Shan-hui Hsu

Cytotoxicity and Immunological Response of Gold and Silver Nanoparticles of Different Sizes

The immunological response of macrophages to physically produced pure Au and Ag nanoparticles (NPs) (in three different sizes) is investigated in vitro. The treatment of either type of NP at > or =10 ppm dramatically decreases the population and increases the size of the macrophages. Both NPs enter the cells but only AuNPs (especially those with smaller diamter) up-regulate the expressions of proinflammatory genes interlukin-1 (IL-1), interlukin-6 (IL-6), and tumor necrosis factor (TNF-alpha). Transmission electron microscopy images show that AuNPs and AgNPs are both trapped in vesicles in the cytoplasma, but only AuNPs are organized into a circular pattern. It is speculated that part of the negatively charged AuNPs might adsorb serum protein and enter cells via the more complicated endocytotic pathway, which results in higher cytotoxicity and immunological response of AuNPs as compared to AgNPS.

Transplantation of bone marrow stromal cells for peripheral nerve repair

Cell transplantation using bone marrow stromal cells (BMSCs) to alleviate neurological deficits has recently become the focus of research in regenerative medicine. Evidence suggests that secretion of various growth-promoting substances likely plays an important role in functional recovery against neurological diseases. In an attempt to identify a possible mechanism underlying the regenerative potential of BMSCs, this study investigated the production and possible contribution of neurotrophic factors by transected sciatic nerve defect in a rat model with a 15 mm gap. Cultured BMSCs became morphologically homogeneous with fibroblast-like shape after ex vivo expansion. We provided several pieces of evidence for the beneficial effects of implanted fibroblast-like BMSCs on sciatic nerve regeneration. When compared to silicone tube control animals, this treatment led to (i) improved walking behavior as measured by footprint analysis, (ii) reduced loss of gastrocnemius muscle weight and EMG magnitude, and (iii) greater number of regenerating axons within the tube. Cultured fibroblast-like BMSCs constitutively expressed trophic factors and supporting substances, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), collagen, fibronectin, and laminin. The progression of the regenerative process after BMSC implantation was accompanied by elevated expression of neurotrophic factors at both early and later phases. These results taken together, in addition to documented Schwann cell-like differentiation, provide evidence indicating the strong association of neurotrophic factor production and the regenerative potential of implanted BMSCs.

Bacterial cellulose and bacterial cellulose-chitosan membranes for wound dressing applications.

Bacterial cellulose (BC) and bacterial cellulose-chitosan (BC-Ch) membranes were successfully produced in large scale. BC was synthesized by Acetobacter xylinum. BC-Ch was prepared by immersing BC in chitosan followed by freeze-drying. The surface morphology of BC and BC-Ch membranes were examined by a scanning electron microscope (SEM). SEM images showed that BC-Ch possessed a denser fibril network with smaller pores than BC. Infrared spectroscopy was used to confirm the incorporation of chitosan in BC-Ch. The swelling behavior, water retention capacity, and mechanical properties of BC and BC-Ch were further evaluated. Results indicated that both membranes maintained proper moisture contents for an extensive period without dehydration. The tensile strength and elongation at break for BC-Ch were slightly lower while the Youngs modulus was higher. Cell culture studies demonstrated that BC and BC-Ch had no cytotoxicity. In the antibacterial test, the addition of chitosan in BC showed significant growth inhibition against Escherichia coli and Staphylococcus aureus. The effects of BC and BC-Ch on skin wound healing were assessed by rat models. Histological examinations revealed that wounds treated with BC-Ch epithelialized and regenerated faster than those treated with BC or Tegaderm. Therefore, BC-Ch was considered as a potential candidate for wound dressing materials.

Preparation of controlled release ophthalmic drops, for glaucoma therapy using thermosensitive poly-N-isopropylacrylamide.

In this study, controlled release ophthalmic agents for glaucoma therapy were developed based on the thermosensitivity of poly-N-isopropylacrylamide (PNIPAAm). The clear solution of PNIPAAm was known to undergo phase transition when the temperature was raised from the room temperature to about 32 degrees C. The drug was entrapped in the tangled polymer chains or encapsulated within the crosslinked polymer hydrogel at room temperature, and released progressively after topical application (i.e., at a higher temperature). Linear PNIPAAm and crosslinked PNIAAm nanoparticles containing epinephrine were prepared. The drug release rate and cytotoxicity were investigated in vitro. Ophthalmic formulations based on either linear PNIPAAm or the mixture of linear PNIPAAm and crosslinked PNIPAAm nanoparticles were administered to rabbits and the intraocular pressure (IOP)-lowering effect was evaluated. The decreased pressure response of the formulation based on linear PNIPAAm lasted six-fold longer than that of the conventional eye drop. Furthermore, for formulation based on the mixture of linear PNIPAAm and crosslinked nanoparticles, the pressure-lowering effect lasted eight times longer. These results suggest the use of thermosensitive polymer solutions or hydrogels is potential in controlled release antiglaucoma ophthalmic drugs.

An Injectable, Self‐Healing Hydrogel to Repair the Central Nervous System

An injectable, self-healing hydrogel (≈1.5 kPa) is developed for healing nerve-system deficits. Neurosphere-like progenitors proliferate in the hydrogel and differentiate into neuron-like cells. In the zebrafish injury model, the central nervous system function is partially rescued by injection of the hydrogel and significantly rescued by injection of the neurosphere-laden hydrogel. The self-healing hydrogel may thus potentially repair the central nervous system.

Spheroid formation of mesenchymal stem cells on chitosan and chitosan-hyaluronan membranes

Stem cells can lose their primitive properties during in vitro culture. The culture substrate may affect the behavior of stem cells as a result of cell-substrate interaction. The maintenance of self-renewal for adult human mesenchymal stem cells (MSCs) by a biomaterial substrate, however, has not been reported in literature. In this study, MSCs isolated from human adipose (hADAS) and placenta (hPDMC) were cultured on chitosan membranes and those further modified by hyaluronan (chitosan-HA). It was observed that the MSCs of either origin formed three-dimensional spheroids that kept attached on the membranes. Spheroid formation was associated with the increased MMP-2 expression. Cells on chitosan-HA formed spheroids more quickly and the size of spheroids were larger than on chitosan alone. The expression of stemness marker genes (Oct4, Sox2, and Nanog) for MSCs on the materials was analyzed by the real-time RT-PCR. It was found that formation of spheroids on chitosan and chitosan-HA membranes helped to maintain the expression of stemness marker genes of MSCs compared to culturing cells on polystyrene dish. The maintenance of stemness marker gene expression was especially remarkable in hPDMC spheroids (vs. hADAS spheroids). Blocking CD44 by antibodies prevented the spheroid formation and decreased the stemness gene expression moderately; while treatment by Y-27632 compound inhibited the spheroid formation and significantly decreased the stemness gene expression. Upon chondrogenic induction, the MSC spheroids showed higher levels of Sox9, aggrecan, and collagen type II gene expression and were stained positive for glycosaminoglycan and collagen type II. hPDMC had better chondrogenic differentiation potential than hADAS upon induction. Our study suggested that the formation of adhered spheroids on chitosan and chitosan-HA membranes may sustain the expression of stemness marker genes of MSCs and increase their chondrogenic differentiation capacity. The Rho/Rho-associated kinase (ROCK) signaling pathway may be involved in spheroid formation.

Improved cell adhesion by plasma-induced grafting of L-lactide onto polyurethane surface.

Lactide-grafted polyurethanes were prepared by exposing the polyurethane films to argon plasma discharge, followed by grafting L-lactide onto the plasma-treated surface. The modified surfaces were characterized by measuring the static contact angle and by electron spectroscopy for chemical analysis (ESCA). The water contact angle of polyurethanes was decreased by L-lactide grafting, indicating hydrophilicity of the modified surface. Grafting also increased the O/C atomic ratio and C(C=O)/Ctotal percentage on the surfaces as detected by ESCA. The grafted surfaces showed enhanced attachment and growth in both 3T3 fibroblast and human umbilical vein endothelial cell culture tests. Platelet adhesion to the modified surfaces was also reduced in vitro. L-Lactide monomers grafted onto polyurethane substrates could therefore be useful in facilitating endothelial cell seeding process in small vascular graft applications.

Antibacterial properties of silver nanoparticles in three different sizes and their nanocomposites with a new waterborne polyurethane

Silver nanoparticles (AgNPs) are strong bactericidal agents but they are also cytotoxic. Embedding them in a polymer matrix may reduce their cytotoxic effect. In the present study, AgNPs in three average sizes were tested for their antibacterial activities and cytotoxicity. Nanocomposites from a new waterborne polyetherurethane (PEU) ionomer and AgNPs were prepared without the use of any crosslinker. It was observed that the antibacterial activity of AgNPs against Escherichia coli started at the effective concentration of 0.1-1 ppm, while that against Staphylococcus aureus started at higher concentrations of 1-10 ppm. Cytotoxicity of AgNPs was observed at the concentration of 10 ppm. AgNPs with smaller average size showed greater antibacterial activity as well as cytotoxicity. The PEU synthesized in this study showed high tensile strength, and the addition of AgNPs at all sizes further increased its thermal stability. The delicate surface features of nanophases, however, were only observed in nanocomposites with either small-or medium-sized AgNPs. PEU-Ag nanocomposites had a strong bacteriostatic effect on the growth of E. coli and S. aureus. The proliferation of endothelial cells on PEU-Ag nanocomposites was enhanced, whereas the platelet adhesion was reduced. The expression of endothelial nitric oxide synthase gene was upregulated on PEU-Ag containing small-sized AgNPs (30 ppm) or medium-sized AgNPs (60 ppm). This effect was not as remarkable in nanocomposites from large-sized AgNPs. Overall, nanocomposites from the PEU and 60 ppm of the medium-sized (5 nm) AgNPs showed the best biocompatibility and antibacterial activity. Addition of smaller or larger AgNPs did not produce as substantial an effect in PEU, especially for the larger AgNPs.

Low-energy laser irradiation increases endothelial cell proliferation, migration, and eNOS gene expression possibly via PI3K signal pathway

The purpose of this study, therefore, was to determine the mechanisms by which low‐energy laser irradiation (LELI) may exert some of its angiogenic effects via the PI3 kinase/eNOS signaling pathway and induce endothelial cell migration and neovascularization, an important and necessary part of wound healing.

Sciatic Nerve Repair by Microgrooved Nerve Conduits Made of Chitosan-Gold Nanocomposites

BACKGROUND To better direct the repair of peripheral nerve after injury, an implant consisting of a multicomponent micropatterned conduit seeded with NSC was designed. METHODS The mechanical properties of the chi-Au nanocomposites were tested. In vitro, the effect of chi-Au on cell behavior (NSC and glial cell line C6) and the influence of micropattern on cell alignment were evaluated. In vivo, the micropatterned conduits with/without the preseeded NSC were implanted to bridge a 10-mm-long defect of the sciatic nerve in 9 male Sprague-Dawley rats. The repair outcome was investigated 6 weeks after the surgery. RESULTS Based on the dynamic modulus, chitosan with 50 ppm or more gold was a stronger material than others. In vitro, gold at 25 or 50 ppm led to better cell performance for NSC; and gold at 50 ppm gave better cell performance for C6. On the microgrooved substrate, the NSC had elongated processes oriented parallel to the grooves, whereas the NSC on the nonpatterned surfaces did not exhibit a particular bias in alignment. In vivo, the number of regenerated axons, the regenerated area, and the number of blood vessels were significantly higher in the NSC-preseeded conduit. CONCLUSION Modification of the chitosan matrix by gold nanoparticles not only provides the mechanical strength but also affects the cellular response. The preliminary in vivo data demonstrated that the biodegradable micropatterned conduits preseeded with NSC provided a combination of physical and biological guidance cues for regenerating axons at the cellular level and offered a better alternative for repairing sciatic nerve transactions.