Higher Alkaline Phosphatase Activity Research Articles

Nanostructured Zirconium‐Oxide Bioceramic Coatings Derived from the Anodized Al/Zr Metal Layers

AbstractHere, zirconium‐oxide ceramic coatings comprising arrays of 3D nanostructures are electrochemically synthesized, ranging in shape, size, spacing, and population density, termed as the nanomounds (ø≈65 nm), nanopillars (ø≈130 nm), and nanostumps (ø≈220 nm). The nanostructured coatings, alongside a flat ZrO2 anodic film, are explored as a potential biomaterial in experiments with Saos‐2 cells. All coatings reveal no cytotoxicity to living cells. The population density and spreading area of the cells, being the largest on the flat film, slightly decrease with increasing nanostructure dimensions. The cells progressively proliferate on all the surfaces, the nanomounds and, especially, nanopillars promoting the best viabilities and proliferation rates. The flat, nanomound, and nanopillar coatings promote the well‐defined organizations of actin filaments across the entire cell bodies with no disruption in the cytoskeletal network and the mature large dash‐shaped focal adhesions. The highest activity of alkaline phosphatase and the biggest deposition of a mineralization‐competent extracellular matrix occur on the nanopillar array, the other nanostructures showing a better result than the flat coating, though. The differences, paradoxes, and regularities in the cell responses are explained through the detailed consideration of the coating morphologies, surface chemistries, and processes at the cell/surface interfaces.

Curculigoside promotes osteogenic differentiation of ADSCs to prevent ovariectomized-induced osteoporosis

BackgroundCurculigoside is a natural phenolic glycoside compound produced by Curculigo orchioides Gaertn. This study aimed to explore the effects of curculigoside in promoting the osteogenic differentiation of adipose-derived stem cells (ADSCs) as well as the underlying mechanism.MethodsADSCs were treated with curculigoside at different concentrations (0 μmol/L, 1 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L, and 20 μmol/L), and cell viability was assessed by CCK-8 assay. Then, the alkaline phosphatase (ALP) activity was determined, and alizarin red S (ARS) staining was performed to measure the extracellular mineralization of curculigoside. Information about protein-chemical interactions is provided by the search tool for interactions of chemicals (STITCH) database. Then, LY294002 was administered to explore the mechanism by which curculigoside promotes the osteogenic differentiation of ADSCs. Western blot assays were performed to assess changes in the expression of osteogenic-related markers and the phosphorylation of PI3K and AKT. Finally, we established an ovariectomized (OVX)-induced osteoporosis mouse model and administered curculigoside to explore the effects of curculigoside in preventing bone loss in vivo.ResultsThe CCK-8 assay indicated that curculigoside did not induce cytotoxicity at a concentration of 5 μmol/L after 48 h. The ALP and ARS results revealed that the induced group had higher ALP activity and calcium deposition than the control group. Moreover, the curculigoside group exhibited increased biomineralization, ALP activity, and ARS staining compared to the induced and control groups, and these effects were partially inhibited by LY294002. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the target genes of curculigoside were mainly involved in the PI3K-Akt signaling pathway. PCR and western blot analysis showed that the expression of RUNX2, ALP, and Osterix was upregulated in curculigoside-treated ADSCs, but this effect was partially reversed by the PI3K inhibitor LY294002. Moreover, the curculigoside-treated group exhibited significantly increased phosphorylation of AKT to P-AKT compared with the osteogenic induction group. After treatment with curculigoside, the mice had a higher bone volume than the OVX mice, suggesting partial protection from cancellous bone loss. In addition, when LY294002 was added, the protective effects of curculigoside could be neutralized.ConclusionsCurculigoside could induce the osteogenic differentiation of ADSCs and prevent bone loss in an OVX model through the PI3K/Akt signaling pathway.

575 An extract of Leontopodium alpinum prolongs anagen phase in human hair follicles ex vivo

Aging and hair follicle disorders, such as telogen effluvium or androgenetic alopecia, lead to premature catagen induction and decreased hair density. Hair loss is an aesthetic problem that may cause major psychological distress in affected individuals. While severe types of hair loss are targeted by the pharmaceutical industry, with drugs exhibiting unwanted side effects, the cosmetic industry is looking for milder, and natural treatment options. Bearing this in mind, we focused on an extract of Leontopodium alpinum (Edelweiss) and investigated its effects on hair growth ex vivo. We treated microdissected anagen VI scalp hair follicles (HFs) from three individuals (two female, one male) with 0.001% edelweiss extract and evaluated hair growth-associated parameters. After 5 days of treatment, we observed significantly more HFs remaining in anagen, and increased hair matrix keratinocyte proliferation. On a molecular level, higher activity of alkaline phosphatase, and up-regulated expression of versican was observed in the dermal papilla of female treated HFs, suggesting improvement of dermal papilla inductivity. In addition, the expression of hair growth-associated growth factors, i.e. IGF-1 or FGF7, was found to be up-regulated in the outer root sheath, and/or dermal papilla of female hair follicles. Our ex vivo results provide preliminary evidence for a beneficial effect of the Leontopodium alpinum extract in limiting hair shedding in vivo.

Enrichment of common carp (Cyprinus carpio) diet with Malic acid: Effects on skin mucosal immunity, antioxidant defecne and growth performance

Abstract The present study investigated possible effects of dietary malic acid on the expression of immunity, antioxidant and growth related genes expression as well as skin mucus immune parameters in common carp. Common carp (Cyprinus carpio) fingerlings were fed diets supplemented with different levels (0 [control], 0.5%, 1%, 2%) of malic acid (MA) for 60 days. The results revealed highest expression levels of immune-related genes (tnf-alpha, il1b, il8 and lyz) in skin of common carp fed 2% MA (P < 0.05). Regarding 1% MA treatment comparison with control group, significant difference was noticed just in case of lyz (P < 0.05). Evaluation of growth related genes expression revealed no significant difference between treatments (P > 0.05). The study of antioxidant related genes (gsta and gpx) in common carp skin fed with MA, showed significant difference between treated groups and control (P < 0.05). Carps fed with 2% MA had highest alkaline phosphatase activity in skin mucus compared other treated groups and control (P < 0.05). There were no significant difference between 0.5% and 1% and control (P > 0.05). The study of total protein and total immunoglobulin (Ig) in common carp skin musus revealed no alteration following MA treatment (P > 0.05). The present data demonstrated that feeding with MA altered immune and antioxidant genes expression in skin mucus of common carp.

Naringenin promotes SDF-1/CXCR4 signaling pathway in BMSCs osteogenic differentiation.

Naringenin, a dihydro-flavonoid compound that shows chemotactic activity, may have a good application prospect in repairing bone tissue, but its specific mechanism in bone regeneration, especially the osteogenic differentiation of stem cells, needs for a further study. The aim of this study was to investigate the effect of naringenin on the osteogenic differentiation and its roles in the C-X-C chemokine receptor type 4/stromal cell-derived factor 1 (SDF-1/CXCR4) signal pathway of bone marrow-derived mesenchymal stem cells (BMSCs). BMSCs were harvested from the femurs and tibias of 4-to-6-week-old male Sprague-Dawley rats. Cell Counting kit-8 assay was used to determine cytotoxicity of naringenin. Alkaline phosphatase (ALP) activity was measured in cell's precipitates and alizarin-red staining was performed to determine the osteogenic differentiation capacity of the BMSCs. Real-time polymerase chain reaction, enzyme-linked immunosorbent assay and western blotting were adopted to determine the expression of genes and proteins. The cellular morphology was spindle-shaped, and arranged in radial and whorled patterns. The flow cytometric analysis have confirmed the presence of characteristic surface proteins in the harvested BMSCs. Different concentrations (0-200 μg/ml) of naringenin have no influence on the viability and proliferation rate of the BMSCs. The highest ALP activity was found at culture day 7 and 9 when the concentration of naringenin was 75 and 100 μg/ml. Positive red or dark red stained cells with mineralized nodules can be observed on day 14. The expression of ALP, Runt-related transcription factor 2, CXCR4 and SDF-1a at the gene and protein levels in naringenin-treated cells were significantly higher than those in the control cells. Moreover, AMD3100, an inhibitor of CXCR4, suppressed the expression of the studied genes and proteins. Naringenin does not show toxic effect on BMSCs. Naringenin promotes the expression of the SDF-1a gene and protein via the SDF-1/CXCR4 signaling pathway. A better understanding of the mechanisms of naringenin action would be helpful for developing specific therapeutic strategies to improve bone regeneration after injuries.

Osteogenic capability of strontium and icariin-loaded TiO2 nanotube coatings in vitro and in osteoporotic rats.

Titanium (Ti) and Ti alloys are widely used biomaterials, but they lack osteogenic capability for rapid bone integration. To improve osseointegration of Ti implants, TiO2 nanotubes were prepared using the anodizing oxidation technique, and strontium (Sr) combined with icariin (ICA) was loaded on TiO2 nanotube coatings. Cell adhesion and proliferation of MC3T3-E1 cells, alkaline phosphatase (ALP) activity, mineralization of extracellular matrix, and bone formation around titanium implants in ovariectomized rats, were examined separately. The results showed that compared with pure Ti, TiO2 and Sr-loaded TiO2 coatings, the coatings loaded with both Sr and ICA showed better effect on cell adhesion and proliferation, higher ALP activity and more red-stained mineralized nodules. Furthermore, more bone was formed around implants loaded with both Sr and ICA in osteoporotic rats. Therefore, coating with Sr and ICA is valuable for clinical application to strengthen the osseointegration of titanium implants, especially in osteoporotic patients.

The effect of Ag substitution on physicochemical and biological properties of sol-gel derived 60%SiO2–31%CaO–4%P2O5–5%Li2O (mol%) quaternary bioactive glass

In the present study, bioactive glasses (BGs) in 60SiO2-(31-x) CaO–4P2O5–5Li2O-xAg2O systems (x = 0, 1, 5, and 10 mol %) with optimum fixed amount of Li2O (5 mol %) were successfully synthesized through sol-gel technique and the effects of Ag2O addition on in-vitro investigations, biological properties, and bactericidal efficiency were evaluated. Structural and morphological evaluations by X-ray diffraction (XRD), Fourier transform infrared (FTIR), transmission electron microscopy (TEM) as well as scanning electron microscopy (SEM) revealed that BG with chemical composition of 60SiO2–30CaO–4P2O5–5Li2O–1Ag2O (mol%) had a higher rate of hydroxyapatite (HA) formation than other synthesized specimens. While, BGs containing 5 and 10 mol% of Ag had a lower bioactivity because of the reduced dissolution rate of Si and Ca. The results obtained from the 3-(4,5dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and evaluation of alkaline phosphatase (ALP) activity exhibited that proliferation and differentiation of MC3T3-E1 osteoblast-like cells and osteoblastic activity were intensified by the presence of Ag in the BGs҆ composition. Meanwhile, BGs containing 1 mol% of Ag not only had no toxic effect on the cells, but also caused a significant enhancement in proliferation and differentiation of the cells within 7 days (**p < 0.01), as well as the highest ALP activity in this specimen (*p < 0.05). Additionally, all the synthesized specimens had significant antibacterial efficiency against E-coli bacteria. Our results showed that addition of 1 mol% of Ag to BG containing Li led to a significant antibacterial efficiency compared to the control (*p < 0.05). Afterwards, DAPI/actin and live/dead cell staining methods were applied, which well confirmed results of MTT assay. Finally, BG with chemical composition (LA01) of 60SiO2–30CaO–4P2O5–5Li2O–1Ag2O was introduced as an optimal novel co-doped BG in biomedical applications due to causing higher differentiation and proliferation of MC3T3-E1 cells and more increase in ALP activity and bactericidal efficiency.

Polycaprolactone-Based Scaffolds Facilitates Osteogenic Differentiation of Human Adipose-Derived Stem Cells in a Co-Culture System.

(1) Background: Stem cells in combination with scaffolds and bioactive molecules have made significant contributions to the regeneration of damaged bone tissues. A co-culture system can be effective in enhancing the proliferation rate and osteogenic differentiation of the stem cells. Hence, the aim of this study was to investigate the osteogenic differentiation of human adipose derived stem cells when co-cultured with human osteoblasts and seeded on polycaprolactone (PCL):hydroxyapatite (HA) scaffold; (2) Methods: Human adipose-derived stem cells (ASC) and human osteoblasts (HOB) were seeded in three different ratios of 1:2, 1:2 and 2:1 in the PCL-HA scaffolds. The osteogenic differentiation ability was evaluated based on cell morphology, proliferation rate, alkaline phosphatase (ALP) activity, calcium deposition and osteogenic genes expression levels using quantitative RT-PCR; (3) Results: The co-cultured of ASC/HOB in ratio 2:1 seeded on the PCL-HA scaffolds showed the most positive osteogenic differentiation as compared to other groups, which resulted in higher ALP activity, calcium deposition and osteogenic genes expression, particularly Runx, ALP and BSP. These genes indicate that the co-cultured ASC/HOB seeded on PCL-HA was at the early stage of osteogenic development; (4) Conclusions: The combination of co-culture system (ASC/HOB) and PCL-HA scaffolds promote osteogenic differentiation and early bone formation.

Alkaline phosphatase activity during a phosphate replete dinoflagellate bloom caused by Prorocentrum obtusidens

Prorocentrum obtusidens Schiller (formerly P. donghaiense Lu), a harmful algal species common in the East China Sea (ECS), often thrives with the depletion of phosphate. Three cruises in the spring of 2013 sampled an entire P. obtusidens bloom process to investigate the dynamics of alkaline phosphatase activity (APA) and phosphorus (P) status of the bloom species using both bulk and cell-specific assays. Unlike previous studies, the bloom of P. obtusidens occurred in a phosphate replete environment. Very high APA, with an average of 76.62 ± 90.24 nmol L–1 h–1, was observed during the early-bloom phase, a value comparable to that in low phosphate environments. The alkaline phosphatase (AP) hydrolytic kinetics also suggested a more efficient AP system with a lower half-saturation constant (Ks), but higher maximum potential hydrolytic velocity (Vmax) in this period. The APA decreased significantly with an average of 24.98 ± 30.98 nmol L–1 h–1 when the bloom reached its peak. The lack of a correlation between dissolved inorganic phosphate (DIP) or dissolved organic phosphate (DOP) concentration and APA suggested that the APA was regulated by the internal P growth demand, rather than the external P availability during the phosphate replete P. obtusidens bloom. These findings facilitate an understanding of the P. obtusidens acclimation strategy with respect to P variations in terms of AP expression during blooms in the ECS.

Feeding history affects the crabseed quality and subsequent culture performance of juvenile Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853 (Brachyura, Varunidae)

Abstract A 6-month feeding trial was conducted to evaluate the hepatopancreas colour, proximate composition, antioxidant capacity, pathogen resistance and growth performance of juvenile Chinese mitten crab Eriocheir sinensis H. Milne Edwards, 1853 fed either formulated diets (FD), traditional diets (TD) or a mixture feeding of traditional diets and frozen trash fish (TF). The results showed that: (1) The hepatopancreas redness (a*) in the FD treatment was significantly higher than that in the other two treatments (). (2) The significantly higher activities of alkaline phosphatase (AKP), acid phosphatase (ACP) and superoxide dismutase (SOD) were observed in the hepatopancreas in the FD treatment, while a significantly higher content of malondialdehyde (MDA) was observed in the haemolymph in the TF treatment (). (3) FD and TD treatment, respectively, had the lowest and the highest mortality after challenging with Aeromonas hydrophila. (4) Females in the FD treatment had a significantly shorter intermoult period in the indoor culture experiment (), no significant difference was observed in weight gain rate, specific growth rate, or survival. In conclusion, formulated diets could enhance the crabseed quality of E. sinensis compared to traditional diets or trash fish.

Effect of Diphosphate on Proliferation of Osteoblasts in Mice

In order to investigate the effect of diphosphate on the proliferation and differentiation of mouse osteoblasts and to further explore the optimal concentration of diphosphate, mouse embryo osteoblasts precursor cells are used as the research object. The expression level of its messenger ribonucleic acid is detected by real-time quantitative polymerase chain reaction (real-time polymerase chain reaction). 3- (4, 5) - dimethylahiazo (-z-yl) -3, 5-di-phenytetrazolium bromide method is used to detect the proliferation of mouse embryo osteoblasts precursor cells and its effect on alkaline phosphatase activity, so as to explore its correlation. The results show that mouse embryo osteoblasts precursor cells have the greatest value-added effect, the most significant gene expression and the highest alkaline phosphatase activity at the concentration of 10-8 M diphosphate. The results are as expected. More accurate, standardized and quantifiable data for mouse embryo osteoblasts precursor cell proliferation are obtained through experiments. There are still some shortcomings in the research process, but the results of this study still provide some guidance for exploring more precise mechanism of action in the future. Therefore, the results of this study provide guidance for exploring more precise mechanisms in the future, which is the value of this study.

Study on proanthocyanidins crosslinked collagen membrane for guided bone tissue regeneration.

The goal of this study is to understand the ability of a newly developed barrier membrane to enhance bone tissue regeneration. Here in this study we present the in vitro characterization of the barrier membrane made from type I collagen and crosslinked by oligomeric proanthocyanidins (OPCs). The effects of the membrane (P-C film) on cell cycle, proliferation, alkaline phosphatase activity, and mineralization were evaluated using the human osteoblast cell line MG-63, while the barrier ability was examined using MG-63 cells, as well as the human skin fibroblast cell line WS-1. The pore size is one of the factors that plays a key role in tissue regeneration, therefore, we evaluated the pore size of the membrane using a capillary flow porometer. Our results showed that the mean pore size of the P-C film was approximately 7-9 µm, the size known to inhibit cell migration across the membrane. The P-C film also demonstrated excellent cell viability and good biocompatibility, since the cell number increased with time, with MG-63 cells proliferating faster on the P-C film than in the cell culture flask. Furthermore, the P-C film promoted osteoblast differentiation, resulting in higher alkaline phosphatase activity and mineralization. Therefore, our results suggest that this P-C film has a great potential to be used in guided bone regeneration during periodontal regeneration and bone tissue engineering.

Biological evaluation of the modified nano-amorphous phosphate calcium doped with citrate/poly-amino acid composite as a potential candidate for bone repair and reconstruction

Large numbers of research works related to fabricating organic–inorganic composite materials have been carried out to mimic the natural structure of bone. In this study, a new modified n-ACP doped with citrate (n-ACP-cit)/poly (amino acids) (PAA) composite (n-ACP-cit/PAA) was synthesized by employing high bioactive n-ACP-cit and the biodegradable and biocompatible PAA copolymer. Its basic structure was characterized by X-ray diffraction spectroscopy, Fourier transformed infrared spectroscopy, and X-ray photoelectron spectroscopy. Moreover, the degradability, bioactivity, biocompatibility, and osteoconductivity of n-ACP-cit/PAA composite were evaluated in vitro and in vivo, using simulated body fluid (SBF) solution soaking test, mouse bone marrow mesenchymal stem cells proliferation and differentiation, morphological observation test, expression of genes associated with osteogenesis, and bone defect model repair test, respectively. The modified n-ACP-cit/PAA composite exhibited a much higher weight loss rate (36.01 wt.%) than that of PAA (23.99 wt.%) after immersing in SBF solution for 16 weeks and the pH values of local environment restored to neutral condition. Moreover, cells co-culturing with composites exhibited higher alkaline phosphatase activity, more calcium nodule-formation, and higher expression levels of osteogenic differentiation-related genes (Bmp-2, Colla I, OCN, OPN, and Runx-2) than that of PAA. Furthermore, the bone defect model repair test revealed that the composite could be intimately incorporated with the surrounding bone without causing any deleterious reaction and capable of guiding new bone formation. Together, these results indicated that the new modified bone repair n-ACP-cit/PAA composite material with specific characteristics may be designed for meeting diverse requirements from biomedical applications.

OSTEOPLASTIC PROPERTIES OF MULTIPOTENT MESENCHYMAL STROMAL CELLS OF ADIPOSE TISSUE

The aim: Determining the ability of samples based on MMSC – AT differentiating in the osteogenic direction. Materials and methods: The study was conducting at Bukovinian State Medical University, Chernivtsi, Ukraine. Adipose tissue samples were obtaining from the neck of 60 experimental animals (white Wistar rats). Multipotent mesenchymal stromal cells of adipose tissue were obtained by grinding adipose tissue of rats in 0.1% collagenase 1A . Alkaline phosphatase activity was assessing by using the Alkaline Phosphatase Detection Kit (Sigma, USA) according to the manufacturer’s protocol. Osteopontin gene expression was determining by immunocytochemical method. To determine the mRNA used the PCR method, which is associated with reverse transcription (RT-PCR) in the area of quantification of gene expression to the marker BGP. Results: On the 21st day of observations, the expression of mRNA encoding the BGP gene decreased in samples № 1 and № 3 to 35,800 ± 420.0 copies and to 35,000 ± 400.0 copies, p1&lt;0.01, p&gt;0.05. Also was observing growth of copies of the BGP gene in samples № 2 and № 4 in 2.1, р&lt;0.01 and 2.2 times, р-р2&lt;0.05, relative to the data in sample № 1. Conclusions: Comparative study of osteoplastic properties samples MMSC-AT showed that a larger number of cells differentiate into the osteoblasts in samples containing MMSC-AT + PRP (№ 2) and MMSC-AT + PRP + «Kolapan» (№ 4). This has been proven higher alkaline phosphatase activity, higher levels osteopontin expression, and higher levels BGP gene expression.

Electrical Stimulation of Adipose-Derived Stem Cells in 3D Nanofibrillar Cellulose Increases Their Osteogenic Potential.

Due to the ageing population, there is a steadily increasing incidence of osteoporosis and osteoporotic fractures. As conventional pharmacological therapy options for osteoporosis are often associated with severe side effects, bone grafts are still considered the clinical gold standard. However, the availability of viable, autologous bone grafts is limited making alternative cell-based strategies a promising therapeutic alternative. Adipose-derived stem cells (ASCs) are a readily available population of mesenchymal stem/stromal cells (MSCs) that can be isolated within minimally invasive surgery. This ease of availability and their ability to undergo osteogenic differentiation makes ASCs promising candidates for cell-based therapies for bone fractures. Recent studies have suggested that both exposure to electrical fields and cultivation in 3D can positively affect osteogenic potential of MSCs. To elucidate the osteoinductive potential of a combination of these biophysical cues on ASCs, cells were embedded within anionic nanofibrillar cellulose (aNFC) hydrogels and exposed to electrical stimulation (ES) for up to 21 days. ES was applied to ASCs in 2D and 3D at a voltage of 0.1 V/cm with a duration of 0.04 ms, and a frequency of 10 Hz for 30 min per day. Exposure of ASCs to ES in 3D resulted in high alkaline phosphatase (ALP) activity and in an increased mineralisation evidenced by Alizarin Red S staining. Moreover, ES in 3D aNFC led to an increased expression of the osteogenic markers osteopontin and osteocalcin and a rearrangement and alignment of the actin cytoskeleton. Taken together, our data suggest that a combination of ES with 3D cell culture can increase the osteogenic potential of ASCs. Thus, exposure of ASCs to these biophysical cues might improve the clinical outcomes of regenerative therapies in treatment of osteoporotic fractures.

Lentivirus-mediated silencing of P75 neurotrophin receptor combined with nerve growth factor overexpression and transfection of bone marrow mesenchymal stem cells combined with demineralized bone matrix for heterotopic osteogenesis

To investigate the effects of silencing P75 neurotrophin receptor (P75NTR) and nerve growth factor (NGF) overexpression on the proliferative activity and ectopic osteogenesis ability of bone marrow mesenchymal stem cells (BMSCs) combined with demineralized bone matrix for heterotopic osteogenesis. BMSCs of Sprague Dawley (SD) rats were cultured and passaged by adherent isolation method. The third generation BMSCs were transfected with lentivirus mediated P75NTR gene silencing (group B), NGF overexpression gene (group C), P75NTR silencing and NGF overexpression double genes (group D), respectively, and untransfected cells as control (group A). After 7 days of transfection, the expression of fluorescent protein of the target gene was observed by fluorescence microscope; cell counting kit 8 method was used to detect the cells activity for 8 days after transfection; the expressions of P75NTR and NGF proteins in each group were detected by Western blot. The adhesion of BMSCs to demineralized bone matrix (DBM) was observed by inverted phase contrast microscope and scanning electron microscope after transfection of p75NTR silencing and NGF overexpression double genes. After transfection, BMSCs and DBM were co-cultured to prepare 4 groups of tissue engineered bone, which were respectively placed in the dorsal subcutaneous tissue of 8-week-old SD rats to construct subcutaneous ectopic osteogenesis model ( n=6). HE staining was performed at 4 and 8 weeks after operation. ALP staining was used to observe the formation of calcium nodules at 8 weeks after operation. The expressions of Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were detected by real-time fluorescent quantitative PCR. At 7 days after transfection, there was no fluorescence expression in group A, red fluorescence expression was seen in group B, green fluorescence expression in group C, and red-green compound fluorescence expression in group D. The fluorescence expression rate of target gene was about 70%. Western blot detection showed that the relative expression of P75NTR protein in groups A and C was significantly higher than that in groups B and D, and the relative expression of NGF protein in groups C and D was significantly higher than that in groups A and B ( P<0.05). With the passage of time, the cell proliferation activity increased in all groups, especially in group D, which was significantly higher than that in group A at 3-8 days ( P<0.05). The results of inverted phase contrast microscope and scanning electron microscope showed that BMSCs could adhere well to DBM. In the subcutaneous ectopic osteogenesis experiment, HE staining showed that at 4 and 8 weeks after operation, the more bone tissue was formed in group D than in the other 3 groups. ALP staining showed that group D had the highest ALP activity and better osteogenic expression. Compared with group A, the relative expressions of Runx2, ALP, and OCN mRNAs in group D were significantly higher than those in group A ( P<0.05). Silencing P75NTR and NGF overexpression double genes co-transfected BMSCs with DBM to construct tissue engineered bone has good ectopic osteogenic ability. By increasing NGF level and closing P75NTR apoptosis channel, it can not only improve cell activity, but also promote bone tissue regeneration.

Activity profile of innate immune-related enzymes and bactericidal of freshwater fish epidermal mucus extract at different pH

The epidermal mucus of fish performs diverse functions from prevention of mechanical abrasion to limit pathogen invasions. The current experiment was designed to extract skin mucus proteins of three freshwater fish, i.e. common carp (Cyprinus carpio), mrigal (Cirrhinus mrigala) and rohu (Labeo rohita) with organic solvent (methanol) and dissolve in different pH of Tris-HCl buffers to examine the significance of pH in the solubilisation of skin mucus proteins. The protein profiles of different pH solubilised methanol fish skin mucus extracts were determined by SDS-PAGE. The non-specific immune enzymes, alkaline phosphatase, lysozyme and protease of fish skin mucus were compared and this present study demonstrated that these enzymes differed in their activity depending on pH buffers. The higher lysozyme and protease activity were observed at the pH of 8.0 and higher alkaline phosphatase activity in the pH 9.0 of C. mrigala fish skin mucus methanol extract. In addition, the bactericidal activity was evaluated against the pathogens Proteus vulgaris and Pseudomonas aeruginosa. The pH 8.0 of C. mrigala skin mucus extract revealed better bactericidal activity than other fish species mucus pH buffers against both P. vulgaris and P. aeruginosa. In the case of protein profile from SDS-PAGE, based on pH buffers and the solubilisation of proteins, differences in the resolution of bands were observed. The higher alkaline pH of 9.0 showed smeared gel bands in all the three fish skin mucus methanol extract. The present study suggests that methanol extracted C. mrigala fish skin mucus at pH 8.0 showed better innate immune enzymes and bactericidal activity. The additional examinations of C. mrigala skin mucus methanol extract in this pH aids in identifying novel bioactive molecules. This is the study of proteome of three fish species skin mucus in the effect of pH. Further analyses are required to evaluate proteins present in fish skin mucus extracted with methanol and the influence of pH on protein solubility. These findings could be helpful in exploring natural alternatives to antibiotics in aquaculture industry against infectious pathogens.

3D printing of PEEK–cHAp scaffold for medical bone implant

The major drawback associated with PEEK implants is their biologically inert surface, which caused unsatisfactory cellular response and poor adhesion between the implants and surrounding soft tissues against proper bone growth. In this study, polyetheretherketone (PEEK) was incorporated with calcium hydroxyapatite (cHAp) to fabricate a PEEK–cHAp biocomposite, using the fused deposition modeling (FDM) method and a surface treatment strategy to create microporous architectures onto the filaments of PEEK lattice scaffold. Also, nanostructure and morphological tests of the PEEK–cHAp biocomposite were modeled and analyzed on the FDM-printed PEEK–cHAp biocomposite sample to evaluate its mechanical and thermal strengths as well as in vitro cytotoxicity via a scanning electron microscope (SEM). A technique was used innovatively to create and investigate the porous nanostructure of the PEEK with controlled pore size and distribution to promote cell penetration and biological integration of the PEEK–cHAp into the tissue. In vivo tests demonstrated that the surface-treated micropores facilitated the adhesion of newly regenerated soft tissues to form tight implant–tissue interfacial bonding between the cHAp and PEEK. The results of the cell culture depicted that PEEK–cHAp exhibited better cell proliferation attachment spreading and higher alkaline phosphatase activity than PEEK alone. Apatite islands formed on the PEEK–cHAp composite after immersion in simulated body fluid of Dulbecco's modified Eagle medium (DMEM) for 14 days and grew continuously with more or extended periods. The microstructure treatment of the crystallinity of PEEK was comparatively and significantly different from the PEEK–cHAp sample, indicating a better treatment of PEEK–cHAp. The in vitro results obtained from the PEEK–cHAp biocomposite material showed its biodegradability and performance suitability for bone implants. This study has potential applications in the field of biomedical engineering to strengthen the conceptual knowledge of FDM and medical implants fabricated from PEEK–cHAp biocomposite materials.

Effects of Plasma Treatment on the Bioactivity of Alkali-Treated Ceria-Stabilised Zirconia/Alumina Nanocomposite (NANOZR).

Zirconia ceramics such as ceria-stabilized zirconia/alumina nanocomposites (nano-ZR) are applied as implant materials due to their excellent mechanical properties. However, surface treatment is required to obtain sufficient biocompatibility. In the present study, we explored the material surface functionalization and assessed the initial adhesion of rat bone marrow mesenchymal stem cells, their osteogenic differentiation, and production of hard tissue, on plasma-treated alkali-modified nano-ZR. Superhydrophilicity was observed on the plasma-treated surface of alkali-treated nano-ZR along with hydroxide formation and reduced surface carbon. A decreased contact angle was also observed as nano-ZR attained an appropriate wettability index. Treated samples showed higher in vitro bovine serum albumin (BSA) adsorption, initial adhesion of bone marrow and endothelial vascular cells, high alkaline phosphatase activity, and increased expression of bone differentiation-related factors. Furthermore, the in vivo performance of treated nano-ZR was evaluated by implantation in the femur of male Sprague–Dawley rats. The results showed that the amount of bone formed after the plasma treatment of alkali-modified nano-ZR was higher than that of untreated nano-ZR. Thus, induction of superhydrophilicity in nano-ZR via atmospheric pressure plasma treatment affects bone marrow and vascular cell adhesion and promotes bone formation without altering other surface properties.

Antioxidant activity and calcium bioaccessibility of Moringa oleifera leaf hydrolysate, as a potential calcium supplement in food.

Moringa oleifera leaf (ML) is rich in vitamins and minerals, specially abundant calcium, therefore it is widely used as a calcium supplement for food. This study aimed to investigate the antioxidant activity and calcium bioaccessibility of M. oleifera leaf hydrolysate (MLH) as a calcium supplement for kimchi. MLH was prepared under three different proteases, two different protease contents, and three different incubation times. Total phenol content (TPC), total flavonoid content (TFC), and antioxidant activities were investigated. Cellular activity and calcium bioaccessibility were also investigated. The highest calcium level of MLH was observed in 3% Protamex treatment for 4h. TPC, TFC, and antioxidant activities of MLH in Protamex and Alcalase treatments were higher than those in Flavourzyme treatment (p < 0.05). Moreover, high cell viability and alkaline phosphatase activity were also observed in C2C12 cells. Kimchi containing MLH showed high calcium accessibility compared to kimchi alone. Taken together, the application of MLH could have potential as a calcium supplement for kimchi production.