Fig 2: In vivo osteodifferentiation and ectopic mineralization of BMSCs-laden HCLS.a1 CLSM images (Live/dead staining) of BMSCs-laden various hydrogels for 14 days. a2 Cell viability of BMSCs-laden various hydrogels at day 3, 7 and 14 (*p = 0.0140, *p = 0.0335, *p = 0.0123, *p = 0.0453, ***p = 1.9112 × 10-7). n = 3 biologically independent replicates. b1 CLSM images (rhodamine-phalloidin/DAPI staining) of BMSCs on day 14. b2 Percentage of cell spreading area after 3, 7, and 14 days (**p = 0.0022, ***p = 8.0407 × 10-7, ***p = 3.1642 × 10-6, **p = 0.0012, *p = 0.0206). c SEM images of BMSCs distributed on gel scaffolds after 14 days incubation. d1, d2) Immunohistochemistry staining of BMP-2 at day 14 and semiquantitative analysis of BMP-2 staining by Image J software (***p = 8.0447 × 10-5, ***p = 1.0591 × 10-4, **p = 0.0032, **p = 0.0026, *p = 0.0124, *p = 0.0221). n = 3 cells examined three independent experiments. e Immunohistochemistry staining of VEGF on day 14. f1–3 Gene expression of OCN, OPN and Runx2 for the BMSCs encapsulated in various hydrogels at day 3, 7 and 14. All gene expressions were normalized to housekeeping gene ß-actin (f1: ***p = 0.0004, ***p = 6.0578 × 10-6, ***p = 1.2233 × 10-7, *p = 0.0156, *p = 0.0147; f2: *p = 0.0464, **p = 0.0067, **p = 0.0014, ***p = 9.8374 × 10-7, ***p = 1.3141 × 10-9, ***p = 2.8964 × 10-10; f3: *p = 0.0461, **p = 0.0031, *p = 0.0139, **p = 0.0069, **p = 0.0023, ***p = 9.2436 × 10-6). n = 3 biologically independent replicates. g Visualization subcutaneous state after 30 days implantation. h1 Gross appearance of the samples before and after implantation. h2) Size change ratio of samples before and after implantation (*p = 0.0448, **p = 0.0056). h3 Dynamic mechanical test after 30 days implantation (***p = 3 × 10-15, ***p = 1 × 10-15, ns = 0.3906, ns = 0.9007). i1 3D reconstruction images of various explants by Micro-CT. i2, i3 Quantitative bone volume analyses (AV: apatite volume. TV: total volume.) i2: **p = 0.0018, ***p = 6.7311 × 10-9; i3: **p = 0.0023, ***p = 5.8921 × 10-9). j SEM images of the inner section of various samples. n = 3 biologically independent replicates. k1 Immunofluorescence staining to detect Runx2 in various BMSCs-loaded hydrogels. k2 Semiquantitative analysis of Runx2 staining by Image J software (**p = 0.0066, ***p = 0.0002). l1 Immunohistochemistry staining to detect VEGF in BMSCs-loaded hydrogels. l2 Semiquantitative analysis of VEGF staining by Image J software (**p = 0.0004, ***p = 5.7513 × 10-5). m1 CD31 staining of various explants. m2 Semiquantitative analysis of CD31 staining by Image J software (**p = 0.0073, ***p = 0.0001). n = 3 cells examined three independent experiments. (Two-sided comparison, error bars represent standard deviation, *p < 0.05, **p < 0.01, and ***p < 0.001).

Fig 3: Representative staining images of regenerated tissue at week 12.a1 H&E staining of regenerated bones induced by different scaffolds at week 12 after operation. a2 Masson’s trichrome staining at week 12 after the operation. a3 CD31 immunofluorescence staining of regenerated vessels at week 12. (Row 1: Overall observation of the cranial defect repair. The red arrow indicates the initial boundary of the defect. Row 2: Magnified view of the center and boundary site of the defects). (N: new bone tissue. S: scaffolds. V: new blood vessels (black arrow). F: fibrous tissue. B: old bone boundary). b1–4 Representative immunohistochemistry images of BMP-2, Runx2, OCN, VEGF at week 12 after implantation. c Quantitative analysis of positive cells at week 12. (all scaffolds without cells. Blank: defect alone with no treatment) (***p = 3.7616 × 10-5, *p = 0.0114; ***p = 2.1035 × 10-5, **p = 0.0032, ***p = 6.41247 × 10-5, *p = 0.0355, ***p = 5.2544 × 10-5, *p = 0.0409). (Two-sided comparison, error bars represent standard deviation, *p < 0.05, **p < 0.01, and ***p < 0.001).

Fig 4: Skull reconstruction by HCLS activating angiogenesis and osteogenesis in beagle dog model.a X-ray images of the regenerated bone tissue. b1 Representative micro-CT images and b2–4 quantitative histomorphometry analyses of bone regeneration in cranial defects in dogs at 4 and 12 weeks (b2: **p = 0.0074, ***p = 1.5931 × 10-7; b3: **p = 0.0008; ***p = 5.4303 × 10-7; b4: **p = 0.0019, ***p = 2.51131 × 10-7). n = 3 biologically independent replicates. c H&E staining of regenerated bones at 4 and 12 weeks after operation. d Masson’s trichrome staining of regenerated bones at 4 and 12 weeks after the operation. (Row 1: overall observation of the cranial defect repair. The red arrow indicates the initial boundary of the defect. Row 2: magnified view of the center and boundary site of the defect). (N: new bone tissue. S: scaffolds. V: new blood vessels. F: fibrous tissue. M: Muscle tissue). e Representative IF staining images of CD31 at 4 and 12 weeks after implantation. f Semiquantitative analysis of CD31 IF staining at 4 and 12 weeks. (all scaffolds without cells. Blank: defect alone with no treatment) (***p = 8.1804 × 10-7, ***p = 9.7494 × 10-8). (two-sided comparison, error bars represent standard deviation, *p < 0.05, **p < 0.01, and ***p < 0.001).