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The binding between receptor-activated nuclear factor kappa B (RANK) and its specific ligand (RANKL) during osteoclast development is an important target for drugs that treat osteoporosis. Recently, the leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) was reported as a negative regulator of RANKL-RANK signaling that suppresses canonical RANK signaling during osteoclast differentiation. Hence, LGR4 agonists may be useful in inhibiting osteoclastogenesis and effectively treating osteoporosis. In this study, we used bone marrow-derived macrophages (BMDM) and a mouse model of RANKL-induced bone loss to investigate the effect of a RANKL-derived mutant, (MT RANKL), which was previously developed based on the crystal structure of the RANKL complex. In the present study, the binding affinity of wild-type (WT) RANKL and MT RANKL for RANK and LGR4 was determined using microscale thermophoresis analysis, and the effect of the ligands on the AKT-GSK-3β-NFATc1 signaling cascade was investigated using western blotting and confocal microscopy. In addition, the immunopositive expression of LGR4 and the colocalization of LGR4 and MT RANKL were analyzed in a mouse model of RANKL-induced bone loss. The results showed that in osteoclast precursor cells, MT RANKL bound with high affinity to LGR4, decreased AKT phosphorylation, and increased GSK-3β phosphorylation, resulting in the inhibition of NFATc1 nuclear translocation. In the mouse model, MT RANKL upregulated LGR4 expression, colocalized with LGR4, and inhibited bone resorption. These results indicate that MT RANKL, which is derived from RANKL and inhibits RANKL-induced osteoclastogenesis through an LGR4-dependent pathway, may be useful in the treatment of osteoporosis.

A small fraction of cancer cells known as cancer stem cells (CSCs) are considered to give rise to differentiated cancer cells and have been proposed to predict cancer recurrence and metastasis. There is further evidence that CSCs may act as metastatic precursors of epithelial-mesenchymal transition (EMT). In the present study, we investigated the key molecules involved in maintaining the stability of CSCs by inducing ectopic overexpression of CD133 to characterize EMT in human prostate cancer cell lines, including PC-3, DU145, and LnCaP cells. Additionally, we investigated whether a specific inhibitor of concomitantly expressed metastasis-related genes could alleviate EMT properties in CD133-overexpressing prostate cancer cells. Ectopic overexpression of CD133 in PC-3, DU145, and LnCaP cells led to an increase in the expression of HDAC9. Moreover, HDAC9 inhibition led to a decrease in EMT properties along with increased E-cadherin expression, a narrower wound gap distance, and enhanced cell invasiveness through the suppression of β-catenin activation and its translocation to the nucleus. Overall, these results suggest that HDAC9 inhibition plays a functional role in the modulation of EMT properties in CSC-like prostate cancer cells. Therefore, these findings could facilitate the development of therapeutic strategies for controlling prostate cancer metastasis.

Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa. RANKL was associated with epithelial–mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Therefore, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of β-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and β-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis. Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCa.

Background: Sphingosine kinase 1 (SPHK1) and heat shock protein 27 (HSP27) are important for antioxidant and anti-inflammatory effects after red light irradiation in an inflammatory model. Objectiv...

Osteoporosis is a common disease that affects millions of patients worldwide and is most common in menopausal women. The main characteristics of osteoporosis are low bone density and increased risk of fractures due to deterioration of the bone architecture. Osteoporosis is a chronic disease that is difficult to treat; thus, investigations into novel effective therapeutic methods are required. A number of studies have focused on determining the most effective treatment options for this disease. There are several treatment options for osteoporosis that differ depending on the characteristics of the disease, and these include both well-established and newly developed drugs. The present review focuses on the various drugs available for osteoporosis, the associated mechanisms of action and the methods of administration.

The use of multi-porous scaffolds for bone tissue engineering has been shown to improve osteogenesis. Although the exact mechanisms by which these scaffolds promote new bone formation have not yet been recognized well, related hypotheses can be found in many previous studies. The aim of this study was to examine the influence of the modulation of the pore size of beta- TCP ceramics on osteogenic differentiation in rats with calvarial defects. Treatments with macro- and meso-sized particles of NaCl, which was used as a porogen, were carried out during scaffold manufacturing, and the pore sizes of the beta-TCP scaffolds were measured by SEM. New bone formation was evaluated by Micro-CT, H&E staining, and immuno-histochemical analysis at week 4 after the operation. It was observed that the multi porosity of beta-TCP ceramics was controlled by treating the scaffolds with NaCl particles of two sizes (500–800 μm and 10–50 μm) during the firing process. Histological and immunohistochemical analysis of type 1 collagen and osteocalcin protein expression showed that the new bone formation and mineralization in case of the multi-porous beta-TCP ceramics comprising macro-and meso-sized pores were significantly higher than those in case of single porous macro- or meso-sized scaffolds. In conclusion, the distribution and size of the pores, as well as the surface structure of the scaffolds, may play an important role for osteogenic differentiation in vivo.

Breast cancer cells often metastasize to bone. Accumulating evidence suggests that inhibiting the receptor activator of nuclear factor-κB ligand (RANKL) not only leads to reduced bone metastasis of breast cancer but also has antitumoral effects. Here, we used mutant receptor activator of nuclear factor-κB ligand (RANKLM) as a vaccine for active immunization to induce antibodies for immunotherapy of bone metastatic cancer. We investigated whether anti-RANKL antibodies inhibit osteolytic bone metastasis in vitro and in a murine model. MC3T3 cells stimulated by MDA-MB-231 culture medium secreted growth differentiation factor-15 (GDF-15), which induced the nuclear factor-κB signaling cascade. In addition, RANKLM treatment-induced reduction of intraosseous growth of MDA-MB-231 cells correlated with decreased GDF-15 expression, a reduced number of osteolytic lesions, and slower tumor progression. In addition, vaccination with RANKLM led to significant improvement in overall survival and skeletal metastasis in tumor-bearing mice. Induction of anti-RANKL antibodies by RANKLM decreased GDF-15 production by deactivating nuclear factor-κB signaling, which in turn inhibited metastasis of MDA-MB-231 cells to bone. Taken together, the results demonstrate a role for RANKLM immunization in preventing bone metastasis of breast cancer.

Background The discovery of receptor activator of nuclear factor‐ĸB ligand (RANKL) as the final effector in the pathogenesis of osteoporosis has led to a better understanding of bone remodeling. When RANKL binds to its receptor (RANK), osteoclastic differentiation and activation are initiated. Herein, we propose a strategy using a novel RANKL variant as a competitive inhibitor for RANKL. The RANKL variant activates LGR4 signaling, which competitively regulates RANK and acts as an immunogen that induces anti‐RANKL antibody production. Methods We modified the RANK‐binding site on RANKL using minimal amino acid changes in the RANKL complex and its counterpart receptor RANK and tried to evaluate the inhibitory effects on osteoclastogenesis. Results The novel RANKL variant did not bind RANK in osteoclast progenitor cells, but activated LGR4 through the GSK3‐β signaling pathway, thereby suppressing activated T cell cytoplasmic nuclear factor calcineurin‐dependent 1 (NFATc1) expression and activity during osteoclastogenesis. Our RANKL variant generated high levels of RANKL‐specific antibodies, blocked osteoclastogenesis, and inhibited osteoporosis in ovariectomized mouse models. Generated anti‐RANKL antibodies showed a high inhibitory effect on osteoclastogenesis in vivo and in vitro. Conclusions We observed that the novel RANKL indeed blocks RANKL via LGR4 signaling and generates anti‐RANKL antibodies, demonstrating an innovative strategy in the development of general immunotherapy., Schematic diagram of mRANKL‐MT3 in dual inhibitory effect against RANKL during osteoclastogenesis. The first effect of mRANKL‐MT3 is induced to RANKL‐LGR4 modulation of the RANKL–NFATc1 signaling cascade by a negative‐feedback mechanism, which controls osteoclast activity. The second effect is induced to anti‐RANKL generation by mutant RANKL, which inhibits osteoclastogenesis and bone erosion.

Background: Prostate cancer (PCa) morbidity in the majority of patients is due to metastatic events, which are a clinical obstacle. Therefore, a better understanding of the mechanism underlying metastasis is imperative if we are to develop novel therapeutic strategies. Receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL) regulates bone remodelling. RANKL was associated with epithelial-mesenchymal transition (EMT) and expression of metastasis-related genes in PC3 cells. Thus, agents that suppress RANKL signalling may be useful pharmacological treatments. Method: In this study, we proposed a strategy to induce anti-cytokine antibodies using mutant RANKL as an immunogen. Here, we used preclinical experimental models to investigate whether an inactive form of RANKL affects bone metastasis in RANKL-induced PCa.Results: RANKL activation was observed in human PCa tissue specimens. RANKL promoted migration and invasion of PC3 cells through EMT, and induced a significant increase in binding of β-catenin to TCF-4, an EMT-induced transcription factor in PCa cells, via mitogen-activated protein kinase and β-catenin/TCF-4 signalling. Thus, RANKL increased EMT and the metastatic properties of PC3 cells, suggesting a role as a therapeutic target to prevent PCa metastasis. Conclusion: Treatment with mutant RANKL reduced EMT and metastasis of PC3 PCa cells in an experimental metastasis model. Thus, mutant RANKL could serve as a potential vaccine to prevent and treat metastatic PCaTrial registration: Chosun University Hospital, CHOSUN 2020-06-001. Registered 01 June 2020-prospectevely registered, https://hosp.chosun.ac.kr/medi_depart/ site=hospital&mn=151&type=view&catename=IRB

In this study, we prepared a beta-cyclodextrin (β-CD)-based carrier consisting of β-CD, polyethylene glycol (PEG) and folic acid (FA) (CDPF) for improved doxorubicin (DOX) delivery to targeted breast cancer in vitro and in vivo. The morphology and size distribution were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements, which revealed in particles ranging from 38 nm to 52 nm in size. DOX from CDPF was released in a sustained manner for 48 h. Cell viability analysis showed the low toxicity of free DOX, whereas CDPF-DOX remarkably exhibited reduced viability after incubation for 7 days. In vivo animal test showed that intravenous injection of CDPF-DOX contributes to decreased tumor volume, along with no systemic toxicity and cardiotoxicity. The results suggested that CDPF can maximize the efficacy of DOX delivery; therefore can be used as a good candidate for developing optimal drug delivery systems.

Precise photothermal cancer therapy still relies on the development of multifunctional theranostic agents to integrate tumor-specific targeting, imaging, and therapy. In this study, we identified the zwitterionic near-infrared (NIR) fluorophore ZW800-Cl, an analog of the well-known ZW800-1, and found that it preferentially accumulated in tumors in various xenograft models. We have demonstrated that the optical and physicochemical properties of ZW800-Cl are similar to those of ZW800-1, but it has a unique tumor targetability. Since ZW800-Cl showed binding selectivity to cancer cells in vitro, it could be specifically targeted to xenograft models of multiple tumor types, including MCF-7, NCI-H460, and HT-29. The in vivo results of photothermal cancer therapy indicated that the xenograft tumors could be effectively ablated by the combination of ZW800-Cl and an 808 nm laser irradiation. In this regard, our finding may provide a new approach for developing multifunctional cancer theranostic agents, setting them apart from conventional nanoparticles based on inorganic and polymeric materials. Therefore, ZW800-Cl may hold promise as a multifunctional theranostic agent for tumor-targeted imaging and effective photothermal cancer therapy.

This study evaluated the near-infrared (NIR) fluorescent probe Sorbitol-ZW800-1 as tracer for targeted tumor imaging of multiple cancer types. Preliminarily, Sorbitol-ZW800-1 conjugate was validated in vivo on breast (MCF-7) cancer cell lines. Subsequently, the tracer was tested in xenograft mouse models with malignant breast (MDA-MB-231), lung (NCI-H460), and colorectal (HT-29) tumors, respectively. Importantly, the Sorbitol-ZW800-1 conjugate showed highly selective tumor uptake in various tumor mice models. Moreover, biodistribution for 24 h exhibited the significant fluorescence signals in tumors and kidneys. Therefore, sorbitol is a promising candidate as a potential cancer targeting agent for the accurate detection of cancer cells.

Background: Evans blue dye (EBD) is the most common indicator to analyze the extent of blood-brain barrier (BBB) breakdown in several neurological disease models. However, the high-dose of EBD (51.9 mg/kg) is usually required for visualization of blue color by the human eye that brings potential safety issues. Methods: To solve this problem, low-dose of EBD was applied for the near-infrared (NIR) fluorescence-assisted quantitation of BBB breakdown in photothrombotic stoke model. Animals were allocated to seven dose groups ranging from 1.35 nmol (5.19 μg/kg) to 13.5 μmol (51.9 mg/kg) EBD. Results: EBD was undetectable in the non-ischemic brain tissue, and the fluorescence signals in the infarcted hemisphere seemed proportional to the injected dose in the dose range. Although the maximum fluorescence signals in brain tissue were obtained with the injections of 1.35 nmol ~ 13.5 μmol EBD, the background signals in the neighboring brain tissues were significantly increased as well. Since the high concentration of EBD is necessary for color-based identification of the infarcted lesion in brain tissues, even 10-fold diluted could not be distinguished visually by naked eye. Conclusions: NIR fluorescence-assisted method could potentially provide new opportunities to study BBB leakage just using small amount of EBD in different pathological conditions and to test the efficacy of various therapeutic strategies to protect the BBB.

Osteopetrosis belongs to a group of rare genetic diseases typically treated with bone marrow transplantation. This approach is not effective in a recently identified form of the disease caused by mutations in the receptor activator of NF-κB ligand (RANKL) gene. In these patients, replacement therapy and RANKL delivery may be a more valid approach than transplantation. Here, we describe the construction of a recombinant gene encoding regions of RANKL (rRANKL), including the biologically active regional loop sequence. We present detailed methods for the cloning, expression, and purification of the recombinant protein. The activity of rRANKL including the active region was assessed in vitro and mature osteoclast generation was evaluated in vivo using a mouse model. We provide a proof of concept for the therapeutic potential of full-length and selected active regions of rRANKL in the treatment of osteopetrosis, warranting clinical assessment.

// Wonbong Lim 1, 2, 3, * , HongMoon Sohn 1, 2, * , Youngjong Ko 1, 2 , Mineon Park 1, 2 , Bora Kim 1, 2 , Danbi Jo 4 , Jin Seok Jung 4 , Dae Hyeok Yang 5 , Jangho Kim 6 , Ok Joon Kim 7 , Donghwi Kim 1, 2 , Young Lae Moon 1, 2 , Jung-Joon Min 8 and Hoon Hyun 4 1 Department of Orthopedic Surgery, Chosun University Hospital, Gwangju 61453, South Korea 2 Laboratory of Orthopedic Research, Chosun University Hospital, Gwangju 61453, South Korea 3 Department of Premedical Program, School of Medicine, Chosun University, Gwangju 61452, South Korea 4 Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju 61469, South Korea 5 Institute of Cell and Tissue Engineering, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea 6 Department of Rural and Biosystems Engineering, Chonnam National University, Gwangju 61186, South Korea 7 Department of Oral Pathology, School of Dentistry, Chonnam National University, Gwangju 61186, South Korea 8 Department of Nuclear Medicine, Chonnam National University Medical School, Gwangju 61469, South Korea * Authors contributed equally to this work Correspondence to: Hoon Hyun, email: hhyun@jnu.ac.kr Keywords: metastatic bone tumor, real-time in vivo imaging, near-infrared fluorescence, targeted fluorophore Received: July 07, 2017 Accepted: August 04, 2017 Published: August 11, 2017 ABSTRACT Tumors of the prostate or breast are particularly likely to metastasize to the bone, and early diagnosis of metastatic bone tumors is important for designing an effective treatment strategy. Imaging modalities for the detection of bone metastasis are limited, and radiation-based techniques are commonly used. Here, we investigated the efficacy of selective near-infrared (NIR) fluorescence detection of metastatic bone tumors and its role in the detection of bone metastasis in prostate and breast cancer cell lines and in a xenograft mouse model. A targeted NIR fluorophore was used to monitor metastatic bone tumors using a NIR fluorescence imaging system in real time, enabling the diagnosis of bone metastasis in vivo by providing the location of the metastatic bone tumor. The NIR fluorescence imaging technique using targeted NIR contrast agents is a potential tool for the early diagnosis of bone tumors.

Prostate cancer (PC) metastasizes to the bone, and a small number of cancer cells, described as cancer stem cells (CSCs), have the ability to differentiate into tumor cells. CSCs are responsible for tumor recurrence and metastases. In the present study, we examined whether ectopic overexpression of CD133, a key molecule maintaining the stability of CSCs in the human PC cell line, LnCaP, caused bone metastasis in a mouse model. Ectopic overexpression of CD133 was induced in LnCaP cells, and CSC-related protein expression was measured. Furthermore, a colony-forming assay was performed to compare results against the blank green fluorescent protein-expressing cells. Furthermore, epithelial to mesenchymal transition-related protein expression, cell migration and wound healing were investigated. To assess the role of CD133 in bone metastasis, CD133-overexpressing LnCaP cells were inoculated into mice via intracardiac injection, and bone metastasis was assessed via histological and immunohistochemical study. In addition, cytokine arrays were used to determine the cytokines involved in bone metastasis. Ectopic overexpression of CD133 in LnCaP cells increased CSC properties such as Oct-4 and Nanog expression and colony-forming ability. Furthermore, epithelial-to-mesenchymal transition (EMT) properties, including decreased E-cadherin and increased vimentin expression, wound gap distance, and cell migration increased. CD133 overexpression led to formation of bone metastatic tumors in mice, consistent with results of hematoxylin and eosin staining. In addition, an increase in expression of the macrophage-migration inhibitory factor was observed at the tumor margin in mice inoculated with CD133+ LNCaP cells. These findings suggest a regulatory role of CD133 in stem cell and EMT properties, and the sustained acquisition of osteolytic features in PC. Therefore, our results may facilitate development of a novel classification system and therapeutic strategies for bone metastasis of PC.

Wnt3a is a glycosylated ligand that activates the β-catenin-dependent signaling pathway. Wnt signaling is also important in the prostate tumor microenvironment, and Wnt proteins secreted by the tumor stroma promote resistance to therapy. Bioactive Wnt3a production requires a number of dedicated factors in the secretory cell, but their coordinated functions are not fully understood. We previously reported transmembrane protein 64 (Tmem64) as a novel regulator of the Wnt/β-catenin signaling pathway, which is correlated with β-catenin regulation. In the present study, the role of Tmem64 in prostate cancer cells was investigated by modulating Wnt3a secretion. Overexpression of Tmem64 inhibited Wnt3a secretion and Lef/Tcf-sensitive transcription. By contrast, a Tmem64 mutation deleting the protein's transmembrane region restored Wnt3a secretion. Notably, Tmem64 protein and mRNA in PC3 cells were significantly overexpressed compared with that observed in LNCaP and DU145 cells. In a mouse metastasis model intracardially injected with PC3 cells, Tmem64 expression was downregulated in the metastatic spine and mandible lesions compared with in the primary injection regions. However, Wnt3a was strongly expressed in the metastatic spine and mandible lesions. Collectively, these findings suggest that Tmem64 is involved in the metastatic progression of prostate cancer cells by regulating Wnt3a secretion.

Bone is the most common site of prostate cancer (PC) metastasis. Studies suggest that cancer stem cells (CSCs) are associated with stemness characteristics, providing some support for the concept that CSCs act as osteosclerotic precursors in bone microenvironmental niches. Here, we asked whether ectopic overexpression of CD133 maintains stability of CSCs in human PC cell lines and induces the changes of molecular features in the bone microenvironment. Ectopic overexpression of CD133 in PC3 or DU145 cells led to increased expression of ALDHA1, OCT4, and NANOG, enhanced colony‐forming ability, and increased ALDH activity. In addition, micro‐CT imaging, confocal microscopy, and H&E staining of mouse tissue confirmed that CD133 overexpression in PC3 and DU145 led to marked osteolytic bone tumor. However, expression of osteoblastic markers such as collagen type I, bone sialoprotein, and osteocalcin (OC) at the tumor margin of CD133‐overexpressing PC3 tumors in mouse tibiae was higher than that of CD133‐overexpressing DU145 tumors with osteosclerotic molecular features. In addition, expression of osteopontin (OPN) mRNA/protein by CD133‐overexpressing PC3 cells was higher than that by DU145 cells. Especially, conditioned medium (CM) from PC3CD133+ cells increased osterix (OSX) activity in bone marrow stromal cells (BMSCs), resulting in increased expression of OC mRNA/protein resulted in increased staining of mineralized matrix by Alizarin red. However, CM from OPN silenced PC3CD133+ cells led to a reduction of OC mRNA and protein expression through OSX activity resulted in reduced amount of mineralized matrix. In conclusion, these findings suggest that CD133 plays a functional role in regulating CSC characteristics in PCs and modulates their abilities in which induce the osteosclerosis of BMSCs. In addition, OPN from CSCs acts as a niche component that promotes osteosclerosis by supporting osteoblastic differentiation of BMSCs. © 2019 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Here, we proposed the use of mutated RANKL as an immunogen for active immunization and to induce anti-cytokine antibodies for osteoporosis treatment. Osteoclasts are responsible for bone resorption in bone-related disorders. Anti-cytokine therapeutic antibodies such as denosumab are effective for the treatment of osteoporosis. However, problems with antibody manufacturing and the immunogenicity caused by multiple antibody doses have led to the use of auto-cytokines as immunogens to induce anti-cytokine antibodies. RANKL was point-mutated based on the crystal structure of the complex of RANKL and its receptor RANK. As a proof of concept, immunization with RANKL produced high levels of specific antibodies and blocked osteoclast development in vitro and inhibited osteoporosis in RANKL-treated or ovariectomized mouse models. The results demonstrate the successful use of mutated RANKL as an immunogen for the induction of anti-RANKL immune response. This strategy is useful in general anti-cytokine immunotherapy to avoid toxic side effects of osteoporosis treatment.

최근 의료 영상 기술의 발전은 진단, 수술계획, 또는 교육에 도움이 되는 수술 시뮬레이션을 만들어 왔다. 개선된 고화질 영상과 3차원 시각화는 의료 영상 가용성을 향상시키고 수술, 교육 분야에서 더 잘 이용할 수 있게 되었다. 실제 인간의 시각은 입체이다. 따라서, 외과의사의 판단을 통해 2차원 영상을 스테레오로 재구성하여 처리하는 것이 함께 필요하다. 이러한 과정을 줄이기 위해, 3차원 (3D) 이미지가 사용되어 왔다. 3D 영상은 복잡한 상황에서 외과 의사가 매우 짧은 시간에 판단할 수 있도록 3D 시각화를 강화하여 제공한다. 3D 화상 데이터 세트에 기초하여, 가상 내시경 수술 계획, 실시간 상호 작용 가상 의료시뮬레이션이 가능하게 되었다. 본 논문은 새로운 이미징 기술의 최근 응용 프로그램을 설명하고 이의 기본과 특별히 주목할 만한 의료 3D 복원 기술에 관한 것이다. 최근 CT, MR 및 기타 영상 양식의 기술발전은 흥미로운 새로운 솔루션과 어깨 영상의 활용 가능성을 넓혀왔다. 특히, 의료 기기에서 파생 된 3차원 (3D) 이미지는 고급 정보를 제공한다. 이 프레젠테이션은 어깨와 팔꿈치의 수술실습에서 원리, 3D 영상기술의 잠재적 응용가능성, 시뮬레이션, 3D프린팅을 설명한다.

One of the theories on cancer stem cells (CSCs) states that these cells initiate most tumors and give rise to more-or-less differentiated tumor cells. Genetic signatures of CSCs are thought to predict tumor recurrence and metastases, thus, supporting the notion that CSCs may be metastatic precursors and induce epithelial-to-mesenchymal transition (EMT). In this study, we tried to examine the association between CSCs and EMT (using specific markers) in the mucoepidermoid carcinoma cell line YD15 and its derivative cell line YD15M (lymph node metastasis). Relative protein expression levels were analyzed by western blotting, flow cytometry, and immunofluorescence assays. In addition, cell cycle assay and aldehyde dehydrogenase (ALDH) activity assay were carried out. Under growth conditions, YD15M cells formed irregular spherical colonies consistent with a stem cell phenotype. YD15M cells demonstrated the low expression of E-cadherin and β-catenin but high expression of vimentin than that in YD15 cells. In the metastatic cells (YD15M), the coexpression of vimentin and CD133 was detected. Weak proliferation based on cell cycle analysis and decreased PCNA expression was also observed. In addition, expression levels of ALDHA1, OCT4, and NANOG (CSC-like properties) were significantly increased in YD15M cells. Taken together, these findings should help to elucidate the interplay between EMT and CSC-like properties during metastasis and may provide useful information for the development of a novel classification system and therapeutic strategies against head and neck cancer.

Macrophage-colony stimulating factor (M-CSF) has been reported to be required for the proliferation and differentiation of macrophages from hematopoietic progenitor cells. Recently, recombinant M-CSF (rM-CSF) became widely used as a biological research reagent in bone marrow stimulations, vaccine development, gene therapy approaches, and stem cell mobilization. rM-CSF is a glycoprotein that activates and enhances the differentiation and survival of macrophages, which play a key role in the osteoclastogenetic response. Here, we describe the construction of the gene encoding rM-CSF, its cloning, and expression in Escherichia coli, as well as the purification of rM-CSF protein, and its activity in a biological assay in mouse bone marrow cells. Our results show that the combination of experimental strategies employed to obtain recombinant rM-CSF can yield a biologically active protein, and may be useful when scaling-up production of other biologically similar proteins.

Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world. In OSCC, CD133 promotes tumor invasion and metastasis by inducing the epithelial-to-mesenchymal transition (EMT). A small subset of cancer cells known as cancer stem cells (CSCs) are thought to give rise to differentiated tumor cells and to predict tumor recurrence and metastases, i.e., CSCs may be metastatic precursors. In this study, we show that ectopic overexpression of CD133 in OSCC cell lines KB, YD9, and YD10B cells significantly promotes the EMT and acquisition of stemness properties. CSC properties were analyzed by colony-formation assay and measurement of OCT4, SOX2, and NANOG expression, and the EMT was monitored by cell migration, a cell invasion assay, and analysis of E-cadherin, N-cadherin, and vimentin expression. CD133 overexpression led to formation of irregular spheroid colonies consistent with a stem cell phenotype and increased the expression of OCT4, SOX2, NANOG, N-cadherin, and vimentin. Taken together, these findings show that elevated levels of CD133 lead to OSCC invasiveness and metastasis, associated with the upregulation of EMT and stemness markers.

Background: Recently, it was reported that leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4, also called GPR48) is another receptor for RANKL and was shown to compete with RANK to bind RANKL and suppress canonical RANK signaling during osteoclast differentiation. The critical role of the protein triad RANK&ndash, RANKL in osteoclastogenesis has made their binding an important target for the development of drugs against osteoporosis. In this study, point-mutations were introduced in the RANKL protein based on the crystal structure of the RANKL complex and its counterpart receptor RANK, and we investigated whether LGR4 signaling in the absence of the RANK signal could lead to the inhibition of osteoclastogenesis., Methods: The effects of point-mutated RANKL (mRANKL-MT) on osteoclastogenesis were assessed by tartrate-resistant acid phosphatase (TRAP), resorption pit formation, quantitative real-time polymerase chain reaction (qPCR), western blot, NFATc1 nuclear translocation, micro-CT and histomorphological assay in wild type RANKL (mRANKL-WT)-induced in vitro and in vivo experimental mice model. Results: As a proof of concept, treatment with the mutant RANKL led to the stimulation of GSK-3&beta, phosphorylation, as well as the inhibition of NFATc1 translocation, mRNA expression of TRAP and OSCAR, TRAP activity, and bone resorption, in RANKL-induced mouse models, and Conclusions: The results of our study demonstrate that the mutant RANKL can be used as a therapeutic agent for osteoporosis by inhibiting RANKL-induced osteoclastogenesis via comparative inhibition of RANKL. Moreover, the mutant RANKL was found to lack the toxic side effects of most osteoporosis treatments.

Bioluminescence imaging is being increasingly utilized in biological research. However, since the most commonly used firefly luciferase generates relatively weak bioluminescent signals, detection of low numbers of luciferase-expressing cells in vivo is challenging. The weak signal makes it difficult to detect cells located in deep tissues, which is problematic for preclinical research in tumor metastasis. In this study, three different types of fluorophores such as D-luciferin, AkaLumine-HCl, and P800SO3 were compared to evaluate the progression of bone metastasis induced by MDA-MB-231 breast cancer cells in vivo. The fluorescent signals for D-luciferin, AkaLumine-HCl, and P800SO3 were differently detected in the chest and knee joint. In particular, the fluorescence signal of P800SO3 was clearly observed in a section of the ribs, where it pointed out fractured bone fragments by tumor mass. Moreover, the P800SO3 signal from the left knee joint also showed a small bone fragment in the distal femur and was highlighted in the proximal tibia. Using targeted NIR fluorophores, metastatic bone tumors were monitored under the NIR fluorescence imaging system in real time, which enabled the in vivo diagnosis of bone metastasis by providing the location of the metastatic bone tumors.

Currently available chemotherapy is associated with serious side effects, and therefore novel drug delivery systems (DDSs) are required to specifically deliver anticancer drugs to targeted sites. In this study, we evaluated the feasibility of visible light-cured glycol chitosan (GC) hydrogels with controlled release of doxorubicin⋅hydrochloride (DOX⋅HCl) as local DDSs for effective cancer therapy in vivo. The storage modulus of the hydrogel precursor solutions was increased as a function of visible light irradiation time. In addition, the swelling ratio of the hydrogel irradiated for 10 s (GC

Background and Objective Bone homeostasis is maintained by a balance between osteoblastic bone formation and osteoclastic bone resorption, where intracellular reactive oxygen species (ROS) are crucial mediators of osteoclastogenesis. Recently, low-level light therapy (LLLT), a form of laser medicine used in various clinical fields, was shown to alleviate oxidative stress by scavenging intracellular ROS. The present study aimed to investigate the impact of 635 nm irradiation from a light-emitting diode (LED) on osteoclastogenesis from receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL)-stimulated mouse bone marrow-derived macrophages (BMMs). Study Design/Materials and Methods The effects of LED irradiation on osteoclastogenesis were assessed in tartrate-resistant acid phosphatase (TRAP), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell viability, and resorption pit formation, respectively. Quantitative real-time polymerase chain reaction (qPCR) and Western blot analyses were also performed to assess mRNA expression of osteoclastogenesis-related genes and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2), p38, and c-Jun-N-terminal kinase (JNK). NF-κB activity was assayed by luciferase reporter assay and Intracellular ROS generation was investigated by the 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) detection method. Results LED irradiation significantly inhibited RANKL-mediated osteoclast differentiation from BMMs and mRNA expression of TRAP, osteoclast-associated immunoglobulin-like receptor (OSCAR), and dendrocyte-expressed seven-transmembrane protein (DC-STAMP). Exposure to LED light likewise significantly decreased RANKL-facilitated NF-κB activity, p38 and ERK phosphorylation and intracellular ROS generation, and increased gene expression of nuclear factor E2-related factor 2 (Nrf2). Conclusions Taken together, the results presented herein show that LED irradiation downregulates osteoclastogenesis by reducing ROS production. Therefore, LED irradiation/LLLT might be useful as an alternative, conservative approach to osteoporosis management. Lasers Surg. Med. 47:745–755, 2015. © 2015 Wiley Periodicals, Inc.

Objectives: Abnormal cellular immune response has been considered to be responsible for oral lesions in recurrent aphthous stomatitis. Zinc has been known to be an essential nutrient metal that is necessary for a broad range of biological activities including antioxidant, immune mediator, and anti-inflammatory drugs in oral mucosal disease. The objective of this study was to investigate the effects of zinc in a phorbol-12-myristate-13-acetate (PMA)-treated inflammatory model on human gingival fibroblast cells (hGFs). Study Design: Cells were pre-treated with zinc chloride, followed by PMA in hGFs. The effects were assessed on cell viability, cyclooxygenease-1,2(COX-1/2) protein expression, PGE2 release, ROS production and cytokine release, Results: The effects were assessed on cell viability, COX1/2 protein expression, PGE2 release, ROS production, cytokine release. The results showed that, in the presence of PMA, zinc treatment leads to reduce the production of ROS, which results in decrease of COX-2 expression and PGE2 release. Conclusions: Thus, we suggest that zinc treatment leads to the mitigation of oral inflammation and may prove to be an alternative treatment for recurrent aphthous stomatitis. Key words:Zinc, inflammatory response, cytokines, phorbol-12-myristate-13-acetate, gingival fibroblasts cells.

Lateral epicondylitis (LE) is difficult to manage and can result in significant patient morbidity. Currently, the clinical use of platelet-rich plasma (PRP) for painful tendons has received attention, but its efficacy remains controversial. This study aimed to investigate the clinical effects of PRP and its biological components. A total of 156 patients with LE were randomly divided into group 1, treated with a single injection of 2-ml autologous PRP, and group 2, treated with a control received only physical therapy without injection. Both groups used a tennis elbow strap and performed stretching and strengthening exercises during 24 weeks' follow-up. Pain and functional improvements were assessed using the visual analog scale (VAS), Modified Mayo Clinic Performance Index for the elbow, and magnetic resonance imaging (MRI). White blood cell count, platelet count, and levels of platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, transforming growth factor-β (TGF-β), vascular endothelial growth factor, epithelial growth factor, and interleukin-1 β in PRP were measured and investigated for statistical correlation with the clinical score. At 24 weeks, all pain and functional variables, including VAS score, Mayo Clinic performance scores, and MRI grade, improved significantly in group 1 (p

Early detection and differential diagnosis of breast microcalcifications are of significant importance in effective treatment of early breast cancer, because mineral composition of breast calcification is directly associated with different pathological states. However, applying image-based modalities for component identification in breast calcification remains challenging, because no calcification-specific contrast agent is available to distinguish between benign and malignant (type I and type II, respectively) calcifications of breast lesions. In this study, real-time near-infrared (NIR) fluorescence imaging of breast microcalcifications using targeted NIR fluorophores in combination with dual-channel NIR fluorescence imaging system is reported. This strategy can be used to solve major problem in mammography and ultrasonography methods for the differentiation of benign and malignant microcalcifications. Thus, this novel technology shows significant potential for breast cancer diagnosis and image-guided surgery performed with increased precision and efficiency by providing differential diagnosis of breast microcalcifications.

Background: Since it is known that serum albumin-bound dyes can cross the blood-brain barrier (BBB) after ischemia, Evans Blue dye is commonly used to assess BBB disruption because of its rapid binding to serum albumin. In addition, indocyanine green (ICG), a clinically available dye, binds to serum proteins that could also be used for assessment of BBB impairment. Unlike these near-infrared (NIR) dyes, zwitterionic NIR fluorophore (ZW800-1) shows no serum binding, ultralow non-specific tissue uptake, and rapid elimination from the body via renal filtration. In this study, we report the use of ZW800-1 as a NIR fluorescence imaging agent for detecting BBB disruption in rat stroke models. Methods: Three types of NIR fluorophores, Evans Blue, ICG, and ZW800-1, were administered intraperitoneally into rat photothrombotic stroke models by using 4% concentration of each NIR dye. The NIR fluorescence signals in the infarcted brain tissue and biodistribution were observed in real-time using the Mini-FLARE® imaging system up to 24 h post-injection. Results: ZW800-1 provided successful visualization of the ischemic injury site in the brain tissue, while the remaining injected dye was clearly excreted from the body within a certain period of time. Although Evans Blue and ICG provided mapping of the infarcted brain lesions, they exhibited high non-specific uptake in most of the tissues and organs and persisted in the body over 24 h post-injection. Conclusion: Our results suggest the promising application of ZW800-1 as a new strategy in BBB experiments and future therapeutic development.

Consistent with cancer stem cell theory, a small fraction of cancer cells, described as cancer stem cells (CSCs), may promote tumor recurrence and anti-cancer drug resistance. Therefore, much effort has been devoted to the development of CSC targeted therapy to vanquish drug resistance. In this study, we have investigated the effect of multiple light-emitting diode (LED) irradiation treatments with conventional anti-cancer drugs on CSC-like oral cancer cells that acquired stemness by ectopic over expression of CD133. To evaluate combined LED irradiation anti-cancer drug effects, we investigated the chemosensitizing effect of 635 nm irradiation on 5-fluorouracil (5FU)-treated KBCD133+ and KBVec cells, interrogating the underlying molecular mechanisms associated with stemness and apoptosis that are responsible for chemopreventive activity. In addition, combination therapy with LED irradiation and 5-FU treatment was carried out in KBCD133+ and KBVec cell-inoculated mouse models. LED irradiation of 635 nm inhibited CSC-like properties consistent with a decrease in OCT4 and NANOG protein expression, reducing colony-forming ability. In addition, LED irradiation enhanced 5-FU-induced cytotoxicity and improved 5-FU chemosensitivity in KBCD133+ via enhancement of apoptosis. These findings were validated in vivo, wherein LED irradiation combined with 5-FU treatment inhibited tumor growth in KBCD133+-inoculated mice. Collectively, our results provide novel evidence for 635 nm irradiation-induced 5-FU chemosensitization of CSC in oral cancer. In addition, this research highlights that 635 nm LED irradiation may serve as an adjunct treatment to conventional chemotherapeutic drugs in patients with oral cancer.

Tumors of the prostate or breast are particularly likely to metastasize to the bone, and early diagnosis of metastatic bone tumors is important for designing an effective treatment strategy. Imaging modalities for the detection of bone metastasis are limited, and radiation-based techniques are commonly used. Here, we investigated the efficacy of selective near-infrared (NIR) fluorescence detection of metastatic bone tumors and its role in the detection of bone metastasis in prostate and breast cancer cell lines and in a xenograft mouse model. A targeted NIR fluorophore was used to monitor metastatic bone tumors using a NIR fluorescence imaging system in real time, enabling the diagnosis of bone metastasis in vivo by providing the location of the metastatic bone tumor. The NIR fluorescence imaging technique using targeted NIR contrast agents is a potential tool for the early diagnosis of bone tumors.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers in the world. Resistance to cytotoxic chemotherapy is a major cause of mortality in patients with HNSCC. A small subset of cancer cells called cancer stem cells (CSCs) may be key contributors to drug resistance and tumor recurrence in HNSCC. The aim of this study was to determine whether CD133, which maintains properties of CSCs, promotes chemoresistance by arresting cell cycle transition and reducing apoptosis in HNSCC cells. CD133 overexpression was examined in KB cells, and colony forming and aldehyde dehydrogenase activity assays were performed. To investigate the role of CD133 in chemoresistance, cell death was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Diff-Quick, flow cytometry, and western blot of apoptosis-related protein expression in fluorouracil (5-FU)- or cisplatin-treated cells. In addition, microarray and related protein expression assessments were performed to investigate the mechanism of chemoresistance against 5-FU and cisplatin in KB cells. Moreover, chemoresistance against 5-FU or cisplatin in a KB-inoculated mouse model was analyzed by hematoxylin and eosin staining, immunohistochemical study of CD133, and immunofluorescence of tumor tissue. In this study, we demonstrate that ectopic overexpression of CD133 significantly promotes properties of stemness in KB cell lines. Furthermore, CD133 promotes chemoresistance by arresting transition of the cell cycle and reducing apoptosis, which results in inhibition of tumor growth in 5-FU- or cisplatin-injected mouse tumor model. Taken together, our findings show that elevated levels of CD133 lead to HNSCC chemoresistance through increased stemness and cell cycle arrest.

Porphyromonas gingivalis causes chronic inflammatory diseases (periodontal diseases) that destroy the periodontal ligament and alveolar bone. Antimicrobial peptides are crucial components of the host defense response required to maintain cellular homeostasis during microbial invasion. Because light-emitting diode (LED) irradiation influences the host defense response against bacterial infections, we investigated its effect on immortalized gingival fibroblasts (IGFs) infected with P. gingivalis. IGFs were incubated with P. gingivalis following LED irradiation at 425, 525, and 625 nm. The dark 1 group comprised noninfected, nonirradiated IGFs, and the dark 2 group comprised nonirradiated IGFs infected with P. gingivalis. These groups served as controls. Infected cells and controls were assayed for reactive oxygen species (ROS) and were subjected to RT-PCR and Western blotting analyses to determine the levels of expression of antimicrobial peptides. LED irradiation enhanced the bactericidal effects of the antimicrobial peptide LL-37 in cells infected with P. gingivalis. Irradiation at 625 nm decreased inflammatory responses involving the release of prostaglandin E2 induced by ROS in P. gingivalis-infected IGFs. LED irradiation at 625 nm induces an anti-inflammatory response that elicits the production of antimicrobial peptides, providing an efficacious method of treatment for periodontal diseases.

Low-level laser therapy (LLLT) has been promoted for its beneficial effects on tissue healing and pain relief. As during laser treatment it is possible to irradiate only a small area of the surface body or wound and, correspondingly, of a very small volume of the circulating blood, it is necessary to explain how its photomodification can lead to a wide spectrum of therapeutic effects. To establish the experimental model for indirect irradiation, irradiation with 635 nm was performed on immortalized human gingival fibroblasts (IGFs) in the presence of Porphyromonas gingivalis lipopolysaccharides (LPS). The irradiated medium was transferred to non-irradiated IGFs which were compared with direct irradiated IGFs. The protein expressions were assessed by Western blot, and prostaglandin E2 (PGE2 ) was measured using an enzyme-linked immunoassay. Reactive oxygen species (ROS) were measured by DCF-DA; cytokine profiles were assessed using a human inflammation antibody array. Cyclooxygenase-2 (COX-2) protein expression and PGE2 production were significantly increased in the LPS-treated group and decreased in both direct and indirect irradiated IGFs. Unlike direct irradiated IGFs, ROS level in indirect irradiated IGFs was decreased by time-dependent manners. There were significant differences of released granulocyte colony-stimulating factor (G-CSF), regulated on activated normal T-cell expressed and secreted (RANTES), and I-TAC level observed compared with direct and indirect irradiated IGFs. In addition, in the indirect irradiation group, phosphorylations of C-Raf and Erk1/2 increased significantly compared with the direct irradiation group. Thus, we suggest that not only direct exposure with 635 nm light, but also indirect exposure with 635 nm light can inhibit activation of pro-inflammatory mediators and may be clinically useful as an anti-inflammatory tool.

One of the theories regarding oral carcinogenesis is that the tumor growth is initiated from cancer stem cells (CSCs) that self-renew and give rise to differentiated tumor cells, like stem cells do in normal tissues. The most common methods of CSC identification are based on CSC marker expression in carcinogenesis. This study examined the expression of CD133 and CD44, the most commonly used CSC biomarkers in oral squamous cell sarcoma (SCC), with the goal of identifying molecular biomarkers whose expression is associated with the multistep oral carcinogenesis. The expression of CD133, CD44, proliferating cell nuclear antigen (PCNA), and Cytokeratin (CK) was examined by Western blot analysis and confirmed by immunohistochemistry in a 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis model. Also, the expression of aldehyde dehydrogenase 1 (ALDH1), OCT-4 and Nanog were investigated for alteration of cancer cell stemness by Western blot. Along with the progress of multistep carcinogenesis, there were slight increases of CD133 and CD44 expression in the dysplasia group compared with normal rats. However, CD133 protein level was significantly overexpressed in SCC. The expression of PCNA and CK were low in normal group, but sequentially increased in SCC. ALDH1, Nanog and OCT-4 expression were significantly increased according to SCC grade during carcinogenesis. The findings indicate that CD133 is useful in identifying oral CSCs, which suggests that CD133 may serve as a predictor to identify CSCs with a high risk of oral cancer development.

Lateral epicondylitis (LE) is difficult to manage and can result in significant patient morbidity. Currently, the clinical use of platelet-rich plasma (PRP) for painful tendons has received attention, but its efficacy remains controversial. This study aimed to investigate the clinical effects of PRP and its biological components. A total of 156 patients with LE were randomly divided into group 1, treated with a single injection of 2-ml autologous PRP, and group 2, treated with a control received only physical therapy without injection. Both groups used a tennis elbow strap and performed stretching and strengthening exercises during 24 weeks' follow-up. Pain and functional improvements were assessed using the visual analog scale (VAS), Modified Mayo Clinic Performance Index for the elbow, and magnetic resonance imaging (MRI). White blood cell count, platelet count, and levels of platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, transforming growth factor-β (TGF-β), vascular endothelial growth factor, epithelial growth factor, and interleukin-1 β in PRP were measured and investigated for statistical correlation with the clinical score. At 24 weeks, all pain and functional variables, including VAS score, Mayo Clinic performance scores, and MRI grade, improved significantly in group 1 (p 0.05). PDGF-AB, PDGF-BB, and TGF-β levels were more significantly increased in PRP than in whole blood. TGF-β level significantly correlated with Mayo Clinic performance score and MRI grade improvement. Thus, TGF-β level in PRP is considered to play a pivotal role in tendon healing. These results may contribute to identifying the best protocol for PRP application in tendinopathies. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:913-920, 2018.