MG63 Osteoblast-Like Cells Exhibit Different Behavior when Grown on Electrospun Collagen Matrix versus Electrospun Gelatin Matrix

Electrospinning is a simple and efficient method of fabricating a non-woven polymeric nanofiber matrix. However, using fluorinated alcohols as a solvent for the electrospinning of proteins often results in protein denaturation. TEM and circular dichroism analysis indicated a massive loss of triple-helical collagen from an electrospun collagen (EC) matrix, and the random coils were similar to those found in gelatin. Nevertheless, from mechanical testing we found the Young's modulus and ultimate tensile stresses of EC matrices were significantly higher than electrospun gelatin (EG) matrices because matrix stiffness can affect many cell behaviors such as cell adhesion, proliferation and differentiation. We hypothesize that the difference of matrix stiffness between EC and EG will affect intracellular signaling through the mechano-transducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MG63 osteoblast-like cells. From the results, we found there was no significant difference between the EC and EG matrices with respect to either cell attachment or proliferation rate. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the EC than in those grown on the EG. In addition, the phosphorylation levels of Y397-FAK, ERK1/2, BSP, and OPN proteins, as well as ALP activity, were also higher on the EC than on the EG. We further inhibited ROCK activation with Y27632 during differentiation to investigate its effects on matrix-mediated osteogenic differentiation. Results showed the extent of mineralization was decreased with inhibition after induction. Moreover, there is no significant difference between EC and EG. From the results of the protein levels of phosphorylated Y397-FAK, ERK1/2, BSP and OPN, ALP activity and mineral deposition, we speculate that the mechanism that influences the osteogenic differentiation of MG63 osteoblast-like cells on EC and EG is matrix stiffness and via ROCK-FAK-ERK1/2

Electrospun Hyaluronan-Gelatin Nanofibrous Matrix for Nerve Tissue Engineering

Schwann cells play a critical role in the repair of the peripheral nerve. The goal of this study was to fabricate electrospun gelatin (Gel) and hyaluronan-gelatin (HA-Gel) composite nanofibers to provide a suitable growth environment for Schwann cells. The fiber diameters of Gel, 0.5 HA-Gel, 1 HA-Gel, and 1.5 HA-Gel were 130 ± 30 nm, 294 ± 87 nm, 362 ± 129 nm, and 224 ± 54 nm, respectively. The biological performance of Gel and HA-Gel was evaluated using an in vitro culture of RT4-D6P2T rat Schwann cells. We found that the cell attachment and proliferation rates were not significantly different on these matrices. However, the Schwann cells displayed better organized F-actin on HA-Gel than on Gel. Moreover, the expression levels of several genes, including Nrg1, GFAP, and P0, were significantly higher on HA-Gel than on Gel. In addition, the levels of Nrg1 and P0 protein expression were also higher on the HA-Gel than on Gel. These results indicate that the hyaluronan-gelatin composite nanofibrous matrix could potentially be used in peripheral nerve repair

Active Pin1 is a key target of all-trans retinoic acid in acute promyelocytic leukemia and breast cancer

A common key regulator of oncogenic signaling pathways in multiple tumor types is the unique isomerase Pin1. However, available Pin1 inhibitors lack the required specificity and potency. Using mechanism-based screening, here we find that all-trans retinoic acid (ATRA)--a therapy for acute promyelocytic leukemia (APL) that is considered the first example of targeted therapy in cancer, but its drug target remains elusive--inhibits and degrades active Pin1 selectively in cancer cells by directly binding to the substrate phosphate- and proline-binding pockets in the Pin1 active site. ATRA-induced Pin1 ablation degrades the fusion oncogene PML-RARα and treats APL in cell and animal models and human patients. ATRA-induced Pin1 ablation also inhibits triple negative breast cancer cell growth in human cells and in animal models by acting on many Pin1 substrate oncogenes and tumor suppressors. Thus, ATRA simultaneously blocks multiple Pin1-regulated cancer-driving pathways, an attractive property for treating aggressive and drug-resistant tumors

Association between Helicobacter pylori infection and cognitive impairment in the elderly

Background/purpose: Helicobacter pylori (H. pylori) infection has been positively associated with cognitive impairment. However, previous studies have shown inconsistent findings. Methods: This cross-sectional study included 587 elderly participants (age ≧ 65) from the annual elderly health checkup program at the National Taiwan University Hospital from 2011 to 2013. Both global and domain-specific cognition were assessed using various neuropsychiatric tests. Multivariable linear regression and logistic regression models were utilized to assess the association between the serum H. pylori IgG level and cognitive impairment. Results: Compared with the lowest quartile of H. pylori IgG (Q1), the highest quartile (Q4) was associated with lower scores on verbal fluency-vegetables (β = −0.24), domain-specific attention [digit span-forward: β = −0.19; odds ratio (OR) = 1.83, 95% confidence interval (CI) = 1.03–3.24], and attention factors (β = −0.20; OR= 2.67, 95% CI = 1.51–4.73). No significant association was observed for global cognition. Stratified analyses revealed that, among men, the highest quartile of serum H. pylori IgG (Q4) was associated with impaired scores on verbal fluency-vegetables (β = −0.38; OR = 3.01, 95% CI = 1.42–6.38). Conclusion: Our findings disclosed a positive association between serum H. pylori level and cognitive impairment, which provides important information for the primary prevention of cognitive impairment through the eradication of H. pylori. Keywords: Helicobacter pylori, Infection, Cognitive impairment, Elder

Electron microscopic images of electrospun collagen matrix (EC) and electrospun gelatin matrix (EG).

<p>Scanning electron microscopic images of (A) electrospun collagen matrix (EC), and (B) electrospun gelatin matrix (EG) at 2,500× magnification. The EC nanofibers had an average diameter of 692±214 nm, and the EG nanofibers had an average diameter of 573±368 nm. Scale bar is 20 µm. Transmission electron microscopic images of (C) electrospun collagen matrix, (D) electrospun gelatin matrix and (E) self-assembled collagen matrix. Scale bar is 0.2 µm. EC nanofibers did not exhibit the characteristic D-periods pattern that was apparent for the native collagen molecules self-assembled into collagen fibrils. Self-assembled collagen was formed by dialyzed collagen solution (in acetic acid) against 0.02 M phosphate buffered-saline (PBS, pH 7) at 4°C. Fibril formation (self-assembled) was initiated by warming the mixture to 37°C for 4 hours.</p

Effects of matrix on BSP and OPN protein expression in MG63 osteoblasts-like cell.

<p>Western blot analysis for BSP and OPN proteins in cells cultured for (A) 14 and (B) 21 days. Densitometric quantitation of the OPN and BSP protein band were performed after normalizing to nucleophosmin B23 levels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031200#pone.0031200.s002" target="_blank">Figure S2</a> shown the images of full western blots). Data are shown as the fold change of the corresponding on the electrospun collagen matrix. The expression level of BSP and OPN proteins showed a significant increase in cells grown on EC at 21 days. Data represent mean ± SD, n = 3. Statistical analysis was compared between EC and EG. (*) denotes a significant difference (<i>p</i><0.05).</p

Matrices regulate the bone-associated genes expressed by MG63 osteoblast-like cells.

<p>Real–time PCR analyses of bone-associated genes expressed by MG63 osteoblast-like cells on various matrix. (A) β-actin levels, (B) type I collagen levels, (C) OPN levels and (D) OCN levels after normalization to 18S ribosomal RNA levels. Data are shown as the fold change relative to the electrospun collagen matrix after 7 days of incubation. On day 7, the cells showed significantly higher expression levels of β-actin, type I collagen, OPN and OCN when grown on EC. And, the expression levels of β-actin, OPN and OCN were significantly higher on day 21 when grown on EC. Data are presented as mean ± SD, n = 4. Statistical analysis was compared between EC and EG. (*) denotes a significant difference (<i>p</i><0.05).</p

Effects of matrix on bone mineralization in MG63 osteoblasts-like cell.

<p>Optical images of ARS staining for mineralization in MG63 osteoblast-like cells for day 28 (A) EC, (B) EG. (C) EC with 10 µM Y27632, (D) EG with 10 µM Y27632. Quantification of mineral deposition by Alizarin Red-S staining. Data represent mean ± SD, n = 4. Statistical analysis was compared between EC and EG with or without Y27632. Different letters represent significance at <i>p</i><0.05 by non-parametric Mann-Whitney U test.</p

Effects of matrix on FAK and phospho-FAK and ERK1/2 activation in MG63 osteoblasts-like cell.

<p>Densitometric quantitation of the protein bands was performed after normalizing to nucleophosmin B23 levels. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031200#pone.0031200.s001" target="_blank">Figure S1</a> shown the images of full western blots). Data are shown as the fold change of the corresponding 2 h of incubation on the EC. The total FAK levels expressed by MG63 osteoblast-like cells were significant higher in cells grown on EC than in those grown on EG. Moreover, the level of FAK phosphorylation at tyrosine 397 was lower in cells grown on EG than in those grown on EC. Data are presented as mean ± SD, n = 3. Statistical analysis was compared between EC and EG. (*) denotes a significant difference (<i>p</i><0.05).</p