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Glycine-spacers influence functional motifs exposure and self-assembling propensity of functionalized substrates tailored for neural stem cell cultures.

Taraballi F, Natalello A, Campione M, Villa O, Doglia SM, Paleari A, Gelain F - Front Neuroeng (2010)

Bottom Line: The understanding of phenomena involved in the self-assembling of bio-inspired biomaterials acting as three-dimensional scaffolds for regenerative medicine applications is a necessary step to develop effective therapies in neural tissue engineering.Accordingly, the longer spacer of glycines, the more effective is the functional motif in both eliciting NSCs adhesion, improving their viability and increasing their differentiation.Therefore, optimized designing strategies of functionalized biomaterials may open, in the near future, new therapies in tissue engineering and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Center for Nanomedicine and Tissue Engineering, A.O. Ospedale Niguarda Ca' Granda Milan, Italy.

ABSTRACT
The understanding of phenomena involved in the self-assembling of bio-inspired biomaterials acting as three-dimensional scaffolds for regenerative medicine applications is a necessary step to develop effective therapies in neural tissue engineering. We investigated the self-assembled nanostructures of functionalized peptides featuring four, two or no glycine-spacers between the self-assembly sequence RADA16-I and the functional biological motif PFSSTKT. The effectiveness of their biological functionalization was assessed via in vitro experiments with neural stem cells (NSCs) and their molecular assembly was elucidated via atomic force microscopy, Raman and Fourier Transform Infrared spectroscopy. We demonstrated that glycine-spacers play a crucial role in the scaffold stability and in the exposure of the functional motifs. In particular, a glycine-spacer of four residues leads to a more stable nanostructure and to an improved exposure of the functional motif. Accordingly, the longer spacer of glycines, the more effective is the functional motif in both eliciting NSCs adhesion, improving their viability and increasing their differentiation. Therefore, optimized designing strategies of functionalized biomaterials may open, in the near future, new therapies in tissue engineering and regenerative medicine.

No MeSH data available.


Related in: MedlinePlus

Phase contrast images of differentiating neural stem cells (7 days in vitro) over the tested scaffolds. (A) 0G-BMHP1, (B) 2G-BMHP1, (C) 4G-BMHP1 SAPeptides. (D) Positive and (E) negative controls. 4G-BMHP1 coaxes NSCs to differentiate and survive most effectively while, in case of 0G-BMHP1, small clusters of poorly adhered NSCs testify a possible poor availability of the BMHP1 functional motif for cell membrane receptor binding and consequently cell differentiation pathways activation. Additionally, the “sinking” of NSCs within the assembled scaffold of 0G-BMHP1 (A) testifies a possible lower mechanical stiffness insufficient to bear the weight of NSCs seeded over the top surface of the substrate. Scale bars are 100 μm. CellTiter results (F) show significant differences for all possible coupled experimental groups (P < 0.05) except for (*) 0G-BMHP1 vs negative control, and (**) 2G-BMHP1 vs 4G-BMHP1. Values are reported as means ± standard error of the mean.
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Figure 6: Phase contrast images of differentiating neural stem cells (7 days in vitro) over the tested scaffolds. (A) 0G-BMHP1, (B) 2G-BMHP1, (C) 4G-BMHP1 SAPeptides. (D) Positive and (E) negative controls. 4G-BMHP1 coaxes NSCs to differentiate and survive most effectively while, in case of 0G-BMHP1, small clusters of poorly adhered NSCs testify a possible poor availability of the BMHP1 functional motif for cell membrane receptor binding and consequently cell differentiation pathways activation. Additionally, the “sinking” of NSCs within the assembled scaffold of 0G-BMHP1 (A) testifies a possible lower mechanical stiffness insufficient to bear the weight of NSCs seeded over the top surface of the substrate. Scale bars are 100 μm. CellTiter results (F) show significant differences for all possible coupled experimental groups (P < 0.05) except for (*) 0G-BMHP1 vs negative control, and (**) 2G-BMHP1 vs 4G-BMHP1. Values are reported as means ± standard error of the mean.

Mentions: We adopted our well-established in vitro protocol (Gelain et al., 2007a) to evaluate differences, if any, occurred in NSC adhesion and proliferation depending on the FP substrate used. Negative control (untreated wells) (Figure 6E) and positive controls (Cultrex) (Figure 6D) were used. At day 0 the absorbance value was 0.21 ± 0.05 (n = 5). One week after seeding NSC proliferated and adhered as expected with 2G-BMHP1 (Figure 6B), less intensively in case of 0G-BMHP1 (Figure 6A), i.e. with isolated clusters of poorly branched cells, and remarkably more for 4G-BMHP1 (Figure 6C) where branched and differentiating cells formed an almost confluent monolayer. Quantification of NSC proliferation and survival confirmed these observations by showing significant differences between FPs with longer spacers and negative controls (Figure 6F). On the other hand the absence of any glycine-spacer between the self-assembling sequence and the functional motifs seems to impair the bioactivity of the latter (no significant difference of absorbance values between 0G-BMHP1 and negative control).


Glycine-spacers influence functional motifs exposure and self-assembling propensity of functionalized substrates tailored for neural stem cell cultures.

Taraballi F, Natalello A, Campione M, Villa O, Doglia SM, Paleari A, Gelain F - Front Neuroeng (2010)

Phase contrast images of differentiating neural stem cells (7 days in vitro) over the tested scaffolds. (A) 0G-BMHP1, (B) 2G-BMHP1, (C) 4G-BMHP1 SAPeptides. (D) Positive and (E) negative controls. 4G-BMHP1 coaxes NSCs to differentiate and survive most effectively while, in case of 0G-BMHP1, small clusters of poorly adhered NSCs testify a possible poor availability of the BMHP1 functional motif for cell membrane receptor binding and consequently cell differentiation pathways activation. Additionally, the “sinking” of NSCs within the assembled scaffold of 0G-BMHP1 (A) testifies a possible lower mechanical stiffness insufficient to bear the weight of NSCs seeded over the top surface of the substrate. Scale bars are 100 μm. CellTiter results (F) show significant differences for all possible coupled experimental groups (P < 0.05) except for (*) 0G-BMHP1 vs negative control, and (**) 2G-BMHP1 vs 4G-BMHP1. Values are reported as means ± standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2821182&req=5

Figure 6: Phase contrast images of differentiating neural stem cells (7 days in vitro) over the tested scaffolds. (A) 0G-BMHP1, (B) 2G-BMHP1, (C) 4G-BMHP1 SAPeptides. (D) Positive and (E) negative controls. 4G-BMHP1 coaxes NSCs to differentiate and survive most effectively while, in case of 0G-BMHP1, small clusters of poorly adhered NSCs testify a possible poor availability of the BMHP1 functional motif for cell membrane receptor binding and consequently cell differentiation pathways activation. Additionally, the “sinking” of NSCs within the assembled scaffold of 0G-BMHP1 (A) testifies a possible lower mechanical stiffness insufficient to bear the weight of NSCs seeded over the top surface of the substrate. Scale bars are 100 μm. CellTiter results (F) show significant differences for all possible coupled experimental groups (P < 0.05) except for (*) 0G-BMHP1 vs negative control, and (**) 2G-BMHP1 vs 4G-BMHP1. Values are reported as means ± standard error of the mean.
Mentions: We adopted our well-established in vitro protocol (Gelain et al., 2007a) to evaluate differences, if any, occurred in NSC adhesion and proliferation depending on the FP substrate used. Negative control (untreated wells) (Figure 6E) and positive controls (Cultrex) (Figure 6D) were used. At day 0 the absorbance value was 0.21 ± 0.05 (n = 5). One week after seeding NSC proliferated and adhered as expected with 2G-BMHP1 (Figure 6B), less intensively in case of 0G-BMHP1 (Figure 6A), i.e. with isolated clusters of poorly branched cells, and remarkably more for 4G-BMHP1 (Figure 6C) where branched and differentiating cells formed an almost confluent monolayer. Quantification of NSC proliferation and survival confirmed these observations by showing significant differences between FPs with longer spacers and negative controls (Figure 6F). On the other hand the absence of any glycine-spacer between the self-assembling sequence and the functional motifs seems to impair the bioactivity of the latter (no significant difference of absorbance values between 0G-BMHP1 and negative control).

Bottom Line: The understanding of phenomena involved in the self-assembling of bio-inspired biomaterials acting as three-dimensional scaffolds for regenerative medicine applications is a necessary step to develop effective therapies in neural tissue engineering.Accordingly, the longer spacer of glycines, the more effective is the functional motif in both eliciting NSCs adhesion, improving their viability and increasing their differentiation.Therefore, optimized designing strategies of functionalized biomaterials may open, in the near future, new therapies in tissue engineering and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Center for Nanomedicine and Tissue Engineering, A.O. Ospedale Niguarda Ca' Granda Milan, Italy.

ABSTRACT
The understanding of phenomena involved in the self-assembling of bio-inspired biomaterials acting as three-dimensional scaffolds for regenerative medicine applications is a necessary step to develop effective therapies in neural tissue engineering. We investigated the self-assembled nanostructures of functionalized peptides featuring four, two or no glycine-spacers between the self-assembly sequence RADA16-I and the functional biological motif PFSSTKT. The effectiveness of their biological functionalization was assessed via in vitro experiments with neural stem cells (NSCs) and their molecular assembly was elucidated via atomic force microscopy, Raman and Fourier Transform Infrared spectroscopy. We demonstrated that glycine-spacers play a crucial role in the scaffold stability and in the exposure of the functional motifs. In particular, a glycine-spacer of four residues leads to a more stable nanostructure and to an improved exposure of the functional motif. Accordingly, the longer spacer of glycines, the more effective is the functional motif in both eliciting NSCs adhesion, improving their viability and increasing their differentiation. Therefore, optimized designing strategies of functionalized biomaterials may open, in the near future, new therapies in tissue engineering and regenerative medicine.

No MeSH data available.


Related in: MedlinePlus