Comparative Gene-Expression Analysis of the Ligamentum Flavum of Patients with Lumbar Spinal Canal Stenosis: Comparison between the Dural and Dorsal Sides of the Thickened Ligamentum Flavum.

Thickening of the ligamentum flavum is the main factor in the development of lumbar spinal canal stenosis (LSCS). Although previous studies have reported factors related to ligamentum flavum thickening, its etiology has not been clarified. Furthermore, it is often difficult to set proper controls to investigate the pathologies of thickening due to differences in patient characteristics, such as age, sex, obesity, and comorbidities. This study aimed to elucidate the pathologies of ligamentum flavum thickening by comparing the dural and dorsal sides of the thickened ligamentum flavum in patients with LSCS. Ligamentum flavum samples were collected from 19 patients with LSCS. The samples were divided into the dural and dorsal sides. The dural side was used as a control to assess the pathologies occurring on the dorsal side. Elastic Masson staining was used to assess the elastic fibres. Gene expression levels were comprehensively assessed using quantitative reverse transcription polymerase chain reaction and DNA microarray analyses. Gene ontology analysis was used to identify biological processes associated with differentially expressed genes. The elastic fibres were significantly decreased on the dorsal side of the thickened ligamentum flavum. Genes related to fibrosis, inflammation, tissue repair, remodeling, and chondrometaplasia, such as COL1A2, COL3A1, COL5A1, TGFB1, VEGFA, TNFA, MMP2, COL10A1, and ADAMTS4, were highly expressed on the dorsal side of the thickened ligamentum flavum. The biological processes occurring on the dorsal side of the thickened ligamentum flavum were extracellular matrix organization, cell adhesion, extracellular matrix disassembly, and proteolysis.These are considered important pathologies of ligamentum flavum thickening.

Comparative proteome analysis of the ligamentum flavum of patients with lumbar spinal canal stenosis.

Background: Thickening of the ligamentum flavum is considered to be the main factor for lumbar spinal canal stenosis (LSCS). Although some mechanisms have been speculated in the thickening of the ligamentum flavum, there are only a few comprehensive approaches to investigate its pathology. The objective of this study was to investigate the pathology of thickened ligamentum flavum in patients with LSCS based on protein expression levels using shotgun proteome analysis. Methods: Ligamentum flavum samples were collected from four patients with LSCS (LSCS group) and four patients with lumbar disc herniation (LDH) as controls (LDH group). Protein mixtures were digested and analyzed by liquid chromatography/mass spectrometry analysis. To compare protein expression levels between the LSCS and LDH groups, the mean Mascot score was compared. Biological processes were assessed using Gene Ontology analysis. Results: A total of 1151 proteins were identified in some samples of ligamentum flavum. Among these, 145 proteins were detected only in the LSCS group, 315 in the LDH group, and 691 in both groups. The demonstrated biological processes occurring in the LSCS group included: extracellular matrix organization, regulation of peptidase activity, extracellular matrix disassembly, and negative regulation of cell growth. Proteins related to fibrosis, chondrometaplasia, and amyloid deposition were found highly expressed in the LSCS group compared with those in the LDH group. Conclusions: Tissue repair via fibrosis, chondrometaplasia, and amyloid deposits may be important pathologies that occur in the thickened ligamentum flavum of patients with LSCS.

Effect of immunosuppressants on a mouse model of osteogenesis imperfecta type V harboring a heterozygous Ifitm5 c.-14C > T mutation.

Osteogenesis imperfecta (OI) type V is an autosomal dominant disorder caused by the c.-14C > T mutation in the interferon-induced transmembrane protein 5 gene (IFITM5), however, its onset mechanism remains unclear. In this study, heterozygous c.-14C > T mutant mice were developed to investigate the effect of immunosuppressants (FK506 and rapamycin) on OI type V. Among the mosaic mice generated by Crispr/Cas9-based technology, mice with less than 40% mosaic ratio of c.-14C > T mutation survived, whereas those with more than 48% mosaic ratio exhibited lethal skeletal abnormalities with one exception. All heterozygous mutants obtained by mating mosaic mice with wild-type mice exhibited a perinatal lethal phenotype due to severe skeletal abnormalities. Administration of FK506, a calcineurin inhibitor, in the heterozygous fetuses improved bone mineral content (BMC) of the neonates, although it did not save the neonates from the lethal effects of the mutation, whereas rapamycin, an mTOR inhibitor, reduced BMC, suggesting that mTOR signaling is involved in the bone mineralization of heterozygous mutants. These findings could clarify certain aspects of the onset mechanism of OI type V and enable development of therapeutics for this condition.

Praja1 RING-finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP-43 aggregate formation.

Transactivation response DNA-binding protein of 43 kDa (TDP-43) is a major constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We have previously shown neuronal cytoplasmic aggregate formation induced by recombinant adenoviruses expressing human wild-type and C-terminal fragment (CTF) TDP-43 under the condition of proteasome inhibition in vitro and in vivo. In the present study, we demonstrated that the formation of the adenoviral TDP-43 aggregates was markedly suppressed in rat neural stem cell-derived neuronal cells by co-infection of an adenovirus expressing heat shock transcription factor 1 (HSF1), a master regulator of heat shock response. We performed DNA microarray analysis and searched several candidate molecules, located downstream of HSF1, which counteract TDP-43 aggregate formation. Among these, we identified Praja 1 RING-finger E3 ubiquitin ligase (PJA1) as a suppressor of phosphorylation and aggregate formation of TDP-43. Co-immunoprecipitation assay revealed that PJA1 binds to CTF TDP-43 and the E2-conjugating enzyme UBE2E3. PJA1 also suppressed formation of cytoplasmic phosphorylated TDP-43 aggregates in mouse facial motor neurons in vivo. Furthermore, phosphorylated TDP-43 aggregates were detected in PJA1-immunoreactive human ALS motor neurons. These results indicate that PJA1 is one of the principal E3 ubiquitin ligases for TDP-43 to counteract its aggregation propensity and could be a potential therapeutic target for ALS and FTLD.

Fatty acid beta oxidation enzyme HADHA is a novel potential therapeutic target in malignant lymphoma.

Cancer cells, including malignant lymphoma cells, alter their metabolism, termed "metabolic reprograming," on initiation of malignant transformation as well as upon accumulation of genetic abnormalities. Here, to identify a novel therapeutic target involved in the metabolic changes during malignant lymphoma, we performed global analyses combined with shotgun proteomics, in silico database analysis, and clinic-pathologic analysis of nonneoplastic lymphoid tissue and malignant lymphoma tissue and verified the molecular functions in vitro. In total, 2002 proteins were detected from both samples and proteins related to fatty acid beta-oxidation (FAO) were detected more frequently in malignant lymphoma tissue. Consequently, the most frequently detected protein, the mitochondrial trifunctional enzyme subunit-alpha (HADHA), was identified as a potential target. Immunohistochemical analyses revealed that HADHA tended to be overexpressed in a high-grade subtype of malignant lymphoma tissue. Clinicopathologic study revealed that HADHA overexpression was correlated with significantly worse overall survival (P = 0.013) and was an independent prognostic predictor in diffuse large B-cell lymphoma (P = 0.027). In vitro, downregulation of HADHA negatively regulated cell growth by causing G0/G1 arrest (P = 0.0008) similar to treatment with etomoxir, an inhibitor of FAO (P = 0.032). Moreover, downregulation of HADHA increased the susceptibility to doxorubicin (P = 0.002) and etoposide (P = 0.004). Moreover, these phenotypes were confirmed in an HADHA knockout system. Thus, we provide a basis for a novel therapeutic strategy through the regulation of HADHA and FAO in patients with refractory malignant lymphoma.

Retained Myogenic Potency of Human Satellite Cells from Torn Rotator Cuff Muscles Despite Fatty Infiltration.

Rotator cuff tears (RCTs) are a common shoulder problem in the elderly that can lead to both muscle atrophy and fatty infiltration due to less physical load. Satellite cells, quiescent cells under the basal lamina of skeletal muscle fibers, play a major role in muscle regeneration. However, the myogenic potency of human satellite cells in muscles with fatty infiltration is unclear due to the difficulty in isolating from small samples, and the mechanism of the progression of fatty infiltration has not been elucidated. The purpose of this study was to analyze the population of myogenic and adipogenic cells in disused supraspinatus (SSP) and intact subscapularis (SSC) muscles of the RCTs from the same patients using fluorescence-activated cell sorting. The microstructure of the muscle with fatty infiltration was observed as a whole mount condition under multi-photon microscopy. Myogenic differentiation potential and gene expression were evaluated in satellite cells. The results showed that the SSP muscle with greater fatty infiltration surrounded by collagen fibers compared with the SSC muscle under multi-photon microscopy. A positive correlation was observed between the ratio of muscle volume to fat volume and the ratio of myogenic precursor to adipogenic precursor. Although no difference was observed in the myogenic potential between the two groups in cell culture, satellite cells in the disused SSP muscle showed higher intrinsic myogenic gene expression than those in the intact SSC muscle. Our results indicate that satellite cells from the disused SSP retain sufficient potential of muscle growth despite the fatty infiltration.

Silver nanoparticles induce tight junction disruption and astrocyte neurotoxicity in a rat blood-brain barrier primary triple coculture model.

BACKGROUND: Silver nanoparticles (Ag-NPs) can enter the brain and induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the BBB and the brain are not well understood. METHOD: To investigate Ag-NP suspension (Ag-NPS)-induced toxicity, a triple coculture BBB model of rat brain microvascular endothelial cells, pericytes, and astrocytes was established. The BBB permeability and tight junction protein expression in response to Ag-NPS, NP-released Ag ions, and polystyrene-NP exposure were investigated. Ultrastructural changes of the microvascular endothelial cells, pericytes, and astrocytes were observed using transmission electron microscopy (TEM). Global gene expression of astrocytes was measured using a DNA microarray. RESULTS: A triple coculture BBB model of primary rat brain microvascular endothelial cells, pericytes, and astrocytes was established, with the transendothelial electrical resistance values >200 Ω·cm(2). After Ag-NPS exposure for 24 hours, the BBB permeability was significantly increased and expression of the tight junction (TJ) protein ZO-1 was decreased. Discontinuous TJs were also observed between microvascular endothelial cells. After Ag-NPS exposure, severe mitochondrial shrinkage, vacuolations, endoplasmic reticulum expansion, and Ag-NPs were observed in astrocytes by TEM. Global gene expression analysis showed that three genes were upregulated and 20 genes were downregulated in astrocytes treated with Ag-NPS. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the 23 genes were associated with metabolic processes, biosynthetic processes, response to stimuli, cell death, the MAPK pathway, and so on. No GO term and KEGG pathways were changed in the released-ion or polystyrene-NP groups. Ag-NPS inhibited the antioxidant defense of the astrocytes by increasing thioredoxin interacting protein, which inhibits the Trx system, and decreasing Nr4a1 and Dusp1. Meanwhile, Ag-NPS induced inflammation and apoptosis through modulation of the MAPK pathway or B-cell lymphoma-2 expression or mTOR activity in astrocytes. CONCLUSION: These results draw our attention to the importance of Ag-NP-induced toxicity on the neurovascular unit and provide a better understanding of its toxicological mechanisms on astrocytes.

The co-transplantation of bone marrow derived mesenchymal stem cells reduced inflammation in intramuscular islet transplantation.

AIMS/HYPOTHESIS: Although the muscle is one of the preferable transplant sites in islet transplantation, its transplant efficacy is poor. Here we attempted to determine whether an intramuscular co-transplantation of mesenchymal stem cells (MSCs) could improve the outcome. METHODS: We co-cultured murine islets with MSCs and then analyzed the morphological changes, viability, insulin-releasing function (represented by the stimulation index), and gene expression of the islets. We also transplanted 500 islets intramuscularly with or without 5 × 105 MSCs to diabetic mice and measured their blood glucose level, the glucose changes in an intraperitoneal glucose tolerance test, and the plasma IL-6 level. Inflammation, apoptosis, and neovascularization in the transplantation site were evaluated histologically. RESULTS: The destruction of islets tended to be prevented by co-culture with MSCs. The stimulation index was significantly higher in islets co-cultured with MSCs (1.78 ± 0.59 vs. 7.08 ± 2.53; p = 0.0025). In terms of gene expression, Sult1c2, Gstm1, and Rab37 were significantly upregulated in islets co-cultured with MSCs. Although MSCs were effective in the in vitro assays, they were only partially effective in facilitating intramuscular islet transplantation. Co-transplanted MSCs prevented an early inflammatory reaction from the islets (plasma IL-6; p = 0.0002, neutrophil infiltration; p = 0.016 inflammatory area; p = 0.021), but could not promote neovascularization in the muscle, resulting in the failure of many intramuscular transplanted islets to engraft. CONCLUSIONS: In conclusion, co-culturing and co-transplanting MSCs is potentially useful in islet transplantation, especially in terms of anti-inflammation, but further augmentation for an anti-apoptosis effect and neovascularization is necessary.

Chondrogenic and fibrotic process in the ligamentum flavum of patients with lumbar spinal canal stenosis.

STUDY DESIGN: A histological, biological, and immunohisto-chemical study of human lumbar ligamentum flavum. OBJECTIVE: To analyze changes in the hypertrophied ligamentum flavum and clarify their etiology. SUMMARY OF BACKGROUND DATA: Hypertrophy of the ligamentum flavum has been considered a major contributor to the development of lumbar spinal canal stenosis (LSCS). Although previous studies have reported some factors related to ligamentum flavum hypertrophy, its etiology is still unclear. METHODS: Ligamentum flavum samples were collected from 20 patients with LSCS (LSCS group) and 10 patients with lumbar disc herniation (LDH group) as a control. The thickness of the ligamentum flavum was measured histologically. The amounts of elastic fibers and proteoglycans were assessed by Elastica-Masson staining and alcian blue staining, respectively. Gene and protein expressions related to fibrosis, inflammation, and chondrogenesis were analyzed by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. The total genes of the 2 groups were compared by DNA microarray analysis. RESULTS: The ligamentum flavum was significantly thicker in the LSCS group, which had a smaller amount of elastic fibers and a larger amount of proteoglycans. The gene expression related to fibrosis was significantly higher in the LSCS group; however, the immunoreactivities of collagen types I and III were weaker on the dorsal side of the ligamentum flavum in the LSCS group. The gene expression related to chondrogenesis and proteoglycan synthesis was significantly higher in the LSCS group. There was no significant difference in the gene expression related to inflammation between the 2 groups. CONCLUSION: Synthesis of the collagenous fibers and degradation of the elastic and collagenous fibers are both accelerated in the ligamentum flavum of patient with LSCS, which may be the reason for hypertrophy of the tissue. In addition, chondrogenesis and proteoglycan synthesis may have critical roles in the pathogenesis of the ligamentum flavum hypertrophy. LEVEL OF EVIDENCE: 5.

Gene chip/PCR-array analysis of tissue response to 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer surfaces in a mouse subcutaneous transplantation system.

To evaluate the in vivo foreign body reaction to bio-inert 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers, MPC polymer-coated porous substrates, with large surface area, were implanted subcutaneously in mice for 7 and 28 days, and the surrounding tissue response and cells infiltrating into the porous structure were evaluated. The MPC polymer surface induced low angiogenesis and thin encapsulation around the porous substrate, and slightly suppressed cell infiltration into the porous substrate. M1-type macrophage specific gene (CCR7) expression was suppressed by the MPC polymer surface after 7 days, resulting in the suppression of inflammatory cytokine/chemokine gene expression. However, the expression of these genes on the MPC polymer surface was higher than on the non-coated surface after 28 days. These findings suggest that MPC polymer surfaces successfully inhibit inflammatory responses during the early stage of tissue response, and seem to retard its occurrence over time.

Comprehensive genetic analysis of early host body reactions to the bioactive and bio-inert porous scaffolds.

To design scaffolds for tissue regeneration, details of the host body reaction to the scaffolds must be studied. Host body reactions have been investigated mainly by immunohistological observations for a long time. Despite of recent dramatic development in genetic analysis technologies, genetically comprehensive changes in host body reactions are hardly studied. There is no information about host body reactions that can predict successful tissue regeneration in the future. In the present study, porous polyethylene scaffolds were coated with bioactive collagen or bio-inert poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (PMB) and were implanted subcutaneously and compared the host body reaction to those substrates by normalizing the result using control non-coat polyethylene scaffold. The comprehensive analyses of early host body reactions to the scaffolds were carried out using a DNA microarray assay. Within numerous genes which were expressed differently among these scaffolds, particular genes related to inflammation, wound healing, and angiogenesis were focused upon. Interleukin (IL)-1ß and IL-10 are important cytokines in tissue responses to biomaterials because IL-1ß promotes both inflammation and wound healing and IL-10 suppresses both of them. IL-1ß was up-regulated in the collagen-coated scaffold. Collagen-specifically up-regulated genes contained both M1- and M2-macrophage-related genes. Marked vessel formation in the collagen-coated scaffold was occurred in accordance with the up-regulation of many angiogenesis-inducible factors. The DNA microarray assay provided global information regarding the host body reaction. Interestingly, several up-regulated genes were detected even on the very bio-inert PMB-coated surfaces and those genes include inflammation-suppressive and wound healing-suppressive IL-10, suggesting that not only active tissue response but also the inert response may relates to these genetic regulations.

Diffraction-unlimited optical imaging of unstained living cells in liquid by electron beam scanning of luminescent environmental cells.

An environmental cell with a 50-nm-thick cathodoluminescent window was attached to a scanning electron microscope, and diffraction-unlimited near-field optical imaging of unstained living human lung epithelial cells in liquid was demonstrated. Electrons with energies as low as 0.8 - 1.2 kV are sufficiently blocked by the window without damaging the specimens, and form a sub-wavelength-sized illumination light source. A super-resolved optical image of the specimen adhered to the opposite window surface was acquired by a photomultiplier tube placed below. The cells after the observation were proved to stay alive. The image was formed by enhanced dipole radiation or energy transfer, and features as small as 62 nm were resolved.

Nm23/nucleoside diphosphate kinase-A as a potent prognostic marker in invasive pancreatic ductal carcinoma identified by proteomic analysis of laser micro-dissected formalin-fixed paraffin-embedded tissue.

BACKGROUND: Pancreatic cancer is among the most lethal malignancies worldwide. This study aimed to identify a novel prognostic biomarker, facilitating treatment selection, using mass spectrometry (MS)-based proteomic analysis with formalin-fixed paraffin-embedded (FFPE) tissue. RESULTS: The two groups with poor prognosis (n = 4) and with better prognosis (n = 4) had been carefully chosen among 96 resected cases of pancreatic cancer during 1998 to 2007 in Tohoku University Hospital. Although those 2 groups had adjusted background (UICC-Stage IIB, Grade2, R0, gemcitabine adjuvant), there was a significant difference in postoperative mean survival time (poor 21.0 months, better 58.1 months, P = 0.0067). Cancerous epithelial cells collected from FFPE tissue sections by laser micro-dissection (LMD) were processed for liquid chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1099 unique proteins were identified and 6 proteins showed different expressions in the 2 groups by semi-quantitative comparison. Among these 6 proteins, we focused on Nm23/Nucleoside Diphosphate Kinase A (NDPK-A) and immunohistochemically confirmed its expression in the cohort of 96 cases. Kaplan-Meier analysis showed high Nm23/NDPK-A expression to correlate with significantly worse overall survival (P = 0.0103). Moreover, in the multivariate Cox regression model, Nm23/NDPK-A over-expression remained an independent predictor of poor survival with a hazard ratio of 1.97 (95% CI 1.16-3.56, P = 0.0110). CONCLUSIONS: We identified 6 candidate prognostic markers for postoperative pancreatic cancer using FFPE tissues and immunohistochemically demonstrated high Nm23/NDPK-A expression to be a useful prognostic marker for pancreatic cancer.

Genotoxicity and molecular response of silver nanoparticle (NP)-based hydrogel.

BACKGROUND: Since silver-nanoparticles (NPs) possess an antibacterial activity, they were commonly used in medical products and devices, food storage materials, cosmetics, various health care products, and industrial products. Various silver-NP based medical devices are available for clinical uses, such as silver-NP based dressing and silver-NP based hydrogel (silver-NP-hydrogel) for medical applications. Although the previous data have suggested silver-NPs induced toxicity in vivo and in vitro, there is lack information about the mechanisms of biological response and potential toxicity of silver-NP-hydrogel. METHODS: In this study, the genotoxicity of silver-NP-hydrogel was assayed using cytokinesis-block micronucleus (CBMN). The molecular response was studied using DNA microarray and GO pathway analysis. RESULTS AND DISCUSSION: The results of global gene expression analysis in HeLa cells showed that thousands of genes were up- or down-regulated at 48 h of silver-NP-hydrogel exposure. Further GO pathway analysis suggested that fourteen theoretical activating signaling pathways were attributed to up-regulated genes; and three signal pathways were attributed to down-regulated genes. It was discussed that the cells protect themselves against silver NP-mediated toxicity through up-regulating metallothionein genes and anti-oxidative stress genes. The changes in DNA damage, apoptosis and mitosis pathway were closely related to silver-NP-induced cytotoxicity and chromosome damage. The down-regulation of CDC14A via mitosis pathway might play a role in potential genotoxicity induced by silver-NPs. CONCLUSIONS: The silver-NP-hydrogel induced micronuclei formation in cellular level and broad spectrum molecular responses in gene expression level. The results of signal pathway analysis suggested that the balances between anti-ROS response and DNA damage, chromosome instability, mitosis inhibition might play important roles in silver-NP induced toxicity. The inflammatory factors were likely involved in silver-NP-hydrogel complex-induced toxic effects via JAK-STAT signal transduction pathway and immune response pathway. These biological responses eventually decide the future of the cells, survival or apoptosis.

Evaluation of mutagenicity and co-mutagenicity of strong static magnetic fields up to 13 Tesla in Escherichia coli deficient in superoxide dismutase.

PURPOSE: To evaluate the biological effects of static magnetic fields (SMFs) up to 13 Tesla (T), with respect to superoxide behavior, by determining the effect on mutagenicity in superoxide dismutase (SOD)-deficient Escherichia coli strain QC774, and its parental strain GC4468. MATERIALS AND METHODS: Experimental strains were exposed to a 5, 10, or 13T SMF for 24 h at 37°C in Luria-Bertani medium. To evaluate mutagenicity after SMF exposure, the mutation frequency in thymine synthesis genes was determined. The effect of exposure to a 5 or 13T SMF on mutagenicity induced by plumbagin was also investigated. RESULTS: No statistically significant differences in the mutation frequency in thymine synthesis genes were observed between SMF-exposed cells and unexposed cells at any of the applied magnetic flux densities. Furthermore, exposure to SMFs up to 13T did not affect mutagenicity induced by plumbagin. CONCLUSION: Exposure to SMFs up to 13T caused neither mutagenicity nor co-mutagenicity in the SOD-deficient E. coli strain QC774 or in its parental strain GC4468, suggesting that exposure to strong SMFs does not affect the behavior of superoxides in these microorganisms.

Effect of interfacial proteins on osteoblast-like cell adhesion to hydroxyapatite nanocrystals.

A quartz crystal microbalance with dissipation (QCM-D) technique was employed to detecting the protein adsorption and subsequent osteoblast-like cell adhesion to hydroxyapatite (HAp) nanocrystals. The interfacial phenomena with the preadsorption of three proteins (albumin (BSA), fibronectin (Fn), and collagen (Col)), the subsequent adsorption of fetal bovine serum (FBS), and the adhesion of the cells were investigated. The QCM-D measured the frequency shift (Δf) and dissipation energy shift (ΔD), and the viscoelastic properties of the adlayers were evaluated using ΔD-Δf plot and Voigt-based viscoelastic model. The Col adsorption significantly showed higher Δf, ΔD, elasticity, and viscosity values as compared to the BSA and Fn adsorption, and the subsequent FBS adsorption depended on the preadsorbed proteins. The ΔD-Δf plot of the cell adhesion also showed a different behavior depending on the surfaces, and the Fn- and Col-modified surfaces showed the rapid mass and ΔD changes by forming the viscous interfacial layers with cell adhesion, indicating that the processes were affected by the cellular reaction through the extracellular matrix (ECM) proteins. The confocal laser scanning microscope images of adherent cells showed a different morphology and pseudopod on the surfaces. The cells adhered to the surfaces modified with the Fn and Col had significantly uniaxially expanded shapes and fibrous pseudopods, and those modified with the BSA had a round shape. Therefore, the different cell-protein interactions would cause the arrangement of the ECM and the cytoskeleton changes at the interfaces, and these phenomena were successfully detected by the QCM-D and Voigt-based model.

Detection of interfacial phenomena with osteoblast-like cell adhesion on hydroxyapatite and oxidized polystyrene by the quartz crystal microbalance with dissipation.

The adhesion process of osteoblast-like cells on hydroxyapatite (HAp) and oxidized polystyrene (PSox) was investigated using a quartz crystal microbalance with dissipation (QCM-D), confocal laser scanning microscope (CLSM), and atomic force microscope (AFM) techniques in order to clarify the interfacial phenomena between the surfaces and cells. The interfacial viscoelastic properties (shear viscosity (η(ad)), elastic shear modulus (µ(ad)), and tan δ) of the preadsorbed protein layer and the interface layer between the surfaces and cells were estimated using a Voigt-based viscoelastic model from the measured frequency (Δf) and dissipation shift (ΔD) curves. In the ΔD-Δf plots, the cell adhesion process on HAp was classified as (1) a mass increase only, (2) increases in both mass and ΔD, and (3) slight decreases in mass and ΔD. On PSox, only ΔD increases were observed, indicating that the adhesion behavior depended on the surface properties. The interfacial µ(ad) value between the material surfaces and cells increased with the number of adherent cells, whereas η(ad) and tanδ decreased slightly, irrespective of the surface. Thus, the interfacial layer changed the elasticity to viscosity with an increase in the number. The tan δ values on HAp were higher than those on PSox and exceeded 1.0. Furthermore, the pseudopod-like structures of the cells on HAp had periodic stripe patterns stained with a type I collagen antibody, whereas those on PSox had cell-membrane-like structures unstained with type I collagen. These results indicate that the interfacial layers on PSox and HAp exhibit elasticity and viscosity, respectively, indicating that the rearrangements of the extracellular matrix and cytoskeleton changes cause different cell-surface interactions. Therefore, the different cell adhesion process, interfacial viscoelasticity, and morphology depending on the surfaces were successfully monitored in situ and evaluated by the QCM-D technique combined with other techniques.

Characterization of the osteoblast-specific transmembrane protein IFITM5 and analysis of IFITM5-deficient mice.

Interferon-inducible transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein whose expression peaks around the early mineralization stage during the osteoblast maturation process. To investigate IFITM5 function, we first sought to identify which proteins interact with IFITM5. Liquid chromatography mass spectrometry revealed that FK506-binding protein 11 (FKBP11) co-immunoprecipitated with IFITM5. FKBP11 is the only protein it was found to interact with in osteoblasts, while IFITM5 interacts with several proteins in fibroblasts. FKBPs are involved in protein folding and immunosuppressant binding, but we could not be sure that IFITM5 participated in these activities when bound to FKBP11. Thus, we generated Ifitm5-deficient mice and analyzed their skeletal phenotypes. The skeletons, especially the long bones, of homozygous mutants (Ifitm5(-/-)) were smaller than those of heterozygous mutants (Ifitm5(+/-)), although we did not observe any significant differences in bone morphometric parameters. The effect of Ifitm5 deficiency on bone formation was more significant in newborns than in young and adult mice, suggesting that Ifitm5 deficiency might have a greater effect on prenatal bone development. Overall, the effect of Ifitm5 deficiency on bone formation was less than we expected. We hypothesize that this may have resulted from a compensatory mechanism in Ifitm5-deficient mice.

Contribution of physicochemical characteristics of nano-oxides to cytotoxicity.

To identify the key physicochemical properties of nano-oxides governing cytotoxicity, we investigate the contribution of the size, shape, morphology, and electronic properties of ten types of insulator (SiO(2), CeO(2) and Al(2)O(3)) and semiconductor (ZnO and CuO) nano-oxides to cytotoxicity using the NIH3T3 and A549 cell lines as models. We find that the shape of the Al(2)O(3) (nanoparticle versus nanowhisker) and the morphology of the SiO(2) (porous versus non-porous nanoparticles) did not have obvious effect on the observed cytotoxicity, and the size of the nano-oxides cannot be regarded as an indicator of cytotoxicity. By contrast, we find that the cell viability exposed to the semiconductor nano-oxides was much lower than that exposed to the insulator nano-oxides. Moreover, the Al-doped ZnO nanoparticle (NP) was more toxic than the non-doped ZnO NP, whereas the Al-doped CuO NP was less toxic than the non-doped CuO NP but more toxic than the Al(2)O(3) NP. Correspondingly, the valence band X-ray photoelectron spectra of the nano-oxides show the density of states of the Al-doped ZnO NP (the Al-doped CuO NP) is higher (lower) than that of the non-doped ZnO NP (the non-doped CuO NP). These results suggest that the electronic properties of nano-oxides may play an important role in the observed cytotoxicity. The results have implications for selectively tailoring the toxic effect and establishing predictive models for the design of various types of nanomaterials with unique properties and for the understanding of interactions between nanomaterials with biological system.

Global gene expression analysis for evaluation and design of biomaterials.

Comprehensive gene expression analysis using DNA microarrays has become a widespread technique in molecular biological research. In the biomaterials field, it is used to evaluate the biocompatibility or cellular toxicity of metals, polymers and ceramics. Studies in this field have extracted differentially expressed genes in the context of differences in cellular responses among multiple materials. Based on these genes, the effects of materials on cells at the molecular level have been examined. Expression data ranging from several to tens of thousands of genes can be obtained from DNA microarrays. For this reason, several tens or hundreds of differentially expressed genes are often present in different materials. In this review, we outline the principles of DNA microarrays, and provide an introduction to methods of extracting information which is useful for evaluating and designing biomaterials from comprehensive gene expression data.