Neurite growth.

<p>Rat dorsal root ganglia were explanted on a positive control substrate (Ctrl, PDL/laminin), on gelatin gel (g gel), and on quattroGel (qGel) and after culturing immunostained for neurites (red, neurofilament) and for cell nuclei (blue, DAPI). Significant neurite outgrowth is evident in the control and on gelatin gel, not on quattroGel. Pictures of individual rows represent corresponding images. Scale bar: 200 µm.</p

Chondrocyte differentiation.

<p>(A-C) Human chondrocytes were cultured on a positive control substrate (Ctrl), on gelatin gel (g gel), and on quattroGel (qGel), and metabolically marked with bromodeoxyuridine (BrdU) to identify mitotic cells and DAPI to display all cell nuclei. (A) Corresponding false color images of chondrocytes on quattroGel (left: proliferating cells; right: total cells). Quantification of cell adhesion (B) and cell proliferation (C) indicated principle cell adhesion on all substrates with an increased adhesion on both hydrogels. No statistically significant differences existed between different substrates with regard to cell proliferation. (D) In-depth confocal images of actin stained chondrocytes with typical ovoid morphologies within a quattroGel matrix. (E) 3-D reconstruction of a rare chondrocyte extending short processes. (F) The viability of chondrocytes incorporated into the hydrogels after 3 days was very high. For negative controls (Ctrl), specimens were exposed to detergent. (G) Gene expression analysis by RT-PCR of chondrocytes enzymatically released from hydrogels after two weeks. A chondrocyte-specific increase of type II collagen synthesis but not of type I collagen used as reference was evident. Values were compared with monolayer control cultures. n = 3, s.d., p<0.05. Scale bars: (A) 50 µm, (D) 10 µm, (E) 6 µm.</p

In vivo stability.

<p>(A) QuattroGel was blue-stained to improve visualization and applied to a rat skin flap. Strong adhesion (arrow) between the pipette tip with quattroGel and mouse tissue allowed the lifting of a skin flap. (B) Subcutaneous situs three months after local injection of quattroGel, which appeared to be stable without adverse clinical symptoms. (C) HE stained tissue section of a three-month implant depicting the skin, the porous quattroGel (qGel) and a thin, dark stained encapsulation layer (arrow). Scale bars: (A) 10 mm, (B) 5 mm, (C) 200 µm.</p

Viscoelastic properties.

<p>(A) Start of gelation. Gelation points deduced from crosspoints of G′ and G″ (circles with arrows); quattroGel showed a delayed gelation (360 sec) compared to gelatin gel (252 sec). (B) End of Gelation. <i>Oscillatory time sweeps</i> to unravel gelation kinetics of the gelatin gel (black) and quattroGel (red) over 2 h at 37°C. Both gels showed typical asymptotic graphs with gelation essentially finalized after about 1 h (plateau phase). (C) Viscoelasticity. <i>Frequency sweep</i> after curing of the gels revealed semi-rigid, elastic properties for both gels as deduced from storage moduli (upper curves) two orders higher than the corresponding loss moduli (lower curves). n = 3.</p

Biocompatibility in

<p>(A) The cell line L929 was cultured on a positive control substrate (Ctrl), on gelatin gel (g gel) and on quattroGel (qGel), metabolically marked with bromodeoxyuridine (BrdU, green) to identify mitotic cells and cytochemically labeled with fluorescent phalloidin (actin, red) and DAPI (blue) to display all cell nuclei. Pictures of individual rows represent corresponding images. Arrows mark cells in different stages of mitosis. Quantification of cell adhesion (B) and cell proliferation (C) indicate that no statistically significant differences exist between different substrates. n = 3, s.d., p<0.05. Scale bar: (A) 50 µm.</p

Microstructure of hydrogels.

<p>(A) The quattroGel forms a macroscopically homogeneous hydrogel. (B) quattroGel with incorporated Alexa488-fibrin and fluorescent latex beads in the gelatin solution. Fibrin and gelatin appear to be equally distributed within quattroGel lamellae. Green – fluorescently labeled fibrin; red – labeled latex beads in gelatin. (C, D) HE stained cryosections of quattroGel (C) and gelatin gel (D). (E, F) Quantification of pore area (E) and pore distribution (F) in both gels indicated a higher porosity in quattroGel. qGel – quattroGel; g gel – gelatin gel. Scale bars: (B) 50 µm, (C and D) 20 µm.</p

Diffusion characteristics.

<p>(A, B) Two-chamber device and scheme. Test gels were inserted as interface between the upper chamber (uCh) containing a marker chromophore and the lower chamber (lCh) filled with colourless buffer. (C) Diffusion kinetics over 24 h with equilibrium reached after one day for both hydrogels. Negative control (Neg Ctrl): parafilm; positive control (Pos. Ctrl: gaze. (D) Permeability coefficients. Gelatin gel (g gel) and quattroGel (qGel) displayed a similar, restricted diffusion when compared with the positive control. n = 3, s.d., p<0.001 compared with positive control.</p

Endothelial differentiation.

<p>(A-C) Human umbilical vascular endothelial cells (HUVEC) were cultured on a positive control substrate (Ctrl), on gelatin gel (g gel) and on quattroGel (qGel), metabolically marked with bromodeoxyuridine (BrdU) to identify mitotic cells and DAPI to display all cell nuclei. (A) Corresponding false color images of HUVEC on quattroGel (left: proliferating cells; right: total cells). Quantification of cell adhesion (B) and cell proliferation (C) indicate principle cell adhesion on all substrates, though reduced on both hydrogels. No statistically significant differences exist between different substrates with regard to cell proliferation. (D) Calcein staining of cultured cells. HUVEC cultured on a positive control substrate (Pos Ctrl, Matrigel™) differentiate vessel-like tubes. No tubes are formed by fibroblasts on Matrigel™ (Neg Ctrl). HUVEC do not differentiate tubes on quattroGel. n = 3, s.d., p<0.05. Scale bar: (A) 50 µm, (D) 200 µm.</p

Additional file 1: of Inflammatory profiles in canine intervertebral disc degeneration

A more detailed representation of included samples in addition to Table  1 in the original article. (DOCX 29 kb

Additional file 2: of Inflammatory profiles in canine intervertebral disc degeneration

Significant differences and confidence intervals of statistical analyses. (DOCX 25 kb