Yuan-Min Lin

Region-specific gene expression profiles in the left atria of patients with valvular atrial fibrillation

BACKGROUND Previous studies have demonstrated that atrial regions contribute differently to atrial fibrillation (AF) substrate. Pulmonary vein and the surrounding left atrial junction (LA-PV junction) are crucial areas in AF substrates and important ablation targets. OBJECTIVE To identify regional differences in the left atria of patients with AF by using a genome-wide approach. METHODS Paired LA-PV junction and left atrial appendage (LAA) specimens were obtained from 16 patients with persistent AF and 3 with sinus rhythm who underwent valvular surgery. The paired specimens were sent for microarray comparison. RESULTS Of 54,675 expressed sequence tags, microarray analysis in patients with AF revealed that 391 genes were differentially expressed between the LA-PV junction and the LAA, including genes related to arrhythmia, cell death, fibrosis, hypertrophy, and inflammation. Microarray and real time-polymerase chain reaction produced parallel results in analyzing the expression of particular genes. In both patients with AF and sinus rhythm, the LA-PV junction exhibited greater paired-like homeodomain-2 and its target protein (short stature homeobox-2) expression than the LAA, which might contribute to arrhythmogenesis. Five genes related to thrombogenesis were upregulated in the LAA of patients with AF, which might implicate for the preferential thrombus formation in the LAA. Genes related to fibrosis were highly expressed in the LAA of patients with AF, which was reflected by intense ultrastructural changes in this region. CONCLUSIONS We used a genome-wide approach to investigate region-specific gene expression in the left atria. Our findings provide important information relevant to region-specific arrhythmogenesis and thrombogenesis in AF pathogenesis.

Bioactive surface modification of polycaprolactone using MG63-conditioned medium can induce osteogenic differentiation of mesenchymal stem cells

We demonstrated an approach that modifies the scaffold surface with a range of molecules, simultaneously conjugated to the scaffold by a single treatment with concentrated conditioned medium (CM), inducing mesenchymal stem cells (MSC) to differentiate into osteogenic lineages. We first show that the CM from MG63 cells is capable of inducing the desired MSC differentiation over 7 days. We then analyze how the biodegradable polymer polycaprolactone (PCL) can be used as the scaffold. Using a CO2 plasma treatment, it is possible to conjugate MG63 CM proteins onto the PCL film surface, and we show a gradual release of protein from such a modified PCL scaffold. Finally, we verified cell differentiation and marker expression of MSCs grown on the modified PCL and show that osteogenic markers, including alkaline phosphatase and Runx2 mRNA, are significantly upregulated. Immunostaining also shows a strong expression of the Runx2 protein. Our study shows that the differentiation effects of a condition medium can be preserved when its content is used to modify the surface of polymer scaffolds. This approach may be further applied for the differentiation of various cell lines, and it provided a first step toward growing MSCs on more complex scaffold shapes aimed at therapeutic uses.

Plasma treatment in conjunction with EGM-2 medium increases endothelial and osteogenic marker expressions of bone marrow mesenchymal stem cells

For many tissue engineering applications, an important goal is to create functional tissues in vitro. In order to make such tissues viable, they have to be vascularized. Bone marrow mesenchymal stem cells (BMSCs) are a promising candidate for vascularization. Growth supplements are commonly used to induce BMSCs differentiation, and further improvements in differentiation conditions can be achieved by modifying the cells’ growth environment, such as pretreating the culture dish with gas plasma to modify the surface functional groups that the cells are seeded on. In this work, we compare the effects of different gas plasmas on the growth and differentiation of BMSCs. We treat the growth dish with different plasmas (CO2, N2, and O2) and induce BMSC differentiation on them with endothelial growth medium-2 (EGM-2). We find that EGM-2 by itself does not have strong effect on endothelial differentiation. However, we find that plasma treatment markedly increases the mRNA expression of endothelial cell markers. Interestingly, we also find that the upregulation of osteogenic markers. The data show that plasma treatment in conjunction with EGM-2 can enhance BMSCs differentiation into both endothelial-like cells and osteogenic-like cells. The effects of EGM-2 on BMSCs differentiation are influenced by the plasma-modified surface of the substrate. Our findings provide a method to enhance EGM-2-based cell differentiation, with consequences for tissue engineering and stem cell biology applications.

Synthesis, Characterization, and Visible Light Curing Capacity of Polycaprolactone Acrylate

Polycaprolactone (PCL) is drawing increasing attention in the field of medical 3D printing and tissue engineering because of its biodegradability. This study developed polycaprolactone prepolymers that can be cured using visible light. Three PCL acrylates were synthesized: polycaprolactone-530 diacrylate (PCL530DA), glycerol-3 caprolactone triacrylate (Glycerol-3CL-TA), and glycerol-6 caprolactone triacrylate (Glycerol-6CL-TA). PCL530DA has two acrylates, whereas Glycerol-3CL-TA and Glycerol-6CL-TA have three acrylates. The Fourier transform infrared and nuclear magnetic resonance spectra suggested successful synthesis of all PCL acrylates. All are liquid at room temperature and can be photopolymerized into a transparent solid after exposure to 470 nm blue LED light using 1% camphorquinone as photoinitiator and 2% dimethylaminoethyl methacrylate as coinitiator. The degree of conversion for all PCL acrylates can reach more than 80% after 1 min of curing. The compressive modulus of PCL530DA, Glycerol-3CL-TA, and Glycerol-6CL-TA is 65.7 ± 12.7, 80.9 ± 6.1, and 32.1 ± 4.1 MPa, respectively, and their compressive strength is 5.3 ± 0.29, 8.3 ± 0.18, and 3.0 ± 0.53 MPa, respectively. Thus, all PCL acrylates synthesized in this study can be photopolymerized and because of their solid structure and low viscosity, they are applicable to soft tissue engineering and medical 3D printing.

Antioxidant N-Acetylcysteine and Glutathione Increase the Viability and Proliferation of MG63 Cells Encapsulated in the Gelatin Methacrylate/VA-086/Blue Light Hydrogel System.

Photoencapsulation of cells inside a hydrogel system can provide a suitable path to establish a gel in situ for soft tissue regeneration applications. However, the presence of photoinitiators and blue or UV light irradiation can result in cell damage and an increase of reactive oxygen species. We here evaluate the benefits of an antioxidant pretreatment on the photoencapsulated cells. We study this by evaluating proliferation and viability of MG63 cells, which we combined with a gelatin methacrylate (GelMA) hydrogel system, using the photoinitiator, VA-086, cured with 440 nm blue light. We found that blue light irradiation as well as the presence of 1% VA-086 reduced MG63 cell proliferation rates. Adding a short pretreatment step to the MG63 cells, consisting of the antioxidant molecules N-acetylcysteine (NAC) and reduced glutathione (GSH), and optimizing the GelMA encapsulation steps, we found that both NAC and GSH pretreatments of MG63 cells significantly increased both proliferation and viability of the cells, when using a 15% GelMA hydrogel, 1% VA-086, and 1-min blue light exposure. These findings suggest that the use of antioxidant pretreatment can counteract the negative presence of the photoinitiators and blue light exposure and result in a suitable environment for photoencapsulating cells in situ for tissue engineering and soft tissue applications.

Differential left-to-right atria gene expression ratio in human sinus rhythm and atrial fibrillation: Implications for arrhythmogenesis and thrombogenesis

BACKGROUND Atrial fibrillation (AF) causes atrial remodeling, and the left atrium (LA) is the favored substrate for maintaining AF. It remains unclear if AF remodels both atria differently and contributes to LA arrhythmogenesis and thrombogenesis. Therefore, we wished to characterize the transcript profiles in the LA and right atrium (RA) in sinus rhythm (SR) and AF respectively. METHODS Paired LA and RA appendages acquired from patients receiving cardiac surgery were used for ion-channel- and whole-exome-based transcriptome analysis. The ultrastructure was evaluated by immunohistochemistry. RESULTS Twenty-two and twenty ion-channels and transporters were differentially expressed between the LA and RA in AF and SR, respectively. Among these, 15 genes were differentially expressed in parallel between AF and SR. AF was associated with increased LA/RA expression ratio in 9 ion channel-related genes, including genes related to calcium handling. In microarray, AF was associated with a differential LA/RA gene expression ratio in 309 genes, and was involved in atherosclerosis-related signaling. AF was associated with the upregulation of thrombogenesis-related genes in the LA appendage, including P2Y12, CD 36 and ApoE. Immunohistochemistry showed higher expressions of collagen-1, oxidative stress and TGF-β1 in the RA compared to the LA. CONCLUSIONS AF was associated with differential LA-to-RA gene expression related to specific ion channels and pathways as well as upregulation of thrombogenesis-related genes in the LA appendage. Targeting the molecular mechanisms underlying the LA-to-RA difference and AF-related remodeling in the LA appendage may help provide new therapeutic options in treating AF and preventing thromboembolism in AF.

The 1.5 GeV operation parameters and performance at SRRC

The storage ring at SRRC is highly promising as an ultraviolet to soft X-ray radiation source for use in basic research and industrial applications. Energy ramping of the storage ring can push the critical photon energy to the edge of hard X-ray. The tune drifting, during the ramping procedure, is expected and should be minimized such that the beam can survive through the ramping process. The betatron frequencies and the ramping function of magnets were carefully monitored in order to avoid the betatron tunes crossing the fatal resonance line. A successful ramping results and lattice parameters were measured and discussed in this paper. The measured photon flux increased by six to seven folds at the X-ray beam line. Life time reaches 9 hours of 200 mA beam current.

Stiffness modification of photopolymerizable gelatin-methacrylate hydrogels influences endothelial differentiation of human mesenchymal stem cells

For stem cell differentiation, the microenvironment can play an important role, and hydrogels can provide a three‐dimensional microenvironment to allow native cell growth in vitro. A challenge is that the stem cells differentiation can be influenced by the matrix stiffness. We demonstrate a low‐toxicity method to create different stiffness matrices, by using a photopolymerizable gelatin methacrylate (GelMA) hydrogel cross‐linked by blue light (440 nm). The stiffness and porosity of GelMA hydrogel is easily modified by altering its concentration. We used human bone marrow mesenchymal stem cells (MSCs) as a cell source and cultured the GelMA‐encapsulated cells with EGM‐2 medium to induce endothelial differentiation. In our GelMA blue light hydrogel system, we found that MSCs can be differentiated into both endothelial‐like and osteogenic‐like cells. The mRNA expressions of endothelial cell markers CD31, von Willebrand factor, vascular endothelial growth factor receptor‐2, and CD34 were significantly increased in softer GelMA hydrogels (7.5% and 10%) compared with stiffer matrices (15% GelMA). On the other hand, the enhancements of osteogenic markers mRNA expressions (Alkaline phosphatase (ALP), Runx2, osteocalcin, and osteopontin) were highest in 10% GelMA. We also found that 10% GelMA hydrogel offered optimal conditions for MSCs to form capillary‐like structures. These results suggest that the mechanical properties of the GelMA hydrogel can influence both endothelial and osteogenic differentiation of MSCs and sequent capillary‐like formation.

Synthesis and Characterization of Polycaprolactone-Based Polyurethanes for the Fabrication of Elastic Guided Bone Regeneration Membrane

The aim of this research is to synthesize polycaprolactone-based polyurethanes (PCL-based PUs) that can be further used for the fabrication of guided bone regeneration (GBR) membranes with higher tensile strength and elongation at break than collagen and PTFE membranes. The PCL-based PUs were prepared by the polymerization of polycaprolactone (PCL) diol with 1,6-hexamethylene diisocyanate (HDI) at different ratios using either polyethylene glycol (PEG) or ethylenediamine (EDA) as chain extenders. The chemical, mechanical, and thermal properties of the synthesized polymers were determined using NMR, FTIR, GPC, DSC, and tensile tester. The PCL and polyurethanes were fabricated as nanofiber membranes by electrospinning, and their mechanical properties and SEM morphology were also investigated. In vitro tests, including WST-1 assay, SEM of cells, and phalloidin cytoskeleton staining, were also performed. It was shown that electrospun membranes made of PCL and PCL-HDI-PEG (2 : 3 : 1) possessed tensile strength of 19.84 MPa and 11.72 MPa and elongation at break of 627% and 362%, respectively. These numbers are equivalent or higher than most of the commercially available collagen and PTFE membrane. As a result, these membranes may have potential for future GBR applications.