To produce an engineered tissue as a treatment for SCI, delivery conditions and appropriate growth factor dosing for promoting ESNPC differentiation into neurons and oligodendrocytes were determined as both of these cells types could potentially contribute to functional recovery after SCI. Neurons could help to restore the circuitry disrupted after injury, allowing for reinnervation (Lee et al., 2007
). Oligodendrocytes could restore the myelination of the spared axons present at and around the injury site and such oligodendrocyte replacement strategies have been shown to promote functional recovery in animal models of SCI (Keirstead et al., 2005
; Liu et al., 2000
; Nistor et al., 2005
). Additionally, minimizing astrocytes production was important as they could contribute to the glial scar and inhibit regeneration (Fawcett and Asher, 1999
Release studies determined the appropriate delivery system conditions need to retain the maximum amount of growth factor using the affinity-based delivery system. The release studies were performed in the absence of cells to quantify the amount of growth factor passively released over time. In the in vitro culture system and in vivo, the remaining growth factor is actively released from the scaffolds when cells activate plasmin, which enzymatically degrades the scaffold. In the in vitro culture system, the growth factors can act upon the ESNPCs seeded inside of the scaffolds and in vivo, the growth factors can be released by both the ESNPCs and the surviving cells that remained after the injury.
Two different heparin concentrations were tested in the context of the delivery system and the 4:1 ratio resulted in maximum growth factor retention for all growth factors tested. These results suggest that the increase in heparin concentration provided more binding sites to retain the growth factors inside of the scaffold. For the 4:1 peptide to heparin ratio, there is ~1300–1700 times excess of binding sites (heparin molecules) per growth factor while the 40:1 ratio only yields ~130–170 times excess of binding sites ().
shows the estimated fraction of growth factor bound at equilibrium calculated from the dissociation constants for each growth factor. The release rates of the growth factors correlate with the estimated bound fraction at equilibrium. The affinity-based delivery system was predicted to retain more PDGF and NT-3 than Shh at equilibrium. This trend was observed in the release studies with the 4:1 ratio, which was confirmed by the 14 day release studies.
For NT-3 and PDGF, the delivery system retained different amounts of growth factor depending on the peptide to heparin ratio. The pattern of growth factor release was consistent with previously published release studies of NT-3 and PDGF-BB, a different PDGF isoform (Taylor et al., 2004
; Thomopoulos et al., 2007
). Only the 4:1 ratio retained Shh inside of the scaffolds. Shh had never been tested previously with the affinity-based delivery system, but it has been reported to have a high affinity for heparin (Rubin et al., 2002
; Zhang et al., 2007
). The quick release of Shh could potentially be due to repulsive interactions between Shh and the scaffold itself. .
An initial burst of growth factor release was observed over the first twenty four hours. In an in vivo setting, this unbound growth factor would be washed away from the scaffold by cerebrospinal fluid present at the injury site. To account for this effect, all scaffolds were washed five times before cell seeding and the addition of the second scaffold layer. Different effects on ESNPC differentiation are observed when the same growth factor dose is polymerized inside of the scaffolds in the presence of delivery system compared to when no delivery system is present.
It is also important to consider the effects of the delivery system on the differentiation of ESNPCs as bioactive peptides have been previously used to promote differentiation of neural progenitors seeded on nanofibers (Silva et al., 2004
). The presence of peptide and heparin, both individually and in combination, reduced the fraction of ESNPCs that differentiated into astrocytes. The reduction in the fraction of cells that differentiated into astrocytes was observed for all of the delivery system groups except when it was used to delivery 10 ng total of PDGF, suggesting that the presence of growth factor does not interfere with this beneficial effect. The presence of heparin alone decreased the fraction of neural progenitors present, but this decrease was not observed when both peptide and heparin were present, suggesting that the peptide may help maintain nestin expression. When looking at the effects of the growth factors on ESNPC differentiation, it is important to view the results in the context of these control experiments. For other tissue engineering applications, it will be important to run similar control experiments to ensure these components do not interfere with the desired stem differentiation.
For each of the growth factors tested, a range of concentrations was selected by combining the results of the release studies and previous studies that looked at the effects of growth factors in the culture media on ESNPC differentiation inside of fibrin scaffolds (Willerth et al., 2007b
). Controlled delivery of 125 ng dose (417 ng/mL) from the affinity-based delivery system produced neuron and oligodendrocyte differentiation comparable to 25 ng/mL of NT-3 in the media. Soluble NT-3 has also been shown to promote the differentiation of human ES cells into neurons in three dimensional settings, suggesting these scaffolds might also be useful for human ES cell lines (Levenberg et al., 2005
). NT-3 also promotes the survival of oligodendrocyte precursors (Kumar et al., 1998
), which could potentially explain the increase in O4 staining observed with controlled delivery of the optimal dose (100 ng NT-3) with delivery system. . Controlled delivery of NT-3 also produced an increase in the percentage of cells staining positive for nestin, which could potentially be due to the delivery system, as this effect was not observed in the previous studies with NT-3 in the media.
From our previous work, the controlled release dose of PDGF (20 ng total) corresponds to between 2 ng/mL and 10 ng/mL of soluble PDGF and replicates the benefits of both doses, which included an increase in the fraction of cells differentiating into oligodendrocytes and a decrease in the fraction of cells differentiating into astrocytes. These results are consistent with other studies that have demonstrated PDGF’s role in promoting oligodendrocyte development (Calver et al., 1998
Controlled delivery of 100 ng of Shh produced similar ESNPC differentiation to both 10 and 25 ng/mL of Shh in the media. The increase in neuronal differentiation was expected as many studies have demonstrated that Shh combined with retinoic acid promotes the differentiation into ES cells into motor neurons (Dutton et al., 1999
; Lee et al., 2007
; Li et al., 2005
; Miles et al., 2004
; Soundararajan et al., 2007
; Soundararajan et al., 2006
). Shh also plays important roles in promoting oligodendrocyte differentiation (Danesin et al., 2006
; Oh et al., 2005
). The poor retention of Shh by the affinity-based delivery system required polymerizing high doses of Shh into the scaffolds to promote differentiation, and more effective retention of Shh by an affinity-based delivery system might be obtained by using different scaffold material.
Controlled delivery of all three growth factor combinations produced significant changes compared to both unmodified fibrin scaffolds with no growth factors present in the media and scaffolds containing the same growth factors combinations with no delivery system. The main difference between the three combinations tested was in the fraction of cells differentiating into neurons. The combination of NT-3 and PDGF produced the largest fraction of cells differentiating into neurons compared to all the groups tested, making it the optimal combination for further testing in the context of an in vivo model of SCI. This result was also confirmed using real time RT-PCR analysis with increases in the expression levels of Map2 and PDGFαR and a decrease in vimentin expression levels compared to the cultures in unmodified fibrin scaffolds with no growth factors present.
These results are consisted with other literature that has shown different situations where combining NT-3 with PDGF produces additive effects. Previous studies have shown that this combination promotes survival of Schwann cell precursors and remyelination in rodent models of multiple sclerosis, suggesting that their signaling pathways may share an intermediate that produces these additional benefits (Fressinaud, 2005
; Lobsiger et al., 2000
). Also, the inclusion of PDGF in combination with other growth factors produced an increase in cell viability. This increase in viability may increase the survival rate of the transplanted cells in vivo
This study has produced an engineered neural tissue consisting of a fibrin scaffold containing an affinity-based delivery system, growth factors and ESNPCs ready for in vivo testing as a potential treatment for SCI. The growth factor delivery system and concentration were optimized to promote the differentiation of the cells seeded within these scaffolds into neurons and oligodendrocytes and to increase cell viability. The concentrations found in this study for promoting the differentiation of ESNPCs into these cell phenotypes can be easily translated to other materials with known release properties for in vivo SCI applications. Such a strategy can be extended to generate replacements for other types of damaged tissues. Finally, this affinity-based delivery system could also be used to study signaling events during development. Its ability to mimic the extracellular matrix and present growth factors in a controlled manner allows for the reproduction of signals present during development in an in vitro setting. Thus, this delivery system allows for generation of tissue engineered scaffolds as well as providing an in vitro system for studying the development biology of stem cells.