Kwanchanok Viravaidya-Pasuwat

Preparation of folate-conjugated pluronic F127/chitosan core-shell nanoparticles encapsulating doxorubicin for breast cancer treatment

A targeting drug delivery system using folate-conjugated pluronic F127/chitosan core-shell nanoparticles was developed to deliver doxorubicin (DOX) to the target cancer cells. First, DOX was encapsulated in pluronic F127 micelle cores in the presence of sodium dodecyl sulfate (SDS) by a self-assembly method. To forma shell, a layer of either chitosan or folate-conjugated chitosan was deposited onto the pluronic micelles. The encapsulation efficiency was approximately 58.1 ± 4.7%. The average size of the core-shell nanoparticles was 37.4±2.0 nm, while the zeta potential was 12.9±2.3mV, indicating the presence of a shell layer and more stable particles. In an in vitro DOX release study, an initial burst release, followed by a sustained release, was observed within 24 hours. In addition, the core-shell nanoparticles showed greater cytotoxicity towards MCF-7 cells than free DOX, suggesting a better therapeutic efficacy in treating cancer.

Construction of a chondrocyte cell sheet using temperature-responsive poly(N-isopropylacrylamide)-co-acrylamide

In this study, a novel temperature-responsive poly(N-isopropylacrylamide)-co-acrylamide was used to prepare a chondrocyte cell sheet. Chondrocytes were isolated from human articular cartilage and plated on the copolymer film grafted tissue culture plates. The cell attachment on the copolymer film was shown to be similar to that of the ungrafted surface. To harvest a cell sheet, the incubation temperature was reduced to 10°C for 30 minutes to allow the polymer chain to fully extend, changing the copolymers phase from hydrophobicity to hydrophilicity. Additional incubation at 20°C for 60 minutes was necessary to activate the cellular metabolism required for cytoskeletal organization and cell detachment. A complete cell sheet recovery was achieved when a PVDF membrane was used as a cell sheet carrier. Unfortunately, the shrinkage of the cell sheet was observed. Nonetheless, the harvested cell sheet was shown to be viable and healthy.

Effect of laser diode light irradiation on growth capability of human hair follicle dermal papilla cells

Low level laser therapy is widely used to relieve pain and inflammation, and to restore cellular functions. The photons of light are absorbed by mitochondria in cells, leading to an increase in the production of adenosine triphosphate (ATP), nitric oxide release, blood flow, and reactive oxygen species (ROS). This study proposed the use of a laser diode array at 808 nm to stimulate the proliferation and to activate the functions of dermal papilla cells, which were an important part of the hair growth cycle. These cells were isolated from human hair follicles and were exposed to 808 nm light at various doses from 0.5, 1, 2.5, 4, and 6 J/cm2. The rate of cell proliferation and the gene expression profile of dermal papilla cells were investigated and compared with the control in which the cells did not received any light treatment. The growth curves of the dermal papilla cells were used to determine the specific growth rates. Higher specific growth rates were observed in the cells exposed to laser at doses higher than 0.5 J/cm2. The effect of the laser light treatment on several gene markers, specifically for dermal papilla cells, was evaluated using real-time polymerase chain reaction (qPCR). Our result shows that collagen type 1 (Col1), alkaline phosphatase (Alp), and versican (Vcan) did not increase when the cells were irradiated by the laser light. Interestingly, sex determining region y-box 2 (Sox2) gene was up-regulated when 0.5 J/cm2, and 1 J/cm2 light was used, while an increase in the level of fibroblast growth factor 7 (Fgf7) gene was observed with light irradiation at 0.5 J/cm2, 1 J/cm2, 2.5 J/cm2, and 4 J/cm2. Too high irradiation dose was shown to yield no effect on the gene expression of dermal papilla cells.

Chondrogenesis and hypertrophy in response to aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface

ObjectivesTo investigate the behaviors of aggregates of human mesenchymal stem cells (hMSCs) on chondrogenesis and chondrocyte hypertrophy using spatiotemporal expression patterns of chondrogenic (type II collagen) and hypertrophic (type X collagen) markers during chondrogenesis.ResultshMSCs were cultured on either a polystyrene surface or polyamidoamine dendrimer surface with a fifth generation (G5) dendron structure in chondrogenic medium and growth medium. At day 7, cell aggregates without stress fibers formed on the G5 surface and triggered differentiation of hMSCs toward the chondrogenic fate, as indicated by type II collagen being observed while type X collagen was undetectable. In contrast, immunostaining of hMSCs cultured on polystyrene, which exhibited abundant stress fibers and did not form aggregates, revealed no evidence of either type II and or type X collagen. At day 21, the morphological changes of the cell aggregates formed on the G5 surface were suppressed as a result of stress fiber formation. Type II collagen was observed throughout the aggregates whereas type X collagen was detected only at the basal side of the aggregates. Change of cell aggregate behaviors derived from G5 surface alone regulated chondrogenesis and hypotrophy, and this was enhanced by chondrogenic medium.ConclusionsIncubation of hMSCs affects the expression of type II and X collagens via effects on cell aggregate behavior and stress fiber formation.

The effect of light-emitting diode irradiation at different wavelengths on calcification of osteoblast-like cells in 3D culture

This study aimed to investigate the effect of four different light-emitting diode (LED) wavelengths on calcification and proliferation of osteoblast-like cells in vitro. MC3T3-E1 cells were seeded within three-dimensional collagen scaffolds and irradiated daily by LED light with peak emission wavelengths of 630-, 680-, 760- and 830-nm at constant fluency of 3.1 J/cm2 (irradiance intensity 2 mW/cm2). Cultures were measured for calcium content at day 0, 7, 14, 21, 28, 35 and 42. The significant enhancement in calcium content was observed at the early stage of culture (days 7 and 14) (p<;0.05). After that, the calcium content of irradiated groups was similar to that of the controls group. This suggests the transient effect of light irradiation on osteoblastic cell calcification. Only 680-nm irradiated samples revealed a significant enhancement of calcium content until the late stages of culture (from days 21 to 42) (p<;0.001). The cyclin D mRNA expression that was investigated 3 hours after stimulation at day3 also show that the 680-nm LED irradiation can enhance cyclin D expression more than others. For enhancing bone mineralization, LED irradiation at the 680-nm is more effective than those at 630-, 760- and 830-nm. Further studies should be investigated in order to obtain the most effective parameters of LLLI on bone regeneration in clinical setting.

Maintenance of human chondrogenic phenotype on a dendrimer-immobilized surface for an application of cell sheet engineering

BackgroundDedifferentiation of chondrocytes during cell expansion is one of the barriers in tissue construction for cartilage repair. To understand chondrocyte behavior and improve cell expansion in monolayer culture, this study investigated the effects of morphological changes and cellular aggregation on the maintenance of chondrogenic capacity by observing the expression patterns of chondrogenic (collagen type II and aggrecan) and dedifferentiation (collagen type I) markers. Primary human chondrocytes were cultured on either a polystyrene surface (PS) or a polyamidoamine dendrimer surface with a fifth-generation (G5) dendron structure to create a one-step process of cell expansion and the maintenance of chondrogenic activities prior to the construction of cell sheets.ResultsDuring the first two passages (P0 - P2), the relative mRNA level of collagen type II decreased in all cultures, while that of collagen type I increased. Remarkably, the level of collagen type II was higher and aggrecan was retained in the chondrocytes, forming cell aggregates and showing some round-shaped cells with less production of stress fibers on the G5 surface compared to fibroblast-like chondrocytes with abundant stress fibers on the PS surface. The numbers of P2 chondrocytes on the G5 and PS surfaces were nearly the same and sufficient for construction of chondrocyte sheets using a temperature-responsive plate. Without a supporting material during cell sheet manipulation, chondrocyte sheets spontaneously detached and exhibited a honeycomb-like structure of stress fibers. Unlike the chondrocyte sheets constructed from cells on the PS surface, the chondrocyte sheets from cells on the G5 surface had higher chondrogenic activities, as evidenced by the high expression of chondrogenic markers and the low expression of dedifferentiation markers.ConclusionsThe one-step process of cell expansion and maintenance of chondrogenic activity could be obtained using the G5 surface. Human chondrocyte sheets were successfully constructed with high chondrogenic activity. These findings may lead to an alternative cultivation technique for human chondrocytes that offers high clinical potential in autologous chondrocyte implantation.

Development of a simple and low-cost automated cell sheet harvesting system for tissue engineering

Recent progress in cell sheet engineering has the potential to overcome many limitations in tissue engineering. However, the cell sheet construction process, currently carried out manually by skilled researchers, has been shown to be labor-intensive and time-consuming. To overcome this problem and improve the efficiency, procedures used in the cell sheet technology should be automated. Here, we described the development of a simple and low-cost automated cell harvesting system for the construction of multi-layered cell sheets. The automatic apparatus consisted of two orthogonally positioned stepper motors, each attached to a ball-screw slide. A holder for a gelatin stamp, which was used as a cell sheet transfer vehicle, was assembled to the vertical slide. The multi-axis movement of the system was controlled using an algorithm in LabView. Our result demonstrated that our automated system provided sufficiently high position accuracy and repeatability. Monolayer and triple-layered chondrocyte cell sheets could be constructed using this automated cell sheet harvesting system.

A miniaturized immunoassay platform to measure neutrophil gelatinase-associated lipocalin (NGAL) for diagnosis of acute kidney injury.

Abstract In this study, we describe the development and evaluation of a slide-based immunoassay platform for the detection of neutrophil gelatinase associated-lipocalin (NGAL) in plasma and urine samples. The capture NGAL antibody was immobilized onto a microscope slide before an analysis of NGAL based on a sandwich immunoassay was further carried out. This assay system exhibited linearity between 50 to 1000 ng/ml of NGAL. The coefficients of variability (CVs) indicated good reproducibility and repeatability of the system. The levels of plasma NGAL measured by the slide-based system were highly correlated with those of ELISA, while this system over-predicted urine NGAL.