Images showing the results obtained when cells were cultured on the smooth surface.

<p>(<b>A</b>) smooth surface at SEM (Calibration Bar = 10 µm); (<b>B</b>) cells, cultured on a smooth surface, under SEM, show an elongated shape (Calibration Bar = 10 µm); (<b>C</b>) confocal image showing the relationship between cells and the smooth surface (Calibration Bar = 10 µm); (<b>D</b>) image showing the relationship between cells and the different surface texturing: (1) concave and (2) smooth.</p

Images showing primary and secondary micro concavities at scanning electron and confocal microscopy.

<p>(<b>A</b>) Primary micro concavity (<u>arrow</u>) of the PLGA surface at SEM. Cells can be completely contained within a primary concavity, due to its dimensions. (Calibration Bar = 10 µm); (<b>B</b>) SEM analysis of primary concavity dimensions (Calibration Bar = 10 µm); (<b>C</b>) SEM analysis of secondary concavity dimensions (Calibration Bar = 10 µm); (<b>D</b>) The interaction between the concave surface, showing primary (<u>white arrow</u>) and secondary (<u>red arrows</u>) micro-concavities at the confocal microscope (in green a cell within a concavity). The intimate adherence of a cell to the polymer surface and its nuclear polarity are clearly observable. The image was been obtained superimposing dark field with light field confocal microscopy (Calibration Bar = 10 µm); (<b>E</b>) Confocal image showing primary (outlined in red) and secondary (outlined in blue) micro-concavities and spider-shaped cellular elongations (Calibration Bar = 10 µm); (<b>F</b>) A gingival fibroblast not showing cellular alterations or nuclear polarity at the confocal microscope (Calibration Bar = 10 µm).</p

Immunofluorescence for HLA-1 Human FITC (green) (A) <i>In vivo</i> concave PLGA scaffold.

<p>(<b>B</b>) <i>In vivo</i> smooth PLGA scaffold. (Calibration Bars = 5 µm). Nuclear staining is obtained with DAPI (blue).</p

Side population analysis.

<p>(A) Cytometric analyses of the side population. The CD133⁺ fraction includes a small subset (0.97%), expressing the characteristic profile of a side population at FACS. (B) ABCG2 expression in SAOS2 cell line, showing an evident positivity; the grey line indicates the isotype control.</p

Cytometric analyses for CD133 on SAOS2, MG63 and U2OS cell lines.

<p>A CD133⁺ cell population can be detected in all the three cell lines.</p

Haematoxilin and Eosin staining.

<p>(<b>A</b>) <i>In vivo</i> sample on concave PLGA scaffold (Calibration Bar = 10 µm). The figures shows that the bone tissue is of great thickness with an evident periosteal layer (red arrow) and containing osteocytes entrapped within the matrix (black arrows). (<b>B</b>) <i>In vivo</i> sample on smooth PLGA scaffold (Calibration Bar = 5 µm) appeared to be more primitive, thin (black arrow) and not as well developed above a connective loose tissue containing vessels (red arrow).</p

Alkaline phosphatase detection during cell differentiation.

<p>The image shows the quantity of ALP during osteoblast differentiation at 24, 48, 72 and 96 hours within the cells cultured on the different surfaces. The data have been rounded to the closest integer value. The error bars are ±SD.*p<0.01. Each experiment was performed in triplicate (n = 3).</p

Immunofluorescence confirming the presence of a mineralized extra cellular matrix on concave texturing.

<p>The panel shows positivity for Collagen I (<b>A</b>) FITC (green) (Calibration Bar = 10 µm), BAP [Bone Alkaline Phosphatase] (<b>B</b>) FITC (green) (Calibration Bar = 5 µm), OC [Osteocalcin] (<b>C</b>) PE (red) (Calibration Bar = 7 µm), ON [Osteonectin] (<b>D</b>) FITC(green) (Calibration Bar = 7 µm) and BSP [Bone Sialoprotein] (<b>E</b>) PE (red) (Calibration Bar = 3 µm). The same analysis confirming the presence of a mineralized extra cellular matrix on smooth texturing. The panel shows positivity for Collagen I (<b>F</b>) FITC (green) (Calibration Bar = 5 µm), BAP (<b>G</b>) FITC (green) (Calibration Bar = 7 µm), OC (<b>H</b>) PE (red) (Calibration Bar = 3 µm), ON (<b>I</b>) FITC (green) (Calibration Bar = 7 µm) and BSP (<b>J</b>) PE (red) (Calibration Bar = 3 µm). Nuclear staining is obtained with DAPI (blue).</p

Spheres assay.

<p>(A) Sphere clusters formed by CD133⁺ cells in semisolid medium after 24 hours (Original Magnification ×100); (B) CD133⁻ cells in semisolid medium after 7 days, do not form spheres. (Original Magnification ×100); (C) Sphere clusters formed by CD133⁺ cells after 48 hours (Original Magnification ×200); (D) Sphere clusters formed by CD133⁺ cells after a new sorting (Original Magnification ×400).</p

CD133 expression in adherent cells and floating spheres.

<p>(A) Immunohistochemical analyses on adherent cells and (B) floating spheres showing the presence of CD133 antigen (<u>arrows</u>). (Original Magnification. ×100); (C) Immunofluorescence analysis on SAOS-2 for CD133 PE, cytoskeleton is stained with phalloidin-FITC, nucleus with DAPI (Original Magnification. ×400); (D) Immunoflurescence analysis on SAOS-2 spheres for CD133 PE after 24 hours in adhesion. (Original Magnification ×200); (E) Confocal analyses on adherent cells and (F) floating spheres confirming the presence of the CD133 antigen. (Original Magnification. ×400).</p