Dai Suzuki

The characteristics of in vitro biological activity of titanium surfaces anodically oxidized in chloride solutions.

Photo-functionalized radical reactions on TiO(2) have been correlated with adsorption of organic impurities and decreasing hydrophilicity of titanium-based biomaterials. Such reactive oxygen species (ROS) spontaneously generated on oxidized titanium surfaces may also have important roles against time-dependent degradation of biological ability and adherent micro-organisms. This study examined in vitro biological ability as a function of time and antimicrobial activity on oxidized titanium surfaces without photo-functionalization. Mechanically polished titanium and thermally oxidized titanium surfaces that had been stored for 4 wks showed adsorbed organic impurities with decreased surface hydrophilicity. Even after the storage period, anodically oxidized titanium surfaces enabled super-hydrophilicity without adsorption of organic impurities, because of the ROS and the hydrophilic functional groups generated on the surfaces. The osteogenic gene expressions of osteoblasts cultured on anodically oxidized titanium surfaces with or without storage were significantly higher than those on thermally oxidized titanium and polished titanium surfaces. Titanium surfaces anodically oxidized in a solution with chloride achieved antimicrobial activity against an oral microorganism due to the amount of ROS generated on the surface. Thus, titanium anodically oxidized in solution with chloride may have potential use for titanium-based internal fixation devices.

Essential mesenchymal role of small GTPase Rac1 in interdigital programmed cell death during limb development.

Developing vertebrate limbs are often utilized as a model for studying pattern formation and morphogenetic cell death. Herein, we report that conditional deletion of Rac1, a member of the Rho family of proteins, in mouse limb bud mesenchyme led to skeletal deformities in the autopod and soft tissue syndactyly, with the latter caused by a complete absence of interdigital programmed cell death. Furthermore, the lack of interdigital programmed cell death and associated syndactyly was related to down-regulated gene expression of Bmp2, Bmp7, Msx1, and Msx2, which are known to promote apoptosis in the interdigital mesenchyme. Our findings from Rac1 conditional mutants indicate crucial roles for Rac1 in limb bud morphogenesis, especially interdigital programmed cell death.

Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development

Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.

Antimicrobial and osteogenic properties of a hydrophilic-modified nanoscale hydroxyapatite coating on titanium

Hydroxyapatite (HA)-coated titanium (Ti) is commonly used for implantable medical devices. This study examined in vitro osteoblast gene expression and antimicrobial activity against early and late colonizers of supra-gingival plaque on nanoscale HA-coated Ti prepared by discharge in a physiological buffered solution. The HA-coated Ti surface showed super-hydrophilicity, whereas the densely sintered HA and Ti surfaces alone showed lower hydrophilicity. The sintered HA and HA-coated Ti surfaces enhanced osteoblast phenotypes in comparison with the bare Ti surface. The HA-coated Ti enabled antimicrobial activity against early colonizers of supra-gingival plaques, namely Streptococcus mitis and Streptococcus gordonii. Such antimicrobial activity may be caused by the surface hydrophilicity, thereby leading to a repulsion force between the HA-coated Ti surface and the bacterial cell membranes. On the contrary, the sintered HA sample was susceptible to infection of microorganisms. Thus, hydrophilic-modified HA-coated Ti may have potential for use in implantable medical devices. From the Clinical Editor: This study establishes that Hydroxyapatite (HA)-coated titanium (Ti) surface of implanted devices may result in an optimal microenvironment to control and prevent infections and may have potential future clinical applications.

BMP2 Differentially Regulates the Expression of Gremlin1 and Gremlin2, the Negative Regulators of BMP Function, During Osteoblast Differentiation

Bone morphogenetic proteins (BMPs) control the expressions of many genes involved in bone formation. On the basis of our hypothesis that BMP2 stimulation-regulated gene expression plays a critical role in osteoblast differentiation, we performed genome-wide screening of messenger RNA from BMP2-treated and -untreated C2C12 cells using a DNA microarray technique. We found that the expressions of Gremlin1 and Gremlin2, which are known BMP antagonists, were bidirectionally regulated by BMP2. Gremlin1 was down-regulated by BMP2, while Gremlin2 was up-regulated in both time- and dose-dependent manners. Ablation of Gremlin1 or Gremlin2 enhanced osteoblast differentiation induced by BMP2. On the other hand, treatment with recombinant Gremlin1 inhibited BMP2-induced osteoblast differentiation. Furthermore, treatment with Smad4 siRNA and the p38 MAPK inhibitor SB203580 suppressed BMP2-induced Gremlin2 gene expression. The differential regulation of Gremlin1 and Gremlin2 gene expressions by BMP2 may explain the critical function of these genes during osteoblast differentiation.

TGF-β suppresses POEM expression through ERK1/2 and JNK in osteoblasts

POEM, also called nephronectin, is an extracellular matrix protein that is considered to play a critical role as an adhesion molecule in the development and functioning of various tissues, such as kidneys and bones. In the present study, we examined the molecular mechanism of POEM gene expression, and found that transforming growth factor‐β (TGF‐β) strongly inhibited POEM expression in the mouse osteoblastic cell line, MC3T3‐E1. TGF‐β‐induced decrease of POEM expression occurred in both time‐ and dose‐dependent manners through the activation of TGF‐β receptor I and extracellular signal‐regulated kinase/c‐Jun N‐terminal kinase pathways.

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-α

POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.

Antioxidant and osteogenic properties of anodically oxidized titanium.

Cells adhering onto implant surfaces are subjected to oxidative stress during wound healing processes. Although titanium and its alloys are among the most frequently used biomaterials in orthopedic and dental implants, titanium surfaces do not have antioxidant properties, and cells grown on these surfaces can show permanent oxidative stress. The present study assessed the antioxidant property and osteogenic properties of titanium samples with or without oxidation treatments. A thick rutile TiO₂ film was observed on thermally oxidized titanium surfaces, while amorphous anatase TiO₂ formed on anodically oxidized titanium surfaces prepared by discharging in 1 M Na₂HPO₄. A resistance to the depletion of reduced glutathione in adherent osteoblasts, which correlates with antioxidant behavior, occurred on anodically oxidized titanium. Enhanced osteogenic gene expressions and nano-biomechanical properties of mineralized tissue were achieved on anodically oxidized titanium, in comparison with thermally oxidized or untreated titanium. Thus, anodic oxidation by discharging in electrolyte is expected to be a useful surface modification for titanium implants.

Cdc42 Is Critical for Cartilage Development During Endochondral Ossification

Cdc42 is a widely expressed protein that belongs to the family of Rho GTPases and controls a broad variety of signal transduction pathways in a variety of cell types. To investigate the physiological functions of Cdc42 during cartilage development, we generated chondrocyte-specific inactivated Cdc42 mutant mice (Cdc42(fl/fl); Col2-Cre). The gross morphology of mutant neonates showed shorter limbs and body as compared with the control mice (Cdc42(fl/fl)). Skeletal preparations stained with alcian blue and alizarin red also revealed that the body and the long bone length of the mutants were shorter than those of the control mice. Furthermore, severe defects were found in growth plate chondrocytes in the femur sections of mutant mice, characterized by a reduced proliferating zone height, wider hypertrophic zone, and loss of columnar organization in proliferating chondrocytes. The expression levels of chondrocyte marker genes, such as Col2, Col10, and Mmp13, in mutant mice were decreased as compared with the control mice. Mineralization of trabecular bones in the femur sections was also decreased in the mutants as compared with control mice, whereas osteoid volume was increased. Together these results suggested that chondrocyte proliferation and differentiation in growth plates in the present mutant mice were not normally organized, which contributed to abnormal bone formation. We concluded that Cdc42 is essential for cartilage development during endochondral bone formation.

Lysyl oxidase like-2 reinforces unsatisfactory ossification induced by bone morphogenetic protein-2: Relating nanomechanical properties and molecular changes

UNLABELLED Bone morphogenetic protein-2 (BMP2) is among the most popular anabolic agents and substantially increase bone volume related to enhanced osteoblast differentiation. Here we demonstrate a remarkable deterioration in the nanomechanical properties of mineralized tissue induced from osteoblasts solely by the function of BMP2. Mineralized tissue of primary osteoblasts cultured with BMP2 shows molecular features of both bone and cartilage, but depletion of lysyl oxidase family members leads to poor nanomechanical properties of the mineralized tissue. Lysyl oxidase like-2 supplementation reinforces the inferior mineralized tissue induced from osteoblasts by BMP2 through intermolecular cross-linking of type II or type X collagen-rich extracellular matrix. This may also mimic a consolidation of bone fracture gaps, despite the fact that the distribution of the bone properties in such microenvironments has been poorly elucidated. These findings confirm the importance of testing newly induced bone down to the microscale and nanoscale in bone tissue engineering. FROM THE CLINICAL EDITOR Bone morphogenetic protein-2 is known to substantially increase bone volume related to enhanced osteoblast differentiation; however, this team of investigators report a remarkable deterioration in the nanomechanical properties of mineralized tissue induced from osteoblasts solely by the function of BMP2.