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Various risk factors and causative genes of osteosarcoma have been reported in the literature; however, its etiology remains largely unknown. Bone formation is a shared phenomenon in all types of osteosarcomas, and sclerostin is an extracellular soluble factor secreted by osteocytes that prevents bone formation by inhibiting the Wnt signaling pathway. We aimed to investigate the antitumor effect of sclerostin against osteosarcoma. Osteosarcoma model mice were prepared by transplantation into the dorsal region of C3H/He and BALB/c-nu/nu mice using osteosarcoma cell lines LM8 (murine) and 143B (human), respectively. Cell proliferations were evaluated by using alamarBlue and scratch assays. The migratory ability of the cells was evaluated using a migration assay. Sclerostin was injected intraperitoneally for 7 days to examine the suppression of tumor size and extension of survival. The administration of sclerostin to osteosarcoma cells significantly inhibited the growth and migratory ability of osteosarcoma cells. Kaplan–Meier curves and survival data demonstrated that sclerostin significantly inhibited tumor growth and improved survival. Sclerostin suppressed the proliferative capacity and migratory ability of osteosarcoma cells. Osteosarcoma model mice inhibited tumor growth and prolonged survival periods by the administration of sclerostin. The effect of existing anticancer drugs such as doxorubicin should be investigated for future clinical applications., Article, Cancers 13(23) : 6015(2021)

Many biomaterials have been evaluated using cultured cells. In particular, osteoblast-like cells are often used to evaluate the osteocompatibility, hard-tissue-regeneration, osteoconductive, and osteoinductive characteristics of biomaterials. However, the evaluation of biomaterial osteogenesis-inducing capacity using osteoblast-like cells is not standardized; instead, it is performed under laboratory-specific culture conditions with different culture media. However, the effect of different media conditions on bone formation has not been investigated. Here, we aimed to evaluate the osteogenesis of MC3T3-E1 cells, one of the most commonly used osteoblast-like cell lines for osteogenesis evaluation, and assayed cell proliferation, alkaline phosphatase activity, expression of osteoblast markers, and calcification under varying culture media conditions. Furthermore, the various media conditions were tested in uncoated plates and plates coated with collagen type I and poly-L-lysine, highly biocompatible molecules commonly used as pseudobiomaterials. We found that the type of base medium, the presence or absence of vitamin C, and the freshness of the medium may affect biomaterial regeneration. We posit that an in vitro model that recapitulates in vivo bone formation should be established before evaluating biomaterials., Article, International Journal of Molecular Sciences. 22(14):7752 (2021)

Various risk factors and causative genes of osteosarcoma have been reported in the literature; however, its etiology remains largely unknown. Bone formation is a shared phenomenon in all types of osteosarcomas, and sclerostin is an extracellular soluble factor secreted by osteocytes that prevents bone formation by inhibiting the Wnt signaling pathway. We aimed to investigate the antitumor effect of sclerostin against osteosarcoma. Osteosarcoma model mice were prepared by transplantation into the dorsal region of C3H/He and BALB/c-nu/nu mice using osteosarcoma cell lines LM8 (murine) and 143B (human), respectively. Cell proliferations were evaluated by using alamarBlue and scratch assays. The migratory ability of the cells was evaluated using a migration assay. Sclerostin was injected intraperitoneally for 7 days to examine the suppression of tumor size and extension of survival. The administration of sclerostin to osteosarcoma cells significantly inhibited the growth and migratory ability of osteosarcoma cells. Kaplan–Meier curves and survival data demonstrated that sclerostin significantly inhibited tumor growth and improved survival. Sclerostin suppressed the proliferative capacity and migratory ability of osteosarcoma cells. Osteosarcoma model mice inhibited tumor growth and prolonged survival periods by the administration of sclerostin. The effect of existing anticancer drugs such as doxorubicin should be investigated for future clinical applications., Article, Cancers 13(23) : 6015(2021)

Takashige Momose,1 Yukio Nakamura,2 Masaki Nakano,2 Takashi Maeda,1 Susumu Morioka,3 Atsushi Sobajima,1 Yukio Nakatsuchi,1 Jun Takahashi,2 Masashi Nawata1 1Department of Orthopaedic Surgery, Marunouchi Hospital, Matsumoto, Nagano, 390-8601, Japan; 2Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Nagano, 390-8621, Japan; 3Department of Orthopaedic Surgery, Chikuma Central Hospital, Chikuma, Nagano, 387– 8512, JapanCorrespondence: Yukio NakamuraDepartment of Orthopaedic Surgery, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano, 390-8621, JapanTel +81-263-37-2659Fax +81-263-35-8844Email yxn14@aol.jpObjective: This study aimed to evaluate the short- to mid-term clinical results of posterior-stabilized trabecular metal total knee arthroplasty (TKA) with cementless fixation of all components and investigate the radiographic changes of tibial and patellar components and cut bone surfaces over time.Methods: We retrospectively collected the data of 128 knees from 88 consecutive patients who had undergone initial TKA with NexGen LPS-Flex TM implants. A total of 66 knees from 45 patients (mean ± standard deviation age: 70.3 ± 7.5 years) met the selection criteria, which had been employed cementless fixation of all parts and at least 3 years of postoperative follow-up duration. Clinical evaluations included range of motion, conventional knee score, function score, postoperative complications, and revision. For radiological evaluations, the bone contact surface of each implant was divided into 7 zones for tibial component and 2 zones for patellar component. Each region was examined immediately after surgery, at 6 and 12 months, and then every year afterwards.Results: The mean observation period of 45 subjects was 4.2 years. Adequate fixation of tibial components was maintained during follow-up, although the patellar components of 2 knees required revision after repeated falls. No loosening was observed in any implants

Background There are many types of therapies for cancer. In these days, immunotherapies, especially immune checkpoint inhibitors, are focused on. Though many types of immune checkpoint inhibitors are there, the difference of effect and its mechanism are unclear. Some reports suggest the response rate of anti-PD-1 antibody is superior to that of anti-PD-L1 antibody and could potentially produce different mechanisms of action. On the other hand, Treg also express PD-1; however, their relationship remains unclear. Methods In this study, we used osteosarcoma cell lines in vitro and osteosarcoma mouse model in vivo. In vitro, we analyzed the effect of IFN gamma for expression of PD-L1 on the surface of cell lines by flowcytometry. In vivo, murine osteosarcoma cell line LM8 was subcutaneously transplanted into the dorsum of mice. Mouse anti-PD-1 antibody was intraperitoneally administered. we analysed the effect for survival of anti-PD-1 antibody and proportion of T cells in the tumour by flowcytometry. Results We discovered that IFN gamma increased PD-L1 expression on the surface of osteosarcoma cell lines. In assessing the relationship between anti-PD-1 antibody and Treg, we discovered the administration of anti-PD-1 antibody suppresses increases in tumour volume and prolongs overall survival time. In the tumour microenvironment, we found that the administration of anti-PD-1 antibody decreased Treg within the tumour and increased tumour-infiltrating lymphocytes. Conclusions Here we clarify for the first time an additional mechanism of anti-tumour effect-as exerted by anti-PD-1 antibody decreasing Treg- we anticipate that our findings will lead to the development of new methods for cancer treatment., Article, BMC CANCER. 20(1):25 (2020)

Scaffolds are essential for bone regeneration due to their ability to maintain a sustained release of growth factors and to provide a place where cells that form new bone can enter and proliferate. In recent years, scaffolds made of various materials have been developed and evaluated. Functionally effective scaffolds require excellent cell affinity, chemical properties, mechanical properties, and safety. Carbon nanotubes (CNTs) are fibrous nanoparticles with a nano-size diameter and have excellent strength and chemical stability. In the industrial field, they are used as fillers to improve the performance of materials. Because of their excellent physicochemical properties, CNTs are studied for their promising clinical applications as biomaterials. In this review article, we focused on the results of our research on CNT scaffolds for bone regeneration, introduced the promising properties of scaffolds for bone regeneration, and described the potential of CNT scaffolds., Article, NANOMATERIALS. 10(4):659 (2020)

We evaluated starfish-derived -tricalcium phosphate (Sf-TCP) obtained by phosphatization of starfish-bone-derived porous calcium carbonate as a potential bone substitute material. The Sf-TCP had a communicating pore structure with a pore size of approximately 10 m. Although the porosity of Sf-TCP was similar to that of Cerasorb M (CM)a commercially available -TCP bone fillerthe specific surface area was roughly three times larger than that of CM. Observation by scanning electron microscopy showed that pores communicated to the inside of the Sf-TCP. Cell growth tests showed that Sf-TCP improved cell proliferation compared with CM. Cells grown on Sf-TCP showed stretched filopodia and adhered; cells migrated both to the surface and into pores. In vivo, vigorous tissue invasion into pores was observed in Sf-TCP, and more fibrous tissue was observed for Sf-TCP than CM. Moreover, capillary formation into pores was observed for Sf-TCP. Thus, Sf-TCP showed excellent biocompatibility in vitro and more vigorous bone formation in vivo, indicating the possible applications of this material as a bone substitute. In addition, our findings suggested that mimicking the microstructure derived from whole organisms may facilitate the development of superior artificial bone., Article, MATERIALS. 12(11):1881 (2019)

Purpose: Multiwalled carbon nanotubes (MWCNTs) have been known to enter the circulatory system via the lungs from inhalation exposure; however, its carcinogenicity and subsequent accumulation in other organs have not been adequately reported in the literature. Moreover, the safety of MWCNTs as a biomaterial has remained a matter of debate, particularly when the material enters the circulatory system. To address these problems, we used carcinogenic rasH2 transgenic mice to intravenously administer highly dispersed MWCNTs and to evaluate their carcinogenicity and accumulation in the organs. Methods: Two types of MWCNTs (thin-and thick-MWCNTs) were intravenously administered at a high dose (approximately 0.7 mg per kg body weight) and low dose (approximately 0.07 mg per kg body weight). Results: MWCNTs showed pancreatic accumulation in 3.2% of mice administered with MWCNTs, but there was no accumulation in other organs. In addition, there was no significant difference in the incidence of tumor among the four MWCNTs-administered groups compared to the vehicle group without MWCNTs administration. Blood tests revealed elevated levels in mean red blood cell volume and mean red blood cell hemoglobin level for the MWCNTs-administered group, in addition to an increase in eotaxin. Conclusion: The present study demonstrated that the use of current technology to sufficiently disperse MWCNTs resulted in minimal organ accumulation with no evidence of carcinogenicity., Article, INTERNATIONAL JOURNAL OF NANOMEDICINE. 14:6465-6480 (2019)

Atsushi Sobajima,1 Hisao Haniu,1,2 Hiroki Nomura,1 Manabu Tanaka,1 Takashi Takizawa,1 Takayuki Kamanaka,1 Kaoru Aoki,1,3 Masanori Okamoto,1 Kazushige Yoshida,1 Jun Sasaki,1 Kumiko Ajima,2 Chika Kuroda,1,2 Haruka Ishida,2 Satomi Okano,2 Katsuya Ueda,2 Hiroyuki Kato,1 Naoto Saito1,21Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Nagano, Japan; 2Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University School of Medicine, Matsumoto, Nagano, Japan; 3Department of Applied Physical Therapy, Shinshu University School of Health Sciences, Matsumoto, Nagano, JapanPurpose: Multiwalled carbon nanotubes (MWCNTs) have been known to enter the circulatory system via the lungs from inhalation exposure; however, its carcinogenicity and subsequent accumulation in other organs have not been adequately reported in the literature. Moreover, the safety of MWCNTs as a biomaterial has remained a matter of debate, particularly when the material enters the circulatory system. To address these problems, we used carcinogenic rasH2 transgenic mice to intravenously administer highly dispersed MWCNTs and to evaluate their carcinogenicity and accumulation in the organs.Methods: Two types of MWCNTs (thin- and thick-MWCNTs) were intravenously administered at a high dose (approximately 0.7 mg per kg body weight) and low dose (approximately 0.07 mg per kg body weight).Results: MWCNTs showed pancreatic accumulation in 3.2% of mice administered with MWCNTs, but there was no accumulation in other organs. In addition, there was no significant difference in the incidence of tumor among the four MWCNTs-administered groups compared to the vehicle group without MWCNTs administration. Blood tests revealed elevated levels in mean red blood cell volume and mean red blood cell hemoglobin level for the MWCNTs-administered group, in addition to an increase in eotaxin.Conclusion: The present study demonstrated tha

Atsushi Sobajima,1 Hisao Haniu,1,2 Hiroki Nomura,1 Manabu Tanaka,1 Takashi Takizawa,1 Takayuki Kamanaka,1 Kaoru Aoki,1,3 Masanori Okamoto,1 Kazushige Yoshida,1 Jun Sasaki,1 Kumiko Ajima,2 Chika Kuroda,1,2 Haruka Ishida,2 Satomi Okano,2 Katsuya Ueda,2 Hiroyuki Kato,1 Naoto Saito1,21Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Nagano, Japan; 2Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University School of Medicine, Matsumoto, Nagano, Japan; 3Department of Applied Physical Therapy, Shinshu University School of Health Sciences, Matsumoto, Nagano, JapanPurpose: Multiwalled carbon nanotubes (MWCNTs) have been known to enter the circulatory system via the lungs from inhalation exposure; however, its carcinogenicity and subsequent accumulation in other organs have not been adequately reported in the literature. Moreover, the safety of MWCNTs as a biomaterial has remained a matter of debate, particularly when the material enters the circulatory system. To address these problems, we used carcinogenic rasH2 transgenic mice to intravenously administer highly dispersed MWCNTs and to evaluate their carcinogenicity and accumulation in the organs.Methods: Two types of MWCNTs (thin- and thick-MWCNTs) were intravenously administered at a high dose (approximately 0.7 mg per kg body weight) and low dose (approximately 0.07 mg per kg body weight).Results: MWCNTs showed pancreatic accumulation in 3.2% of mice administered with MWCNTs, but there was no accumulation in other organs. In addition, there was no significant difference in the incidence of tumor among the four MWCNTs-administered groups compared to the vehicle group without MWCNTs administration. Blood tests revealed elevated levels in mean red blood cell volume and mean red blood cell hemoglobin level for the MWCNTs-administered group, in addition to an increase in eotaxin.Conclusion: The present study demonstrated tha

Kazushige Yoshida,1 Masanori Okamoto,1 Jun Sasaki,1 Chika Kuroda,2 Haruka Ishida,2 Katsuya Ueda,2 Satomi Okano,2 Hirokazu Ideta,1 Takayuki Kamanaka,1 Atsushi Sobajima,1 Takashi Takizawa,1 Munehisa Kito,1 Kaoru Aoki,3 Takeshi Uemura,2 Hisao Haniu,2 Hiroyuki Kato,1 Naoto Saito2 1Department of Orthopaedic Surgery, Shinshu University School of Medicine, Matsumoto, Japan; 2Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Japan; 3Physical Therapy Division, School of Health Sciences, Shinshu University School of Medicine, Matsumoto, Japan Purpose: Although both anti-PD-1 antibody and treatments using anti-PD-L1 antibody are currently in clinical use, their therapeutic effects vary according to cancer type. One of the factors accounting for this variability is the expression level of the immune checkpoint molecule that differs between cancer types; thus, it is important to clarify the relationship between clinical outcomes and immune checkpoint molecules for all types of human cancer. The purpose of this study is to evaluate the clinical outcome of osteosarcoma in relation to PD-L1, PRF, GZMB, and IFNγ expression. Methods: Using 19 clinical specimens of osteosarcoma, we examined the expression of PD-L1, PRF, GZMB, and IFNγ in relation to their clinical outcomes. Results: PD-L1 expression correlated with early metastatic formation in clinical specimens of osteosarcoma, and the group with highly expressed functional markers for T cells such as PRF and GZMB resulted in a long overall survival time. Conclusion: This is the first study to elucidate the clinical outcomes of osteosarcoma in relation to PD-L1, PRF, GZMB, and IFNγ expression. This study provides valuable information regarding the clinical indication and prediction of effect for anti-PD-1 antibody in osteosarcoma. Keywords: anti-PD-1 antibody, perforin, granzyme B, IFNγ, osteosarcoma, clinical outcome

Chika Kuroda,1,2,* Katsuya Ueda,1,3,* Hisao Haniu,1,3–5 Haruka Ishida,1,4 Satomi Okano,1,4 Takashi Takizawa,5 Atsushi Sobajima,5 Takayuki Kamanaka,5 Kazushige Yoshida,5 Masanori Okamoto,5 Tamotsu Tsukahara,6 Yoshikazu Matsuda,7 Kaoru Aoki,8 Hiroyuki Kato,5 Naoto Saito1,3–5 1Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 2Department of Orthopaedic Surgery, Graduate School of Medicine, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 3Department of Biomedical Engineering, Graduate School of Science and Technology, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 4Department of Biomedical Engineering, Graduate School of Medicine, Science and Technology, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 5Department of Orthopaedic Surgery, Shinshu University School of Medicine, Asahi, Matsumoto, Nagano, Japan; 6Department of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, Bunkyo-machi, Nagasaki, Japan; 7Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Komuro, Ina-machi, Saitama, Japan; 8Physical Therapy Division, School of Health Sciences, Shinshu University, Asahi, Matsumoto, Nagano, Japan *These authors contributed equally to this work Introduction: Carbon nanotubes (CNTs) have various shapes, including needle-like shapes and curled shapes, and the cytotoxicity and carcinogenicity of CNTs differ depending on their shapes and surface modifications. However, the biological responses induced by CNTs and related mechanisms according to the dispersion state of CNTs have not been extensively studied.Materials and methods: We prepared multiwalled CNTs (MWCNTs) showing different dispersions and evaluated these MWCNTs in RAW264 cells to determine cytotoxicity, cellular uptake, and immune responses. Furthermore, RAW264 cells were also used to compare the cellular uptake and cyto

Chika Kuroda,1,2,* Katsuya Ueda,1,3,* Hisao Haniu,1,3–5 Haruka Ishida,1,4 Satomi Okano,1,4 Takashi Takizawa,5 Atsushi Sobajima,5 Takayuki Kamanaka,5 Kazushige Yoshida,5 Masanori Okamoto,5 Tamotsu Tsukahara,6 Yoshikazu Matsuda,7 Kaoru Aoki,8 Hiroyuki Kato,5 Naoto Saito1,3–5 1Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 2Department of Orthopaedic Surgery, Graduate School of Medicine, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 3Department of Biomedical Engineering, Graduate School of Science and Technology, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 4Department of Biomedical Engineering, Graduate School of Medicine, Science and Technology, Shinshu University, Asahi, Matsumoto, Nagano, Japan; 5Department of Orthopaedic Surgery, Shinshu University School of Medicine, Asahi, Matsumoto, Nagano, Japan; 6Department of Molecular Pharmacology and Neuroscience, Nagasaki University Graduate School of Biomedical Sciences, Bunkyo-machi, Nagasaki, Japan; 7Clinical Pharmacology Educational Center, Nihon Pharmaceutical University, Komuro, Ina-machi, Saitama, Japan; 8Physical Therapy Division, School of Health Sciences, Shinshu University, Asahi, Matsumoto, Nagano, Japan *These authors contributed equally to this work Introduction: Carbon nanotubes (CNTs) have various shapes, including needle-like shapes and curled shapes, and the cytotoxicity and carcinogenicity of CNTs differ depending on their shapes and surface modifications. However, the biological responses induced by CNTs and related mechanisms according to the dispersion state of CNTs have not been extensively studied.Materials and methods: We prepared multiwalled CNTs (MWCNTs) showing different dispersions and evaluated these MWCNTs in RAW264 cells to determine cytotoxicity, cellular uptake, and immune responses. Furthermore, RAW264 cells were also used to compare the cellular uptake and cyto

The unidirectional porous hydroxyapatite HAp (UDPHAp) is a scaffold with continuous communicated pore structure in the axial direction. We evaluated and compared the ability of the UDPHAp as a three-dimensional (3D) bone tissue engineering scaffold to the interconnected calcium porous HAp ceramic (IP-CHA). To achieve this, we evaluated in vitro the compressive strength, controlled rhBMP-2 release behavior, adherent cell morphology, cell adhesion manner, and cell attachment of UDPHAp. As a further in vivo experiment, UDPHAp and IP-CHA with rhBMP-2 were transplanted into mouse calvarial defects to evaluate their bone-forming ability. The Results demonstrated that the maximum compressive strengths of the UDPHAp was 7.89 +/- 1.23 MPa and higher than that of IP-CHA (1.92 +/- 0.53 MPa) (p = 0.0039). However, the breaking energies were similar (8.99 +/- 2.72 vs. 13.95 +/- 5.69 mJ, p = 0.055). The UDPHAp released rhBMP-2 more gradually in vivo. Cells on the UDPHAp adhered tightly to the surface, which had grown deeply into the scaffolds. A significant increase in cell number on the UDPHAp was observed compared to the IP-CHA on day 8 (102,479 +/- 34,391 vs. 32,372 +/- 29,061 estimated cells per scaffold, p = 0.0495). In a mouse calvarial defect model, the percentages of new bone area (mature bone + trabecular bone) in the 2x field were 2.514% +/- 1.224% for the IP-CHA group and 7.045% +/- 2.055% for the UDPHAp group, and the percentage was significantly higher in the UDPHAp group (p = 0.0209). While maintaining the same strength as the IP-CHA, the UDPHAp with 84% porosity showed a high cell number, high cell invasiveness, and excellent bone formation. We believe the UDPHAp is an excellent material that can be applied to bone regenerative medicine., Article, MATERIALS. 10(1):33 (2017)

Carbon nanotubes (CNTs) have attracted a great deal of attention for the biological and medical science fields because of their characteristic physical and biological properties. In this study, we investigated the capacity of the 3D porous CNT scaffold (CNT porous block; CNTp) for bone regenerative medicine. Surface observations using a scanning electron microscope (SEM), crystal depositions on the surface of CNTps immersed in simulated body fluid (SBF), and evaluations of protein adsorption and controlled releasing were conducted to assess physical properties. The cell proliferation and cell morphology were observed using SEM and fluorescent microscopy. CNTps were implanted into critical-size mouse calvarial defects and evaluated for their osteoconductive ability and in vivo controlled release of recombinant human BMP-2 (rhBMP-2). Interconnected porous HA ceramics (IP-CHAs) were used for comparison. CNTps have multiporous structures with interporous connections with networks of multiwalled CNTs. Crystals containing calcium and phosphate were deposited in CNTps and on the surface of the CNT networks by immersing CNTps in SBF. CNTps adsorbed more significantly and released protein more gradually than IP-CHAs. Preosteoblasts seeded onto CNTps filled pores with stretched actin filaments and filopodia. Compared with IP-CHAs, CNTps showed significantly higher cell proliferation, better osteoconduction, and more bone generation with rhBMP-2. In this study, CNTps demonstrated good osteoconductive ability, cell attachment and proliferation capacity, and growth factor retaining ability. CNTps have the potential not only as artificial bones for the treatment of bone defects, but also as scaffolds for regenerative medicine using tissue engineering approaches., Article, NANOMATERIALS. 7(2):46 (2017)