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A Pyrococcus furiosus Argonaute (PfAgo)-based biosensor is presented for alkaline phosphatase (ALP) activity detection in which the ALP-catalyzed hydrolysis of 3′-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon mediates the fluorescent reporter cleavage as a guide sequence of PfAgo. Under the dual amplification mode of PfAgo-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied to ALP activity determination with a detection limit (LOD) of 0.0013 U L−1 (3σ) and a detection range of 0.0025 to 1 U L−1 within 90 min. The PfAgo-based method exhibits satisfactory analytic performance in the presence of potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapid analysis, high sensitivity, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications. [ABSTRACT FROM AUTHOR]

Abstract Background As calcium silicate-based cements (CSCs) have found success in various vital pulp therapy applications, several new CSC products have emerged. This study aimed to assess the genotoxicity, cytotoxicity, and bioactivity of four CSCs by comparing the newly introduced materials Bio MTA+ and MTA Cem with previously studied materials, Biodentine and NeoMTA. Methods Genotoxicity was evaluated using the micronucleus (MN) assay in human peripheral blood lymphocyte cells, measuring MN frequency and nuclear division index (NDI). Cytotoxicity was assessed in human dental pulp stem cells through the Water-Soluble Tetrazolium Salt-1 (WST-1) colorimetric assay. Bioactivity was determined by ELISA, measuring the levels of angiogenic and odontogenic markers (BMP-2, FGF-2, VEGF, and ALP). Statistical analyses included ANOVA, Dunnet and Sidak tests, and Wald chi-square test. (p

Background: As calcium silicate-based cements (CSCs) have found success in various vital pulp therapy applications, several new CSC products have emerged. This study aimed to assess the genotoxicity, cytotoxicity, and bioactivity of four CSCs by comparing the newly introduced materials Bio MTA+ and MTA Cem with previously studied materials, Biodentine and NeoMTA. Methods: Genotoxicity was evaluated using the micronucleus (MN) assay in human peripheral blood lymphocyte cells, measuring MN frequency and nuclear division index (NDI). Cytotoxicity was assessed in human dental pulp stem cells through the Water-Soluble Tetrazolium Salt-1 (WST-1) colorimetric assay. Bioactivity was determined by ELISA, measuring the levels of angiogenic and odontogenic markers (BMP-2, FGF-2, VEGF, and ALP). Statistical analyses included ANOVA, Dunnet and Sidak tests, and Wald chi-square test. (p <.05). Results: The MN frequency in the groups was significantly lower than that in the positive control group (tetraconazole) (p <.05). NDI values decreased with increasing concentration (p <.05). Bio MTA+ and NeoMTA showed decreased cell viability at all concentrations in 7-day cultures (p <.01). All materials increased BMP-2, FGF-2, and VEGF levels, with Biodentine and NeoMTA showing the highest levels of BMP-2 and FGF-2 on day 7. Biodentine displayed the highest VEGF levels on day 7. Biodentine and NeoMTA groups exhibited significantly higher ALP activity than the Bio MTA+ and MTA Cem groups by day 7. Conclusion: Bio MTA+ and MTA Cem demonstrated no genotoxic or cytotoxic effects. Moreover, this study revealed bioactive potentials of Bio MTA+ and MTA Cem by enhancing the expression of angiogenic and osteogenic growth factors. [ABSTRACT FROM AUTHOR]

Abstract Background To support bone regeneration, 3D-printed templates function as temporary guides. The preferred materials are synthetic polymers, due to their ease of processing and biological inertness. Poly(lactide-co-trimethylene carbonate) (PLATMC) has good biological compatibility and currently used in soft tissue regeneration. The aim of this study was to evaluate the osteoconductivity of 3D-printed PLATMC templates for bone tissue engineering, in comparison with the widely used 3D-printed polycaprolactone (PCL) templates. Methods The printability and physical properties of 3D-printed templates were assessed, including wettability, tensile properties and the degradation profile. Human bone marrow-derived mesenchymal stem cells (hBMSCs) were used to evaluate osteoconductivity and extracellular matrix secretion in vitro. In addition, 3D-printed templates were implanted in subcutaneous and calvarial bone defect models in rabbits. Results Compared to PCL, PLATMC exhibited greater wettability, strength, degradation, and promoted osteogenic differentiation of hBMSCs, with superior osteoconductivity. However, the higher ALP activity disclosed by PCL group at 7 and 21 days did not dictate better osteoconductivity. This was confirmed in vivo in the calvarial defect model, where PCL disclosed distant osteogenesis, while PLATMC disclosed greater areas of new bone and obvious contact osteogenesis on surface. Conclusions This study shows for the first time the contact osteogenesis formed on a degradable synthetic co-polymer. 3D-printed PLATMC templates disclosed unique contact osteogenesis and significant higher amount of new bone regeneration, thus could be used to advantage in bone tissue engineering.

Bone defects lead to the structural loss of normal architecture, and those in the field of bone tissue engineering are searching for new alternatives to aid bone regeneration. Dental pulp-mesenchymal stem cells (DP-MSC) could provide a promising alternative to repair bone defects, principally due to their multipotency and capacity to fabricate three-dimensional (3D) spheroids. The present study aimed to characterize the 3D DP-MSC microsphere and the osteogenic differentiation capacity potential cultured by a magnetic levitation system. To achieve this, the 3D DP-MSC microsphere was grown for 7, 14, and 21 days in an osteoinductive medium and compared to 3D human fetal osteoblast (hFOB) microspheres by examining the morphology, proliferation, osteogenesis, and colonization onto PLA fiber spun membrane. Our results showed good cell viability for both 3D microspheres with an average diameter of 350 μm. The osteogenesis examination of the 3D DP-MSC microsphere revealed the lineage commitment, such as the hFOB microsphere, as evidenced by ALP activity, the calcium content, and the expression of osteoblastic markers. Finally, the evaluation of the surface colonization exhibited similar patterns of cell-spreading over the fibrillar membrane. Our study demonstrated the feasibility of forming a 3D DP-MSC microsphere structure and the cell-behavior response as a strategy for the applications of bone tissue guiding. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) have the potential to differentiate into a variety of mature cell types and are a promising source of regenerative medicine. The success of regenerative medicine using MSCs strongly depends on their differentiation potential. In this study, we sought to identify marker genes for predicting the osteogenic differentiation potential by comparing ilium MSC and fibroblast samples. We measured the mRNA levels of 95 candidate genes in nine ilium MSC and four fibroblast samples before osteogenic induction, and compared them with alkaline phosphatase (ALP) activity as a marker of osteogenic differentiation after induction. We identified 17 genes whose mRNA expression levels positively correlated with ALP activity. The chondrogenic and adipogenic differentiation potentials of jaw MSCs are much lower than those of ilium MSCs, although the osteogenic differentiation potential of jaw MSCs is comparable with that of ilium MSCs. To select markers suitable for predicting the osteogenic differentiation potential, we compared the mRNA levels of the 17 genes in ilium MSCs with those in jaw MSCs. The levels of 7 out of the 17 genes were not substantially different between the jaw and ilium MSCs, while the remaining 10 genes were expressed at significantly lower levels in jaw MSCs than in ilium MSCs. The mRNA levels of the seven similarly expressed genes were also compared with those in fibroblasts, which have little or no osteogenic differentiation potential. Among the seven genes, the mRNA levels of IGF1 and SRGN in all MSCs examined were higher than those in any of the fibroblasts. These results suggest that measuring the mRNA levels of IGF1 and SRGN before osteogenic induction will provide useful information for selecting competent MSCs for regenerative medicine, although the effectiveness of the markers is needed to be confirmed using a large number of MSCs, which have various levels of osteogenic differentiation potential.

ABSTRACT Nematode predation plays an essential role in determining changes in the rhizosphere microbiome. These changes affect the local nutrient balance and cycling of essential nutrients by selectively structuring interactions across functional taxa in the system. Currently, it is largely unknown to what extent nematode predation induces shifts in the microbiome associated with different rates of soil phosphorous (P) mineralization. Here, we performed an 7-year field experiment to investigate the importance of nematode predation influencing P availability and cycling. These were tracked via the changes in the alkaline phosphomonoesterase (ALP)-producing bacterial community and ALP activity in the rhizosphere of rapeseed. Here, we found that the nematode addition led to high predation pressure and thereby caused shifts in the abundance and composition of the ALP-producing bacterial community. Further analyses based on cooccurrence networks and metabolomics consistently showed that nematode addition induced competitive interactions between potentially keystone ALP-producing bacteria and other members within the community. Structural equation modeling revealed that the outcome of this competition induced by stronger predation pressure of nematodes was significantly associated with higher diversity of ALP-producing bacteria, thereby enhancing ALP activity and P availability. Taken together, our results provide evidence for the importance of predator-prey and competitive interactions in soil biology and their direct influences on nutrient cycling dynamics. IMPORTANCE Nematode predation plays an essential role in determining the rhizosphere microbiome. In doing so, predation dynamically affects the soil nutrient cycling, for instance, by shifting the availability of phosphorus (P) for plant uptake. However, the role of nematode predation inducing selective changes in the microbiome and affecting rates of P mineralization remains still largely unknown. Here, we used a field site treated with different fertilizers to investigate the importance of nematode predation influencing P availability and plant productivity, via changes in bacterial taxa producing alkaline phosphomonoesterases (ALP) and ALP activity in the rhizosphere of rapeseed. We integrated field and laboratory experiments to show that nematode predation induces bacterial keystone taxa to compete with the connected members and results in the modulation of ALP-producing bacterial populations and ALP activity in the rhizosphere. Taken together, our study provides novel insights into microbially mediated mechanisms of competitive interaction induced by nematode predation in enhancing P availability in the plant rhizosphere.

Objective: Ferula spp. have many applications in complementary medicine and are recognized as the most important sources of natural products for bone health and regeneration especially in postmenopausal women. Therefore, the aim of this study was to investigate the effects of the extracts from three Ferula species on proliferation and osteogenesis potential of human menstrual blood-derived mesenchymal stem cells (MenSCs). Materials and Methods: The possible cytotoxic activity of three members of Ferula spp. (at concentrations of 5 to 100 μg/ml) was determined using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay. Alkaline phosphatase (ALP) activity assay, Alizarin Red-S staining, and the expression analysis of an osteoblastic gene were performed to evaluate osteogenic differentiation potential. Results: The extracts of F. flabelliloba and F. szowitsiana decreased the viability and growth of MenSCs while F. foetida increased the proliferation of cells after 72 hr incubation. Treatment of MenSCs with selected plant extracts revealed that F. foetida and F. szowitsiana could enhance the osteogenic potential of MenSCs in terms of ALP activity. The Runx-2 expression in the presence of F. foetida was significantly greater than observed following treatment with 17β-estradiol (as positive control). Conclusion: The results of this study suggest that F. foetida and F. szowitsiana may have therapeutic values as a nutraceutical with respect to their considerable influence on osteogenic potential of mesenchymal stem cells.

Bone defects lead to the structural loss of normal architecture, and those in the field of bone tissue engineering are searching for new alternatives to aid bone regeneration. Dental pulp-mesenchymal stem cells (DP-MSC) could provide a promising alternative to repair bone defects, principally due to their multipotency and capacity to fabricate three-dimensional (3D) spheroids. The present study aimed to characterize the 3D DP-MSC microsphere and the osteogenic differentiation capacity potential cultured by a magnetic levitation system. To achieve this, the 3D DP-MSC microsphere was grown for 7, 14, and 21 days in an osteoinductive medium and compared to 3D human fetal osteoblast (hFOB) microspheres by examining the morphology, proliferation, osteogenesis, and colonization onto PLA fiber spun membrane. Our results showed good cell viability for both 3D microspheres with an average diameter of 350 μm. The osteogenesis examination of the 3D DP-MSC microsphere revealed the lineage commitment, such as the hFOB microsphere, as evidenced by ALP activity, the calcium content, and the expression of osteoblastic markers. Finally, the evaluation of the surface colonization exhibited similar patterns of cell-spreading over the fibrillar membrane. Our study demonstrated the feasibility of forming a 3D DP-MSC microsphere structure and the cell-behavior response as a strategy for the applications of bone tissue guiding.

Simulated microgravity (SMG) inhibits osteoblast differentiation (OBD) and induces bone loss via the inhibition of the Wnt/β-catenin pathway. However, the mechanism by which SMG alters the Wnt/β-catenin pathway is unknown. We previously demonstrated that SMG altered the focal adhesion kinase (FAK)-regulated mTORC1, AMPK and ERK1/2 pathways, leading to the inhibition of tumor cell proliferation/metastasis and promoting cell apoptosis. To examine whether FAK similarly mediates SMG-dependent changes to Wnt/β-catenin in osteoblasts, we characterized mouse MC3T3-E1 cells cultured under clinostat-modeled SMG (µg) conditions. Compared to cells cultured under ground (1 g) conditions, SMG reduces focal adhesions, alters cytoskeleton structures, and down-regulates FAK, Wnt/β-catenin and Wnt/β-catenin-regulated molecules. Consequently, protein-2 (BMP2), type-1 collagen (COL1), alkaline-phosphatase activity and matrix mineralization are all inhibited. In the mouse hindlimb unloading (HU) model, SMG-affected tibial trabecular bone loss is significantly reduced, according to histological and micro-computed tomography analyses. Interestingly, the FAK activator, cytotoxic necrotizing factor-1 (CNF1), significantly suppresses all of the SMG-induced alterations in MC3T3-E1 cells and the HU model. Therefore, our data demonstrate the critical role of FAK in the SMG-induced inhibition of OBD and bone loss via the Wnt/β-catenin pathway, offering FAK signaling as a new therapeutic target not only for astronauts at risk of OBD inhibition and bone loss, but also osteoporotic patients. [ABSTRACT FROM AUTHOR]

To promote and enhance the biological responses of implant hydroxyapatite (HAP) coatings, these are modified by substitution with various ions for the development of bioactive bone implants. In the present work, we have fabricated a MgO/Tb,Eu-HAP dual substituted hydroxyapatite coating on borate passivated 316L stainless steel (316L SS) by the electrodeposition method. The MgO/Tb,Eu-HAP dual substituted hydroxyapatite coating on borate passivated 316L SS was characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray diffractometry (XRD), high resolution scanning electron microscopy (HRSEM) with an energy dispersive X-ray (EDAX) analysis. Also, the mechanical property of the MgO/Tb,Eu-HAP dual layer coating was evaluated using adhesion and Vickers micro-hardness tests. The corrosion performance of MgO/Tb,Eu-HAP dual layer coating was investigated in Ringer's solution. The antibacterial activity of dual layer coating was evaluated against Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria) bacterial strains. The alkaline phosphate activity of MC3T3-E1 osteoblasts on the MgO/Tb,Eu-HAP dual layer coating was tested in order to examine their differentiation phase, which is a quantitative marker of osteogenesis. In addition, the in-vitro biocompatibility of cell proliferation and Live/Dead assay of MgO/Tb,Eu-HAP dual layer coating were investigated on MC3T3-E1 cells. Hence these results improved the anti-corrosion performance, bioactivity and biocompatibility of MgO/Tb,Eu-HAP coating on passivated 316L SS as well. Finally, it is concluded that the MgO/Tb,Eu-HAP dual layer coating can be considered to be a capable candidate for biomedical applications.

Skeletal reconstruction is necessary in cases of bone defects created by tumors, trauma, and abnormalities. Regeneration of bone defects remains a critical problem, and current approaches are based on biocompatible scaffolds. Spheroids represent a simple 3D system since no supporting material is required for cell growth. Different techniques are used to generate spheroids, such as hanging drop, low-attachment plates, and magnetic nanoparticles. The idea of using magnetic nanoparticles is to cross-link through cell membrane overnight to create complex 3D cellular spheroid by using magnets to guide the cellular response. Herein, the current study aimed to achieve 3D human fetal osteoblast (hFOB) spheroid under magnetic levitation. Formation of 3D spheroid culture under magnetic levitation was evaluated by cell viability at 3, 7, and 14 days. Morphology of the 3D hFOB spheroid was analyzed by SEM and fluorescence microscopy and the differentiation towards mineralized lineage by ALP assay, qPCR, and alizarin red staining. The cell viability indicated that the 3D hFOB spheroid still viable after 14 days of culture. ALP assay, qPCR analysis expression of Col1, ALP, and Itg-β1 molecules, and calcium deposition with alizarin red showed a high level of bioactivity of the 3D hFOB spheroid. SEM images allowed the morphological analysis of the 3D microtissue-like spheroid with the presence of matrix deposition. These results indicate that magnetic levitation culture enables 3D stable osteoblast spheroids and could be a promising strategy for engineering application in the 3D construct in surgery regeneration of mineralized tissue.

Objective: Ferula spp. have many applications in complementary medicine and are recognized as the most important sources of natural products for bone health and regeneration especially in postmenopausal women. Therefore, the aim of this study was to investigate the effects of the extracts from three Ferula species on proliferation and osteogenesis potential of human menstrual bloodderived mesenchymal stem cells (MenSCs). Materials and Methods: The possible cytotoxic activity of three members of Ferula spp. (at concentrations of 5 to 100 µg/ml) was determined using MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay. Alkaline phosphatase (ALP) activity assay, Alizarin Red-S staining, and the expression analysis of an osteoblastic gene were performed to evaluate osteogenic differentiation potential. Results: The extracts of F. flabelliloba and F. szowitsiana decreased the viability and growth of MenSCs while F. foetida increased the proliferation of cells after 72 hr incubation. Treatment of MenSCs with selected plant extracts revealed that F. foetida and F. szowitsiana could enhance the osteogenic potential of MenSCs in terms of ALP activity. The Runx-2 expression in the presence of F. foetida was significantly greater than observed following treatment with 17ß-estradiol (as positive control). Conclusion: The results of this study suggest that F. foetida and F. szowitsiana may have therapeutic values as a nutraceutical with respect to their considerable influence on osteogenic potential of mesenchymal stem cells. [ABSTRACT FROM AUTHOR]

Simulated microgravity (SMG) inhibits osteoblast differentiation (OBD) and induces bone loss via the inhibition of the Wnt/β-catenin pathway. However, the mechanism by which SMG alters the Wnt/β-catenin pathway is unknown. We previously demonstrated that SMG altered the focal adhesion kinase (FAK)-regulated mTORC1, AMPK and ERK1/2 pathways, leading to the inhibition of tumor cell proliferation/metastasis and promoting cell apoptosis. To examine whether FAK similarly mediates SMG-dependent changes to Wnt/β-catenin in osteoblasts, we characterized mouse MC3T3-E1 cells cultured under clinostat-modeled SMG (µg) conditions. Compared to cells cultured under ground (1 g) conditions, SMG reduces focal adhesions, alters cytoskeleton structures, and down-regulates FAK, Wnt/β-catenin and Wnt/β-catenin-regulated molecules. Consequently, protein-2 (BMP2), type-1 collagen (COL1), alkaline-phosphatase activity and matrix mineralization are all inhibited. In the mouse hindlimb unloading (HU) model, SMG-affected tibial trabecular bone loss is significantly reduced, according to histological and micro-computed tomography analyses. Interestingly, the FAK activator, cytotoxic necrotizing factor-1 (CNF1), significantly suppresses all of the SMG-induced alterations in MC3T3-E1 cells and the HU model. Therefore, our data demonstrate the critical role of FAK in the SMG-induced inhibition of OBD and bone loss via the Wnt/β-catenin pathway, offering FAK signaling as a new therapeutic target not only for astronauts at risk of OBD inhibition and bone loss, but also osteoporotic patients.

Wei He,1 Dina A Mosselhy,2,3 Yudong Zheng,1 Qingling Feng,4 Xiaoning Li,4 Xing Yang,4 Lina Yue,1 Simo-Pekka Hannula2 1School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing, People’s Republic of China; 2Department of Chemistry and Materials Science, School of Chemical Engineering, Aalto University, Espoo, Finland; 3Microbiological Unit, Fish Diseases Department, Animal Health Research Institute, Giza, Egypt; 4State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing, People’s Republic of China Introduction: In recent years, there has been an increasing interest in silica (SiO2) nanoparticles (NPs) as drug delivery systems. This interest is mainly attributed to the ease of their surface functionalization for drug loading. In orthopedic applications, gentamicin-loaded SiO2 NPs (nanohybrids) are frequently utilized for their prolonged antibacterial effects. Therefore, the possible adverse effects of SiO2–gentamicin nanohybrids on osteogenesis of bone-related cells should be thoroughly investigated to ensure safe applications. Materials and methods: The effects of SiO2–gentamicin nanohybrids on the cell viability and osteogenic differentiation of human osteoblast-like SaOS-2 cells were investigated, together with native SiO2 NPs and free gentamicin.Results: The results of Cell Count Kit-8 (CCK-8) assay show that both SiO2–gentamicin nanohybrids and native SiO2 NPs reduce cell viability of SaOS-2 cells in a dose-dependent manner. Regarding osteogenesis, SiO2–gentamicin nanohybrids and native SiO2 NPs at the concentration range of 31.25–125 µg/mL do not influence the osteogenic differentiation capacity of SaOS-2 cells. At a high concentration (250 µg/mL), both materials induce a lower expression of alkaline phosphatase (ALP) but an enhanced mineralization. Free gentamicin at concentrations of 6.26 and 9.65 µg/mL does not significantly influence the cell viability and osteogenic differentiation capacity of SaOS-2 cells. Conclusions: The results of this study suggest that both SiO2–gentamicin nanohybrids and SiO2 NPs show cytotoxic effects to SaOS-2 cells. Further investigation on the effects of SiO2–gentamicin nanohybrids on the behaviors of stem cells or other regular osteoblasts should be conducted to make a full evaluation of the safety of SiO2–gentamicin nanohybrids in orthopedic applications. Keywords: SiO2 NPs, gentamicin, cytotoxicity, ALP activity, mineralization

Magnesium is well known as a biodegradable biomaterial that has been reported to promote bone remodeling in several studies; however, the underlying biological mechanism remains unclear. In the present study, the role of magnesium ions in the migration of U-2 OS cells, which are osteoblast-like cell lines, was investigated. Magnesium treatment did not significantly alter the global transcriptome of U-2 OS cells, but increased the protein expression level of SNAI2, an epithelial–mesenchymal transition (EMT) marker. In addition, it was confirmed that the junctional site localization of Zona-occludens 1 (ZO-1), a representative tight junction protein, was destroyed by magnesium treatment; furthermore, it was determined that cytoplasmic localization increased, and alkaline phosphatase (ALP) activity increased. The obtained results on the mechanism by which magnesium is involved in osteoblast migration, which is important for fracture healing, will contribute to the understanding of the bone-formation process in patients with osteoporosis and musculoskeletal injury. • MgCl 2 treatment promoted cell migration in the osteoblast-like cell line U-2 OS. • MgCl 2 treatment did not affect transcriptional gene expression. • The protein expression level of the EMT marker SNAI2 was increased by MgCl 2 treatment. • The tight junction localization of ZO-1 was destroyed and relocated to the cytoplasm by MgCl 2 treatment. • ALP activity was enhanced by MgCl 2 treatment. [ABSTRACT FROM AUTHOR]

Fluorine may only have negative effects on bone metabolism if the optimum concentration of fluorine would not be determined, especially in osteoporosis. In this study a nano-porous and uniform fluorhydroxyapatite (FA) coating in different concentration of fluorine could be deposited on the 316 L substrate with a thickness of 50 μm. Fluorine can have influence on cell proliferation, matrix production, ALP releasing, coating dissolution rate, bio activity, and corrosion resistivity. The dissolution rate of the coating evaluated by ICP and found with an increase in fluorine due to the elimination of CaO, Ca2+ releasing decreased. The results of SBF and MTT exhibited the fluorine within the apatite structure enhanced the growth of natural apatite and function of human osteoblast like cell (MG-63). Furthermore, cell density and attachment showed an enhancement while fluorine increased. Surprisingly the ALP activity test showed the mineralization ability of coating would be improved significantly in presence of fluorine. Furthermore potentiodynamic polarization showed with increasing fluorine, the current density decreased from 112 to 13 nA/cm2. Corrosion property of FA is higher because it is too stable but can also persuade to severe adverse effects like osteomalacia. According to the achieved results, 75% partially-substituted fluorine within apatite structure was introduced as a proper combination of biocompatibility while 100% substituted fluorine could be a better candidate in corrosion resistant performance. Image 1 • The full ranges of fluorhydroxyapatite were synthesized by substitution of OH- with F- within the HA structure. • Measuring of FA coating dissolution rate by dissolved Ca, P ions versus fluorine concentration within the apatite structure. • Better biocompatibility and corrosion performance were obtained by 75% partially and 100% substituted fluorine, respectively. [ABSTRACT FROM AUTHOR]

• A novel SERS readout biosensor has been developed to assay ALP activity in serum. • The signal amplification strategy is used to improve sensitivity. • The magnetic substrate is recyclable, consistent with the green chemistry concept. • Ag-4-MPBA-AAP is used both as a graft mediator and a Raman signal indicator. As an important enzyme in human tissues, abnormally alkaline phosphatase (ALP) is strongly associated with many diseases, particularly breast and prostate cancer. In this study, we proposed a rapid and sensitive surface-enhanced Raman scattering (SERS)-based biosensor for ALP detection. The biosensor utilized Fe 3 O 4 @Au@TiO 2 nanoparticles (FAT NPs) as magnetic separation and enrichment materials to enrich grafted mediator Ag-4-mercaptophenylboronic acid (4-MPBA)-ascorbic acid 2-phosphate (AAP) NPs to form FAT-(AAP-4-MPBA-Ag) NPs, and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) was used as the internal standard tag molecule to prepare Au@DTNB@Ag as the signal source. The phosphate group in AAP was cleaved by ALP to form ascorbic acid (AA), which acts as a receptor for the boric acid to adsorb the 4-MPBA-Au@DTNB@Ag NPs. When the switch was "Off" to "On", the Au@DTNB@Ag NPs were used to significantly improve the sensitivity of the proposed SERS platform, enabling detection as low as 1.17U/L of target ALP with a good linear range (R 2 = 0.991) of 2–10 U/L. Furthermore, the platform successfully detected ALP from 2 % human serum. The recoveries were 99.5–106 %. In addition, FAT-(AAP-4-MPBA-Ag) NPs could be transformed into FAT NPs to achieve recyclability by ultrasonication in acidic environments. Overall, this sensitive and fast SERS platform has promising potential for accurate and efficient identification of ALP from human serum. [ABSTRACT FROM AUTHOR]

Abstract The aim of this study was to find an optimum dose of the synthetic amorphous calcium phosphate (ACP) nanoparticles to be incorporated in resin-modified glass ionomer cements (RMGICs) for triggering the release of PO 4 3−/Ca2+, alkaline phosphatase (ALP) activity and osteogenic differentiation of mesenchymal stem cells (hMSCs) without significantly affecting the essential properties of the cements. RMGICs were formulated from the powder composed of melt-derived strontium fluoro-aluminosilicate glass (SFAG) and synthetic ACP nanoparticles (0–20 wt%), as well as commercial polyalkenoic acid liquid. The effect of ACP incorporation on the workability, microstructure, Ca2+/PO 4 3−/F− ion release and compressive strength was investigated. The response of hMSCs to the optimized cements was assessed by MTT cytotoxicity, ALP activity, and staining tests. The working time of the formulated RMGICs decreased significantly upon increase of ACP content from 5 to 20%. ACP (5%)–incorporated RMGICs showed improved photopolymerization and setting. An insignificant reduction was recorded in the compressive strength of RMGICs with addition of 1.5–5% ACP. The fluoride release didn't significantly decrease due to addition of 5% ACP. Upon incorporating 5% ACP, the biocompatibility of RMGICs rose to about 20%. In addition, ALP activity and osteogenic differentiation of hMSCs noticeably increased after exposure to ACP–incorporated RMGIC. ACP (5%)–incorporated RMGICs could be promising candidates for both restorative and regenerative dentistry owing to the optimum mechanical strength, prolonged ion release, and their effective role in the cell differentiation and biomineralization demanded for pulp regeneration. Highlights • ACP nanoparticles were synthesized in choline chloride-urea solvent. • SFAG microparticles were prepared by the melt-quenching method. • RMGICs were formulated from mixed SFAG-ACP (0–20 wt%) powder and polyacid liquid. • ACP (5.0 wt%)–incorporated RMGICs showed improved setting and ion release behavior. • Differentiation of hMSCs increased after exposure to ACP–incorporated RMGICs. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • PU thin films coated on Ti substrates showed superior corrosion resistance. • Alkaline treated Ti substrates coated with plain PU film showed better cell attachment and spreading. • PU coated and alkaline-treated Ti substrates induced the highest adhesion performance. • Mg particles/PU coated treated Ti can support cell proliferation and differentiation. Abstract This study aimed to design an adhesive biodegradable polymer layer on the surface of titanium (Ti) implants for enhanced surface bioactivity. To this end, a coating of magnesium-particles doped biodegradable polyurethane (PU) was introduced as a composite layer on Ti surfaces using a simple spin coating technique. The coating's performance and characteristics were investigated in terms of the surface topography, composition, surface roughness, wettability, adhesion and electrochemical behavior of composite coatings on untreated (polished) and alkaline treated Ti substrates. Interestingly, the Ti samples coated with the composite layers showed superior corrosion resistance compared to the uncoated samples. Additionally, the coating on alkali-treated Ti surfaces demonstrated enhanced adhesion (5B, measured by cross-cut test) compared to the coating on untreated Ti (1B), indicating the vital role of the alkaline-treatment step. A composite thin layer coated on alkaline-treated Ti enhanced osteoblastic-like (MC3T3-E1) cellular adhesion and cell proliferation and was found to support osteoblastic differentiation compared to uncoated alkaline-treated Ti. Surface modification of alkaline-treated Ti with a biodegradable Mg-particles/PU thin layer appears to be a promising strategy for developing surface bioactivity of orthopedic devices. [ABSTRACT FROM AUTHOR]

Two benzimidazole‐based zinc complexes [(Zn)2(L2)2Cl2(DMSO)2] (R1) and [Zn(L2)2(NO3)2(H2O)2] (R2) were synthesized and characterized with various spectroscopic data. The single X‐ray structure determination reveals that complex R1 is dinuclear and tetrahedral in geometry, while complex R2 is mononuclear and octahedral in geometry. Further, both zinc complexes were investigated for pyrophosphate sensing in an aqueous medium. Complex R1 is found to be selective towards pyrophosphate; it leads to 5.5‐fold enhancement in the emission intensity due to aggregation‐induced emission. However, complex R2 has shown binding with all, ATP, AMP, ADP, and pyrophosphate, which is attributed to the chelate effect. Consequently, complex R1 was utilized for the intracellular detection of pyrophosphate in HeLa cells. Furthermore, the PPi based zinc complex R1 is also used as a bio‐analytical tool to construct a real‐time fluorescence assay for the enzymatic activity of alkaline phosphatases (ALP). We have designed and synthesized two benzimidazole‐based zinc complexes for pyrophosphate sensing in aqueous media. One of the complexes also shows pyrophosphate sensing in Hela cells. It is also used as a bio‐analytical tool to construct a real‐time fluorescence assay for the enzymatic activity of alkaline phosphatases (ALP). [ABSTRACT FROM AUTHOR]

In this study, we assessed the biocompatibility and bioactivity of various pulp capping materials—ProRoot MTA (Dentsply Tulsa Dental Specialties), Biodentine (Septodont), TheraCal LC (Bisco), and Dycal (Dentsply Caulk)—on human dental pulp stem cells (hDPSCs). Experimental disks (diameter, 7 mm; height, 4 mm) were stored in a humified incubator at 37 °C for 48 h. Then, the pulp capping materials were tested for cytotoxic effects by methyl-thiazoldiphenyl-tetrazolium and scratch wound healing assays, and for mineralization potential by Alizarin red S (ARS) staining assay and alkaline phosphatase enzyme (ALP) activity. Cell viability and cell migration did not significantly differ between ProRoot MTA, Biodentine, and control (p > 0.05). TheraCal LC exhibited slower cell migration on days 2–4 compared to control (p < 0.05), and Dycal showed no cell migration. ALP activity was highest with Biodentine on days 10 and 14, and was lowered with TheraCal LC and Dycal (p < 0.05). In the ARS assay, hDPSCs grown in ProRoot MTA and TheraCal LC eluates showed significantly increased mineralized nodule formation on day 21 compared to Biodentine, Dycal, and control (p < 0.05). These findings indicate that ProRoot MTA, Biodentine, and TheraCal LC exhibit better biocompatibility and bioactivity than Dycal.

Bone tissue engineering using in situ forming 3D scaffolds can be an alternative to surgically treated scaffolds. This work aimed to develop in situ forming scaffolds using poly (lactic-co-glycolic acid) and a bone synthesizing drug (risedronate) with or without the porogenic agent (collagen). Hybrid scaffolds were formed through solvent-induced phase inversion technique and were morphologically evaluated using scanning electron microscopy (SEM). The effect of scaffolds on Saos-2 cell line viability using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test besides their effect on cell growth using fluorescence microscope was assessed. Furthermore, alkaline phosphatase (ALP) activity as well as Ca2+ deposition on the scaffolds was evaluated. SEM images revealed the porous structure for collagen-based scaffolds. Saos-2 cell proliferation was significantly enhanced with risedronate-loaded scaffolds compared to those lacking the drug. Porous collagen-based scaffolds were more favorable for both the cell growth and the promotion of ALP activity. Furthermore, collagen-based scaffolds promoted the Ca2+ deposition compared to their counterparts without collagen. Such results suggest that collagen-based scaffolds offer excellent biocompatibility for bone regeneration, where this biocompatible nature of scaffold leads to the proliferation of cells that lead to the deposition of mineral on the scaffold. Such in situ forming 3D scaffolds provide a promising noninvasive approach for bone tissue engineering. [ABSTRACT FROM AUTHOR]

As an essential enzyme, alkaline phosphatase (ALP) has attracted considerable attention for its regulating effect to the dephosphorylation process in living organisms. Here, we present a colorimetric and surface enhanced Raman scattering (SERS) dual-readout approach for assaying ALP activity. The major advantage of dual-readout assay is the perfect combination of the merit of colorimetric and SERS, which not only allow rapid preliminary discrimination of ALP activity by the naked eye but also greatly improved the detection sensitivity by SERS. Specifically, 4-mercaptophenylboronic acid modified Ag coated gold nanoparticles (4-MPBA-Au@Ag NPs) are employed as the colorimetric and SERS bifunctional reporting nanoprobes. Upon the presence of ALP, the phosphate group in the ascorbic acid 2-phosphate (AAP) is cleaved to produce ascorbic acid (AA), which acted as boronic acid moieties receptors to control the aggregation of 4-MPBA-Au@Ag NPs. The color of 4-MPBA-Au@Ag NPs solutions had changed from bright orange to light brown to dark gray, simultaneously accompanied by a substantial enhancement of SERS-readouts for the strong Raman hot-spots between the aggregation of 4-MPBA-Au@Ag NPs. A distinguishable change in the color was observed at an ALP activity of 5.0 U/L, meanwhile, SERS-readout sensing method showed a good linear relationship from 0.50 to 10.0 U/L (R = 0.997) with an exciting detection limit of 0.10 U/L (signal-to-noise ratio of 3). In addition, the dual-readout approach developed here was applied for ALP inhibitor evaluation. With the simple, rapid/direct readout yet outstanding sensitivity, we anticipate that this method would greatly promote practical application in ALP-related early-stage diseases diagnosis. [ABSTRACT FROM AUTHOR]

The structural modifications of pregnenolone have been described via the introduction of heterocyclic moieties at C-17 position by limiting the acyl group. Novel heterocyclic analogues of pregnenolone have been synthesized by using Friedlander and Claisen-Schmidt reactions, and the synthesized compounds were evaluated for their osteogenic activity. Among the synthesized derivatives, four compounds showed significantly increased ALP activity. Among all four active compounds, the novel compound 3a has shown significant bone matrix mineralization and mRNA expressions of osteogenic marker genes, BMP2, RUNX-2 and OCN at 1 pM concentration. [ABSTRACT FROM AUTHOR]

Osteoporosis is a common skeletal disorder, resulting from an imbalance in bone resorption relative to formation. Bone morphogenetic protein (BMP) is a key regulator in bone formation and osteoblastic differentiation. Hence, compounds that promote BMP expression may be suitable candidates for osteoporosis treatment. This study examined the effects of the traditional Chinese medicinal agent, Kuei-Lu-Er-Xian-Jiao (KLEXJ), on BMP-2 production in osteoblasts. We found that KLEXJ extract promoted osteoblastic differentiation marker ALP activity and increased BMP-2 production; pretreatment with PI3K and Akt inhibitors, or small interfering RNA (siRNA), reduced these effects. KLEXJ also enhanced PI3K and Akt phosphorylation. Treatment of osteoblastic cells with NF-κB inhibitors (TPCK or PDTC) markedly inhibited KLEXJ-enhancement of ALP activity and BMP-2 production. KLEXJ also significantly promoted p65 phosphorylation, while treatment with PI3K and Akt inhibitors antagonized KLEXJ-enhanced p65 phosphorylation. Thus, KLEXJ enhances ALP activity and BMP-2 production of osteoblasts through the PI3K/Akt/NF-κB signaling pathway and hence may be suitable in the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

In order to use a simple and ecofriendly method to prepare porous silk scaffolds, aqueous silk fibroin solution (ASF) was extracted from silk gland of 7-day-old fifth instar larvae of Bombyx mori (B. mori). SDS-page analysis indicated that the obtained fibroin had a molecular weight higher than 200 kDa. The fabrication of porous scaffolds from ASF was achieved by using the freeze-drying method. The pore of porous scaffolds is homogenous and tends to become smaller with an increase in the concentration of ASF. Conversely, the porosity is decreased. The porous scaffolds show impressive compressive strength which can be as high as 6.9 ± 0.4 MPa. Furthermore, ASF has high cell adhesion and growth activity. It also exhibits high ALP activity. This implies that porous scaffolds prepared from ASF have biocompatibility. Therefore, the porous scaffolds prepared in this study have potential application in tissue engineering due to the impressive compressive strength and biocompatibility.

PURPOSE. In this study, the aim of this study was to evaluate the effect of implant surface treatment on cell differentiation of osteoblast cells. For this purpose, three surfaces were compared: (1) a modified SLA (MSLA: sand-blasted with large grit, acid-etched, and immersed in 0.9% NaCl), (2) a laser treatment (LT: laser treatment) titanium surface and (3) a laser and acid-treated (LAT: laser treatment, acid-etched) titanium surface. MATERIALS AND METHODS. The MSLA surfaces were considered as the control group, and LT and LAT surfaces as test groups. Alkaline phosphatase expression (ALP) was used to quantify osteoblastic differentiation of MC3T3-E1 cell. Surface roughness was evaluated by a contact profilometer (URFPAK-SV; Mitutoyo, Kawasaki, Japan) and characterized by two parameters: mean roughness (Ra) and maximum peak-to-valley height (Rt). RESULTS. Scanning electron microscope revealed that MSLA (control group) surface was not as rough as LT, LAT surface (test groups). Alkaline phosphatase expression, the measure of osteoblastic differentiation, and total ALP expression by surface-adherent cells were found to be highest at 21 days for all three surfaces tested (P<.05). Furthermore, ALP expression levels of MSLA and LAT surfaces were significantly higher than expression levels of LT surface-adherent cells at 7, 14, and 21 days, respectively (P<.05). However, ALP expression levels between MSLA and LAT surface were equal at 7, 14, and 21 days (P>.05). CONCLUSION. This study suggested that MSLA and LAT surfaces exhibited more favorable environment for osteoblast differentiation when compared with LT surface, the results that are important for implant surface modification studies. [ABSTRACT FROM AUTHOR]