Search

Ginseng-derived extracellular nanovesicles effectively blocked bone loss both in vitro and in vivo by inhibiting osteoclast differentiation through RANKL-induced IκBα, JNK, and ERK activation.

In the present study, a novel ELISA method used recombinant nucleocapsid protein (rNP) as the coating agent. Recombinant Newcastle disease virus (NDV) protein was cloned and expressed in Escherichia coli. Though the rNP-ELISA results were consistent with commercial ELISA results for the NDV-negative sera samples, qualitatively and quantitatively variable (often reduced) results were obtained with NDV-positive sera. Although the rNP-ELISA results for NDV detection were inconclusive, further improvement and standardization of the rNP-ELISA approach, such as using multiple recombinant proteins as the ELISA coating agent and performing comprehensive statistical analyses of combined recombinant protein ELISA, should help counter Newcastle disease outbreaks by improving NDV detection.

Scoparone (SCOP), an active and efficient coumarin compound derived from Artemisia capillaris Thunb, has been used as a traditional Chinese herbal medicine. Herein, we investigated the effects of SCOP on the osteogenic processes using MC3T3-E1 pre-osteoblasts in in vitro cell systems. SCOP (C

Despite advances in the bio-tissue engineering area, the technical basis to directly load hydrophobic drugs on chitosan (CTS) electrospun nanofibers (ENs) has not yet been fully established. In this study, we fabricated CTS ENs by using an electrospinning (ELSP) system, followed by surface modification using succinyl-beta-cyclodextrin (β-CD) under mild conditions. The β-CD-modified CTS (βCTS) ENs had slightly increased hydrophobicity compared to pristine CTS ENs as well as decreased residual amine content on the surface. Through FTIR spectroscopy and thermogravimetric analysis (TGA), we characterized the surface treatment physiochemically. In the drug release test, we demonstrated the stable and sustained release of a hydrophobic drug (e.g., dexamethasone) loaded on β-CD ENs. During in vitro biocompatibility assessments, the grafting of β-CD was shown to not reduce cell viability compared to pristine CTS ENs. Additionally, cells proliferated well on β-CD ENs, and this was confirmed by F-actin fluorescence staining. Overall, the material and strategies developed in this study have the potential to load a wide array of hydrophobic drugs. This could be applied as a drug carrier for a broad range of tissue engineering applications.

Objectives: As biomaterial‐induced modulation of mediators of the immune response may be a potential therapeutic approach to enhance wound healing events, the aim of this study was to delineate the effects of titanium surface modification on macrophage phenotype and function. Material and methods: Rodent bone marrow‐derived macrophages were polarized into M1 and M2 phenotypes and cultured on micro‐rough (SLA) and hydrophilic modified SLA (modSLA) titanium discs. Macrophage phenotype and cytokine secretion were subsequently assessed by immunostaining and ELISA, respectively. Osteoblast gene expression in response to culture in the M1 and M2 macrophage conditioned media was also evaluated over 7 days by RT‐PCR. Results: M1 macrophage culture on the modSLA surface promoted an M2‐like phenotype as demonstrated by marked CD163 protein expression, Arg1 gene expression and the secretion of cytokines that significantly upregulated in osteoblasts the expression of genes associated with the TGF‐s/BMP signalling pathway and osteogenesis. In comparison, M2 macrophage culture on SLA surface promoted an inflammatory phenotype and cytokine profile that was not conducive for osteogenic gene expression. Conclusions: Macrophages are able to alter or switch their phenotype according to the signals received from the biomaterial surface. A hydrophilic micro‐rough titanium surface topography elicits a macrophage phenotype associated with reduced inflammation and enhanced pro‐osteogenic signalling.

Background: Patient safety is considered an important issue in the field of healthcare, and most advanced countries. Purpose: This study was designed to evaluate a patient safety education program among hospitalized patients. Of the 69 participants, 33 completed the patient safety education program while the 36 remaining participants were given educational booklets. The program was used to measure knowledge about patient safety, patient safety perception, and willingness to participate in patient safety. Methods: Patient safety education was developed by the analysis–design–development–implementation–evaluation model considering expert advice, patient needs, and an extensive literature review. Data were collected from 20 July to 13 November 2020. Data were analyzed using SPSS statistical program. The effectiveness of the experimental and control groups before and after education was analyzed using paired t-tests, and the difference in the amount of increase in the measured variables for each group was analyzed using independent t-tests. Results: The experimental group had significantly higher patient safety scores (t = 2.52, p = 0.014) and patient safety perception (t = 2.09, p = 0.040) than those of the control group. However, there was no significant difference between the two groups regarding the willingness to participate in patient safety. Conclusion: The patient safety education program developed using mobile tablet PCs could be an effective tool to enhance patient involvement in preventing events that may threaten the safety of patients. Further studies are recommended to develop a variety of educational interventions to increase patient safety knowledge and perceptions of patients and caregivers.

Three-dimensional (3D) printing shows potential for use as an advanced technology for forming biomimetic tissue and other complex structures. However, there are limits and restrictions on selection of conventional bioinks. Here we report the first 3D-printable platelet lysate (PLMA)-based hydrogel, which consists of platelet lysate from whole blood of humans that can simulate the 3D structure of tissues and can be formed into a crosslinked hydrogel layer-by-layer to build cell-laden hydrogel constructs through methacrylated photo-polymerization. Furthermore, it can be customized for use with various tissues by controlling the physical properties according to irradiation time and concentration. In particular, different cells can be mixed and printed, and the integrity of the 3D printed structure can maintain its shape after crosslinking. The bio-ink exhibits excellent cell diffusion and proliferation at low concentrations, which improves moldability and biocompatibility. The 3D-printable PLMA bioinks may constitute a new strategy to create customized microenvironments for the repair of various tissues in vivo using materials derived from the human body.

Exosomes are nanosized vesicles (30–140 nm) of endocytic origin that play important roles in regenerative medicine. They are derived from cell membranes during endocytic internalization and stabilize in biological fluids such as blood and synovia. Temporomandibular joint osteoarthritis (TMJ OA) is a degenerative disease, which, in addition to chronic pain, is characterized by progressive cartilage breakdown, condylar bone remodeling, and synovitis. However, traditional clinical treatments have limited symptom- and structure-modifying effects to restore damaged cartilage and other TMJ tissues. This is due to the limited self-healing capacity of condylar cartilage. Recently, stem-cell-derived exosomes have been studied as an alternative therapeutic approach to tissue repair and regeneration. It is known that trophic regulation of mesenchymal stem cells (MSCs) has anti-inflammatory and immunomodulatory effects under pathological conditions, and research on MSC-derived exosomes is rapidly accumulating. MSC-derived exosomes mimic the major therapeutic effects of MSCs. They affect the activity of immune effector cells and possess multilineage differentiation potential, including chondrogenic and osteogenic differentiation. Furthermore, exosomes are capable of regenerating cartilage or osseous compartments and restoring injured tissues and can treat dysfunction and pain caused by TMJ OA. In this review, we looked at the uniqueness of TMJ, the pathogenesis of TMJ OA, and the potential role of MSC-derived exosomes for TMJ cartilage and bone regeneration.

Ensuring the formation of a robust trans-mucosal soft-tissue seal at the dental abutment surface is crucial towards protecting the underlying dental implant associated tissues from the external microbial-rich oral environment. The ability to mechanically enhance fibroblast functions at the dental abutment-mucosa interface, without the use of bioactive agents, holds great promise towards reducing the ingress of oral pathogens into the dental implant microenvironment. We hereby propose fabrication of unique anisotropic titania nanopores (TNPs) on the surface of titanium (via electrochemical anodization, EA) towards enhancing the soft-tissue integration and wound healing abilities of the conventional abutments. Using optimized EA, mechanically robust TNPs of varied diameters were fabricated on Ti surfaces with preserved underlying substrate micro-features: dual micro-nanostructured surfaces. Next, we evaluated the mechanical stability of such structures and demonstrated the ease of fabrication on commercial abutment geometries. The functions of primary human gingival fibroblasts (GFs) cultured on these surfaces in vitro were evaluated from 1 h to 7 days, and were compared between TNPs and clinically relevant titanium controls: as-received irregular rough Ti (Rough Ti) and mechanically prepared micro-rough Ti (Micro Ti). Improved cell viability was observed on TNPs as compared to controls. Additionally, cellular spreading morphology indicated cell alignment along the direction of the nanopores with strong anchoring evident by enhanced filopodia and stress fibers. RT-PCR showed improved wound healing, cell migration/adhesion and angiogenesis related mRNA, especially for TNPs with large diameters. This study provides a proof-of-concept towards using anodization for improving soft-tissue sealing around dental abutment surfaces, with implications towards reducing implant failure/peri-implantitis and achieving long-term success, especially in compromised patient conditions.

Bone defects can occur from many causes, including disease or trauma. Bone graft materials (BGMs) have been used to fill damaged areas for the reconstruction of diseased bone tissues since they are cost effective and readily available. However, BGMs quickly disperse around the tissue area, which ultimately leads to it migrating away from the defect after transplantation. We tested chitosan hydrogels as a useful carrier to hold BGMs in the transplantation area. In this study, we synthesized succinylated chitosan (SCS)-based hydrogels with a high decomposition rate and excellent biocompatibility. We confirmed that BGMs were well distributed inside the SCS hydrogel. The SCS-B hydrogel showed a decrease in mechanical properties, such as compressive strength and Young’s modulus, as the succinylation rate increased. SCS-B hydrogels also exhibited a high cell growth rate and bone differentiation rate. Moreover, the in vivo results showed that the SCS hydrogel resorbed into the surrounding tissues while maintaining the BGMs in the transplantation area for up to 6 weeks. These data support the idea that SCS hydrogel can be useful as a bioactive drug carrier for a broad range of biomedical applications.

Statement of problem Acrylic resin materials for interim restoration may adversely affect pulp tissue during the polymerization phase. Purpose The purpose of this in vitro study was to determine the cytotoxic and proinflammatory cytokine production effects induced by interim resin materials in primary cultured human dental pulp cells (hDPCs). Material and methods Five interim resin materials were evaluated: 3 types of chemically activated products, 1 light-activated product, and 1 computer-aided design and computer-aided manufacturing (CAD-CAM) product. After obtaining eluates from interim resin materials that either were in the process of polymerizing or were already polymerized, these extracts were cocultured with hDPCs under serially diluted conditions (50%, 25%, 12.5%, 6.25%, and 3.125%) for 24 hours with positive (1% phenol) and negative (distilled water) controls. A cell viability assay with tetrazolium was used to evaluate toxic effects on the cells, and images of both live and dead cells were captured using confocal microscopy. Proinflammatory cytokine levels were measured using cytokine antibody arrays. All experiments were independently repeated 3 times, and data were analyzed using 1-way ANOVA and post hoc Tukey honest significant differences test (α=.05). Results Cell viabilities less than 70% were observed from the eluates of the 3 chemically activated products under the 50% conditions. Among the chemically activated products, the adverse effects were significantly greater with eluates derived from the polymerizing phase compared than those that had already polymerized, as shown by confocal microscopy images of live and dead cells. However, the light-activated and CAD-CAM-fabricated products did not adversely affect the hDPCs. Significantly increased levels of proinflammatory cytokines were not detected in 12.5% of extract from polymerizing compared with distilled water control. Conclusions The 50% eluates derived from chemically activated interim resin during the polymerizing phase were cytotoxic to hDPCs and may adversely affect pulp tissue. Recommendations such as excess washing are necessary during fabrication.

Titanium implants (Ti) have been widely used in several medical fields. In clinical practice, Ti can become contaminated with bacteria through a variety of mechanisms. This contamination can lead to implant failure and serious infections. In this study, we aimed to develop a new, hybrid Ti with good biocompatibility and antibacterial properties by immobilizing ceftazidime (CFT) onto the Ti surface through polydopamine (PDA) and polyethyleneimine (PEI) chemistry. Hybrid Ti was confirmed by assessing the cell proliferation of human adipose-derived stem cells using a cell counting. The biofilm formation across the Ti surface of two bacterial strains associated with nosocomial infections, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus, was evaluated by scanning electron microscopy. The viability of the bacteria exposed to Ti surface was evaluated by cell counting. Our results clearly demonstrate that the bacterial biofilm formation as well as bacterial viability was significantly reduced on the hybrid Ti as compared to the control, Ti alone. Collectively, the Ti surface was successfully modified to form the hybrid Ti exhibiting good biocompatibility and antibacterial properties through PDA, PEI, and CFT grafting. Within the limitations of this in vitro study, we conclude that the hybrid Ti may be useful for successful implant treatment., This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (No. 2017M3A9E4048170).

Titanium surface mediated immunomodulation may address compromised post-implantation bone healing in diabetes mellitus. To assess in vitro phenotypic changes, M1 and M2 polarised Type 2 diabetic rat (Goto Kakizaki, GK) macrophages were cultured on micro-rough (SLA) or hydrophilic nanostructured SLA (modSLA) titanium. The in vivo effects of the SLA and modSLA surfaces on macrophage phenotype, wound-associated protein expression and bone formation were investigated using a critical-sized calvarial defect model. Compared to healthy macrophages, GK M2 macrophage function was compromised, secreting significantly lower levels of the anti-inflammatory cytokine IL-10. The modSLA surface attenuated the pro-inflammatory cellular environment, reducing pro-inflammatory cytokine production and promoting M2 macrophage phenotype differentiation. ModSLA also suppressed gene expression associated with macrophage multinucleation and giant cell formation and stimulated pro-osteogenic genes in co-cultured osteoblasts. In vivo, modSLA enhanced osteogenesis compared to SLA in GK rats. During early healing, proteomic analysis of both surface adherent and wound exudate material showed that modSLA promoted an immunomodulatory pro-reparative environment. The modSLA surface therefore successfully compensated for the compromised M2 macrophage function in Type 2 diabetes by attenuating the pro-inflammatory response and promoting M2 macrophage activity, thus restoring macrophage homeostasis and resulting in a cellular environment favourable for enhanced osseous healing.

Osteoporosis, a major skeletal disorder, is an increasing worldwide social burden among the aging society, which develops when the ratio of bone resorption regulated by osteoclasts reverses the bone formation in the bone remodeling cycle. Basically, osteoporosis is commonly treated using bone resorption inhibitors such as bisphosphonates. However, long-term intake of bisphosphonates exposes patients to traumatic side effects such as digestive disorders and medication-related osteonecrosis of the jaw (MRONJ). Therefore, an alternative solution is required in the area of bone tissue engineering. Hence, to solve this issue, in this study, we designed GNPs and functionalized GNPs’ surface through conjugation with SH-PEG-vitamin D (SPVD) via the Au-S binding system. Vitamin D-conjugated GNPs (VGNPs) were successfully synthesized and confirmed by various physicochemical analyses. These conjugated nanoparticles significantly increased cell viability and decreased tartrate-resistant acid phosphatase (TRAP) activity and actin ring formation. In addition, VGNPs suppressed the expression of genes associated with osteoclast differentiation. Finally, VGNPs also significantly inhibited the expression of reactive oxygen species (ROS). These results demonstrate the inhibitory effect of VGNPs on osteoclast differentiation and suggest that the developed nanocarriers could play a key role as a bone resorption inhibitor in the field of bone tissue engineering.

Drug-loaded hydrogel-based controllable drug delivery systems (DDS) have received considerable attention since they may overcome the drawbacks of conventional drug administration. The desirable hydrogels can be injected in any part of the body and are easily-available. To develop DDS, a succinylated chitosan (SCS)-based hydrogel was manufactured using facile and water-based conditions. This ideal and ecofriendly hydrogel showed rapid gelation and efficient release of gold nanoparticles (GNP) as representative small particles and drug substitutes. Moreover, the SCS hydrogel showed outstanding GNP release at low pH, with 100% degradation after a week at a pH of 5.5. Additionally, the drug release rate of SCS hydrogel could be controlled by adjusting succinylation and temperature. The SCS hydrogel exhibited good biocompatibility for applications in DDS. On the basis of these advantages, the SCS hydrogel can be used as a drug-loaded vehicle, which can be released at the appropriate time (important for cancer and inflammation treatments) and injected at the appropriate site.

Donghyun Lee,1 Dong Nyoung Heo,2 Ha Ram Nah,3 Sang Jin Lee,1 Wan-Kyu Ko,4 Jae Seo Lee,3 Ho-Jin Moon,1 Jae Beum Bang,5 Yu-Shik Hwang,6 Rui L Reis,1,7 Il Keun Kwon1 1Department of Dental Materials, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea; 2Department of Engineering Science and Mechanics, Pennsylvania State University, Pennsylvania 16802, USA; 3Department of Detistry, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea; 4Department of Neurosurgery, CHA Bundang Medical Center, CHA University, Seongnam-si, Gyeonggi-do 13496, Republic of Korea; 5Department of Dental Education, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea; 6Department of Maxillofacial Biomedical Engineering, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea; 7The Discoveries Centre for Regenerative and Precision Medicine, Headquarters at University of Minho, Barco, Guimarães, Portugal Background: For effective bone regeneration, it is necessary to implant a biocompatible scaffold that is capable of inducing cell growth and continuous osteogenic stimulation at the defected site. Here, we suggest an injectable hydrogel system using enzymatic cross-linkable gelatin (Gel) and functionalized gold nanoparticles (GNPs). Methods: In this work, tyramine (Ty) was synthesized on the gelatin backbone (Gel-Ty) to enable a phenol crosslinking reaction with horseradish peroxidase (HRP). N-acetyl cysteine (NAC) was attached to the GNPs surface (G-NAC) for promoting osteo-differentiation. Results: The Gel-Ty hydrogels containing G-NAC (Gel-Ty/G-NAC) had suitable mechanical strength and biocompatibility to embed and support the growth of human adipose derived stem cells (hASCs) during a proliferation test for three days. In addition, G-NAC promoted osteo-differentiation both when it was included in Gel-Ty and when it was used directly in hASCs. The osteogenic effects were demonstrated by the alkaline phosphatase (ALP) activity test. Conclusion: These findings indicate that the phenol crosslinking reaction is suitable for injectable hydrogels for tissue regeneration and G-NAC stimulate bone regeneration. Based on our results, we suggest that Gel-Ty/G-NAC hydrogels can serve both as a biodegradable graft material for bone defect treatment and as a good template for tissue engineering applications such as drug delivery, cell delivery, and various tissue regeneration uses. Keywords: nanomaterial, gelatin, enzymatic cross-linking, osteogenesis, N-acetyl cysteine

The immunomodulatory effects of lithium have been reported across a range of models and contexts. Lithium appears to have a positive effect on osteogenesis in vivo, while in vitro outcomes throughout the literature are varied. Tissue engineering approaches have rarely targeted local lithium delivery within a regenerative setting. We hypothesized that part of the positive effects of lithium in vivo may be due to an immunomodulatory effect manifesting in a local environment. To achieve a sustained lithium release from scaffold constructs, we blended lithium carbonate, a soluble salt of lithium, with the biomaterial polymer polycaprolactone (PCL). We printed constructs of PCL alone, and with 5% (5Li) and 10% (10Li) lithium carbonate. Mechanical testing revealed that mechanical properties were largely retained with lithium carbonate incorporation, and we measured a consistent release of the ion over a 7 day period. The efficacy of our construct system was then assessed using a primary mouse macrophage culture, and a differentiated osteoclast culture. We found that the lithium released from constructs had a great effect on macrophage polarization, resulting in pronounced upregulation of immunomodulatory (M2) genes, and a decrease in pro-inflammatory (M1) genes. This was reflected in cytokine expression, and illustrated through immunofluorescent staining. Osteoclast activity was greatly suppressed by the lithium incorporation, with a marked effect on gene expression and actin ring formation. Our work demonstrated an effective system for local lithium delivery, confirmed the pronounced effects that lithium has on macrophage and osteoclast response, and sets the stage for further innovations in ion release for targeted tissue engineering.

Initiated chemical vapor deposition (iCVD) was utilized to generate a 200nm thick, uniform, functionalized polymer nanolayer comprised of glycidyl methacrylate (GMA) on the surface of titanium implants as a means to improve cellular attachment. Dot-patterned GMA-coated specimens were prepared as well as fully coated specimens. In vitro cellular responses, including cell morphology, protein adsorption, cell proliferation assays, alkaline phosphate activity (ALP) assays, and calcium deposition assays were studied using adipose derived stem cells. The mechanical stability of the thin film was investigated by XPS and FE-SEM analysis of the GMA-coated implant after implantation to an extracted bone from a pig. The GMA-coated specimens displayed increased protein adsorption, higher alkaline phosphatase activities, and higher calcium deposition as compared to control sample with no cytotoxicity. Additionally, no defect was observed in the test of mechanical stability. Notably, dot-patterned GMA-coated samples displayed higher alkaline phosphatase activities than others. Functionalized polymer nanolayer deposition via iCVD is a flexible and robust technique capable of mass production of biocompatible layers. These properties make this technique very suitable for implant applications in a variety of ways.

This letter describes a simple surface modification strategy based on a single-step electrochemical anodization towards generating dual micro- and nano-rough horizontally-aligned TiO2 nanopores on the surface of clinically utilized micro-grooved titanium implants. Primary macrophages, osteoblasts and fibroblasts were cultured on the nano-engineered implants, and it was demonstrated that the modified surfaces selectively reduced the proliferation of macrophages (immunomodulation), while augmenting the activity of osteoblasts (osseo-integration) and fibroblasts (soft-tissue integration). Additionally, the mechanically robust nanopores also stimulated osteoblast and fibroblast adhesion, attachment and alignment along the direction of the pores/grooves, while macrophages remained oval-shaped and sparsely distributed. This study for the first time reports the use of cost-effectively prepared nano-engineered titanium surface via anodization, with aligned multi-scale micro/nano features for selective cellular bioactivity, without the use of any therapeutics.

Gold nanoparticles (GNPs) have been previously reported to inhibit osteoclast (OC) formation. However, previous research only confirmed the osteoclastogenesis inhibitory effect under in vitro conditions. The aim of this study was to develop a therapeutic agent for osteoporosis based on the utilization of GNPs and confirm their effect both in vitro and in vivo. We prepared β-cyclodextrin (CD) conjugated GNPs (CGNPs), which can form inclusion complexes with curcumin (CUR-CGNPs), and used these to investigate their inhibitory effects on receptor activator of nuclear factor-κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). The CUR-CGNPs significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells in BMMs without inducing cytotoxicity. The mRNA expressions of genetic markers of OC differentiation including c-Fos, nuclear factor of activated T cells 1 (NFATc1), TRAP, and osteoclast associated receptor (OSCAR) were significantly decreased in the presence of CUR-CGNPs. In addition, the CUR-CGNPs inhibited OC differentiation of BMMs through suppression of the RANKL-induced signaling pathway. Additionally, CUR-CGNPs caused a decrease in RANKL-induced actin ring formation, which is an essential morphological characteristic of OC formation allowing them to carry out bone resorption activity. Furthermore, the in vivo results of an ovariectomy (OVX)-induced osteoporosis model showed that CUR-CGNPs significantly improved bone density and prevented bone loss. Therefore, CUR-CGNPs may prove to be useful as therapeutic agents for preventing and treating osteoporosis.

Existing influenza vaccines protect mostly homologous subtypes and acted most effectively only when well matched to the circulating strain. Immunization with an updated vaccine is therefore necessary to maintain long-term protection and the development of a broadly protective influenza vaccine against the threat of pandemic outbreak. The highly conserved HA2 glyco-polypeptide (HA2 gp) is a promising new candidate for such an influenza vaccine. Helical domain and the fusion peptide (residues 15-137) of surface antigen from influenza A subtype A/EM/Korea/W149/06 (H5N1) was used to assess the potentiality of HA2 vaccination against multiple subtypes of the influenza viruses. The construct, named H5HA2 was expressed in Escherichia coli and allowed to refold from inclusion bodies. Purified proteins were used to investigate the immunogenicity of H5HA2 and its potential for cross protection. The immunization of mice with H5HA2 induced HA2 antibodies, HA2 specific T-cell responses, and protection against homologous A/EM/Korea/W149/06 (H5N1) influenza. Immunized mice were also protected from two distinct heterosubtypes of influenza: A/Puerto Rico/1/34(H1N1) and bird/Korea/w81/2005(H5N2). Results suggest that recombinant proteins based on the highly conserved residues within HA2 are candidates for the development of vaccines against pandemic outbreaks of emergent influenza variants.

Since the 17th century Spica Prunella has been used as a medicinal herb. Dried and pulverized Spica Prunella samples were extracted and used in these experiments. In this study, the effects of Spica Prunella extract (SPE) on RANKL (receptor activator of nuclear factor κB ligand)-induced osteoclastogenesis were examined. Actin ring formation, a typical marker of osteoclastogenesis, was inhibited by SPE without any toxicity. There was also a marked inhibition in the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in bone marrow-derived monocytes (BMMs). SPE also suppressed phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinases (ERK) both of which are signals of the mitogen-activated protein kinases (MAPKs) signaling pathway. Additionally, SPE inhibited IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) signaling pathway, which is an important factor in osteoclastogenesis. These results indicate that SPE might suppress osteoclast differentiation by inhibiting the phosphorylation of JNK and ERK in MAPK and NF-κB signaling pathways which act as messengers in the RANKL-induced osteoclast differentiation pathway. This means that SPE could potentially have great therapeutic usage in treating bone erosive diseases such as rheumatoid arthritis or in preventing metastasis associated with bone loss.

Canine parvovirus (CPV2) is one of the most virulent virus causing acute hemorrhagic enteritis and myocarditis in dogs. Infection mainly caused by the ingestion of virus through the mucosal route. Therefore, induction of mucosal immunity is essential in prevention of Canine Parvovirus (CPV2) infection. For safe and effective delivery of viral antigens to the mucosal immune system, a novel surface antigen display system for lactic acid bacteria using the poly-γ-glutamic acid synthetase A protein (pgsA) of Bacillus subtilis as an anchoring matrix was applied in order to display CPV2 antigen on the surface of the recombinant L. casei. Recombinant fusion proteins comprised of pgsA and the capsid protein (VP2-S1) showed stable expression in Lactobacillus casei. Surface localization of the fusion protein was verified by cellular fractionation analyses. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA, as demonstrated by ELISA using recombinant VP2-S1 proteins. Mice receiving intranasal immunization mounted higher antibody response than those receiving oral immunization. These results indicate that mucosal immunization with recombinant L. casei expressing CPV2 VP2-S1 protein on its surface provides an effective means for elicitation of strong antibody responses against CPV 2 VP2-S1.

Coenzyme Q10 (CoQ10), a powerful antioxidant, is a key component in mitochondrial bioenergy transfer, generating energy in the form of ATP. Many studies suggest that antioxidants act as inhibitors of osteoclastogenesis and we also have previously demonstrated the inhibitory effect of CoQ10 on osteoclast differentiation. Despite the significance of this effect, the molecular mechanism when CoQ10 is present at high concentrations in bone remodeling still remains to be elucidated. In this study, we investigated the inhibitory effect of CoQ10 on osteoclastogenesis and its impact on osteoblastogenesis at concentrations ranging from 10 to 100 μM. We found that nontoxic CoQ10 markedly attenuated the formation of receptor activator of nuclear factor κB ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone-marrow-derived monocytes (BMMs) and RAW 264.7 cells. Osteoclastogenesis with CoQ10 was significantly suppressed the gene expression of NFATc1, TRAP, and osteoclast-associated immunoglobulin-like receptor, which are genetic markers of osteoclast differentiation and scavenged intracellular reactive oxygen species, an osteoclast precursor, in a dose-dependent manner. Furthermore, CoQ10 strongly suppressed H2 O2 -induced IκBα, p38 signaling pathways for osteoclastogenesis. In bone formation study, CoQ10 acted to enhance the induction of osteoblastogenic biomarkers including alkaline phosphatase, type 1 collagen, bone sialoprotein, osteoblast-specific transcription factor Osterix, and Runt-related transcription factor 2 and, also promoted matrix mineralization by enhancing bone nodule formation in a dose-dependent manner. Together, CoQ10 acts as an inhibitor of RANKL-induced osteoclast differentiation and an enhancer of bone-forming osteoblast differentiation. These findings highlight the potential therapeutic applications of CoQ10 for the treatment of bone disease.

Purpose We evaluated the activities of both osteoblastic and osteoclastic differentiation on sandblasted/acid etched (SLA), hydrophilic SLA surfaces (modSLA) and pretreatment titanium (PT). Material and methods The osteoblastic differentiation was evaluated by alkaline phosphatase analysis and Alizarin Red S staining, and the expression of bone-related proteins, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN), was investigated by reverse transcriptase–polymerase chain reaction (RT-PCR). Primary mice monocytes were expanded and differentiated in the presence of macrophage-colony stimulating factor (M-CSF), and osteoclastic differentiation was evaluated by actin ring formation assay and tartrate-resistant acid phosphatase (TRAP) activity assay. Real-time PCR tests were performed to investigate the expression of gene mRNA expression levels in osteoclast cells. Result Differentiation of osteoblasts in the Alizarin Red S test staining and ALP assay was significantly increased in the modSLA surface. The preceding results were supported by the result of RT-PCR for the expression of Runx2, OPN, and OCN. As for osteoclastic activity, differentiated osteoclasts rarely existed on the SLA and modSLA surface with actin ring. The results of real-time PCR and TRAP activity supported the preceding results. Conclusion It may be concluded that the modSLA surface promotes osteogenic effect and prevents osteoclastic differentiation. Promotion of osteoblastic proliferation after a short-term cell culture might be responsible for stimulated bone regeneration implying that early loading may be possible. Also, the anti-osteoclastic effect of the modSLA surface may contribute to maintenance of the marginal bone level of dental implants, implying long-term stability would be provided by this surface technology. The modSLA surface may not only make early loading possible but possibly reduce marginal bone loss during the maintenance phase.

Neural stem cells (NSCs) have a high potency for differentiation to neurons and glial cells for replacement of damaged cells and paracrine effects for the regeneration and remyelination of host axons. Dental pulp is known to have a potential to differentiate into neural-like cells; therefore, dental pulp may be used as an autologous cell source for neural repair. In this study, we selectively expanded stem cells from human dental pulp in an initial culture using NSC media under xeno- and serum-free conditions. At the initial step of primary culture, human dental pulp was divided into two groups according to the culture media: 10% fetal bovine serum medium group (FBS group) and NSC culture medium group (NSC group). In the NSC group relative to the FBS group, the expression of NSC markers and the concentrations of leukemia inhibitory factor, nerve growth factor, and stem cell factor were higher, although their expression levels were lower than those of human fetal NSCs. The transplanted cells of the NSC group survived well within the normal brain and injured spinal cord of rats and expressed nestin and Sox2. Under the xeno- and serum-free conditions, autologous human dental pulp-derived stem cells might prove useful for clinical cell-based therapies to repair damaged neural tissues.

The threat of a highly pathogenic avian influenza (HPAI) H5N1 virus causing the next pandemic remains a major concern. In this study, we evaluated the immunogenicity and efficacy of an inactivated whole-virus H5N1 pre-pandemic vaccine (MG1109) formulated by Green Cross Co., Ltd containing the hemagglutinin (HA) and neuraminidase (NA) genes of the clade 1 A/Vietnam/1194/04 virus in the backbone of A/Puerto Rico/8/34 (RgVietNam/04xPR8/34). Administration of the MG1109 vaccine (2-doses) in mice and ferrets elicited high HI and SN titers in a dose-dependent manner against the homologous (RgVietNam/04xPR8/34) and various heterologous H5N1 strains, (RgKor/W149/06xPR8/34, RgCambodia/04xPR8/34, RgGuangxi/05xPR8/34), including a heterosubtypic H5N2 (A/Aquatic bird/orea/W81/05) virus. However, efficient cross-reactivity was not observed against heterosubtypic H9N2 (A/Ck/Korea/H0802/08) and H1N1 (PR/8/34) viruses. Mice immunized with 1.9 μg HA/dose of MG1109 were completely protected from lethal challenge with heterologous wild-type HPAI H5N1 A/EM/Korea/W149/06 (clade 2.2) and mouse-adapted H5N2 viruses. Furthermore, ferrets administered at least 3.8 μg HA/dose efficiently suppressed virus growth in the upper respiratory tract and lungs. Vaccinated mice and ferrets also demonstrated attenuation of clinical disease signs and limited virus spread to other organs. Thus, this vaccine provided immunogenic responses in mouse and ferret models even against challenge with heterologous HPAI H5N1 and H5N2 viruses. Since the specific strain of HPAI H5N1 virus that would potentially cause the next outbreak is unknown, pre-pandemic vaccine preparation that could provide cross-protection against various H5 strains could be a useful approach in the selection of promising candidate vaccines in the future.

In addition to development of vaccines and synthetic antiviral drugs, recent studies have advocated the use of natural substances that inhibit or prevent viral infections. High-molecular-weight poly-γ-glutamate (HM-γ-PGA) produced by Bacillus subtilis chungkookjang was evaluated for anti-influenza virus activity. HM-γ-PGA induced type I interferons (IFNs), which in turn stimulated expression of Myxovirus resistant 1 protein and IFN-related proteins in vitro. In the B6.A2G-Mx1 mouse model, which mimics the innate immune system of humans, treatment with HM-γ-PGA enhanced the antiviral state of mice and protected them against highly pathogenic influenza A virus. Naturally synthesized HM-γ-PGA has potent anti-influenza activity and may be a useful means for control of influenza virus.

Safflower seed has been reported to have a protective effect against bone loss diseases. However, the precise molecular mechanisms underlying the inhibitory effect of safflower seed in osteoclast differentiation remain unclear. In this study, we investigated the inhibitory action of safflower seed extract (SSE) on the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in cultured mouse-derived bone marrow macrophages (BMMs). We found that SSE significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in BMMs without cytotoxicity. The gene expressions of nuclear factor of activated T-cells (NFATc1) and TRAP, which are genetic markers of osteoclast differentiation, were substantially decreased by SSE in a dose-dependent manner. Also, SSE diminished RANKL-mediated intracellular reactive oxygen species (ROS) generation on osteoclastogenesis in a dose-dependent manner. The SSE thereafter suppressed RANKL-induced p38 mitogen-activated protein kinase and IκBα kinase signalling activities which were activated by ROS generation for osteoclastogenesis. Additionally, SSE was found to decrease RANKL-induced actin ring formation, which is required for bone resorption activity. Taken together, our results suggest that SSE acts as a RANKL-induced osteoclastogenesis inhibitor by suppression of ROS generation. This induces a remarkable suppression of the p38 and IκBα kinase pathways, thereby suppressing the gene expression of NFATc1 in osteoclast precursors.

Coenzyme Q10 (CoQ10), selenium, and curcumin are known to be powerful antioxidants. Osteoclasts are capable of resorbing mineralized bone and excessive bone resorption by osteoclasts causes bone loss-related diseases. During osteoclast differentiation, the reactive oxygen species (ROS) acts as a secondary messenger on signal pathways. In this study, we investigated whether antioxidants can inhibit RANKL-induced osteoclastogenesis through suppression of ROS generation and compared the relative inhibitory activities of CoQ10, sodium selenite, and curcumin on osteoclast differentiation. We found that antioxidants markedly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in both bone marrow-derived monocytes (BMMs) and RAW 264.7 cells. Antioxidants scavenged intracellular ROS generation within osteoclast precursors during RANKL-stimulated osteoclastogenesis. These also acted to significantly suppress the gene expression of NFATc1, TRAP, and osteoclast-associated immunoglobulin-like receptor (OSCAR), which are genetic markers of osteoclast differentiation in a dose-dependent manner. These antioxidants also suppressed ROS-induced IκBα signaling pathways for osteoclastogenesis. Specially, curcumin displayed the highest inhibitory effect on osteoclast differentiation when concentrations were held constant. Together, CoQ10, selenite, and curcumin act as inhibitors of RANKL-induced NFATc1 which is a downstream event of NF-κB signal pathway through suppression of ROS generation, thereby suggesting their potential usefulness for the treatment of bone disease associated with excessive bone resorption.

We describe in this study whether the gold nanoparticle (AuNP) surface-functionalized with PEG, biotin, paclitaxel (PTX) and rhodamine B linked beta-cyclodextrin (β-CD) (AuNP-5') can be useful as a theranostic agent for cancer therapy without the cytotoxic effect on normal cells. Prior to surface-functionalizing AuNPs, the cytotoxicity of the nanoparticles was evaluated, followed by their cytocompatibility. PTX, an anti-cancer agent, formed inclusion complexations with β-CD conjugated AuNPs, and effectively released from the AuNP-2' surface-functionalized with PEG, beta-cyclodextrin (β-CD) and paclitaxel (PTX) using the intracellular glutathione (GSH) level (10 mm). Two types of AuNP-4 surface-functionalized with PEG and rhodamine B linked β-CD and AuNP-5 surface-functionalized PEG, biotin and rhodamine B linked β-CD were used for evaluating their specific interaction on cancer cells such as HeLa, A549 and MG63. These were also tested against normal NIH3T3 cell, determining that the AuNP-5 was more effectively involved with the cancer cells. Confocal laser scanning microscopy (CLSM), fluorescence-activated cell-sorting (FACS) and cell viability analyses showed that the AuNP-5' plays a significant role in the diagnosis and therapy of the cancer cells, and may be used in theranostic agents.

To investigate whether currently circulating H9N2 avian influenza viruses (AIVs) in domestic poultry have evolved in Korean poultry since 2007, genetic and serological comparisons were conducted of H9N2 isolates from poultry slaughterhouses from January 2008 to December 2009. The isolation rate was relatively low in 2008 but increased gradually from January 2009 onwards. Genetic and phylogenetic analyses revealed that reassortant viruses had emerged, generating at least five novel genotypes, mostly containing segments of a previously prevalent domestic H9N2 virus lineage (Ck/Korea/04116/04-like). It was noteworthy that the N2 genes of some H9N2 isolates (genotypes D, E and F) were derived from those of H3N2-like viruses commonly isolated among domestic ducks in live-poultry markets. Animal challenge studies demonstrated that the pathogenicity of Ck/Korea/SH0906/09 (genotype B) and Ck/Korea/SH0912/09 (genotype F) in domestic avian species was altered due to reassortment. Furthermore, serological analysis revealed that the isolates were antigenically distinct from previous Korean H9N2 viruses including Ck/Korea/01310/01. Such antigenic diversity was illustrated further in experiments using H9N2-immunized chickens, which could not inhibit the replication and transmission of challenge viruses from each genotype. These results suggest that H9N2 viruses from domestic poultry have undergone substantial evolution since 2007 by immune selection as a result of vaccinal and natural immunity, coupled with reassortment. Taken together, this study demonstrates that periodical updating of vaccine strains, based on continuous surveillance data, is an important issue in order to provide sufficient protectivity against AIV infections.