PfAgo-based dual signal amplification biosensor for rapid and highly sensitive detection of alkaline phosphatase activity.

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.

Evaluation of the genotoxicity, cytotoxicity, and bioactivity of calcium silicate-based cements.

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.

Fluorescence Turn-on Detection of Alkaline Phosphatase Activity and Al3+ Using Vitamin B6 Cofactor Conjugated GSH Capped Mn-doped ZnS Quantum Dots.

The glutathione (GSH) functionalized Mn-doped ZnS quantum dots (GSH_Mn_ZnS QDs) was conjugated with pyridoxal 5'-phosphate (PLP). The -CHO group of vitamin B6 cofactor PLP interacted with the -NH2 group of GSH functionalized Mn_ZnS QDs. The conjugation of PLP quenched the fluorescence emission of GSH_Mn_ZnS QDs at 601 nm. Addition of alkaline phosphatase (ALP) catalytically dephosphorylated the PLP into pyridoxal that restored the fluorescence emission of GSH_Mn_ZnS QDs. With a sensitivity of 0.035 U/L, the PLP conjugated GSH_Mn_ZnS QDs was applied to quantify ALP activity in human serum and plasma. Further, the developed nanoprobe PLP conjugated GSH_Mn_ZnS QDs was also applied to detect Al3+. The complexation-induced fluorescence enhancement was observed at 492 nm upon the interaction of Al3+ with the PLP conjugated GSH_Mn_ZnS QDs. Without any interference from other tested metal ions, this nanoprobe can be employed to detect Al3+ down to 2.30 µM.

Multiple proliferation signaling pathways are modulated by octacalcium phosphate in osteoblasts.

Octacalcium phosphate (OCP), a type of bioactive ceramics, may be associated with dentine, tooth apatite, and especially bone generation, and promotes wound healing after fracture. Recently, commercial bone grafting products containing a large amount of OCP material have been released because OCP can be synthesized in large quantities. It is reported to increase cell proliferation, but the interaction between OCP and cell signaling pathways is still unclear. In this study, first, we demonstrated OCP mediated cell signaling pathways with only purified OCP materials. OCP regulated P38, JNK (c-Jun N-terminal kinase), Src, and AKT (protein kinase B) signaling pathways. OCP crystals appeared in the characteristic ribbon shape but varied by several tens of micrometers in size. The X-ray diffraction pattern was the same as previously reported. We studied two concentrations of OCP (10 mg/ml and 20 mg/ml) to understand whether the effect of OCP on cell signaling pathways is dose dependent. We confirmed that OCP treatment affected cell proliferation and alkaline phosphatase and disrupted Src phosphorylation but did not change the total protein level. P38 phosphorylation was activated with OCP treatment and inhibited by SB203580, but P38 total protein level did not change. OCP inhibited JNK phosphorylation signaling, whereas PD98509 inhibited JNK phosphorylation with or without OCP. Interestingly, the AKT total level decreased after OCP treatment, but AKT phosphorylation increased considerably. Our results demonstrate that OCP materials modulate cell signaling pathways and increase cell proliferation.

Activation of Focal Adhesion Kinase Restores Simulated Microgravity-Induced Inhibition of Osteoblast Differentiation via Wnt/Β-Catenin Pathway.

Simulated microgravity (SMG) inhibits osteoblast differentiation (OBD) and induces bone loss via the inhibition of the Wnt/ß-catenin pathway. However, the mechanism by which SMG alters the Wnt/ß-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/ß-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/ß-catenin and Wnt/ß-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/ß-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.

The Identification of Marker Genes for Predicting the Osteogenic Differentiation Potential of Mesenchymal Stromal Cells.

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.

Biochemical activity of magnesium ions on human osteoblast migration.

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.

Two new secondary metabolites isolated from Avena sativa L. (Oat) seedlings and their effects on osteoblast differentiation.

Seedlings of natural crops are valuable sources of pharmacologically active phytochemicals. In this study, we aimed to identify new active secondary metabolites in Avena sativa L. (oat) seedlings. Two new compounds, avenafuranol (1) and diosgenoside (2), along with eight known compounds (3-10) were isolated from the A. sativa L. seedlings. Their chemical structures were elucidated via 1D and 2D NMR spectroscopy, high-resolution ESIMS, IR spectroscopy, optical rotation analysis, and comparisons with the reported literature. The effect of each isolated compound on alkaline phosphatase (ALP) activity for osteoblast differentiation induced by bone morphogenetic protein-2 (BMP-2) was investigated using the C2C12 immortal mouse myoblast cell line. Compounds 1, 4, 6, 8, and 9 induced dose-dependent increases in ALP expression relative to ALP expression in cells treated with only BMP-2, and no cytotoxicity was observed. These results suggest that A. sativa L. seedlings are a natural source of compounds that may be useful for preventing bone disorders.

Synthesis and biological evaluation of heterocyclic analogues of pregnenolone as novel anti-osteoporotic agents.

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 1pM concentration.

Osteogenic growth peptide promotes osteogenic differentiation of mesenchymal stem cells mediated by LncRNA AK141205-induced upregulation of CXCL13.

The aim of present study was to characterize long non-coding RNA (lncRNA) AK141205 as a cellular regulator of osteogenic differentiation of mice mesenchymal stem cells (MSCs) towards osteogenic growth peptide (OGP) stimulation. Mice MSCs cells were isolated, transfected with si-AK141205, pcDNA-AK141205 or control, and stimulated with OGP. The AK141205, CXC chemokine ligand-13 (CXCL13), and osteogenic differentiation-associated parameters were determined by western blotting or quantitative RT-PCR. To determine the role of AK141205/CXCL13 in SMCs osteogenic differentiation, SMCs subjected to co-transfection of pcDNA-AK141205 and si-CXCL13 or si-AK141205 and pcDNA-CXCL13, and were submitted for osteogenic differentiation-associated parameters analyses. The results showed that stimulation of SMCs with OGP induced upregulation of both AK141205 and CXCL13, and osteogenic differentiation of MSCs. Transfection of si-AK141205 partly suppressed OGP-induced formation of calcium salt nodules, alkaline phosphatase (ALP) activity and osteogenic differentiation-associated gene expression, suggesting key regulatory role of AK141205. Analysis of CXCL13 expression in SMCs(pcDNA-AK141205) revealed that AK141205 positively promoted CXCL13 expression via acetylation of H4 histone in the promoter region. This signal transduction was demonstrated to be essential for OGP-induced osteogenic differentiation of MSCs through osteogenic differentiation analysis in simultaneously AK141205/CXCL13 controlled SMCs. In summary, we report a completely novel role of AK141205/CXCL13 as a regulator of OGP-induced osteogenic differentiation of SMCs. Our finding provides a potential therapeutic targeting of AK141205 for enhancing disease-treatment effect of SMCs.

Expression analysis of zinc-metabolizing enzymes in the saliva as a new method of evaluating zinc content in the body: two case reports and a review of the literature.

BACKGROUND: The activity level of alkaline phosphatase, a zinc-requiring enzyme in the serum, is used to indicate zinc nutritional status; however, it does not correlate with serum zinc levels or subjective symptoms of taste disorder in many cases. Hence, this study focused on the total activity of alkaline phosphatase, a zinc-requiring enzyme. The total alkaline phosphatasa activity level in the saliva was measured before and after zinc supplementation, and the results were compared with serum zinc levels. CASE PRESENTATION: This study included patients with hypozincemia, specifically a patient with zinc-deficient taste disorder (patient 1: a 69-year-old Japanese woman) and a patient with glossodynia with zinc deficiency (patient 2: an 82-year-old Japanese woman). Saliva samples were collected, and blood tests were performed before and after zinc supplementation. Subjective symptoms and serum zinc levels were simultaneously evaluated. Zinc supplementation was performed using zinc acetate hydrate or Polaprezinc. CONCLUSIONS: Total alkaline phosphatase activity levels were found to be associated with serum zinc levels and subjective symptoms. A further study with a higher number of patients is necessary to confirm whether total alkaline phosphatase activity levels more accurately reflect the amounts of zinc in the body than serum zinc levels.

In Vitro Bone Differentiation of 3D Microsphere from Dental Pulp-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.

Preparation of porous scaffolds from silk fibroin extracted from the silk gland of Bombyx mori (B. mori).

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.

Status Epilepticus due to Asfotase Alfa Interruption in Perinatal Severe Hypophosphatasia.

BACKGROUND: Hypophosphatasia (HPP), an inherited, metabolic disorder caused by loss-of-function mutations in the ALPL gene, affects not only bone and tooth mineralization but also central nervous system (CNS) function, resulting in vitamin B6/pyridoxine-responsive seizures. Asfotase alfa treatment mainly improves the skeletal manifestations of HPP. As of yet, there are no reports demonstrating seizure exacerbation caused by asfotase alfa interruption. CASE: The patient was a 2-year and 8-month-old female with clinical and genetic diagnosis of perinatal severe HPP. Genetic analysis of ALPL identified compound heterozygous variants. Asfotase alfa and pyridoxine administration begun on postnatal day 2 restored normal development and suppressed seizures except for simple febrile seizures. From age 2 years when her asfotase alfa injections became irregular, she began experiencing seizure exacerbation, including status epilepticus, leading to acute encephalopathy and severe sequelae. The seizure exacerbations always coincided with low alkaline phosphatase (ALP) activity caused by the interruption of asfotase alfa administration. DISCUSSION: The clinical course of the present case demonstrated the effect of asfotase alfa on CNS symptoms and a clear correlation between low serum ALP activity and seizure exacerbation. Serum ALP activity measurements were useful as a therapeutic marker in the present case. Furthermore, the risk of seizure exacerbation in the patient could have been predicted, given the genotype-phenotype correlation related to the ALPL gene in the Japanese population. CONCLUSION: Regular asfotase alfa injections are needed to prevent seizure exacerbation in patients with HPP. Educating patients and their family about the need for regular asfotase alfa treatment is crucial to preventing disease exacerbation.

Nematode Predation and Competitive Interactions Affect Microbe-Mediated Phosphorus Dynamics.

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.

Contact osteogenesis by biodegradable 3D-printed poly(lactide-co-trimethylene carbonate).

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.

Case Report: Formation of 3D Osteoblast Spheroid Under Magnetic Levitation for Bone Tissue Engineering.

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-ß1 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.

Nano-octahedral bimetallic Fe/Eu-MOF preparation and dual model sensing of serum alkaline phosphatase (ALP) based on its peroxidase-like property and fluorescence.

Herein a nano-scale bimetallic Fe/Eu-MOF with a regular octahedral structure was synthesized for the first time. The synthesized Fe/Eu-MOF has both peroxidase-like activity and fluorescence properties. Fe/Eu-MOF can catalyze H2O2 to oxidize the chromogenic substrate TMB to produce blue oxTMB, which has ultraviolet absorption at 652 nm. Unexpectedly, the generated oxTMB can effectively quench the fluorescence of the catalyst Fe/Eu-MOF at 450 nm. The quenching mechanism is mainly the internal filtration effect (IFE), accompanied by static quenching (SQE), Förster resonance energy transfer (FRET) and photoelectron transfer (PET). Fe/Eu-MOF has a high affinity for sodium pyrophosphate (PPi). PPi can be adsorbed to the surface of Fe/Eu-MOF, destroying the structure of Fe/Eu-MOF and inhibiting its catalytic activity, resulting in a decrease in UV absorbance and the decline of fluorescence quenching. In contrast, phosphoric acid (Pi) has almost no effect on the reaction system. Alkaline phosphatase (ALP) can catalyze the hydrolysis of PPi to Pi, thereby reducing the inhibitory effect of PPi. Based on this, we successfully constructed a dual-mode ALP sensor with high selectivity. The linear ranges based on the 652 nm absorption or the fluorescence detection are from 1 to 200 U/L, and the detection limits are 0.6 for the absorption method and 0.9 U/L for the fluorescence method, respectively.

Interplay between surface chemistry and osteogenic behaviour of sulphate substituted nano-hydroxyapatite.

Surface potential and chemical compositions of the bioceramics are the core of therapeutic effect and are key factors to trigger the interfacial interactions with surrounding hard and soft tissues to repair and regeneration. Ionic substitution in hydroxyapatite (Hap) lattice significantly influences the zeta potential from -16.46 ± 0.66 mV to -6.01 ± 0.68 mV as well as an average nano-rod length from ~40 nm to ~26 nm with respect to SO42- ion content. Moreover, the surface chemistry of Hap is mainly inter-related to SO42- substitution rate at PO42- site. Specifically, nano-sized feature with lowered negative surface potential influences the protein adsorption via their weak repulsive or attractive forces. Bovine serum albumin (BSA) and lysozyme (LSZ) adsorption studies confirmed the increased affinity to active binding sites of Hap's surface. Further, SO42- ion substituted Hap (SNHA) showed improved in vitro biomineralization activity and alkaline phosphatase activity. Expression of osteogenic biomarkers such as collagen I, V, osteopontin and osteocalcin were evaluated in Saos-2 and MC3T3-E1 cells. Gene expression of these markers was influenced by SO42- ion content in Hap (maximum with 10SNHA). Altogether, these data emphasizes that chemical composition and surface properties are dominant aspect in bioceramic development towards bone regeneration.

Bioactive Zinc(II) complex incorporated PCL/gelatin electrospun nanofiber enhanced bone tissue regeneration.

Bone tissue regeneration is augmented by biocompatible nanofiber scaffolds, that supports reliable and enhanced bone formation. Zinc is an essential mineral that is vital for routine skeletal growth and it emerges to be able to improve bone regeneration. Phytochemicals, particularly flavonoids have achieved prominent interest for their therapeutic ability, they have demonstrated promising effects on bone by encouraging osteoblastogenesis, which finally leads to bone formation. In this study, we have synthesized bioactive zinc(II) quercetin complex material and used for nanofibers scaffold fabrication to enhance bone tissue regeneration property. Two derivatives of zinc(II) quercetin complexes [(Zn(quercetin) (H2O)2) (Zn+Q), and Zn(quercetin)(phenanthroline) (Zn+Q(PHt)) have been synthesized and characterized using UV-Visible spectrophotometer and Fourier Transform-IR spectroscopy. The UV-Visible absorption and IR spectra prove the B-ring chelation of the flavonoid quercetin to zinc(II) rather C-ring chelation. The potential ability of the above synthesized metal complexes on osteogenesis and angiogenesis have been studied. Besides the bioactivity of the metal complexes, the control quercetin has also been examined. The chick embryo chorioallantoic membrane (CAM) assay demonstrated that the angiogenic parameters were increased by the (Zn+Q(PHt)) complex. Amongst, (Zn+Q(PHt)) complex showed significant activity and thereby this complex has been further examined for the bone tissue activity by incorporating the complex into a nanofiber through electrospinning method. At the molecular level, Runx2, mRNA and protein, ALP and type 1 collagen mRNAs, and osteoblast-specific microRNA, pre-mir-15b were examined using real time RT-PCR and Western blot assay. Histology studies showed that the (PCL/gelatin/Zn+Q(PHt)) was biocompatibility in-ovo. Overall, the present study showed that quercetin-zinc complex (Zn+Q(PHt)) incorporated into PCL/gelatin nanofiber can act as a pharmacological agent for treating bone associated defects and promote bone regeneration.