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In this study, we created a hydrogel composed of glycol chitosan (gC) and oxidized hyaluronate (oHA). Gold nanoparticles (GNPs) were conjugated with ursodeoxycholic acid (UDCA). The GNP-UDCA complex was embedded into gC-oHA (CHA) hydrogels to form a CHA-GNP-UDCA gel. This CHA-GNP-UDCA gel was injected once into an epicenter of an injured region in SCI rats. Near-infrared (NIR) irradiation was then applied to the lesion as a means of local therapy. To optimize the viscosity for injection into a lesion, several volume ratios of gC and oHA were investigated using scanning electron microscopy and a rotating rheometer. The optimally synthesized CHA-GNP-UDCA gel under NIR irradiation suppressed the production of inflammatory cytokines in vitro. In addition, the optimized CHA-GNP-UDCA gel under NIR irradiation inhibited the cystic cavity of the lesion and significantly improved the hindlimb function. The production of inflammatory cytokines following SCI was significantly inhibited in the CHA-GNP-UDCA gel + NIR group. CHA-GNP-UDCA gels with NIR irradiation can therefore have therapeutic effects for those with spinal cord injuries.

We investigate the anti-inflammatory effects of injectable hydrogel containing tauroursodeoxycholic acid (TUDCA) in a spinal cord injury (SCI) model. To this end, TUDCA-hydrogel (TC gel) is created by immersing the synthesized hydrogel in a TUDCA solution for 1 h. A mechanical SCI was imposed on rats, after which we injected the TC gel. After the SCI and injections, motor functions and lesions were significantly improved in the TC gel group compared with those in the saline group. The TC gel significantly decreased pro-inflammatory cytokine levels compared with the saline; TUDCA and glycol chitosan-oxidized hyaluronate were mixed at a ratio of 9:1 (CHA) gel independently. In addition, the TC gel significantly suppressed the phosphorylation of extracellular signal-regulated kinase (p-ERK) and c-Jun N-terminal kinase (p-JNK) in the mitogen-activated protein kinase (MAPK) pathway compared with the saline, TUDCA, and CHA gel independently. It also decreased tumor necrosis factor-α (TNF-α) and glial fibrillary acidic protein (GFAP), inflammatory marker, at the injured sites more than those in the saline, TUDCA, and CHA gel groups. In conclusion, the results of this study demonstrate the neuroinflammatory inhibition effects of TC gel in SCI and suggest that TC gel can be an alternative drug system for SCI cases.

The purpose of this study is to elucidate the anti-inflammatory effect of lobeglitazone (LOBE) in lipopolysaccharide (LPS)-induced bone-marrow derived macrophages (BMDMs). We induced nitric oxide (NO) production and pro-inflammatory gene expression through LPS treatment in BMDMs. The changes of NO release and expression of pro-inflammatory mediators by LOBE were assessed via NO quantification assay and a real-time quantitative polymerase chain reaction (RT-qPCR), respectively. In addition, the regulatory effect of LOBE on activation of mitogen-activated protein kinase (MAPK) signaling pathway was investigated by measuring the phosphorylation state of extracellular regulatory protein (ERK) and c-Jun N-terminal kinase (JNK) proteins by Western blot. Our results show that LOBE significantly reduced LPS-induced NO production and pro-inflammatory gene expression of interleukin-1β (IL-1β), IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and monocyte chemoattractant protein-1 (MCP-1). Moreover, LOBE reduced phosphorylation levels of ERK and JNK of MAPK signaling pathway. In conclusion, LOBE exerts an anti-inflammatory effect in LPS-induced BMDMs by suppression of NO production and pro-inflammatory gene expression, and this effect is potentially through inhibition of the MARK signaling pathway.

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 In this study, we study the transplantation of tauroursodeoxycholic acid (TUDCA)‐induced M2‐phenotype (M2) macrophages and their ability to promote anti‐neuroinflammatory effects and functional recovery in a spinal cord injury (SCI) model. Methods To this end, compared to the granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), we evaluated whether TUDCA effectively differentiates bone marrow–derived macrophages (BMDMs) into M2 macrophages. Results The M2 expression markers in the TUDCA‐treated BMDM group were increased more than those in the GM‐CSF‐treated BMDM group. After the SCI and transplantation steps, pro‐inflammatory cytokine levels and the mitogen‐activated protein kinase (MAPK) pathway were significantly decreased in the TUDCA‐induced M2 group more than they were in the GM‐CSF‐induced M1 group and in the TUDCA group. Moreover, the TUDCA‐induced M2 group showed significantly enhanced tissue volumes and improved motor functions compared to the GM‐CSF‐induced M1 group and the TUDCA group. In addition, biotinylated dextran amine (BDA)–labelled corticospinal tract (CST) axons and neuronal nuclei marker (NeuN) levels were increased in the TUDCA‐induced M2 group more than those in the GM‐CSF‐induced M1 group and the TUDCA group. Conclusions This study demonstrates that the transplantation of TUDCA‐induced M2 macrophages promotes an anti‐neuroinflammatory effect and motor function recovery in SCI. Therefore, we suggest that the transplantation of TUDCA‐induced M2 macrophages represents a possible alternative cell therapy for SCI., Bone marrow–derived monocytes were differentiated into M2‐phenotype (M2) macrophages using tauroursodeoxycholic acid (TUDCA). TUDCA‐induced M2 macrophages were directly transplanted to a lesion in an injured spinal cord. The pro‐inflammatory cytokine levels were significantly decreased in the TUDCA‐induced M2 group. We suggest that the transplantation of TUDCA‐induced M2 macrophages represents a possible alternative cell therapy for SCI.

Neuroinflammation forms a glial scar following a spinal cord injury (SCI). The injured axon cannot regenerate across the scar, suggesting permanent paraplegia. In this study, we report that d-chiral glutathione (D-GSH) suppresses the inflammatory response after SCI and leads to axon regeneration of the injured spinal cord to a greater extent than l-chiral glutathione (L-GSH). After SCI, axon regrowth in D-GSH-treated rats was significantly increased compared to that in L-GSH-treated rats (***p < 0.001). Secondary damage and motor function were significantly improved in D-GSH-treated rats compared to those outcomes in L-GSH-treated rats (**p < 0.01). Moreover, D-GSH significantly decreased pro-inflammatory cytokines and glial scar via inhibition of the mitogen-activated protein kinase (MAPK) signaling pathway compared to L-GSH (***p < 0.001). In primary cultured macrophages, we found that D-GSH undergoes more intracellular interaction with activated macrophages than L-GSH (***p < 0.001). These findings reveal a potential new regenerative function of chiral GSH in SCI and suggest that chiral GSH has therapeutic potential as a treatment of other diseases.

Gold nanoparticles (GNPs) are widely used in diagnostics, drug delivery, biomedical imaging, and photo-thermal therapy due to their surface plasmon resonance, fluorescence, and easy-surface functionalization. According to recent studies, GNPs display a positive effect on the osteogenic differentiation of mesenchymal stem cells (MSCs) and MC3T3-E1 osteoblast-like cells. The aim of this study was to develop a new approach for bone tissue regeneration based on the utilization of a biodegradable hydrogel loaded with GNPs. We have used photo-curable gelatin hydrogels (Gel) in order to provide a proof of principle of GNPs in regeneration strategies for bone tissue repair. We have investigated the effects of these Gel–GNP composite hydrogels both in vitro and in vivo. The in vitro results showed that the hydrogels loaded with GNPs promote proliferation, differentiation, and alkaline phosphate (ALP) activities of human adipose-derived stem cells (ADSCs) as they differentiate towards osteoblast cells in a dose-dependent manner. Moreover, the in vivo results showed that these hydrogels loaded with high concentrations of GNPs had a significant influence on new bone formation. Through these in vitro and vivo tests, we found that the Gel–GNP can be a useful material for bone tissue engineering.

To treat neuropathic pain, dexamethasone (DEX, an anti-inflammatory agent) and ropivacaine (RVC, a local anesthetic) are injected into the epidural space of the patient's spine in clinics. This combinatorial drug treatment is short-acting and subjected to uncontrolled drug flow to the motor nerve. To overcome these limitations, we developed thiolate poly(lactic-co-glycolic acid) (PLGA-SH) nanofibers conjugated with mono-(6-mercapto-6-deoxy)-β-cyclodextrin (SH-β-CD) containing DEX and RVC. The anti-inflammatory effect in the PGLA-CD-DEX-RVC nanofibers was assessed using bone marrow-derived macrophages (BMMs) in vitro. We injured the sciatic nerve in Sprague Dawley (SD) rats to create a chronic constriction injury (CCI) model for an in vivo assessment. Neuropathic pain was evaluated by testing the cold allodynia response, and by immunofluorescence (IF) staining of transient receptor potential vanilloid 1 (TRPV1, a nociceptor marker) and ionized calcium-binding adaptor molecule 1 (iba1, a microglia marker). In this study, the synthesized PLGA-CD-DEX-RVC nanofibers sustainably released RVC and DEX for more than 48 h. The PLGA-CD-DEX-RVC nanofibers suppressed polarized M1 macrophages and increased polarized M2 macrophages. The allodynia cold sensitivity was consistently relieved in the PLGA-CD-DEX-RVC group for 14 days. Moreover, the expressions of the TRPV1 and iba1 were remarkably reduced in the PLGA-CD-DEX-RVC group on day 14. PLGA-CD-DEX-RVC nanofibers can restrict drugs flow to motor nerves and of relieving pain for extended periods. We suggest that PLGA-CD-DEX-RVC nanofibers can serve as a useful treatment for neuropathic pain in clinics.

Recently, implantable neural electrodes have been developed for recording and stimulation of the nervous system. However, when the electrode is implanted onto the nerve trunk, the rigid polyimide has a risk of damaging the nerve and can also cause inflammation due to a mechanical mismatch between the stiff polyimide and the soft biological tissue. These processes can interrupt the transmission of nerve signaling. In this paper, we have developed a nerve electrode coated with PEG hydrogel that contains poly(lactic-co-glycolic) acid (PLGA) microspheres (MS) loaded with anti-inflammatory cyclosporin A (CsA). Micro-wells were introduced onto the electrode in order to increase their surface area. This allows for loading a high-dose of the drug. Additionally, chemically treating the surface with aminopropylmethacrylamide can improve the adhesive interface between the electrode and the hydrogel. The surface of the micro-well cuff electrode (MCE) coated with polyethylene glycol (PEG) hydrogel and drug loaded PLGA microspheres (MS) were characterized by SEM and optical microscopy. Additionally, the conductive polymers, poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT/PSS), were formed on the hydrogel layer for improving the nerve signal quality, and then characterized for their electrochemical properties. The loading efficiencies and release profiles were investigated by High Performance Liquid Chromatography (HPLC). The drug loaded electrode resulted in a sustained release of CsA. Moreover, the surface coated electrode with PEG hydrogel and CsA loaded MP showed a significantly decreased fibrous tissue deposition and increased axonal density in animal tests. We expect that the developed nerve electrode will minimize the tissue damage during regeneration of the nervous system. Statement of Significance The nerve electrodes are used for interfacing with the central nervous system (CNS) or with the peripheral nervous system (PNS). The interface electrodes should facilitate a closed interconnection with the nerve tissue and provide for selective stimulation and recording from multiple, independent, neurons of the neural system. In this case, an extraneural electrodes such as cuff and perineural electrodes are widely investigated because they can completely cover the nerve trunk and provide for a wide interface area. In this study, we have designed and prepared a functionalized nerve cuff electrode coated with PEG hydrogel containing Poly lactic-co-glycol acid (PLGA) microspheres (MS) loaded with cyclosporine A (CsA). To our knowledge, our findings suggest that surface coating a soft-hydrogel along with an anti-inflammatory drug loaded MS can be a useful strategy for improving the long-term biocompatibility of electrodes.

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

α-asarone, a bioactive compound found in Acorus plant species, has been shown to exhibit neuroprotective, anti-oxidative, anti-inflammatory, and cognitive-enhancing effects. However, the effects of α-asarone on spinal cord injury (SCI) have not yet been elucidated. The present study investigated the effects of α-asarone on the mRNA of pro-inflammatory cytokines, macrophage polarization toward an anti-inflammatory M2 phenotype, and angiogenesis in rats with compressive SCI. α-Asarone was orally administered (10 mg/kg) once per day for 14 days following moderate static compression SCI. Compared to controls, α-asarone treatment significantly improved locomotor score, prevented neuroinflammation, and facilitated angiogenesis in the spinal cord at 14 days after SCI. Furthermore, α-asarone significantly reduced the TNF-α, IL-1β, IL-6, monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 2 (MIP-2), and inducible nitric oxide synthase (iNOS) levels but increased the IL-4, IL-10, and arginase 1 levels at 24 h after SCI. At 7 and 14 days after SCI, immunohistochemistry showed reduced reactive gliosis and neuroinflammation and an increased expression of M2 macrophage markers and angiogenesis. The results suggest that the inhibition of pro-inflammatory cytokines, macrophage polarization toward an anti-inflammatory M2 phenotype, and angiogenesis by α-asarone may be some of the mechanisms underlying the α-asarone-mediated neuroprotective effects on an injured spinal cord.

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.

In the last decade, titanium has been effectively used in the dental field for oral surgery as an implant material. However, disinfected Ti can be easily re-infected by the surrounding environment. Thus, a novel anti-fouling treatment for Ti implants is currently necessary. In this study, we designed an anti-fouling surface comprised of poly N-isopropylacylamide (PIPAAM) grafted Ti by introducing poly glycidyl methacrylate (pGMA) coating via an initiated chemical vapor deposition (iCVD) system to prevent bacterial infection. The results indicate that pristine Ti was well coated with pGMA with a film thickness of approximately 60 nm and uniformly grafted with PIPAAM. The bacteria were effectively detached after rinsing with a buffer solution at room temperature, while hADSCs were well attached on the surface treated Ti surface at oral temperature. All tests clearly confirm that our strategy may be a useful means of imparting anti-fouling characteristics to Ti in order to prevent bacterial adhesion and resultant peri-implantitis.

The aim of this study was to investigate the anti-inflammatory effects by ursodeoxycholic acid (UDCA) in rats with a spinal cord injury (SCI). A moderate mechanical compression injury was imposed on adult Sprague-Dawley (SD) rats. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale and the tissue volume of the injured region was analyzed using hematoxylin and eosin staining. The pro-inflammatory factors were evaluated by immunofluorescence (IF) staining, a quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). The phosphorylation of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 in mitogen-activated protein kinase (MAPK) signaling pathways related to inflammatory responses were measured by Western blot assays. UDCA improved the BBB scores and promoted the recovery of the spinal cord lesions. UDCA inhibited the expression of glial fibrillary acidic protein (GFAP), tumor necrosis factor-α (TNF-α), ionized calcium-binding adapter molecule 1 (iba1), and inducible nitric oxide synthase (iNOS). UDCA decreased the pro-inflammatory cytokines of TNF-α, interleukin 1-β (IL-1β), and interleukin 6 (IL-6) in the mRNA and protein levels. UDCA increased the anti-inflammatory cytokine interleukin 10 (IL-10) in the mRNA and protein levels. UDCA suppressed the phosphorylation of ERK, JNK, and the p38 signals. UDCA reduces pro-inflammatory responses and promotes functional recovery in SCI in rats. These results suggest that UDCA is a potential therapeutic drug for SCI.

Osseointegration is important in osteopenia and osteoporosis patients due to their low bone densities. Gold nanoparticles (GNPs) are greatly beneficial materials as osteogenic agents. The aim of this study is to investigate osseointegration between bones and double layers of GNP-immobilized titanium (Ti) implants. The physicochemical properties of the Ti surface were evaluated by scanning electron microscopy, by atomic force microscopy, by means of the contact angle using water drops, and by x-ray photoelectron spectroscopy. Osteogenic differentiation of human bone-marrow-derived mesenchymal stem cells was analyzed and showed the higher values in double layers of GNP (GNP2) groups. In addition, we performed an in vivo study using hydroxyapatite (HA) and GNP2 spine pedicle screws in ovariectomized (OVX) and SHAM rabbits. Osseointegration parameters also showed higher values in GNP2 than in HA groups. These findings suggest that implants with double layers of GNPs can be a useful alternative in osteoporotic patients.

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.

Spinal cord injury (SCI) causes permanent nerve damage by disturbing axon regeneration from pro-inflammations and still remains a serious worldwide problem. Ursodeoxycholic acid (UDCA) has been reported that it can serve as an anti-neuroinflammatory drug such as Methylprednisolone (MP). However, the therapeutic effect of UDCA is limited due to its hydrophobic property. In this study, we aimed to investigate the anti-neuroinflammatory effects of gold nanoparticles (GNPs) loading UDCA (GNP-UDCA complex) following SCI. For this, we conjugated with β-cyclodextrin (β-CD) on the GNP’s surface and included UDCA in β-CD by sonicating. We confirmed that the GNP-UDCA complex successfully synthesized. A mechanical SCI was imposed on rats and we injected GNP-UDCA complex three times after injury. After SCI, motor function and the lesions were significantly improved in the GNP-UDCA complex group than those in MP group and UDCA group. This complex significantly decreased pro-inflammatory cytokines and increased anti-inflammatory cytokines in terms of mRNA and protein levels than MP and UDCA. In addition, GNP-UDCA complex significantly suppressed the phosphorylation of extracellular signal-regulated kinase (p-ERK) and c-Jun N-terminal kinase (p-JNK) in the mitogen-activated protein kinase (MAPK) pathway than MP and UDCA. Finally, the complex significantly exhibited the expression of inducible nitric oxide synthase (iNOS). It also induced the expression of arginase-1 (Arg-1), anti-inflammatory marker, at the injured sites more than that in the saline group. In conclusion, this study demonstrates the anti-neuroinflammatory effects of GNP-UDCA complex in SCI and suggests that the GNP-UDCA complex can be an alternative drug system for SCI cases.

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.

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.

Poly (L-lactic acid) (PLLA), a biodegradable and biocompatible polyester, has been used as material of for tissue engineered scaffold and regenerative medicine. In this study, hydroxyapatite (HAp) and PLLA composite was electrospun. PLLA fibers were treated with an amino groups containing base in order to fabricate amino groups modified polymeric microcylinders. Simvastatin was loaded into the PLLA/HAp microcylinders. Our study focuses on the incorporation HAp and Simvastatin into the fibrous and cylindrical structure, its effects on the loading and release of simvastatin, and biological responses to osteoblast cells. The aspect ratio of microcylinders was tunable by varying the aminolysis treatment time and density of the base. The effects of the PLLA/HAp composites on osteoblast cell proliferation were evaluated. The PLLA/HAp may interact with osteoblast and obtain desired effects that were stimulating osteoblast function and restraining the osteoclast function for bone tissue regeneration. The potential of the developed composite microcylinders could be a promising way to fabricate the microfibrous structural scaffold that accelerates cell proliferation and functions for bone tissue engineering.

Purpose The aim of this study was to investigate the anti-inflammatory effects of Ursodeoxycholic acid (UDCA) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Methods We induced an inflammatory process in RAW 264.7 macrophages using LPS. The anti-inflammatory effects of UDCA on LPS-stimulated RAW 264.7 macrophages were analyzed using nitric oxide (NO). Pro-inflammatory and anti-inflammatory cytokines were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The phosphorylations of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 in mitogen-activated protein kinase (MAPK) signaling pathways and nuclear factor kappa-light polypeptide gene enhancer in B-cells inhibitor, alpha (I kappa B alpha) signaling pathways were evaluated by western blot assays. Results UDCA decreased the LPS-stimulated release of the inflammatory mediator NO. UDCA also decreased the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin 1-alpha (IL-1 alpha), interleukin 1-beta (IL-1 beta), and interleukin 6 (IL-6) in mRNA and protein levels. In addition, UDCA increased an anti-inflammatory cytokine interleukin 10 (IL-10) in the LPS-stimulated RAW 264.7 macrophages. UDCA inhibited the expression of inflammatory transcription factor nuclear factor kappa B (NF-kappa B) in LPS-stimulated RAW 264.7 macrophages. Furthermore, UDCA suppressed the phosphorylation of ERK, JNK, and p38 signals related to inflammatory pathways. In addition, the phosphorylation of I kappa B alpha, the inhibitor of NF-kappa B, also inhibited by UDCA. Conclusion UDCA inhibits the pro-inflammatory responses by LPS in RAW 264.7 macrophages. UDCA also suppresses the phosphorylation by LPS on ERK, JNK, and p38 in MAPKs and NF-kappa B pathway. These results suggest that UDCA can serve as a useful anti-inflammatory drug.

In recent years, gold nanoparticles (GNPs) have been reported to affect the regeneration of bone tissue. The goal of this study was to improve bone tissue regeneration by using targeted GNPs. We fabricated a functionalized GNPs conjugated with alendronate (ALD), of the bisphosphonate group. Subsequently, the ALD, GNPs, and ALD conjugated GNPs (GNPs-ALD) were analyzed by ultraviolet-visible absorbance (UV-vis) spectrophotometer, Attenuated total reflectance Fourier transform infrared spectrometer (ATR-FTIR), and thermo gravimetric analysis (TGA). The prepared GNPs-ALD were used to investigate their inhibitory effects on the receptor activator of nuclear factor- κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). Additionally, the GNPs-ALD were applied to ovariectomy (OVX)-induced osteoporotic mice and the experiments were evaluated. ALD was found to be successfully conjugated to the GNPs surface, and it displayed significant adhesion onto the bone surface. The in-vitro study indicated that the GNPs, ALD and GNPs-ALD suppressed osteoclast formation in a dose-dependent manner. Furthermore, in the OVX mouse model, the mice treated GNPs-ALD had higher bone density as compared to other OVX mice groups. The results from these tests indicated that GNPs-ALD can be useful agents for preventing and treating osteoporosis.

Baicalin (BC) has been used for cancer therapy due to its multiple effects as an anti-cancer drug. However, the effective delivery of this molecule to targeted cells is difficult. Gold nanoparticles (AuNPs) conjugated with thiolated beta cyclodextrin (AuNP-S-β-CD) were used as a delivery vector in this study. Cell viability tests were evaluated by cell counting kit-8 (CCK) and live/dead cell assay. To demonstrate the proliferation inhibition effect on Michigan Cancer Foundation-7 (MCF-7) cells by BC, we analyzed using Hoechst 33342 staining assay and gel electrophoresis. The S-β-CD conjugated AuNPs were characterized by transmission electron microscopy (TEM), 1H nuclear magnetic resonance ((1)H NMR), and ultraviolet visible (UV-vis) spectroscopy. AuNP-S-β-CD with approximately 40 μM of BC loaded by inclusion complex showed an inhibition effect on MCF-7 cells by inducing apoptosis. Apoptosis test results were evaluated by analyzing the expression of typical apoptic markers such as cleaved caspase-3, full length caspase-3, and apaf-1 in western blot assay. These results demonstrated that AuNP-S-β-CD-BC inhibited the proliferation of cancerous MCF-7 cells by inducing apoptosis. These findings suggested that AuNP-S-β-CD-BC could be a promising agent for chemotherapeutic usage.

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.

Gold nanoparticles (GNPs) are quite attractive materials for use as osteogenic agents due to their potential effects on the stimulation of osteoblast differentiation. In this study, an osseo-integrated titanium (Ti) implant surface coated with GNPs was used for promotion of bone regeneration. We prepared a silanized Ti surface by chemical treatment of (3-Mercaptopropyl) trimethoxysilane (MPTMS) and immobilized the GNP layer (Ti-GNP) on their surfaces via Au-S bonding. The GNP layer is uniformly immobilized on the surface and the layer covers the titanium oxide surface well, as confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The Ti-GNP was used to investigate the effectiveness of this system both in vitro and in vivo. The in vitro results showed that the Ti-GNP significantly enhances the osteogenic differentiation with increased mRNA expression of osteogenic differentiation specific genes in human adipose-derived stem cells (ADSCs). Furthermore, the in vivo results showed that Ti-GNP had a significant influence on the osseous interface formation. Through these in vitro and vivo tests, we found that Ti-GNP can be useful as osseo-integration inducing dental implants for formation of an osseous interface and maintenance of nascent bone formation.

Electrospun chitosan (CTS) nanofibers have been well known for use as a wound dressing in the biomedical field. Nevertheless, fatal bacterial infections are still a serious problem when CTS nanofibers are used for wound treatment. In this study, we designed a novel wound dressing based on blending the chitosan with polyurethane (CTS/PU) containing silver sulfadiazine (AgSD) in order to enhance both antibacterial activity and mechanical strength. This fiber sheet was produced using the electrospinning (ELSP) technique. The CTS/PU containing AgSD fiber sheet was characterized by energy-dispersive X-ray spectroscopy (EDX). The physicochemical properties of the CTS/PU/AgSD fiber sheets were also characterized by thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FT-IR). The electrospun fibers were morphologically characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For an in vitro evaluation, the CTS/PU/AgSD fiber sheets were tested for their antibacterial activity against gram-negative Pseudomonas aeruginosa (P. aeruginosa), gram-positive Staphylococcus aureus (S. aureus) and Methicillin-resistant Staphylococcus aureus (MRSA). The results indicate that CTS/PU/AgSD fiber sheets have strong antimicrobial activity as displayed by inhibition of bacterial growth and prevention of infection during the healing process. These results indicate that this material would be good for use as a wound dressing material.

There have been many medical applications based on gold nanoparticles (GNPs) over the past several centuries. Recently, researchers have focused on bone tissue engineering applications utilizing GNPs. The effect of various sizes of gold nanoparticles on the differentiation of human adipose-derived stem cells (ADSCs) into osteoblasts was investigated. The concentration of gold nanoparticles was fixed at 1 μM and varying sizes of 15, 30, 50, 75 and 100 nm (spherical GNPs) were used. The lack of cytotoxicity was confirmed by establishing viability of ADSCs using cell counting kit-8 (CCK-8) and live/dead assays. The results showed that each size of GNPs had no significant toxicity on ADSCs during 1 week of incubation. Osteogenic differentiation of ADSCs was confirmed by alkaline phosphatase (ALP) staining, ALP activity, calcium deposition, and real time PCR experiments. It was found, through dark field assays and microscope cell images, that 30 nm and 50 nm GNPs were preferentially up taken into the ADSCs. As expected, all sizes of gold nanoparticles promoted the differentiation of ADSCs toward osteoblasts more than control. Among all sizes, 30 and 50 nm GNPs appeared to have the highest differentiation rates. The data consistently demonstrated that 30 and 50 nm GNPs are the most effective in promoting osteogenic differentiation of ADSCs.

The ideal wound dressing would have properties that allow for absorption of exudates, and inhibition of microorganism for wound protection. In this study, we utilized an electrospinning (ELSP) technique to design a novel wound dressing. Chitosan (CTS) nanofibers containing various ratios of silver nanoparticles (AgNPs) were obtained. AgNPs were generated directly in the CTS solution by using a chemical reduction method. The formation and presence of AgNPs in the CTS/AgNPs composite was confirmed by x-ray diffraction (XRD), ultraviolet-visible spectroscopy (UV) and thermogravimetric analysis (TGA). The electrospun CTS/AgNPs nanofibers were characterized morphologically by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). These nanofibers were subsequently tested to evaluate their antibacterial activity against gram-negative Pseudomonas aeruginosa (P. aeruginosa) and gram-positive Methicillin-resistant Staphylococcus aureus (MRSA). Results of this antibacterial testing suggest that CTS/AgNPs nanofibers may be effective in topical antibacterial treatment in wound care.

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.

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.