Osteogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells in Electrospun Silica Nonwoven Fabrics

Silica nonwoven fabrics (SNFs) with enough mechanical strength are candidates as implantable scaffolds. Culture of cells therein is expected to affect the proliferation and differentiation of the cells through cell–cell and cell–SNF interactions. In this study, we examined three-dimensional (3D) SNFs as a scaffold of mesenchymal stem cells (MSCs) for bone tissue engineering applications. The interconnected highly porous microstructure of 3D SNFs is expected to allow omnidirectional cell–cell interactions, and the morphological similarity of a silica nanofiber to that of a fibrous extracellular matrix can contribute to the promotion of cell functions. 3D SNFs were prepared by the sol–gel process, and their mechanical properties were characterized by the compression test and rheological analysis. In the compression test, SNFs showed a compressive elastic modulus of over 1 MPa and a compressive strength of about 200 kPa. These values are higher than those of porous polystyrene disks used for in vitro 3D cell culture. In rheological analysis, the elastic modulus and fracture stress were 3.27 ± 0.54 kPa and 25.9 ± 8.3 Pa, respectively. Then, human bone marrow-derived MSCs were cultured on SNFs, and the effects on proliferation and osteogenic differentiation were evaluated. The MSCs seeded on SNF proliferated, and the thickness of the cell layer became over 80 μm after 14 days of culture. The osteogenic differentiation of MSCs on SNFs was induced by the culture in the commercial osteogenic differentiation medium. The alkaline phosphatase activity of MSCs on SNFs increased rapidly and remained high up to 14 days and was much higher than that on two-dimensional tissue culture-treated polystyrene. The high expression of RUNX2 and intense staining by alizarin red s after differentiation supported that SNFs enhanced the osteogenic differentiation of MSCs. Furthermore, permeation analysis of SNFs using fluorescein isothiocyanate-dextran suggested a sufficient permeability of SNFs for oxygen, minerals, nutrients, and secretions, which is important for maintaining the cell viability and vitality. These results suggested that SNFs are promising scaffolds for the regeneration of bone defects using MSCs, originated from highly porous and elastic SNF characters

Cartilage Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells in Three-Dimensional Silica Nonwoven Fabrics

In cartilage tissue engineering, three-dimensional (3D) scaffolds provide native extracellular matrix (ECM) environments that induce tissue ingrowth and ECM deposition for in vitro and in vivo tissue regeneration. In this report, we investigated 3D silica nonwoven fabrics (Cellbed®) as a scaffold for mesenchymal stem cells (MSCs) in cartilage tissue engineering applications. The unique, highly porous microstructure of 3D silica fabrics allows for immediate cell infiltration for tissue repair and orientation of cell–cell interaction. It is expected that the morphological similarity of silica fibers to that of fibrillar ECM contributes to the functionalization of cells. Human bone marrow-derived MSCs were cultured in 3D silica fabrics, and chondrogenic differentiation was induced by culture in chondrogenic differentiation medium. The characteristics of chondrogenic differentiation including cellular growth, ECM deposition of glycosaminoglycan and collagen, and gene expression were evaluated. Because of the highly interconnected network structure, stiffness, and permeability of the 3D silica fabrics, the level of chondrogenesis observed in MSCs seeded within was comparable to that observed in MSCs maintained on atelocollagen gels, which are widely used to study the chondrogenesis of MSCs in vitro and in vivo. These results indicated that 3D silica nonwoven fabrics are a promising scaffold for the regeneration of articular cartilage defects using MSCs, showing the particular importance of high elasticity

Initial colonization of BipA mutant in murine lungs.

<p>Groups of three BALB/c mice were intranasally infected with the BipA mutant BH2Sm^r-ΔBipA or its parental strain BH2Sm^r. The colonization levels were assessed after 3, 6, and 24 h. Each symbol represents one mouse, and horizontal lines represent the mean number of CFU recovered. Statistical significance was determined using Student’s <i>t</i>-test. The dashed line indicates the lower limit of detection (100 CFU/lung). The lung CFUs in two mice infected with BH2Sm^r-ΔBipA were below the limit of detection at 24 h post-infection.</p

Lack of biofilm formation by <i>Bordetella holmesii</i> isolate BH7.

<p>The clinical isolates BH2 and BH7 were cultured statically on vertically submerged glass slides in mSS broth. After 5 and 72 h, the cell adherence and the biofilm growth at the air-liquid interface were visualized by SEM. Scale bars, 1 μm</p

Autoagglutination of BipA mutant.

<p>(A) BipA mutant BH2Sm^r-ΔBipA and its parental strain, BH2Sm^r, were suspended in casamino acid solution and incubated for 4 h at 36°C under static conditions. (B) Time course analysis of the turbidity (OD₆₅₀) of the bacterial suspensions. Data are presented as the means ± standard deviations of results obtained from 3 separate experiments performed in triplicate. (C) Microscopic analysis of the morphology of the bacterial suspensions. The suspensions were incubated for 4 h at 36°C on glass slides, and observed by phase-contrast microscopy (objective magnification, 100×).</p

Autoagglutination of <i>Bordetella holmesii</i> isolate BH7.

<p>(A) The clinical isolates BH2, BH6, BH7, and BH8 were suspended in casamino acid solution and incubated for 4 h at 36°C under static conditions. (B) Time course analysis of the turbidity (OD₆₅₀) of the bacterial suspensions. Data are presented as means ± standard deviations of results obtained from 3 separate experiments performed in triplicate. (C) Microscopic analysis of the morphology of the bacterial suspensions. The suspensions were incubated for 4 h at 36°C on glass slides, and observed by phase-contrast microscopy (objective magnification, 100×).</p

BipA Is Associated with Preventing Autoagglutination and Promoting Biofilm Formation in <i>Bordetella holmesii</i>

<div><p><i>Bordetella holmesii</i> causes both invasive and respiratory diseases in humans. Although the number of cases of pertussis-like respiratory illnesses due to <i>B</i>. <i>holmesii</i> infection has increased in the last decade worldwide, little is known about the virulence factors of the organism. Here, we analyzed a <i>B</i>. <i>holmesii</i> isolate that forms large aggregates and precipitates in suspension, and subsequently demonstrated that the autoagglutinating isolate is deficient in Bordetella intermediate protein A (BipA) and that this deletion is caused by a frame-shift mutation in the <i>bipA</i> gene. A BipA-deficient mutant generated by homologous recombination also exhibited the autoagglutination phenotype. Moreover, the BipA mutant adhered poorly to an abiotic surface and failed to form biofilms, as did two other <i>B</i>. <i>holmesii</i> autoagglutinating strains, ATCC 51541 and ATCC 700053, which exhibit transcriptional down-regulation of <i>bipA</i> gene expression, indicating that autoagglutination indirectly inhibits biofilm formation. In a mouse intranasal infection model, the BipA mutant showed significantly lower levels of initial lung colonization than did the parental strain (<i>P</i> < 0.01), suggesting that BipA might be a critical virulence factor in <i>B</i>. <i>holmesii</i> respiratory infection. Together, our findings suggest that BipA production plays an essential role in preventing autoagglutination and indirectly promoting biofilm formation by <i>B</i>. <i>holmesii</i>.</p></div

Lack of BipA production in <i>Bordetella holmesii</i> isolate BH7.

<p>(A) Comparison of the protein expression profiles of the clinical isolates BH2, BH6, BH7, and BH8. Total protein (20 μg) was subjected to SDS-PAGE followed by CBB staining (left panel). The dashed-boxed area is enlarged (right panel). The arrows indicate the bands that were identified as BipA by nano-LC-MS/MS analysis. (B) Analysis of BipA production in <i>B</i>. <i>holmesii</i> isolates. Total protein (2 μg) was subjected to immunoblot analysis with anti-BipA antisera. (C) Comparison of BipA sequences among the isolates. The <i>bipA</i> gene of BH7 encodes a deletion of a guanine (G) at nucleotide position 2039 (gray box). The deletion mutation generates a premature stop codon at nucleotide position 2066. The translation stop site (amino acid 689) is shown in the deduced amino acid sequence (BH7, gray box).</p