Shiao-Wen Tsai

Tunable micropatterned substrates based on poly(dopamine) deposition via microcontact printing.

A simple technique was developed to fabricate tunable micropatterned substrates based on mussel-inspired surface modification. Polydopamine (PDA) was developed on polydimethylsiloxane (PDMS) stamps and was easily imprinted to several substrates such as glass, silicon, gold, polystyrene, and poly(ethylene glycol) via microcontact printing. The imprinted PDA retained its unique reactivity and could modulate the chemical properties of micropatterns via secondary reactions, which was illustrated in this study. PDA patterns imprinted onto a cytophobic and nonfouling substrates were used to form patterns of cells or proteins. PDA imprints reacted with nucleophilic amines or thiols to conjugate molecules such as poly(ethylene glycol) for creating nonfouling area. Gold nanoparticles were immobilized onto PDA-stamped area. The reductive ability of PDA transformed silver ions to elemental metals as an electroless process of metallization. This facile and economic technique provides a powerful tool for development of a functional patterned substrate for various applications.

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 Youngs 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.

Beads of collagen-nanohydroxyapatite composites prepared by a biomimetic process and the effects of their surface texture on cellular behavior in MG63 osteoblast-like cells.

The aim of this work was to develop a novel method for preparing a three-dimensional bone-like matrix comprising nanohydroxyapatite crystals and fibrous collagen and to apply it for bone tissue engineering. Hydroxyapatite and collagen are the major components of natural hard bone. Therefore, they have been used extensively in orthopedic surgery as bone-filling materials. According to the principle of complex coacervation, three-dimensional collagen beads can be formed by extruding collagen solution into chondroitin sulfate A (CSA) solution. Subsequently, the collagen beads thus formed are soaked in simulated body-fluid solution to biomimic the formation process of natural bone matrix via the fabrication of collagen-nanohydroxyapatite beads. We also investigate the effect of the collagen-nanohydroxyapatite matrix on the proliferation and differentiation of MG63 cells. The presence of crystalline hydroxyapatite structure on the surface of fibrous collagen was confirmed by X-ray diffraction. MG63 cells cultured on the collagen-nanohydroxyapatite beads proliferate at the normal rate. Moreover, alkaline phosphatase (ALP) activity and the expression levels of three osteogenic genes, namely, type I collagen osteopontin and osteocalcin, in MG63 cells were significantly higher when the cells were cultured on collagen-nanohydroxyapatite beads than when they were cultured on collagen alone. The results of this study reveal that, in the presence of nanohydroxyapatite, the three-dimensional cell beads not only provide a substrate for cell growth but could also enhance the osteoblast-like cell differentiation of MG63 cells.

Surface-Modified Gold Nanoparticles with Folic Acid as Optical Probes for Cellular Imaging

In this study, we demonstrate that the uptake rate of the surface-modified gold nanoparticles (GNPs) with folic acid by specific cells can be increased significantly, if the membranes of these cells have sufficient folic-acid receptors. Two human breast cancer cell lines were studied; one is MDA-MB-435S cell, and the other T-47D cell. The expression of the folic acid receptors of the former is much higher than that of the latter. These cells were incubated with media containing bare GNPs or GNPs conjugated with folic acid individually. Due to the unique optical behavior (i.e. surface plasmon resonance) of GNPs, the uptake amount of GNPs by cells can be identified by using the laser scanning confocal microscopy. Our experiments show that the uptake amount of GNPs in MDA- MB-435S cells is higher than that in T-47D cells for the same culture time, if the culture medium contains bare GNPs. Moreover, if the GNPs conjugated with folic acid are used for the culture, the uptake rate of GNPs by MDA-MB-435S cells is improved more. In contrast, the uptake rates of both GNPs are almost the same by T-47D cells. The phenomenon indicates that the uptake rate of GNPs can be improved via the ligand- receptor endocytosis, compared with the nonspecific endocytosis. Therefore, the uptake rate of GNPs conjugated with folic acid by MDA-MB-435S cells is higher than that of bare GNPs.

Internalized Gold Nanoparticles Do Not Affect the Osteogenesis and Apoptosis of MG63 Osteoblast-Like Cells: A Quantitative, In Vitro Study

The long-term toxicity effects of gold nanoparticles (GNPs) on the proliferation and differentiation of a progenitor cell line, MG63 osteoblast-like cells, was investigated. These cells were treated for 20 hours with two media that contained 10 nm GNPs at concentrations of 1 ppm and 10 ppm. The mitosis of the GNP-treated MG63 was observed after at least 21 hours using dark-field and fluorescence microscopy. The TEM, LSCM and dark-field hyperspectral images indicated that the late endosomes in cells that contained aggregated GNPs were caused by vesicle fusion. Subsequently, after 21 days of being cultured in fresh medium, the specific nodule-like phenotypes and bone-associated gene expression of the treated MG63 cells exhibited the same behaviors as those of the control group. Statistically, after 21 days, the viability of the treated cells was identical to that of the untreated ones. During the cell death program analysis, the apoptosis and necrosis percentages of cells treated for 8 or fewer days were also observed to exhibit no significant difference with those of the untreated cells. In summary, our experiments show that the long-term toxicity of GNPs on the osteogenetic differentiation of MG63 is low. In addition, because of their low toxicity and non-biodegradability, GNPs can potentially be used as biomarkers for the long-term optical observation of the differentiation of progenitor or stem cells based on their plasmonic light-scattering properties.

Paper-based microfluidic sensing device for label-free immunoassay demonstrated by biotin–avidin binding interaction

Efficient diagnosis is very important for the prevention and treatment of diseases. Rapid disease screening in ambulatory environment is one of the most pressing needs for disease control. Despite there are many methods to detect the results of immunoassays, quantitative measurement for rapid disease screening is still a great challenge for point-of-care applications. In this study, a fabrication method for depositing carbon nanotube bundles has been successfully developed for realization of functional paper-based microfluidic sensing device. Quantitative detection of label-free immunoassay, i.e., biotin-avidin binding interaction, was demonstrated by direct measurement of the current change of the biosensor after single application of the target analyte. Sensitivity of 0.33 μA/ng mL(-1) and minimal detectable analyte concentration of 25 ng/mL were achieved. The time necessary for the detection was 500 s which is a large reduction compared with the conventional immunoassay. Such paper-based biosensor has the benefits of portability, fast response, simple operation, and low cost and has the potential for the development of rapid disease screening devices.

Influence of topography of nanofibrils of three-dimensional collagen gel beads on the phenotype, proliferation, and maturation of osteoblasts

Our objectives were to fabricate three-dimensional collagen/chondroitin sulfate beads using mild conditions and no chemical reagents, and subsequently to investigate the influence of the nanotopography of these beads on primary osteoblast and MG63 cell responses, including cell morphology, proliferation rate, and gene expression. The major principle used to prepare beads was complex coacervation, with which we could obtain a three-dimensional collagen matrix with or without a characteristic D-pattern. Therefore, we utilized primary osteoblasts and MG63, an osteoblast-like cell line, to examine the effects of specific structures on cellular behaviors. We found that the phenotype of primary osteoblasts grown on D-periodic beads was a polygonal shape with extending lamellipodia; however, a greater number of cells displayed a circular morphology on the non-D-periodic carriers. After 7 and 14 days, MG63 cells on D-periodic beads expressed higher levels of osteopontin, alkaline phosphatase activity, type I collagen, and osteocalcin than those on the non-D-periodic beads. Together, these data demonstrate that the unique D-pattern of collagen not only enhances the gene expression and mineralization of osteoblasts but also induces the cells to display the normal phenotype, which indicates that the nanotopography of carriers may regulate cellular responses through the spatial control of downstream signaling.

Hyaluronan-cisplatin conjugate nanoparticles embedded in Eudragit S100-coated pectin/alginate microbeads for colon drug delivery.

Hyaluronan–cisplatin conjugate nanoparticles (HCNPs) were chosen as colon-targeting drug-delivery carriers due to the observation that a variety of malignant tumors overexpress hyaluronan receptors. HCNPs were prepared by mixing cisplatin with a hyaluronan solution, followed by dialysis to remove trace elements. The cells treated with HCNPs showed significantly lower viability than those treated with cisplatin alone. HCNPs were entrapped in Eudragit S100-coated pectinate/alginate microbeads (PAMs) by using an electrospray method and a polyelectrolyte multilayer-coating technique in aqueous solution. The release profile of HCNPs from Eudragit S100-coated HCNP-PAMs was pH-dependent. The percentage of 24-hour drug release was approximately 25.1% and 39.7% in pH 1.2 and pH 4.5 media, respectively. However, the percentage of drug released quickly rose to 75.6% at pH 7.4. Moreover, the result of an in vivo nephrotoxicity study demonstrated that Eudragit S100-coated HCNP-PAMs treatment could mitigate the nephrotoxicity that resulted from cisplatin. From these results, it can be concluded that Eudragit S100-coated HCNP-PAMs are promising carriers for colonspecific drug delivery.

Preparation and characterization of microspheres comprised of collagen, chondroitin sulfate, and apatite as carriers for the osteoblast-like cell MG63

Numerous studies about bone matrix fabrication focus on how the species and concentrations of components affect the cellular response. However, there are few studies that investigate how the related spatial arrangement of the components influences cellular activity. The aim of this work was to develop a novel method to biomimetically manufacture a three-dimensional mineral bone matrix and study the effect of apatite-collagen-chondroitin sulfate (CS) microspheres on the adhesion rate and activity of osteoblast-like cells. Although previous studies used a crosslinking agent or lyophilized methods to fabricated three-dimensional collagen microspheres, we produced beads composed of collagen and CS under mild reaction conditions. This process not only maintains collagen self-assembly into fibrils with a D-periodic pattern ability but also simultaneously introduces two major native bone matrix elements, collagen and CS, into the beads. Furthermore, we mimic the native in vivo bone matrix formation process by the direct nucleation and growth of apatite crystals on collagen fibrils. The apatite crystals are similar in composition to human bone mineral via X-ray diffraction and energy-dispersive X-ray spectrometric analysis. The cellular attachment rate of MG63 osteoblast-like cells is significantly higher for collagen-CS-apatite gel beads than for collagen-CS gel beads. In addition, with regard to the osteoblast bioactivity, we observed that alkaline phosphatase activity of MG63 cells on the collagen-CS-apatite gel beads higher than on the collagen-CS gel beads on day 14.

Photothermal effects of laser-activated surface plasmonic gold nanoparticles on the apoptosis and osteogenesis of osteoblast-like cells.

The specific properties of gold nanoparticles (AuNPs) make them a novel class of photothermal agents that can induce cancer cell damage and even death through the conversion of optical energy to thermal energy. Most relevant studies have focused on increasing the precision of cell targeting, improving the efficacy of energy transfer, and exploring additional functions. Nevertheless, most cells can uptake nanosized particles through nonspecific endocytosis; therefore, before hyperthermia via AuNPs can be applied for clinical use, it is important to understand the adverse optical–thermal effects of AuNPs on nontargeted cells. However, few studies have investigated the thermal effects induced by pulsed laser-activated AuNPs on nearby healthy cells due to nonspecific treatment. The aim of this study is to evaluate the photothermal effects induced by AuNPs plus a pulsed laser on MG63, an osteoblast-like cell line, specifically examining the effects on cell morphology, viability, death program, and differentiation. The cells were treated with media containing 50 nm AuNPs at a concentration of 5 ppm for 1 hour. Cultured cells were then exposed to irradiation at 60 mW/cm2 and 80 mW/cm2 by a Nd:YAG laser (532 nm wavelength). We observed that the cytoskeletons of MG63 cells treated with bare AuNPs followed by pulsed laser irradiation were damaged, and these cells had few bubbles on the cell membrane compared with those that were not treated (control) or were treated with AuNPs or the laser alone. There were no significant differences between the AuNPs plus laser treatment group and the other groups in terms of cell viability, death program analysis results, or alkaline phosphatase and calcium accumulation during culture for up to 21 days. However, the calcium deposit areas in the cells treated with AuNPs plus laser were larger than those in other groups during the early culture period.