Orientation-Adjustable Metal-Organic Framework Nanorods for Efficient Oxygen Evolution Reaction.

A series of orientation-adjustable metal-organic framework (MOF) nanorods, CoFe(dobpdc)-I to CoFe(dobpdc)-III (dobpdc = 4,4'-dihydroxybiphenyl-3,3'-dicarboxylate), is developed on a 3D nickel foam (NF) template. By modulating the solvent composition for synthesis, the feature of MOF nanorods on the template can be varied from disorganized to a unidirectional orientation perpendicular to the NF. Well-aligned, vertically oriented CoFe(dobpdc)-III nanorods are hydrophilic and have more exposed active sites and interfacial charge transfer ability. Consequently, they exhibit a superior activity for oxygen evolution reaction (OER) with ultralow overpotentials of 176 and 240 mV at 10 and 300 mA cm-2 in 1.0 M KOH (aq), respectively. CoFe(dobpdc)-III also shows a record low overpotential of 204 mV at J10 mA cm-2 among the electrocatalysts based on CoFe MOF and an excellent overpotential at a high current density (100 mA cm-2) of 312 mV in 0.1 M KOH (aq). This is the first report of a convenient method to straighten up MOF nanorods on a template for highly efficient OER.

Healing of osteoporotic bone defects by baculovirus-engineered bone marrow-derived MSCs expressing MicroRNA sponges.

Fractures associated with osteoporosis are a worldwide health problem. To augment osteoporotic bone healing, we aimed to develop a cell/gene therapy approach in combination with miRNA manipulation. We unraveled aberrant overexpression of miR-140* and miR-214 in the bone marrow-derived MSCs isolated from ovariectomized (OVX) rats (OVX-BMSCs). To suppress the miRNA levels, we constructed hybrid baculovirus vectors expressing miRNA sponges to antagonize miR-140* or miR-214. Engineering OVX-BMSCs with the hybrid vectors persistently attenuated the cellular miR-140*/miR-214 levels, which promoted the OVX-BMSCs osteogenesis and augmented the ability of OVX-BMSCs to repress osteoclast maturation in vitro. Notably, suppressing miR-214 exerted more potent osteoinductive effects. In the osteoporotic rat models with a critical-size bone defect at the femoral metaphysis, implanting the OVX-BMSCs ectopically expressing BMP2 failed to heal the defect, which underscored the difficulty to heal osteoporotic bone defects. Nonetheless, allotransplantation of the miR-214 sponges-expressing OVX-BMSCs healed the defect and ameliorated the bone quality (density, trabecular number, trabecular thickness and trabecular space) at 4 weeks post-implantation. Co-expressing BMP2 and miR-214 sponges in OVX-BMSCs further synergistically substantiated the healing. The baculovirus-engineered OVX-BMSCs that expressed miR-214 sponge, with or without BMP2 expression, thus paved a new avenue to the treatment of osteoporotic bone defects.

Healing of massive segmental femoral bone defects in minipigs by allogenic ASCs engineered with FLPo/Frt-based baculovirus vectors.

Adipose-derived stem cells (ASCs) hold promise for bone regeneration but possess inferior osteogenesis potential. Allotransplantation of ASCs engineered with the BMP2/VEGF-expressing baculoviruses into rabbits healed critical-size segmental bone defects. To translate the technology to clinical applications, we aimed to demonstrate massive bone healing in minipigs that more closely mimicked the clinical scenarios, using a new hybrid baculovirus system consisting of BacFLPo expressing the codon-optimized FLP recombinase (FLPo) and the substrate baculovirus harboring the transgene flanked by Frt sequences. Co-transduction of minipig ASCs (pASCs) with BacFLPo and the substrate baculovirus enabled transgene cassette excision, recombination and minicircle formation in ≈73.7% of pASCs, which substantially prolonged the transgene (BMP2 and VEGF) expression to 28 days. When encoding BMP2, the FLPo/Frt-based system augmented the pASCs osteogenesis. Allotransplantation of the BMP2/VEGF-expressing pASCs into minipigs healed massive segmental bone defects (30 mm in length) at the mid-diaphysis of femora, as evaluated by computed tomography, positron emission tomography, histology, immunohistochemical staining and biochemical testing. The defect size was ≈15% of femoral length in minipigs and was equivalent to ≈60-70 mm of femoral defect in humans, thus the healing using pASCs engineered with the FLPo/Frt-based baculovirus represented a remarkable advance for the treatment of massive bone defects.

Efficient gene delivery into cell lines and stem cells using baculovirus.

Baculovirus is a promising vector for transducing numerous types of mammalian cells. We have developed hybrid baculovirus vectors and protocols for the efficient transduction of a variety of cell lines, primary cells and stem cells, including bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ASCs). The hybrid vector enables intracellular minicircle formation and prolongs transgene expression. The advantages of this transduction protocol are that baculovirus supernatant alone needs to be added to cells growing in medium, and transduction occurs after only 4-6 h of incubation at room temperature (25 °C) with gentle shaking. The entire procedure, from virus generation to transduction, can be completed within 4 weeks. Compared with other transduction procedures, this protocol is simple and can confer efficiencies >95% for many cell types. This protocol has potential applications in tissue regeneration, as transduced cells continue to express transgenes after implantation. For example, transduction of rabbit ASCs (rASCs) with growth factor-encoding hybrid baculovirus vectors, as described as an example application in this protocol, enables robust and sustained growth factor expression, stimulates stem cell differentiation and augments tissue regeneration after implantation.

Osteogenic differentiation of adipose-derived stem cells and calvarial defect repair using baculovirus-mediated co-expression of BMP-2 and miR-148b.

Repair of large calvarial bony defect remains a challenge for orthopedic surgeons. Since microRNAs (miRNAs) modulate the osteogenesis of osteoprogenitor cells, we aimed to engineer human adipose-derived stem cells (hASCs), a promising cell source for bone engineering, with miRNA-expressing baculovirus vectors. We constructed 4 baculoviruses each expressing 1 human miRNA (miR-26a, miR-29b, miR-148b, miR-196a) and verified that the miRNA-expressing baculovirus vectors augmented hASCs osteogenesis. Among these 4 miRNAs, miR-148b and miR-196a exerted more potent osteoinductive effects than miR-26a and miR-29b. Furthermore, we unveiled that co-transduction of hASCs with miR-148b-expressing and bone morphogenetic protein 2 (BMP-2)-expressing baculovirus vectors enhanced and prolonged BMP-2 expression, and synergistically promoted the in vitro osteogenic differentiation of hASCs. Implantation of the hASCs co-expressing BMP-2/miR-148b into critical-size (4 mm in diameter) calvarial bone defects in nude mice accelerated and potentiated the bone healing and remodeling, filling ≈94% of defect area and ≈89% of defect volume with native calvaria-like flat bone in 12 weeks, as judged from micro computed tomography, histology and immunohistochemical staining. Altogether, this study confirmed the feasibility of combining miRNA and growth factor expression for synergistic stimulation of in vitro osteogenesis and in vivo calvarial bone healing.

Baculovirus-transduced, VEGF-expressing adipose-derived stem cell sheet for the treatment of myocardium infarction.

Cell sheet technology has been widely employed for the treatment of myocardial infarction (MI), but cell sheet fabrication generally requires the use of thermo-responsive dishes. Here we developed a method for the preparation of adipose-derived stem cell (ASC) sheet that obviated the need of thermo-responsive dishes. This method only required the seeding of rabbit ASC onto 6-well plates at an appropriate cell density and culture in appropriate medium, and the cells were able to develop into ASC sheet in 2 days. The ASC sheet allowed for transduction with the hybrid baculovirus at efficiencies >97%, conferring robust and prolonged (>35 days) overexpression of vascular endothelial growth factor (VEGF). The ASC sheet was easily detached by brief (10 s) trypsinization and saline wash, while retaining the extracellular matrix and desired physical properties. The ASC sheet formation and VEGF expression promoted cell survival under hypoxia in vitro. Epicardial implantation of the VEGF-expressing ASC sheet to rabbit MI models reduced the infarct size and improved cardiac functions to non-diseased levels, as judged from the left ventrical ejection fraction/myocardial perfusion. The VEGF-expressing ASC sheet also effectively prevented myocardial wall thinning, suppressed myocardium fibrosis and enhanced blood vessel formation. These data implicated the potential of this method for the preparation of genetically engineered ASC sheet and future MI treatment.

Regenerating cartilages by engineered ASCs: prolonged TGF-ß3/BMP-6 expression improved articular cartilage formation and restored zonal structure.

Adipose-derived stem cells (ASCs) hold promise for cartilage regeneration but their chondrogenesis potential is inferior. Here, we used a baculovirus (BV) system that exploited FLPo/Frt-mediated transgene recombination and episomal minicircle formation to genetically engineer rabbit ASCs (rASCs). The BV system conferred prolonged and robust TGF-ß3/BMP-6 expression in rASCs cultured in porous scaffolds, which critically augmented rASCs chondrogenesis and suppressed osteogenesis/hypertrophy, leading to the formation of cartilaginous constructs with improved maturity and mechanical properties in 2-week culture. Twelve weeks after implantation into full-thickness articular cartilage defects in rabbits, these engineered constructs regenerated neocartilages that resembled native hyaline cartilages in cell morphology, matrix composition and mechanical properties. The neocartilages also displayed cartilage-specific zonal structures without signs of hypertrophy and degeneration, and eventually integrated with host cartilages. In contrast, rASCs that transiently expressed TGF-ß3/BMP-6 underwent osteogenesis/hypertrophy and resulted in the formation of inferior cartilaginous constructs, which after implantation regenerated fibrocartilages. These data underscored the crucial role of TGF-ß3/BMP-6 expression level and duration in rASCs in the cell differentiation, constructs properties and in vivo repair. The BV-engineered rASCs that persistently express TGF-ß3/BMP-6 improved the chondrogenesis, in vitro cartilaginous constructs production and in vivo hyaline cartilage regeneration, thus representing a remarkable advance in cartilage engineering.

The use of ASCs engineered to express BMP2 or TGF-ß3 within scaffold constructs to promote calvarial bone repair.

Calvarial bone healing is difficult and grafts comprising adipose-derived stem cells (ASCs) and PLGA (poly(lactic-co-glycolic acid)) scaffolds barely heal rabbit calvarial defects. Although calvarial bone forms via intramembranous ossification without cartilage templates, it was suggested that chondrocytes/cartilages promote calvarial healing, thus we hypothesized that inducing ASCs chondrogenesis and endochondral ossification involving cartilage formation can improve calvarial healing. To evaluate this hypothesis and selectively induce osteogenesis/chondrogenesis, rabbit ASCs were engineered to express the potent osteogenic (BMP2) or chondrogenic (TGF-ß3) factor, seeded into either apatite-coated PLGA or gelatin sponge scaffolds, and allotransplanted into critical-size calvarial defects. Among the 4 ASCs/scaffold constructs, gelatin constructs elicited in vitro chondrogenesis, in vivo osteogenic metabolism and calvarial healing more effectively than apatite-coated PLGA, regardless of BMP2 or TGF-ß3 expression. The BMP2-expressing ASCs/gelatin triggered better bone healing than TGF-ß3-expressing ASCs/gelatin, filling ≈ 86% of the defect area and ≈ 61% of the volume at week 12. The healing proceeded via endochondral ossification, instead of intramembranous pathway, as evidenced by the formation of cartilage that underwent osteogenesis and hypertrophy. These data demonstrated ossification pathway switching and significantly augmented calvarial healing by the BMP2-expressing ASCs/gelatin constructs, and underscored the importance of growth factor/scaffold combinations on the healing efficacy and pathway.

Development of fibroblast culture in three-dimensional activated carbon fiber-based scaffold for wound healing.

This work developed a novel bi-layer wound dressing composed of 3D activated carbon fibers that allows facilitates fibroblast cell growth and migration to a wound site for tissue reconstruction, and the gentamicin is incorporated into a poly(γ-glutamic acid)/gelatin membrane to prevent bacterial infection. In an in vitro, field emission scanning electron microscopy shows that rat skin fibroblasts appeared and spread on the surface of activated carbon fibers, and penetrated the interior and exterior of the 3D activated carbon fiber construct to a depth of roughly 200 µm. An in vivo analysis shows that fibroblast cells containing the proposed 3D scaffold had the potential of a biologically functionalized dressing to accelerate wound closure. Additionally, fibroblasts migrated to the wound site in a bi-layer wound dressing containing fibroblasts, enhancing fibronectin and type I collagen expression, resulting in faster skin regeneration than that achieved with a Tegaderm™ hydrocolloid dressing or gauze.

Purpura, cutaneous necrosis, and antineutrophil cytoplasmic antibodies associated with levamisole-adulterated cocaine.

OBJECTIVE: To describe the clinical and serologic abnormalities in 6 patients who presented with retiform purpura and extensive cutaneous necrosis after exposure to levamisole-adulterated cocaine. METHODS: All patients were evaluated at San Francisco General Hospital or the University of California San Francisco Medical Center. Each underwent standard screening for substances of abuse and had urine tested for the presence of levamisole by liquid chromatography tandem mass spectrometry. Routine laboratory, autoantibody, and antiphospholipid antibody testing was performed in the hospitals' clinical or reference laboratories. Testing for atypical antineutrophil cytoplasmic antibodies (ANCAs) was performed separately using commercially available enzyme-linked immunosorbent assay kits. RESULTS: The patients were women ages 39-50 years who presented with retiform purpura and cutaneous necrosis. Skin biopsies revealed a predominantly small-vessel thrombotic vasculopathy with varying degrees of vasculitis. Four patients were neutropenic. All tested positive for lupus anticoagulant, had IgM antibodies to cardiolipin, and tested strongly positive for ANCAs in a perinuclear pattern by immunofluorescence. Each patient had antibodies to multiple components of neutrophil granules, including neutrophil elastase, lactoferrin, cathepsin G, proteinase 3, and myeloperoxidase. CONCLUSION: Rheumatologists should be aware of this distinctive form of necrotic purpura, its associated autoantibodies, and its link to levamisole-adulterated cocaine.

Nanoparticles incorporated in pH-sensitive hydrogels as amoxicillin delivery for eradication of Helicobacter pylori.

A variety of approaches have been studied to overcome the problems encountered with using antibiotics, which are ineffective in treating Helicobacter pylori infections. In our study, chitosan/poly-gamma-glutamic acid nanoparticles incorporated into pH-sensitive hydrogels were developed as an efficient carrier for amoxicillin delivery. Our results indicate that hydrogels are pH-sensitive, leading to protecting nanoparticles from being destructed by gastric acid. The results of drug releasing in vitro study clearly indicate that the amount of amoxicillin released from nanoparticles incorporated in hydrogels at pH 1.2 was relatively low (14%), compared to that from only nanoparticles (50%). Confocal laser scanning microscopy revealed that nanoparticles could infiltrate cell-cell junctions and interact with H. pylori infection sites in the intercellular spaces. Additionally, the incorporation of amoxicillin-loaded nanoparticles in a hydrogel protected the drug from the actions of the gastric juice and facilitated amoxicillin interaction specifically with intercellular spaces, the site of H. pylori infection.

Endoscopic treatment in a patient with obstructive jaundice caused by pancreatic pseudocyst.

We report a case of chronic alcoholic pancreatitis with obstructive jaundice due to compression of the common bile duct by pancreatic pseudocyst. Ultrasonography and computed tomographic scan on admission demonstrated an 8 cm cystic lesion located at the head of the pancreas. Endoscopic retrograde cholangiopancreatography (ERCP) revealed a segment of narrowing at the distal common bile duct which was compressed by the pancreatic pseudocyst. Communication between the cyst and pancreatic duct was also noted. Bacteriological examination of cystic contents yielded the growth of Proteus vulgaris, Morganella morganii, Stenotrophomonas maltophilia and Pseudomonas aeruginosa. The patient was treated with broad-spectrum antibiotic, endoscopic sphincterotomy, endoscopic nasopancreatic duct drainage, and transpapillary pancreatic endoprosthesis. Jaundice subsided gradually during admission and a marked reduction of pancreatic pseudocyst was found 18 months after discharge.

Control of acid gases using a fluidized bed adsorber.

During incineration, secondary pollutants such as acid gases, organic compounds, heavy metals and particulates are generated. Among these pollutants, the acid gases, including sulfur oxides (SO(x)) and hydrogen chloride (HCl), can cause corrosion of the incinerator piping and can generate acid rain after being emitted to the atmosphere. To address this problem, the present study used a novel combination of air pollution control devices (APCDs), composed of a fluidized bed adsorber integrated with a fabric filter. The major objective of the work is to demonstrate the performance of a fluidized bed adsorber for removal of acid gases from flue gas of an incinerator. The adsorbents added in the fluidized bed adsorber were mainly granular activated carbon (AC; with or without chemical treatment) and with calcium oxide used as an additive. The advantages of a fluidized bed reactor for high mass transfer and high gas-solid contact can enhance the removal of acid gases when using a dry method. On the other hand, because the fluidized bed can filter particles, fine particles prior to and after passing through the fluidized bed adsorber were investigated. The competing adsorption on activated carbon between different characteristics of pollutants was also given preliminary discussion. The results indicate that the removal efficiencies of the investigated acid gases, SO(2) and HCl, are higher than 94 and 87%, respectively. Thus, a fluidized bed adsorber integrated with a fabric filter has the potential to replace conventional APCDs, even when there are other pollutants at the same time.