Applications of Carbon Nanotubes in Bone Regenerative Medicine

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.ArticleNANOMATERIALS. 10(4):659 (2020)journal articl

In Vitro and In Vivo Evaluation of Starfish Bone-Derived -Tricalcium Phosphate as a Bone Substitute Material

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.ArticleMATERIALS. 12(11):1881 (2019)journal articl

Physico-Chemical, In Vitro, and In Vivo Evaluation of a 3D Unidirectional Porous Hydroxyapatite Scaffold for Bone Regeneration

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.ArticleMATERIALS. 10(1):33 (2017)journal articl

Evaluation of MC3T3-E1 Cell Osteogenesis in Different Cell Culture Media

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.ArticleInternational Journal of Molecular Sciences. 22(14):7752 (2021)journal articl

Antitumor Effect of Sclerostin against Osteosarcoma

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.ArticleCancers 13(23) : 6015(2021)journal articl

Anti-PD-1 antibody decreases tumour-infiltrating regulatory T cells

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.ArticleBMC CANCER. 20(1):25 (2020)journal articl

Organ accumulation and carcinogenicity of highly dispersed multi-walled carbon nanotubes administered intravenously in transgenic rasH2 mice

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.ArticleINTERNATIONAL JOURNAL OF NANOMEDICINE. 14:6465-6480 (2019)journal articl

Prevalence of hypothyroidism in patients with hyponatremia: A retrospective cross-sectional study.

OBJECTIVE:Hypothyroidism has been suggested to be an uncommon cause of hyponatremia. However, little is known about the prevalence of hypothyroidism in patients with different levels of hyponatremia. The objective of this study was to investigate the prevalence of hypothyroidism among patients with hyponatremia of varying severity while taking into consideration potential confounders associated with thyroid function. METHODS:All data on thyrotropin (TSH), free thyroxine (T4), and serum sodium (Na) levels were retrospectively collected from medical records at two Japanese tertiary hospitals. The main outcome measure was overt hypothyroidism, defined as TSH > 10.0 μIU/mL and free T4 < 1.01 ng/dL. RESULTS:Of 71,817 patients, 964 patients (1.3%) had overt hypothyroidism. The prevalence of overt hypothyroidism in each category of hyponatremia (Na ≥136, 130-135, and ≤129 mEq/L) was 1.2% (787/65,051), 2.4% (124/5,254) and 3.5% (53/1,512), respectively. A significant increase in prevalence was observed as the severity of hyponatremia increased (P < 0.001 for trend). Multivariate logistic regression with adjustment for age, sex, kidney function, and serum albumin level showed that the odds ratios for overt hypothyroidism increased with increasing severity of hyponatremia when compared with Na ≥ 136 mEq/L (130-135 mEq/L: 1.43, 95% confidence interval [CI], 1.15 to 1.78, P = 0.001; ≤129 mEq/L: 1.87, 95% CI, 1.32 to 2.63, P < 0.001; P< 0.001 for trend). CONCLUSION:The prevalence of overt hypothyroidism was significantly higher as the severity of hyponatremia progressed, even after adjusting for potential confounders. Hypothyroidism should be differentiated in patients with hyponatremia