of bone repair and regeneration strategies due to its superior biocompatibility and tunable mechanical properties. Recently, Nb was reported to strongly promote the bone regeneration process via an accelerated osteoblast-modulated alkaline phosphatase activity mechanism. In particular, Nb2C MXenes have drawn widespread attention due to their excellent biocompatibility and ability to induce bone formation. However, the easy agglomeration of Nb2C nanosheets and subsequent low cell endocytosis efficacy greatly suppressed the osteogenesis effect. In this study, a subtractive nanopore-engineered Nb2C MXene was prepared through a microwave combustion method, gelatin methacrylate was used as the carrier hydrogel, and the photo-triggered Porous-Nb2C@GelMA hydrogel was fabricated by a photo-triggered process. The pore-forming strategy not only successfully improved the distribution of Nb2C and formed more homogenous Porous-Nb2C@GelMA hydrogels but also guided bone marrow mesenchymal stem cells (BMSCs) toward osteoblast differentiation. Porous Nb2C provided convenient cellular grasping and endocytosis for BMSCs, which further created a favorable environment for differentiation and osteogenesis. This, in turn, leads to an increase in the expression of osteogenic markers, such as ALP and ARS, as well as osteogenic factors, such as BMP-2, COL-1, OCN, and OPN. Consequently, enhancing the regenerative microenvironment by incorporating porous Nb2C composite hydrogels shows promise for application in bone regeneration.

Role of vitamin K2 and vitamin K cycle enzymes in aortic valvular interstitial cell calcification

Abstract Introduction Calcific aortic valve disease (CAVD) is the most common valve disease. Currently, there is no medical treatment available for CAVD. Vitamin K antagonists promote CAVD while population-based studies suggest that dietary intake of vitamin K reduces the incidence of CAVD in adults. However, randomized trials have so far not proven a clear benefit of vitamin K supplementation on the progression of AVS. Consequently, there is a need for a deeper understanding of the role of vitamin K in valvular calcification. This study aims to further investigate mechanisms of action of vitamin K during valvular calcification. Methods and Results Aortic valve samples were obtained from patients undergoing surgical aortic valve replacement for CAVD and valvular interstitial cells (VIC) were isolated by collagenase II digestion (Fig. 1). VIC expressed typical markers like Vimentin and α-SMA and were cultured in a control medium (CM) or further calcified in a pro-calcifying medium (PCM) containing sodium dihydrogen phosphate and ascorbic acid. PCM led to upregulation of Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP) expression, and ALP-activity after 7 and 21 days of culture. Moreover, alizarin red staining visualized significant calcium mineral deposition after 21 days of culture (Fig. 2). The addition of the most potent vitamin K2 derivative (Menaquinone-7, MK-7) improved cell viability under both control and calcifying conditions (Fig. 3). Moreover, MK-7 protected VIC from ferroptotic cell death (Fig. 4) and increased expression of the vitamin K-dependent Matrix-Gla-protein (MGP), a major inhibitor of ectopic calcification (Fig. 5). To fulfill its function as an activator of vitamin K-dependent proteins like MGP, vitamin K needs to be reduced to vitamin K hydroquinone by the vitamin K epoxide reductase enzymes VKORC1 and VKORC1L1. In VIC, using our calcification protocol, treatment with PCM led to the downregulation of VKORC1, but not of VKORC1L1, an enzyme with known anti-oxidative properties (Fig. 6). We next performed siRNA-mediated knockdown of VKORC1 and VKORC1L1 in VIC and observed calcification after 7 and 21 days of culture by measuring expression of ALP, RUNX2, quantifying ALP activity, and staining for calcium minerals (alizarin red). Intriguingly, VKORC1L1-knockdown sharply increased the expression of ALP. To further elucidate these findings, we will perform overexpression experiments to analyze, if VKORC1L1 can alleviate PCM-induced calcification Conclusion: Vitamin K2 inhibits in vitro calcification of valvular interstitial cells and upregulates the calcification inhibitor Matrix-Gla-Protein. Our results suggest the involvement of the vitamin K cycle enzyme VKORC1L1, which is known to exhibit anti-oxidative and cytoprotective effects. Further studies are warranted to shed light on the regulation of the enzymes to improve our understanding.

Investigating the Biological Efficacy of Albumin-Enriched Platelet-Rich Fibrin (Alb-PRF): A Study on Cytokine Dynamics and Osteoblast Behavior.

The development of effective biomaterials for tissue regeneration has led to the exploration of blood derivatives such as leucocyte- and platelet-rich fibrin (L-PRF). A novel variant, Albumin-Enriched Platelet-Rich Fibrin (Alb-PRF), has been introduced to improve structural stability and bioactivity, making it a promising candidate for bone regeneration. This study aimed to evaluate Alb-PRF's capacity for cytokine and growth factor release, along with its effects on the proliferation, differentiation, and mineralization of human osteoblasts in vitro. Alb-PRF membranes were analyzed using histological, scanning electron microscopy, and fluorescence microscopy techniques. Cytokine and growth factor release was quantified over seven days, and osteoinductive potential was evaluated with MG-63 osteoblast-like cells. Structural analysis showed Alb-PRF as a biphasic, highly cellularized material that releases lower levels of inflammatory cytokines and higher concentrations of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) compared to L-PRF. Alb-PRF exhibited higher early alkaline phosphatase activity and in vitro mineralization (p < 0.05) and significantly increased the OPG/RANKL mRNA ratio (p < 0.05). These results indicate that Alb-PRF has promising potential as a scaffold for bone repair, warranting further in vivo and clinical assessments to confirm its suitability for clinical applications.

The Impact of Acute Ammonia Nitrogen Stress on Serum Biochemical Indicators and Spleen Gene Expression in Juvenile Yellowfin Tuna (Thunnus albacares).

The presence of ammonia nitrogen in water has a significant impact on the serum and spleen of fish, potentially leading to changes in substances such as proteins in the serum while also causing damage to the immune function of the spleen. To investigate the effects of ammonia nitrogen (NH3-N) stress on juvenile yellowfin tuna (Thunnus albacares), this study established three NH3-N concentrations, 0, 5, and 10 mg/L, denoted as L0, L1, and L2, respectively. Serum and spleen samples were collected at 6, 24, and 36 h. The effects of different NH3-N concentrations and exposure times on the physiological status of juvenile fish were investigated by analyzing serum biochemical indices and splenic gene expression. The results indicate that in the L1 group, the serum high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), complement 3 (C3) and complement 4 (C4) levels, and acid phosphatase (ACP) activity showed a trend of initially increasing and then decreasing. In the L2 group, the serum low-density lipoprotein cholesterol (LDL-C), HDL-C, and C4 levels and ACP activity also displayed an initially rising and then declining trend, while TG, C3, and creatinine (CRE) levels and alkaline phosphatase (AKP) activity showed an upward trend. In the L1 group, glutathione peroxidase 1b (GPX1b), interleukin 10 (IL-10), interleukin 6 receptor (IL-6r), and tumor necrosis factor alpha (TNF-α) gene expression levels in the spleen exhibited a trend of first increasing and then decreasing. In the L2 group, IL-10, IL-6r, tumor necrosis factor beta (TNF-β), caspase 2 (casp2), and caspase 9 (casp9) gene expression levels in the spleen also showed an initial increase followed by a decrease. When NH3-N levels are below 5 mg/L, it is recommended to limit stress exposure to no more than 36 h for the juvenile fish. For concentrations ranging from 5 to 10 mg/L, stress should be strictly controlled to within 24 h. Exposure to high NH3-N levels may affect biochemical indicators such as serum lipid metabolism, immunity, and metabolism in juvenile fish, and may damage the expression of antioxidant, immune gene, and apoptosis factors in the spleen. This study aims to deepen our understanding of the effects of NH3-N on juvenile tuna, with the goal of establishing effective water quality monitoring and management strategies. This will ensure the quality of aquaculture water, reduce the harm caused by NH3-N to juvenile yellowfin tuna, and enhance aquaculture efficiency and product quality.

Innovative 3D-Printed Titanium Specimens Favor a Modulation of Inflammation in Dental Pulp Stem Cells During Liposome-Triggered Mineralization.

developing customized titanium specimens, with innovative surfaces, is a suitable strategy to overcome implant failure. Additionally, a faster and efficient osteogenic commitment assists tissue regeneration. To investigate the interplay between inflammation and differentiation upon implantation, Dental Pulp Stem Cells (DPSCs) were cultured on 3D-printed titanium owning an internal open cell form, administering osteogenic factors by a liposomal formulation (LipoMix) compared to traditional delivery of differentiation medium (DM). osteogenic differentiation was evaluated by western blot, by measuring β1 integrin expression, and by and real-time RT-PCR, by measuring SP7 and Collagen I gene expression; while.angiogenesis was characterized by measuring VEGF secretion levels. Matrix mineralization was assessed by means of Alizarin Red Staining, cell adhesion and inflammation responses through western blot, enzymatic and ELISA assays evaluating Nrf2 expression, catalase activity and Prostaglandin E2 secretion, respectively. LipoMix enhances cell proliferation and adhesion, as revealed by increased integrin β1 expression. Mineralized matrix deposition, SP7 gene expression, Collagen I release and Alkaline Phosphatase activity appear increased in LipoMix condition. Additionally, the redox-sensitive transcription factor Nrf2 is overexpressed at the earliest experimental times, triggering the catalase activity. data reported confirm that internal topography and post-production treatments on titanium surfaces dynamically and positively condition the DPSC progress towards the osteogenic phenotype, moreover, the combination with LipoMix fastens the positive modulation of inflammation under osteogenic conditions. Therefore, the development of customized surfaces along with the administration of differentiating factors enclosed in a liposomal delivery system, could represent a promising and innovative tool in regenerative dentistry.

Vancomycin-Loaded Silk Fibroin/Calcium Phosphate/Methylcellulose-Based In Situ Thermosensitive Hydrogel: A Potential Function for Bone Regeneration

This study explores the efficacy of a vancomycin-loaded silk fibroin/calcium phosphate/methylcellulose-based in situ thermosensitive hydrogel (VM-SF/CaP/MC) in promoting the osteogenic differentiation of preosteoblast cells. Three VM-SF/CaP/MC formulations with varying low (L) and high (H) concentrations of silk fibroin (SF) and calcium phosphate (CaP) were prepared: VM-HSF/LCaP/MC, VM-LSF/HCaP/MC, and VM-HSF/HCaP/MC. These hydrogels significantly enhanced MC3T3-E1 cell migration and proliferation in a dose- and time-dependent manner, achieving complete cell migration within 48 h. In addition, they significantly promoted alkaline phosphatase activity, collagen content, and mineralization in MC3T3-E1 cells, indicating their potential for osteogenesis. Among the hydrogel formulations, the VM-HSF/HCaP/MC hydrogel, with high SF and CaP content, demonstrated superior potential in promoting the osteogenic differentiation of MC3T3-E1 cells. It exhibited the highest ALP activity (11.13 ± 0.91 U/mg protein) over 14 days, along with increased collagen content (54.00 ± 1.71 µg/mg protein) and mineralization (15.79 ± 1.48 mM) over 35 days. Therefore, this formulation showed a promising candidate for clinical application in localized bone regeneration, particularly in treating osteomyelitis.

Potential of Cranberry to Stimulate Osteogenesis: An In Vitro Study

This study investigated the potential of Cranberry extract to stimulate osteogenesis in vitro. The total phenolic and monomeric anthocyanin contents in the Cranberry were determined. Liquid chromatography coupled with mass spectrometry was used to identify the Cranberry’s constituents. To assess the Cranberry’s cytotoxicity, a thiazolyl blue tetrazolium bromide assay was employed. Concerning the osteogenesis potential of Cranberry, alkaline phosphatase (ALP) activity, bone morphogenetic protein 2 (BMP-2) expression, and extracellular matrix mineralization were evaluated. The total phenolic content was 522.72 ± 9.80 mg GAE g-1 ES and 364.95 ± 12.49 mg GAE/g detected by the Fast Blue BB and Folin–Ciocalteu method, respectively. For monomeric anthocyanin, the content was 460 ± 30 mg ECG g-1 ES. Moreover, Cranberry concentrations ranged from 62.5 to 500 mg/mL and were found to be biocompatible with osteoblasts and mesenchymal stromal cells. Regarding osteogenesis, 20 mg/mL of Cranberry promoted 2-fold more ALP activity and almost 1.5-fold more BMP-2 than compared to the positive control group. Additionally, 200 mg/mL of Cranberry stimulated a 1.7-fold increase in extracellular matrix mineralization compared to the positive control group. In conclusion, Cranberry displayed potential in stimulating early and late markers of osteogenesis. Its ability to promote osteogenesis and its biocompatibility at higher concentrations hold promise for future application into biomaterials for bone regeneration.

Real-Time Fluorescence Visualization of the Dynamic Distribution of Zn2+ Ions during Osteoblast Differentiation.

The differentiation and maturation of osteoblasts are essential for bone formation. Zn2+ plays a crucial role in cell differentiation and is involved in osteogenic differentiation. The concentration and distribution of Zn2+ in the nucleus and cytoplasm indicate the differentiation states of osteoblasts. However, there is an absence of a real-time method for monitoring the dynamic fluctuations of endogenous Zn2+ within the nucleus. Here, a novel Zn2+ fluorescent probe (NTAD-N1) with nuclear membrane permeability was designed and developed, allowing for distribution throughout the entire cell, including the nucleus. The NTAD-N1 probe successfully showed the dynamic distribution and concentration changes of Zn2+ in the nucleus and cytoplasm of preosteoblast MC3T3-E1 during the 21-day differentiation period. The results showed that free Zn2+ increased significantly during differentiation of osteoblasts (2-21 days). Importantly, after 4 days of differentiation, osteoblasts are mainly distributed in the nucleus, which is confirmed by metallothionein expression. Subsequently, the level of free Zn2+ in the cytoplasm remained at a high level, which promoted the increase in alkaline phosphatase activity and inhibited the activity of cis-aconitase in the tricarboxylic acid cycle, resulting in the accumulation of citric acid. This series of events promotes the formation of mineralized nodules. In the process of osteoblast differentiation, the detection time of Zn2+ (≤7 days) is ahead of the late marker of alkaline phosphatase (14 days) and mineralized nodules (14-21 days). This indicates that Zn2+ can be used as a biomarker and an intervention point for early differentiation of osteoblasts.