Stimulation of Early Osteochondral Differentiation of Human Mesenchymal Stem Cells Using Binary Colloidal Crystals (BCCs)

A new surface based on self-assembly of two colloids into well-defined nanostructures, so-called binary colloidal crystals (BCCs), was fabricated for stem cell culture. The facile fabrication process are able to cover large surface areas (>3 cm-diameter, i.e. > 7 cm²) with ordered surface nanotopographies that is often a challenge particularly in biomaterials science. From our library, four different combinations of BCCs were selected using mixtures of silica, polystyrene and poly­(methyl methacrylate) particles with sizes in the range from 100 nm to 5 μm. Cell spreading, proliferation, and surface-induced lineage commitment of human adipose-derived stem cells (hADSCs) was studied using quantitative real time polymerase chain reaction (qRT-PCR) and immunostaining. The results showed that BCCs induced osteo- and chondro- but not adipo-gene expression in the absence of induction medium suggesting that the osteochondral lineage can be stimulated by the BCCs. When applying induction media, higher osteo- and chondro-gene expression on BCCs was found compared with tissue culture polystyrene (TCPS) and flat silica (Si) controls, respectively. Colony forming of chondrogenic hADSCs was found on BCCs and TCPS but not Si controls, suggesting that the differentiation of stem cells is surface-dependent. BCCs provide access to complex nanotopographies and chemistries, which can find applications in cell culture and regenerative medicine

Modulation of Human Mesenchymal Stem Cell Behavior on Ordered Tantalum Nanotopographies Fabricated Using Colloidal Lithography and Glancing Angle Deposition

Ordered surface nanostructures have attracted much attention in biotechnology and biomedical engineering because of their potential to modulate cell–surface interactions in a controllable manner. However, the ability to fabricate large area ordered nanostructures is limited because of high costs and low speed of fabrication. Here, we have fabricated ordered nanostructures with large surface areas (1.5 × 1.5 cm²) using a combination of facile techniques including colloidal self-assembly, colloidal lithography and glancing angle deposition (GLAD). Polystyrene (722 nm) colloids were self-assembled into a hexagonally close-packed (hcp) crystal array at the water–air interface, transferred on a biocompatible tantalum (Ta) surface and used as a mask to generate an ordered Ta pattern. The Ta was deposited by sputter coating through the crystal mask creating approximately 60-nm-high feature sizes. The feature size was further increased by approximately 200-nm-height respectively using GLAD, resulting in the fabrication of four different surfaces (FLAT, Ta60, GLAD100, and GLAD200). Cell adhesion, proliferation, and osteogenic differentiation of primary human adipose-derived stem cells (hADSCs) were studied on these ordered nanostructures for up to 2 weeks. Our results suggested that cell spreading, focal adhesion formation, and filopodia extension of hADSCs were inhibited on the GLAD surfaces, while the growth rate was similar between each surface. Immunostaining for type I collagen (COL1) and osteocalcin (OC) showed that there was higher osteogenic components deposited on the GLAD surfaces compared to the Ta60 and FLAT surfaces after 1 week of osteogenic culture. After 2 weeks of osteogenic culture, alkaline phosphatase (ALP) activity and the amount of calcium was higher on the GLAD surfaces. In addition, osteoblast-like cells were confluent on Ta60 and FLAT surfaces, whereas the GLAD surfaces were not fully covered suggesting that the cell–cell interactions are stronger than cell–substrate interactions on GLAD surfaces. Visible extracellular matrix deposits decorated the porous surface can be found on the GLAD surfaces. Depth profiling of surface components using a new Ar cluster source and X-ray photoelectron spectroscopy (XPS) showed that deposited extracellular matrix on GLAD surfaces is rich in nitrogen. The fabricated ordered surface nanotopographies have potential to be applied in diverse fields, and demonstrate that the behavior of human stem cells can be directed on these ordered nanotopographies, providing new knowledge for applications in biomaterials and tissue engineering

Modulation of PEI-Mediated Gene Transfection through Controlling Cytoskeleton Organization and Nuclear Morphology via Nanogrooved Topographies

The effect of nanotopographies on cell adhesion, migration, proliferation, differentiation, and/or apoptosis have been studied over the last two decades. However, the effect of nanotopography on gene transfection of adhered cells is far from understood. One key phenomenon of using nanotopography is mimicry of native cell morphology in vitro such as in alignment of skeletal myoblasts on nanogrooves. The formation of focal adhesions, the cytoskeleton, and the morphology of cell nuclei are altered by underlying nanogrooves, but the role of these changes in gene transfection are not well understood. In this study, C2C12 skeletal myoblasts were transfected using polyethylenimine (PEI)/DNA complexes on nanogrooved patterns of two groove widths (400 and 800 nm) at three depths (50 nm and 400 or 500 nm). The results showed that the deep nanogrooved surfaces (i.e., 400/400 and 800/500) induced formation of aligned, parallel F-actin and elongated nucleus morphology. Gene transfection was also reduced on the deep nanogrooved surfaces. Disruption of F-actin organization using Cytochalasin D (Cyto-D) restored the nuclear morphology accompanied by higher transfection efficiency, demonstrating that the reduction in gene expression on deep nanogrooves was due to cytoskeletal stretching and nucleus elongation. Spatiotemporal images of fluorescent-labeled PEI/DNA complexes showed that endocytosis of PEI/DNA complexes was retarded and DNA trafficking into the cell nucleus was reduced. This study demonstrates for the first time the important role of cytoskeletal organization and nuclear morphology in PEI-mediated gene transfection to skeletal myoblasts using nanogrooved patterns. These findings are informative for in vitro studies and could potentially be useful in in vivo intramuscular (IM) administration

Blood BT assessment based on EGFP labeled tracer bacteria.

<p>Peritonitis was induced in animals loaded with EGFP-BL21 <i>E</i>. <i>coli</i>. Twenty-four hours later, blood was collected from the animals. Plasma samples from animals receiving different treatment were subjected to an FCM assay. The fluorescent counts are shown in the bar chart. Typical FCM count curves are shown at the top of each column. ^a<i>P</i> < 0.05 compared with sham.</p

Colony-forming unit (CFU) counts in the animals.

<p>Very dilute (1: 1 × 10⁶) feces samples from individual mice were cultured on agar plates containing ampicillin (100 μg/mL). Ampicillin-resistant microbes (presumably the exogenous tracer <i>E</i>. <i>coli</i>) formed clones. The numbers of clones were represented in this chart. There is no statistically significant difference between the three groups.</p

Western blotting to detect bacterial enhanced green fluorescent protein in liver and lung tissues.

<p>At 24 hr after the induction of peritonitis, the mice were euthanized. Fresh protein samples were extracted from lung or liver tissue and were subsequently processed for Western blotting assays. <b>A:</b> Western blotting was performed with enhanced green fluorescent protein (EGFP)-targeted antibodies using protein from liver tissue; <b>B:</b> EGFP Western blotting in tissue. Three repeated experiments showed similar results. IP: Injective peritonitis; IAP: intestinal alkaline phosphatase. <b>C:</b> The quantification of Liver Western blotting data; blots were analyzed using the Image J software. <b>D:</b> The quantification of Lung Western blotting data; blots were analyzed using the Image J software.</p

Intestinal epithelial barrier function was enhanced by intestinal alkaline phosphatase.

<p>Caco-2 cells were plated in the upper wells of transwell chambers. Once confluent monolayers formed, serial doses of intestinal alkaline phosphatase (0, 2, 4, 8, or 16 mIU) were added to the medium for 24 h. <b>A:</b> TEERs of treated monolayers were measured. <b>B:</b> Medium from the basal chamber was collected for HPLC measurements of the paracellular flux tracer FD-40. Data denote three independent experiments. *P<0.05 compared with controls.</p

The change of VEGF level in Serum of mouse after treating with IAP.

<p>IAP decreased the level of serum VEGF in injective peritonitis mice partially. *P<0.01 compared with Control. **P<0.05 compared with IP.</p

Intestinal alkaline phosphatase prevented the increase in permeability following peritonitis.

<p>At 24 h after the induction of peritonitis, intestinal permeability was examined using plasma FD40. ^a<i>P</i> < 0.05 compared with sham, ^b<i>P</i> < 0.05 compared injective peritonitis (IP). Experiments were repeated in at least three mice. IAP: Intestinal alkaline phosphatase.</p

In vivo imaging assay showing the intestinal alkaline phosphatase-induced reduction in bacterial translocation.

<p><b>A:</b> At 24 h after the induction of peritonitis, mice were subjected to in vivo imaging, and red fluorescence was recorded. <b>B:</b> The fluorescence intensities in each organ area. (a) Fluorescence intensity in the liver, (b) fluorescence intensity in the lungs; ^a/c<i>P</i> < 0.05 <i>vs</i>. sham, ^b/d<i>P</i> < 0.05 <i>vs</i>. IP. The experiments were repeated in at least three individuals. IP: Injective peritonitis; IAP: intestinal alkaline phosphatase.</p