The effects of proteoglycan surface patterning on neuronal pathfinding

Abstract

Protein micropatterning techniques are increasingly applied in cell choice assays to investigate fundamental biological phenomena that contribute to the host response to implanted biomaterials, and to explore the effects of protein stability and biological activity on cell behavior for in vitro cell studies. In the area of neuronal regeneration the protein micropatterning and cell choice assays are used to improve our understanding of the mechanisms directing nervous system during development and regenerative failure in the central nervous system (CNS) wound healing environment. In these cell assays, protein micropatterns need to be characterized for protein stability, bioactivity, and spatial distribution and then correlated with observed mammalian cell behavior using appropriate model system for CNS development and repair. This review provides the background on protein micropatterning for cell choice assays and describes some novel patterns that were developed to interrogate neuronal adaptation to inhibitory signals encountered in CNS injuries.

Figure 1

Microfabrication steps for soft lithography: 1 - Exposure of 1^st mask with pattern generator, 2 - Development and etch of pattern, 3 - 5x Image reduction of 1^st pattern, 4 - Development and etch, 5 - UV exposure through 2^nd mask onto SU-8 spin coated on silicon wafer, 6 - SU-8 development, 7 - PDMS curing step, 8 -Removal of PDMS stamp, or alternatively 5a - PDMS curing step, and 6a - Removal of PDMS stamp.

Figure 2

Protein deposition by μCP stamping on a bare substratum (panel A), on a substratum already covered with an adsorbed protein layer (panel B), and by a combination of the μCP and μFN (panel C).

Figure 3

Stamped 80 μm stripes of FITC-labeled human serum albumin separated by 40 μm-wide gaps with a Alexafluor488 dye-laminin and albumin adsorbed from two protein solution mixture. The image is the combination of brightfield (DRGs) and fluorescence images. From ref. .

Figure 4

The method of protein deposition determines how DRGs react to the combination of permissive (laminin) and inhibitory (aggrecan) signals. The upper panel shows DRGs on the pattern consisting of μCP stamped aggrecan and μFN deposited laminin (scheme C from Fig 2) and the lower panel shows the DRG on the pattern made of μCP stamped aggrecan on the uniform adsorbed laminin (scheme B in Fig 2). The image is the combination of brightfield (DRGs) and fluorescence images. Aggrecan was stained with AlexaFluor-594 dye. From ref. .

Figure 5

Fluorescence images of μCP stamped aggrecan patterns on uniform laminin adsorbed layer. The aggrecan island coverages were 2 % (left), 7 % (middle) and 31 % (right) coverage. Aggrecan was stained with the AlexaFluor-488 dye. Scale bar: 10 μm. From ref. .

Figure 6

Combined phase contrast and fluorescence time-lapse microscopy images showing that a narrow growth cone of a DRG chooses an outgrowth pathway predominately between aggrecan islands (brighter dots) after the filopodia sample the area inbetween aggrecan islands. Substratum: uniform laminin field covered by μCP stamped aggrecan micrometer-sized islands (11% average aggrecan surface coverage). Scale bar −10 μm, time in minutes as indicated. [120]