Proliferation and differentiation of MC3T3-E1 cells on polymethyl methacrylate cements containing Fe3O4 and TiO2 for hyperthermic treatment of metastatic bone tumors.

Polymethyl methacrylate (PMMA) bone cement is widely used to relieve pain caused by metastatic bone tumors. We previously found that PMMA bone cement containing 15 mass% or more of TiO2 showed good apatite-forming ability, and 25 mass% or more of Fe3O4 generated sufficient heat for hyperthermia under an alternating current (AC) magnetic field. In this study, the cytocompatibility of PMMA bone cement with Fe3O4:TiO2 weight ratios of 25:15 (F25T15-3/2-42) and 30:15 (F30T15-3/2-42) was evaluated using osteoblastic cells (MC3T3-E1). The proliferation and differentiation of MC3T3-E1 cells were suppressed for F25T15-3/2-42 and F30T15-3/2-42 compared to PMMA bone cement without Fe3O4 and TiO2 (F0T0-3/2-42). The release of methyl methacrylate (MMA) monomers from F25T15-3/2-42 and F30T15-3/2-42 at 7 days was about 33 and 50 times higher than that from F0T0-3/2-42, respectively. The remarkable release of MMA monomers from F25T15-3/2-42 and F30T15-3/2-42 may be responsible for the suppressed proliferation and differentiation of MC3T3-E1 cells. The release of MMA monomers was not reduced when the MMA/PMMA weight ratio was decreased from 3/2 to 1/1, however, it was significantly reduced by increasing the content of benzoyl peroxide (BPO) and N, N-dimethyl-p-toluidine (DMPT) to 8 and 4 mass% against MMA, respectively. Proliferation and differentiation of MC3T3-E1 cells on PMMA-type cements containing Fe3O4 and TiO2 with increased BPO and DMPT contents need to be investigated in the future; however, our findings will be useful for designing PMMA cements for the hyperthermic treatment of metastatic bone tumors.

Visible-Light-Enhanced Antibacterial Activity of Silver and Copper Co-Doped Titania Formed on Titanium via Chemical and Thermal Treatments.

Dental implants made of titanium (Ti) are used in dentistry, but peri-implantitis is a serious associated problem. Antibacterial and osteoconductive Ti dental implants may decrease the risk of peri-implantitis. In this study, titania (TiO2) co-doped with silver (Ag) at 2.5 at.% and copper (Cu) at 4.9 at.% was formed on Ti substrates via chemical and thermal treatments. The Ag and Cu co-doped TiO2 formed apatite in a simulated body fluid, which suggests osteoconductivity. It also showed antibacterial activity against Escherichia coli, which was enhanced by visible-light irradiation. This enhancement might be caused by the synergistic effect of the release of Ag and Cu and the generation of •OH from the sample. Dental implants with such a Ag and Cu co-doped TiO2 formed on their surface may reduce the risk of peri-implantitis.

Indirect cytotoxicity evaluations of antibacterial raw silk fabric doped with calcium, copper and zinc on fibroblasts and osteoblasts.

Antibacterial materials are widely used to prevent hospital-acquired infections. In our previous report, metal (calcium, copper or zinc)-doped raw silk fabrics were shown to possess strong antibacterial activities against Escherichia coli. However, antibacterial materials may occasionally be harmful to the human body; thus, in this study, we investigated the cytotoxicities of extracts from metal-doped raw silk fabrics with respect to fibroblasts and osteoblasts indirectly. Calcium-doped raw silk fabric demonstrated cytocompatibility with fibroblasts. Contrarily, copper- and zinc-doped raw silk fabrics remarkably decreased the cell densities of fibroblasts, indicating their cytotoxic effects. This observation could be attributed to the high concentrations of the released copper or zinc ions. However, calcium-, copper- and zinc-doped raw silk fabrics did not demonstrate any cytotoxic effects on osteoblasts because a high concentration of the serum alleviated the effects of these metal ions released from the fabrics. Thus, calcium-doped raw silk fabric is a promising antibacterial material that does not induce strong cytotoxicity. This study will facilitate the design of materials that are both antibacterial and safe.

Proteomic identification of serum proteins to induce osteoconductivity of hydroxyapatite.

We performed proteomic analysis of rat serum proteins adsorbed on hydroxyapatite (HAp) and α-alumina (α-Al2O3) in order to identify proteins that specifically adsorb onto HAp and control cellular responses. Proteins with either or both molecular weight of 22-32 kDa and computed isoelectric point of 5.0-5.5 were preferentially adsorbed on HAp. In total, 182 proteins were adsorbed on both HAp and α-Al2O3, of which 14 were highly enriched on HAp, whereas 68 were adsorbed only on HAp. Therefore, 82 (14+68) proteins were further evaluated by bioinformatics and literature-based analyses. We predicted that hepatocyte growth factor and angiopoietin-like protein 3 (ANGPTL3) are candidate proteins responsible for the osteoconductivity of HAp. Although ANGPTL3 promoted the attachment and spreading of MC3T3-E1 cells, it did not promote their proliferation and differentiation. Our results suggest that specific adsorption of ANGPTL3 on HAp induced osteoconductivity by enhancing the attachment and spreading of osteoblasts.

Cytotoxicity evaluation of iron nitride nanoparticles for biomedical applications.

Magnetic nanoparticles are widely studied for their use in various therapeutic and diagnostic purposes. As biomaterials, their biocompatibility is as important as their magnetic properties. Iron nitride (Fex Ny ) has excellent magnetic properties, and thus Fex Ny nanoparticles could be useful as potential biomaterials. However, the biocompatibility of Fex Ny nanoparticles is yet to be investigated. In this study, we assessed the biocompatibility of Fex Ny nanoparticles by evaluating their direct-contact cytotoxicity compared with that of magnetite nanoparticles (MNPs). Rat fibroblasts were incubated with the nanoparticle samples dispersed in culture medium at concentrations of 10, 50, and 100 µg/ml. The DNA concentration measurement, MTT assay, and trypan blue exclusion test were conducted after days 1 and 3 of incubation. After day 1, the cell viability decreased, and cell death increased with increasing sample concentration when compared with the control. However, after day 3, there were no significant differences when compared with the control, irrespective of the sample concentrations. Further, there were no significant differences between the Fex Ny nanoparticles and MNPs at the same concentrations in all the cytotoxicity evaluation tests. Therefore, it is suggested that Fex Ny nanoparticles might be as cytocompatible as the conventional MNPs.

Apatite formation and bacterial growth on raw silk fabric heated in argon gas.

Raw silk has the potential to be a flexible, osteoconductive material because it forms bone-like apatite on its surface in acellular simulated body fluid with ion concentrations nearly 1.5 times greater than that of human plasma (1.5SBF). It has been reported that silk-which has many similarities to raw silk-develops antibacterial properties when heated in inert gas, which may be advantageous in preventing bacterial infection. Hence, raw silk heated in inert gas may be a flexible, osteoconductive material with antibacterial activity. Thus, we examined the effect of the heat treatment of raw silk fabric on its apatite-forming ability in 1.5SBF and on the growth of Escherichia coli. Raw silk fabric was heated in argon gas at several temperatures, to a maximum of 500 °C. The results of soaking tests in 1.5SBF indicate that the apatite-forming ability of raw silk decreases with increasing temperature. This may be because favourable structures for apatite formation, such as carboxyl groups, are thermally decomposed. The results of bacterial tests indicate that raw silk fabrics heated to 300 °C or 500 °C exhibit reduced bacterial growth compared to those that were not heated or were heated only to 100 °C. This might be because hydrophobic surfaces inhibit bacterial adhesion, or because the thermal decomposition of sericin-a component of raw silk-leads to a lack of available nutrients for the bacteria. Although this study did not demonstrate the expected material properties needed for clinical applications, this research contributes to a better understanding of silk biomaterials.

Interventional nephrology: current status and clinical impact in Japan.

BACKGROUND: Current status and clinical significance of interventional nephrology has not been reported from Japan. METHODS: Questionnaires were mailed twice to the directors of all 534 Japanese certificated nephrology training institutions in 2014. The main questions were current performance, categorized annual procedure volume and managers of peritoneal dialysis (PD) access, vascular access (VA) surgery, endovascular intervention, and kidney biopsy. Frequencies of nephrologist involvement between high volume center and low volume center and association between the level of nephrologists' involvement to each procedure and annual procedure volume were examined. RESULTS: 332 (62.2%) institutions answered performance of all procedures and 328 (61.4%) institutions answered all procedure volume. Kidney biopsy, VA surgery, endovascular intervention and PD access surgery were performed by any doctors in 94.2, 96.3, 88.4, and 76.2% and each involvement of nephrologist was 93.9, 54.1, 53.1 and 47.6%, respectively. Cochran-Armitage analyses demonstrated significant increases in all 4 procedure volume with greater management by nephrologists (p < 0.01). Nephrologists involvement to VA surgery associated with procedure volume increase in not only VA surgery, but also PD catheter insertion (p < 0.01) and kidney biopsy (p < 0.05). And nephrologists involvement to PD catheter insertion also associated with surgical volume increase in both VA surgery (p < 0.01) and endovascular intervention (p < 0.05). CONCLUSIONS: Main manager of all 4 procedures was nephrologist in Japan. Each procedure volume increased as nephrologists become more involved. Acquisition of one specific procedure by nephrologist associated with increase not only in this specific procedure volume, but also the other procedure volume.

TiO2 microspheres containing magnetic nanoparticles for intra-arterial hyperthermia.

Magnetic microspheres measuring 15-35 µm in diameter are believed to be useful for intra-arterial hyperthermia. In this study, we attempted to prepare titanium dioxide (TiO2 ) microspheres containing magnetic nanoparticles (MNPs). MNP-containing TiO2 microspheres with diameters of approximately 30 µm were successfully obtained by sol-gel reaction of titanium tetraisopropoxide in a water-in-oil emulsion with added cosurfactant of 1-butanol and subsequent heat treatment at 200°C. The microspheres showed ferrimagnetism owing to high content of MNPs in approximately 60 wt % and had a low-crystalline TiO2 matrix. Furthermore, the agar phantom was heated to above 43°C after approximately 1 min under an alternating magnetic field of 100 kHz and 300 Oe and showed in vitro biocompatibility similar to that of MNP-free TiO2 microspheres. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2308-2314, 2017.

Formation of bioactive N-doped TiO2 on Ti with visible light-induced antibacterial activity using NaOH, hot water, and subsequent ammonia atmospheric heat treatment.

Titanium (Ti) treated with NaOH and hot water, and heated in an ammmonia (NH3) gas atmosphere for 1 or 3h exhibited in vitro apatite formation within 7days when soaked in simulated body fluid (SBF). Moreover, the treated Ti decomposed methylene blue and showed excellent bactericidal activity against Escherichia coli under visible light irradiation. The surface treatment resulted in the formation of a fine network of N-doped anatase-type titania (TiO2-xNx) on the Ti surface, which was responsible for both the apatite formation in SBF and the visible light-induced antibacterial activity. These preliminary results highlight the efficacy of our simple method for producing novel bioactive Ti with visible light-induced antibacterial activity, which could be applied to orthopaedic and dental implants without the risk of infection.

Clinical and pathological features of donor/recipient body weight mismatch after kidney transplantation.

BACKGROUND: Previous studies have shown that a donor/recipient body weight mismatch affects long-term graft survival and graft function after kidney transplantation. However, the mechanisms are not fully understood. AIM: To address the mechanisms, we compared the pathological and physiological features between patients with a donor/recipient body weight mismatch and those without a mismatch 1 yr after kidney transplantation. Furthermore, we investigated the correlation with the donor/recipient body weight ratio. METHODS: We examined allograft biopsy specimens from 10 recipients with stable kidney function, with body weight mismatch (donor/recipient body weight ratio [D/R BWR] < 0.9), and compared them with samples from 13 patients without mismatch. We measured glomerular volume (GV) using the Weibel-Gomez method and glomerular density (GD) defined by nonsclerotic glomerular number/renal cortical area as pathological findings. The physiological parameters included estimated glomerular filtration rate and proteinuria (mg/day). These data were evaluated to identify a correlation with D/R BWR. RESULTS: The pathological features showed that GV and GD were identical in the two groups. However, when glomerular enlargement was defined by ΔGV (GV at the 1-yr biopsy minus GV at baseline biopsy), ΔGV was higher in mismatch cases compared with that in cases without a mismatch (10.6 ± 4.6 vs. 5.5 ± 7.1 × 10(5) µm(3) ; P = 0.049). Furthermore, D/R BWR was significantly correlated with ΔGV (P = 0.03, r = -0.436). eGFR values were physiologically identical between the two groups, but the mismatch cases had significantly higher proteinuria levels than that of the cases without a mismatch at 1 yr after kidney transplantation. CONCLUSION: A donor/recipient body weight mismatch could affect glomerular enlargement and increased proteinuria 1 yr after kidney transplantation. How these two features affect long-term graft survival and function must be addressed in the future.

Probable C4d-negative accelerated acute antibody-mediated rejection due to non-HLA antibodies.

We report a case of probable C4d-negative accelerated acute antibody-mediated rejection due to non-HLA antibodies. A 44 year-old male was admitted to our hospital for a kidney transplant. The donor, his wife, was an ABO minor mismatch (blood type O to A) and had Gitelman syndrome. Graft function was delayed; his serum creatinine level was 10.1 mg/dL at 3 days after transplantation. Open biopsy was performed immediately; no venous thrombosis was observed during surgery. Histology revealed moderate peritubular capillaritis and mild glomerulitis without C4d immunoreactivity. Flow cytometric crossmatching was positive, but no panel-reactive antibodies against HLA or donor-specific antibodies (DSAbs) to major histocompatibility complex class I-related chain A (MICA) were detected. Taken together, we diagnosed him with probable C4d-negative accelerated antibody-mediated rejection due to non-HLA, non-MICA antibodies, the patient was treated with steroid pulse therapy (methylprednisolone 500 mg/day for 3 days), plasma exchange, intravenous immunoglobulin (40 g/body), and rituximab (200 mg/body) were performed. Biopsy at 58 days after transplantation, at which time S-Cr levels were 1.56 mg/dL, found no evidence of rejection. This case, presented with a review of relevant literature, demonstrates that probable C4d-negative accelerated acute AMR can result from non-HLA antibodies.

Acute T cell-mediated rejection accompanied by C4d-negative acute antibody-mediated rejection and cell debris in tubulus: A case report.

Herein, we report a complicated case of acute T-cell-mediated rejection (ACR) accompanied by C4d-negative acute antibody-mediated rejection (AMR) and cell debris in tubulus. A 32 year-old male was admitted for an episode biopsy with a serum creatinine (S-Cr) level of 1.83 mg/dL and pyuria (20-29 white blood cells per high power field) 49 days following kidney transplantation. Histological features included three distinct entities, mainly, in one of the three specimens: 1) focal aggressive tubulointerstitial inflammatory cell infiltration with moderate tubulitis, 2) inflammatory cell infiltration in peritubular capillaries (including neutrophils) and glomerular capillaries, and 3) cell debris consisting mainly of neutrophils in tubulus. Laboratory examination revealed evidence of non-human leukocyte antigen donor-specific antibodies. However, urinary culture and gram staining were negative. Considering both the histological and laboratory findings, the patient was diagnosed with ACR accompanied by C4d-negative AMR and suspicion of a urinary tract infection (UTI). The patient was treated for three consecutive days with steroid pulse therapy. The patient's S-Cr level decreased to ~1.5 mg/dL following treatment and did not increase thereafter. A second biopsy 133 days following kidney transplantation showed an excellent response to treatment and revealed no evidence of rejection. This case report demonstrates the difficulty in the diagnosis of, and therapy for, the complicated pathological findings of ACR, AMR and suspicion of a UTI.

Plasma cell-rich rejection accompanied by acute antibody-mediated rejection in a patient with ABO-incompatible kidney transplantation.

We report a case of plasma cell-rich rejection accompanied by acute antibody-mediated rejection in a patient with ABO-incompatible kidney transplantation. A 33-year-old man was admitted for an episode biopsy; he had a serum creatinine (S-Cr) level of 5.7 mg/dL 1 year following primary kidney transplantation. Histological features included two distinct entities: (1) a focal, aggressive tubulointerstitial inflammatory cell (predominantly plasma cells) infiltration with moderate tubulitis; and (2) inflammatory cell infiltration (including neutrophils) in peritubular capillaries. Substantial laboratory examination showed that the patient had donor-specific antibodies for DQ4 and DQ6. Considering both the histological and laboratory findings, we diagnosed him with plasma cell-rich rejection accompanied by acute antibody-mediated rejection. We started 3 days of consecutive steroid pulse therapy three times every 2 weeks for the former and plasma exchange with intravenous immunoglobulin (IVIG) for the latter histological feature. One month after treatment, a second allograft biopsy showed excellent responses to treatment for plasma cell-rich rejection, but moderate, acute antibody-mediated rejection remained. Therefore, we added plasma exchange with IVIG again. After treatment, allograft function was stable, with an S-Cr level of 2.8 mg/dL. This case report demonstrates the difficulty of the diagnosis of, and treatment for, plasma cell-rich rejection accompanied by acute antibody-mediated rejection in a patient with ABO-incompatible kidney transplantation. We also include a review of the related literature.

Role of the Msh2 gene in genome maintenance and development in mouse fetuses.

In an attempt to evaluate the roles of the mismatch repair gene Msh2 in genome maintenance and in development during the fetal stage, spontaneous mutations and several developmental indices were studied in Msh2-deficient lacZ-transgenic mouse fetuses. Mutation levels in fetuses were elevated at 9.5 dpc (days post coitum) when compared to wild-type mice, and the level of mutations continued to increase until the fetuses reached the newborn stage. The mutation levels in 4 different tissues of newborns showed similar magnitudes to those in the whole body. The levels remained similar after birth until 6 months of age. The molecular nature of the mutations examined in 12.5 dpc fetuses of Msh2(+/+) and Msh2(-/-) revealed unique spectra which reflect errors produced during the DNA replication process, and those corrected by a mismatch repair system. Most base substitutions and simple deletions were reduced by the presence of the Msh2 gene, whereas G:C to A:T changes at CpG sequences were not affected, suggesting that the latter change was not influenced by mismatch repair. On the other hand, analysis of developmental indices revealed that there was very little effect, including the presence of malformations, resulting from Msh2-deficiencies. These results indicate that elevated mutation levels have little effect on the development of the fetus, even if a mutator phenotype appears at the organogenesis stage.

Effects of calorie restriction on the age-dependent accumulation of mutations in the small intestine of lacZ-transgenic mice.

To understand the effect of calorie restriction on genome maintenance systems, the age-dependent accumulation of mutations in animals maintained on high and low calorie diets was examined using lacZ-transgenic mice. Mice were fed a diet of 95 kcal/w or 65 kcal/w from 2 to 17 months of age. The mutation frequencies in the lacZ gene in epithelial tissues from the small intestine were examined at 12 and 17 months. Mutation frequencies were found to be lower in mice fed with a low calorie diet than in mice fed with a high calorie diet at the two age points. The molecular nature of the mutations was examined with DNA sequencing. It showed a predominance of transversions from G:C to T:A, and this is a typical type of mutation induced by reactive oxygen species. The fraction of this type of mutation among the different types of mutations detected was not affected by calorie restriction. The percentage of the other types of mutation was not influenced either. These results suggest that calorie restriction reduces the age-dependent accumulation of mutations by stimulating or inducing various types of DNA protection and repair systems rather than protecting cells against any specific type of DNA alteration.

XPC is involved in genome maintenance through multiple pathways in different tissues.

In an attempt to evaluate the role of the Xpc gene in maintaining genomic stability in vivo under normal conditions, the age-dependent accumulation of spontaneous mutations in different tissues was analyzed in Xpc-deficient lacZ-transgenic mice. Brain, testis, and small intestine revealed no effects from the Xpc-deficiency, whereas liver, spleen, heart, and lung showed an enhanced age-related accumulation of mutations in Xpc-deficient mice. In the spleen, the effect was not obvious at 2 and 12 months of age, but became apparent at 23 months. The magnitude of the observed effect at an advanced age was similar in the liver, spleen and heart, but was comparatively smaller in the lung. Haploinsufficiency was observed in liver and spleen but not in heart and lung. Analysis of DNA sequences in the mutants revealed that the frequency of G:C to T:A changes were elevated in the liver and heart of Xpc-deficient aged mice, supporting the possible involvement of XPC in base excision repair of oxidized guanine. The occurrence of two or more mutations within a single lacZ gene was termed a multiple mutation and was also elevated in old Xpc-deficient mice. Among the clones examined, two mutant clones showed as many as four mutations within a short stretch of DNA. This is the first demonstration to support suggestions for the existence of a role for XPC in the suppression of multiple mutations. These multiple mutations could conceivably be generated by error-prone trans-lesional DNA synthesis. Overall, these results indicate that there may be diverse roles or mechanisms through which XPC participates in genome maintenance in different tissues.