Detection Of Alkaline Phosphatase Research Articles

A ligand-free up-conversion nanoplatform based on enzyme cascade amplification strategy for highly sensitive detection of alkaline phosphatase

An upconversion signal readout platform for the detection of alkaline phosphatase (ALP) was constructed by combining the inner filter effect (IFE) and the cascade signal amplification strategy. First, 3, 3′, 5, 5′ -tetramethylbenzidine (TMB) is oxidized by Ag+ to the oxidative product (TMBox), which quenches the up-conversion luminescence (UCL) of the ligand-free up-conversion nanoparticles (UCNPs) through the IFE principle. However, when in the presence of ascorbic acid 2-phosphate (AAP) and ALP, AAP is catalytically hydrolyzed by ALP to produce ascorbic acid (AA), which could not only decrease TMBox formation by reducing Ag+, but also reduce the TMBox generated to TMB, thereby resulting in fluorescence recovery of UCNP. Under optimal experimental conditions, ALP was linearly correlated in the range of 0.15∼8 U/L with a LOD of 0.032 U/L (S/N=3). In addition, the developed method was applied to the detection of ALP in actual serum samples, and the detection result was close to that of the traditional pNPP-based standard assay, which showed that the UCNP-TMB nanosystem has great potential to accurately detect ALP in the clinical bioassay.

Nanozyme Rich in Oxygen Vacancies Derived from Mn-Based Metal-Organic Gel for the Determination of Alkaline Phosphatase.

Vacancy engineering as an effective strategy has been widely employed to regulate the enzyme-mimic activity of nanomaterials by adjusting the surface, electronic structure, and creating more active sites. Herein, we purposed a facile and simple method to acquire transition metal manganese oxide rich in oxygen vacancies (OVs-Mn2O3-400) by pyrolyzing the precursor of the Mn(II)-based metal-organic gel directly. The as-prepared OVs-Mn2O3-400 exhibited superior oxidase-like activity as oxygen vacancies participated in the generation of O2•-. Besides, steady state kinetic constant (Km) and catalytic kinetic constant (Ea) suggested that OVs-Mn2O3-400 had a stronger affinity toward 3,3',5,5'-tetramethylbenzidine and possessed prominent catalytic performance. By taking 2-phospho-l-ascorbic acid as the substrate, which can be converted into reducing substance ascorbic acid in the presence of alkaline phosphatase (ALP), OVs-Mn2O3-400 can be applied as an efficient nanozyme for ALP colorimetric analysis without the help of destructive H2O2. The colorimetric sensor established by OVs-Mn2O3-400 for ALP detection showed a good linearity from 0.1 to 12 U/L and a lower limit of detection of 0.054 U/L. Our work paves the way for designing enhanced enzyme-like activity nanozymes, which is of significance in biosensing.

An Optimized Melanin Depigmentation Method for Histopathological and Immunohistochemical Staining of Formalin-Fixed Paraffin-Embedded Tissues.

Introduction. The difficulty in diagnosis of severe melanocytic lesions is a problem to be overcome in pathological practice. Melanin bleaching is an effective approach to ameliorate melanin disturbances in severely pigmented lesions. Although various methods for improving melanin pigmentation in immunohistochemical staining have been reported, these depigmentation methods still need to be optimized and standardized. In this study, the coloring efficiency of 3,3'-diaminobenzidine (DAB) and alkaline phosphatase (AP) after melanin depigmentation was compared under the automatic immunohistochemical staining platform. Methods. The applicability of the optimized depigmentation method was validated in 10 formalin-fixed paraffin-embedded (FFPE) blocks of ocular melanoma tissues. Specimens were demelaninized with 10% hydrogen peroxide at 60°C for immunohistochemical staining (Melan-A and SOX10), and tissue chromogenic staining was performed with DAB and AP detection systems, respectively. Results. The optimized depigmentation method including immunohistochemistry (IHC) could be completed in 3 h, effectively preserving cell morphology and immunoreactivity. Among these, the color-rendering effect and contrast of AP are better than DAB. Conclusion. This optimized method can effectively remove melanin and improve the accuracy of IHC staining interpretation. AP staining has better visibility and readability without the interference of residual melanin. The comparison results showed that after melanin depigmentation, the immunohistochemical staining agent was replaced with red AP, which avoided the misjudgment caused by brown DAB when melanin depigmentation was incomplete. This improved method can be applied to future histopathological and immunohistochemical staining of melanin-deposited tissues.

In situ formation of fluorescence species for the detection of alkaline phosphatase and organophosphorus pesticide via the ascorbate oxidase mimetic activity of AgPd bimetallic nanoflowers

Sensitive detection strategies for enzymes and their inhibitors not only have critical significance in clinical diagnosis but can be extended to food and environment analysis. Here, bimetallic AgPd alloy nanoflowers were synthesized using carbon dots as the reductant via a two-step reduction method. The AgPd bimetallic nanoflowers exhibit ascorbate oxidase mimetic activity, which can rapidly catalyze the oxidization of ascorbic acid to dehydroascorbic acid (DHA). The DHA can then react with o-phenylenediamine (OPD) to produce 3-(1, 2-dihydroxyethyl)furo[3,4-b] quinoxalin-1(3H)-one (DFQ), accompanying with the characteristic fluorescence peak at 440 nm for DFQ. Alkaline phosphatase catalyzes l-ascorbyl-2-phosphate to generate AA, while organic phosphorus pesticides (OPPs) such as chlorpyrifos can inhibit the activity of alkaline phosphatase. By utilizing the enzyme-nanozyme tandem catalytic reaction, the fluorescence of the system varies in the presence of different concentrations of ALP or OPPs, which can be used to detect ALP in serum and OPPs in vegetables and fruits.

Construction of Aggregation-Induced Emission Molecule-MnO2 Composite Nanoprobe and Its Application in Alkaline Phosphatase Detection.

Alkaline phosphatase (ALP) is among the most studied enzymes by far, playing an important role in the metabolism of organisms and the regulation of protein activity. Herein, a label-free composite nanoprobe is constructed by combining inorganic nanomaterials and aggregation-induced emission (AIE) molecule to achieve highly sensitive and selective detection of ALP. Negatively charged 9,10-bis [2-(6-sulfonatopropoxyl) naphthylethenyl] anthracene (BSNVA) molecule is synthesized, which has the AIE performance and can be assembled on the surface of amino-SiO2 nanoparticles through electrostatic interaction for fluorescence enhancement. MnO2 nanosheets are rich in negative charges, enabling them to be wrapped on the surface of the amino-SiO2 nanosphere to shield the positive charge on its surface, making it impossible for BSNVA to accumulate on the surface and then weakening the bio-fluorescence of the system. Furthermore, with catalyzed substrates induced by ALP, generating ascorbic acid and the redox reaction between ascorbic acid and MnO2, the nanoprobe helps in realizing the high-sensitivity detection of ALP with a detection limit of 0.38 mU/mL. The proposed strategy requires no complex cleaning and modification processes and can overcome the quenching effect caused by the aggregation of traditional organic dyes, proving to be a simple, low-cost and "turn-on" fluorescent detection method for ALP.

Time-resolved luminescent nanoprobes based on lanthanide nucleotide self-assemblies for alkaline phosphatase detection

Time-resolved luminescent nanoprobes based on lanthanide nucleotide self-assemblies for alkaline phosphatase detection

Study on the Osteogenic Differentiation of hUCMSCs in Silk Fibroin/Polycaprolactone Membrane Under the Intervention of Photobiomodulation

Fractures, as the most common traumatic injuries, were a significant health problem worldwide. Presently, the application of tissue engineering in the treatment of bone defects has received massive attention. Herein, we verified that photobiomodulation (PBM) could accelerate the osteogenic differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) on the silk fibroin/polycaprolactone (SF/PCL) membrane, providing a new insight into tissue engineering. HUCMSCs were divided into non-irradiated group and experimental groups. The laser of 635 and 808 nm were emerged in this study. The output energy density and power density of the laser were settled as 1-12 J/cm² and 10, 20 and 40 mW/cm², respectively. Dexamethasone acted as an inducer for osteogenic differentiation. Alkaline phosphatase (ALP) activity was detected by ALP detection kit. Runt-related transcription factor 2 (Runx2), ALP and osteocalcin (OCN) gene expressions were determined by RT-PCR. Cytokines bone morphogenetic protein 2 (BMP-2), collagen-1 (COL-1) and OCN were measured by ELISA. It was demonstrated that PBM efficiently promoted the proliferation and differentiation of hUCMSCs. When hUCMSCs were inoculated on SF/PCL membrane and treated with PBM, the activity of ALP significantly increased, and the expression levels of osteogenic-related genes Runx2, ALP, OCN and osteogenic related cytokines such as BMP-2, Col-1 and OCN all presented massive increasing with dose-dependent. Notably, the effect of PBM in promoting osteogenic differentiation would also be inhibited when the energy density exceeded the threshold.

Carbon dot/Co-MOF nanocoral mediated fluorescence-scattering ratiometric sensor for highly sensitive detection of alkaline phosphatase

Abnormal expression of alkaline phosphatase (ALP) in serum has received considerable attention in health monitoring and disease diagnosis. However, conventional optical analysis based on a single signal must compromise background interference and limited sensitivity in trace analysis. As an alternative candidate, the ratiometric approach depends on the self-calibration of two independent signals in a single test to minimize interferences from the background for accurate identification. Here, a carbon dot/cobalt-metal organic framework nanocoral (CD/Co-MOF NC) mediated fluorescence-scattering ratiometric sensor has been developed for simple, stable, and highly sensitive detection of ALP. ALP-responsive phosphate production was used to coordinate cobalt ion and collapse the CD/Co-MOF NC, resulting in the recovery of fluorescence signal from dissociative CDs and the decrease of second-order scattering (SOS) signal from the cracked CD/Co-MOF NC. The ligand-substituted reaction and the optical ratiometric signal transduction provide a rapid and reliable chemical sensing mechanism. The ratiometric sensor effectively converted ALP into a ratio signal of fluorescence-scattering dual emission throughout a wide linear concentration range of six orders of magnitude with a detection limit of 0.6 mU/L. In addition, self-calibration of fluorescence-scattering ratiometric method can reduce background interference and improve sensitivity in serum, approaching recoveries of ALP from 98.4% to 101.8%. Due to the above advantages, the CD/Co-MOF NC mediated fluorescence-scattering ratiometric sensor readily provides rapid and stable quantitative detection of ALP as a promising in vitro analytical method for clinical diagnostics.

Synergistic effect-triggered fluorescence quenching enables rapid and sensitive detection of alkaline phosphatase

The development of biosensors mediated by synergistic quenching effect is of great significance for rapid and accurate clinical diagnosis. Hence, we prepared a cyan-emitting fluorescent Si dots for alkaline phosphatase (ALP) detection through the synergistic quenching effect of inner filter effect (IFE) and photo-induced electron transfer (PET). Si dots were prepared by microwave-assisted method, which displayed high quantum yield (28.7%), as well as good physiochemical properties, such as photo-stability, pH stability, and chemical stability. As the hydrolysate of 4-nitrophenyl phosphate disodium salt hexahydrate catalyzed by ALP, both IFE and PET of 4-nitrophenyl to Si dots were used for the turn-off mode detection of ALP. The linear relationships were established between the change of fluorescence intensity and ALP concentration in the range of 0.05 U L−1 to 5.0 U L-1, and 5.0 U L−1 to 80.0 U L−1, respectively. The detection limit was 0.01 U L−1. The synergistic quenching effect caused the turn-off mode detection to be more sensitive, and it can also be used for the accurate detection of ALP in human serum, thereby showing great anti-interference ability in complex environments.

Fluorescent assay for alkaline phosphatase by integrating strand displacement amplification with DNAzyme-catalytic recycling cleavage of molecular beacons

A fluorescence sensing method for the quantification of alkaline phosphatase (ALP) was developed by integrating the strand displacement amplification with DNAzyme-catalytic recycling cleavage of molecular beacons. ALP can hydrolyze a 3′-phosphoralated primer into a 3′-hydroxy primer which can initiate the strand displacement amplification to produce the Mg2+-dependent DNAzyme. The DNAzyme can then catalyze the cleavage of the DNA molecular beacon labeled with FAM fluorophore at its 5′-end and BHQ1 quencher at its 3′-end, turning on the fluorescence of FAM fluorophore. The content of ALP in a sample can be deduced from the measured fluorescence intensity. Due to the cascading nature of its amplification strategy, the proposed method achieved sensitive and specific ALP detection in human serum samples. Its results were in good consistent with the corresponding values obtained by a commercial ALP detection kit. The limit of detection of the proposed method for ALP is about 0.015 U/L, lower than some methods recently reported in literature, demonstrating its potential for ALP detection in biomedical research and clinical diagnosis.

Relaxin treatment stimulates the differentiation of mesenchymal stem cells into osteoblasts

Background/purposeSeveral studies have determined that relaxin stimulates differentiation and regulates the activity of mature osteoclasts, but little is known about its effect on the differentiation of mesenchymal cells towards the osteogenic lineage. Therefore, this study aimed to determine the effect of relaxin on the proliferation and differentiation of the osteoblastic lineage of mesenchymal cells derived from human dental pulp (hDPSC). Materials and methodsIn this in vitro study, hDPSC were characterized and treated with relaxin at different doses (10–80 ng/ml) and times (1–21 days). Morphology was assessed by microscopy, and proliferation was assessed using a resazurin assay. Osteoblastic differentiation was evaluated by Alizarin Red staining, alkaline phosphatase (ALP) labeling, and changes in the expression of the osteoblastic differentiation genes RUNX2 and BMP2. ResultsRelaxin treatment did not induce changes in the proliferation or viability of hDPSCs; however, larger cells and increased cytoplasmic prolongation were observed. Relaxin treatment (20 and 80 ng/ml) significantly increased calcified nodule formation on days 14 and 21. The cytochemical signals for ALP, RUNX2, and BMP2 gene expression were significantly (P < 0.05) increased by the relaxin treatment. ConclusionRelaxin treatment does not induce changes in hDPSC proliferation but induces morphological changes, increases ALP detection, calcified nodule formation, and increases expression of RUNX2 and BMP2, suggesting the induction of osteoblastic differentiation of hDPSC.

A turn-on chemiluminescent assay for alkaline phosphatase using two-dimensional Fe-centered metal-organic frameworks as the signaling probe.

Alkaline phosphatase (ALP) is an essential enzyme involved in cell phosphorus metabolism. Developing sensitive and accurate ALP quantitative assays is significant. In this study, a turn-on chemiluminescence (CL) analysis platform for the detection of ALP activity in human serum was established based on two-dimensional (2D) Fe-centered metal-organic frameworks with 1,3,5-benzene tricarboxylic acid as ligands (denoted as 2D Fe-BTC). The 2D Fe-BTC as the signaling probe reacts with ascorbic acid forming reduced Fe-BTC which catalyzes the luminol CL reaction producing a strong CL signal. The 2D Fe-BTC-based luminol CL reaction exhibited good CL responses when the concentration of ascorbic acid was in the range of 5-500nM. By employing magnesium ascorbyl phosphate (MAP) as the substrate which can be hydrolyzed by ALP to generate ascorbic acid, a turn-on CL assay for the detection of ALP was established. Under optimal conditions, as low as 0.00046UL-1 of ALP could be sensitively detected with a linear range of 0.001-0.1UL-1. ALP in human serum can be detected after a simple dilution process without any other pretreatment.

Functionalized pyrite nanozyme probe and imprinted polymer modified with hydrophilic layer for rapid colorimetric analysis of glycoprotein in serum

The biological molecules used in the sandwich detection method have problems such as complex extraction processes, high costs, and uneven quality. Therefore we integrated glycoprotein molecularly controllable-oriented surface imprinted magnetic nanoparticles (GMC-OSIMN) and boric acid functionalized pyrite nanozyme probe (BPNP) to replace the traditional antibody and horseradish peroxidase for sensitive detection of glycoproteins through sandwich detection.In this work, a novel nanozyme functionalized with boric acid was used to label glycoproteins that were captured by GMC-OSIMN. The substrate in the working solution catalyzed by the nanozyme labeled on the protein underwent visible color changes to the naked eye, and the generated signal can be quantitatively detected by a spectrophotometer, and the best color development conditions of the novel nanozyme under the influence of many factors were determined through multi-dimensional investigation. The optimum conditions of sandwich are optimized with ovalbumin (OVA), and it was extended to the detection of transferrin (TRF) and alkaline phosphatase (ALP) in the application. The detection range for TRF was 2.0 × 10−1-1.0 × 104 ng mL−1 with a detection limit of 1.32 × 10−1 ng mL−1, The detection range for ALP was 2.0 × 10−3-1.0 × 102 U L−1 with the detection limit of 1.76 × 10−3 U L−1. This method was subsequently used to detect TRF and ALP levels in 16 liver cancer patients, and the standard deviation of the test results of each patient was less than 5.7%.

Docking guided phase display to develop fusion protein with novel scFv and alkaline phosphatase for one-step ELISA salbutamol detection.

Salbutamol (SAL) is a β2 adrenergic receptor agonist which has potential hazardous effects for human health. It is very important to establish a sensitive and convenient method to monitor SAL. Here we introduce a method to combine the information from docking and site specific phage display, with the aim to obtain scFv with high affinity to SAL. First, single chain variable fragment (scFv) antibodies against SAL were generated through phage display. By using molecular docking approach, the complex structure of SAL with antibody was predicted and indicated that H3 and L3 contribute to the binding. Then new libraries were created by randomization specific residues located on H3 and L3 according to the docking results. Anti-SAL scFv antibodies with high efficiency were finally identified. In addition, the selected scFv was fused with alkaline phosphatase and expressed in E coli to develop a rapid and low-cost one step ELISA to detect SAL.

Pd(II)-based coordination polymer nanosheets for ratiometric colorimetric and photothermal dual-mode assay of serum alkaline phosphatase

Fabrication of a multi-signal readout assay with high sensitivity and selectivity is highly desirable for clinical and biochemical analysis, but remains a challenge due to laborious procedures, large-scale instruments, and inadequate accuracy. Herein, a straightforward, rapid, and portable detection platform based on palladium(II) methylene blue (MB) coordination polymer nanosheets (PdMBCP NSs) was unveiled for the ratiometric dual-mode detection of alkaline phosphatase (ALP) with temperature and colorimetric signal readout properties. The sensing mechanism is the ALP-catalyzed generation of ascorbic acid for competitive binding and etching PdMBCP NSs to release free MB in a quantitive means for detection. Specifically, ALP addition led to the decrease of temperature signal readout from the decomposed PdMBCP NSs under 808 nm laser excitation, and simultaneous increase of the temperature from the generated MB with a 660 nm laser, together with the corresponding absorbance changes at both wavelengths. Notably, this ratiometric nanosensor exhibited a detection limit of 0.013 U/L (colorimetric) and 0.095 U/L (photothermal) within 10 min, respectively. The reliability and satisfactory sensing performance of the developed method were further confirmed by clinic serum samples. Therefore, this study provides a new insight for the development of dual-signal sensing platforms for convenient, universal, and accurate detection of ALP.

Gelatinous lanthanide coordination polymer with aggregation-enhanced antenna effect for ratiometric detection of endogenous alkaline phosphatase

Aggregation-induced emission (AIE) and antenna effect (AE) are two significant behaviors that have attracted increasing attention. However, it is challenging to achieve the synergistic effect of AIE and AE in luminescent materials for more extensive applications. Here, four gelatinous Ln3+ coordination polymers (Ln-CPs) are synthesized by self-assembly of ciprofloxacin (CIP), adenosine monophosphate (AMP), and Ln3+ ions in aqueous medium. Encouragingly, a remarkable increase in the characteristic fluorescence of Ln3+ and a significant decrease in CIP are observed along with increasing concentration of Ln-CPs, which is attributed to the large aggregates formed by self-assembly that strictly constrain the intramolecular motions of antenna ligands, thereby achieving the aggregation-enhanced AE. More meaningfully, Eu-CP not only shows a rice-like morphology at high aggregation state, but also provides an opportunity for the selective detection of alkaline phosphatase (ALP). A new flower-like polymer is formed upon incubating Eu-CP with ALP, accompanied by the fluorescence quenching of Eu3+ and recovery of CIP, a ratiometric determination of ALP in the range of 0.1–6.0 U·L−1 is thus achieved. Additionally, ALP assay in human serum and bioimaging in living cells have been successfully performed. This research opens a new horizon for the fabrication of Ln3+-based luminescent materials with promising applications.

Laser-induced in situ synthesis of nano-composite Co–Co3O4–rGO on paper: miniaturized biosensor for alkaline phosphatase detection

Recent progress in the in situ synthesise of various nanomaterials has gained tremendous interest and wide applications in various fields. For the first time to the best of our knowledge, this work reports a methodology of ultra-fast in situ synthesis of cobalt–cobalt oxide-reduced graphene oxide (Co−Co3O4−rGO (CC–rGO)) composite by laser ablation. The photothermal reduction technique was leveraged to develop the CC–rGO. For this, a low-cost 450 nm blue diode laser was irradiated onto a grade 1 filter paper in the presence of cobalt ions readily patterns the carbon matrix of paper to the composite material. Moreover, the variation of cobalt concentrations from 0.1–0.5 M led to structural and morphological changes. Standard techniques were adopted for thorough characterizations of developed sensor material for conductivity analysis, specific surface area, crystal-structural information, surface morphology, and chemical composition. The observed results were highly promoting towards the electrochemical sensing applications. Further, the developed sensor was found to be highly selective toward detecting a vital bio analyte alkaline phosphatase (ALP). The sensors performance was highly significant in the linear range of 10–800 mU l−1 with a detection limit of 10.13 mU l−1. The sensors applicability was further validated in actual human serum samples via a recovery-based approach. In the future, the developed in situ material methodology can begin a rapid composite material synthesis at a larger scale.

MoO2 and N-Doped Carbon Nano-Octahedron Nanozymes for Sensitive Detection of Alkaline Phosphatase

As a typical biomarker for clinical diagnosis of diseases, alkaline phosphatase (ALP) plays a crucial role in regulating the dephosphorylation of biomolecules. Consequently, it is necessary to develop an effective and simple method for the determination of ALP activity. In this work, we report a catalytic reaction-based colorimetric method to monitor ALP activity by using a nanostructured MoO2@NP-doped carbon nano-octahedron (MoO2@NPC) as a favorable oxidase mimetic. Because the C–O bond generated in the nano-octahedron MoO2@NPC regulates the electronic valence state of Mo between +5 and +6, the as-prepared MoO2@NPC regular octahedron with good peroxide-like (POD-like) properties and colorless 3,3′,5,5′-tetramethylbenzidine dihydrochloride hydrate (TMB) can be catalyzed to dark-blue oxTMB. When ALP hydrolyzes the substrate AAP, the produced AA with a certain reducing ability can not only reduce oxTMB to TMB but also consume hydroxyl radicals (•OH) generated in the MoO2@NPC/H2O2/TMB system, which significantly inhibits the color reaction of TMB. On this basis, a simple and efficient colorimetric method for ALP was constructed based on carbon-based nanozymes. Furthermore, the system goes for the detection of ALP with high sensitivity for the first time. The linearity is good in the range of 0.5–30 U/L, and the detection limit was 0.02 U/L and had good stability and reproducibility. Reliable and accurate measurements of analytes in real samples were also validated by our analytical method, showing that it can provide a reliable, convenient, and efficient nanoplatform for clinical diagnosis of ALP activity.

A facile and sensitive magnetic relaxation sensing strategy based on the conversion of Fe3+ ions to Prussian blue precipitates for the detection of alkaline phosphatase and ascorbic acid oxidase

Herein, a novel magnetic relaxation sensing strategy based on the change in Fe3+ content has been proposed by utilizing the conversion of Fe3+ ions to Prussian blue (PB) precipitates. Compared with the common detection approach based on the valence state change of Fe3+ ions, our strategy can cause a larger change in the relaxation time of water protons and higher detection sensitivity since PB precipitate can induce a larger change in the Fe3+ ion concentration and has a weaker effect on the relaxation process of water protons relative to Fe2+ ions. Then, we employ alkaline phosphatase (ALP) as a model target to verify the feasibility and detection performance of the as-proposed strategy. Actually, ascorbic acid (AA) generated from the ALP-catalyzed L-ascorbyl-2-phosphate hydrolysis reaction can reduce potassium ferricyanide into potassium ferrocyanide, and potassium ferrocyanide reacts with Fe3+ to form PB precipitates, leading to a higher relaxation time. Under optimum conditions, the method for ALP detection has a wide linear range from 5 to 230 mU/mL, and the detection limit is 0.28 mU/mL, sufficiently demonstrating the feasibility and satisfactory analysis performance of this strategy, which opens up a new path for the construction of magnetic relaxation sensors. Furthermore, this strategy has also been successfully applied to ascorbic acid oxidase detection, suggesting its expansibility in magnetic relaxation detection.

SOX9 Inhibits the Progression of Osteonecrosis of the Femoral Head via the Activation of the Wnt/Beta-Catenin Pathway

Background: In this study, we aimed to explore whether the SRY-box transcription factor 9 (SOX9) can play protective roles against the occurrence and development of osteonecrosis of the femoral head (ONFH) by regulating the proliferation, apoptosis, and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) via the Wnt/β-catenin pathway. Methods: We used 1600 mg of glucocorticoid (GC) to induce hBMSCs to establish an ONFH cell model and performed various experiments. Reverse transcription-quantitative polymerase chain reaction and western blotting assays were used to determine the expression levels of SOX9 and osteoblast markers, such as the RUNX family transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osterix, Wnt3a, and β-catenin. An ALP detection kit was used to measure the ALP activity. Flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to determine the cell viability. Results: GC treatment decreased the expression levels of RUNX2, ALP, and osterix, suppressed ALP activity, and inhibited SOX9 expression. SOX9 overexpression promoted GC-induced cell proliferation and decreased cell apoptosis. Additionally, hBMSCs were transfected with SOX9-small interfering RNA in GC treatment, and SOX9 knockdown was found to suppress the osteogenic differentiation of cells and decrease their viability. Conclusion: Our results revealed that SOX9 is related to the Wnt/β-catenin pathway in ONFH. Moreover, SOX9 participated in ONFH development by activating the Wnt/β-catenin pathway.