Fig 2: The intervertebral disc and degenerative disc disease. Schematic and a hematoxylin/eosin-stained histological sections of a healthy (A, B) and degenerated (C, D) canine intervertebral disc. (E) Human lumbar intervertebral disc obtained postmortem. Appropriate tissue selection for Tie2 fishing is indicated by dashed black lines, while degenerated or AF tissue areas are indicated by dashed red lines. (F) Human intervertebral disc (IVD)-derived tissues during discectomy are selected based on morphology, color and stiffness. Only gelatinous, white, and transparent tissue should be used for Tie2 fishing. Yellow and stiff tissue should be disregarded in order to enhance Tie2 detection

Fig 3: (a) Expression levels of VEGFR-2, Tie2, and Angiopoietin-1 genes in hDPSCs seeded on PCL/CS and PCL/CS/MTA scaffolds after 7 days. (b) Immunoblotting assay for the proteins of these markers showed increased levels of these proteins in hDPSCs, too. *P = 0.05, **P = 0.01, ***P = 0.001, and ****P = 0.000.

Fig 4: Expression levels of VEGFR-2, Tie-2, and Angiopoietin-1 in hDPSCs seeded on PCL/CS/bFGF scaffolds by western blotting after seven days. The seeding of hDPSCs on these scaffolds significantly increased the expression of evaluated angiogenic markers in protein levels. *P = 0.05 and **P = 0.01.

Fig 5: In vivo subcutaneous angiogenesis and osteogenic differentiation in HUVECs after OCT4-30Kc19 and BMP4 treatment. (A) Schematics of procedure of in vivo experiments, both for subcutaneous model and cranial defect model. HUVECs treated with OCT4-30Kc19 and BMP4 were seeded into gelatin-heparin scaffold. After cell-seeded scaffold were delivered into subcutaneous tissue, angiogenesis and osteogenic differentiation capacity was analyzed. Also, cell-seeded scaffolds were put in the defect area of sagittal crest of mice, bone regeneration capacity was analyzed. (B) Masson’s trichrome staining (MTC) of cell seeded gelatin-heparin scaffolds. Cells without treatment, only OCT4-30Kc19 treatment, only BMP4 treatment, both OCT4-30Kc19 and BMP4 treatment were each seeded on the scaffold and delivered into mouse subcutaneous tissue. After 6 weeks, MTC staining was performed. The sample treated with OCT4-30Kc19 and BMP4 showed the highest accumulation of collagen. Collagen is stained in blue. Scale bar, 200 µm. (C) Immunofluorescence staining of angiogenesis marker, TIE-2. TIE-2 was detected by anti-TIE 2-antibody (1:400). Both treatments of OCT4-30Kc19 and BMP4 on HUVECs elevated the expression of TIE-2, which indicates angiogenesis. Blue, green represent the nucleus and TIE-2 protein, respectively. Scale bar, 100 µm