Jyh-Ping Chen

Surface modification of electrospun PLLA nanofibers by plasma treatment and cationized gelatin immobilization for cartilage tissue engineering.

Electrospun poly(lactic acid) (PLLA) nanofibers (NF) were modified with cationized gelatin (CG) to improve their compatibility with chondrocytes and to show in vitro and in vivo the potential applications of CG-grafted PLLA nanofibrous membranes (CG-PLLA NFM) as a cartilage tissue engineering scaffold. PLLA NF were first treated with oxygen plasma to introduce -COOH groups on the surface, followed by covalent grafting of CG molecules onto the fiber surface, using water-soluble carbodiimide as the coupling agent. The effects of CG grafting and properties of NFM were characterized by scanning electron microscopy (SEM), transmission electron microscopy, thermogravimetric analysis, atomic force microscope, X-ray photoelectron spectra and Fourier transform infrared spectroscopy. In vitro studies indicated that CG-PLLA NFM could enhance viability, proliferation and differentiation of rabbit articular chondrocytes compared with pristine PLLA NFM. SEM observations of the cell-scaffold construct confirmed the tight attachment of chondrocytes to CG-PLLA NF and in-growth of cells into the interior of the membrane with proper maintenance of cell morphology. Improved cell differentiation in CG-PLLA NFM was confirmed by enhanced glycoaminoglycan and collagen secretion, histological analysis and reverse transcription-polymerase chain reaction studies, which showed that the cells were able to maintain the expression of characteristic markers (collagen II, aggregan and SOX 9) of chondrocytes. Subcutaneous implantation of the cell-scaffold constructs with autologous chondrocytes also confirmed the formation of ectopic cartilage tissues after 28 days by histological examination and immunostaining.

Dual targeted delivery of doxorubicin to cancer cells using folate-conjugated magnetic multi-walled carbon nanotubes.

By combining the advantage of multi-walled carbon nanotubes (MWCNTs) and iron oxide magnetic nanoparticles (MNs), we develop a magnetic dual-targeted nanocarrier for drug delivery. MWCNTs were functionalized with poly(acrylic acid) through free radical polymerization, decorated with MNs, conjugated with a targeting ligand folic acid (FA), for loading of an anti-cancer drug doxorubicin (DOX). The proposed methodology provides dual targeted delivery of the anti-cancer drug to cancer cells under the guidance of a magnetic field and through ligand-receptor interactions. The chemico-physical properties of the nanocarrier were characterized, in addition to its drug loading efficiency and drug releasing characteristics. Doxorubicin could be loaded to MWCNTs with high efficiency via π-π stacking and hydrogen bonding and showed enhanced cytotoxicity toward U87 human glioblastoma cells compared with free DOX. From transmission electron microscopy and confocal laser scanning microscopy, we confirmed that DOX-FA-MN-MWCNT could be efficiently taken up by U87 cells with subsequent intracellular release of DOX, followed by transport of DOX into the nucleus with the nanocarrier left in the cytoplasm. These properties make the magnetic nanocarrier a potential candidate for targeted delivery of DOX for cancer treatment.

Magnetically targeted thrombolysis with recombinant tissue plasminogen activator bound to polyacrylic acid-coated nanoparticles.

We investigated the feasibility and efficacy of target thrombolysis with recombinant tissue plasminogen activator (rtPA) covalently bound to magnetic nanoparticle (MNP) and retained to the target site in vivo by an external magnet. Polyacrylic acid-coated magnetite (PAA-MNP, 246 nm) was synthesized and characterized; rtPA was immobilized to PAA-MNP through carbodiimide-mediated amide bond formation. The enzyme activities of the bound rtPA, as measured by a chromogenic substrate assay and (125)I-fibrinolysis assay, were 87+/-1% and 86+/-3% of that of free rtPA. Under guidance with the magnet moving back and forth along the iliac artery, the thrombolytic activity of PAA-MNP-rtPA with rtPA equivalent to 0.2mg/kg was determined by flowmetry in a rat embolic model. Intra-arterial administration of PAA-MNP-rtPA restored the iliac blood flow within 75 min to 82% of that before the clot lodging, whereas equivalent amount of PAA-MNP or free rtPA exerted no improvement on hemodynamics. At the end of 2-h period, PAA-MNP-rtPA did not alter levels of hemoglobin, hematocrit, or blood cell count. In conclusion, immobilization of rtPA to PAA-MNP with covalent binding resulted in a stable rtPA preparation and predictable amount of rtPA around the target site under magnetic guidance; this approach may achieve reproducible and effective target thrombolysis with <20% of a regular dose of rtPA.

Preparation and characterization of composite nanofibers of polycaprolactone and nanohydroxyapatite for osteogenic differentiation of mesenchymal stem cells

Nanocomposites of nanohydroxyapatite (nHAP) dispersed in poly(ɛ-caprolactone) (PCL) were prepared by electrospinning (ES) to obtain PCL/nHAP nanofibers. Nanofibers with similar diameters (340 ± 30 nm) but different nHAP concentrations (0-50%) were fabricated and studied for growth and osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). The nanofibrous membranes were subjected to detailed analysis for its physicochemical properties by scanning electron microscopy (SEM), thermogravimetric analysis, X-ray diffraction, Fourier-transform infrared spectroscopy, and mechanical tensile testing. nHAP particles (~30 nm diameter) embedded in nanofibers increased the nanofibrous membranes ultimate stress and the elastic modulus, while decreased the strain at failure. When cultured under an osteogenic stimulation condition on nanofibers, MSCs showed normal phenotypic cell morphology, and time-dependent mineralization and osteogenic differentiation from SEM observations and alkaline phosphatase activity assays. The nanofibers could support the growth of mesenchymal stem cells without compromising their osteogenic differentiation capability up to 21 days and the enhancement of cell differentiation by nHAP is positively correlated with its concentration in the nanofibers. Energy dispersive X-ray analysis of Ca and P elements indicated mineral deposits on the cell surface. The mineralization extent was significantly raised in nanofibers with 50% nHAP where a Ca/P ratio similar to that of bone was found. The present study indicated that electrospun composite PCL/nHAP nanofibrous membranes are suitable for mineralization of MSCs intended for bone tissue engineering.

A poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-2-hydroxyethyl methacrylate) composite hydrogel membrane for urease immobilization to enhance urea hydrolysis rate by temperature swing☆☆

A composite membrane made of cross-linked poly(N-isopropylacrylamide-co-N-acryloxysuccinimide-co-2-hydroxyethyl methacrylate) (p(NIPAAm-NAS-HEMA)) hydrogel on polyester nonwoven support has been synthesized. The composite membrane shows temperature-responsive properties similar to conventional PNIPAAm hydrogels beads, which reversibly swells below and de-swells above the lower critical solution temperature of PNIPAAm (around 32 to 33 degrees C). Diffusion of urea through the membrane was temperature-dependent with the effective diffusion coefficient at 20 degrees C being 18 times that at 60 degrees C. Urease was immobilized directly to the membrane by forming covalent bonds between its amino groups and the succinimide ester groups of the membrane. Membrane prepared with NIPAAm to NAS molar ratio of 9, and then reacted in pH 7 buffer with 6 mg of urease gave the best immobilized enzyme, where 0.102 mg protein and 5.71 U activity per cm(2) membrane, and 55% relative specific activity could be obtained. There was negligible internal mass transfer resistance for this preparation judging from the calculated effectiveness factor. Urease shows enhanced thermal stability after immobilization with the first-order inactivation rate constant at 70 degrees C decreased to 1/8 of that of free urease. Membrane-immobilized urease could be utilized in a two-compartment membrane reactor with temperature swing to substantially enhance urea hydrolysis rate. The best operating condition of the membrane reactor was with temperature cycling between 60 to 20 degrees C and with temperature change every 10 min, where concentration of product ammonia after 3 h reaction increased 3.8-folds when compared with isothermal operation at 60 degrees C.

Novel magnetic/ultrasound focusing system enhances nanoparticle drug delivery for glioma treatment

Malignant glioma is a common and severe primary brain tumor with a high recurrence rate and an extremely high mortality rate within 2 years of diagnosis, even when surgical, radiological, and chemotherapeutic interventions are applied. Intravenously administered drugs have limited use because of their adverse systemic effects and poor blood-brain barrier penetration. Here, we combine 2 methods to increase drug delivery to brain tumors. Focused ultrasound transiently permeabilizes the blood-brain barrier, increasing passive diffusion. Subsequent application of an external magnetic field then actively enhances localization of a chemotherapeutic agent immobilized on a novel magnetic nanoparticle. Combining these techniques significantly improved the delivery of 1,3-bis(2-chloroethyl)-1-nitrosourea to rodent gliomas. Furthermore, the physicochemical properties of the nanoparticles allowed their delivery to be monitored by magnetic resonance imaging (MRI). The resulting suppression of tumor progression without damaging the normal regions of the brain was verified by MRI and histological examination. This noninvasive, reversible technique promises to provide a more effective and tolerable means of tumor treatment, with lower therapeutic doses and concurrent clinical monitoring.

Composite chitosan/silk fibroin nanofibers for modulation of osteogenic differentiation and proliferation of human mesenchymal stem cells

Nanofibrous membrane scaffolds of chitosan (CS), silk fibroin (SF) and CS/SF blend were prepared by electrospinning and studied for growth and osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). The morphology and physico-chemical properties of all membrane scaffolds were compared. The influence of CS and SF on cell proliferation was assessed by the MTS assay, whereas osteogenic differentiation was determined from the Alizarin Red staining, alkaline phosphatase activity and expression of osteogenic marker genes. The osteogenic differentiation and proliferation of hMSCs were enhanced by CS and SF nanofibers, respectively. Blending CS with SF retained the osteogenesis nature of CS without negatively influencing the cell proliferative effect of SF. By taking advantage of the differentiation/proliferation cues from individual components, the electrospun CS/SF composite nanofibrous membrane scaffold is suitable for bone tissue engineering.

Latex Particles with Thermo-Flocculation and Magnetic Properties for Immobilization of α-Chymotrypsin

Core‐shell‐type latex particles composed of styrene, N‐isopropylacrylamide (NIPAAm), and N‐acryloxysuccinimide (NAS) were synthesized by surfactant‐free emulsion polymerization. The latex particles show thermo‐flocculation behavior due to the presence of temperature‐sensitive monomer NIPAAm and could be used for immobilization of α‐chymotrypsin through covalent bonding with the reactive ester groups of NAS. Enzyme recycle could be accomplished in this immobilized enzyme system by sedimentation of the thermo‐flocculated latex particles in 20 min at 30 °C by raising the salt (NaCl) concentration to 0.5 M. To further enhance the sedimentation rate, ultrafine magnetite particles were prepared and included during polymerization to produce magnetic temperature‐sensitive latex particles (MTLP), which could be recovered 6 times faster after thermo‐flocculation by applying a low magnetic field. However, a higher salt concentration was necessary to flocculate the MTLP under the same condition as a result of its increased surface hydrophilicity, which originates from different polymerization conditions and the incorporation of magnetite. The immobilized enzyme shows high activity even against macromolecular substrates (hemoglobin and casein) owing to limited diffusion resistance, with full activity retention for nonmagnetic latex but one‐half reduction in activity if the magnetic property was introduced. Optimal enzyme immobilization pH and enzyme loading were determined, and properties of the immobilized enzyme were characterized. The immobilized enzyme was used in 10 repeated batch hydrolyses of casein with successive flocculation/dispersion cycles and showed less than 15% activity decrease at the end. Overall, introducing the magnetic property to the latex could effectively enhance the solid‐liquid separation rate after thermo‐flocculation and maintain enzyme activity after repeated use but adversely influence the activity of the immobilized enzyme.

Preparation and characterization of gelatin/hyaluronic acid cryogels for adipose tissue engineering: in vitro and in vivo studies.

Macroporous elastic scaffolds containing gelatin (4% or 10%) and 0.25% hyaluronic acid (HA) were fabricated by cryogelation for application in adipose tissue engineering. These cryogels have interconnected pores (∼200 μm), high porosity (>90%) and a high degree of cross-linking (>99%). The higher gelatin concentration reduced the pore size, porosity and swelling ratio of the cryogel but improved its swelling kinetics. Compressive mechanical testing of cryogel samples demonstrated non-linear stress-strain behavior and hysteresis loops during loading-unloading cycles, but total recovery from large strains. The presence of more gelatin increased the elastic modulus, toughness and storage modulus and yielded a cryogel that was highly elastic, with a loss tangent equal to 0.03. Porcine adipose-derived stem cells (ADSCs) were seeded in the cryogel scaffolds to assess their proliferation and differentiation. In vitro studies demonstrated a good proliferation rate and the adipogenic differentiation of the ADSCs in the cryogel scaffolds, as shown by their morphological change from a fibroblast-like shape to a spherical shape, decreased actin cytoskeleton content, growth arrest, secretion of the adipogenesis marker protein leptin, Oil Red O staining for triglycerides and expression of early (LPL and PPARγ) and late (aP2 and leptin) adipogenic marker genes. In vivo studies of ADSCs/cryogel constructs implanted in nude mice and pigs demonstrated adipose tissue and new capillary formation, the expression of PPARγ, leptin and CD31 in immunostained explants, and the continued expression of adipocyte-specific genes. Both the in vitro and in vivo studies indicated that the gelatin/HA cryogel provided a structural and chemical environment that enabled cell attachment and proliferation and supported the biological functions and adipogenesis of the ADSCs.

Preparation and characterization of biomimetic silk fibroin/chitosan composite nanofibers by electrospinning for osteoblasts culture.

In this study, we have successfully fabricated electrospun bead-free silk fibroin [SF]/chitosan [CS] composite nanofibers [NFs] covering the whole range of CS content (0%, 25%, 50%, 75%, and 100%). SF/CS spinning solutions were prepared in a mixed solvent system of trifluoroacetic acid [TFA] and dichloromethane. The morphology of the NFs was observed by scanning electron microscope, and the average fiber diameter ranges from 215 to 478 nm. Confocal laser scanning microscopy confirms the uniform distribution of SF and CS within the composite NFs. To increase biocompatibility and preserve nanostructure when seeded with cells in culture medium, NFs were treated with an ethanol/ammonia aqueous solution to remove residual TFA and to change SF protein conformation. After the chemical treatment, SF/CS NFs could maintain the original structure for up to 54 days in culture medium. Properties of pristine and chemically treated SF/CS NFs were investigated by Fourier transform infrared spectroscopy [FT-IR], X-ray diffraction [XRD], and thermogravimetry/differential scanning calorimetry [TG/DSC]. Shift of absorption peaks in FT-IR spectra confirms the conformation change of SF from random coil to β-sheet by the action of ethanol, which is also consistent with the SF crystalline diffraction patterns measured by XRD. From TG/DSC analysis, the decomposition temperature peaks due to salt formation from TFA and protonated amines disappeared after chemical treatment, indicating complete removal of TFA by binding with ammonium ions during the treatment. This was also confirmed with the disappearance of F1s peak in X-ray photoelectron spectroscopy spectra and disappearance of TFA salt peaks in FT-IR spectra. The composite NFs could support the growth and osteogenic differentiation of human fetal osteoblastic [hFOB] cells, but each component in the composite NF shows distinct effect on cell behavior. SF promotes hFOB proliferation while CS enhances hFOB differentiation. The composite SF/CS NFs will be suitable for bone tissue engineering applications by choosing a suitable blend composition.PACS: 87.85.jf; 87.85.Rs; 68.37.Hk.