Fig 2: Constructive remodeling of NGO-crosslinked scaffolds by M2c macrophages.Gross images (a) of harvested scaffolds and the box plot (b) showing cross-sectional area of harvested scaffolds after 35 days of transplantation. In each box plot, the center line within the box indicates the median, the edges of the box indicate the 25th and 75th percentile, and the whiskers indicate outliers outside the 10th and 90th percentile (n = 5). Representative confocal images of the implants on day 35 in each group stained with the following antibodies; DAPI (blue), F4/80 (red), MMP-9 (green) and CCR7 (white) (c), and TIMP-1 (green) and CD206 (white) (d). Scale bar, 400 µm. e Quantification of infiltrating M1 and M2 macrophages normalized to F4/80+ cells within the implants (n = 5). f Ratio of M1+ cells to M2+ cells in each group (n = 5). qRT-PCR of M1 (g), M2 (h), and M2c (i) -related genes in each scaffold harvested on day 35. (n = 5) j Mean fluorescence intensity (MFI) of MMP-9 and TIMP-1 in the stained sections of each group (n = 5). qRT-PCR analysis of mouse Timp subtypes (k) and Mmp subtypes (l) in each scaffold harvested on day 35 (n = 5). Quantitative data were presented as a mean ± SD. Statistical differences between the groups were determined by ordinary one-way ANOVA with post-hoc Tukey test (*p < 0.05, **p < 0.01, ***p < 0.001 versus CTL. ###p < 0.001 versus GA. ns; not statistically significant). Source data are provided as a Source Data file.

Fig 3: Transplantation of MBLs into a mouse model of chronic liver failure.a Schematic diagram of the strategy for establishing thioacetamide (TAA)-induced chronic liver failure model and subsequent transplantation of mouse bioengineered livers produced by using scaffolds without crosslinking (CTL-MBLs) and crosslinked with nano-graphene oxide (NGO-MBLs). b Gross images of remaining CTL-MBLs and NGO-MBLs at 2 weeks after transplantation. c The residual amounts of CTL-MBLs and NGO-MBLs, characterized by cross-sectional area and weight of residual transplants (n = 5). d Immunostaining of harvested CTL-MBLs and NGO-MBLs probed with following antibodies: F4/80 (red), DAPI (blue), MMP-9 (green) and CCR7 (white), TIMP-1 (green) and CD206 (white), and streptavidin (green) and MMP-2 (red). Scale bar, 100 µm. Quantification of M1 macrophages and M2 macrophages normalized to F4/80+ cells (e), and MMP-2 expression (f) in each group (n = 5). g Cytometric bead array performed with the serum collected at 2 weeks from the normal mice, the mice in which chronic liver injury was induced (Sham), the mice receiving CTL-MBL and NGO-MBL transplantation. In each box plot, the center line indicates the median, the edges of the box indicate the 25th and 75th percentile, and the whiskers indicate the 10th and 90th percentile (n = 5). h Immunostaining of harvested CTL-MBLs and NGO-MBLs. ALB (green), HNF4a (red) and DAPI (blue). Scale bar, 100 µm. Picrosirius red staining (i) of livers in each group and quantification (j) of fibrotic area. Scale bar, 40 µm (n = 5). k qRT-PCR analysis of liver fibrosis-related genes in each group (n = 5). Serum ALT (l) and AST (m) levels of mice in each group, before and after transplantation (n = 5). Quantitative data were presented as a mean ± SD. Statistical differences between the groups were determined by unpaired, two-tailed student’s t test (c, e, f), Ordinary one-way ANOVA with post-hoc Tukey test (g, j, k) and two-way ANOVA with post-hoc Tukey test (l, m) (*p < 0.05, **p < 0.01, ***p < 0.001 versus CTL-MBL, ##p < 0.05, ###p < 0.001 versus Sham, ns; not statistically significant). Source data are provided as a Source Data file.

Fig 4: In vitro resistance of NGO-crosslinked scaffolds to enzymatic degradation.a Raman spectra of scaffold crosslinked with 5 µg/mL nano-graphene oxide (NGO-5) after MMP-1 treatment (top) and degradation products of MMP-treated scaffold (bottom). b,c Fourier transform infrared spectroscopy (FTIR) spectra of non-crosslinked scaffolds (CTL) and NGO-5 scaffolds were measured before (black) and after (red) MMP-1 treatment. d Scanning electron microscopy (SEM) images of the crosslinked scaffolds after MMP-1 treatment for 2 h. Scale bar, 1 µm. e Picrosirius red staining of the scaffolds degraded by MMP-1. Scale bar, 200 µm. f Area quantification of collagen fibers stained by picrosirius red (n = 4). g Weight loss measurements of each scaffold after treatment of MMP-1, MMP-2 and MMP-9 (n = 4). h Ninhydrin assay quantifying the crosslinking degree of the scaffolds after MMP treatment (n = 4). i Quantification of the amount of retained insoluble collagen of the scaffolds exposed to MMPs normalized to that of intact scaffolds (n = 4). Quantitative data were presented as a mean ± SD. Statistical differences between the groups were determined by ordinary one-way ANOVA with post-hoc Tukey test (***p < 0.001 versus CTL, ns; not statistically significant). Source data are provided as a Source Data file.

Fig 5: Transplantation of MBLs into a mouse model of acute liver failure.a Immunostaining of normal mouse livers and mouse bioengineered livers (MBLs) fabricated using scaffolds without crosslinking (CTL-MBLs), crosslinked with glutaraldehyde (GA-MBLs), and crosslinked with nano-graphene oxide (NGO-MBLs). ALB (green) and DAPI (nuclei, blue). Scale bar, 100 µm. b Quantification of ALB+ cells (n = 4). c Immunostaining of CD31 (green) and DAPI (nuclei, blue). Scale bar, 100 µm. d Quantification of CD31+ cells covering vascular lumen (n = 4). Quantification of secreted albumin (e) and urea (f) from CTL-MBLs, NGO-MBLs and GA-MBLs during bioreactor culture on day 4 and 8 (n = 4). g Schematic diagram of transplantation of MBLs into the mice receiving 70% partial hepatectomy. h Gross images of transplanted CTL-MBLs and NGO-MBLs on day 5. i Residual amounts of transplanted CTL-MBLs and NGO-MBLs, characterized by cross-sectional area and weight of transplants (n = 5). j Immunostaining of harvested CTL-MBLs and NGO-MBLs probed with following antibodies: F4/80 (red), DAPI (blue), MMP-9 (green) and CCR7 (white), TIMP-1 (green) and CD206 (white), streptavidin (green) and MMP-2 (red). Scale bar, 100 µm. Quantification of M1 macrophages and M2 macrophages normalized to F4/80+ cells (k), and MMP-2 expression (l) within CTL-MBLs and NGO-MBLs (n = 5). m Cytometric bead array of serum samples from the following groups; Normal mice, the mice receiving partial hepatectomy (PHx Sham), the mice receiving CTL-MBL and NGO-MBL transplantation after PHx. In each box plot, the center line indicates the median, the edges of the box indicate the 25th and 75th percentile, and the whiskers indicate the 10th and 90th percentile (n = 5). n Transplanted CTL-MBLs and NGO-MBLs stained with ALB (green) and HNF4a (red). Scale bar, 100 µm. o qRT-PCR analysis of liver regeneration-related genes (n = 5). p Serum levels of ALT and AST in each mouse on day 5 (n = 5). Quantitative data were presented as a mean ± SD. Statistical differences between the groups were determined by ordinary one-way ANOVA with post-hoc Tukey test (b, d–f, m, o, p) and unpaired, two-tailed student’s t test (i, k, l) (*p < 0.05, **p < 0.01, ***p < 0.001, ns; not statistically significant). Source data are provided as a Source Data file.