Fig 2: TAZ is upregulated during osteogenesis while it is downregulated during adipogenesis in human ADSCs. a Osteogenic differentiation of ADSCs was induced in vitro and monitored by Alizarin Red and von Kossa staining at the indicated time points (days 7, 14, 21, and 28 after induction). Scale bar = 50 μm. b,c Transcriptional coactivator with PDZ-binding motif (TAZ) protein was significantly increased during the osteogenic differentiation process of ADSCs, concomitant with markedly upregulated expression of the osteogenic markers runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN). Representative images of Western blots from three independent experiments are shown. d The mRNA levels of TAZ and the osteogenic markers were significantly increased during ADSC osteogenic differentiation in vitro as measured by quantitative RT-PCR. e Adipogenic differentiation of ADSCs was induced in vitro and determined by Oil Red O staining at the indicated time points (days 7, 14, and 21 after induction). Scale bar = 50 μm. f TAZ protein was significantly downregulated during the adipogenic differentiation process of ADSCs. Representative images of Western blots from three independent experiments are shown. g The mRNA levels of TAZ decreased while the adipogenic marker increased during ADSC adipogenic differentiation in vitro as measured by quantitative RT-PCR. Data shown here are mean ± SD from three independent experiments; #P ˃ 0.05, *P < 0.05, **P < 0.01, by ANOVA. ALP alkaline phosphatase, GAPDH glyeraldehyde-3-phosphate dehydrogenase, PPARγ peroxisome proliferator-activated receptor-γ

Fig 3: Exposure of TAZ activator TM-25659 promotes osteogenic differentiation of ADSCs in vitro. a Cell viability was determined after ADSCs were cultured with diverse concentrations of TM-25659 (0–50 μM) at 24 h and 48 h, respectively. B Enhanced osteogenic differentiation of ADSCs was detected and quantified by alkaline phosphatase (ALP) activity assay following ADSC culture with osteoinductive medium and TM-25659 at day 7. c, d Increased mineralization in ADSCs cultured in osteoinductive medium and TM-25659 was observed and quantified via Alizarin Red staining at days 7 and 14. Scale bar = 50 μm. e Significantly increased expression of the osteogenic markers runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) was observed in ADSCs cultured in osteoinductive medium and TM-25659 at days 7 and 14, respectively. Representative images of Western blots from three independent experiments are shown. Data shown here are mean ± SD from three independent experiments; **P < 0.01, by Student’s t test. GAPDH glyeraldehyde-3-phosphate dehydrogenase, OD optical density

Fig 4: TAZ knockdown impairs osteogenic differentiation of ADSCs in vitro. a Transcriptional coactivator with PDZ-binding motif (TAZ) knockdown (KD) in ADSCs infected with shRNA lentiviral particles targeting human TAZ was verified by Western blot. Representative images of Western blots from three independent experiments are shown. b TAZ and its downstream targets CTGF and Cyr61 were downregulated in TAZ-knockdown ADSCs as determined by quantitative RT-PCR. c Cell proliferation was compromised in TAZ-knockdown ADSCs compared with control cells infected with nontargeting lentiviral vectors with scrambled sequence. d Compromised osteogenic differentiation of ADSCs was detected and quantified by alkaline phosphatase (ALP) activity assay following TAZ knockdown of ADSCs induced in vitro at day 7. e Reduced mineralization in TAZ-knockdown ADSCs cultured in osteoinductive medium was observed via Alizarin Red staining at days 7, 14, and 21. f Significantly reduced expression of TAZ as well as the osteogenic markers osteopontin (OPN) and osteocalcin (OCN) in TAZ-knockdown ADSCs cultured in osteoinductive medium at days 7, 14, and 21 was detected by Western blot. Representative images of Western blots from three independent experiments are shown. Data shown here are mean ± SD from three independent experiments, #P ˃ 0.05, *P < 0.05, **P < 0.01, by Student’s t test. GAPDH glyeraldehyde-3-phosphate dehydrogenase, NC normal control, OD optical density, Runx2 runt-related transcription factor 2

Fig 5: Transplantation of the 3D printed hydrogel scaffold with NSs promotes rapid regeneration of critical-sized calvarial defect.a, b Illustration of the implantation of NSs-loaded 3D printing gel scaffold before and after calvarial defect surgery. c, d μ-CT evaluation of bone regeneration in Defect control (Defect), 3D printed scaffold (Scaffold), 3D printed scaffold containing NSs (Scaffold + NSs) groups at 4 weeks and 3 12 weeks post-surgery of calvarial defect (the yellow area indicates new bone formation), respectively. n = 3 rats for per group and per time point. e Bone volume fraction (BV/TV) (Exact p value calculated with one-way ANOVA Tukey’s multiple comparisons test: *p = 0.0218, *p = 0.0391); and f Bone mass density (BMD) (Exact p value calculated with one-way ANOVA Tukey’s multiple comparisons test: *p = 0.0113, *p = 0.0214) of regenerated tissues in defect area at 4 weeks after calvarial defect. g Bone volume fraction (BV/TV) (Exact p value calculated with one-way ANOVA Tukey’s multiple comparisons test: **p = 0.0076, p = 0.0532); and h Bone mass density (BMD) (Exact p value calculated with one-way ANOVA Tukey’s multiple comparisons test: **p = 0.0045, p = 0.0582) of regenerated tissues in defect area 12 weeks after calvarial defect; n = 3 biologically independent rats. i, j HE staining and Masson’ Trichromic staining of paraffin sections in Defect, 3D Scaffold, Scaffold + NSs groups at 4 weeks after calvarial defect (bar = 500 μm at low magnification and bar = 50 μm at high magnification). k, l HE staining and Masson’ Trichromic staining of paraffin sections in Defect, 3D Scaffold, Scaffold + NSs groups at 12 weeks after calvarial defect (bar = 500 μm at low magnification and bar = 50μm at high magnification). m Co-immunofluorescent staining of NGFR and OCN expression in Defect, 3D Scaffold, Scaffold + NSs groups at 4 and 12 weeks after calvarial defect (bar = 50 μm), respectively. n Immunofluorescent staining of CD31 expression in Defect, 3D Scaffold, Scaffold + NSs groups at 4 and 12 weeks after calvarial defect (bar = 200 μm), respectively. The white dashed line indicates the border between the newly formed bone-like tissues. All data represent mean ± SD, and source data are provided as a Source Data file. At least three times of experiments were repeated independently.