Exploring the memory of the extracellular matrix using MASH-derived decellularized scaffolds

Abstract

Emerging evidence suggests that the extracellular matrix (ECM) possesses a “memory” that can influence cell physiology and recellularization outcomes. Understanding this memory is essential to allow the use of bioengineered organs derived from diseased ECM, offering a solution to the critical organ shortage. To address this, we investigated whether the memory of ECM derived from metabolic dysfunction-associated steatohepatitis (MASH) livers impacts disease establishment following transplantation. Partial orthotopic transplantation of decellularized MASH-derived ECM was performed in control and MASH recipients. Histological analysis confirmed complete recellularization; however, molecular and metabolomic analyses revealed that MASH ECM stimulated de novo lipogenesis and fibrogenesis, inducing impaired lipid oxidation and mitochondrial dysfunction, which contributed to disease progression by promoting altered lipid turnover and inflammatory signalling. In vitro analysis revealed that MASH-ECM disrupted calcium signalling and promoted the maintenance of a pathological phenotype. Although derived from diseased livers, human ECM can promote cell survival and permissiveness. In conclusion, diseased ECM memory impacts cell physiology, suggesting that the scaffold can drive disease progression independently of the cellular environment. Thus, further studies are needed to develop strategies capable of reversing the pathological memory associated with ECM to allow its use in liver transplantation.

Graphical Abstract.

1. Introduction

Liver diseases account for 4% of all deaths annually, comprising over two million deaths worldwide. ¹ Liver transplantation (LT) is the most effective life-saving treatment for end-stage liver disease (ESLD), and the increasing burden of ESLD heightens the importance of this treatment for chronic liver disease (CLD) patients. ² The history of LT began in 1967, when the first successful deceased donor LT was performed by Thomas Starzl and colleagues. Since then, the success rates of this procedure have steadily increased over the decades due to innovations in organ preservation strategies, surgical techniques, perioperative care, and immunosuppression.^3,4 However, the discrepancy between the increased demand for organs and the available supply from deceased donors has created an organ shortage scenario, reflected in large transplantation waiting lists, in which insufficient functioning results in high mortality rates of patients depending on transplants.^3–5 To address this disparity, various alternatives are being scientifically explored, including the development of acellular liver scaffolds (ALS) based on decellularized ECMs derived from decellularization techniques.^6–8

The in vivo ALS exploration burden began in 2015, when the first successful orthotopic transplantation of ALS into mice was reported, confirming that decellularized matrices can be transplanted into healthy recipient animals. ⁹ This was followed by the successful transplantation of ALS in rats after partial hepatectomy, highlighting the availability of in vivo recellularization of decellularized grafts. ¹⁰ In addition to the observation of the interaction between cells and the ECM of healthy scaffolds, In vitro experiments reported by Miyauchi and colleagues showed that the decellularized ECM obtained from cirrhotic rat livers promotes the acceleration of the progression of hepatocellular carcinoma cells to a more aggressive and proliferative profile. ¹¹ Similar results were observed by Mazza and colleagues when human cirrhotic scaffolds were recellularized with HepG2 cells. ¹² In addition to the liver, the influence of a fibrotic ECM on cells has also been observed in scaffolds originating from other organs. Booth and colleagues also observed that fibroblasts were activated and differentiated, acquiring a myofibroblast phenotype when cultured in human fibrotic lung scaffolds. ¹³

In this context, the combination of biochemical and structural components of the ECM can alter the phenotype and genotype of the cell. In 2023, data from our group in rat models showed that decellularized liver ECM derived from healthy animals can be successfully transplanted into cirrhotic recipient rats, enabling in vivo regeneration by stimulating the recruitment and proliferation of endogenous cells. ⁷ Interestingly, it was found that the transplanted healthy liver ECM was not affected by the diseased liver and created an environment that stimulated healthy cell growth, regeneration, and repair. ⁷ Together, all these data pointed out the peculiar characteristics of ECM suggested by us as “ECM memory”. The extracellular matrix encodes biochemical and structural signals, in addition to matrix-bound molecules, which might include growth factors, that shape cellular genotype, phenotype, and physiology. These ECM-mediated cues or “zip codes” direct the behaviour of resident and interacting cells, such that homeostatic matrix properties promote normal tissue development and function,^14,15 whereas altered or diseased matrix states drive maladaptive cellular responses and diseased tissue formation.^11,13,16

Taking into account the clinical reality, the possible source of organs for ALS generation would be discarded marginal livers, which represent organs that do not have any potential direct application for transplantation.^7,9,17 Epidemiological data have shown that the global prevalence of Metabolic dysfunction-associated steatotic liver disease (MASLD) in the period from 1990-2019 was about 30.1%, representing an increase of 50.4% over the past 3 decades.^18,19 In the period from 2002-2019, Metabolic dysfunction-associated steatohepatitis (MASH) was the leading non-hepatocellular carcinoma (HCC) cause of CLD among patients in the LT waiting list registered in the Scientific Registry of Transplant Recipients (SRTR) system.^18,20

Considering the important contribution of MASLD and MASH complications for the rising in LT indication rates, it is possible to hypothesize that steatotic marginal livers derived from ESLD patients discarded after LT could be a relevant organ source for ALS manufacturing, with the aim of being further utilized for the development of artificial bioengineered organs, which could enable a solution for the liver supply shortage issue.^17,18 Nevertheless, bioengineering approaches for the development of functional ALS derived from diseased organs are still under investigation, and the impact of the transplantation of diseased-derived ALS in diseased recipients is unknown. Thus, to continue the progress on the development of this promising technology for LT-indicated patients’ treatment it is necessary to investigate whether a diseased ECM can be used as an ALS in transplantation.

In addition, in this study we also sought to expand the understanding of ECM memory through a new set of questions: a) whether a MASH ECM can be transplanted into a diseased recipient; b) whether a MASH ECM can be recellularized when transplanted into a diseased recipient; c) whether the diseased ECM impacts in recipient liver metabolism, functional and injury markers; d) whether a MASH ECM can retain memory and impact in vivo recellularization and promote steatohepatitis; e) whether a MASH ECM can impact healthy cell phenotypes, inducing steatohepatitis-like features.

2. Materials and methods

2.1. Experimental design

Liver steatohepatitis was induced in female Wistar rats through high-fat diet administration in association with sucrose and intraperitoneal injections of carbon tetrachloride (CCl₄) (see section 2.3 below). Steatohepatitis-induced animals were divided into two cohorts, donor and recipient rats (Figure 1). Donor animals underwent a total hepatectomy procedure. Excised livers were decellularized to obtain MASH ECM. Recipient animals were submitted to a partial hepatectomy of the median lobe (10%), and a MASH ECM or a Healthy ECM fragment was transplanted into the excised site. Healthy animals were also submitted to MASH ECM orthotopic transplantation. After 30 days, recipient animals were euthanized. Transplanted liver and recellularized MASH ECM samples were submitted to multiple analyses, including histology, RT-qPCR, metabolomics, and ex vivo analysis.

Figure 1.

Experimental design. Liver steatohepatitis was induced in female Wistar rats through high-fat diet administration in association with intraperitoneal injections of carbon tetrachloride (CCl₄). The steatohepatitis-induced animals were divided into two cohorts: donor and recipient rats. The donor animals underwent total hepatectomy. The excised livers were decellularized to obtain MASH ECM. Recipient animals underwent partial hepatectomy of the median lobe (10%), and a MASH ECM or healthy ECM fragment was transplanted into the excised site. Healthy animals were also subjected to MASH-ECM orthotopic transplantation. After 30 days, the recipient animals were euthanized. Transplanted liver and recellularized MASH ECM samples were subjected to multiple analyses, including histology, metabolomics, qRT-PCR, and in vitro and ex vivo analyses. Human MASH/HCC ECM was used to evaluate whether it would provide a supportive microenvironment for cellular development, despite being a pathological ECM.

2.2. Animals

All animal procedures carried out in this study were approved and followed the animal care guidelines of the Animal Use Ethics Committee of the Health Science Center of the Federal University of Rio de Janeiro, Brazil (CEUA CCS UFRJ 69/24). Female Wistar rats, ranging in age from 8 to 16 weeks, were randomly assigned into the following groups: MASH liver donor rats (n=15), healthy liver donor rats (n=15), MASH recipient rats (n=20) and healthy recipient rats (n=10). Animals were kept on a 12 h light/dark cycle, 25 ◦C mean ambient temperature, and 55 +/- 5% humidity. The rats were fed with a high-fat diet, specific for steatohepatitis induction, and water ad libitum. Their body weight was monitored weekly. Anesthesia was induced by inhalation of 3-4% isoflurane (Isoforine®, Cristália, São Paulo, Brazil) and maintained by inhalation of 1-2% isoflurane, associated with an oxygen dose of 0.3-0.5 L/min.

2.3. Steatohepatitis induction

Steatohepatitis was induced by a high-fat diet containing 45% kcal from palm oil derived fat, 20% kcal from fructose, and 2% cholesterol (Pragsoluções Biociências, São Paulo, Brazil) to female Wistar rats aged from 8 to 16 weeks for 4 months. In addition to the diet, the animals received 6% sucrose in their drinking water ad libitum throughout the entire protocol (Figure 2(a)). One week prior to the ending, animals received intraperitoneal injections of carbon tetrachloride (CCl₄; 1 mL/kg in olive oil, 1:1) on alternate days for a total of three injections.

Figure 2.

Characterization of MASH rat livers. (a) MASH livers were obtained after high-fat diet administration in Wistar rats for 120 days. (b) Body weights of Wistar rats during the MASH induction protocol. (c) HDL, LDL, TGC, Albumin, ALT and AST levels in the serum of Wistar rats before and after MASH induction (n= 20). (d) Blood glucose levels in healthy and MASH rats. (e) Macroscopic and microscopic views of healthy (control) and MASH livers. Hematoxylin and Eosin, Sirius red, and Oil red O-stained sections from control and MASH livers. Scale bars: 50 μm. (f) Percentage of microvesicular, macrovesicular, and total steatosis in MASH livers. (g) PCA score plot showing the separation between CTRL_Liver and MASH_Liver samples based on metabolomic profiles. (h) Heatmap of discriminant metabolites illustrating the hierarchical clustering of samples and metabolites (red: higher abundance; blue: lower abundance). (i) Bean plots of representative metabolites (PE(18:1 (9Z)/0:0), palmitoleic acid, 9,10-DiHOME, and N-arachidonoyl-L-serine) highlighting differences in relative abundance between the CTRL_Liver (n=5) and MASH_Liver (n=4) groups; dots represent individual samples, and diamonds indicate group means The results are presented as mean ± SD. Statistical significance between the control and MASH groups was determined using the Student’s t-test where *p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001. CTRL, Control group; MASH, metabolic dysfunction-associated steatohepatitis.

2.4. Liver procurement

2.4.1. Rat liver procurement

Animals from both the MASH and healthy donor groups (n=10 each) were euthanized by isoflurane inhalation overdose (4% isoflurane, associated with an oxygen dose of 1-2 L/min). A transverse abdominal incision followed by laparotomy was executed and the portal vein (PV) was identified, isolated, and cannulated with a 24-gauge catheter (Angiocatch®, BD, São Paulo, Brazil). The liver was subsequently excised and transferred to a sterile 60-mm Petri dish for subsequent decellularization process (Figure 1).

2.4.2. Human liver procurement

Liver specimens (n=3) were obtained from patients with hepatocellular carcinoma (HCC) arising from the setting of MASH. Those samples were from patients who underwent a curative intent surgical resection or from patients who underwent liver transplantation. The use of liver specimens was approved by the medical ethical council of the Clementino Fraga Filho University Hospital (HUCFF/UFRJ; CAAE: 16079319.00000.5257). After tissue collection, liver specimens were washed with saline solution 3 times for 5 min each and then stored in -80C until decellularization steps.

2.5. Decellularization

2.5.1. MASH rat livers decellularization

MASH-rat livers (n=10) were decellularized using a peristaltic pump (Masterflex Cole Parmer L/S, model 7522-20) at a flow rate of 7 mL/min. Livers were first subjected to continuous perfusion with distilled water for 2 h, followed by perfusion of 1% (v/v) Triton X-100 (Sigma-Aldrich, Saint Louis, MO, USA) through the PV for 2 h. Subsequently, a 1% (w/v) sodium dodecyl sulfate (SDS; Synth, São Paulo, Brazil) solution was perfused for 48 h. At the end of the process, MASH ECM-rat livers were perfused with distilled water overnight, followed by sterilization: 0.1% (v/v) sterile peracetic acid (PAA; SigmaAldrich 433241) at 3 mL/min for 1 h and by two washing cycles with 1% (v/v) sterile phosphate-buffered saline (PBS; Nova Biotecnologia 13-30262-05; São Paulo, Brazil) and stored in 0.1% (v/v) sterile PAA (SigmaAldrich 433241) at 4 °C until transplantation. Macroscopic and microscopic comparisons were made with healthy ECM obtained from the decellularization of healthy livers (n=10) in accordance with the protocol described by Dias et al., 2023. ⁷ To verify the efficiency of the decellularization process, DNA quantification was performed. DNA was isolated from 25 mg of wet tissue (n=4) using the DNeasy® Blood & Tissue Kit (Qiagen, Hilden, Germany), and total DNA content was measured withNanoDrop Lite Plus Spectrophotometer (Thermo Fisher Scientific; Massachusetts, USA).

2.5.2. Human liver decellularization

The decellularization protocol was based on the procedure described by Thanapirom et al. (2021). ²¹ First, liver specimens were cut into 48 cubes and stored individually at -80 C in 2 mL microtubes. They were then subjected to a water bath at 37 °C for 45 min. The microtubes were then filled with approximately 1.5 mL of 1X phosphate-buffered saline (PBS) and subjected to a water bath again for 15 min at 37 °C. After complete thawing, human liver cubes were subjected to the physical and chemical decellularization process using Tissue Lyser III (QIAGEN), which consists of short agitation cycles with a frequency from 15 to 20 Hz. During cycles, the microtubes were filled with 1.5 mL of ultrapure water, alternating with 1.5 mL of a specific RM (reagent mix) decellularization solution that consists of 4.3% (w/v) sodium chloride, 0.5% (w/v) sodium lauryl sulfate, 0.003% (v/v) Triton X-100, 3% (w/v) sodium deoxycholate and trypsin. EDTA 0.0025% (v/v). To verify the efficiency of the decellularization process, DNA quantification was performed as described above.

2.6. ECM orthotopic transplantation

Animals in the recipient cohort underwent partial median-lobe hepatectomy (10%). A partial ECM graft was then transplanted into the resected site and sutured with a continuous 6-0 silk suture. Transplanted animals were euthanized 30 days after the procedure. Partial orthotopic transplantation was performed in the following groups: Tx MASH ECM-MASH (n=10), Tx MASH ECM-CTRL (n=10), and Tx CTRL ECM-MASH (n=10).

2.7. Recellularization

CTRL ECM (n=4), MASH ECM (n=4) and human MASH/HCC ECM (n=3) were cut into 48 cubes (each group) and then subjected to prior sterilization with 0.1% (v/v) peracetic acid and 4% ethanol (Proquimios, Rio de Janeiro, Brazil) in ultrapure water. Sterile 1X PBS was used to wash the scaffolds. Sterilization was performed using an orbital shaker at 700 RPM in different cycles, followed by the transfer of the sterilized scaffolds to a non-adherent 48-well-plate previously filled with 1 mL of DMEM-High glucose culture medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (5,000 Units/mL Penicillin; 5,000 µg/mL Streptomycin). Overnight incubation was performed prior recellularization to ensure aseptic handling. For recellularization, HepG2 cells were resuspended in DMEM-High glucose culture medium (433241 Sigma-Aldrich™; Saint Louis, Missouri, USA) supplemented with 10% fetal bovine serum (12657-029 Sigma-Aldrich™) - FBS - and 1% Penicillin-Streptomycin 5,000 Units/mL Penicillin; 5,000 µg/mL Streptomycin (15070063 Sigma-Aldrich™;). Recellularization of each scaffold was performed from cell grafting at a density of 5 × 10⁵ cells in 20 µL - with the scaffold positioned in the center of a cell-repellent surface 96-well-plate (655970 Greiner Bio-One™; Frickenhausen, Germany). Recellularization then consisted of 20 µL of cell suspension grafted onto the top of the scaffold with incubation at 37 °C for 30 min. The step was repeated four times to ensure better cell adhesion to the scaffold. Finally, 140 µL of the complete culture medium were added to each well for overnight incubation at 37 °C. Subsequently, the recellularized scaffolds with HepG2 cells were transferred into a cell-repellent surface 48-well-plate (677970 Greiner Bio-One™) 48-well-plate previously filled with 1 mL of complete medium.

2.8. ECM-conditioned medium preparation

Liver specimens derived from MASH and Healthy rats (n=24 from 3 MASH and CTRL livers) were obtained during orthotopic liver transplantation performed in recipient animals. The excised tissue was collected during partial hepatectomy and immediately preserved at −80 °C. Following the protocol previously described by Thanapirom et al. (2021), ²¹ the preserved tissue samples were sectioned into cubic fragments 5mm × 5 mm × 5mm) and individually transferred into microcentrifuge tubes. Cubes (n = 24) were subjected to two sequential thawing steps in a water bath: first at 37 °C for 45 min, followed by immersion in 1× PBS for 15 min at 37 °C. The procedures for decellularization and sterilization were performed in accordance with the protocols previously described for human tissue samples. MASH ECM-rat cubes were then placed in a tissue-treated 6-well-culture plate (Corning 3506) filled with 3 mL complete culture medium/well at 37 °C in an incubator under 5% CO₂. After ten days, both MASH ECM and Healthy ECM conditioned medium were collected. Subsequently, HepG2 cells were seeded onto a 24-well culture-plate (Corning 3524) with sterile coverslips. Media was exchanged every 2-3 days and cells were cultured until 75–80% confluence.

2.9. Staining to trace lipids

BDP® 630/650 lipid stain (Lumiprobe Corporation Westminster, Maryland) was used as a fluorescent dye for visualization of neutral lipid droplets in HepG2 cell line cultured with control medium, MASH and Healthy ECM-conditioned medium. ²² The dye was dissolved in dimethyl sulfoxide (DMSO; SigmaAldrich D8418). For staining, the stock solution (10 mM) was diluted in 1X PBS (Nova Biotecnologia 13-30262-05; São Paulo, Brazil) to a final working concentration of 10 µM. HepG2 cells grown on glass coverslips to 75–80% confluence were incubated with 100 µL of the working solution at room temperature for 30 min, then washed thrice with 1X PBS for 5 min each and imaged by fluorescence microscopy using ​Invitrogen EVOS M5000 (Thermo Fisher Scientific). HepG2 cell line cultured with complete growth medium as control were separated into negative - growth medium only - and positive - 30 µM oleic acid - control groups. The positive control group was incubated 30 μM oleic acid (Thermo Scientific AC270290050) for 24 h before staining.

2.10. Biochemical analysis

Blood samples (500 μL) were collected by cardiac puncture into clot-activator gel microtubes (Vacuplast, São Paulo, Brazil). After collection, samples were centrifuged at 1,400 × g for 10 min, and the resulting serum was stored at −20 °C. Serum levels of albumin (ALB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), high density lipoprotein (HDL), low density lipoprotein (LDL), and triglycerides (TGC) were quantified using a semi-automatic biochemical analyzer (Bio 200, Bioplus, Rio de Janeiro, Brazil). Analyses were performed with the following kits: ALB (Ref. 19), AST (Ref. 109), ALT (Ref. 108) (Labtest, Minas Gerais, Brazil), HDL (Ref. K071-23), LDL (Ref. K088-1), and TGC (Ref. K117-3) (Bioclin, Minas Gerais, Brazil). For blood glucose test, the animals underwent 12 hours fast, then a glucose solution (1g/Kg/body mass) was administered by gavage, and the blood was collected from the tail 30, 60, 90 and 120 min after gavage.

2.11. Intracellular Ca²⁺ signalling imaging in recellularized scaffolds

HepG2-recellularized ECM (MASH ECM-rat and human, and CTRL ECM) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and kept in culture conditions until imaging experiments were performed. Prior to Ca²⁺ imaging, scaffolds were gently rinsed with pre-warmed DMEM, and excess medium was removed. Care was taken to preserve the structural integrity of the recellularized matrices and to avoid detachment of the HepG2 cells. Each scaffold was incubated with 12 µM Fluo-4 AM (Invitrogen, Grand Island, NY, USA) for 30 minutes at 37°C in a humidified 5% CO₂ incubator. After loading, scaffolds were washed with HEPES 1X buffer to remove excess dye and mounted onto glass coverslips for imaging. The recellularized scaffolds were transferred to a custom-built perfusion chamber positioned on the stage of a ZEISS-LSM 880 Airyscan Confocal Microscope. Fluorescence excitation was achieved using a 488-nm laser, and emitted fluorescence was collected using a 515/30-nm bandpass filter. Imaging was performed using a 20× objective lens, and intracellular Ca²⁺ dynamics were monitored in individual HepG2 cells using time-lapse acquisition. Baseline fluorescence was recorded for 30 seconds in HEPES 1X buffer. Scaffolds were then perfused with 50 µM ATP (Sigma Aldrich for 5 minutes to assess intracellular Ca²⁺ changes. To data analysis, individual regions of interest corresponding to single HepG2 cells within the scaffold were analyzed with ImageJ, and time-dependent changes in fluorescence were quantified across the entire recording period. Data acquisition and analysis were performed using standardized protocols and predefined criteria to minimize potential bias. Fluorescence intensity values following stimulation (F) were normalized to baseline fluorescence (F₀), and Ca²⁺ responses were expressed as (F/F₀) × 100.

2.12. Metabolomics analysis

2.12.1. Sample preparation

Liver tissue samples (CTRL Liver, n=5; MASH Liver, n=4; Tx MASH ECM- MASH, n=5; Tx MASH ECM- CTRL, n=9) were processed as previously described with minor modifications. ²³ Briefly, 200 mg of intact liver tissue was homogenized in phosphate-buffered saline (PBS) at a ratio of 1:3 (w/v). Homogenization was performed using a bead ruptor porcelain bead for three 40 s cycles at 4 m/s, with samples kept on ice between cycles. For metabolite extraction, 100 µL of homogenate were mixed with 1.5 mL of ice-cold isopropanol: methanol (3:1, v/v) containing the internal standards furosemide and p-fluoro-DL-phenylalanine (0.2 µg/mL). Samples were vortexed, centrifuged at 15,000 rpm for 15 min at 4 °C, and the supernatant was collected. After a second centrifugation under identical conditions, the supernatant was dried under vacuum and reconstituted in 100 µL of acetonitrile: water (3:1, v/v). To improve metabolite recovery, a second extraction was performed on the remaining protein pellet using methanol: water (1:1, v/v). After vortexing and centrifugation, the supernatant was collected, dried under vacuum, and reconstituted in acetonitrile: water (3:1, v/v). Extracts from both steps were combined, centrifuged to remove particulates, and transferred to LC-MS vials for analysis.

2.12.2. LC–HRMS analysis

Untargeted metabolomic analyses were performed using a Vanquish UHPLC system coupled to an Orbitrap Exploris 480 high-resolution mass spectrometer (Thermo Scientific), equipped with a heated electrospray ionization source operating with fast polarity switching between positive and negative modes. Data was acquired over an m/z range of 50–1000. Chromatographic separation was achieved on an Acquity HSST3 C18 column (100 × 2.1 mm, 1.8 µm; Waters Corporation). The column temperature was maintained at 35 °C and the autosampler at 4 °C. The flow rate was set to 400 µL/min, and 3 µL of each sample were injected. The mobile phases consisted of ultrapure water (A) and acetonitrile (B), both containing 0.1% formic acid. The gradient elution was performed as follows: 85% A and 15% B at 0 min; the composition was linearly changed to 60% A and 40% B at 3.0 min; then to 5% A and 95% B at 13.0 min, which was maintained until 16.0 min; the system was returned to 85% A and 15% B at 16.5 min and re-equilibrated under these conditions until 20.0 min. The ionization parameters were as follows: spray voltage was set to 3500 V in positive mode and 2500 V in negative mode. The ion transfer tube temperature was set to 325 °C for both modes. Sheath gas flow was set to 50, auxiliary gas to 10, and sweep gas to 3 for both polarities. The maximum spray current was set to 10 in both positive and negative modes. The vaporizer temperature was maintained at 0 °C for both modes. The Automatic gain control (AGC) detection time was set to 20.0 ms in standard mode and 4.0 ms in High Mass (HM) mode. AGC and injection times were optimized to ensure high mass accuracy and sensitivity throughout the analysis.

2.12.3. LC-MS data processing and statistics

LC-MS raw data files were converted to mzML format using MSConvert. Data Processing, including peak picking, deconvolution, grouping and alignment was performed using MS-DIAL (v4.9.22). A peak list was extracted with a signal-to-noise threshold of 10 and with minimum peak height of 5e5. Missing values were filtered prior to statistical analysis and only features detected in 75% of the samples within at least one experimental group were preserved for downstream analysis. Remaining missing values were assumed to originate from below detection limit and imputed by replacing them with half of the minimum observed value for each feature. Data was normalized using median normalization, scaled using Auto Scaling and log₂-transformed to reduce systematic variation between samples. Detailed processing parameters are provided in supplemental table 2. Processed data was submitted to MetaboAnalyst (v6.0) for univariate and multivariate statistical analysis and visualization, including fold change, ANOVA, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), hierarchical clustering heatmap and violin plots. Metabolites were putatively annotated by MS level 2 by matching exact mass (MS1 tolerance of 0.005 Da; MS2 tolerance of 0.05 Da) and spectra against internal library and NIST database. ²⁴ The identification of shared metabolites and Venn diagram was analyzed using the LC–MS intensity matrix generated in MS-DIAL and analyzed in LibreOffice Calc. Features were considered present when detected (intensity >0) in at least 50% of the samples within each group (≥5/10 for Tx MASH ECM-CTRL and ≥3/5 for Tx MASH ECM-MASH). Shared metabolites between groups were identified using conditional counting functions. The resulting lists were used to generate a Venn diagram in Intervene to visualize unique and overlapping metabolic features.

2.13. Histology analysis

For histological evaluation, human samples, biopsies from transplanted livers, decellularized and recellularized ECM were formalin-fixed (4%, 24h), paraffin-embedded and sectioned (4µm). Tissue morphology was assessed using hematoxylin and eosin (H&E) staining (Merck, São Paulo, Brazil), ECM deposition was evaluated using Sirius Red staining (365548; Sigma-Aldrich), and lipid accumulation was assessed using Oil Red O staining (O0625-25G; Sigma-Aldrich). Images were obtained using EVOS microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific).

2.13.1. Oil red staining

The Oil Red O (O0625; Sigma-Aldrich) stock solution was prepared by dissolving 0.25 g of Oil Red O powder in 50 mL of isopropanol. The solution was subsequently filtered through a 0.2 μm membrane and stored at 4°C. The working solution was freshly prepared by mixing the stock solution with distilled water (dH2O) at a 6:4 volume ratio (e.g., 3 mL of stock solution and 2 mL of dH2O). This mixture was incubated at room temperature for 20 minutes and filtered twice through a 0.2 μm filter prior to application.

To investigate ECM-bounded lipids, fresh healthy ECM and MASH ECM were formalin-fixed (4%, 24h), embedded in OCT (Optimal Cutting Temperature compound) media and stored overnight at -80C. Frozen tissue sections (4µm) were obtained and then washed twice with 1X phosphate-buffered saline (PBS). Samples were then incubated with 1 mL of the Oil Red O working solution per well for 10 minutes at room temperature. Following incubation, the staining solution was removed, and the tissue samples were immediately washed four times with dH2O. To prevent the tissues from drying out, they were kept submerged in dH2O, and microscopic images were captured immediately.

2.14. Immunohistochemistry analysis

For the immunohistochemistry analyses, following deparaffinization and rehydration, the sections were immersed in Tris-buffered saline with 0.1% Tween (TBS-T) for 10 minutes. Then, the slides were submitted to the antigen retrieval step. Antigen retrieval was performed by immersing the slides in pre-heated sodium citrate buffer (pH 6) in a microwave (MTO30; Electrolux) (lower power) at 90-95 °C for 10 min. The slides were then allowed to return to room temperature for approximately 1 hour, followed by a wash in distilled water for 2 min. After that, the slides were exposed to hydrogen peroxide (3%) for 15 min. Excess peroxide was removed with TBS-T for 3 min. Then, the sections were incubated in 2.5 % horse serum albumin for 15 min. Subsequently, the slides were incubated with primary antibodies against desmin (1:50; D1033; Merck) and α-smooth muscle actin (α-SMA) (1:100; Ab7817; Abcam, Cambridge, MA, USA) for 2h at 4 °C. Sections processed in absence of the primary antibodies were used as negative controls. After 2 h, the slides were washed with TBS-T (3 min) and incubated with the Novolink Polymer Detection System (RE7260-CE, Leica Biosystems Newcastle upon Tyne, UK). First, the slides were incubated with post-primary antibody for 30 min. After that, the excess of post-primary solution was removed with TBS-T for 3 min. Then, the slides were incubated with novocastra polymer solution for 30 min and washed with TBS-T for 3 min. The reaction was developed using 3,3-diaminobenzidine (DAB) (ImmPACT DAB Substrate Kit; REF SK-4105) and subsequently stopped with water. Finally, the sections were counterstained with hematoxylin and mounted with Entellan. Images were acquired using an EVOS microscope (Invitrogen EVOS M5000, Thermo Fisher Scientific).

2.15. Scanning electron microscopy

MASH ECM sections were washed three times with 0.1 M sodium cacodylate buffer (pH 7.2) and fixed in 2.5% glutaraldehyde for 24 hours. Subsequently, the samples were washed three times in 0.1 M sodium cacodylate buffer (pH 7.2) and post-fixed for 1 hour in 1% osmium tetroxide (OsO₄) solution in 0.1 M sodium cacodylate buffer (pH 7.2); then, they were dehydrated in an increasing series of ethanol (30%, 50%, 70%, 90%, and 100%). The fragments were subsequently dried in a critical point dryer (Autosamdri-815; Tousimis, Cambridge, MA, USA), coated with gold in an ion sputtering apparatus (Cressington 108, Watford, UK) and observed under a scanning electron microscope (JEOL-JSM-6390-LV, Akishima, Tokyo, Japan).

2.16. Second harmonic generation

Second harmonic generation (SHG) signals recorded at 470 nm were used to capture architectural features of extracellular matrix collagen fibrils (n=3 per group) through laser excitation at 940 nm, while two-photon excited fluorescence (TPEF) signals recorded were used to visualize tissue structures. Images were acquired at 20× magnification with 512×512-pixel resolution, each image had a dimension of 200 µm × 200 µm.

2.17. Quantification of extracellular matrix fiber length and orientation

Quantitative analysis of ECM fibers was performed using ImageJ/Fiji (NIH, USA), following an image-processing workflow adapted from previously validated approaches for automated fiber analysis, including the General Image Fiber Tool (GIFT) methodology adapted from Huling et al. ²⁵ Briefly, fluorescence images were acquired under identical acquisition parameters and analyzed in a blinded manner. Images were converted to grayscale by extracting the red fluorescence channel, corresponding to ECM fiber labeling. Spatial calibration was performed using the embedded scale bar (50 µm), and this calibration was applied uniformly across all images prior to analysis. To reduce background noise while preserving fiber morphology, images were smoothed using a Gaussian blur filter (σ = 1 pixel). Fiber structures were segmented using automatic global thresholding (Otsu method), generating binary images in which ECM fibers were represented as foreground objects. Small, isolated particles and background artifacts were excluded using size-based filtering to ensure that only continuous fibrous structures were retained for quantitative analysis. Binary images were subsequently processed using the Skeletonize function in ImageJ, reducing fibers to one-pixel-wide representations while preserving their topology. This approach is consistent with skeleton-based fiber analysis strategies commonly employed for fibrous networks and is conceptually aligned with the edge-based and morphology-driven framework implemented in the GIFT macro. Quantification of individual fibers was performed using the Analyze Skeleton plugin, which enables the identification of individual fiber segments within the skeletonized image. For each detected fiber, the following parameters were extracted:

• (a) Fiber length, calculated as the cumulative length of the skeletonized segment and converted from pixels to micrometers using the calibrated image scale;

• (b) Fiber orientation (angle), determined from the principal axis of each fiber segment and expressed in degrees.

Consistent with the statistical strategy recommended for automated fiber analysis workflows, fiber-level measurements were averaged per image, such that each image represented one independent biological replicate. Group-level comparisons between control and fibrotic (MASH) liver samples were therefore performed using image-level mean values (n = 3 per group), thereby avoiding pseudo-replication arising from the large number of fibers detected within individual images. This adapted ImageJ-based pipeline enables reproducible and unbiased quantification of ECM fiber length and orientation and follows the same core principles of automated, morphology-driven fiber analysis validated by Huling et al. for fibrous biomaterials. ²⁵

2.18. Hidroxyproline assay

Hydroxyproline content in both MASH and CTRL ECM (n=3 per group) was assessed using a commercial Hydroxyproline Assay Kit (MAK008; Sigma-Aldrich, St. Louis, MO, USA) in accordance with the manufacturer’s protocol. Briefly, 10 mg of each ECM sample was homogenized in 100 µL of ultrapure water and transferred to pressure-tight polypropylene vials. Samples were subjected to acid hydrolysis by adding an equal volume of concentrated hydrochloric acid (000154.06; Vetec Química Fina, RJ, Brazil) followed by incubation at 120°C for 3 hours. Post-hydrolysis, the homogenates were thoroughly mixed and centrifuged at 10,000 × g for 3 minutes. The clarified supernatant (5 µL) was plated in technical duplicates onto a 96-well flat-bottom plate (3590; Corning, NY, USA). Hydroxyproline concentration was evaluated via oxidation with Chloramine-T, followed by a colorimetric reaction with 4-(dimethylamino)benzaldehyde (DMAB) to generate a measurable chromophore. Absorbance was obtained at 560 nm using a Varioskan microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The amount of hydroxyproline present in the samples was determined from the standard curve as follows: Concentration of Hydroxyproline: Sa/Sv = C, where Sa = Amount of hydroxyproline in sample (µg) from standard curve; Sv = Sample volume (µL) added into the wells; and C = Concentration of hydroxyproline in sample.

2.19. qRT-PCR

Pro-fibrogenic, tissue remodeling and metabolic targets were assessed by real-time quantitative PCR (RT-qPCR) targeting Col1a1, Timp, Acta2, Acaca, ApoB, PPARα, CPT1α, Srebp1c and GAPDH. Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany). RNA concentration and purity were determined with NanoDrop Lite Plus spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the High-Capacity Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) following the manufacturer’s instructions. Quantitative PCR reactions were performed on a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems). Each reaction was conducted in duplicate with a final volume of 15 µL, containing 10 ng of cDNA, 7.5 µL of 2×Power SYBR Green Master Mix (Applied Biosystems), RNase-free water and 0.5 µL each of forward and reverse primers (10 µM). Thermal cycling conditions consisted of an initial incubation at 50 °C for 2 min, followed by enzyme activation at 95 °C for 10 min, and 40 cycles of denaturation at 95 °C for 15 s, primer annealing at gene-specific temperatures (listed in Supplemental Table S1) for 30 s, and extension at 72 °C for 30s. Gene expression levels were normalized to GAPDH as the endogenous control. Relative transcript abundance was calculated using the 2 -(ΔΔCt)) method, where ΔΔCt was determined by comparing experimental samples with control livers. Data are presented on a logarithmic scale (base 10). Primer sequences for the analyzed genes are listed in Supplemental Table S1.

2.20. Statistical analysis

Statistical analyses were performed through GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA). Descriptive data are expressed as means ± standard deviation (SD). Group comparisons were assessed using paired or unpaired student’s t-tests with Welch’s correction. One-way ANOVA followed by Tukey’s post hoc test, or two-way ANOVA followed by Šídák’s multiple comparisons test were applied. Statistical significance was set at p < 0.05.

3. Results

3.1. High fat diet + CCl₄ induces steatohepatitis in rats

The first set of experiments was performed to obtain rats with steatohepatitis. Wistar rats subjected to a multiple hit MASH induction (Figure 2(a)) showed differences in body weight when compared to the control group (Figure 2(b)). Biochemical analysis showed that rats submitted to steatohepatitis protocol significantly elevated serum ALB, ALT, and AST (Figure 2(c)). In addition, a significant increase in TGC, HDL, LDL, were observed similar to the biochemical alterations described in human liver steatohepatitis elsewhere (Figure 2(c)). No differences were observed in serum glucose levels between groups (Figure 2(d)).

In contrast with control liver, macroscopic analysis showed that our protocol generates yellowish livers that were lighter in color with white regions suggestive of lipid deposition (Figure 2(e)). While hepatocytes aligned to sinusoids and absence of steatosis was observed in the control liver tissue, histological analysis of MASH livers stained with H&E confirmed inflammation, hepatocyte ballooning, macrovesicular and microvesicular steatosis (Figure 2(e)). After quantitative analysis we confirmed that most of the liver surface was occupied by macrovesicular steatosis (Figure 2(f)). This intense lipid deposition was confirmed by Oil red O staining (Figure 2(e)). In addition to that, Sirius red staining confirmed the presence of fibrosis distributed throughout the liver parenchyma (Figure 2(e)).

A comprehensive untargeted metabolomic analysis was performed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) in both positive and negative electrospray ionization modes (ESI⁺/ESI^-) to characterize metabolic alterations associated with MASH. Multivariate statistical analyses were applied to evaluate global metabolic differences between CTRL and MASH liver samples. Partial Component Analysis (PCA) (Figure 2(g)) revealed a clear separation between CTRL Liver and MASH Liver groups, indicating a robust disease-associated metabolic signature. Component 1 (32.7%) and Component 2 (23.4%) jointly explained a substantial proportion of the variance related to disease status. Model performance metrics demonstrated high classification accuracy (1.0), strong goodness-of-fit (R² up to 0.97), and satisfactory predictive ability (Q² ≈ 0.78–0.83). Permutation testing (1,000 permutations) confirmed the statistical robustness of the model, with the observed class separation significantly exceeding that obtained from randomized labels, minimizing the risk of overfitting. Hierarchical clustering analysis of discriminant m/z features further supported these findings, revealing distinct metabolomic profiles between CTRL Liver and MASH Liver groups (Figure 2(h)) based on combined criteria of statistical significance (p < 0.05) and magnitude of change (|log₂FC| ≥ 1), this approach allowed prioritizing biologically relevant metabolites, reducing the impact of variations of low pathophysiological relevance. The heatmap analysis (Figure 2(h)) highlights a predominant cluster of metabolites increased in MASH Liver samples, largely composed of lipid-related species such as phospholipids, lysophospholipids, fatty acids, and bioactive lipid mediators. The coordinated accumulation of these species is consistent with dysregulation of hepatic lipid metabolism, remodeling of cell membranes, lipotoxicity, and activation of inflammatory pathway processes central to the pathogenesis of MASH. Consistent with the heatmap analysis, the beanplot analysis (Figure 2(i)) highlights some specific lipids classes, such as PE (18:1 (9Z)/0:0) and palmitoleic acid increased in MASH samples, supporting enhanced phospholipid turnover and activation of de novo lipogenesis. In contrast, other lipid-derived mediators, including 9,10-DiHOME and N-arachidonoyl-L-serine, are significantly reduced in MASH samples. 9,10-DiHOME is an oxylipin derived from linoleic acid metabolism and participates in oxidative lipid signalling, whereas N-arachidonoyl-L-serine belongs to the family of endocannabinoid-related lipids involved in inflammatory and metabolic regulation. Their depletion suggests a disruption of lipid mediator signalling pathways that normally contribute to metabolic homeostasis and resolution of inflammation. These metabolites represent distinct lipid classes, indicating broad alterations in hepatic lipid metabolism associated with disease status.

3.2. MASH promotes biochemical and ultrastructural alterations in the ECM

MASH livers were submitted to decellularization using a detergent-based perfusion protocol adapted from our previous report (Dias et al., 2023). ⁷ While decellularization of healthy livers generated a translucent ALS, the decellularization of MASH livers originated a dense, opaque and white-colored MASH ECM (Figure 3(a)). The success of decellularization was confirmed by DNA quantification (MASH liver 294.8±30.8 ng/µL vs. MASH ECM 18.0±3.3 ng/µL) (Figure 3(b)). Weight quantification analysis showed that MASH ECM is significantly heavier than control ALS (Figure 3(c)). The collagen content was significantly higher in the MASH ECM than in CTRL ECM (Figure 3(d)). In addition to macroscopic, weight, and collagen content differences, histological analysis also showed important differences between control and MASH ECM (Figure 3(a)). Histological sections stained with H&E showed that MASH ECM is composed of a dense ECM, while a less abundant ECM is present in control ECM (Figure 3(a)). Sirius red staining showed that MASH ECM is composed of abundant collagen content. In contrast, control ECM showed less intense Sirius red staining (Figure 3(a)). SEM analysis also revealed important differences between control and MASH ECM (Figure 3(e)). While an ECM with preserved collagen fibers, following general micro and ultra-structure tissue preservation was observed in the control ECM, MASH ECM showed a dense ECM structure, with a different arrangement and conservation of the three-dimensional liver ECM (Figure 3(e)). These differences were also explored by SHG analysis (Figure 3(e)). A distinct pattern of organization and disposition of collagen fibers were observed in MASH ECM when compared to the control ECM (Figure 3(e)). The collagen network exhibited bundling in MASH ECM whereas the network appeared to be more organized with thinner fibres in control ECM. Significant differences were observed when the fiber length was compared between the groups (Figure 3(f)). No differences were observed regarding fiber angle when MASH ECM and CTRL ECM were compared (Figure 3(g)).

Figure 3.

Characterization of decellularized MASH ECM. (a) Macroscopic and microscopic views of decellularized control ECM and MASH ECM obtained after decellularization. Hematoxylin and Eosin, Sirius red-stained and Oil Red O sections from control and MASH decellularized ECM. Scale bars: 50 μm. (b) DNA content quantification in MASH Liver and MASH ECM (n=4). (c) Weights of decellularized control and MASH ECM (n= 4). (d) Hydroxyproline content quantification in control ECM and MASH ECM (n=3). (e) Ultrastructural characterization of control and MASH ECM. Scanning electron microscopy images at 800x and 1500x magnification obtained from control and MASH ECM. Scale bars: 20 and 10 μm, respectively. Second Harmonic Generation (SHG) images of control and MASH ECM (n=3). Scale bars: 50 μm. (f) Quantification of fiber length, expressed as the mean fiber length per image (n = three images per group). (g) Quantification of fiber orientation (angle), calculated as the mean fiber angle per image (n = 3 images per group). Statistical comparisons were performed using the image-level averages to avoid pseudo-replication. (h) Triglyceride content (ng/mg) in the hepatic tissue before and after decellularization (n= 5). (I) Triglyceride content (mg/dL) in the supernatant of MASH ECM, where PBS was used as a control (n= 5). The results are represented as mean ± SD. Statistical significance between the groups was determined using the Student’s t-test where *p <0.05, **p <0.01, ***p <0.001, and ****p <0.0001.

3.2.1. Lipids remain present in the MASH ECM even after decellularization

Given the nature of MASH disease, highlighted by lipid accumulation in the liver tissue, our next question focused on evaluating the presence of triglycerides in the MASH ECM. Oil Red O analysis revealed a higher presence of lipid droplets linked to the MASH ECM than in the control ECM (Figure 3(a)). To directly test the presence of triglycerides in the ECM, MASH ECM was subjected to triglyceride quantification after decellularization. The TGC assay showed that the MASH ECM contained triglyceride content at similar levels found in the MASH liver before decellularization (106.0±13.31 ng/mg; 101.4±9.72 ng/mg, respectively) (Figure 3(h)). Considering this result, we also evaluated the presence of triglyceride content in the supernatant (PBS) where MASH ECM was stored previously to the transplantation procedure to further explore the possibility that the triglyceride embedded in the MASH ECM could be released in the storage media. TGC assay analysis confirmed the presence of triglyceride content in the supernatant where MASH ECM was stored for 24h and the data demonstrated a significant release of triglyceride content in the media of MASH ECM when compared to control media (7.25±0.16 ng/mg; 25.35±10.09 ng/mg, respectively) (Figure 3(i)).

3.2.2. MASH ECM maintains steatohepatitis after transplantation

From a translational perspective and to evaluate the use of an ECM derived from MASH liver in transplantation we promoted a MASH ECM partial orthotopic transplantation in recipient MASH rats. Exploratory laparotomy after euthanasia 30 days after transplantation revealed the assembly of a soft textured, yellowish colored tissue connected to the recipient rat median lobe (Figure 4(a)). Histological analyses were performed to investigate whether MASH ECM was able to be recellularized after transplantation in MASH recipients. Histological sections stained with H&E revealed that MASH ECM was repopulated by a large number of cells, including mononuclear cells (Figure 4(a)). Notably, we observed that the recellularization was followed by the detection of vesicular structures similar to those found in the MASH liver before transplantation suggesting the presence of macrovesicular steatosis into MASH ECM 30 days after transplantation (Figure 4(a)). Sirius red staining revealed a colocalization between steatosis and collagen fibers (Figure 4(a)).

Figure 4.

Microscopic, macroscopic, and molecular analyses of MASH ECM after transplantation in healthy and MASH recipients. (a) Macroscopic and microscopic views of biopsies from the MASH ECM 30 days after transplantation in MASH recipients. Histological sections (20x, 40x magnification) were stained with hematoxylin and eosin and Sirius red. Scale bars: 50 µm and 20 μm, respectively. (b) Macroscopic and microscopic views of biopsies from MASH ECM 30 days after transplantation in control recipients. Histological sections (20x, 40x magnification) were stained with hematoxylin and eosin and Sirius red. Scale bars: 50 µm and 20 μm, respectively. (c) Macroscopic and microscopic views of biopsies from healthy ECM 30 days after transplantation in MASH recipients. Histological sections (20x, 40x magnification) were stained with hematoxylin and eosin and Sirius red. Scale bars: 50 µm and 20 μm, respectively. (d) Immunohistochemical staining of desmin and α-SMA – positive cells in MASH liver (positive control), MASH ECM transplanted in MASH recipients (Tx MASH ECM -MASH), MASH ECM transplanted in control recipients (Tx MASH ECM-CTRL), and healthy (CTRL) ECM transplanted in MASH recipients 30 days after transplantation. Scale bars: 75 μm. (e) Expression of genes linked to lipid metabolism and tissue fibrosis, Acaca, ApoB, CPT1α, PPARα, Srebp1c, Acat2, Col1a1, and Timp1, in the MASH liver and MASH ECM transplanted in MASH recipients. Gene expression levels were normalized to GAPDH as the endogenous control, and the data are presented on a logarithmic scale (base 10). Statistical significance between groups was determined using an unpaired Student’s t-test with Welch’s correction, where *p <0.05 and **p <0.01 (n= 7).

Given these results, our next question was to determine whether the steatosis maintenance could be promoted by the MASH ECM. To directly test the effect of a MASH ECM, we performed MASH ECM transplantation in recipient healthy rats. Histological analysis performed 30 days after transplantation revealed similar findings even when MASH ECM is transplanted in recipient healthy rats (Figure 4(b)). H&E-stained sections confirmed the presence of vesicular structures suggesting the presence of macrovesicular steatosis into MASH ECM 30 days after transplantation in healthy recipient rats (Figure 4(b)). Colocalization between steatosis and collagen fibers was also observed (Figure 4(b)).

To investigate the impact of MASH ECM on steatosis maintenance, an inverse experiment was performed in which a healthy ECM was transplanted into a MASH recipient rat (Figure 4(c)). Histological analysis performed 30 days after transplantation revealed that the cells in the healthy ECM did not maintain steatosis, as no vesicular structures were observed in the cells present in the healthy transplanted ECM. Sirius red-stained sections confirmed the presence of collagen fibers around the resident cells (Figure 4(c)).

As we observed ECM depositions in both transplanted MASH ECM, we performed immunohistochemistry analysis to confirm the presence of hepatic stellate cells and myofibroblasts into the MASH ECM after transplantation. We observed cells expressing both desmin and alpha smooth muscle actin (α-SMA) in MASH ECM after transplantation in MASH and CTRL recipients. The presence of α-SMA- and desmin-positive cells was also detected in the transplanted healthy ECM. MASH recipient liver was used as a positive control (Figure 4(d)).

In addition to histological evaluation, biochemical analysis was performed to investigate whether the MASH ECM could impact liver metabolism, function and injury parameters (Supplemental material). HDL levels significantly increased, while LDL levels significantly decreased 30 days after transplantation. No significant differences were observed in triglycerides content before and after transplantation (Supplemental material). In addition, MASH ECM transplantation significantly decreased albumin levels and did not promote changes in ALT and AST levels (Supplemental material 1A). Histological analysis of the recipient liver 30 days post-MASH ECM transplantation showed that the transplant did not reverse the histopathological characteristics of MASH. Macrovesicular and microvesicular steatosis and inflammatory infiltrates, in addition to fibrosis, remained in the native liver after transplantation (Supplemental material 1B). Therefore, although we observed a decrease in LDL and an increase in HDL levels after MASH ECM transplantation, these two parameters alone are not sufficient to confirm a good prognosis or evaluate transplantation success. Besides that, histological analysis confirmed that histopathological characteristics of MASH remain in the native liver after MASH ECM transplantation.

3.2.3. MASH ECM maintains the expression of genes involved in lipid metabolism and induces fibrosis

In light of findings suggesting that MASH ECM promotes steatohepatitis maintenance after transplantation, we investigated this aspect at the molecular level. To address this issue, the mRNA expression levels for lipid metabolism and pro-fibrogenic and tissue remodelling genes were also investigated. Our data showed no significant differences in Acaca, Apob, Cpt1a, Ppara, Acat2 and Timp1 gene expression when MASH livers and MASH ECM transplanted in MASH recipients were compared. However, a significant increase in Screbp1c, which plays a central role in controlling the expression of genes involved in de novo lipogenesis, and Col1a1, which is involved in ECM production and fibrosis, were observed (Figure 4(e)), confirming our previously obtained data from histological analysis.

3.2.4. MASH ECM acquires a metabolic profile similar to that of the MASH liver after transplantation

To explore whether de MASH ECM can impact the metabolic profile of resident cells and promote steatohepatitis, we collected MASH ECM transplanted into MASH recipient rats (Tx MASH ECM-MASH) for metabolomic analysis (Figure 5). The metabolomic data was compared with MASH ECM transplanted into healthy recipient rats (Tx MASH ECM-CTRL).

Figure 5.

Metabolomic similarities and differences between MASH ECM transplanted in MASH and Control recipients. (a) PCA score plot showing the separation between MASH ECM transplanted into control recipient rats (Tx MASH ECM-CTRL, n=9) and MASH ECM transplanted into MASH recipient rats (Tx MASH ECM-MASH, n=5) Liver samples based on metabolomic profiles. (b) Heatmap of discriminant metabolites illustrating the hierarchical clustering of samples and metabolites (red: higher abundance; blue: lower abundance). (c) Bean plots of representative metabolites (oleoyl-L-carnitine, LPE 18:3 and 5-Androsten-3. beta.,16. alpha.-diol-17-one) highlighting differences in relative abundance between Tx MASH ECM-CTRL and Tx MASH ECM-MASH; dots represent individual samples, and diamonds indicate group means. (d) Donut charts showing the chemical subclass distribution of VIP metabolites identified in the comparisons between CTRL Liver (n=5) and MASH Liver (n=4) and Tx MASH ECM-CTRL and Tx MASH ECM-MASH. Each segment represents the relative proportion of metabolites within each chemical subclass among the selected VIP features. (e) Venn diagram showing the overlap of metabolic features between Tx MASH ECM-CTRL and Tx MASH ECM-MASH samples. A substantial number of shared metabolites (1615) indicates a largely conserved metabolic composition of the transplanted MASH ECM, while a smaller subset of unique metabolites suggests selective metabolic modulation by the hepatic microenvironment. (f) Comparative pathway enrichment analysis between control and MASH liver tissue and transplanted ECM conditions.

First, we attempted to investigate the metabolomic profile of MASH ECM to understand whether it can be different or similar to that of the recipient liver. We observed that MASH ECM acquired a similar metabolic profile even when transplanted in healthy or MASH recipient rats (Figure 5). PCA of transplanted MASH ECM samples showed a less pronounced separation (Figure 5(a)) when compared to PCA of CTRL livers x MASH livers (see Figure 2(g)). Tx MASH ECM-CTRL and Tx MASH ECM-MASH samples clustered separately along PC1 (20.2%) and PC2 (12.9%), although partial overlap was observed. The MASH ECM transplantation analysis suggests that the host microenvironment may partially influence the molecular profile of the transplanted scaffold (Figure 5(a) and (d)); however, some metabolites are common, independent of whether MASH ECM is transplanted in healthy or MASH recipients (Figure 5(a)). The greater dispersion observed in the Tx MASH ECM-MASH group suggests that the diseased hepatic microenvironment promotes heterogeneous ECM remodelling, potentially reflecting differences in fibrosis progression, inflammatory activity, or stellate cell activation.

Hierarchical clustering analysis of the transplanted MASH ECM samples (Figure 5(b)) revealed distinct molecular patterns between Tx MASH ECM-CTRL and Tx MASH ECM-MASH groups. Most features displayed higher relative abundance in Tx MASH ECM-CTRL samples and reduced levels in Tx MASH ECM-MASH samples, suggesting a global reduction of several ECM-associated molecules in the MASH hepatic microenvironment. Conversely, a smaller cluster of features exhibited the opposite trend, with increased abundance in Tx MASH ECM-MASH samples, potentially reflecting ECM remodeling processes induced by the diseased liver environment. The beanplot analysis (Figure 5(c)) of selected discriminant metabolites between Tx MASH ECM-MASH and Tx MASH ECM-CTRL revealed distinct metabolic signatures associated with the hepatic microenvironment in which the MASH ECM scaffold was transplanted. Notably, Oleoyl-L-carnitine and lysophosphatidylethanolamine (LPE 18:3) were more abundant in MASH ECM samples recovered from control livers, whereas 5-Androsten-3β,16α-diol-17-one was elevated in MASH ECM transplanted into MASH livers. Oleoyl-L-carnitine is an acylcarnitine involved in mitochondrial fatty acid transport and β-oxidation, and its reduced levels in the Tx MASH ECM-MASH group may reflect impaired lipid oxidation and mitochondrial dysfunction, metabolic features commonly associated with MASH. Similarly, the decreased abundance of LPE (18:3), a lysophospholipid generated during membrane phospholipid remodeling, may indicate altered lipid turnover and inflammatory signaling within the diseased hepatic microenvironment. In contrast, the increase in the steroid-derived metabolite 5-Androsten-3β,16α-diol-17-one in ECM exposed to MASH livers suggests alterations in steroid metabolism, consistent with the known role of the liver in steroid biotransformation and the endocrine disturbances reported in metabolic liver disease. Together, these findings support the concept that the MASH hepatic microenvironment actively reprograms the metabolic composition of transplanted ECM scaffolds, reflecting the systemic metabolic disturbances characteristic of steatohepatitis.

After these observations, we explored the similarities and differences between CTRL Liver, MASH Liver, Tx MASH ECM-CTRL and Tx MASH ECM-MASH by analysing the chemical nature of metabolites found in metabolomic analysis. The donut charts (Figure 5(d)) illustrate the chemical subclass distribution of VIP metabolites identified in the comparisons CTRL Liver vs MASH Liver and Tx MASH ECM- CTRL vs Tx MASH ECM-MASH. In the CTRL Liver vs MASH Liver comparison, the metabolite profile was strongly dominated by bile acids, alcohols and derivatives, indicating that metabolites associated with bile acid metabolism represent the main discriminant class between healthy and MASH liver samples. Other subclasses were present at lower proportions, including fatty acids and conjugates, amino acids, benzoic acid derivatives, benzenesulfonamides, and glycerophosphocholines. In contrast, the Tx MASH ECM-CTRL vs Tx MASH ECM-MASH comparison exhibited a more heterogeneous distribution of metabolite classes, with notable contributions from monoradylglycerols, amino acids, glycerophosphocholines, fatty acids and conjugates, linoleic acid derivatives, and glycerophosphoethanolamines. This pattern suggests that lipid-related metabolites and phospholipid subclasses play an important role in the metabolic profile observed in the transplanted MASH ECM, potentially reflecting lipid remodeling and metabolic signaling processes within the MASH hepatic microenvironment. In addition to the differences, we also observed similarities between Tx MASH ECM-CTRL and Tx MASH ECM-MASH groups. The donut charts showed that the chemical class distribution revealed a highly similar metabolic composition between Tx MASH ECM-CTRL and Tx MASH ECM-MASH, with both groups predominantly enriched in lipid-related metabolites such as monoradylglycerols, glycerophosphocholines and fatty acid derivatives. This profile becomes more evident when we analyze the Venn diagram of the comparison between Tx MASH ECM-CTRL and Tx MASH ECM-MASH. A total of 1,615 metabolites were shared between the two conditions, whereas only 44 and 446 metabolites were uniquely detected by the groups (Figure 5(e)). This large intersection highlights a strong similarity in the metabolic composition of the transplanted ECM regardless of the hepatic microenvironment. Such overlap suggests that the MASH ECM retains a conserved biochemical signature, likely reflecting its structural components and lipid-associated metabolites.

Then, we investigated the lipid-related metabolic pathways in MASH ECM transplanted in healthy and MASH recipients. Pathway enrichment analysis revealed that lipid-related metabolic pathways were prominently altered in both comparisons (Figure 5(f)). In CTRL Liver vs MASH Liver samples, significant pathways included glycerophospholipid metabolism, linoleic acid metabolism, glycerolipid metabolism, and biosynthesis of unsaturated fatty acids, highlighting profound alterations in hepatic lipid metabolism associated with MASH. Interestingly, several of these pathways were also enriched in the ECM transplantation analysis (Tx MASH ECM-CTRL vs Tx MASH ECM-MASH), suggesting that the metabolic environment of the MASH liver influences the biochemical composition of the transplanted ECM. In addition to lipid metabolism, ECM-specific enrichment of arginine and proline metabolism and arachidonic acid metabolism was observed, pathways closely related to extracellular matrix remodeling, inflammatory signaling, and fibrogenesis.

3.2.5. MASH ECM promotes lipid accumulation in vitro

We next evaluated the impact of MASH ECM in vitro. MASH ECM and Healthy ECM (n=24 from 3 MASH and CTRL livers) were first submitted to culture with a basal medium for 10 days (Figure 6(a)). Lipids droplets were observed in the MASH ECM medium during all the culture time (Figure 6(b)). Conditioned medium was collected every other day. Then, HepG2 cells were cultured with the medium collected from MASH ECM and Healthy ECM cultures to investigate whether ECM-associated factors and lipids associated with MASH ECM could solely induce lipid accumulation in HepG2 (Figure 6(c)). Next, both phase-contrast imaging and fluorescence imaging where BDP® 630/650 was used to stain lipid droplets were obtained. Stained HepG2 cells revealed lipid accumulation after MASH ECM conditioned medium culturing similarly to the positive control where HepG2 cells were cultured with 30 μM oleic acid (positive control). In contrast, less intense staining was observed in HepG2 cells cultured with control medium and Healthy ECM conditioned medium (Figure 6(c)). These qualitative observations were confirmed by a quantitative analysis of fluorescence intensity (Figure 6(f)), demonstrating that Healthy ECM conditioned medium presented a statistically significant difference in contrast to the MASH ECM conditioned medium group. Then MASH ECM was recellularized with HepG2 cells to investigate whether the MASH ECM could promote lipid accumulation in vitro when cells directly interact with the ECM (Figure 6(d)). H&E-stained sections showed that HepG2 cells also accumulated lipids after culture in MASH ECM for 10 days (Figure 6(e)).

Figure 6.

In vitro analysis of MASH ECM and MASH ECM-associated factors on lipid accumulation by HepG2 cells. (a) Experimental design used to investigate the impact of MASH ECM-associated factors on HepG2 cells in vitro. (b) Macroscopic view of MASH ECM in culture without cells. Black arrows indicate lipid droplets in the wells in which MASH ECM was cultured for 10 days. (c) Phase-contrast and BDP-stained HepG2 cells after culture with control medium, conditioned medium derived from MASH ECM, Healthy ECM and 30 μM oleic acid (positive control). Scale bars: 150 μm. (f) Fluorescence intensity (a.u.) of BDP-stained HepG2 cells after culture with control medium, conditioned medium derived from MASH ECM, Healthy ECM and 30μM oleic acid. (d) MASH ECM after recellularization with HepG2 cells for 10 days in culture. Black arrows indicate lipid droplets. (e) Hematoxylin and eosin-stained section from MASH ECM recellularized with HepG2 cells. Black arrows indicate cells with lipid accumulation. Scale bars: 50 μm.

3.2.6. MASH ECM reduces calcium signalling amplitude after recellularization

After in vivo and in vitro exploration, MASH ECM was also explored in ex vivo experiments. MASH ECM cubes were recellularized with HepG2 cells and then submitted to intracellular Ca²⁺ signaling analysis to investigate whether MASH ECM environment could impact on cell physiology properties (Figure 7). Time-lapse confocal images of Fluo-4–loaded HepG2 cells within MASH ECM or CTRL ECM showed that in both conditions’ cells were able to be responsive to ATP stimulation (Figure 7(a)). However, the quantification of ATP-evoked Ca²⁺ signals revealed that HepG2 cells cultured in MASH ECM were less responsive to ATP than observed in CTRL ECM (Figure 7(b)). In addition to that, we also observed that the calcium signalling amplitude was significantly reduced in MASH ECM recellularized with HepG2 when compared to recellularized CTRL ECM (Figure 7(c)). Moreover, in MASH ECM a change in the pattern of the Ca²⁺ transient was also observed, with a high number of cells responding in a wave manner (Figure 7(d)). Immunohistochemistry analysis for intracellular calcium channel isoforms 1, 2 e 3 (ITPR1, 2 e 3) were also evaluated to demonstrate the presence and spatial distribution of ITPR isoforms within the scaffolds (Figure 7(e)). All isoforms were identified in HepG2 cultured in both CTRL and MASH ECM. However, a more intense staining was observed for ITPR3 isoform in HepG2 cultured in MASH ECM (Figure 7(e)).

Figure 7.

Ca²⁺ dynamics in HepG2-recellularized MASH ECM derived from rats. (a) Representative time-lapse confocal images of Fluo-4/AM–loaded HepG2 cells within rat decellularized scaffolds at baseline and following ATP stimulation. The images show fluorescence frames at +115, +125, and +135 s after the onset of stimulation. The pseudocolor scale (right) indicates the minimum to maximum intracellular Ca²⁺ fluorescence intensity. Scale bar: 100 nm. (b) Quantification of ATP-evoked Ca²⁺ signals in HepG2 cells, with individual traces representing single-cell recordings in mouse scaffolds under MASH or CTRL conditions. (c) The Amplitude of Ca²⁺ signaling in mice scaffolds induced by ATP was reduced in steatotic scaffolds (p<0.001, n=cells from three individual scaffolds in each group). (d) Summary of the ATP-evoked Ca²⁺ profile across the experimental groups. (e) Hematoxylin and eosin-stained sections of control ECM and MASH ECM recellularized with HepG2 cells. Immunohistochemical staining of ITPR1, ITPR2, and ITPR3– positive cells in control ECM and MASH ECM recellularized with HepG2 cells. Scale bars: 50 µm and 20 μm.

In addition to performing calcium assay on rat MASH ECM, we also performed this analysis on human ECM from livers of patients who had MASH and progressed to HCC. Our main objective was to understand whether a diseased human derived ECM could still provide an environment that allows cell survival and development. So, human MASH/neoplastic livers were decellularized (supplemental material) and then, human MASH/neoplastic ECM were also recellularized with HepG2 cells for 14 days and submitted to intracellular Ca²⁺ signaling analysis (Figure 8). Time-lapse confocal images of Fluo-4–loaded HepG2 cells within Human MASH/neoplastic ECM showed that the cells were able to be responsive to ATP stimulation (Figure 8(a)). We were able to detect specific responses of different cells into the human MASH/neoplastic ECM after ATP stimulation (Figure 8(b)). We also observed that the profile relates to the Ca²⁺ pattern after ATP stimulation was diverse with distinct cells responding in wave, compound, transient and oscillatory manner (Figure 8(c)). Immunohistochemistry analysis for intracellular calcium channel isoforms 1, 2 e 3 (ITPR1, 2 e 3) were also evaluated (Figure 8(d)). All isoforms were identified in HepG2 cultured in human MASH/neoplastic ECM. Together, these results suggest that, although the ECM is diseased, it is still able to promote a physiological environment that ensures cell survival.

Figure 8.

Ca²⁺ dynamics in HepG2-recellularized human scaffolds. (a) Representative time-lapse confocal images of intracellular Ca²⁺ changes (Fluo-4/AM) in HepG2 cells seeded on human decellularized scaffolds. Fluorescence frames are shown at baseline and +120, +130, and +140 s after ATP stimulation. The pseudocolor scale (right) represents the minimum to maximum Ca²⁺ fluorescence intensity. (b) Quantification of ATP-evoked Ca²⁺ signals in HepG2-recellularized human scaffolds, with individual traces representing single-cell recordings. Rescaled representative tracing of individual cells (1–7) that responded sequentially to ATP. (c) Summary of the ATP-evoked Ca²⁺ signaling profile of HepG2 cells seeded on human decellularized scaffolds. (d) Hematoxylin and eosin-stained sections from human MASH/HCC ECM recellularized with HepG2 cells. Immunohistochemical staining of ITPR1, ITPR2, and ITPR3– positive cells in human MASH/HCC ECM recellularized with HepG2 cells. Scale bars: 50 and 20 μm.

4. Discussion

The concept of ECM memory was previously demonstrated by our group, ⁷ showing that transplantation of a healthy Acellular Liver Scaffold (ALS) into partially hepatectomized cirrhotic rats led to complete graft recellularization by healthier cells. Notably, the transplanted ALS remained unaffected by the diseased hepatic environment, and although repopulated by cells of cirrhotic origin, it enabled the formation of physiologically normal tissue, providing an effective template for cell growth and supporting the development of functional tissue with normal biochemical, histological, and ultrasonographic features. Based on these findings, the present study sought to expand the understanding of ECM memory through the use of MASH-derived ECM.

The rising prevalence of MASH has profoundly affected liver transplantation, reducing the quality of available donor organs and exacerbating organ shortage. ²⁶ In the present study, we aimed to advance the translational applicability of decellularized ECM from marginal livers. To do this, we investigated the mechanisms of MASH ECM transplantation within steatotic and healthy recipients and assessed how a pathological graft affects cell physiology. To our knowledge, this is the first study to evaluate a partial MASH ECM transplantation, offering new insights into how diseased ECM can promote disease maintenance in vivo and in vitro.

The generated MASH ECM were macroscopically and microscopically distinct from healthy ECM controls, maintaining fibrotic features associated with MASH disease. After observing these differences and given the nature of MASH disease, highlighted by lipid accumulation in the liver tissue, we investigated the presence of lipids in the MASH ECM. Our histological and quantitative analyses revealed that the MASH ECM retains bound lipids, suggesting that these molecules may confer a biochemical “memory” to the diseased ECM. The presence of fat content after decellularization was also observed by Acun et al., 2024 ¹⁶ after steatotic liver decellularization. Taking together with the data confirming our successful decellularization, these findings demonstrate that lipid content is not a cellular remnant but rather a component bound to the ECM itself. The presence of ECM-bound lipids has been demonstrated previously but remains largely unexplored. ¹⁶

Currently, there are different methods to promote decellularization, including chemical, physical, biological, and mixed methods. ²⁷ Different decellularization techniques have been shown to differentially affect ECM composition.^28,29 Perfusion decellularization has been shown to achieve qualitative elimination of DNA along with substantially greater phospholipid reduction compared to conventional agitation, even when the same decellularizing solution is used for both methods.^27,28,30 The reason for this higher efficiency is mainly because access to the vascular network allows better organ perfusion. When vascular access is not possible, the agitation/immersion method is the most common alternative approach.

In this study, we employed two distinct decellularization strategies for two different applications: agitation/immersion, applied to small human liver fragments intended for recellularization, based on a protocol previously described by Mazza et al. ¹² for healthy and cirrhotic liver scaffolds, and vascular perfusion, used to generate a whole MASH liver scaffold intended for transplantation. While both approaches generate scaffolds that meet standard decellularization criteria, the underlying mechanisms of cell removal differ and may not preserve ECM components, including structural proteins and lipids, to the same extent. However, it is worth noting that our data show that the structures obtained using the two techniques provided an environment conducive to cell development in vivo or ex vivo. MASH ECM obtained from perfusion and agitation/immersion promoted the steatosis phenotype when applied in vivo and ex vivo. Although both techniques induce changes in ECM composition, the structures retain specific components that can still influence cellular metabolism and physiology. Future studies directly comparing both techniques under matched conditions will help clarify the extent to which differential ECM and lipid retention contribute to the observed tissue responses.

After detecting the presence of ECM-bound lipids, we wondered whether these molecules might confer a memory effect on the ECM when transplanted. To investigate this, we performed MASH ECM transplantations under different conditions. First, to establish a translational scenario wherein patients with MASH could eventually benefit from scaffold transplantation, we transplanted MASH ECM into animal recipients with the same condition. As expected, our results showed that the cells present in the MASH ECM retained the same characteristics as those of the native liver that received transplantation. These characteristics were not limited to histological changes but also included hepatic stellate cell activation, and molecular alterations, in which genes involved in fibrosis and de novo lipogenesis were expressed in the MASH ECM at levels like those in MASH livers. Second, we investigated the contribution of the MASH ECM in dictating these cellular metabolic alterations that could result in steatosis. Interestingly, we observed that the MASH ECM had a significant effect on the cells, resulting in the maintenance of steatohepatitis characteristics and the perpetuation of the disease even when transplanted in healthy recipients. Finally, after these observations, we performed an inverse experiment to investigate the impact of MASH ECM on steatosis maintenance versus the contribution of the microenvironment of the native recipient liver. We did not observe the same pattern. No vesicular structures were detected in the healthy ECM transplanted into MASH recipients.

To the best of our knowledge, our results are the first to show that diseased ECM derived from MASH livers has a memory that can determine cell metabolism and promote disease progression. These findings are consistent with important observations previously reported in pre-clinical and clinical trials.^31–33 Many studies involving the use of bone marrow-derived mesenchymal stromal cells for the treatment of liver cirrhosis have yielded negative results, in which treatment with the cells did not improve the cirrhotic condition. These findings may be consistent with the results of the present study, which showed that a diseased ECM can affect cells, promoting a pathological state. Similar conclusions were obtained by Miyauchi and colleagues after recellularization of fibrotic livers with HepG2 cells ¹¹ and by Booth and colleagues when fibroblasts were activated and differentiated in myofibroblasts after culture in fibrotic lung scaffolds. ¹³

Evidence that MASH ECM can drive steatosis was also supported by metabolomics analysis. In addition to the differences between healthy and MASH recipients, we observed that both MASH ECM transplanted into healthy and MASH animals exhibited similar metabolic signatures, confirming that MASH ECM also directs cellular metabolism, inducing steatohepatitis-like features in resident cells. Our data showed that the MASH ECM modulates important pathways, including lipid, arginine and proline, and arachidonic acid metabolism. These pathways are closely related to extracellular matrix remodelling, inflammatory signalling, and fibrogenesis. As expected, our data also showed that metabolic signatures associated with the native liver that received the transplant were observed in MASH ECM. Therefore, the metabolic profile of cells repopulating the MASH ECM is driven by the recipient liver microenvironment alongside the influences of the diseased ECM. Future studies are needed to characterize the entire metabolic signature of MASH ECM after transplantation.

After in vivo analysis, we next investigated MASH-derived ECM in vitro. Since the ECM acts as a reservoir for cytokines and the release of TGF-β has already been demonstrated, ¹² we sought to determine whether matrix-bound lipids could also be released into the microenvironment and subsequently modulate cell physiology in vitro.

Both HepG2 cells recellularized within MASH ECM and those exposed to MASH ECM-supplemented medium displayed lipid accumulation patterns comparable to the oleic acid-treated positive control, while cells exposed to low-fat control medium and to a healthy ECM-supplemented medium exhibited minimal lipid staining suggesting that not only the MASH ECM, but also the molecules released by it, can promote lipid accumulation in HepG2 cells. One potential explanation for this observation could be the existence of different lipids associated with the MASH ECM. According to Chait, A et al., 2000, different lipid classes carried by lipoproteins can bind to the extracellular matrix, such as triacylglycerols (TAG), diacylglycerols (DAG), cholesteryl esters (CE), ceramides, and retinyl esters. ³⁴ This interaction is important for the development of several diseases, including atherosclerosis and diabetes. ³⁵ In addition, Chen et al., 2025 reported that lipid droplets containing TAG, DAG, CE, ceramides, and retinyl esters are determinants of cell fate. ³⁶ Therefore, further studies exploring the different class of lipids bound to the MASH ECM following decellularization are necessary to support this hypothesis.

We also observed that MASH ECM directly reprograms intracellular Ca²⁺ signalling by altering both signal amplitude and response dynamics, indicating impaired intercellular communication and aberrant Ca²⁺ handling, which favors non-canonical signalling pathways. As calcium regulates several intracellular events, including cell metabolism, our data suggest that MASH ECM disrupts calcium signalling and consequently affects metabolism, promoting disease perpetuation.

Previous studies have demonstrated that disruption of calcium homeostasis compromises mitochondrial oxidative metabolism and ATP synthesis, impairs hepatocyte metabolic regulation, and contributes to ER stress and cell injury.^37–39 In chronic liver diseases, altered calcium signalling has additionally been associated with inflammation, defective regeneration, and progression toward apoptosis or necrosis.^40,41 Thus, attenuation of calcium amplitude in our model may reflect reduced functional capacity of cells interacting with the pathological ECM microenvironment rather than simply a quantitative reduction in fluorescence intensity. At the same time, the heterogeneous and disorganized calcium signalling patterns observed in the human scaffold experiments may also reflect pathological features of the diseased ECM microenvironment. However, because all human scaffolds used in this study were derived from HCC tissues and no healthy human ECM control group was available, we cannot definitively determine whether these signalling patterns are disease specific.

On the other hand, our data with human MASH/HCC ECM demonstrate that, despite its pathological origin, the ECM can sustain a physiologically permissive microenvironment that supports cell survival. This finding represents an initial step toward the translational application of human disease-derived ECM as functional bioengineered platforms. One limitation of this study is that we did not investigate whether the specific metabolic profiles of cells cultured within the MASH/HCC ECM were modified. Therefore, although our findings indicate that this human diseased ECM supports basic cell viability and attachment, the metabolic and fibrosis pathways in these cells when cultured in a MASH/HCC environment should be investigated.

Taken together, our in vivo and in vitro data suggests that the ECM memory encoded by lipids bound to the ECM and other biochemical compounds may lead to steatosis-like phenotype. Understanding how pathological memory is retained and transmitted by diseased ECM is a critical step toward developing strategies to reprogram these scaffolds into healthy-like matrices. ⁴² The clinical use of healthy-like ECM in liver disease patients could underline the improvement of diseased organs functionality, enabling the reuse of marginal donor livers through ECM decellularization and pathological memory reprogramming generating bioengineered grafts capable of promoting tissue repair into diseased recipient organs. To achieve ECM reprogramming, different strategies can be used, including treatments with enzymes, such as lipase, ¹³ or organic reagents, such as isopropanol. ⁴³ These strategies could expand the therapeutic possibilities for patients with end-stage liver disease, improving organ functionality to extend survival while waiting for liver transplantation, and helping to reduce mortality on liver transplant waiting lists. Moreover, modifying disease-associated ECM memory may allow novel tissue engineering strategies, facilitating whole ECM recellularization through IPSc-derived liver cells or spheroids, and the consequent creation of functional bioengineered livers, thereby contributing to long-term solutions for organ shortage.

5. Conclusion

In conclusion, we show here that ECM obtained from MASH livers retained a memory based on biochemical, and structural cues that promoted steatohepatitis maintenance. Our findings show that ECM obtained from marginal livers should be treated prior to use in liver transplantation to minimize the adverse effects of residual fat in the scaffold.

ORCID iDs

Gabriel Reis Pinto https://orcid.org/0009-0004-4877-3333

Luana Diniz Guerra Braz https://orcid.org/0009-0002-6535-7863

Yasmin Pestana https://orcid.org/0009-0007-1340-0926

Giulia Roldão B. Freire https://orcid.org/0000-0002-1855-8972

Maria Isabel Moraes do Amaral Candido Gomes https://orcid.org/0009-0004-3966-643X

Julia Helena Oliveira de Barros https://orcid.org/0000-0003-3701-1558

Thamires Siqueira de Oliveira https://orcid.org/0000-0002-8987-3822

Barbara Fidelix Santana https://orcid.org/0009-0001-6819-8637

Hernandes F. Carvalho https://orcid.org/0000-0002-3080-9447

Cherley Borba Vieira Andrade https://orcid.org/0000-0002-4320-4252

Lucas Pires Guarnier https://orcid.org/0000-0002-0733-1975

Érica Almeida Amorim https://orcid.org/0000-0002-9562-673X

Cibele Ferreira Pimentel https://orcid.org/0000-0003-4827-5436

M. Fátima Leite https://orcid.org/0000-0001-9709-8865

Marina Amaral Alves https://orcid.org/0000-0002-8188-5554

Regina Coeli dos Santos Goldenberg https://orcid.org/0000-0002-0886-9603

Marlon Lemos Dias https://orcid.org/0000-0001-9354-7280