Fig 2: PACAP and/or mechanical load altered the collagen production of HDC. PACAP 1–38 at 100 nM was applied continuously from day one. Short time, transient MS, was applied on days two and three for 30 min on both days. (A) Collagen production of HDC was determined by 3H-collagen incorporation; (B) RNA expression of collagen type II (Col2a1), collagen type IX (Col9a1) and collagen type X (Col10a1) on day three. GAPDH was used as a control; (D) Protein expression of collagen type II, collagen type IX, collagen type X on day three. Actin was used as a control. For panels (B,D) numbers below signals represent integrated densities of signals determined by ImageJ software. Representative data of three independent experiments are shown; (C,E) Statistical analysis of RT-PCR and Western blot data. All data presented are the averages of at least three different experiments. Statistical analysis was performed by Student’s t-test. All data were normalized on GAPDH/Actin and data are expressed as mean ± SEM. (C, control; P1–38, PACAP 1–38; MS; mechanical stimulus). Statistically significant difference between collagen production rate of cells in cultures that received the loading regime and/or PACAP 1–38 vs. control cultures is marked by asterisk (* p < 0.05) in all panels.

Fig 3: The normal early postnatal disc.The normal structure of the postnatal day four (P4) lumbar IVD are shown. All sections are mid-coronal. (A) shows the general structure of the disc, stained with H&E. B-M show the distribution of molecular markers stained with specific antibodies (red in each case): Brachyury in cell nuclei of the NP (B), Sox 9 in cell nuclei of the NP (C), AF and EP (D), Keratin 19 in the cytoplasm of NP cells (E and E'), collagen 1 (Col1) in NP and outer part of the AF (F), collagen 2 (Col2) in the superficial region of the EP and outer AF (G), chondroitin sulfate (Ch SO4) in both the NP, AF and EP (H), keratin sulfate (Ker SO4) in the outer AF (I), aggrecan in the NP only (J), Shh in the NP only (K), patched-1 (L) and Gli1 (M) in many regions of the IVD and growth plate. Scale bars indicate magnifications used. Green = wheat germ agglutinin, blue = nuclei stained with POPO-3, NP = nucleus pulposus, AF = annulus fibrosus, IAF = inner AF, OAF = outer AF, EP = endplate. For details, see text.

Fig 4: Representative images of four BMSCs/hydrogel before and after 14/28 days’ implantation in vivo. (A) HE staining and (B) TB staining. Scale bars represent 100 µm. (C) ARS staining and (D) IHC staining of COL2. Scale bars represent 250 µm.

Fig 5: Matrix viscoelasticity mediates long-term commitment of MSC chondrogenesis through regulating cell survival via adjusting ROCK-dependent apoptosis.(A) TUNEL staining images of MSCs treated with blebbistatin or Y-27632 (day 2). Scale bars, 50 µm. (B) Quantification of TUNEL-positive cells (n = 3, *P < 0.05; data are shown as means ± SD). (C) Quantification of DNA of MSCs treated with blebbistatin or Y-27632 (day 6; two independent experiments; one representative experiment is shown; n = 3; *P < 0.05; data are shown as means ± SD). (D) Representative images of type II collagen immunofluorescent staining of MSCs treated with blebbistatin or Y-27632 (day 6). Scale bars, 25 µm. (E and F) Quantification of type II collagen fluorescence intensity of MSCs treated with blebbistatin (E) or Y-27632 (F) (n = 3; *P < 0.05; data are shown as means ± SD). (G and H) Gene expression of ACAN and Col2a1 of MSCs treated with blebbistatin (G) or Y-27632 (H) (day 6; three independent experiments; one representative experiment is shown; n = 3; *P < 0.05; data are shown as means ± SD). (I and J) Quantification of GAGs produced by MSCs treated with blebbistatin (I) or Y-27632 (J) [day 6; two independent experiments; one representative experiment is shown; n = 3; *P < 0.05; a and b indicate a significant difference (*P < 0.05) compared with the slower and faster hydrogels in the control group, respectively; data are shown as means ± SD].