Nanofibers with good alignment has been successfully developed from copolymer
P(LLA/CL, 75:25) and seeded with smooth muscle cells (SMC).

Proton Beam Writing is a new lithography developed in the Centre for Ion Beam Applications (CIBA) Dept of Physics. In Proton Beam Writing, a focused beam of million electron volt (MeV) protons is written into a suitable resist (e.g. PMMA or SU-8) in a direct write process. Proton beam writing is used to produce high aspect ratio 3D microstructures with sub-100 nm feature sizes. The depth of the microstructures can be controlled by adjusting the energy of the protons very accurately between 5 and 160 mm. Complicated 3D microstructures can be machined and have arbitrary shapes with 90o sidewalls. The proton beam has uniform energy deposition along its path and practically no proximity effects (unanted exposure due to secondary electrons). SU-8 & PMMA direct written microstructures can be successfully converted to metallic Ni components using electroplating with the same sidewall quality as the original polymer structures.

Fig. 1 Schematics showing AFM oxidation of OTS SAM: (a) before and (b) after oxidation.

                                    3(a)                                        3(b)
                    

3(c)                                     3(d)

Fig. 3 (a) and (b): AFM height images of protruded oxide patterns fabricated on OTS surface at negative probe bias. (c) and (d): AFM height and deflection images of depressed patterns generated on OTS surface at positive probe bias. (e) and (f): Schematics showing the oxide growth and the scratch of OTS molecules at negative and positive probe bias, respectively.

(a)                                     (b)

AFM pictures of Fe particles (a) before and (b) after ion beam modification

CNTs grown on modified (Region M) and unmodified (Region U) at low temperature

Fig. 1 Transmission electron micrograph of drug loaded nanoparticles

Fig. 2  Release of cis-platin from Au-Au2S nanoparticles as a function of NIR irradiation time and simple heating

The primary goal of this project is to conduct a systematic study to understand, predict and control the effects of nano-scale changes in the synthetic ECM on cell morphology and functions. Hence, this research will directly test the underlying hypothesis that the interplay between cells and nano-scale synthetic ECM is critical to the morphological and functional development of cells. Although it is well characterized that gross changes in ECM impact on cell behavior, little is know on how cell morphology and functions would be affected by fine changes (at the nanometer scale) in the synthetic ECM. An in-depth understanding of the molecular effects of synthetic nanofiber ECM on cell behavior will aid in designing superior biomaterials for tissue engineering with specific applications. 

Synthetic polymeric nanofibrous scaffolds prepared by electrospinning and phase separation techniques are designed and modified to mimic the native ECM, and used for elucidating the molecular and biochemical interactions of the following cells when cultured on these synthetic polymeric nanofibrous ECM scaffolds: human coronary artery smooth muscle cells (HCASMCs) and endothelial cells (HCAECs), the two major cell types of blood vessels; human dermal fibroblasts (HDFs) isolated from the skin; and the mouse neural cell line c17.2. 

The following issues are being addressed to identify molecular interactions of cell and synthetic ECM:

1. cell morphology – as an initial screen for ECM properties that would enhance cell spreading in the synthetic matrix.

2. gene expression and function – examining tissue-specific gene expression using cDNA microarray.

3. cell signaling – monitoring dynamic protein kinase activities by fluorescence resonance energy transfer (FRET).

4. characterization of cell surface heparan sulfate-like glycosaminoglycans (HSGAGs) which play an important role in regulating cell proliferation and migration.

The biodegradable synthetic polymer poly (L-lactic acid) (PLLA), poly (e-caprolactone) (PCL), and co-polymer PLLA/PCL were used for this study because of biocompatibility, thermal and biochemical stability, reproducibility, controlled porosity and pore size, and generally non-toxic.

For morphological study, it has been found that the metabolic fluorescent dye CMFDA is able to stain all cells homogenously even for long term culture and not the nanofibers (Fig. 1).

HCASMCs and HCAECs Interactions with Synthetic Nanofibrous ECM

Vascular smooth muscle cells form the middle layer (tunica media) of the blood vessels and are circumferentially arranged to provide the mechanical and elastic properties of the blood vessels. We have demonstrated that human coronary artery smooth muscle cells (HCASMCs) cultured on synthetic co-polymer PLLA/PCL nanofibrous scaffolds showed the normal morphology and good proliferation, and the cells organized along the aligned nanofibers in a directional manner typified by the orientation of the cytoskeletal protein a-actin suggesting that nanofiber orientation can impart a functional development on the cells   (Fig. 2). 

Electrospun synthetic polymer PLLA scaffolds with nano-scale dimensions which mimic the native ECM environment were found to improve the morphology, adhesion and proliferation of HCASMCs over submicron/micron-dimension fibers (Fig. 3).

Endothelial cells form the inner wall (tunica intima) of blood vessels during biogenesis and serve to prevent thrombosis and leakage. In our study, we found that human coronary artery endothelial cells (HCAECs) do not adhere and proliferate very well on unmodified nanofibers taking on a more rounded morphology. However, on collagen-modified nanofibers, HCAECs take on cobbled-stone morphology, typical of endothelial cells cultured on tissue culture polystyrene surface (TCPS) (Fig. 4), with comparable adhesion and proliferation rates. 

             
Fig. 1 Laser scanning confocal micrographs of CMFDA-stained endothelial cells. For initial screening of synthetic ECM properties that would enhance cell spreading in the synthetic matrix, the fluorescent metabolic dye 5-chloromethylfluorescein diacetate (CMFDA) which is able to stain cells homogenously and for relatively long term culture of up to 8 days was used. CMFDA was found to be non-toxic at 15 mM final concentration and did not affect cell adhesion and proliferation as determined by MTS assays. The lipid dye, FM-143, which stains cell membrane, was found to stain the polymer nanofibers. Green fluorescent protein (EGFP) was found to be cytotoxic following liposome transfection of EGFP plasmid in HCASMCs and HCAECs, which are primary cells.
 

Fig. 2 Laser scanning confocal micrographs of immunostained a-actin filaments in HCASMCs cultured on co-polymer PLLA/PCL (a). aligned nanofibrous scaffold, and (b). aligned nanofibrous scaffold overlaid image of the aligned fibers. The cells organized along the aligned nanofibers following the orientation of the nanofibers, and exhibiting spiral morphology very similar to vascular SMCs in their native environment. The orientation of the cytoskeletal protein a-actin in the direction of the aligned nanofibers suggests that nanofiber orientation can influence the functional development of the cells.


 

  

  

Fig. 3 Laser scanning confocal micrographs of immunostained a-actin filaments in HCASMCs cultured on PLLA (a). nanofibers, (b). sub-micron fibers, (c). micron fibers, and (d). tissue culture polystyrene surface as control. HCASMCs proliferated better on nanofibers than on sub-micron and micron fibers with improved morphology, higher adhesion level and proliferation rate, which were comparable to cells cultured on tissue culture polystyrene surface.

Synthetic Nanofibrous PLLA Scaffold Interactions with Neural Cell Line c17.2

C17.2 is an undifferentiated multipotent neural cell line derived from mouse cerebellar progenitor cells by retrovirus-mediated v-myc gene transfer, and can differentiate to replace neurons undergoing targeted apoptotic degeneration in adult mouse neocortex.

We have demonstrated that PLLA nanofibrous scaffolds prepared by phase separation and electrospinning are able to support neural cell c17.2 growth and neurites extension (Fig. 5).

On aligned nanofibers, the neural cells c17.2 adhere and elongate along the fibers and neurites extend along the direction of the aligned fibers (Fig. 6).

HDFs Interactions with Collagen Nanofibers and Collagen-Modified PCL Nanofibers

Fibroblasts are one of the major cell types found in the dermis of skin and are responsible for the synthesis of collagen and elastic fibers, and glycosaminoglycans such as dermatan sulfate, chondroitin sulfate and hyaluronate, into the dermis which serves to support the epidermis and hypodermis of the skin. 

Human dermal fibroblasts (HDFs) have been demonstrated to grow better on collagen nanofibrous scaffold (Fig. 7) than tissue culture polystyrene surface with good potentials for clinical applications.

Fig. 5 Scanning electron micrographs of the neural cells, c17.2, cultured on PLLA (5% weight/volume in tetrahydrofuran) nanofibrous scaffold prepared by phase separation. The nanofibrous scaffold was able to support neural cell growth and on day 2 of culture in differentiation-conducive medium, neurite growth was observed. On the third day of culture, the neurite extended more than twice the length of the cell body.

Fig. 6 Laser scanning confocal micrograph of immunostained neural filament 200 kD (NF200) in the neural cells, c17.2, cultured on aligned PLLA nanofibers. The cells adhered to the nanofibers in an orientation parallel to the nanofiber alignment and exhibited bi-polar shape with two extended neurites which emerged from the regions of the cell body parallel to the nanofiber alignment.

Abstract
Although organic semiconductors have received the most attention, the development of compatible passive elements, such as interconnects and electrodes, is also central to plastic electronics. For this, ligand-protected metal-cluster films have been shown to anneal at low temperatures below 250 ^°C to highly conductive metal films, but they suffer from cracking and inadequate substrate adhesion. Here, we report printable metal-cluster–polymer nanocomposites that anneal to a controlled-percolation nanostructure without complete sintering of the metal clusters. This overcomes the previous challenges while still retaining the desired low transformation temperatures. Highly water- and alcohol-soluble gold clusters (75 mg ml^-1) were synthesized and homogeneously dispersed into poly(3,4-ethylenedioxythiophene) to give a material with annealed d.c. conductivity tuneable between 10^-4 and 10⁵ S cm^-1. These composites can inject holes efficiently into all-printed polymer organic transistors. The insulator–metal transformation can also be electrically induced at 1 MV cm^-1, suggesting possible memory applications.

We have fabricated well-ordered organic donor/acceptor nanojunction arrays comprising p-sexiphenyl (6P) and C₆₀ via self-assembly of C₆₀ on the molecular nanotemplate of 6P nanostripes on Ag(111). This paper has made the cover of Applied Physices Letters, a Tier 1, physics journal on 12 May 2008 issue.