Series of images to show the fabrication of the biphasic scaffold an intervertebral disc.

<p>(A) A CG core was encapsulated in the first collagen layer before crosslinking. (B) Some of the photochemically-crosslinked CG cores were then encapsulated in a further 1 to 9 layers of collagen. (C) Immersion of the CG core with collagen layers in the photosensitizer, rose Bengal. (D) Irradiation of the construct with an argon laser at 514 nm for photochemical crosslinking. (E) Dehydration of the CG containing the 1^st collagen layer by rolling it on absorbent filter paper. (F) Dehydration of the CG core now encapsulated in the 2^nd to 10^th collagen layers. (G) The rehydrated CG core in one of the AF-like collagen lamella (E1). (H) Rehydrated CG core in two to ten collagen lamellae (E2 to E10). (I) Condensed biphasic scaffold in top view. (J) Condensed biphasic scaffold in side view. (K) Rabbit disc harvest. (L) Native rabbit disc in top view. (M) Native rabbit disc in side view.</p

A representative stress-strain curve showing the four stage-loading protocol used for the native discs and biphasic constructs.

<p>Samples were subjected to: pre-load (A) at 0.6 MPa for 150 sec and then 0.1 MPa for 600 sec; creep (B) at 0.6 MPa for 12,000 sec; dynamic load (C) at sine stresses between 0.3 and 0.9 MPa at 0.1, 0.32, 1, 3.2 and 10 Hz (linear log scale); and recovery (D) at 0.1 MPa for 12,000 sec.</p

Histomorphometric and ultrastructural characterization of the fabricated biphasic scaffold and native disc.

<p>(A-D) Fabricated biphasic scaffold and (E-H) native disc; (A&E) Alcian blue staining and (B&F) immunohistochemistry of type I collagen. (C-D & G&H) SEM images. (C) AF-like collagen lamellae; (D) NP-like core with compaction; (D1) NP-like core without compaction; (G) AF lamellae in the native disc; (H) NP core in the native disc. Scale bars are 500 μm (for A-B & E-F); 10 μm (for C&G); 1 μm (for G1) and 500 nm (for D-D1&H).</p

Dynamic stiffness and damping factor of samples during the dynamic mechanical analysis (DMA).

<p>(A) Line chart of the dynamic stiffness against the log loading frequency. (B) Bar chart showing the slope values measured for the dynamic stiffness-log loading frequency curves (mean+-2SE, n = 2–4). (C) Line charts of the damping factor (tangent delta) against log loading frequency; D: Bar chart showing the slopes of the tangent delta-log loading frequency curves (mean+-2SE, n = 2–4).</p

Gross appearance and dimension analysis of the samples in the different groups before and after biomechanical testing.

<p>(A-D) Gross appearance of the biphasic scaffolds from side (A-B) and top (C-D) views before (A,C) and after (B,D) the mechanical tests. (E-G) Dimension analysis of the biphasic scaffolds (with 1, 2, 4 and 10 layers of collagen lamellae) and the native disc controls at different stages during the mechanical test (i.e., before and after pre-load, after the creep, and after the test). Bar graphs show: (E) disc diameter, (F) disc height, and (G) percentage of disc height recovery. Data are expressed as mean+-2SE of n = 2–4 experiments.</p

Bar charts showing the mechanical properties of samples during the creep and recovery phases of the mechanical tests.

<p>The mechanical properties of the fabricated biphasic scaffolds and native IVD (A-D) during the creep and (E-H) recovery phases. (A&E) Elastic compliance (E, mm/N); (B&F) Viscous compliance (V, mm/N); (C&G) Time constants (T, seconds) and (D&H) Stretch constants (B). Data are expressed as mean±2SE of n = 2–4 experiments.</p

Human MSC-collagen microspheres under cultures.

<p>(A) Temporal change in the size of hMSC-collagen microspheres (n = 10); (B1-5) Live and Dead staining of hMSCs in collagen microspheres at day 21 during chondrogenic differentiation (Green: Live cells labeled by cacein AM; Red: Dead cells labeled ethidium homodimer-1); (C1-5) sox 9 immunofluorescence; (D1-5) Alcian blue staining; (E1-5) Type II collagen immunohistochemistry; (F1-5) Aggrecan immunohistochemistry; (G1-5) Type I collagen immunohistochemistry; (H1-5) Type X collagen immunohistochemistry; 1: Negative control (Normal medium); 2: Positive control (Chondrogenic medium with TGFβ alone); 3: ED (Chondrogenic medium with TGFβ and intracellular protease inhibitor E64D); 4: GM (Chondrogenic medium with TGFβ and extracellular matrix protease inhibitor GM6001); 5: ED+GM (Chondrogenic medium with TGFβ and both intracellular protease inhibitor E64D and extracellular protease inhibitor GM6001).</p

Bar charts showing the extracellular matrix composition of hMSC-collagen microspheres after chondrogenesis.

<p>(A) Hydroxyproline (collagen) content (n = 3); (B) Type II collagen content, measured using ELISA (n = 4); (C) Glycosaminoglycan (GAG) content (n = 3); and (D) DNA content (cellularity) (n = 3).</p

Collagen remodeling in hMSC in rat collagen microspheres.

<p>(A-H) Collagen type I remodeling by microencapsulated-hMSCs after 24 hours of incubation. Green: Bovine DQ FITC collagen type I 10 mg/ml, Orange: Human type I collagen, Red: Rat type I collagen 1 mg/ml and Blue: DAPI. (A) Fluorescent staining of bovine DQ FITC type I collagen, which was mixed with rat type I collagen during fabrication of microspheres and it fluoresces if degraded; (B) Immunofluorescent staining of human type I collagen, which should be synthesized by hMSC; (C) Immunofluorescent staining of rat type I collagen; (D) DAPI, which labelled the nuclei; (E) Merged panels A+B+D (cell nuclei in degrading DQ collagen and newly synthesized human type I collage); (F) Merged panels B+C+D (cell nuclei in starting material rat collagen and newly synthesized human type I collagen); (G) Merged panels A+C+D (starting materials rat type I collagen and DQ collagen are largely co-localizing); (H) Merged panels A+B+C+D (cells synthesizing human type I collagen in starting materials, which are undergoing degradation); (I-N) Collagen type II deposition in MSC-collagen type I microsphere after chondrogenic differentiation for 21 days; (I) Immunofluorescent staining of rat type I collagen, which is the starting material of the microsphere; (J) Immunohistochemistry of human type II collagen (DAB: substrated), which is newly synthesized by MSC during chondrogenic differentiation; (K) Merged panels (I+J); (L) Alexa fluor 488 labelled rat type I collagen; (M) Immunofluorescent staining of human type II collagen; (N) Merged panels (L+M) showing co-localization in some regions.</p

Customized two degree-of-freedom (i.e., compression and torsion) bioreactor to maintain disc explants alive and to apply repeated mechanical loading for two days.

<p> A. close-up view of single station harboring a bovine coccygeal IVD soaked in culture media. Biocompatible materials are enox aluminium (black), polyoxymethylene (POM, white parts) and glass with a “press-fit” design and silicon rings (black rings) to ensure no leakage between glass and POM. <b>B.</b> 4-unit design arranged in 5% CO₂ and 60% humidity incubator. <b>C.</b> Scheme of control of uniaxial compression and axial torsion using fluidic muscle and servo-controlled valve. <b>D.</b> Close-up view of serrated titanium plate surface, which grasps IVD and keeps it in place and ensures nutrition diffusion to the bony endplate.</p