Enhancement of the chondrogenic differentiation capacity of human dental pulp stem cells via chondroitin sulfate-coated polycaprolactone-MWCNT nanofibers.

Most of the conditions involving cartilaginous tissues are irreversible and involve degenerative processes. The aim of the present study was to fabricate a biocompatible fibrous and film scaffolds using electrospinning and casting techniques to induce chondrogenic differentiation for possible application in cartilaginous tissue regeneration. Polycaprolactone (PCL) electrospun nanofibrous scaffolds and PCL film were fabricated and incorporated with multi-walled carbon nanotubes (MWCNTs). Thereafter, coating of chondroitin sulfate (CS) on the fibrous and film structures was applied to promote chondrogenic differentiation of human dental pulp stem cells (hDPSCs). First, the morphology, hydrophilicity and mechanical properties of the scaffolds were characterized by scanning electron microscopy (SEM), spectroscopic characterization, water contact angle measurements and tensile strength testing. Subsequently, the effects of the fabricated scaffolds on stimulating the proliferation of human dental pulp stem cells (hDPSCs) and inducing their chondrogenic differentiation were evaluated via electron microscopy, flow cytometry and RT‒PCR. The results of the study demonstrated that the different forms of the fabricated PCL-MWCNTs scaffolds analyzed demonstrated biocompatibility. The nanofilm structures demonstrated a higher rate of cellular proliferation, while the nanofibrous architecture of the scaffolds supported the cellular attachment and differentiation capacity of hDPSCs and was further enhanced with CS addition. In conclusion, the results of the present investigation highlighted the significance of this combination of parameters on the viability, proliferation and chondrogenic differentiation capacity of hDPSCs seeded on PCL-MWCNT scaffolds. This approach may be applied when designing PCL-based scaffolds for future cell-based therapeutic approaches developed for chondrogenic diseases.

Evaluation of incipient enamel-carious-like lesion treated with hydroxyapatite-chitosan nanocomposite hydrogel.

BACKGROUND: Non-invasive restoration of tooth enamel is a topic of high relevance in dental material science. Multiple approaches have been proposed to reach optimum reconstruction results. The current study was performed to evaluate the cross-sectional microhardness besides mineral quality and content in artificially induced carious enamel after treatment with hydroxyapatite-chitosan (HAp-CS) nanocomposite gel. METHODS: Artificially carious lesions were induced by immersion of teeth in acidic carboxymethyl cellulose gel (pH 4.95-5) for 24- and 72-hours periods. Two different compositions of HAp-CS nanocomposite hydrogel were prepared with two different ratios 50/50 (%) and 70/30 (%), respectively. Additionally, sodium fluoride gel (1000 ppm concentration) was prepared and used as reference. Gels were applied to carious lesions twice/day for 3 min/each. After 45 days of application, surface morphology, energy dispersive x-ray spectroscopy, micro-Raman analysis in addition to cross-sectional microhardness were evaluated. Statistical analysis was performed using two-way ANOVA and Tukey's post hoc statistical tests. RESULTS: Surface morphological evaluation of treated surfaces showed obliteration of surface irregularities. Groups demineralized for 24 hours and treated with 70/30 (HAp-CS) showed highest significant cross-sectional-microhardness (P ≤ 0.05). Evaluated subsurface cross-sectional microhardness showed better mineral quality for groups demineralized for 24 hours and treated with HAp-CS nanocomposite gels. CONCLUSIONS: Nanocomposite gel with 70/30 (HAp-CS) could efficiently improve cross-sectional microhardness and both minerals composition and quality for lesions demineralized for 24 hours. More severely induced lesions, as demineralized for 72 hours, need more powerful agent compositions and/or prolonged application protocols for improvement.

Estradiol and zinc-doped nano hydroxyapatite as therapeutic agents in the prevention of osteoporosis; oxidative stress status, inflammation, bone turnover, bone mineral density, and histological alterations in ovariectomized rats.

Osteoporosis (OP) is a serious health problem, and the most popular therapeutic strategy for OP is hormone replacement (estrogen); however, it increases the risk of reproductive cancers. Hydroxyapatite (HA) nanoparticles have a similar chemical structure to the bone mineral component and can be used as a new remedy for OP. This study was designed to investigate the osteoporosis-protective potential of nano zinc hydroxyapatite (ZnHA-NPs) and/or estradiol (E2) combined therapy. A total of 35 adult female rats were assigned into five groups (n = 7): 1) control group; 2) ovariectomized group (OVX); 3) OVX received oral estradiol replacement therapy (OVX/E2); 4) OVX received ZnHA replacement therapy (OVX/ZnHA); and 5) OVX received both estradiol and ZnHA-NPs combined therapy (OVX/E2+ZnHA). After 3 months of treatment, serum bone markers and estrogen level, oxidative/antioxidant, and inflammatory cytokines were determined. Additionally, femoral expression of estrogen receptors alpha and beta (ESR1; ESR2), receptor activator of nuclear factor-kappa B (RANKL) ligand, osteoprotegerin (OPG), bone mineral density (BMD), histological alterations, and immunohistochemical expression of vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) were assessed. ALP, PINP, Ca, and P concentrations improved significantly (p < 0.05) in all treatment groups, especially in the OVX/E + ZnHA group. MDA and NO were higher in OVX rats, while SOD activity and GSH were lower (p < 0.05). E2 alone or with ZnHA-NPs restored the estimated antioxidant molecules and cytokines toward normal levels in OVX rats (p < 0.05). On the other hand, E2 and ZnHA increased OPG and OC expression in femurs while decreasing ESR1, ESR2, and NF-kB expression (p < 0.05). The combination treatment was superior in the restoration of normal femoral histoarchitecture and both cortical and trabecular BMD (p < 0.05). Overall, the combined therapy of OVX/E2+ZnHA was more effective than the individual treatments in attenuating excessive bone turnover and preventing osteoporosis.

Combating Bacterial Biofilm Formation in Urinary Catheter by Green Silver Nanoparticle.

Urinary catheters are commonly associated with urinary tract infections. This study aims to inhibit bacterial colonisation and biofilm of urinary tract catheters. Silicon catheter pieces were varnished with green silver nanoparticles (AgNPs) using Pistacia lentiscus mastic to prevent bacterial colonisation. Pomegranate rind extract was used to synthesize AgNPs. AgNPs were characterized by UV-Vis spectroscopy, X-ray crystallography, and transmission electron microscopy (TEM). Results obtained revealed that the size of most AgNPs ranged between 15-25 nm and they took crystallised metal and oxidised forms. The amounts of released silver ions from 1 cm pieces of catheters coated with AgNPs were estimated for five days and ranged between 10.82 and 4.8 µg. AgNPs coated catheters significantly inhibited the colonisation of catheters by antibiotic-resistant clinical Gram-positive (Staphylococcus epidermidis and Staphylococcus aureus) and Gram-negative (Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa) bacteria. AgNPs-varnish was more active against Gram-negative bacteria than Gram-positive bacteria. The significant inhibitory effect of coated catheters lasted for 72 h for both Gram-positive and Gram-negative bacteria. Varnishing catheters with AgNPs may help to prevent bacterial colonisation and infections.

Bond strength of demineralized dentin after synthesized collagen/hydroxyapatite nanocomposite application.

Treatment the deeper and remineralizable carious zone (DRCZ) in dentin with various remineralizing methods, either with classic top-down or biomimetic bottom-up remineralization approaches, has remained a constant main issue to enhance dentin substrate bonding quality. The concern of remineralizing the remaining, partially demineralized and physiologically re-mineralizable collagen fibrils was the optimum target. However, applying already mineralized type I collage fibrils which have the ability to chemically cross-link with remaining collagen and minerals did not gain much interest. Synthesis of collagen/hydroxyapatite (Col/Hap) nanocomposite was done with self-assembling Hap in situ onto Col fibrils with different % (70/30, 50/50, 30/70% of Col/Hap, respectively). Micro-tensile bond strength (µTBS) was evaluated after pre-treatment of artificially demineralized dentin with these suggested protocols [nanocomposite together with grape seed extract (GSE; 6.5%) cross-linker for two periods, 10min and 1 h] then applying self-adhesive bonding system. Applied Col/Hap (30/70%) together with GSE (6.5%) gave the significantly highest µTBS (25.04 ± 5.47 and 25.53 ± 7.64 MPa, for 10min and 1 h application times, respectively). After thermocycling for 10,000 cycles at 5 and 55 °C, µTBS for all protocols and both application times substantially decreased especially for the two control groups. Using the suggested dentin pre-treatment protocols, in chair-side, may possibly enhance the bond strength to DRCZ and its durability.

Actinomycete strain type determines the monodispersity and antibacterial properties of biogenically synthesized silver nanoparticles.

BACKGROUND: Bio-nanotechnology is considered as one of the low-cost approaches that have been utilized in production of nanomaterials. The current research aimed at investigating the influence of different types of Actinomycete strains on the final properties of silver nanoparticles (AgNPs) such as size, shape, polydispersity, and antibacterial properties. For this purpose, the following techniques were employed UV spectrophotometer, SDS-PAGE electrophoresis, TEM, FTIR, antibacterial agar diffusion test, and Zetasizer. RESULTS: It was found that among 34 Streptomyces isolates collected from the soil, Streptomyces spiralis and Streptomyces rochei were able to reduce silver nitrate into sliver nanoparticles. The diversity and molecular weights of extracellular proteins secreted by these stains were different as proved by SDS-PAGE technique. This consequently resulted in differences in polydispersity of AgNPs which indicate that the sizes of AgNPs were highly dependent on the amount, molecular sizes, and diversity of extracellular matrix proteins of the microorganism. CONCLUSION: This article might give an insight about the importance of molecular sizes of biomacromolecules such as proteins on the physical properties of biogenic synthesized nanoparticles.

A synthetic biology approach for the fabrication of functional (fluorescent magnetic) bioorganic-inorganic hybrid materials in sponge primmorphs.

During evolution, sponges (Porifera) have honed the genetic toolbox and biosynthetic mechanisms for the fabrication of siliceous skeletal components (spicules). Spicules carry a protein scaffold embedded within biogenic silica (biosilica) and feature an amazing range of optical, structural, and mechanical properties. Thus, it is tempting to explore the low-energy synthetic pathways of spiculogenesis for the fabrication of innovative hybrid materials. In this synthetic biology approach, the uptake of multifunctional nonbiogenic nanoparticles (fluorescent, superparamagnetic) by spicule-forming cells of bioreactor-cultivated sponge primmorphs provides access to spiculogenesis. The ingested nanoparticles were detected within intracellular vesicles resembling silicasomes (silica-rich cellular compartments) and as cytosolic clusters where they lent primmorphs fluorescent/magnetic properties. During spiculogenesis, the nanoparticles initially formed an incomplete layer around juvenile, intracellular spicules. In the mature, extracellular spicules the nanoparticles were densely arranged as a surface layer that rendered the resulting composite fluorescent and magnetic. By branching off the conventional route of solid-state materials synthesis under harsh conditions, a new pathway has been opened to a versatile platform that allows adding functionalities to growing spicules as templates in living cells, using nonbiogenic nanoscale building blocks with multiple functionalities. The magnet-assisted alignment renders this composite with its fluorescent/magnetic properties potentially suitable for application in biooptoelectronics and microelectronics (e.g., microscale on-chip waveguides for applications of optical detection and sensing).

Silver nanoparticles stimulate osteogenesis of human mesenchymal stem cells through activation of autophagy.

Aim: Previously, different results have been achieved regarding effects of silver nanoparticles (Ag NPs) on osteogenesis of stem cells and the mechanisms have not been disclosed yet, which are quite important for potential application of Ag NPs in bone reconstruction. Materials & methods: Effects of Ag NPs on osteogenesis of human mesenchymal stem cells (hMSCs) with underlying mechanisms were investigated. Results: Ag NPs at 2.5 and 5 µg/ml increased osteogenic proteins expression and mineralization of hMSCs. Meanwhile, autophagy was activated by Ag NPs and it could be inhibited by 3-methyladenine. Furthermore, osteogenesis induced by Ag NPs could also be reversed by 3-methyladenine. Conclusion: These findings suggest that autophagy is involved in stimulating osteogenesis of hMSCs induced by Ag NPs.

Effect of Biotitania and Titania Addition on Bioactivity and Antibacterial Properties of Calcium Silicate Cement.

Introduction: Nanoparticles are gaining more interest in dentistry for their antimicrobial, physical as well as other properties. This study aimed to evaluate the effect of adding two types of nanoparticles (NPs) on calcium silicate hydraulic cement's (CSHC) unique bioactivity and antibacterial properties. Methods and Materials: Biotitania/AgCl NPs were synthetized and characterized for its morphology, types of formed functional groups and crystalline AgCl using field emission scanning electron microscope (FE-SEM) equipped with energy-dispersive X-ray spectroscopy (EDS), X-ray diffractometer (XRD), Fourier transformation infrared spectroscopy (FT-IR) and thermo-gravimetric analysis (TGA). The former NPs and commercial titania (TiO2) NPs were added (0.5, 1.5 and 3-weight %) to commercial CSHS powder. A total of 140 disk-shaped specimens (10 mm×1 mm) were prepared (seven material groups per each test in addition to the eighth cell control group) to evaluate cell viability and alkaline phosphatase activity (ALP) after 3 and 12 days, respectively. All were incubated with mesenchymal stem cells. Antibacterial efficacy against Streptococcus mutans (S. mutans) was evaluated through the bacterial growth curve slopes while being in direct contact with the tested material groups for 18 h. One-way analysis of variance (ANOVA) and post hoc Tukey's tests were used to analyze the obtained data. Results: Addition of all NPs percentages had no significant effect (P 0.05) on cell viability in comparison to positive control CSHC. Commercial TiO2 NPs (0.5 weight %) had statistically significant lower values (P≤0.05) for bacterial growth curve slope. However, addition of all NPs percentages had significantly improved (P≤0.05) the ALP activity of CSHC with the most prominent effect to 3-weight% biotitania/AgCl NPs. Conclusion: Based on this in vitro study, addition of biotitania/AgCl NPs up to 3-weight% significantly improved the bioactivity of CSHC without having a significant negative impact on its antibacterial efficacy. Interestingly, the addition of commercial TiO2 even in small amounts can significantly improve CSHC antibacterial efficacy.

Facile coating of urinary catheter with bio-inspired antibacterial coating.

Formation of bacterial biofilm on indwelling urinary catheters usually causes catheter-associated urinary tract infections (CAUTIs) that represent high percent of nosocomial infections worldwide. Therefore, coating urinary catheter with antibacterial and antifouling coating using facile technique is in great demand. In this study, commercial urinary catheter was coated with a layer of the self-polymerized polydopamine which acts as active platform for the in situ formation of silver nanoparticle (AgNPs) on catheter surface. The formed coating was intensively characterized using spectroscopic and microscopic techniques. The coated catheter has the potential to release silver ion in a sustained manner with a concentration of about 2-4 µg ml-1. Disk diffusion test and colony forming unites assay verified the significant bactericidal potential of the AgNPs coated catheter against both gram-positive and gram-negative bacteria as a consequence of silver ion release. In contrast to commercial catheter, the AgNPs coated catheter prevented the adherence of bacterial cells and biofilm formation on their surfaces. Interestingly, scanning electron microscope investigations showed that AgNPs coated catheter possess greater antifouling potential against gram-positive bacteria than against gram-negative bacteria. Overall, the remarkable antibacterial and antifouling potential of the coated catheter supported the use of such facile approach for coating of different medical devices for the prevention of nosocomial infections.

Reduced inflammatory response by incorporating magnesium into porous TiO2 coating on titanium substrate.

The implant materials with proper anti-inflammatory and osteogenic properties may be promising for orthopedic applications. The inflammatory response induced by biomaterials has been regarded as one of the critical factors in determining in vivo fate of implants. Therefore, a novel bone biomaterial should have inflammation regulatory effects instead of being completely bio-inert. In the present work, the inflammation regulatory effects of exogenous magnesium (Mg) ions were investigated. Under the stimulation of lipopolysaccharide (LPS), macrophages exposed to Mg2+ exhibited down-regulated gene expressions of M1 markers (CD86, CD11c and inducible nitric oxide synthase (iNOS)) and pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1ß), up-regulated gene expression of M2 marker CD163 and decreased TNF-α release, indicating that Mg2+ could switch macrophages from M1 to M2 phenotype. Thereafter, micro-arc oxidation (MAO) technique was employed to fabricate Mg-containing ceramic coatings on titanium substrates. Macrophages grown on Mg-containing surface were switched from M1 to M2 phenotype with the stimulation of LPS, evidenced by suppressed gene expressions of M1 markers (CD86, CD11c and iNOS) and pro-inflammatory cytokines (TNF-α and IL-1ß), promoted gene expression of M2 marker CD163 and decreased TNF-α release. Moreover, gene expressions of bone morphogenetic protein-2 (BMP-2), BMP-6 and vascular endothelial growth factor (VEGF) were up-regulated on Mg incorporated MAO surface without LPS stimulation. Together, Mg could be used as an anti-inflammatory agent for suppressing inflammation and mediating osteogenesis. The integration of Mg in biomaterials could endow bone biomaterials with anti-inflammatory property.

The Cu-containing TiO2 coatings with modulatory effects on macrophage polarization and bactericidal capacity prepared by micro-arc oxidation on titanium substrates.

The implant materials with both osteogenic and anti-bacterial properties are promising for orthopedic and dental applications. Moreover, the inflammatory response induced by biomaterials has been recently recognized as one of the critical factors in determining implantation fate. A new generation of implant materials should have modulatory effects on the local inflammatory environment such that it favors osteogenesis and osteointegration instead of being bio-inert. In this study, the micro-arc oxidation (MAO) technique was employed to fabricate Cu-containing ceramic coatings on titanium substrates. The macrophages cultured on Cu-containing MAO-fabricated surfaces were polarized to M1 phenotype, evidenced by the high expression levels of inducible nitric oxide synthase (iNOS), low expression levels of arginase1 (Arg1), enhanced pro-inflammatory cytokine interleukin-6 (IL-6) release and inhibited IL-4 and IL-10 (anti-inflammatory cytokines) release. The MAO-treated surface incorporated with larger amounts of Cu (referred as Cu(h)-MAO) could modulate a favorable inflammatory microenvironment for osteoblast-like cell differentiation. Moreover, the macrophages cultured on Cu(h)-MAO surface exhibited enhanced bacteria uptake and killing rate, indicating that the Cu(h)-MAO surface promoted the bactericidal capacity of macrophages. Together, Cu could be used as a promising modulatory agent for macrophage functions. The integration of Cu in biomaterials could lead to enhanced macrophage-mediated osteogenesis and bactericidal capacity.

The osteogenic, inflammatory and osteo-immunomodulatory performances of biomedical Ti-Ta metal-metal composite with Ca- and Si-containing bioceramic coatings.

It is known that good mechanical properties, low modulus to reduce stress-shielding effect, favorable osteogenic activity and limited inflammatory response are critical factors for orthopedic implants to induce excellent osteointegration. In this study, Ti-20% Ta metal-metal composite (referred as Ti-Ta) which consisted of Ti- and Ta-rich phases was fabricated via the strategy of powder metallurgy. Micro-arc oxidation (MAO) was employed to modify the surface of Ti-Ta composite. The surfaces of Ti-Ta composite after MAO treatment at an applied voltage of 250 (referred as MAO-250 V) or 300 V (referred as MAO-300 V) exhibited three distinct zones with significantly different morphological features and surface chemistry. Osteoblast-like SaOS-2 cells were found to be preferential to attach on the Ta-rich phase and its surrounding areas, exhibiting an area-dependent adhesion tendency. However, the attachment of Raw 264.7 macrophages was found to be insensitive to the surface characteristics. The proliferation and differentiation of SaOS-2 cells cultured on various surfaces basically followed the trend: MAO-modified surfaces > Ti-Ta surface > Ti surface. The Ti-Ta and MAO-modified surfaces were found to inhibit the inflammatory response and polarize macrophages to anti-inflammatory M2 phenotype compared to Ti surface. Moreover, the microenvironments created by Ti-Ta, MAO-250 V and MAO-300 V/macrophage interactions promoted the proliferation and differentiation of SaOS-2 cells compared to that created by Ti/macrophage interactions. MAO-300 V surface exhibited further enhanced positive osteo-immunomodulatory effects compared to Ti-Ta surface. Together, the Ti-20% Ta metal-metal composite modified by MAO at an applied voltage of 300 V is considered as a promising implant material for orthopedic applications.

Effects of titanium surface roughness on the mediation of osteogenesis via modulating the immune response of macrophages.

Osteoblastic lineage cells are commonly used to evaluate the in vitro osteogenic ability of bone biomaterials. However, contradictory results obtained from in vivo and in vitro studies are not uncommon. With the increasing understanding of osteoimmunology, the immune response has been recognized as playing an important role in bone regeneration. In this study, we examined the effect of submicron-scaled titanium surface roughness (ranging from approximately 100 to 400 nm) on the response of osteoblasts and macrophages. The results showed that osteoblast differentiation enhanced with increased surface roughness of titanium substrates. The cytoskeleton of macrophages altered with the variation in titanium surface roughness. The production of cytokines (TNF-α, IL-6, IL-4 and IL-10) could be regulated by titanium surface roughness. Moreover, macrophages cultured on titanium surfaces exhibited a tendency to polarize to M1 phenotype with the increase of surface roughness. Material/macrophage conditioned medium tended to promote osteoblast differentiation with the increase of surface roughness. The results indicate that increasing surface roughness in the submicron range is beneficial for osteogenesis via modulating the immune response of macrophages. Modifying biomaterial surfaces based on their immunomodulatory effects is considered as a novel strategy for the improvement of their biological performance.

Effects of the hierarchical macro/mesoporous structure on the osteoblast-like cell response.

To improve the success of medical devices, implants with strong surface bioactivity are urgently required. Coatings with a macroporous structure produced by micro-arc oxidation possess advantages, such as strong adhesion to substrate and excellent resistance to wear and corrosion. Mesoporous structures contain pores with sizes of 2-50 nm, which can endow the biomaterials with the ability to enhance osteogenesis and to be loaded with diverse drugs. Thus, in this study, we aimed to evaluate the effects of both macroporous and mesoporous structures using a hierarchical macro/mesoporous structure to modify the titanium implant surface. The behaviors of SaOS-2 human osteosarcoma cells on the macro/mesoporous structure, including initial adhesion, proliferation, alkaline phosphatase (ALP) activity, and collagen secretion, were investigated. Cells that attached on the macro/mesoporous surface showed the highest cell numbers and greatest spreading area after incubation for 1, 2, and 4 h compared with the polished smooth substrate and macroporous surface in the presence of fetal bovine serum (FBS). However, in the absence of FBS, cell adhesion on the polished substrate, macroporous structure, and macro/mesoporous structure did not differ significantly. Cell proliferation on the macroporous and macro/mesoporous surfaces increased compared with that on the smooth substrate surface. Furthermore, ALP activity and collagen secretion were enhanced on the macro/mesoporous structure. Our findings provided important insights into the cellular responses to macro/mesoporous structures in the field of implant surface modification. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1896-1902, 2018.

An evolutionary perspective on the role of mesencephalic astrocyte-derived neurotrophic factor (MANF): At the crossroads of poriferan innate immune and apoptotic pathways.

The mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to a recently discovered family of neurotrophic factors. MANF can be secreted but is generally resident within the endoplasmic reticulum (ER) in neuronal and non-neuronal cells, where it is involved in the ER stress response with pro-survival effects. Here we report the discovery of the MANF homolog SDMANF in the sponge Suberites domuncula. The basal positioning of sponges (phylum Porifera) in the animal tree of life offers a unique vantage point on the early evolution of the metazoan-specific genetic toolkit and molecular pathways. Since sponges lack a conventional nervous system, SDMANF presents an enticing opportunity to investigate the evolutionary ancient role of these neurotrophic factors. SDMANF shares considerable sequence similarity with its metazoan homologs. It also comprises a putative protein binding domain with sequence similarities to the Bcl-2 family of apoptotic regulators. In Suberites, SDMANF is expressed in the vicinity of bacteriocytes, where it co-localizes with the toll-like receptor SDTLR. In transfected human cells, SDMANF was detected in both the organelle protein fraction and the cell culture medium. The intracellular SDMANF protein level was up-regulated in response to both a Golgi/ER transport inhibitor and bacterial lipopolysaccharides (LPS). Upon LPS challenge, transfected cells revealed a decreased caspase-3 activity and increased cell viability with no inducible Bax expression compared to the wild type. These results suggest a deep evolutionary original cytoprotective role of MANF, at the crossroads of innate immune and apoptotic pathways, of which a neurotrophic function might have arisen later in metazoan evolution.

A dual-layer macro/mesoporous structured TiO2 surface improves the initial adhesion of osteoblast-like cells.

A dual-layer TiO2 surface with hierarchical macro and mesoporous structure was prepared by a combinational approach of micro-arc oxidation followed by evaporation-induced self-assembly of nano-crystallites. The mesoporous layer contains pores with an average size of <10nm and consists of anatase TiO2 nanocrystallites. The dual-layer hierarchical macro/mesoporous structured TiO2 surface improves the hydrophilicity and fibronectin adsorption ability in comparison with the sole macroporous or smooth TiO2 surface. With the formation of an additional mesoporous layer on macroporous TiO2 surface, the attached number of human osteogenic sarcoma cells (SaOS-2) increases in the initial incubation of 4h but it does not show significant difference after 24h compared to that attached on the macroporous or smooth surfaces. Whereas, it was noticed that SaOS-2 cells have larger spread area and more stress fibers on the macro/mesoporous structured surface than those on the other surfaces. To understand the intracellular mechanism of the initial cell adhesion on the macro/mesoporous surface, the Rho/ROCK pathway was investigated to reveal the topography-induced biological functions by introducing the ROCK inhibitor Y-27632 during cell culture. In the presence of Y-27632, cells on the macroporous surface and macro/mesoporous surface both show stellate appearance, with poor assembly stress fibers and long cell membrane protrusions. Cells on the smooth surface have larger spread areas compared to the former two surfaces. And the attached cells significantly reduced but there are no differences among the three surfaces. It reveals that the ROCK inhibitor invalidates the promotion of initial cell adhesion on the macro/mesoporous structure. This study may shed light on the mechanism behind the enhancement effect of macro/mesoporous structure for initial cell adhesion.

In Vitro Effect of 30 nm Silver Nanoparticles on Adipogenic Differentiation of Human Mesenchymal Stem Cells.

With the combined use of silver nanoparticles (Ag NPs) and human bone marrow derived mesenchymal stem cells (hMSCs) in bone tissue engineering, more knowledge of the effects of Ag NPs on hMSCs is required. Up to date, researches mainly focused on the cytotoxicity and genotoxicity of Ag NPs, only few studies discussed their influence on the differentiation of stem cells, especially adipogenic differentiation. In the present study, we investigated the in vitro uptake of 30 nm PVP-coated Ag NPs in hMSCs and their effects on cell viability, cell morphology and adipogenic differentiation of hMSCs. HMSCs were exposed to Ag NPs at concentrations of 25 and 50 µg/mL for 24 hours and at concentrations of 5 and 10 µg/mL throughout the whole differentiation period. Results of cell viability showed that Ag NPs caused time- and dose-dependent toxicity in hMSCs. Transmission electron microscopy (TEM) confirmed the uptake of Ag NPs into cytoplasm of hMSCs. No influence on cell morphology was observed. The 30 nm sized Ag NPs had no effects on adiponectin secretion, lipid droplet formation and the expression of adipogenic marker genes. It is concluded that under our experimental conditions, 30 nm PVP-coated Ag NPs do not influence the adipogenic differentiation of hMSCs in vitro. The present results provide a reference for the usage of 30 nm Ag NPs in the presence of hMSCs in bone tissue engineering.

SaOS-2 cell response to macro-porous boron-incorporated TiO2 coating prepared by micro-arc oxidation on titanium.

The aims of the present study were to develop boron-incorporated TiO2 coating (B-TiO2 coating) through micro-arc oxidation (MAO) and subsequently evaluate the effect of boron incorporation on the in vitro biological performance of the coatings. The physicochemical properties of B-TiO2 coating and its response to osteoblast like cells (SaOS-2) were investigated compared to the control group without boron (TiO2 coating). The morphological and X-ray diffraction results showed that both coatings exhibited similar surface topography and phase composition, respectively. However, the incorporation of B led to an enhancement in the surface hydrophilicity of B-TiO2 coating. The spreading of SaOS-2 cells on B-TiO2 coating was faster than that on TiO2 coating. The proliferation rate of SaOS-2 cells cultured on B-TiO2 decreased after 5days of culture compared to that on TiO2 coating. SaOS-2 cells cultured on B-TiO2 coating exhibited an enhanced alkaline phosphatase (ALP) activity, Collagen I synthesis and in vitro mineralization compared to those on TiO2 coating. The present findings suggest that B-TiO2 coating is a promising candidate surface for orthopedic implants.

Effects of hierarchical micro/nano-topographies on the morphology, proliferation and differentiation of osteoblast-like cells.

Coating the surfaces of titanium-based implants with appropriate hierarchical micro/nano-topographies resembling the structure of natural bone significantly enhances their biological performance. However, the relationship between nanostructures surfaces and their effects on modulating cellular response is not clearly understood. Moreover, it is not clear whether the surface chemistry or topography is the main factor on modulating cellular behavior, because the commonly used surface modification techniques for titanium-based implants simultaneously modify surface topography and chemistry. The aim of this study is to investigate osteoblast-like cell adhesion, proliferation and differentiation on hierarchical micro/nano-topographies with similar surface chemistry but different nano-scale features. Micro-arc oxidation and post hydrothermal treatment were employed to fabricate micro/nano-topographies on titanium. According to the morphological features, they were classified as microcrater (micro-topography), nanoplate (hierarchical topography with nanoplates) and nanoleaf (hierarchical topography with nanoleaves). The response of osteoblast like cells (SaOS-2) was studied on each surface after sputtering with a thin layer of gold (Au) to minimize the influence of surface chemistry. The morphological evaluation after histochemical staining revealed that the adherent cells were polygonal-shaped on microcrater surface, roundish on nanoplate surface and elongated on nanoleaf surface. Additionally, compared to microcrater surface, nanoplate surface slowed down cell proliferation and exhibited no enhancement on cell differentiation. However, nanoleaf surface supported cell proliferation and promoted cell differentiation. The results indicate that tuning morphological features of nanostructures on micro-topography can serve as a promising strategy to specifically modulate cellular response, such as cell morphology, proliferation, differentiation and mineralization.