Alkaline Phosphatase Secretion Research Articles

The combination of hexagonal microfluidic devices and cell-based reporter cells allows detection of cytokine-producing cells at the single-cell level

Cytokine production analysis at the single-cell level, such as detection of type I interferon (IFN-I), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) producing cells, is beneficial in clinical diagnostics and drug discovery. In this work, we developed a microfluidic device with hexagonal microchambers capable of generating single-cell/multicellular systems, oil-droplet compartmentalization, and performing functional assays based on green fluorescent protein (GFP) expression or secreted embryonic alkaline phosphatase (SEAP) secretion for cytokine secretion detection at single-cell level. Since the droplets are stationary, cell/cell-pairs are identified, and real-time monitoring and end-point analysis can be performed. At optimized conditions, up to 60% single-cell chamber occupancy can be achieved. Cytokines such as IFN-I, TNF-α, and IL-6 secreted by human immune cells (THP1 cells, plasmacytoid dendritic cells (pDCs), T-cells, B-cells, and monocytes) can be detected at the single-cell level upon pairing with GFP and SEAP expressing Human Embryonic Kidney (HEK)− 293 reporter cells. This proof-of-concept device has the potential to evaluate single-cell behaviors and could benefit the field of single-cell cytokine secretion monitoring.

Physical, mechanical, and biological performance of chitosan-based nanocomposite coating deposited on the polycaprolactone-based 3D printed scaffold: Potential application in bone tissue engineering

Recently, coating on composite scaffolds has attracted many researchers' attention to improve scaffolds' properties. In this research, a 3D printed scaffold was fabricated from polycaprolactone (PCL)/magnetic mesoporous bioactive glass (MMBG)/alumina nanowire (Al2O3, Optimal percentage 5 %) (PMA) and then coated with chitosan (Cs)/multi-walled carbon nanotubes (MWCNTs) by an immersion coating method. Structural analyses such as XRD and ATR-FTIR confirmed the presence of Cs and MWCNTs in the coated scaffolds. The SEM results of the coated scaffolds showed homogeneous three-dimensional structures with interconnected pores compared to the uncoated scaffolds. The coated scaffolds exhibited an increase in compression strength (up to 16.1 MPa) and compressive modulus (up to 40.83 MPa), improved surface hydrophilicity (up to 32.69°), and decrease in degradation rate (68 % remaining weight) compared to the uncoated scaffolds. The increase in apatite formation in the scaffold coated with Cs/MWCNTs was confirmed by SEM, EDAX, and XRD tests. Coating the PMA scaffold with Cs/MWCNTs leads to the viability and proliferation of MG-63 cells and more secretion of alkaline phosphatase and Ca activity, which can be introduced as a suitable candidate for use in bone tissue engineering.

Biological evaluation and osteogenic potential of polyhydroxybutyrate-keratin/Al2O3 electrospun nanocomposite scaffold: A novel bone regeneration construct

In this study, the effect of alumina nanowire on the physical and biological properties of polyhydroxybutyrate-keratin (PHB-K) electrospun scaffold was investigated. First, PHB-K/alumina nanowire nanocomposite scaffolds were made with an optimal concentration of 3 wt% alumina nanowire by using the electrospinning method. The samples were examined in terms of morphology, porosity, tensile strength, contact angle, biodegradability, bioactivity, cell viability, ALP activity, mineralization ability, and gene expression. The nanocomposite scaffold provided a porosity of >80 % and a tensile strength of about 6.72 MPa, which were noticeable for an electrospun scaffold. AFM images showed an increase in surface roughness with the presence of alumina nanowires. This led to an improvement in the degradation rate and bioactivity of PHB-K/alumina nanowire scaffolds. The viability of mesenchymal cells, alkaline phosphatase secretion, and mineralization significantly increased with the presence of alumina nanowire compared to PHB and PHB-K scaffolds. In addition, the expression level of collagen I, osteocalcin, and RUNX2 genes in nanocomposite scaffolds increased significantly compared to other groups. In general, this nanocomposite scaffold could be a novel and interesting construct for osteogenic induction in bone tissue engineering.

Hybrid Cell Membrane-Functionalized Matrixes for Modulating Inflammatory Microenvironment and Improving Bone Defect Repair.

Cell membranes from different sources retain the integrity of the original membrane structure and special membrane protein functions. However, the diversity function of membrane surface proteins underutilized in bone defect repair. The current study creatively prepared a hybrid membrane (HM) (diameter, 92.25 ± 16.47nm) and coated it on a poly-ε-caprolactone (PCL) (diameter, 313.79 ± 4.69nm) electrospinning membrane for cell membrane-functionalized matrixes (MFMs) (diameter, 368.76 ± 5.90nm) preparation. The effects of the HM and MFMs on inflammatory regulation and bone formation were further explored in vitro and in vivo. The results showed that MFMs could regulate macrophage into anti-inflammatory phenotype and promote alkaline phosphatase secretion and mineralization deposition of mesenchymal stem cells (MSCs) in vitro. The foreign body response was alleviated and bone regeneration was facilitated in rat calvarial critical-size defect in vivo. The study provides an innovative approach to applicate the cell membrane functions from different sources for immunomodulatory and osteogenic differentiation and provides a promising choice for designing bone repair materials. This article is protected by copyright. All rights reserved.

Enhanced Cell Osteogenic Differentiation in Alendronate Acid and Flufenamic Acid Drug-Impregnated Nanoparticles of Mesoporous Bioactive Glass Composite Calcium Phosphate Bone Cement In Vitro.

This study aims to compare the anti-osteoporotic drugs alendronic acid (ALN) and flufenamic acid (FA) alone impregnate into nanoparticles of mesoporous bioactive glass (nMBG), which further composites calcium phosphate cement (CPC) and investigates their in vitro performance. The drug release, physicochemical properties, and biocompatibility of nMBG@CPC composite bone cement are tested, and the effect of the composites on improving the proliferation and differentiation efficiency of mouse precursor osteoblasts (D1 cells) is also investigated. Drug release shows that FA impregnates nMBG@CPC composite, a large amount of FA is released rapidly within 8 h, gradually reaching a stable release within 12 h, followed by a slow and sustained release within 14 days, and then reaches a plateau within 21 days. The release phenomenon confirms that the drug-impregnated nBMG@CPC composite bone cement effectively achieves slow drug delivery. The working time and setting time of each composite are within 4-10 min and 10-20 min, respectively, meeting the operational requirements of clinical applications. The addition of nMBG nanoparticles in the CPC matrix did not prevent the aggregation phenomenon under microstructural observation, thus resulting in a decrease in the strength of the nMBG@CPC composite. However, after 24 h of immersed reaction, the strength of each 5 wt.% nMBG impregnated with different concentrations of FA and ALN is still greater than 30 MPa, which is higher than the general trabecular bone strength. The drug-impregnated nMBG@CPC composites did not hinder the product formation and exhibit biocompatibility. Based on the proliferation and mineralization of D1 cells, the combination of nMBG with abundant FA and ALN in CPC is not conducive to the proliferation of D1 cells. However, when D1 cells are contact cultured for 21 days, alkaline phosphatase (ALP) enzyme activity shows higher ALP secretion from drug-impregnated nMBG@CPC composites than drug-free composites. Accordingly, this study confirms that nMBG can effectively impregnate the anti-osteoporosis drugs FA and ALN, and enhance the mineralization ability of osteoblasts. Furthermore, drug-impregnated nMBG applications can be used alone or in combination with CPC as a new option for osteoporotic bone-filling surgery.

PhoD Harboring Microbial Community and Alkaline Phosphatase as Affected by Long Term Fertilization Regimes on a Calcareous Soil

Organic phosphorus (Po) may play a vital role in phosphorus availability via its mineralization by alkaline phosphatase (ALP), being encoded by phoD gene, in calcareous soil. Understanding the effects of long-term fertilization on the community of phoD harboring bacteria and the related alteration of the P availability owing to the changes in ALP secretion may offer a chance to elucidate the Po contribution to soil available P. Based on a long-term experiment, we analyzed the phoD gene harboring microbial diversity, abundance and composition, ALP and Po forms, and their relationship. The treatments involved were control without any fertilizers (CK), synthetic nitrogen and potassium (NK), synthetic nitrogen, phosphorus and potassium (NPK), NPK and crop stalk return (SNPK), and NPK plus organic manure (MNPK). Fertilization increased the abundance and diversity of phoD gene harboring microbial over CK. Those receiving NPK and NPK treatments integrated with organic supplements significantly improved the relative abundance of Proteobacteria but decreased Gemmatimonadetes at the phylum level, while all fertilized treatments appreciably increased the relative abundance of Lysobacter but decreased that of Gemmatirosa and Afipia, at the genus level. SNPK and MNPK treatments noticeably increased the relative abundance of Methylobacter but reduced Pseudomonas and Streptomyces relative to those receiving synthetic fertilizer treatments. Long-term fertilization markedly raised ALP activity, which was significantly and positively correlated with the relative abundance of the phylum Proteobacteria as represented by the genera Methylobacterium and Lysobacter. ALP was closely associated with moderately labile Po, followed by enzyme P, recalcitrant Po, and labile Po. The changes in phoD bacteria and ALP were mainly driven by soil organic carbon, Olsen P and pH. We concluded that the long-term fertilization, especially the addition of organic supplements, profoundly modified the soil properties and subsequently changed the diversity and relative abundance of phoD gene harboring bacteria, which promoted the activity of ALP, and thus the mineralization of various forms of Po (mainly moderately labile Po) to enhance the P availability.

Investigating of physical, mechanical, and biological properties of polyhydroxybutyrate-keratin/alumina electrospun scaffold utilized in bone tissue engineering

Electrospun nanofiber scaffolds have a similar structure to extracellular matrix, leading to increased cell adhesion, proliferation, and migration. In this study, 1–5wt % of alumina nanowires were added to polyhydroxybutyrate-keratin (PHB–K) solution, and the morphology of the electrospun scaffolds in terms of fiber diameter, porosity percentage, and the uniformity of the alumina nanowires distribution was evaluated by SEM. FTIR and raman tests were also used to evaluate the chemical bonds of nanofibers and the presence of alumina and keratin in electrospun scaffolds. The crystallinity of the scaffolds was also measured using DSC and confirmed by XRD. The increase of alumina augmented the crystallinity of scaffolds because alumina is a nucleating agent. The tensile strength of PHB-K scaffolds increased up to 3 fold in the presence of 3 wt % of alumina.The MG63 cells survived, and the secretion of alkaline phosphatase and mineralization due to the presence of alumina was significantly higher than PHB and PHB-K scaffolds. Based on the results, the electrospun PHB-K/AL2O3 scaffolds are potential candidates for bone tissue engineering.

In vitro evaluation of electrospun polyvinylidene fluoride hybrid nanoparticles as direct piezoelectric membranes for guided bone regeneration

A polyvinylidene fluoride (PVDF) piezoelectric membrane containing carbon nanotubes (CNTs) and graphene oxide (GO) additives was prepared, with special emphasis on the piezoelectric activity of the aligned fibers. Fibroblast viability on membranes was measured to study cytotoxicity. Osteoprogenitor D1 cells were cultured, and mineralization of piezoelectric composite membranes was assessed by ultrasound stimulation. Results showed that the electrospun microstructures were anisotropically aligned fibers. As the GO content increased to 1.0 wt% (0.2 wt% interval), the β phase in PVDF slightly increased but showed the opposite trend with the increase in CNT. Excessive addition of GO and CNT hindered the growth of the β phase in PVDF. The direct piezoelectric activity and mechanical properties showed the same trend as the β phase in PVDF. Moreover, GO/PVDF with the same nanoparticle content showed better performance than CNT/PVDF composites. In this study, a comparison of the generated piezoelectric specific voltage (unit: 10−3 Vg−1 cm−2, linear stretch, g33) with control PVDF only (0.55 ± 0.16) revealed that the two composites containing 0.8 wt% GO- and 0.2 wt% CNT- with 15 wt% PVDF exhibited excellent piezoelectric voltages, which were 3.37 ± 1.05 and 1.45 ± 0.07 (10−3 Vg−1 cm−2), respectively. In vitro cultures of these two groups in contact with D1 cells showed significantly higher alkaline phosphatase secretion than the PVDF only group within 1–10 days of cell culture. Further application of ultrasound stimulation showed that the piezoelectric membrane differentiated D1 cells earlier than without ultrasound and induced higher proliferation and mineralization. This developing piezoelectric effect is expected to generate voltage through activities to enhance microcurrent stimulation in vivo.

Galangin induces the osteogenic differentiation of human amniotic mesenchymal stem cells via the JAK2/STAT3 signaling pathway

The regulation of stem cell directional differentiation is a core research topic in regenerative medicine, and modulating the fate of stem cells is a promising strategy for precise intervention through the utilization of naturally small molecule compounds. The present study aimed to explore the potential pro-osteogenic differentiation effect of galangin, a flavonoid derived from Alpinia officinarum, on human amniotic mesenchymal stem cells (hAMSCs) and the underlying molecular mechanism. The results showed that galangin had no cytotoxicity towards hAMSCs when the concentration was less than 50 μM. Treatment with 10 μM galangin significantly increased alkaline phosphatase (ALP) secretion and calcium deposition in hAMSCs. Meanwhile, galangin upregulated the mRNA and protein expression of early osteoblast-specific markers, namely ALP, RUNX2, and OSX, and late osteoblast-specific markers, CoL1α1, OPN, and OCN, in hAMSCs. Furthermore, signaling pathway screening studies showed that galangin enhanced the phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). In addition, molecular docking results suggest there is a promising interaction between galangin and JAK2. Finally, treatment with the JAK2 specific inhibitor AG490 effectively reversed the induction of osteogenic differentiation, upregulation of osteoblast-specific marker expression, and activation of JAK2/STAT3 signaling induced by galangin. These results show that galangin induces the osteogenic differentiation of hAMSCs through the JAK2/STAT3 signaling pathway and could serve as a promising small molecular osteoinducer for application to hAMSCs in regenerative medicine.

Biomimetic anti-inflammatory and osteogenic nanoparticles self-assembled with mineral ions and tannic acid for tissue engineering

Bone healing involves complex processes including inflammation, induction, and remodeling. In this context, anti-inflammatory and osteoconductive multi-functional nanoparticles have attracted considerable attention for application in improved bone tissue regeneration. In particular, nanoparticles that promote suppression of inflammatory response after injury and direction of desirable tissue regeneration events are of immense interest to researchers. We herein report a one-step method to prepare multi-functional nanoparticles using tannic acid (TA) and simulated body fluid (SBF) containing multiple mineral ions. Mineral-tannic acid nanoparticles (mTNs) were rapidly fabricated in 10 min, and their size (around 250–350 nm) and chemical composition were controlled through the TA concentration. In vitro analysis using human adipose derived stem cells (hADSCs) showed that mTNs effectively scavenged reactive oxygen species (ROS) and enhanced osteogenesis of hADSCs by inducing secretion of alkaline phosphatase. mTNs also increased osteogenic marker gene expression even in the presence of ROS, which can generally arrest osteogenesis (OPN: 1.74, RUNX2: 1.90, OCN: 1.47-fold changes relative to cells not treated with mTNs). In vivo analysis using a mouse peritonitis model revealed that mTNs showed anti-inflammatory effects by decreasing levels of pro-inflammatory cytokines in blood (IL-6: 73 ± 4, TNF-α: 42 ± 2%) and peritoneal fluid (IL-6: 78 ± 2, TNF-α: 21 ± 6%). We believe that this one-step method for fabrication of multi-functional nanoparticles has considerable potential in tissue engineering approaches that require control of complex microenvironments, as required for tissue regeneration.

Scutellarin Loaded on Ultradeformable Nanoliposome Scutellarin EDTMP (S-UNL-E) Promotes Osteogenesis in Osteoporotic Rats.

Scutellarin is known as a safe, effective, and low-cost traditional Chinese medicine and has a variety of biological activities. Studies reported that the scutellarin loaded on ultradeformable nanoliposome scutellarin EDTMP (S-UNL-E) could promote osteoblast differentiation and bone formation in vitro. However, its effect on promoting osteogenesis in vivo is still unclear. In this study, pharmacology network and transcriptome sequencing were used to screen the potential targets and pathways of scutellarin in treating osteoporosis. The female Sprague-Dawley (SD) rats were operated on with bilateral oophorectomy and femoral defect to establish an osteoporosis model and then treated separately with bone dust, single scutellarin, 40 mg/kg ultradeformable nanoliposome scutellarin (S-UNL), and the optimal concentration of 40 mg/kg S-UNL-E for a total of 56 d to detect the parameters of trabecular bones. And qRT-PCR and western blot were performed to determine the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), alkaline phosphatase (ALP), transcription factor 4 (TCF4), and β-catenin. Results of microscopic computed tomography (Micro-CT) of trabecular bones showed that single scutellarin, S-UNL, and S-UNL-E all promoted the bone formation of osteoporotic rats, in which S-UNL-E manifested the most remarkable therapeutic effect. And it is found that 40 mg/kg of S-UNL-E increased the expression of PTGS2, ALP, TCF4, and β-catenin, which indicated that S-UNL-E stimulated the secretion of ALP in bone defect areas to promote bone healing, and increased PTGS2 expression thereby enhancing the transcription and translation of key gene β-catenin and TCF4 in the Wnt/β-catenin signaling pathway to treat osteoporotic rats.

Autophagy Is Possibly Involved in Osteoblast Responses to Mechanical Loadings.

Both mechanical loading and autophagy play important roles in regulating bone growth and remodeling, but the relationship between the two remains unclear. In this study, we examined bone structure with micro-CT imaging and measured bone mechanical properties with three-point bending experiments using bones from wild-type (WT) mice and conditional knockout (cKO) mice with Atg7 deletion in their osteoblasts. We found that the knockout mice had significantly less bone volume, bone thickness, bone ultimate breaking force, and bone stiffness compared to wild-type mice. Additionally, bone marrow cells from knockout mice had reduced differentiation and mineralization capacities in terms of alkaline phosphatase and calcium secretion, as well as Runx2 and osteopontin expression. Knockout mice also had significantly less relative bone formation rate due to mechanical loading. Furthermore, we found that the osteoblasts from wild-type mice had stronger responses to mechanical stimulation compared to autophagy-deficient osteoblasts from knockout mice. When inhibiting autophagy with 3 MA in wild-type osteoblasts, we found similar results as we did in autophagy-deficient osteoblasts. We also found that mechanical loading-induced ATP release is able to regulate ERK1/2, Runx2, alkaline phosphatase, and osteopontin activities. These results suggest that the ATP pathway may play an important role in the possible involvement of autophagy in osteoblast mechanobiology.

Mussel-inspired polydopamine decorated alginate dialdehyde-gelatin 3D printed scaffolds for bone tissue engineering application.

This study utilized extrusion-based 3D printing technology to fabricate calcium-cross-linked alginate dialdehyde-gelatin scaffolds for bone regeneration. The surface of polymeric constructs was modified with mussel-derived polydopamine (PDA) in order to induce biomineralization, increase hydrophilicity, and enhance cell interactions. Microscopic observations revealed that the PDA layer homogeneously coated the surface and did not appear to induce any distinct change in the microstructure of the scaffolds. The PDA-functionalized scaffolds were more mechanically stable (compression strength of 0.69 ± 0.02 MPa) and hydrophilic (contact angle of 26) than non-modified scaffolds. PDA-decorated ADA-GEL scaffolds demonstrated greater durability. As result of the 18-days immersion in simulated body fluid solution, the PDA-coated scaffolds showed satisfactory biomineralization. Based on theoretical energy analysis, it was shown that the scaffolds coated with PDA interact spontaneously with osteocalcin and osteomodulin (binding energy values of −35.95 kJ mol−1 and −46.39 kJ mol−1, respectively), resulting in the formation of a protein layer on the surface, suggesting applications in bone repair. PDA-coated ADA-GEL scaffolds are capable of supporting osteosarcoma MG-63 cell adhesion, viability (140.18% after 7 days), and proliferation. In addition to increased alkaline phosphatase secretion, osteoimage intensity also increased, indicating that the scaffolds could potentially induce bone regeneration. As a consequence, the present results confirm that 3D printed PDA-coated scaffolds constitute an intriguing novel approach for bone tissue engineering.

Engineered 3D-Printed Polyvinyl Alcohol Scaffolds Incorporating β-Tricalcium Phosphate and Icariin Induce Bone Regeneration in Rat Skull Defect Model.

The skull defects are challenging to self-heal, and autologous bone graft repair has numerous drawbacks. The scaffolds for the rapid and effective repair of skull defects have become an important research topic. In this study, polyvinyl alcohol (PVA)/β-tricalcium phosphate(β-TCP) composite scaffolds containing icariin (ICA) were prepared through direct-ink three-dimensional (3D) printing technology. β-TCP in the composite scaffold had osteoconductive capability, and the ICA molecule had osteoinductive capacity. The β-TCP and ICA components in the composite scaffold can enhance the capability to repair skull defects. We show that ICA exhibited a slow-release behaviour within 80 days. This behaviour helped the scaffold to continuously stimulate the formation of new bone. The results of in vitro cell compatibility experiments showed that the addition of ICA molecules contributed to the adhesion and proliferation of MC-3T3-E1 cells. The level of alkaline phosphatase secretion demonstrated that the slow release of ICA can promote the osteogenic differentiation of MC-3T3-E1 cells. The introduction of ICA molecules accelerated the in situ bone regeneration in in vivo. It is concluded that the 3D-printed PVA scaffold with β-TCP and ICA has a wide range of potential applications in the field of skull defect treatment.

Phytochemical Analysis, Antioxidant and Bone Anabolic Effects of Blainvillea acmella (L.) Philipson.

Blainvillea acmella (L.) Philipson [Asteraceae] (B. acmella) is an important medicinal plant native to Brazil, and it is widely known as a toothache plant. A plethora of studies have demonstrated the antioxidant activities of B. acmella and few studies on the stimulatory effects on alkaline phosphatase (ALP) secretion from bone cells; however, there is no study on its antioxidant and anabolic activity on bone cells. The study aimed to evaluate the phytochemical contents of aqueous and ethanol extracts of B. acmella using gas chromatography mass spectrometry (GCMS) and liquid chromatography time of flight mass spectrometry (LCTOFMS) along with the total phenolic (TPC) and flavonoid (TFC) contents using Folin-Ciocalteu and aluminum colorimetric methods. The extracts of B. acmella leaves were used to scavenge synthetic-free radicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays. The bone anabolic effects of B. acmella extracts on MC3T3-E1 cells were measured with 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoium bromide (MTT) at 1, 3, 5, and 7 days, Sirius-red and ALP at 7 and 14 days, and Alizarin Red S at 14 and 21 days. Comparatively, ethanol extract of B. acmella (BaE) contributed higher antioxidant activities (IC50 of 476.71 µg/ml and 56.01 ± 6.46 mg L-ascorbic acid/g against DPPH and FRAP, respectively). Anabolic activities in bone proliferation, differentiation, and mineralization were also higher in B. acmella of ethanol (BaE) than aqueous (BaA) extracts. Positive correlations were observed between phenolic content (TPC and TFC) to antioxidant (ABTS and FRAP) and anabolic activities. Conversely, negative correlations were present between phenolic content to antioxidant (DPPH) activity. These potential antioxidant and bone anabolic activities in BaE might be due to the phytochemicals confirmed through GCMS and LCTOFMS, revealed that terpenoids of α-cubebene, cryophyllene, cryophyllene oxide, phytol and flavonoids of pinostrobin and apigenin were the compounds contributing to both antioxidant and anabolic effects in BaE. Thus, B. acmella may be a valuable antioxidant and anti-osteoporosis agent. Further study is needed to isolate, characterize and elucidate the underlying mechanisms responsible for the antioxidant and bone anabolic effects.

Three-dimensional porous reduced graphene oxide/hydroxyapatite membrane for guided bone regeneration

The guided bone regeneration (GBR) membrane is intended to provide sufficient space for alveolar bone regeneration and meanwhile prevent the invasion of gingival epithelium. In this study, three-dimensional porous reduced graphene oxide/hydroxyapatite (3D rGO/HA) membrane with two different sides was prepared using a two-step electrochemical method. One side of this composite membrane facing the bone defect was formed by 3D porous rGO with HA deposited on the frame of the 3D structure, and the other side of the membrane presented a dense 2D rGO surface to prevent the invasion of the gingival epithelium. The morphology, phase composition, and physical properties of the 3D rGO/HA composite membrane were characterized. Then the cell morphology, viability, and proliferation of pre-osteoblasts (MC3T3-E1 cells) on the 3D porous structure surface of membranes were evaluated and alkaline phosphatase (ALP) secretion as an indication of osteogenic differentiation was also investigated. Meanwhile, cell morphology, viability, and proliferation of HUVEC and L929 cells on the dense structure surface were examined. Finally, a cranial defect model of rat was employed to evaluate the effect of 3D rGO/HA as a GBR membrane in vivo. The results revealed the 3D rGO/HA membrane had good biocompatibility for MC3T3-E1 and HUVEC cells and could significantly enhance ALP secretion. Furthermore, this membrane also promoted the repair of calvarial defects in vivo. These results demonstrated that 3D porous rGO/HA composite membrane with a porous side and another dense side represents great application potential as an ideal GBR membrane.

Phosphorus deficiency stimulates dominance of Cylindrospermopsis through facilitating cylindrospermopsin-induced alkaline phosphatase secretion: Integrating field and laboratory-based evidences

Potentially toxic Cylindrospermopsis raciborskii blooms are of emerging concerns, as its scale is spreading from tropical regions to high latitudes, increasing the risk of aquatic biota being exposed to cylindrospermopsin (CYN). So far, CYN-producing C. raciborskii strains have only been reported in tropical waters which are commonly phosphorus (P)-deficient, where they can dominate phytoplankton communities. However, the influence of CYN on phytoplankton communities under different P status remains unclear. In this study, we first analyzed the summer observations of 120 tropical reservoirs in Guangdong Province. The proportion of potential CYN-producers was significantly higher in P-deficient and CYN-present reservoirs than that in P-sufficient or CYN-absent ones. This suggested that in P-deficient condition, the potential CYN producers might gain more advantages by the help of CYN. Then, in laboratory experiments we found that upon P deprivation, CYN did not inhibit the cell growth of other algal cells, but significantly stimulates them to secret more alkaline phosphatase (ALP) than in P-sufficient condition. Through transcriptomics, we further revealed that under such P-deficient condition, CYN remarkably induced intracellular nitrogen allocation and protein export system by activating the PIK3/Akt-cGMP/PKG signaling pathways in Scenedesmus bijugatus, thus enhancing its ALP secretion. Our study implies that CYN-induced ALP secretion is facilitated upon P deficiency, thus supporting the dominance of its producers C. raciborskii.

In vitro Studies of Polycaprolactone Nanofibrous Scaffolds Containing Novel Gehlenite Nanoparticles.

Background:Recently, many studies have been done on the physicochemical properties and biocompatibility of polycaprolactone (PCL) scaffolds containing ceramic reinforcers in the field of bone tissue engineering. In this study, the physical, mechanical and biological properties of electrospined-fabricated PCL scaffolds containing gehlenite (GLN) nanoparticles (NPs) as a novel bioceramic were investigated.Methods:To obtain the appropriate mechanical properties, the solution contains 3%, 5%, 7%, and 10% wt. of GLN NPs were prepared. Fiber morphology was investigated by scanning electron microscopy. In order to evaluate the NPs distribution, Energy Dispersive X-Ray Spectroscopy, X-ray diffraction, and Fourier Transform Infrared Spectroscopy spectroscopy were used. The scaffold hydrophilicity was measured by the water contact angle test. The tensile test was used to check the mechanical strength of the scaffold. The proliferation of MG-63 cells was evaluated by the MTT test. Alkaline phosphatase (ALP) activity of MG-63 cells was also examined.Results:Average fibers' diameters and porosity of PCL/GLN7% were obtained 150–500 nm and 80%, respectively. An increase in the scaffold hydrophilicity was observed by the addition of GLN NPs. The strength of PCL/GLN7% was higher than the blank PCL scaffold. Cell proliferation of scaffolds containing GLN was higher than the blank PCL scaffold. A significant increase in the secretion of ALP for GLN-loaded scaffolds was seen.Discussion:The results showed that PCL/GLN7% composite scaffold could be a good candidate for bone tissue engineering.Conclusion:The overall results indicate that the scaffold (PCL /GLN7%) has suitable mechanical properties, a great cell compatibility for bone tissue regeneration.

Hydrogel-Assisted 3D Model to Investigate the Osteoinductive Potential of MC3T3-Derived Extracellular Vesicles.

Effective and rapid regeneration of bone defects often pose substantial challenges in severe accidental injuries and disabilities occurring due to diseases and/or advanced age, especially in patients having reduced tissue regeneration competence. The success of mesenchymal stromal cell (MSC)-based research strategies in improving bone regeneration was hampered not only due to the limited knowledge of therapeutic actions of MSCs but also due to difficulties as well as expenses related to cell manufacturing and time taken for ethical approvals for clinical use of living cells and engineered tissues. The recent trend indicated that there is a shift from the direct usage of MSCs toward the application of paracrine factors and extracellular vesicles (EVs) isolated from their MSC secretome in bone tissue regeneration. This shift has directed research into the development of "cell-free" therapeutics, which could be a better alternative due to its several advantages over the use of their parental MSCs. Furthermore, accumulating evidence suggested that the 3D microenvironment influences the paracrine effects of MSCs. Although the osteogenic role of EVs has been explored recently, the current study showed, for the first time, that encapsulation of EVs along with MC3T3 cells in a 3D hydrogel-assisted culture with a distinct porous microenvironment having meso and macro (0.05-200 μm) pore size distribution resulted in an improved osteogenic response in vitro. The present work was primarily focused on investigating the influence of EVs isolated under distinct priming conditions to enhance the osteogenic potential. In addition, in the current work, the osteogenic ability of different types of EVs (microvesicles and exosomes) and total EVs isolated at different time points was also examined when encapsulated with MC3T3 cells in an alginate gel. Using various biochemical assays, such as alkaline phosphatase (ALP) production and calcium secretion, it was observed that both microvesicles and exosomes collected from MC3T3 cells independently had osteogenic potential; however, their collective activity was found to be superior. We further showed that EVs induce an early osteogenic response in MC3T3 cells as indicated by ALP and calcium secretion at a much earlier time point, compared to the controls. Our data suggested that this 3D hydrogel-assisted system provides close proximity of cells and EVs, and thus, mimics the in vivo scenario, making it clinically useful for bone tissue engineering.

Composite Coating of Graphene Oxide/TiO₂ Nanotubes/HHC-36 Antibacterial Peptide Construction and an Exploration of Its Bacteriostat and Osteogenesis Effects.

Graphene oxide (GO), a kind of polymer, is often selected as a controlled released agent, whereas titanium dioxide (TiO₂) nanotubes are commonly used as a drug-coated carrier. This study was conducted to develop methods for manufacturing the GO/TiO₂/HHC-36 composite coating and exploring its bacteriostat and osteogenesis properties. The GO/TiO₂ nanotubes were prepared by electrochemical methods and HHC-36 was then adsorbed to GO/TiO₂to obtain GO/TiO₂/HHC-36. Sustained release of HHC-36 was analyzed and the antibacterial effect was examined by the inhibition zone test. The biocompatibility and osteogenesis in vitro of GO/TiO₂/HHC-36 were explored. Finally, the osteogenesic property of the composite coating was investigated in a rat femoral defect model in vivo. GO/TiO₂/HHC-36 was successfully prepared and had good controlled released performance in vitro. The inhibit zone size of S. aureus was 2.1 mm and that of E. coli was 3.0 mm. GO/TiO₂/HHC-36 showed good biocompatibility with mesenchymal stem cells (MSCs) and promoted their adhesion, migration, and differentiation. In addition, the secretion of alkaline phosphatase, collagen, mineralized matrix and osteoblast-related nutrient factors of MSCs was increased after treatment with GO/TiO₂/HHC-36. Furthermore, GO/TiO₂/HHC-36 also stimulated endotheliocytes to secrete VEGF, leading to angiogenesis. Finally, implantation of GO/TiO₂/HHC-36 in the rat femur defect model resulted in MSC migration and increased expression of osteoblast related proteins. The composite coating with controlled released of HHC-36 showed distinct antibacterial properties and promoted osteogenesis in vitro and in vivo.