Biosafety of mesoporous silica nanoparticles: a combined experimental and literature study.

Mesoporous silica (MS) particles have been explored for various healthcare applications, but universal data about their safety and/or toxicity are yet to be well-established for clinical purposes. Information about general toxicity of hollow MS (HMS) particles and about immunotoxicity of MS particles are significantly lacked. Therefore, acute toxicity and immunotoxicity of HMS particles were experimentally evaluated. A systematic and objective literature study was parallelly performed to analyze the published in vivo toxicity of MS particles. Lethal acute toxicity of MS particles is likely to arise from their physical action after intravenous and intraperitoneal administrations, and only rarely observed after subcutaneous administration. No clear relationship was identified between physicochemical properties of MS particles and lethality as well as maximum tolerated dose with some exceptions. At sub-lethal doses, MS particles tend to accumulate mainly in lung, liver, and spleen. The HMS particles showed lower inflammation-inducing ability than polyinosinic-polycytidylic acid and almost the same allergy-inducing ability as Alum. Finally, the universal lowest observed adverse effect levels were determined as 0.45, 0.81, and 4.1 mg/kg (human equivalent dose) for intravenous, intraperitoneal, and subcutaneous administration of MS particles, respectively. These results could be helpful for determining an appropriate MS particle dose in clinical study.

Cell attachment area of rat mesenchymal stem cells correlates with their osteogenic differentiation level on substrates without osteoconductive property.

Cell morphology is related to proliferation and differentiation. We previously reported that cell attachment area of rat mesenchymal stem cells (MSCs) is negatively correlated with their osteogenic differentiation level on osteoconductive hydroxyapatite (HAp) with various microstructures. In this study, the correlation between the cell attachment area and osteogenic differentiation level was investigated on substrates without osteoconductive property using tissue culture polystyrene (TCPS), and 3 mol% yttria-stabilized zirconia (3Y-TZP) with or without surface periodic microstructures. It was found that the osteogenic differentiation level after 3 weeks of culture increased with a decrease in cell attachment area after 3 h of culture. The square of the correlation coefficient between cell attachment area and osteocalcin secretion content was 0.845 among the three types of substrates. Thus, the negative correlation between cell attachment area and differentiation level is confirmed even when cultured on substrates without osteoconductive property. These findings suggest that the correlation between the cell attachment area of rat MSCs and osteogenic differentiation level could also apply to various types of substrate, regardless of osteoconductive property.

Tailoring inorganic nanoadjuvants towards next-generation vaccines.

Vaccines, one of the most effective and powerful public health measures, have saved countless lives over the past century and still have a tremendous global impact. As an indispensable component of modern vaccines, adjuvants play a critical role in strengthening and/or shaping a specific immune response against infectious diseases as well as malignancies. The application of nanotechnology provides the possibility of precisely tailoring the building blocks of nanoadjuvants towards modern vaccines with the desired immune response. The last decade has witnessed great academic progress in inorganic nanomaterials for vaccine adjuvants in terms of nanometer-scale synthesis, structure control, and functionalization design. Inorganic adjuvants generally facilitate the delivery of antigens, allowing them to be released in a sustained manner, enhance immunogenicity, deliver antigens efficiently to specific targets, and induce a specific immune response. In particular, the recent discovery of the intrinsic immunomodulatory function of inorganic nanomaterials further allows us to shape the immune response towards the desired type and increase the efficacy of vaccines. In this article, we comprehensively review state-of-the-art research on the use of inorganic nanomaterials as vaccine adjuvants. Attention is focused on the physicochemical properties of versatile inorganic nanoadjuvants, such as composition, size, morphology, shape, hydrophobicity, and surface charge, to effectively stimulate cellular immunity, considering that the clinically used alum adjuvants can only induce strong humoral immunity. In addition, the efforts made to date to expand the application of inorganic nanoadjuvants in cancer vaccines are summarized. Finally, we discuss the future prospects and our outlook on tailoring inorganic nanoadjuvants towards next-generation vaccines.

Hollow ZnO Nanospheres Enhance Anticancer Immunity by Promoting CD4+ and CD8+ T Cell Populations In Vivo.

Appropriate adjuvant aiding in generating robust anticancer immunity is crucial for cancer immunotherapy. Herein, hollow ZnO (HZnO) nanospheres are synthesized by a facile method using carbon nanospheres as the template. The HZnO nanospheres significantly promote the cellular uptake of a model antigen, and cytokine secretion by antigen-presenting cells in vitro. HZnO loaded with ovalbumin and polyinosinic-polycytidylic acid (poly(I:C)) inhibits cancer growth and metastasis to inguinal lymph node in a cancer cell challenge model. Moreover, HZnO loaded with autologous cancer antigens inhibits cancer cell growth in a cancer cell re-challenge model. HZnO nanospheres significantly improve the CD4+ and/or CD8+ T cell population in splenocytes of mice in both cancer cell challenge model and re-challenge model. The HZnO nanospheres can be used for cancer immunotherapy as adjuvant.

Hollow Structure Improved Anti-Cancer Immunity of Mesoporous Silica Nanospheres In Vivo.

Hollow and non-hollow mesoporous silica nanospheres are synthesized and used for cancer vaccine adjuvants. The hollow structure of mesoporous silica nanospheres significantly promote cellular uptake of a model cancer antigen by macrophage-like cells in vitro, improve anti-cancer immunity, CD4(+) and CD8(+) T cell populations in splenocytes of mice in vivo.

Effects of gatifloxaine content in gatifloxacine-loaded PLGA and ß-tricalcium phosphate composites on efficacy in treating osteomyelitis.

Composites of gatifloxacin (GFLX)-loaded poly (lactic-co-glycolic) acid (PLGA) and ß-tricalcium phosphate (ßTCP) containing 0, 1, and 10 wt % GFLX (0, 1, and 10 wt % GFLX composites), and GFLX-loaded PLGA containing 1, 5, and 10 wt % GFLX (1, 5, and 10wt % GFLX-PLGA) as controls were fabricated and characterized in vitro and in vivo. On in vitro evaluation, the 10 wt % GFLX composite released GFLX over at least 28 days in Hanks' balanced solution and exhibited clinically sufficient bactericidal activities against Streptococcus milleri and Bacteroides fragilis from 1 h to 10 days. The 0, 1, and 10 wt % GFLX composites and 10 wt % GFLX-PLGA were implanted in bone defects created by debridement of osteomyelitis lesions induced by S. milleri and B. fragilis in the mandible of rabbits (n = 5). Four weeks after implantation of the 10 wt % GFLX composite, inflammation in the debrided area disappeared in all the rabbits, while inflammation remained in all the rabbits after implantation of the 0 wt % GFLX composite and 10 wt % GFLX-PLGA, and in three rabbits after implantation of the 1 wt % GFLX composite. Bone formation appears to be less intense for the 10 wt % GFLX composite than for the 1 wt % GFLX composite probably owing to the rapid degradation of the 10 wt % GFLX composite. These findings show that the GFLX composite is effective for the local treatment of osteomyelitis.

Stimulation of In Vivo Antitumor Immunity with Hollow Mesoporous Silica Nanospheres.

The use of appropriate adjuvants that support the generation of robust and long-lasting antitumor immune responses is crucial for tumor immunotherapy owing to the immunosuppressive environment of the growing tumor. However, the most commonly used adjuvant, aluminum hydroxide, is ineffective for generating such immune responses and therefore not suitable for cancer immunotherapy. It is now shown that plain hollow mesoporous silica nanospheres markedly improve the antitumor immunity, the Th1 and Th2 immunity, and the CD4(+) and CD8(+) effector memory T cell population in bone marrow in vivo and may thus be used as immunoadjuvants to treat cancer in humans.

Area-specific cell stimulation via surface-mediated gene transfer using apatite-based composite layers.

Surface-mediated gene transfer systems using biocompatible calcium phosphate (CaP)-based composite layers have attracted attention as a tool for controlling cell behaviors. In the present study we aimed to demonstrate the potential of CaP-based composite layers to mediate area-specific dual gene transfer and to stimulate cells on an area-by-area basis in the same well. For this purpose we prepared two pairs of DNA-fibronectin-apatite composite (DF-Ap) layers using a pair of reporter genes and pair of differentiation factor genes. The results of the area-specific dual gene transfer successfully demonstrated that the cells cultured on a pair of DF-Ap layers that were adjacently placed in the same well showed specific gene expression patterns depending on the gene that was immobilized in the underlying layer. Moreover, preliminary real-time PCR results indicated that multipotential C3H10T1/2 cells may have a potential to change into different types of cells depending on the differentiation factor gene that was immobilized in the underlying layer, even in the same well. Because DF-Ap layers have a potential to mediate area-specific cell stimulation on their surfaces, they could be useful in tissue engineering applications.

The calcium phosphate matrix of FGF-2-apatite composite layers contributes to their biological effects.

The purpose of the present study was to fabricate fibroblast growth factor (FGF)-2-apatite composite layers on titanium (Ti) pins in one step at 25 °C using a supersaturated calcium phosphate (CaP) solution, and to evaluate the physicochemical characteristics and biological effects of the coated Ti pins compared with coated Ti pins fabricated at 37 °C. Ti pins were immersed in a supersaturated CaP solution containing 0.5, 1.0, or 2.0 µg/mL FGF-2 at 25 °C for 24 h (25F0.5, 25F1.0, and 25F2.0) or containing 4.0 µg/mL FGF-2 at 37 °C for 48 h (37F4.0). Except for the 25F0.5, the chemical compositions and the mitogenic activity levels of FGF-2 of the composite layers formed by these two methods were similar, except for the Ca/P molar ratio, which was markedly smaller at 25 °C (1.55-1.56±0.01-0.02, p=0.0008-0.0045) than at 37 °C (1.67±0.11). Thus, either the apatite was less mature or the amount of amorphous calcium phosphate was higher in the composite layer formed at 25 °C. In vivo, the pin tract infection rate by visual inspection for 37F4.0 (45%) was lower than that for 25F1.0 (80%, p=0.0213), and the rate of osteomyelitis for 37F4.0 (35%) was lower than that for 25F0.5 (75%, p=0.0341). The extraction torque for 37F4.0 (0.276±0.117 Nm) was higher than that for 25F0.5 (0.192±0.117 Nm, p=0.0142) and that for 25F1.0 (0.176±0.133 Nm, p=0.0079). The invasion rate of S. aureus for 37F4.0 (35%) was lower than that for 25F0.5 (75%, p=0.0110). On the whole, the FGF-2-apatite composite layer formed at 25 °C tended to be less effective at improving fixation strength in the bone-pin interface and resisting pin tract infections. These results suggest that the chemistry of the calcium phosphate matrix that embeds FGF-2, in addition to FGF-2 content and activity, has a significant impact on composite infection resistance and fixation strength.

Potential of Titanium Pins Coated with Fibroblast Growth Factor-2-Calcium Phosphate Composite Layers to Reduce the Risk of Impaired Bone-Pin Interface Strength in the External Fixation of Distal Radius Fractures.

Background: The risk of impaired bone-pin interface strength in titanium (Ti) pins coated with fibroblast growth factor (FGF)-calcium phosphate (CP) composite layers is yet to be evaluated in a clinical study. This retrospective study used Weibull plot analysis to evaluate bone-pin interface strength in Ti pins coated with FGF-CP layers for external distal radius fracture fixation. Methods: The distal radial fractures were treated with external fixation. The FGF-CP group comprised five patients (all women, aged 70.4 ± 5.9 (range: 62-77) years), and the uncoated pin group comprised ten patients (eight women and two men, aged 64.4 ± 11.7 (range: 43-83) years). The pins were removed after six weeks. The insertion and extraction peak torques were measured. The extraction peak torque was evaluated using Weibull plot analysis. Results: We compared the extraction torque of the two groups at or below 506 Nmm for a fair comparison using Weibull plot analysis. The Weibull plots were linear for both the FGF-CP and uncoated pin groups. The slope of the regression line was significantly higher in the FGF-CP group (1.7343) than in the uncoated pin group (1.5670) (p = 0.011). The intercept of the regression line was significantly lower in the FGF-CP group (-9.847) than in the uncoated pin group (-8.708) (p = 0.002). Thus, the two regression lines significantly differed. Conclusions: Ti pins coated with FGF-CP layers exhibit the potential to reduce the risk of impaired bone-pin interface strength in the external fixation of distal radius fractures.

Femtosecond Laser Irradiation to Zirconia Prior to Calcium Phosphate Coating Enhances Osteointegration of Zirconia in Rabbits.

Calcium phosphate (CaP) coating of zirconia and zirconia-based implants is challenging, due to their chemical instability and susceptibility to thermal and mechanical impacts. A 3 mol% yttrium-stabilized tetragonal zirconia polycrystal was subjected to femtosecond laser (FsL) irradiation to form micro- and submicron surface architectures, prior to CaP coating using pulsed laser deposition (PLD) and low-temperature solution processing. Untreated zirconia, CaP-coated zirconia, and FsL-irradiated and CaP-coated zirconia were implanted in proximal tibial metaphyses of male Japanese white rabbits for four weeks. Radiographical analysis, push-out test, alizarin red staining, and histomorphometric analysis demonstrated a much improved bone-bonding ability of FsL-irradiated and CaP-coated zirconia over CaP-coated zirconia without FsL irradiation and untreated zirconia. The failure strength of the FsL-irradiated and CaP-coated zirconia in the push-out test was 6.2-13.1-times higher than that of the CaP-coated zirconia without FsL irradiation and untreated zirconia. Moreover, the adhesion strength between the bone and FsL-irradiated and CaP-coated zirconia was as high as that inducing host bone fracture in the push-out tests. The increased bone-bonding ability was attributed to the micro-/submicron surface architectures that enhanced osteoblastic differentiation and mechanical interlocking, leading to improved osteointegration. FsL irradiation followed by CaP coating could be useful for improving the osteointegration of cement-less zirconia-based joints and zirconia dental implants.

Correlation between cell attachment areas after 2 h of culture and osteogenic differentiation activity of rat mesenchymal stem cells on hydroxyapatite substrates with various surface properties.

The initial attachment of mesenchymal stem cells (MSCs) to substrates and osteogenic differentiation are supported by culture on a hydroxyapatite substrate. Cell attachment areas of rat MSCs after 2 h of culture on hydroxyapatite substrates with various microstructures and the osteogenic differentiation activity thereafter were measured. The perceived outcome was that, after 2 h of culture, rat MSCs with a small attachment area would have a high osteogenic differentiation activity, whereas those with a large attachment area would have a low osteogenic differentiation activity. Furthermore, rat MSCs with a small attachment area had many cytoplasmic processes, while those with a large attachment area revealed clear stress fibers and focal contacts. These results suggest that cell attachment area of rat MSCs after 2 h of culture has a strong effect on the osteogenic differentiation of rat MSCs. Thus, the measurement of cell attachment area after 2 h of culture could become valuable for estimating the osteogenic differentiation activity of rat MSCs thereafter.

Angiogenesis therapy for brain infarction using a slow-releasing drug delivery system for fibroblast growth factor 2.

Although fibroblast growth factor 2 (FGF2) is a promising agent for treating brain infarction, current methods of FGF2 administration are associated with a short circulating half-life. An FGF2 apatite coating was developed as a slow-releasing drug delivery system (DDS) by forming an FGF2/calcium phosphate composite layer. Hydroxyapatite was coated with high or low doses of FGF2, denoted as FGF-high and FGF-low. This study investigated the efficacy of the coating as angiogenesis therapy for brain infarction. Rats were subjected to permanent occlusion of the middle cerebral artery, an FGF2 apatite-coated implant was inserted, and the rat brains were removed 2 weeks after implantation. Rats in groups treated with FGF-high had significantly smaller areas of brain infarction, particularly in the external capsule and the lateral side of the putamen, and better capillary density than rats in groups treated with non-FGF2 apatite-coated implants. Histologic analysis indicated that the new vessels were larger and had thicker walls in the FGF2 apatite-coated groups than in the non-FGF2 groups. Fluorescence immunohistochemistry of the peri-infarction region showed that FGF2 released from FGF2 apatite-coated implants might have biological activity. Moreover, fluorescence immunohistochemistry showed that released FGF2 influenced microglia cells. This new FGF2 DDS involving an FGF2 apatite coating can prevent infarction of the penumbra through the multipotential effects of FGF2.

Contaminants in tracked seabirds showing regional patterns of marine pollution.

Ocean-scale monitoring of pollution is challenging. Seabirds are useful indicators because they travel over a broad foraging range. Nevertheless, this coarse spatial resolution is not fine enough to discriminate pollution in a finer scale. Previous studies have demonstrated that pollution levels are higher in the Sea of Japan and South and East China Seas than the Northen Pacific Ocean. To test these findings in a wide-ranging animal, we tracked streaked shearwaters (Calonectris leucomelas) from four islands in Japan using global positioning system (GPS) and measured persistent organic pollutants (POPs) in the oil of their preen glands. The POPs did not change during 6 to 21 days when birds from Awashima were foraging only in the Sea of Japan, while it increased when they crossed to the Pacific through the Tsugaru Strait and foraged along the eastern coast of Hokkaido where industrial cities occur. These results indicate that POPs in the oil reflect relatively short-term exposure. Concentrations of POPs displayed greater variation among regions. Total polychlorinated biphenyls were highest in birds foraging in a small area of the semiclosed Seto Inland Sea surrounded by urbanized coast, p,p'-dichlorodiphenyltrichloroethane (DDT) was highest in birds foraging in the East China Sea, and total hexachlorocyclohexanes were highest in birds foraging in the Sea of Japan. All were lowest in birds foraging in the Pacific. This distribution of POPs concentration partly agrees with previous findings based on mussels, fish, and seawater and possibly reflects the mobility and emission sources of each type of POP. These results highlight the importance of information on the foraging area of highly mobile top predators to make them more effective monitors of regional marine pollution.

Tissue-engineered endothelial cell layers on surface-modified Ti for inhibiting in vitro platelet adhesion.

A tissue-engineered endothelial layer was prepared by culturing endothelial cells on a fibroblast growth factor-2 (FGF-2)-l-ascorbic acid phosphate magnesium salt n-hydrate (AsMg)-apatite (Ap) coated titanium plate. The FGF-2-AsMg-Ap coated Ti plate was prepared by immersing a Ti plate in supersaturated calcium phosphate solutions supplemented with FGF-2 and AsMg. The FGF-2-AsMg-Ap layer on the Ti plate accelerated proliferation of human umbilical vein endothelial cells (HUVECs), and showed slightly higher, but not statistically significant, nitric oxide release from HUVECs than on as-prepared Ti. The endothelial layer maintained proper function of the endothelial cells and markedly inhibited in vitro platelet adhesion. The tissue-engineered endothelial layer formed on the FGF-2-AsMg-Ap layer is promising for ameliorating platelet activation and thrombus formation on cardiovascular implants.

Formation of apatite coatings on an artificial ligament using a plasma- and precursor-assisted biomimetic process.

A plasma- and precursor-assisted biomimetic process utilizing plasma and alternate dipping treatments was applied to a Leed-Keio artificial ligament to produce a thin coating of apatite in a supersaturated calcium phosphate solution. Following plasma surface modification, the specimen was alternately dipped in calcium and phosphate ion solutions three times (alternate dipping treatment) to create a precoating containing amorphous calcium phosphate (ACP) which is an apatite precursor. To grow an apatite layer on the ACP precoating, the ACP-precoated specimen was immersed for 24 h in a simulated body fluid with ion concentrations approximately equal to those in human blood plasma. The plasma surface modification was necessary to create an adequate apatite coating and to improve the coating adhesion depending on the plasma power density. The apatite coating prepared using the optimized conditions formed a thin-film that covered the entire surface of the artificial ligament. The resulting apatite-coated artificial ligament should exhibit improved osseointegration within the bone tunnel and possesses great potential for use in ligament reconstructions.

Improved bonding of partially osteomyelitic bone to titanium pins owing to biomimetic coating of apatite.

Increased fixation strength of the bone-pin interface is important for inhibiting pin loosening after external fixation. In a previous study, an apatite (Ap) layer was formed on anodically oxidized titanium (Ti) pins by immersing them in an infusion fluid-based supersaturated calcium phosphate solution at 37 °C for 48 h. In the present study, an Ap layer was also successfully formed using a one-step method at 25 °C for 24 h in an infusion fluid-based supersaturated calcium phosphate solution, which is clinically useful due to the immersion temperature [corrected]. After percutaneous implantation in a proximal tibial metaphysis for four weeks in rabbits (n = 20), the Ti pin coated with the Ap layer showed significantly increased extraction torque compared with that of an uncoated Ti screw even with partial osteomyelitis present, owing to dense bone formation on the Ap layer in the cortical and medullary cavity regions. When the infection status was changed from "no osteomyelitis" to "partial osteomyelitis," the extraction torque in the Ap group with "partial osteomyelitis" was almost identical to that for "no osteomyelitis" cases. These results suggest that the Ap layer formed by the room temperature process could effectively improve the fixation strength of the Ti pin for external fixation clinically even with partial osteomyelitis present.

Clinical Trial for the Safety and Feasibility of Pedicle Screws Coated with a Fibroblast Growth Factor-2-Apatite Composite Layer for Posterior Cervical Fusion Surgery.

To solve the instrument loosening problem, we developed a fibroblast growth factor-2-calcium phosphate composite layer as a novel coating material to improve screw fixation strength. The primary aim of the present study was to demonstrate the safety and feasibility of screws coated with the FGF-2-calcium phosphate composite layer for posterior instrumented surgery of the cervical spine. The trial design was a single-arm, open-label, safety and feasibility study. Patients receiving fusion of the cervical spine from C2 (or C3) to C7 (or T1) were recruited. The primary endpoint to confirm safety was any screw-related adverse events. Seven patients who underwent posterior fusion surgery of the cervical spine were enrolled in the present study. The coated pedicle screws were inserted bilaterally into the lowest instrumented vertebrae. There was only one severe adverse event unrelated with the coated screw. Three out of the fourteen coated screws showed loosening. The present results prove the safety and feasibility of pedicle screws coated with the FGF-2-calcium phosphate composite layer for fusion surgery in the cervical spine. This is the first step to apply this novel surface coating in the field of spine surgery.

Fabrication of a DNA-lipid-apatite composite layer for efficient and area-specific gene transfer.

A surface-mediated gene transfer system using biocompatible apatite-based composite layers has great potential for tissue engineering. Among the apatite-based composite layers developed to date, we focused on a DNA-lipid-apatite composite layer (DLp-Ap layer), which has the advantage of relatively high efficiency as a non-viral system. In this study, various lipid transfection reagents, including a newly developed reagent, polyamidoamine dendron-bearing lipid (PD), were employed to prepare the DLp-Ap layer, and the preparation condition was optimized in terms of efficiency of gene transfer to epithelial-like CHO-K1 cells in the presence of serum. The optimized DLp-Ap layer derived from PD had the highest gene transfer efficiency among all the apatite-based composite layers prepared in this study. In addition, the optimized DLp-Ap layer demonstrated higher gene transfer efficiency in the presence of serum than the conventional particle-mediated systems using commercially available lipid transfection reagents. It was also shown that the optimized DLp-Ap layer mediated the area-specific gene transfer on its surface, i.e., DNA was preferentially transferred to the cells adhering to the surface of the layer. The present gene transfer system using the PD-derived DLp-Ap layer, with the advantages of high efficiency in the presence of serum and area-specificity, would be useful in tissue engineering.

Fabrication of DNA-antibody-apatite composite layers for cell-targeted gene transfer.

Surface-mediated gene transfer systems using apatite (Ap)-based composite layers have received increased attention in tissue engineering applications owing to their safety, biocompatibility and relatively high efficiency. In this study, DNA-antibody-apatite composite layers (DA-Ap layers), in which DNA and antibody molecules are immobilized within a matrix of apatite nanocrystals, were fabricated using a biomimetic coating process. They were then assayed for their gene transfer capability for application in a specific cell-targeted gene transfer. A DA-Ap layer that was fabricated with an anti-CD49f antibody showed a higher gene transfer capability to the CD49f-positive CHO-K1 cells than a DNA-apatite composite layer (D-Ap layer). The antibody facilitated the gene transfer capability of the DA-Ap layer only to the specific cells that were expressing corresponding antigens. When the DA-Ap layer was fabricated with an anti-N-cadherin antibody, a higher gene transfer capability compared with the D-Ap layer was found in the N-cadherin-positive P19CL6 cells, but not in the N-cadherin-negative UV♀2 cells or in the P19CL6 cells that were pre-blocked with anti-N-cadherin. Therefore, the antigen-antibody binding that takes place at the cell-layer interface should be responsible for the higher gene transfer capability of the DA-Ap than D-Ap layer. These results suggest that the DA-Ap layer works as a mediator in a specific cell-targeted gene transfer system.