Sirtuin 6 Overexpression Improves Rotator Cuff Tendon-to-Bone Healing in the Aged.

Aging is an independent risk factor for recurrent tearing after surgical repair of rotator cuff ruptures around the tendon-to-bone area. However, aging signature factors and related mechanisms involved in the healing of the rotator cuff are still unknown. We hypothesized that differences in proteins involved in the rotator cuff according to age may affect tendon-to-bone healing. The proteome analysis performed to identify the signature aging proteins of the rotator cuff confirmed the sirtuin signal as an age-specific protein. In particular, the expression of SIRT6 was markedly down-regulated with age. Ingenuity pathway analysis of omics data from age-dependent rat rotator cuffs and linear regression from human rotator cuffs showed SIRT6 to be closely related to the Wnt/ß-catenin signal. We confirmed that overexpression of SIRT6 in the rotator cuff and primary tenocyte regulated canonical Wnt signaling by inhibiting the transcriptional expression of sclerostin, a Wnt antagonist. Finally, SIRT6 overexpression promoted tendon-to-bone healing after tenotomy with reconstruction in elderly rats. This approach is considered an effective treatment method for recovery from recurrent rotator cuff tears, which frequently occur in the elderly.

Sirt6 Activation Ameliorates Inflammatory Bone Loss in Ligature-Induced Periodontitis in Mice.

Periodontitis is an inflammatory disease caused by microorganisms that induce the destruction of periodontal tissue. Inflamed and damaged tissue produces various inflammatory cytokines, which activate osteoclasts and induce alveolar bone loss and, eventually, tooth loss. Sirt6 expression suppresses inflammation and bone resorption; however, its role in periodontitis remains unclear. We hypothesized that Sirt6 has a protective role in periodontitis. To understand the role of Sirt6 in periodontitis, we compared periodontitis with ligature placement around the maxillary left second molar in 8-week-old control (C57BL/6J) male mice to Sirt6-overexpressing Tg (Sirt6Tg) mice, and we observed the resulting phenotypes using micro-CT. MDL801, a Sirt6 activator, was used as a therapy for periodontitis through oral gavage. Pro-inflammatory cytokines and increased osteoclast numbers were observed in alveolar bone tissue under periodontitis surgery. In the same condition, interestingly, protein levels from Sirt6 were the most downregulated among sirtuins in alveolar bone tissue. Based on micro-CT and CEJ-ABC distance, Sirt6Tg was observed to resist bone loss against ligature-induced periodontitis. Furthermore, the number of osteoclasts was significantly reduced in Sirt6Tg-ligated mice compared with control-ligated mice, although systemic inflammatory cytokines did not change. Consistent with this observation, we confirmed that bone loss was significantly reduced when MDL801, a Sirt6 activator, was included in the ligation mouse model. Our findings demonstrate that Sirt6 activation prevents bone loss against ligature-induced periodontitis. Thus, a Sirt6 activator may provide a new therapeutic approach for periodontitis.

Overexpression of COMP-Angiopoietin-1 in K14-Expressing Cells Impairs Hematopoiesis and Disturbs Erythrocyte Maturation.

Numerous studies highlight the potential benefits potentials of supplemental cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) through improved angiogenic effects. However, our recent findings show that excessive overexpression of COMP-Ang1 induces an impaired bone marrow (BM) microenvironment and senescence of hematopoietic stem cells (HSCs). Here, we investigated the underlying mechanisms of how excessive COMP-Ang1 affects the function of BM-conserved stem cells and hematopoiesis using K14-Cre;inducible-COMP-Ang1-transgenic mice. Excessive COMP-Ang1 induced peripheral egression and senescence of BM HSCs and mesenchymal stem cells (MSCs). Excessive COMP-Ang1 also caused abnormal hematopoiesis along with skewed differentiation of HSCs toward myeloid lineage rather than lymphoid lineage. Especially, excessive COMP-Ang1 disturbed late-stage erythroblast maturation, followed by decreased expression of stromal cell-derived factor 1 (SDF-1) and globin transcription factor 1 (GATA-1) and increased levels of superoxide anion and p-p38 kinase. However, transplantation with the mutant-derived BM cells or treatment with rhCOMP-Ang1 protein did not alter the frequency or GATA-1 expression of erythroblasts in recipient mice or in cultured BM cells. Together, our findings suggest that excessive COMP-Ang1 impairs the functions of BM HSCs and MSCs and hematopoietic processes, eventually leading to abnormal erythropoiesis via imbalanced SDF-1/CXCR4 axis and GATA-1 expression rather than Ang1/Tie2 signaling axis alterations.

Genetic overexpression of COMP-Ang1 impairs BM microenvironment and induces senescence of BM HSCs.

Supplemental Angiopoietin 1 (Ang1) exerts its therapeutic potential on microvascular regression-associated diseases, and this potential is linked with the function of hematopoietic stem cells (HSCs). However, the underlying mechanisms of the effect of enhanced angiogenesis on the modulation of HSCs are not yet defined. Here, we generated transgenic mice expressing Cartilage Oligomeric Matrix Protein (COMP)-Ang1 in keratin 14-expressing cells. The mutant animals expressed excessive angiogenic characteristics in the skin and bone marrow (BM) along with redder skin with more numerous and branched vessels compared with their wild-type (WT) littermates. The mutants displayed reduced long bone formation and osteoclast activity than did WT littermates and had fewer CD150+CD48-Lineage-Sca-1+c-Kit+ (LSK) cells in the BM. The mutants also exhibited greater senescence-associated (SA) ß-gal activity, p16INK4a protein expression, and superoxide anion levels in CD150+CD48-LSK cells in the BM. Furthermore, transplantation assay revealed that the mutant-derived LSK cells were inferior to the cells derived from WT littermate in inducing competitive repopulating capacity in the recipients. Collectively, our results demonstrate that persistent and prolonged administration of COMP-Ang1 by inducible transgenic expression mediates excessive angiogenesis in the body and impairs BM microenvironment, eventually leading to senescence of BM HSCs.

Constitutive Activation of Smoothened in the Renal Collecting Ducts Leads to Renal Hypoplasia, Hydronephrosis, and Hydroureter.

Sonic Hedgehog (Shh) signaling plays a major role in and is essential for regulation, patterning, and proliferation during renal development. Smoothened (Smo) plays a pivot role in transducing the Shh-glioma-associated oncogene Kruppel family member. However, the cellular and molecular mechanism underlying the role of sustained Smo activation in postnatal kidney development is still not clearly understood. Using a conditional knockin mouse model that expresses a constitutively activated form of Smo (SmoM2) upon Homeobox-B7-mediated recombination (Hoxb7-Cre), the effects of Shh signaling were determined in postnatal kidney development. SmoM2;Hoxb7-Cre mutant mice showed growth retardation with a reduction of body weight. Constitutive activation of Smo in the renal collecting ducts caused renal hypoplasia, hydronephrosis, and hydroureter. The parenchymal area and glomerular numbers were reduced, but the glomerular density was increased in SmoM2;Hoxb7-Cre mutant mice. The expression of Patched 1, the receptor of Shh and a downstream target gene of the Shh signaling pathway, was highly restricted and it was upregulated in the inner medullary collecting ducts of the kidney. The proliferative cells in the mesenchyme and collecting ducts were decreased in SmoM2;Hoxb7-Cre mutant mice. This study showed for the first time that sustained Smo inhibits postnatal kidney development by suppressing the proliferation of the mesenchyme and medullary collecting ducts in mice.

Activation of canonical Wnt/ß-catenin signaling inhibits H2O2-induced decreases in proliferation and differentiation of human periodontal ligament fibroblasts.

Human periodontal ligament fibroblasts (hPLFs) are exposed to oxidative stress during periodontal inflammation and dental treatments. It is hypothesized that hydrogen peroxide (H2O2)-mediated oxidative stress decreases survival and osteogenic differentiation of hPLFs, whereas these decreases are prevented by activation of the Wnt pathway. However, there has been a lack of reports that define the exact roles of canonical Wnt/ß-catenin signaling in H2O2-exposed hPLFs. Treatment with H2O2 reduced viability and proliferation in hPLFs in a dose- and time-dependent manner and led to mitochondria-mediated apoptosis. Pretreatment with lithium chloride (LiCl) or Wnt1 inhibited the oxidative damage that occurred in H2O2-exposed hPLFs. However, knockout of ß-catenin or treatment with DKK1 facilitated the H2O2-induced decreases in viability, mitochondrial membrane potential, and Bcl-2 induction. Osteoblastic differentiation of hPLFs was also inhibited by combined treatment with 100 µM H2O2, as evidenced by the decreases in alkaline phosphatase (ALP) activity and mineralization. H2O2-mediated inhibition of osteoblast differentiation in hPLFs was significantly attenuated in the presence of 500 ng/ml Wnt1 or 20 mM LiCl. In particular, H2O2 stimulated the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) at protein and mRNA levels in hPLFs, whereas the induction was almost completely suppressed in the presence of Wnt1 or LiCl. Furthermore, siRNA-mediated silencing of Nrf2 blocked H2O2-induced decreases in ALP activity and mineralization of hPLFs with the concomitant restoration of runt-related transcription factor 2 and osteocalcin mRNA expression and ALP activity. Collectively, these results suggest that activation of the Wnt/ß-catenin pathway improves proliferation and mineralization in H2O2-exposed hPLFs by downregulating Nrf2.

Carbon Nanotube/Magnesium Composite as a Hydrogen Source.

Hydrogen produced using the steam reforming process contains sulfur and carbon monoxide that are harmful to the Pt catalyst in proton-exchange-membrane fuel cells (PEMFCs). However, CO-free hydrogen can be generated from the hydrolysis of either Al in strongly alkaline water or Mg in neutral water with chlorides such as sea water. The hydrogen generation rate from the hydrolysis of Mg is extremely slow and linearly proportional to the corrosion rate of Mg in chloride water. In this work, we fabricated a carbon nanotube (CNT)--reinforced Mg--matrix composite by Spark Plasma Sintering as a fast hydrogen generation source for a PEMFC. The CNTs distributed in the Mg matrix act as numerous local cathodes, and hence cause severe galvanic corrosion between the Mg-matrix anode and CNT-cathode in NaCl solution. It was found that the hydrogen generation rate from the hydrolysis of the 5 vol.% CNT/Mg composite is 3300 times faster than that of the Mg without CNTs due primarily to the galvanic corrosion effect.

COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways.

Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

Field emission behavior of carbon nanotube yarn for micro-resolution X-ray tube cathode.

Carbon nanotube (CNT) has excellent electrical and thermal conductivity and high aspect ratio for X-ray tube cathode. However, CNT field emission cathode has been shown unstable field emission and short life time due to field evaporation by high current density and detachment by electrostatic force. An alternative approach in this direction is the introduction of CNT yarn, which is a one dimensional assembly of individual carbon nanotubes bonded by the Van der Waals force. Because CNT yarn is composed with many CNTs, CNT yarns are expected to increase current density and life time for X-ray tube applications. In this research, CNT yarn was fabricated by spinning of a super-aligned CNT forest and was characterized for application to an X-ray tube cathode. CNT yarn showed a high field emission current density and a long lifetime of over 450 hours. Applying the CNT yarn field emitter to the X-ray tube cathode, it was possible to obtain micro-scale resolution images. The relationship between the field emission properties and the microstructure evolution was investigated and the unraveling effect of the CNT yarn was discussed.

Constitutive activation of smoothened leads to impaired developments of postnatal bone in mice.

Sonic hedgehog (Shh) signaling regulates patterning, proliferation, and stem cell self-renewal in many organs. Smoothened (Smo) plays a key role in transducing Shh signaling into the nucleus by activating a glioma family of transcription factors; however, the cellular and molecular mechanisms underlying the role of sustained Smo activation in postnatal development are still unclear. In this study, we explored the effects of Shh signaling on bone development using a conditional knock-in mouse model that expresses a constitutively activated form of Smo (SmoM2) upon osteocalcin (OCN)-Cre-mediated recombination (SmoM2; OCN-Cre mice). We also evaluated the expression pattern of bone formation-related factors in primary calvarial cultures of mutant and control mice. The SmoM2; OCN-Cre mutant showed growth retardation and reduction of bone mineral density compared to control mice. Constitutively activated SmoM2 also repressed mRNA expression of Runx2, osterix, type I collagen, and osteocalcin. Further, sustained SmoM2 induction suppressed mineralization in calvarial primary osteoblasts cultures, whereas such induction did not affect cell proliferation in the mutant cultures as compared with SmoM2 only control cultures. These results suggest that sustained Smo activation inhibits postnatal development of bone by suppressing gene expression of bone formation regulatory factors in mice.

Synthesis and characterization of vertically aligned carbon nanotube forest for solid state fiber spinning.

Continuous carbon nanotubes (CNT) fibers were directly spun from a vertically aligned CNT forest grown by a plasma-enhanced chemical vapor deposition (PECVD) process. The correlation of the CNT structure with Fe catalyst coarsening, reaction time, and the CNTs bundling phenomenon was investigated. We controlled the diameters and walls of the CNTs and minimized the amorphous carbon deposition on the CNTs for favorable bundling and spinning of the CNT fibers. The CNT fibers were fabricated with an as-grown vertically aligned CNT forest by a PECVD process using nanocatalyst an Al2O3 buffer layer, followed by a dry spinning process. Well-aligned CNT fibers were successfully manufactured using a dry spinning process and a surface tension-based densification process by ethanol. The mechanical properties were characterized for the CNT fibers spun from different lengths of a vertically aligned CNT forest. Highly oriented CNT fibers from the dry spinning process were characterized with high strength, high modulus, and high electrical as well as thermal conductivities for possible application as ultralight, highly strong structural materials. Examples of structural materials include space elevator cables, artificial muscle, and armor material, while multifunctional materials include E-textile, touch panels, biosensors, and super capacitors.

Fabrication and characterization of a 3D-structured field emitter using carbon nanotube.

Carbon nanotube has good electrical properties and a high aspect ratio, which enable it to obtain a high current at a low voltage due to its high field. Due to the life and uniformity of their emission tips, carbon nanotube field emitters are hard to commercialize. A field emitter with a three-dimensional (3D) structure was fabricated in this study to overcome such problems. In the 3D-structured field emitter, the field emission tips are located only at the vertical plane, where an enlarged field emission area can be attained. To fabricate the tip of the 3D-structured field emitter, carbon nanotube/silver nanocomposite powders were fabricated via molecular-level mixing and were sprayed at a substrate with good attachment and homogeneous dispersion between the CNT tips and the silver. The field emission properties of the 3D-structured field emitter were then determined and compared with those of a flat field emitter. The field emission area of the 3D-structured field emitter was found to be 4.5 times larger than that of the flat field emitter, with six times higher current density. Moreover, the 3D-structured field emitter had better stability than the flat field emitter. At a high gate field, the emission images of the 3D-structured field emitter showed light spots expanded towards the gate direction.