NSC/PC, indicating a faster differentiation process. In particular, after 6 days of
differentiation in the 3D scaffold, NSC/PC from the postnatal brain had generated up
to 70% neurons, compared with 14% in a two-dimensional (2D) culture. The reason
for the relatively high percentage of apoptosis is most likely dead cells being trapped
in the 3D scaffold, compared with 2D culture, in which dead cells are detached from
the surface and washed out during medium exchange. Another contributing factor
could be a difficulty with exchange of gases and nutrients into the core of the matrix,
compared with the corresponding 2D culture, which is only one cell layer thick.
However, NSC/PC from other ages gave rise to approximately the same proportion
of neurons in 3D as in 2D (9–26% depending on the source for NSC/PC). In the
postnatal NSC/PC cultures, the majority of βIII-tubulin-positive cells expressed
glutamate, g-aminobutyric acid, and synapsin I after 11 days of differentiation,
indicating differentiation to mature neurons in the biocompatible hydrogel (Fig. 1).
Bozkurt and co-workers compared in vitro a cross-linked porcine collagen
scaffold with a fibrin hydrogel-based system for the ability to support neurite
outgrowth in the rat dorsal root ganglion (DRG) [20]. It is important to highlight
that although DRG studies deal with the peripheral nervous system (PNS), they can
provide valuable data for regeneration of the relevant adult neurons. The nerve guide
described in this work showed a high degree of porosity and, more importantly, a
remarkable degree of orientation, with channel sizes between 20 and 50 μm. Such
collagen guidance channels were manufactured using a series of chemical and
mechanical treatments with a patented unidirectional freezing process (Fig. 2).
Hemisected rat DRGs were positioned such that neural and non-neural elements
could migrate into the collagen scaffold (Fig. 3). After 21 days, S100-positive
Schwann cells (SCs) migrated into the scaffold and aligned within the guidance
channels in a columnar fashion, resembling “Bands of Büngner.” Overall, the
microstructural properties of collagen scaffold and the in vitro data after DRG
loading make this scaffold a good candidate to be considered for promoting oriented
nerve fiber regeneration in the PNS.
Around the same time, a study on DRG carried out by Blewitt and co-workers
[21] revealed that hydrogels with low collagen concentrations (0.4–1.0 mg/mL)
helped to achieve the longest neurite extension. Hydrogels with higher collagen
concentration and thus higher stability required modifications with adhesive recognition peptides to exhibit similar properties. The aim of this study was to investigate
the effects of inhibitory molecules on nerve growth in 3D environments as compared
to 2D surfaces. Thus, soluble peptide sequences were used as competitive inhibitors
of neurite extension in collagen gels. In order to determine the effect of collagen gel
properties on neurite extension, dissociated DRG cells were seeded into an array of
collagen gel concentrations. E9 chick dorsal root ganglion cells were seeded within
collagen gels as well as onto collagen-coated glass and were exposed, for 24 h, to
one of three experimental peptide sequences, namely, arginine-glycine-aspartic
acid-threonine (RGDT), cyclo(RGD-D-Phe-Val) (cRGD), or aspartic acid-glycineglutamic acid-alanine (DGEA) (Fig. 4). In 3D collagen gels, only the cRGD peptide
sequence reduced neurite extension across a variety of gel concentrations. In contrast, on 2D surfaces, both RGD peptides reduced the number of cells expressing
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R. de la Cruz and D. D. Díaz
differentiation in the 3D scaffold, NSC/PC from the postnatal brain had generated up
to 70% neurons, compared with 14% in a two-dimensional (2D) culture. The reason
for the relatively high percentage of apoptosis is most likely dead cells being trapped
in the 3D scaffold, compared with 2D culture, in which dead cells are detached from
the surface and washed out during medium exchange. Another contributing factor
could be a difficulty with exchange of gases and nutrients into the core of the matrix,
compared with the corresponding 2D culture, which is only one cell layer thick.
However, NSC/PC from other ages gave rise to approximately the same proportion
of neurons in 3D as in 2D (9–26% depending on the source for NSC/PC). In the
postnatal NSC/PC cultures, the majority of βIII-tubulin-positive cells expressed
glutamate, g-aminobutyric acid, and synapsin I after 11 days of differentiation,
indicating differentiation to mature neurons in the biocompatible hydrogel (Fig. 1).
Bozkurt and co-workers compared in vitro a cross-linked porcine collagen
scaffold with a fibrin hydrogel-based system for the ability to support neurite
outgrowth in the rat dorsal root ganglion (DRG) [20]. It is important to highlight
that although DRG studies deal with the peripheral nervous system (PNS), they can
provide valuable data for regeneration of the relevant adult neurons. The nerve guide
described in this work showed a high degree of porosity and, more importantly, a
remarkable degree of orientation, with channel sizes between 20 and 50 μm. Such
collagen guidance channels were manufactured using a series of chemical and
mechanical treatments with a patented unidirectional freezing process (Fig. 2).
Hemisected rat DRGs were positioned such that neural and non-neural elements
could migrate into the collagen scaffold (Fig. 3). After 21 days, S100-positive
Schwann cells (SCs) migrated into the scaffold and aligned within the guidance
channels in a columnar fashion, resembling “Bands of Büngner.” Overall, the
microstructural properties of collagen scaffold and the in vitro data after DRG
loading make this scaffold a good candidate to be considered for promoting oriented
nerve fiber regeneration in the PNS.
Around the same time, a study on DRG carried out by Blewitt and co-workers
[21] revealed that hydrogels with low collagen concentrations (0.4–1.0 mg/mL)
helped to achieve the longest neurite extension. Hydrogels with higher collagen
concentration and thus higher stability required modifications with adhesive recognition peptides to exhibit similar properties. The aim of this study was to investigate
the effects of inhibitory molecules on nerve growth in 3D environments as compared
to 2D surfaces. Thus, soluble peptide sequences were used as competitive inhibitors
of neurite extension in collagen gels. In order to determine the effect of collagen gel
properties on neurite extension, dissociated DRG cells were seeded into an array of
collagen gel concentrations. E9 chick dorsal root ganglion cells were seeded within
collagen gels as well as onto collagen-coated glass and were exposed, for 24 h, to
one of three experimental peptide sequences, namely, arginine-glycine-aspartic
acid-threonine (RGDT), cyclo(RGD-D-Phe-Val) (cRGD), or aspartic acid-glycineglutamic acid-alanine (DGEA) (Fig. 4). In 3D collagen gels, only the cRGD peptide
sequence reduced neurite extension across a variety of gel concentrations. In contrast, on 2D surfaces, both RGD peptides reduced the number of cells expressing
360
R. de la Cruz and D. D. Díaz