The differential influence of colocalized and segregated dual protein signals on neurite outgrowth on surfaces

Abstract

We present an in vitro micropatterning approach in which the density and spatial presentation of two separate protein layers can be independently controlled to form cell stripe assays through (1) the simultaneous application of microcontact printing (μCP) and microfluidic network patterning (μFN) to generate alternating stripes of pure single protein layers or (2) through μCP onto a pre-adsorbed homogeneous protein layer to generate alternating single and dual protein stripes. This approach enabled the creation of choice boundaries in which protein-protein interactions were limited and the effects of spatially segregated or colocalized dual protein signals on model primary neuronal behavior could be readily interrogated and compared on both glass and tissue culture polystyrene substrates. Dorsal root ganglion (DRG) cell body attachment was dictated largely by non-specific cell adhesion interactions and interactions between the guidance molecules laminin and aggrecan were insufficient to explain aggrecan inhibition on neurite outgrowth. The presentation of a specific laminin epitope stabilized by interactions with aggrecan and destabilized by μCP was a strong predictor of neurite promoting activity. These observations provide evidence that aggrecan is intrinsically inhibitory and that laminin-aggrecan interactions do not diminish laminin growth promoting properties.

The effects of proteoglycan surface patterning on neuronal pathfinding

“…In addition, it appears that little information is available to accurately predict the effect of laminin deposition through soft lithography techniques on its growth promoting activity. In an effort to address these shortfalls, we have developed a set of cell choice stripe assays in order to separate CSPG-laminin interactions from possible intrinsic effects that CSPGs may exert on neuronal cell attachment and neurite outgrowth [120,125]. Additionally, antibody attachment to a laminin epitope known to promote neuronal cell attachment and neurite outgrowth was used to relate the method of laminin deposition to resulting bioactivity [120,125].…”

“…It was found that the activity of substrate bound laminin on glass differed whether it was adsorbed from a solution, using microfluidic networks patterning, or transferred by microcontact printing, and that laminin conformation, as measured by integrin presentation, was a strong predictor of neuronal cell adhesion and neurite outgrowth behavior [120,125]. It was additionally observed that the order in which laminin and the CSPG aggrecan were adsorbed onto glass clearly affected laminin activity, suggesting interactions between CSPGs and laminin.…”

“…To improve the understanding of the competing signals between growth promoting molecules such as laminin and inhibitory proteoglycans we and other labs have developed in vitro assays to test how the chondroitin sulfate containing proteoglycan signals presented in a mixed CSPG-laminin surface-attached layer are affecting neuronal cell adhesion and growth cone pathfinding [120][121][122][123][124][125]. It is known that neurons possess the ability to integrate multiple signals in their environment [120,[125][126][127][128], and it has been demonstrated that the ratio of substratum bound laminin and CSPGs such as aggrecan can be varied to yield net neurite outgrowth promoting or inhibiting signals [120,[129][130][131]. It has also been found that aggrecan affects laminin activity on neuron cell body substratum attachment depending on the order in which the two molecules are adsorbed, providing evidence that the effect of aggrecan is not simply additive [132].…”

“…In an effort to address these shortfalls, we have developed a set of cell choice stripe assays in order to separate CSPG-laminin interactions from possible intrinsic effects that CSPGs may exert on neuronal cell attachment and neurite outgrowth [120,125]. Additionally, antibody attachment to a laminin epitope known to promote neuronal cell attachment and neurite outgrowth was used to relate the method of laminin deposition to resulting bioactivity [120,125].It was found that the activity of substrate bound laminin on glass differed whether it was adsorbed from a solution, using microfluidic networks patterning, or transferred by microcontact printing, and that laminin conformation, as measured by integrin presentation, was a strong predictor of neuronal cell adhesion and neurite outgrowth behavior [120,125]. It was additionally observed that the order in which laminin and the CSPG aggrecan were adsorbed onto glass clearly affected laminin activity, suggesting interactions between CSPGs and laminin.…”

Optimization of protein patterns for neuronal cell culture applications

“…14,36,37 This study is part of a larger project aimed at understanding neuronal pathfinding dynamics on patterned biomaterials displaying two or more ECM proteins to modulate axonal growth. Allowing the axons to choose between more permissive and less permissive substrates may be a way to control the nerve's transition across an unfavorable boundary (e.g., the glial scar), which may be encountered by a regenerating neuron after a spinal cord injury (SCI).…”