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Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2.

Tamplenizza M, Lenardi C, Maffioli E, Nonnis S, Negri A, Forti S, Sogne E, De Astis S, Matteoli M, Schulte C, Milani P, Tedeschi G - J Nanobiotechnology (2013)

Bottom Line: Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF.We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIMAINA and Dipartimento di Fisica, Università degli Studi di Milano, via Celoria 16, Milano 20133, Italy. paolo.milani@mi.infn.it.

ABSTRACT

Background: Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified.

Results: To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.

Conclusion: Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

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AFM images and photoemission spectra. (A) AFM images of reference and nanostructured samples. Reference samples: a) coverslip (1 x 1 μm2), rms roughness is 0.343 ± 0.004 nm; b) Poly-L-Lysine coated coverslip (1 x 1 μm2), rms roughness 0.271 ± 0.020 nm; c) flat TiO2 (2 x 1 μm2), rms roughness of 0.229 ± 0.004 nm. The vertical color scales range between 0 and 10 nm. Nanostructured samples: d) and e) high resolution SEM images and f) and g) AFM topographies (2 x 1 μm2) of 50 nm and 200 nm thick nanostructured TiO2, respectively. The roughness of the thinner film has been evaluated 20.2 ± 0.5 nm, whereas of the thicker film 29.1 ± 1 nm. The ns-TiO2 films clearly appear to have a fine raster of nanometer-sized grains with porosity at the subnanometer scale and with the thicker film showing larger height fluctuations. (B). O1s and Ti 2p photoemission spectra before and after thermal annealing of flat and ns-TiO2 films. Ti 2p1/2 and Ti 2p3/2 peaks fall at 465.3 eV and 459.6 eV respectively (Ti(IV) bound to oxygen) and O 1 s peak shows two components one at 531.1 eV (oxygen bound to Ti(IV)) and the other at 533.5 eV (oxygen bound to contaminants).
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Figure 1: AFM images and photoemission spectra. (A) AFM images of reference and nanostructured samples. Reference samples: a) coverslip (1 x 1 μm2), rms roughness is 0.343 ± 0.004 nm; b) Poly-L-Lysine coated coverslip (1 x 1 μm2), rms roughness 0.271 ± 0.020 nm; c) flat TiO2 (2 x 1 μm2), rms roughness of 0.229 ± 0.004 nm. The vertical color scales range between 0 and 10 nm. Nanostructured samples: d) and e) high resolution SEM images and f) and g) AFM topographies (2 x 1 μm2) of 50 nm and 200 nm thick nanostructured TiO2, respectively. The roughness of the thinner film has been evaluated 20.2 ± 0.5 nm, whereas of the thicker film 29.1 ± 1 nm. The ns-TiO2 films clearly appear to have a fine raster of nanometer-sized grains with porosity at the subnanometer scale and with the thicker film showing larger height fluctuations. (B). O1s and Ti 2p photoemission spectra before and after thermal annealing of flat and ns-TiO2 films. Ti 2p1/2 and Ti 2p3/2 peaks fall at 465.3 eV and 459.6 eV respectively (Ti(IV) bound to oxygen) and O 1 s peak shows two components one at 531.1 eV (oxygen bound to Ti(IV)) and the other at 533.5 eV (oxygen bound to contaminants).

Mentions: Figure 1(A) (a)-(c) report the AFM characterization of glass and flat TiO2 substrates: Poly-L-Lysine coated glass has a calculated rms roughness of 0.271 ± 0.020 nm, whereas flat TiO2 films show a rms roughness of 0.229 ± 0.004 nm. Figure 1(d)-(g) show SEM and AFM images of cluster-assembled ns-TiO2 films with roughness of 20.2 ± 0.5 nm and 29.1 ± 1 nm respectively (corresponding to 50 nm and 200 nm film thickness). The random stacking of nanoparticles on substrates resulting from SCBD produces films with a homogeneous nanoscale porosity and roughness: the nanoparticles landing on the substrate stick on the surface of the growing film without any relevant diffusion or re-arrangement as it is typical of a ballistic deposition regime [36,47]. Ns-TiO2 substrates have been evaluated in terms of the reproducibility and control of their structural (morphology) and physico-chemical properties by accurate statistical intraslide/interslide data, showing an extremely good reproducibility among different production batches [37].


Nitric oxide synthase mediates PC12 differentiation induced by the surface topography of nanostructured TiO2.

Tamplenizza M, Lenardi C, Maffioli E, Nonnis S, Negri A, Forti S, Sogne E, De Astis S, Matteoli M, Schulte C, Milani P, Tedeschi G - J Nanobiotechnology (2013)

AFM images and photoemission spectra. (A) AFM images of reference and nanostructured samples. Reference samples: a) coverslip (1 x 1 μm2), rms roughness is 0.343 ± 0.004 nm; b) Poly-L-Lysine coated coverslip (1 x 1 μm2), rms roughness 0.271 ± 0.020 nm; c) flat TiO2 (2 x 1 μm2), rms roughness of 0.229 ± 0.004 nm. The vertical color scales range between 0 and 10 nm. Nanostructured samples: d) and e) high resolution SEM images and f) and g) AFM topographies (2 x 1 μm2) of 50 nm and 200 nm thick nanostructured TiO2, respectively. The roughness of the thinner film has been evaluated 20.2 ± 0.5 nm, whereas of the thicker film 29.1 ± 1 nm. The ns-TiO2 films clearly appear to have a fine raster of nanometer-sized grains with porosity at the subnanometer scale and with the thicker film showing larger height fluctuations. (B). O1s and Ti 2p photoemission spectra before and after thermal annealing of flat and ns-TiO2 films. Ti 2p1/2 and Ti 2p3/2 peaks fall at 465.3 eV and 459.6 eV respectively (Ti(IV) bound to oxygen) and O 1 s peak shows two components one at 531.1 eV (oxygen bound to Ti(IV)) and the other at 533.5 eV (oxygen bound to contaminants).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: AFM images and photoemission spectra. (A) AFM images of reference and nanostructured samples. Reference samples: a) coverslip (1 x 1 μm2), rms roughness is 0.343 ± 0.004 nm; b) Poly-L-Lysine coated coverslip (1 x 1 μm2), rms roughness 0.271 ± 0.020 nm; c) flat TiO2 (2 x 1 μm2), rms roughness of 0.229 ± 0.004 nm. The vertical color scales range between 0 and 10 nm. Nanostructured samples: d) and e) high resolution SEM images and f) and g) AFM topographies (2 x 1 μm2) of 50 nm and 200 nm thick nanostructured TiO2, respectively. The roughness of the thinner film has been evaluated 20.2 ± 0.5 nm, whereas of the thicker film 29.1 ± 1 nm. The ns-TiO2 films clearly appear to have a fine raster of nanometer-sized grains with porosity at the subnanometer scale and with the thicker film showing larger height fluctuations. (B). O1s and Ti 2p photoemission spectra before and after thermal annealing of flat and ns-TiO2 films. Ti 2p1/2 and Ti 2p3/2 peaks fall at 465.3 eV and 459.6 eV respectively (Ti(IV) bound to oxygen) and O 1 s peak shows two components one at 531.1 eV (oxygen bound to Ti(IV)) and the other at 533.5 eV (oxygen bound to contaminants).
Mentions: Figure 1(A) (a)-(c) report the AFM characterization of glass and flat TiO2 substrates: Poly-L-Lysine coated glass has a calculated rms roughness of 0.271 ± 0.020 nm, whereas flat TiO2 films show a rms roughness of 0.229 ± 0.004 nm. Figure 1(d)-(g) show SEM and AFM images of cluster-assembled ns-TiO2 films with roughness of 20.2 ± 0.5 nm and 29.1 ± 1 nm respectively (corresponding to 50 nm and 200 nm film thickness). The random stacking of nanoparticles on substrates resulting from SCBD produces films with a homogeneous nanoscale porosity and roughness: the nanoparticles landing on the substrate stick on the surface of the growing film without any relevant diffusion or re-arrangement as it is typical of a ballistic deposition regime [36,47]. Ns-TiO2 substrates have been evaluated in terms of the reproducibility and control of their structural (morphology) and physico-chemical properties by accurate statistical intraslide/interslide data, showing an extremely good reproducibility among different production batches [37].

Bottom Line: Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF.We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: CIMAINA and Dipartimento di Fisica, Università degli Studi di Milano, via Celoria 16, Milano 20133, Italy. paolo.milani@mi.infn.it.

ABSTRACT

Background: Substrate nanoscale topography influences cell proliferation and differentiation through mechanisms that are at present poorly understood. In particular the molecular mechanism through which cells 'sense' and adapt to the substrate and activate specific intracellular signals, influencing cells survival and behavior, remains to be clarified.

Results: To characterize these processes at the molecular level we studied the differentiation of PC12 cells on nanostructured TiO2 films obtained by supersonic cluster beam deposition.Our findings indicate that, in PC12 cells grown without Nerve Growth Factor (NGF), the roughness of nanostructured TiO2 triggers neuritogenesis by activating the expression of nitric oxide synthase (NOS) and the phospho-extracellular signal-regulated kinase 1/2 (pERK1/2) signaling. Differentiation is associated with an increase in protein nitration as observed in PC12 cells grown on flat surfaces in the presence of NGF. We demonstrate that cell differentiation and protein nitration induced by topography are not specific for PC12 cells but can be regarded as generalized effects produced by the substrate on different neuronal-like cell types, as shown by growing the human neuroblastoma SH-SY5Y cell line on nanostructured TiO2.

Conclusion: Our data provide the evidence that the nitric oxide (NO) signal cascade is involved in the differentiation process induced by nanotopography, adding new information on the mechanism and proteins involved in the neuritogenesis triggered by the surface properties.

Show MeSH
Related in: MedlinePlus