Reduced Cell Migration in Human Chondrocyte Sheets Increases Tissue Stiffness and Cartilage Protein Production.

BACKGROUND: Chondrogenic differentiation medium (CDM) is usually used to maintain chondrogenic activity during chondrocyte sheet production. However, tissue qualities remain to be determined as to what factors improve cell functions. Moreover, the relationship between CDM and cell migration proteins has not been reported. METHOD: In this study, the effect of CDM on the behavior of chondrocyte sheets was investigated. Structural analysis, mechanical testing and proteomics were performed to observe tissue qualities. The relationship between CDM and cell migration proteins were investigated using time-lapse observations and bioinformatic analysis. RESULTS: During 48 h, CDM affected the chondrocyte behaviors by reducing cell migration. Compared to the basal medium, CDM impacted the contraction of monolayered chondrocyte sheets. At day 7, the contracted sheets increased tissue thickness and improved tissue stiffness. Cartilage specific proteins were also upregulated. Remarkedly, the chondrocyte sheets in CDM displayed downregulated proteins related to cell migration. Bioinformatic analysis revealed that TGFß1 was shown to be associated with cartilage functions and cell migration. Pathway analysis of chondrocyte sheets in CDM also revealed the presence of a TGFß pathway without activating actin production, which might be involved in synthesizing cartilage-specific proteins. Cell migration pathway showed MAPK signaling in both cultures of the chondrocyte sheets. CONCLUSION: Reduced cell migration in the chondrocyte sheet affected the tissue quality. Using CDM, TGFß1 might trigger cartilage protein production through the TGFß pathway and be involved in cell migration via the MAPK signaling pathway. Understanding cell behaviors and their protein expression would be beneficial for developing high-quality tissue-engineered cartilage.

Translational application of human keratinocyte-fibroblast cell sheets for accelerated wound healing in a clinically relevant type 2 diabetic rat model.

BACKGROUND AIMS: Despite advancements in wound care, wound healing remains a challenge, especially in individuals with type 2 diabetes. Cell sheet technology has emerged as an efficient and promising therapy for tissue regeneration and wound repair. Among these, bilayered human keratinocyte-fibroblast cell sheets constructed using temperature-responsive culture surfaces have been shown to mimic a normal tissue-like structure and secrete essential cytokines and growth factors that regulate the wound healing process. METHODS: This study aimed to evaluate the safety and therapeutic potential of human skin cell sheets to treat full-thickness skin defects in a rat model of type 2 diabetes. RESULTS: Our findings demonstrate that diabetic wounds transplanted with bilayered cell sheets resulted in accelerated re-epithelialization, increased angiogenesis, enhanced macrophage polarization and regeneration of tissue that closely resembled healthy skin. In contrast, the control group that did not receive cell sheet transplantation presented characteristic symptoms of impaired and delayed wound healing associated with type 2 diabetes. CONCLUSIONS: The secretory cytokines and the upregulation of Nrf2 expression in response to cell sheet transplantation are believed to have played a key role in the improved wound healing observed in diabetic rats. Our study suggests that human keratinocyte-fibroblast cell sheets hold great potential as a therapeutic alternative for diabetic ulcers.

Expansion of fibroblast cell sheets using a modified MEEK micrografting technique for wound healing applications.

Cell sheet engineering, a scaffold-free approach to fabricate functional tissue constructs from several cell monolayers, has shown promise in tissue regeneration and wound healing. Unfortunately, these cell sheets are often too small to provide sufficient wound area coverage. In this study, we describe a process to enlarge cell sheets using MEEK micrografting, a technique extensively used to expand skin autografts for large burn treatments. Human dermal fibroblast cell sheets were placed on MEEK's prefolded gauze without any use of adhesive, cut along the premarked lines and stretched out at various expansion ratios (1:3, 1:6 and 1:9), resulting in regular distribution of many square islands of fibroblasts at a much larger surface area. The cellular processes essential for wound healing, including reattachment, proliferation, and migration, of the fibroblasts on expanded MEEK gauze were superior to those on nylon dressing which served as a control. The optimal expansion ratio with the highest migration rate was 1:6, possibly due to the activation of chemical signals caused by mechanical stretching and an effective intercellular communication distance. Therefore, the combination of cell sheet engineering with the MEEK micrografting technique could provide high quality cells with a large coverage area, which would be particularly beneficial in wound care applications.

Controlled Release Mechanism of Vancomycin from Double-Layer Poly-L-Lactic Acid-Coated Implants for Prevention of Bacterial Infection.

Implantation failure due to bacterial infection incurs significant medical expenditure annually, and treatment tends to be complicated. This study proposes a method to prevent bacterial infection in implants using an antibiotic delivery system consisting of vancomycin loaded into poly-L-lactic acid (PLLA) matrices. A thin layer of this antibiotic-containing polymer was formed on stainless steel surfaces using a simple dip-coating method. SEM images of the polymeric layer revealed a honeycomb structure of the PLLA network with the entrapment of vancomycin molecules inside. In the in vitro release study, a rapid burst release was observed, followed by a sustained release of vancomycin for approximately 3 days. To extend the release time, a drug-free topcoat of PLLA was introduced to provide a diffusion resistance layer. As expected, the formulation with the drug-free topcoat exhibited a significant extension of the release time to approximately three weeks. Furthermore, the bonding strength between the double-layer polymer and the stainless steel substrate, which was an important property reflecting the quality of the coating, significantly increased compared to that of the single layer to the level that met the requirement for medical coating applications. The release profile of vancomycin from the double-layer PLLA film was best fitted with the Korsmeyer-Peppas model, indicating a combination of Fickian diffusion-controlled release and a polymer relaxation mechanism. More importantly, the double-layer vancomycin-PLLA coating exhibited antibacterial activity against S. aureus, as confirmed by the agar diffusion assay, the bacterial survival assay, and the inhibition of bacterial surface colonization without being toxic to normal cells (L929). Our results showed that the proposed antibiotic delivery system using the double-layer PLLA coating is a promising solution to prevent bacterial infection that may occur after orthopedic implantation.

Evaluation of osteochondral-like tissues using human freeze-dried cancellous bone and chondrocyte sheets to treat osteochondral defects in rabbits.

Human freeze-dried cancellous bone combined with human chondrocyte sheets have recently been used to construct an osteochondral-like tissue, which resembled a cartilage layer on a subchondral bone layer. Nevertheless, the efficacy of these human tissues in a xenogeneic model has been rarely reported. Therefore, this study aimed to evaluate the potential of human freeze-dried cancellous bones combined with human chondrocyte sheets for the treatment of osteochondral defects in rabbits. The key roles of the extracellular matrix (ECM) and released cytokines in these tissues in osteochondral repair were also assessed. Triple-layered chondrocyte sheets were constructed using a temperature-responsive culture surface. Then, they were placed onto cancellous bone to form chondrocyte sheet-cancellous bone tissues. The immunostaining of collagen type II (COL2) and the proteomic analysis of the human tissues were carried out before the transplantation. In our in vitro study, the triple-layered chondrocyte sheets adhered well on the cancellous bone, and the COL2 expression was apparent throughout the tissue structures. From the proteomic analysis results, it was found that the major function of the secreted proteins found in these tissues was protein binding. The distinct pathways were focal adhesion and the ECM-receptor interaction pathways. Among the highly expressed proteins, laminin-alpha 5 (LAMA5) and fibronectin (FN) not only played roles in the protein binding and ECM-receptor interaction, but also were involved in the cytokine-mediated signaling pathway. At 12 weeks after xenogeneic transplantation, compared to the control group, the defects treated with the chondrocyte sheets showed more hyaline-like cartilage tissue, as indicated by the abundance of safranin-O and COL2 with a partial collagen type I (COL1) expression. At 4, 8, and 12 weeks, compared to the defects treated with the cancellous bone, the staining of safranin-O and COL2 was more apparent in the defects treated with the chondrocyte sheet-cancellous bone tissues. Therefore, the human chondrocyte sheets and chondrocyte sheet-cancellous bone tissues provide a potential treatment for rabbit femoral condyle defect. LAMA5 and FN found in these human xenografts and their culture media might play key roles in the ECM-receptor interaction and might be involved in the cytokine-mediated signaling pathway during tissue repair.

Construction and Evaluation of Osteochondral-Like Tissue Using Chondrocyte Sheet and Cancellous Bone.

The manipulation of human chondrocyte sheets in target areas frequently results in their tearing because they are thin and fragile. In this study, human cancellous bones were used as a supporting material to create chondrocyte sheet-cancellous bone tissues, and their properties were evaluated. Using cell sheet technology, human chondrocytes were constructed into triple-layered chondrocyte sheets that displayed chondrogenic properties. After transferring the chondrocyte sheets onto cancellous bones, the top area of the chondrocyte sheet-cancellous bone tissues exhibited a smooth surface topography without cell sheet floating within 7 days of culture. The immunofluorescence staining of collagen type II (COL2A1) and fibronectin (FN1) was also performed and examined. Using the shotgun proteomic analysis, the proteins associated with cell adhesion, extracellular matrix (ECM) organization, cell-substrate junction assembly, and cell adhesion mediated by integrin were observed in the chondrocyte sheets, cancellous bones, and chondrocyte sheet-cancellous bone tissues. Three integrin members, including integrin ß4 (ITGB4), ITGB6, and ITGB8, were found in the chondrocyte sheets. Only ITGB8 was found in the chondrocyte sheets and chondrocyte sheet-cancellous bone tissues. During 48 h, the mean velocity of the individual cell migration was low, which did not affect the structure and chondrogenic properties of the chondrocyte sheets. Staining of the filamentous actin (F-actin) cytoskeleton in the migratory cells also provided a better understanding of the dynamic communication between the cell cytoskeleton and adhesion molecules through ITGB8, which may play a key role in the attachment of the chondrocyte sheets and the synthesis of the cartilage ECM. Therefore, we suggest that cancellous bone could be used as a supporting material to construct chondrocyte sheet-cancellous bone tissues for potential treatment of osteochondral lesions. Impact Statement We proposed a method to construct an osteochondral-like tissue by placing human chondrocyte sheets onto cancellous bone. The stationary chondrocyte sheets and the low mean velocity of the individual cell migration on the cancellous bone with the expression of COL2A1 indicated that the cancellous bone served as an appropriate supporting material. Moreover, the cellular mechanism for the adhesion of the chondrocyte sheets on the cancellous bone based on ITGB8-mediated adhesion through the rearrangement of filamentous actin provided a better understanding to improve the construction of osteochondral-like tissues, and to predict the repair mechanism in osteoarthritis therapy.

Inclusion of IR-820 into Soybean-Phosphatides-Based Nanoparticles for Near-Infrared-Triggered Release and Endolysosomal Escape in HaCaT Keratinocytes at Insignificant Cytotoxic Level.

PURPOSE: The degradation of drugs within endolysosomes has been widely addressed as a cause of poor bioavailability. One of the strategies to allow molecules to escape from a destructive fate is to introduce a photosensitizing moiety into a drug carrier enabling the permeabilization of endosomes and endolysosomes upon irradiation. This paper presents an alternative delivery nanosystem composed of cost-effective soybean phosphatides mixed with IR-820, a near-infrared (NIR) sensitizer, to load various active compounds and trigger an endolysosomal escape with a low cytotoxic effect. METHODS: IR-820-incorporated phosphatides-based nanoparticles were formulated using a thin-film hydration method to encapsulate different molecular probes and a drug model. The nanoparticles were characterized in vitro using dynamic light scattering, transmission electron microscopy, as well as ultraviolet-visible and fluorescence spectroscopy techniques. The NIR-corresponding generation of the photochemical products, the content release, and the cytotoxicity toward the HaCaT keratinocyte cell line were evaluated. The cellular internalization and endolysosomal escape were monitored using a cytochemical marker and fluorescent probes with a colocalization analysis. RESULTS: The IR-820-combined nanoparticles revealed the NIR-triggered changes in the singlet oxygen presence, nanoparticle architecture, and release rate without being cytotoxic. Additionally, the nanoplatform appeared to enhance cellular uptake of the macromolecules. The localization of the cytochemical marker and the colocalization analysis on the fluorescence signals of the encapsulated fluorophore and the lysosome-labeling reporter implied the transient endolysosomal escape of the cargo within the HaCaT cells after NIR irradiation. CONCLUSION: The inclusion of IR-820 into a soybean-phosphatides base ingredient provides NIR responsiveness, particularly the endolysosomal escape of the payload, to the formulated nanoparticles, while preserving the beneficial properties as a drug carrier. This alternative delivery nanomedicine system has future potential to provide high bioavailability of cytosolic drugs utilizing time- and spatial-controllable NIR triggerability as well as the synergistic therapeutic effects with NIR-biomodulation.

Preparation and characterization of human keratinocyte-fibroblast cell sheets constructed using PNIAM-co-AM grafted surfaces for burn wound healing.

Autologous skin grafting, the standard treatment for severe burns, is sometimes not possible due to the limited available skin surfaces for the procedure. With advances in tissue engineering, various cell-based skin substitutes have been developed to serve as skin replacements and to promote tissue regeneration and healing. In this work, we propose the use of cell sheet technology to fabricate keratinocyte-fibroblast tissue constructs from the temperature-responsive poly(N-isoproprylacrylamide-co-acrylamide) (PNIAM-co-AM) grafted surfaces for the treatment of burn wounds. The characteristics of the human keratinocyte and fibroblast cell sheets harvested using PNIAM-co-AM grafted surfaces were similar to those cell sheets detached from the commercially-available UpCellTM plates. Upon lowering the incubation temperature, confluent keratinocytes and fibroblasts could be detached as intact sheets, consisting of biologically active cells, as indicated by their high cell viability and their reattachment, migratory, and proliferative activities. A histological analysis of the stratified keratinocyte-fibroblast cell sheets revealed the evidence of cell migration and tissue reorganization to form two distinct epidermal and dermal layers, quite similar to the skin tissue's structure. In addition, the keratinocyte-fibroblast sheets could synthesize and release significant amounts of essential cytokines and growth factors involved in regulating the wound healing process, including IL-1α, IL-6, TNF-α, VEGF, and bFGF, implying the therapeutic effect of these cell sheets, which could be beneficial to accelerate tissue repair and regeneration, leading to faster wound healing.

Injectable Poly(ethylene glycol) Hydrogels Cross-Linked by Metal-Phenolic Complex and Albumin for Controlled Drug Release.

Injectable hydrogel is advantageous as a drug reservoir for controlled drug release since its injectability provides minimally invasive access to internal tissues and irregular-shaped target sites. Herein, we fabricated pH-responsive injectable hydrogels constructed of a supramolecular cross-link network, which contained tannic acid (TA), Fe(III), poly(ethylene glycol) (PEG), and bovine serum albumin (BSA) for controlled drug release. The hydrogel precursors rapidly turned into a gel when co-injected with NaOH in a time scale of seconds. The hydrogel properties and drug release profiles are all tunable by adjusting the concentrations of BSA, NaOH, and doxorubicin (DOX). The Young's moduli range from 3.19 ± 0.93 to 43.24 ± 1.37 kPa that match internal soft tissues. The hydrogel lasts more than 3 weeks and gradually releases doxorubicin up to 123.6 ± 1.7 µg at pH 6.4. The results of the physical properties and drug release suggest supramolecular interactions that correspond to Fourier transform infrared (FTIR) results. In vitro cytotoxicity was also assessed using L929 cells, and the results demonstrated the material biocompatibility. The tunable properties, controlled release profiles, and biocompatibility of injectable poly(ethylene glycol) hydrogels support that they have great potential as a drug-releasing material for localized treatments.

Validation of uniformity-optimized irradiance distribution on a well-plate platform from a light-emitting-diode array.

The computational optimization of irradiance distribution uniformity has been conducted in several studies to obtain the evenness of photoresponses on an irradiated surface using light-emitting-diode (LED) arrays. However, there has been little discussion on the precision of predictive simulations. This study aims to validate the simulated irradiance predicted by a mathematical model on the working area of a six-well plate and investigate the spatial consistency of the photobleaching of methylene blue and IR-820 photosensitizers on the bottom of the different wells illuminated by using the local-search-optimized LED configurations. The validation signified the negative deviation of both the measured irradiance and irradiance uniformity as compared to the simulated data. Despite the coefficients of variation observed as low as 1.9% and 7.4% for red-light and infrared irradiance, respectively, the photobleaching responses were found to be spatially diverse. The implications of this study are opportunities for further enhancements to the predictability of the simulations for the design of prospective illumination setups.

Development of anti-HER2-targeted doxorubicin-core-shell chitosan nanoparticles for the treatment of human breast cancer.

Purpose: Doxorubicin (DOX) encapsulated O-succinyl chitosan graft Pluronic® F127 (OCP) copolymer nanoparticles conjugated with an anti-HER2 monoclonal antibody were developed as targeted drug delivery vehicles for the treatment of HER2-overexpressing breast cancer. Methods: Five percent and 10% (w/w) of O-succinyl chitosan was grafted onto Pluronic® F127 using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as mediated cross-linking agents. DOX was added to the copolymer solution to form DOX-nanoparticles before conjugation with anti-HER2 on the surface of the nanoparticles. Results: DOX was encapsulated within the NP matrices at an encapsulation efficiency of 73.69 ± 0.53% to 74.65 ± 0.44% (the initial DOX concentration was 5 µg/mL). Anti-HER2 was successfully conjugated onto the surface of the nanoparticles at a moderately high conjugation efficiency of approximately 57.23 ± 0.38% to 61.20 ± 4.42%. In the in vitro DOX dissolution study, the nanoparticle formulations exhibited a biphasic drug release with an initial burst release followed by a sustained release profile at both pH 5.0 and pH 7.4. The drug was rapidly and completely released from the nanoparticles at pH 5.0. In the in vitro cytotoxicity, the anti-HER2 conjugated OCP copolymer nanoparticles showed the lowest IC50, which indicated an increase in the therapeutic efficacy of DOX to treat human breast cancer cells with the HER2 overexpression. Conclusion: Our study shows that anti-HER2 conjugated OCP copolymer nanoparticles have the potential for the development of anticancer drug carriers.

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

BACKGROUND: Dedifferentiation 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. RESULTS: During 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. CONCLUSIONS: The 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.

Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II and Stress Fiber Formation in Human Chondrocyte Sheets.

Cell sheet technology is applied to human articular chondrocytes to construct a tissue-like structure as an alternative treatment for cartilage defect. The effect of a gelatin manipulator, as a cell sheet transfer system, on the quality of the chondrocyte sheets was investigated. The changes of important chondrogenic markers and stress fibers, resulting from the cell sheet manipulation, were also studied. The chondrocyte cell sheets were constructed with patient-derived chondrocytes using a temperature-responsive polymer and a gelatin manipulator as a transfer carrier. The properties of the cell sheets, including sizes, expression levels of collagen type II and I, and the localization of the stress fibers, were assessed and compared with those of the cell sheets harvested without the gelatin manipulator. Using the gelatin manipulator, the original size of the chondrocyte cell sheets was retained with abundant stress fibers, but with a decrease in the expression of collagen type II. Without the gelatin manipulator, although the cell shrinkage occurred, the cell sheet with suppressed stress fiber formation showed significantly higher levels of collagen type II. These results support our observations that stress fiber formation in chondrocyte cell sheets affected the production of chondrogenic markers. These densely packed tissue-like structures possessed a good chondrogenic activity, indicating their potential for use in autologous chondrocyte implantation to treat cartilage defects.

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

OBJECTIVES: To 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. RESULTS: hMSCs 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. CONCLUSIONS: Incubation 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/cm(2) (irradiance intensity 2 mW/cm(2)). 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.

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 copolymer's 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.

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

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.

Controlled transdermal iontophoresis of sulfosalicylic acid from polypyrrole/poly(acrylic acid) hydrogel.

A conductive polymer-hydrogel blend between sulfosalicylic acid-doped polypyrrole (PPy) and poly(acrylic acid) (PAA) was used as a carrier/matrix for the transdermal drug delivery under applied electrical field. PAA films and the blend films were prepared by solution casting with ethylene glycol dimethacrylate (EGDMA) as a cross-linking agent, followed by the blending of PPy particles and the PAA matrix. The effects of cross-linking ratio and electric field strength on the diffusion of the drug from PAA and PPy/PAA hydrogels were investigated using a modified Franz-diffusion cell with an acetate buffer of pH 5.5 and at 37 degrees C, for a period of 48h. The diffusion coefficient of the drug is calculated using the Higuchi equation, with and without an electric field, at various cross-linking ratios. The drug diffusion coefficient decreases with increasing drug size/mesh size ratio, irrespective of the presence of the conductive polymer as the drug carrier. The diffusion coefficient, at the applied electric field of 1.0V, becomes larger by an order of magnitude relative to those without the electric field.