D.O. Meredith

3D polymer scaffolds for tissue engineering

This review discusses some of the most common polymer scaffold fabrication techniques used for tissue engineering applications. Although the field of scaffold fabrication is now well established and advancing at a fast rate, more progress remains to be made, especially in engineering small diameter blood vessels and providing scaffolds that can support deep tissue structures. With this in mind, we introduce two new lithographic methods that we expect to go some way to addressing this problem.

ENHANCEMENT OF IMMUNOGOLD-LABELLED FOCAL ADHESION SITES IN FIBROBLASTS CULTURED ON METAL SUBSTRATES: PROBLEMS AND SOLUTIONS

Visualisation of cell adhesion patterns by scanning electron microscopy requires special preparation and labelling. The membranes and cytoplasm must be removed, without damaging the antigen, to facilitate antibody access to vinculin in the focal adhesions. Low beam energy imaging is used to visualise the cell undersurface (embedded in resin after staining with osmium tetroxide) and immunogold‐labelled adhesion sites. The gold probe, must be large enough (>40nm) for detection, while viewing the whole cell, but large gold markers increase steric hindrance and decrease labelling efficiency. This problem can be overcome by using small gold probes (1–5nm) followed by enlargement with silver enhancement, but osmium tetroxide stain etches the silver. We demonstrated that metal substrates increased this etching. Reducing the concentration of osmium tetroxide and incubation time reduced the amount of etching. We have demonstrated that gold enhancement was not etched by osmium tetroxide, irrespective of the substrate. Therefore, comparative studies of cell adhesion to different biomaterial substrates can be performed using immunogold‐labelling with gold enhancement.

Simultaneously identifying S-phase labelled cells and immunogold-labelling of vinculin in focal adhesions.

A new combination of autoradiography and immunolabelling techniques is presented that allows the simultaneous identification of both S‐phase cells and their focal adhesions using scanning electron microscopy. The technique allows both labels to be discerned visually by their unique shapes and location within and on the cell. S‐phase cells were radio‐labelled with a pulse of tritiated thymidine, selectively incorporated into synthesizing DNA. The cells were then immunogold‐labelled for the focal adhesion protein, vinculin, prepared for autoradiography, and embedded in resin. The resin was then polymerized before removing the substrate, to expose the embedded cell undersurface. Electron‐energy ‘sectioning’ of the sample by varying the accelerating voltage of the electron beam allowed separate S‐phase cell identification in one electron‐energy ‘section’ and visualization of immunogold label in another ‘section’, within the same cell. As a result of applying this technique it was possible to positively identify S‐phase cells and immunogold‐labelled focal adhesions on the same cell simultaneously, which could be used to quantify focal adhesion sites on different substrates.

Steps toward a model nanotopography

A natural lithography technique is employed to create an irregular, submonolayer colloidal topography. Epitenon cells were cultured on these colloidal surfaces, and cell morphology investigations using scanning electron microscropy were conducted. Preliminary experiments brought into question the stability of the colloidal nanotopography, and it was unsure if the surface was presented to cells as a static structure. Investigations using secondary electron and backscattered electron imaging, and also X-ray microanalysis, indicated that the colloidal structure was in fact stable, and cells were capable of direct interactions at the peripheral membrane with the colloids.

A Hierarchical Response of Cells to Perpendicular Micro- and Nanometric Textural Cues

In this paper, we report on the influence of shallow micro- and nanopatterned substrata on the attachment and behavior of a human fibroblast [human telomerase transfected immortalized (hTERT)] cells. We identify a hierarchy of textural guidance cues with respect to cell alignment on these substrates. Cells were seeded and cultured for 48 h on silicon substrates patterned with two linear textures overlaid at 90°, both with 24 ¿m pitch: a micrograting and a nanopattern of rows of 140- nm-diameter pits arranged in a rectangular array with 300 nm centre-to-centre spacing. The cell response to these textures was shown to be highly dependent on textural feature dimensions. We show that cells align to the stripes of nanopits. Stripes of 30-nm deep nanopits were also shown to elicit a stronger response from cells than 160-nm deep nanopits.

A Hybrid Three-Dimensional Nanofabrication Method for Producing Vascular Tissue Engineering Scaffold

There is a trend towards the production of lithographically defined materials for biological applications. The field of nanobio technology is rapidly growing and so is demand for materials nanostructured in three dimensions. Here we present a hybrid approach where we use electron beam lithography, photolithography and hot embossing to produce membranes patterned on both sides. The double sided patterned membranes were subsequently rolled to create the three dimensional tissue construct aimed at vascular repair. The mechanical properties such as bending and bursting pressure were also investigated.

Correlating cell morphology and osteoid mineralization relative to strain profile for bone tissue engineering applications

A number of bone tissue engineering strategies use porous three-dimensional scaffolds in combination with bioreactor regimes. The ability to understand cell behaviour relative to strain profile will allow for the effects of mechanical conditioning in bone tissue engineering to be realized and optimized. We have designed a model system to investigate the effects of strain profile on bone cell behaviour. This simplified model has been designed with a view to providing insight into the types of strain distribution occurring across a single pore of a scaffold subjected to perfusion–compression conditioning. Local strains were calculated at the surface of the pore model using finite-element analysis. Scanning electron microscopy was used in secondary electron mode to identify cell morphology within the pore relative to local strains, while backscattered electron detection in combination with X-ray microanalysis was used to identify calcium deposition. Morphology was altered according to the level of strain experienced by bone cells, where cells subjected to compressive strains (up to 0.61%) appeared extremely rounded while those experiencing zero and tensile strain (up to 0.81%) were well spread. Osteoid mineralization was similarly shown to be dose dependent with respect to substrate strain within the pore model, with the highest level of calcium deposition identified in the intermediate zones of tension/compression.

CORRELATING CELL MORPHOLOGY AND OSTEOID MINERALIZATION RELATIVE TO STRAIN PROFILE FOR BONE TISSUE ENGINEERING APPLICATIONS

A number of bone tissue engineering strategies use porous three-dimensional scaffolds in combination with bioreactor regimes. The ability to understand cell behaviour relative to strain profile will allow for the effects of mechanical conditioning in bone tissue engineering to be realized and optimized. We have designed a model system to investigate the effects of strain profile on bone cell behaviour. This simplified model has been designed with a view to providing insight into the types of strain distribution occurring across a single pore of a scaffold subjected to perfusion–compression conditioning. Local strains were calculated at the surface of the pore model using finite-element analysis. Scanning electron microscopy was used in secondary electron mode to identify cell morphology within the pore relative to local strains, while backscattered electron detection in combination with X-ray microanalysis was used to identify calcium deposition. Morphology was altered according to the level of strain experienced by bone cells, where cells subjected to compressive strains (up to 0.61%) appeared extremely rounded while those experiencing zero and tensile strain (up to 0.81%) were well spread. Osteoid mineralization was similarly shown to be dose dependent with respect to substrate strain within the pore model, with the highest level of calcium deposition identified in the intermediate zones of tension/compression.