Osteogenic ASCs on NiTi-sheet, half-coated with NiTi-nanoparticles.

<p>Light microscopic overview shows no visual differences between coated and uncoated parts, whereas live/dead stainings revealed higher rates of vital cells on coated parts. A: light microscopic overview, coated part indicated by arrow; scale bar 2 mm. B: specimen of part A in fluorescence; green: vital cells, red: dead cells; scale bar 2 mm. C: coated part, detail; green: vital cells, red: dead cells; scale bar 100 µm. D: uncoated part, detail; green: vital cells, red: dead cells; scale bar 100 µm.</p

Metabolic activity after 48 hrs stimulation.

<p>A: ASCs on NiTi sheets (1), NiTi-nanoparticle coated NiTi-sheets (2) and cell culture plastic (3) showed no significant differences in metabolic activity. B: ASCs incubated with nanoparticle colloids and/or additives on cell culture plastic (1–14) and Nickel sheets (15). C: macrophages incubated with nanoparticle colloids and/or additives on cell culture plastic (1–14) and Nickel sheets (15). B & C: 1: H2O, 2 Citrate (Ci), 3 Cystein (Cy), 4 NiTi H2O, 5 NiTi Ci, 6 NiTi Cy, 7 Ti H2O, 8 Ti Ci, 9 Ti Cy, 10 Ni H2O, 11 Ni Cy, 12 Ni Cy, 13 cell culture medium, 14 DMSO, 15 Ni sheet. Statistical analysis of metabolic assay was performed with graph pad prism software by using one way ANOVA, followed by Dunnetts post hoc test. Only control without stimulation (B13) compared to DMSO control (B14) and stimulation with nickel (B15) showed significant changes in metabolic activity (indicated by asterisk).</p

ASCs on NiTi, verification of osteogenic differentiation by staining of calcium depositions and osteocalcin.

<p>A: alizarin red staining, partly coated NiTi-sheet (coated part indicated by arrow, border indicated by dot line); scale bar 2 mm. B: detail of A, uncoated part; scale bar 200 µm. C: detail of A, coated part; scale bar 200 µm. D: NiTi-nanocoated structure with osteogenic ASCs; green: osteocalcin (indicated by arrows), blue: nuclei; scale bar 50 µm.</p

Macroscopic comparisons of alizarin red stainings.

<p>Comparison of stainings demonstrated that calcium deposition occurred only under long time cultivation of ASCs with osteogenic medium (B and C), while cells on A were kept under same conditions without osteogenic medium. Scale bar 1 cm. A: ASCs on cover glass, cultivated for 6 weeks with medium. B: ASCs on cover glass, cultivated for 6 weeks with osteogenic medium. C: ASCs on NiTi-sheet, cultivated for 6 weeks with osteogenic medium.</p

ASCs on cover glasses, 6 weeks cultivation without osteogenic medium.

<p>Alizarin red staining as well as immunofluorescence demonstrated that there did not occur osteogenesis or mineralization without osteogenic medium as the stainings were negative for the analyzed osteo markers. A: alizarin red staining; scale bar 50 µm. B: red: osteopontin, green: fibronectin, blue: nuclei; scale bar 50 µm.</p

Osteogenic ASCs on NiTi-nanocoated NiTi secrete fibronectin as part of the extracellular matrix.

<p>blue: nuclei, green: fibronectin (indicated by arrows); scale bar 50 µm.</p

SEM pictures of ASCs on NiTi for 48 hrs.

<p>More cells were found on the coated parts of the sheets. There grew with a dimensional shape whereas only few cells were found on the uncoated parts. There cells appeared in a very flat shape. A: NiTi-nanoparticle coated part, overview; scale bar 500 µm. B: coated part, detail; scale bar 20 µm. C: uncoated part, overview; scale bar 500 µm. D: uncoated part, detail; scale bar 20 µm.</p

Immunofluorescence of undifferentiated ASCs on cover glasses with or without NiTi-nanoparticle coating of 50 µg/cm².

<p>Actin and tubulin arrangement is comparable on uncoated and coated glass. Integrins α₅ and ß₁ are involved in adhesion on both surface types. Cells secrete fibronectin as part of the extracellular matrix. A: uncoated glass; blue: nuclei, green: actin, red: integrin α₅, scale bar 10 µm. B: coated glass; green: actin, red: integrin α₅, scale bar 20 µm. C: uncoated glass; blue: nuclei, green: actin, red: integrin ß₁, scale bar 20 µm. D: coated glass; blue: nuclei, green: actin, red: integrin ß₁, scale bar 20 µm. E: uncoated glass; blue: nuclei, green: tubulin, scale bar 20 µm. F: coated glass; blue: nuclei, green: tubulin, scale bar 20 µm. G: coated glass; blue: nuclei, red: fibronectin, scale bar 20 µm.</p

Matrix secretion of osteogenic ASCs on NiTi.

<p>SEM and violet laser scan of osteogenic ASCs revealed no visual differences in matrix secretion, whereas 3-d displays showed an enhanced morphology of cells and matrix on the coated parts of the sheets. A: SEM of half-coated NiTi-sheet (NiTi-nanoparticle coated part indicated by arrow, border indicated by dot line) with osteogenic ASCs; scale bar 500 µm. B: violet laser surface scan (VK-9700, Keyence) of half-coated NiTi-sheet (NiTi-nanoparticle coated part indicated by arrow, border indicated by dot line) with osteogenic ASCs; scale bar 10 µm. C: 3d-display of B, uncoated part. D: 3d-display of B, coated part.</p

Axon counts and demonstration of myelinated of myelinated profiles proximal and distal to either the autologous transplanted nerve or the implanted spider silk construct.

<p>(A) Axon counts lesion site and at regions proximal and distal were compared between both groups. Plastic embedded coronal sections of autologous nerve transplant (B and D) and spider silk construct (C and E) at 10 months post surgery. The improved organization of myelinated axons after transplantation of the spider silk construct could be demonstrated by light (C) and transmission electron microscope (E). All spider fibers were degraded in a subtle manner, since an inflammation has never been seen. Scale bar for C and D in D = 3 µm, for E and F in F = 1 µm.</p