Application of hepatocyte-derived exosome-assisted 3D bioprinted skin organoids in wound regeneration

Abstract

The application provides application of a hepatocyte-derived exosome-assisted 3D bioprinted skin organoid in wound regeneration. The 3D bioprinted skin organoid scaffold wraps keratinocytes, fibroblasts and endothelial cells in a GelMa-ECM hydrogel containing HC-exos, simulating the structure of natural skin. After adding HC-exos, the construct plays important roles such as antioxidation, up-regulation of the regeneration process of cells and regulation of signal pathways, and realizes efficient tissue regeneration in the wound healing microenvironment, providing a promising solution for the treatment of large-area full-thickness skin damage. The construct has excellent biocompatibility and effectiveness, and has broad prospects in clinical transformation.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of hepatocyte conditioned medium and hepatocyte-derived exosomes in wound regeneration, and more specifically, to the application of hepatocyte-derived exosome-assisted 3D bioprinted skin organoids in wound regeneration. Background Technology

[0002] Skin healing is a complex and dynamic process involving multiple cells within the skin and other organs through the secretome (including proteins, exosomes, and metabolites), collectively regulating biological processes such as cell proliferation, angiogenesis, and gene regulation. Skin healing is mainly divided into four stages: hemostasis, inflammation, proliferation, and remodeling. Large-area third-degree skin wounds caused by thermal injury, trauma, and surgery are prone to necrosis and cannot be sutured, imposing a heavy socioeconomic burden on patients and severely impacting their quality of life. Although autologous skin transplantation is the gold standard for treating such injuries, limited graft availability and transplant failure due to infection or insufficient perfusion remain challenges.

[0003] Therefore, there is an urgent need for alternatives to skin grafting to heal complex wounds that require long-term recovery and to eliminate / reduce hypertrophic scarring. Summary of the Invention

[0004] The present invention aims to overcome the above-mentioned defects and provide a treatment solution for large-area full-thickness skin damage.

[0005] This invention provides the application of hepatocyte conditioned medium in the preparation of skin regeneration / repair / healing products.

[0006] This invention provides the application of hepatocyte conditioned medium in the preparation of products that promote angiogenesis.

[0007] This invention provides the application of hepatocyte-derived exosomes in the preparation of skin regeneration / repair / healing products.

[0008] This invention provides the application of hepatocyte-derived exosomes in the preparation of products that promote angiogenesis.

[0009] This invention provides the application of hepatocyte-derived exosomes in the preparation of scar remodeling products.

[0010] This invention provides the application of hepatocyte-derived exosomes in the preparation of products for preventing scar formation.

[0011] This invention provides the application of hepatocyte-derived exosomes in the preparation of products for the regeneration of aging wounds.

[0012] This invention provides the application of hepatocyte-derived exosomes in the preparation of products for the regeneration of diabetic wounds.

[0013] This invention provides the application of hepatocyte-derived exosome-assisted 3D bioprinted skin organoids in wound regeneration.

[0014] This invention provides a method for preparing 3D bioprinted skin organoids assisted by hepatocyte-derived exosomes, characterized in that:

[0015] Keratinocytes, fibroblasts, and endothelial cells were encapsulated in GelMa-ECM hydrogel containing hepatocyte-derived exosomes, and skin organoids were 3D bioprinted to simulate the structure of natural skin.

[0016] The bottom layer contains endothelial cells, the middle layer contains fibroblasts, and the top layer contains keratinocytes. Attached Figure Description

[0017] Figure 1 Schematic diagram of skin organoid design and bioprinting for wound healing, using hepatocyte-derived exosomes (HC-exos) embedded with bio-ink to construct multicellular spatial distribution structures. A) Preparation of bio-ink for skin microtissue printing. B) Transplantation of active scaffolds in a full-thickness wound animal model. C) Cellular and molecular mechanisms by which skin organoids promote wound healing through the regulation of signaling pathways, key gene and protein expression, and angiogenesis.

[0018] Figure 2 Effects of hepatocyte conditioned medium (CM) on the proliferation of different skin cell types. a) Schematic diagram of hepatocyte conditioned medium (CM) preparation and skin cell culture process; b, c, d) Proliferative activity of keratinocytes, fibroblasts, and endothelial cells after 1, 3, and 5 days of culture (n=4). Data are expressed as mean ± standard deviation (*p≤0.05; **p≤0.01; ***p≤0.001, compared with the control group; ns: no significant difference).

[0019] Figure 3 Effects of hepatocyte conditioned medium (CM) on gene expression in different skin cells. RT-PCR was used to detect gene expression of K5 and IGBT in keratinocytes (a) and (b); FN and Col1A1 in fibroblasts (c) and (d); and eNOS and PDGF in endothelial cells (e) and (f) after 2 days of culture (n=9). Data are expressed as mean ± standard deviation (*p≤0.05; **p≤0.01; ***p≤0.001, compared with the control group; ns: no significant difference).

[0020] Figure 4Effects of conditioned medium (CM) on hepatocyte angiogenesis: a) Images of endothelial cell angiogenesis; b, c, d) Quantitative analysis of node number, segment number, and tube length (n=3). Data are expressed as mean ± standard deviation (*p≤0.05; **p≤0.01; ***p≤0.001, compared with the control group; ns: no significant difference).

[0021] Figure 5 Characterization of exosomes released by hepatocytes (HC-exos) and their uptake by skin cells. a) Schematic diagram of exosome isolation process. b) Morphological observation of HC-exos under transmission electron microscopy (TEM, scale bar: 200 nm). c) Particle size distribution obtained by nanoparticle tracking analysis (NTA). d) Exosome characteristic surface markers CD63, CD9, and selenop as detected by Western blotting. e) Internalization of DIL-labeled HC-exos by keratinocytes, fibroblasts, and endothelial cells after 24 hours of incubation (detected by fluorescence). Red, blue, and green signals represent HC-exos, nucleus, and cytoskeletal proteins, respectively (scale bar: 50 μm).

[0022] Figure 6 Biological effects of HC-exos on skin cells in a two-dimensional environment. a) Cell proliferation of keratinocytes, b) fibroblasts, and c) endothelial cells after co-culturing with different concentrations of HC-exos on days 1, 3, and 5 (n=3). Results of cell scratch assay after HC-exos treatment: d) keratinocytes treated for 18 hours, e) fibroblasts treated for 6 hours, f) endothelial cells treated for 12 hours (scale bar: 100 μm). Quantitative analysis of cell scratch assay: g) keratinocytes, h) fibroblasts, i) endothelial cells (n=3). RT-qPCR was used to verify the relative mRNA expression levels of GPX4 (j), (l), and (m) in the three skin cell types induced by HC-exos treatment, k) the expression level of IGBT in keratinocytes, and n) the expression level of PDGF in endothelial cells (n=9). o) Angiogenesis of endothelial cells cultured in HC-exos (scale bar: 100 μm); pr) Quantitative analysis of the number of vessel nodes, segments, and total length in the angiogenesis experiment (n=4). Data are expressed as mean ± standard deviation (p≤0.05; p≤0.01; p≤0.001, compared with PBS group; ns: no statistical significance). s) Immunofluorescence imaging of selenop (red), DAPI (blue), and FITC (green) after HC-exos treatment.

[0023] Figure 7Characterization of bio-ink and fabrication of 3D bioprinted organoid scaffolds. a) Schematic diagram of the decellularization process of native porcine skin. b) Macroscopic images (scale bar: 1 cm) and histological analysis results of native skin and extracellular matrix (ECM), including H&E staining (scale bar: 50 μm), DAPI staining (scale bar: 100 μm), Masson trichomorph staining (scale bar: 50 μm) and scanning electron microscopy (SEM, scale bar: 100 μm). c) Collagen content of tissues before and after decellularization (n = 3); d) Quantitative analysis of DNA content (n = 3). Reported data are mean ± standard deviation (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns: no statistical significance). e) Solution-gel phase transition process of bio-ink composed of ECM and Gelma (GECM) from room temperature (RT) to 37°C and pH ≈ 7. f) Schematic diagram of GECM printing. g) Dynamic curves of storage modulus (G') and loss modulus (G'') of GECM. h) Shear thinning behavior of bio-ink as shear rate increases from 0.1 s⁻¹ to 10⁴ s⁻¹. i) The scaffold can be flexibly printed into various shapes. j) SEM image of the printed scaffold (scale bar: 100 μm). k) Degradation of the scaffold by 0.5 mg·ml⁻¹ collagenase under oscillation at 37°C (n=4). l) Swelling rate of the scaffold after immersion in PBS solution at 37°C (n=6). m) Spatial observation of the 3D printed skin organoid: purple represents the keratinocyte layer, green represents the fibroblast layer, and red represents the endothelial layer; followed by a three-dimensional (3D) merged view under a confocal laser scanning microscope (CLSM, scale bars 500 μm and 200 μm, respectively).

[0024] Figure 8 Biocompatibility of bio-ink with three different skin cells on days 1, 3, and 7.

[0025] Figure 9. Regulatory effect of HC exosome encapsulated bio-ink on skin cells. a) Visual and CLMS observations of scaffolds printed using bio-ink containing DIL-labeled HC exosomes (scale bars 500 μm and 200 μm, respectively). b) Exosome release assay (n=3). c) Calcein-stained fluorescence images of keratinocytes, e) fibroblasts, and g) endothelial cells cultured in GECM scaffolds containing or without HC exosomes on days 1, 7, and 14 (image brightness adjusted for qualitative observation); d, f, h) proliferation rates of the three skin cell types in this construct (n=3). Data are expressed as mean ± standard deviation (*, p≤0.05; **, p≤0.01; ***, p≤0.001; ns: no statistical significance). i, j) Hemolysis analysis of HC-exos@GECM, Org and HC-exos@Org extracts was performed using Triton-X and PBS as controls (n=4).

[0026] Figure 10 Evaluation of wound healing effects after in vivo implantation of 3D-printed skin organoids using HC-Exos-containing bio-ink. a) Schematic diagram of animal experimental procedures and tissue collection; b) Visualization of splint dimensions and its fixation on the wound, scaffold implantation, and subsequent 3M filler filling; c) Digital photographs showing flowcharts and corresponding simulation charts at various time points from day 0 to day 14; d) Quantitative data on wound healing area in different groups (n=6); e) H&E staining images of major organs of different groups of animal models collected on day 14 (scale bar: 50 μm).

[0027] Figure 11 Histological staining and analysis of skin samples from different treatment groups. a) Hematoxylin-eosin (HE) staining and Masson's triangular staining results on day 7 (top scale bar 1 mm), the black dashed area is the magnified image below (scale bar 250 μm), and the red inverted triangle indicates the re-epithelialization area. b) HE staining and Masson's triangular staining results on day 14. Quantitative analysis was performed on c) tissue epidermal thickness on days 7 and 14 and d) wound width (n = 4); e) tissue collagen content on day 14 (n = 4). Data are expressed as mean ± standard deviation (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ns: no statistical significance). f) HE-stained sections for hair follicle assessment (scale bar 1 mm), with magnified images within the black dashed lines (scale bar 250 μm); g) Immunohistochemical staining results of type I and type III collagen on day 21 (scale bar 50 μm).

[0028] Figure 12 Immunofluorescence staining results of wound tissue samples. Skin tissue sections were stained on day 7 with the following antibodies: a) CD31 (scale bar: 50 μm), b) KI67 (scale bar: 100 μm), c) α-SMA (scale bar: 50 μm), d) CK10 (scale bar: 100 μm), e) selenop (scale bar: 100 μm). On day 14, the relative fluorescence intensity (RIF) of f) CD31, g) KI67, h) α-SMA, i) CK10, and j) selenop was quantitatively analyzed (n=4). Data are expressed as mean ± standard deviation (p≤0.05; p≤0.01; p≤0.001; ns: no statistical significance).

[0029] Figure 13 Transcriptome analysis of HC-exos@Org-induced wound healing. a) Heatmap of differentially expressed genes in wound skin samples collected from the control and HC-exos@Org groups; b) Volcano plot showing upregulated and downregulated gene expression levels; c) Principal component analysis (PCA) of the two gene groups; d) GO enrichment analysis of upregulated biological processes; e) KEGG enrichment analysis of upregulated and downregulated pathways; f) Reactome database annotation of downregulated pathways; GESA analysis of gene sets related to g) AMPK signaling pathway and h) inflammatory response signaling pathway. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] In this study, we discovered that 3D printing technology can be used to construct organoid scaffolds, enabling more precise and large-scale simulation of spatiotemporal synergies between different cells. These models are crucial for studying the dynamic behavior of cells in 3D environments similar to the complex microenvironment of the human body, contributing advanced biological insights for the treatment of long-term, complex wounds. Using this technology, researchers have constructed 3D skin models in both normal and pathological states and conducted drug testing, which is of significant clinical translational potential. Orthotopic implantation of such skin organoids is emerging as a promising approach to skin regeneration, providing biomimetic tissue by modulating the healing microenvironment and promoting efficient regeneration. However, the clearance of cells within the scaffold by activated immune cells and excessive reactive oxygen species (ROS) may hinder the repair process.

[0032] We also found that the liver plays a crucial role in maintaining homeostasis, exhibiting strong regenerative capacity even after 70% removal. Exosomes released by healthy liver cells are essential for organ regeneration, primarily through the synthesis of sphingosine 1-phosphate (S1P), an important sphingolipid involved in cell proliferation, migration, immune regulation, and angiogenesis. The liver is the only organ responsible for assembling and metabolizing the absorbed essential trace element selenium (Se) into selenoproteins (SELENOP). These selenoproteins are transported to various organs via exosomes, which is crucial for maintaining homeostasis, enhancing immune responses, upregulating potent antioxidant enzymes, and exerting anticancer activity. Due to the beneficial roles of selenium in supporting multiple biological functions, researchers have attempted to optimize the delivery of synthetic selenium nanoparticles for the treatment of atopic dermatitis, androgenetic alopecia, and to promote skin and bone healing. However, the toxicity and low bioavailability of high-concentration inorganic selenium nanoparticles remain issues.

[0033] Inspired by the liver's powerful regenerative capacity and the fact that hepatocyte exosomes carry highly bioavailable organic selenium, this study investigated the regenerative effects of exosomes on three types of skin cell lines. The treated cells exhibited enhanced regenerative function and increased expression of potent antioxidants such as glutathione peroxidase. Subsequently, we constructed [a specific cytokine / exosome / etc.]. Figure 1 This novel 3D-printed skin organoid scaffold demonstrates a novel approach. The design combines three types of skin cells with GelMa-ECM bio-ink carrying hepatocyte exosomes, employing a layer-by-layer extrusion bioprinting process followed by photocuring to mimic the skin's microstructure. In vivo validation of its efficacy and molecular mechanisms revealed significant proliferation, angiogenesis, and antioxidant effects, optimizing the wound healing microenvironment. The results also indicate a potential role in scar remodeling by accelerating the conversion of type III collagen to type I collagen. This innovative skin organoid exhibits high biocompatibility, high efficiency, and adaptability, showing great promise for clinical translation. This study is the first to explore the impact of hepatocyte exosomes on skin regeneration, and the findings can pave new avenues for future research into the treatment of more complex wounds and skin diseases.

[0034] The specific research methods are as follows:

[0035] Example 1. Preparation of hepatocyte supernatant, i.e., conditioned medium (CM)

[0036] Human liver epithelial cells (THLE-2) were transformed and cultured in THLE-specific medium (Procell, China) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. When cell confluence reached 80%, the cells were washed three times with sterile PBS and then cultured for 48 hours in complete medium supplemented with 5% exosome-free FBS. The supernatant was collected and subjected to a series of low-temperature centrifugations (Thermo Scientific, USA): 300 g for 10 min, 2000 g for 20 min, and 10000 g for 30 min to remove cell debris. The mixture was then filtered through a 0.22 μm filter (Millipore Sigma, USA). The hepatocyte conditioned medium was co-cultured with three types of skin cell (fibroblasts, endothelial cells, and keratinocytes) to investigate their proliferation rate, gene regulation, and angiogenesis effects.

[0037] This experiment investigates whether the hepatocyte secretome can regulate cellular functions essential for tissue regeneration, such as proliferation, migration, and angiogenesis. Figure 2 As shown in Figure a, hepatocyte supernatant (conditioned medium, CM) was added to cultured skin cells, with normally cultured skin cells serving as a control.

[0038] The proliferation of CMs was detected using a cell counting kit (CCK-8), such as... Figure 2 As shown in bd, the results showed that the number of fibroblasts increased significantly on days 1, 3, and 5, while the proliferation of keratinocytes and endothelial cells increased significantly on days 5 and 3, respectively.

[0039] Subsequently, gene expression regulation in CM-cultured cells was assessed using real-time quantitative PCR (RT-PCR). Figure 3 As shown in figure a, CM-treated keratinocytes showed high expression of cytokeratin 5 (K5, a basal cell marker), such as... Figure 3 As shown in b, integrin β (IGBT, a cell adhesion and signaling marker) expression showed no significant change. Similarly, in the treated group, the expression of genes such as type I collagen A (COL1A1, essential for collagen formation) and fibronectin (FN, involved in cell adhesion and migration) in fibroblasts was as follows: Figure 3 The cd value showed a significant increase. Furthermore, as... Figure 3 As shown in ef, CM also upregulated the gene expression of endothelial nitric oxide synthase (eNOS, which is essential for angiogenesis and vascular homeostasis) and platelet-derived growth factor (PDGF, which is essential for angiogenesis, proliferation and vascular remodeling in wound healing and tissue repair).

[0040] like Figure 4As shown in Figure 1, a classic endothelial cell tube formation experimental model was used to simulate the processes of endothelial cell migration, proliferation, lumen formation, and vascular network construction. Angiogenesis experiments demonstrated that endothelial cells (CMs) promote rapid angiogenesis, significantly increasing the number of nodes, segments, and tube length.

[0041] Example 2. Hepatocyte exosomes (HC-exos)

[0042] Based on the significant effect of hepatocyte CM on skin cells in Example 1, we hypothesize that exosomes also have beneficial effects on skin cells.

[0043] 2.1. Isolation of hepatocyte exosomes (HC-exos)

[0044] like Figure 5 As shown in a, HC-exos were isolated from THLE-2 cells CM using ultracentrifugation (Optima XPN-100, Beckman Coulter, USA), centrifuged at 100,000 g for 120 minutes. The exosome pellet was washed with pre-cooled PBS and then ultracentrifuged again to obtain pure exosomes, which were stored at -80°C.

[0045] 2.2. Characterization of HC-exos

[0046] Exosomal protein concentrations were determined using the quinolinic acid (BCA) method, and exosomal-specific surface markers CD63, CD9, and SELENOP were detected by Western blot. HC-exos samples were dropped onto a carbon-coated copper mesh (200 μm), negatively stained with 2% phosphotungstic acid (pH≈6), and their morphology was observed using a transmission electron microscope (JEM-1230, JOEL Corporation, Japan). Exosomal particle size distribution was determined using nanoparticle tracking analysis (ZetaView, Particle Metrix, Germany).

[0047] like Figure 5 As shown in bc, transmission electron microscopy (TEM) revealed that negatively stained HC-exos exhibited a circular, bilayered cup-shaped structure. Nanoparticle tracking analysis (NTA) showed that the exosome particle size ranged from 50 to 250 nm, with a peak concentration of 6 × 10⁻⁶. 6 The number of particles per mL is 114 nm, and the average hydrodynamic diameter is 114 nm. Figure 5 As shown in d, Western blot analysis revealed positive expression of exosome surface markers CD63, CD9, and SELENOP, confirming the successful isolation of pure HC-exos containing SELENOP.

[0048] 2.3. HC-exos uptake experiment

[0049] HC-exos were labeled with DIL (Beyotime, China) and co-cultured with skin cells for 24 hours. After washing with PBS, F-actin was stained green to label the cytoskeleton, and DAPI was stained blue to label the nuclei. The slides were then mounted and observed under a confocal laser microscope (Phoenix Optima, Germany).

[0050] The results are as follows Figure 5 As shown in Figure e, HC-exos labeled with DIL were co-cultured with skin cells. Fluorescence imaging showed that all three types of skin cells endocytosed exosomes within 24 hours, indicating that HC-exos could be successfully taken up by skin cells.

[0051] 2.4. The repair potential of hepatocyte exosomes on skin cells

[0052] 2.4.1. Cell viability assay

[0053] To further confirm whether HC-exos can exert a regenerative effect on skin cells similar to hepatocyte secretion, different concentrations (0.5 μg / mL, 5 μg / mL, 50 μg / mL) of purified exosomes were added to keratinocytes, fibroblasts, and endothelial cells, with PBS as a control group. Cell viability analysis showed that HC-exos exhibited high biocompatibility and non-toxicity regardless of concentration. Figure 6 As shown in ac, on day 5, 50 μg / mL HC-exos significantly promoted the activity and proliferation rate of the three skin cell types. Therefore, this concentration was selected as the optimal concentration for subsequent analysis.

[0054] 2.4.2. Scratch Test

[0055] Skin cells were seeded into 2-well culture inserts (Ibidi, Germany) in 24-well plates. After cell adhesion, the inserts were removed, creating a 500 μm wide scratch. Cells were washed with PBS and then cultured in exosome-free serum medium, with or without HC-exos. Images were taken under a bright-field microscope at different time points, and the results were analyzed and compared using ImageJ software.

[0056] like Figure 6 As shown in di, exosomes also significantly promote the migration ability of keratinocytes and fibroblasts.

[0057] 2.4.3. RT-PCR analysis

[0058] Glutathione peroxidase 4 (GPX4) is a potent antioxidant enzyme that inhibits ferroptosis and ultimately mediates tissue regeneration. Figure 6As shown in the figure, RT-PCR analysis revealed that GPX4 gene expression was significantly upregulated in all skin cells of the HC-exos group. Simultaneously, genes closely related to cell regeneration (including ITGB and PDGF) showed significantly increased expression in keratinocytes and endothelial cells, respectively. The wound area undergoes angiogenesis to achieve neovascularization, providing nutrients and oxygen perfusion for regeneration.

[0059] 2.4.4. Angiogenesis Experiment

[0060] Employing a classic model of wound angiogenesis, neovascularization is achieved in the wound area through angiogenesis, providing nutrition and oxygen perfusion for regeneration. For example... Figure 6 As shown in the figure, angiogenesis experiments confirmed that HC-exos significantly enhances the pro-angiogenic activity of endothelial cells.

[0061] 2.4.5. Immunofluorescence analysis

[0062] like Figure 6 As shown in Figure s, immunofluorescence analysis revealed a significant increase in SELENOP levels in fibroblasts after 24 hours of co-culturing with HC-exos. As a key regulator of selenium metabolism, SELENOP's core function is to exert anti-inflammatory and antioxidant effects through GPX, providing a molecular basis for subsequent wound healing.

[0063] Therefore, the above experiments show that HC-exos can positively alter the regenerative characteristics of skin cells and optimize their regenerative capacity by regulating proliferation, migration, and key genes and proteins. At the same time, it can enhance the angiogenesis capacity of endothelial cells and play a dual regulatory role in wound healing.

[0064] Example 4. 3D Bioprinted Skin Organoids

[0065] 4.1 GelMa Preparation

[0066] The method described in *Synthesis, properties, and biomedical applications of gelatinmethacryloyl (GelMA) hydrogels. Biomaterials, 2015, 73:254–271* was employed. Gelatin powder (Sigma-Aldrich, USA) was dissolved in deionized water by continuous stirring until completely dissolved. Methacrylic anhydride was added dropwise with stirring, and the reaction was terminated by adding deionized water. The mixture was dialyzed at 40°C for one week, and the product was lyophilized for three days to obtain a white, dry foam, which was stored at -20°C for later use.

[0067] 4.2 Decellularization and Characterization of Skin Tissue

[0068] The extracellular matrix (ECM) is an important component of connective tissue and various organs, providing physical support for cell growth and communication. Since the components of ECM derived from porcine skin are highly similar to those of human tissue, this study prepared tissue-specific ink raw materials through decellularization. Details are as follows:

[0069] Fresh pigskin was obtained from a local slaughterhouse and decellularized according to existing procedures. Figure 7 The specific steps were as follows: the epidermis was manually removed, and the dermis was sequentially treated with 0.25% trypsin solution, 0.1% sodium dodecyl sulfate (SDS) in 70% isopropanol solution, and 1% Triton X-100 in 70% isopropanol solution at 37°C with shaking. The tissue was thoroughly rinsed with sterile PBS to remove cell debris and detergent, and then lyophilized to dryness. Decellularized tissue samples and native tissue were stained with hematoxylin-eosin (HE) and 4',6-diamidinyl-2-phenylindole (DAPI). Cell residue was observed under bright-field and immunofluorescence microscopy (Olympus BX45, Japan). Decellularization efficiency and collagen retention were observed using HE and Masson's trichrome staining, showing complete removal of cells from the native tissue. 4',6-diamidinyl-2-phenylindole (DAPI) staining further confirmed the absence of cell nuclei in the decellularized tissue. Scanning electron microscopy (SEM) showed that the tissue was dense before decellularization, but became loose, porous, and fibrous after decellularization, as shown in the results. Figure 7 As shown in b.

[0070] like Figure 7 As shown in c, although the decellularization process slightly reduced the collagen content in the ECM, there was no significant difference compared with the natural tissue, indicating that there was no significant loss of collagen after treatment.

[0071] DNA was quantified from natural skin and ECM using a DNA extraction kit (TIANamp Genomic DNA Kit, China). DNA concentration was determined using an IMPLEM spectrophotometer, and ng / mg dry weight was calculated. Collagen content was calculated using a hydroxyproline quantification kit (Nanjing Jiancheng Biotechnology Co., Ltd., China) at 550 nm absorbance. Results are as follows: Figure 7 As shown in d, quantitative detection of deoxyribonucleic acid (DNA) showed that the DNA content of the sample was approximately 16 ng / mg dry weight, confirming high decellularization efficiency, significantly reduced DNA content in the tissue, and no obvious damage to the collagen structure.

[0072] After adjusting the pH of the liquefied ECM to approximately 7 and balancing the ion concentration, a sol-gel transition occurred at 37°C, confirming its suitability as a component for printing bio-inks.

[0073] 4.3. Preparation of Bio-ink

[0074] like Figure 7As shown in Figure e, a mixture of 10 mg / mL ECM and 6% methacrylic anhydride-modified gelatin (GelMa) underwent a sol-gel transition at pH 7 and 37°C, confirming that this mixture can be used as a component of printing bio-inks. Therefore, the following experiments are as follows... Figure 7 As shown in f, the printing performance of the composite bio-ink GECM was investigated by mixing 10 mg / mL dissolved ECM with 6% methacrylic anhydride gelatin (GelMa).

[0075] like Figure 7 As shown in g, composite modulus analysis revealed that when the temperature rose above 25°C, the storage modulus of the bio-ink was lower than its loss modulus, until a gel-sol transition occurred at 27°C. As the temperature continued to rise, the storage modulus exceeded the loss modulus, leading to re-gelation. This indicates that GECM possesses excellent printing performance and structural integrity, making it suitable for transplantation at physiological body temperatures. Furthermore, as... Figure 7 As shown in h, the viscosity of the bio-ink changes with shear rate from 0.1 s⁻¹. -1 Rise to 100 s -1 The shear stress is significantly reduced, exhibiting shear-thinning properties, which is crucial for extrusion bioprinting because the low shear stress during the extrusion process is beneficial for cell survival.

[0076] At the same time, such as Figure 7 As shown in figure i, GECM exhibits excellent printing performance, capable of constructing scaffolds of various shapes to fit different wound surfaces. For example... Figure 7 SEM observation shown in figure j reveals a distinct macroporous structure within the hydrogel, which is conducive to cell adhesion and growth. Figure 7 As shown in k, when co-incubated with collagenase at 37°C, the scaffold gradually degrades within 18 hours, making it suitable for normal tissue formation. Figure 7 As shown in Figure 1, the freeze-dried scaffold can absorb water and expand up to 900% within 1 hour and maintain its volume for up to 6 hours, which is beneficial for the 3D construct to absorb culture medium to nourish and encapsulate cells.

[0077] To investigate the toxicity of the composite ink to skin cells, an ECM solution was prepared: ECM was dissolved in dilute hydrochloric acid solution (containing 1 mg / mL pepsin) at a concentration of 1 mg / mL and stirred continuously at room temperature. Before bioprinting, NaOH solution was added dropwise with stirring to adjust the pH of the ECM solution to 7.4. Then, it was mixed with 6% GelMa (6 g of lyophilized gelma dissolved in 100 ml of DD water) and cell suspension (the cell volume was 10... 6(cells / mL) were mixed. Scaffolds were fabricated using aseptic extrusion 3D bioprinting with bio-inks encapsulating different skin cells and incubated at 37°C. The bioprinted scaffolds were stained with calcein (Beyotime, China) according to the manufacturer's instructions to analyze biocompatibility and proliferation in the 3D environment. Under an immunofluorescence microscope, calcein stained live cells green and dead cells red. Images were taken on days 1, 3, and 7. Results are as follows: Figure 8 As shown, calcein staining on days 1, 3, and 7 revealed good cell proliferation within the scaffold, confirming the biocompatibility of GECM ink for bioprinting.

[0078] 4.4.3 Preparation of Skin Organoids for Bioprinting

[0079] Inspired by the microstructure and layered characteristics of skin, this study combined keratinocytes, fibroblasts, and endothelial cells with GECM to prepare and design in vitro 3D-printed skin organoids. The specific method is as follows: Human keratinocytes (HaCat), human dermal fibroblasts, and human umbilical vein endothelial cells (HUVECs) were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C. Primary cells from passages 4 to 6 were used for experiments. Keratinocytes were labeled purple with DID (1,1'-bis(octadecyl-3,3,3',3'-tetramethylindole) carbocyanine perchlorate, fibroblasts were labeled green with DIO (3,3'-bis(octadecyloxacarbocyanine) perchlorate), and endothelial cells were labeled red with DIL (1,1'-bis(octadecyl-3,3,3',3'-tetramethylindole) carbocyanine perchlorate). 10 6 Cell suspensions of 1 cell / mL were mixed with sterile GECM bio-ink, with each 100ml of ink containing 1g ECM and 6g GelMa. The mixture was then loaded into a printing cartridge for 3D organoid scaffold printing. Bioprinting parameters: fiber thickness 0.15 mm, extrusion pressure 0.05 MPa, printing speed 5 mm / s, cartridge temperature 20–25℃, printing platform temperature 10℃. The endothelial cell layer was printed first, followed by a fibroblast cell layer, and then a keratinocyte layer. The organoids were cross-linked under 405 nm UV light for 10 seconds, and the spatial distribution of cells in each layer was observed using a confocal laser microscope. Results are as follows: Figure 7 As shown in m, confocal laser microscopy (CLSM) observation revealed that the cells were distributed in the upper, middle, and lower layers, respectively.

[0080] 4.5. Exosome-assisted 3D bioprinting of skin organoids derived from hepatocytes

[0081] HC-exos labeled with octadecyl-tetramethylindolecarbonylcyanine perchlorate (DIL) was mixed with GECM and printed to form a scaffold. Visibility was observed under CLSM, as shown... Figure 9As shown in Figure a, the exosomes are uniformly distributed within the scaffold. Figure 9 As shown in b, the release of exosomes within the scaffold increased gradually, reaching a stable release state after day 4. Cells continued to grow within the 3D bioprinted scaffold, while exosomes were released from the scaffold in a controlled manner.

[0082] Based on the significant effects of HC exosomes on skin cells in two-dimensional culture demonstrated in the above experiments, we used bioprinting technology to culture cells at 37°C and observed them under a fluorescence microscope using calcein staining to analyze the proliferation-promoting effect of HC exosomes@GECM on three types of skin cells within the scaffold. Figure 9 As shown in c, e, and g, on days 7 and 14, the number of cells within the scaffold in the HC exosomes@GECM group was relatively high in all three cell types. Furthermore, as... Figure 9 As shown in d, f, and h, quantitative analysis of CCK-8 further confirmed that the cell proliferation rate of the HC exosomes@GECM group was significantly increased compared with the control group, which is consistent with our previous CCK-8 experimental results in two-dimensional culture.

[0083] Based on the above results, we believe that the bio-ink with added HC exosomes exhibits high biocompatibility with the three types of skin cell lines and can effectively induce cell proliferation in a three-dimensional culture environment. Subsequently, we performed a hemolysis assay using mouse erythrocytes to verify the biocompatibility of HC-exos@GECM (a scaffold without cell suspension), Org (3D-printed skin organoids with bio-ink containing HC-exos), and Org@HC-exos (3D-printed skin organoids with bio-ink containing HC-exos), with Triton-X as a positive control and PBS as a negative control. Figure 9 As shown in i and j, the hemolysis rate in all experimental groups was less than 1%, confirming its high biocompatibility. Therefore, all bioprinted structures were considered suitable for implantation in mouse models for subsequent experiments.

[0084] 4.6. Healing effect of 3D-printed skin organoids equipped with hepatocyte exosomes in a mouse full-thickness wound model

[0085] To further verify the in vivo effects of skin organoids, the following methods were used: Figure 10For the method shown in a-b, a 10-mm full-thickness wound was created on the back of mice and fixed with a silicone splint of the same size. Wound healing in mice mainly occurs through contraction. To mimic the way of human healing through granulation tissue and re-epithelialization and prevent wound contraction, an excisional wound model with splint fixation was established, randomly grouped, and different constructs were transplanted. All animal experiments were conducted in accordance with the animal care and laboratory animal protocols approved by the Animal Care Committee of Tongji University in Shanghai (Ethical Approval Number: TJAA15125101), and the animal license number was SYXK (Shanghai) 2020-0002. Female BLAB / c mice at 5-6 weeks of age were purchased and acclimated for 1 week in an environment with a 12-hour day-night cycle and free access to food and water. The specific process of modeling was as follows: Anesthetize by inhalation of 10% isoflurane and maintain at 5%, and remove the back hair. After disinfecting the hairless area, a full-thickness wound was created using a sterile 10-mm biopsy punch, and a sterile splint was sutured and fixed to prevent contractile healing. The mice were randomly divided into four groups: 1) Wound without scaffold (blank group); 2) HC-exos@GECM group; 3) Org group; 4) HC-exos@Org group, and transplantation was performed. Finally, it was bandaged with sterile 3M Tegaderm, and gross photographs of the wounds were taken at different time points to compare the healing efficiency.

[0086] The results showed that the healing effect of the HC-exos@Org group was significant, and there was no residual scab on the 14th day. Statistical analysis was as Figure 10 shown in c-d. The percentage of wound area in the HC-exos@Org group decreased from 100% to 1% on average, and the healing speed was significantly faster than the other three groups, confirming its superior healing ability. Since multiple toxic reagents were used in the ink preparation process, improper operation may lead to systemic toxicity and organ failure. To confirm the in vivo biocompatibility and safety of the constructs, histopathological staining of the main organs (heart, liver, spleen, lung, kidney) was performed, and the results were as Figure 10 shown in e. There were no abnormal manifestations in the treatment group and the blank group, confirming that the organoids are suitable for in vivo application and are expected to be used for future clinical translation.

[0087] To clarify the microscopic changes caused by different treatments, HE and Masson staining were performed on the skin samples of each group on the 7th and 14th days. As Figure 11 shown in a-d, on the 7th day, the wound area of the HC-exos@Org group was completely re-epithelialized, while the HC-exos@GECM and Org groups were mainly covered with scabs and the re-epithelialization was incomplete, and the control group was the worst. On the 7th and 14th days, the epidermal thickness of the HC-exos@Org group was significantly thicker and the wound width was relatively smaller. As Figure 11 shown in e, Masson staining showed that the arrangement of collagen fibers in the HC-exos@Org group was more regular, and the collagen content was 79%, while those in the Org group, HC-exos@GECM group, and control group were 56.9%, 61%, and 49% respectively.

[0088] During the wound healing and remodeling phase, the regeneration of skin appendages faces significant challenges due to scar formation. To investigate the impact of microtissue on hair follicle regeneration, tissue samples from day 21 were stained with hematoxylin and eosin (HE). The results are as follows: Figure 11 As shown in f, the HC-exos@GECM and HC-exos@Org groups had a relatively large number of hair follicles, with the HC-exos@Org group having the most hair follicles in the wound area, demonstrating its potential in hair follicle regeneration.

[0089] During wound healing, collagen remodeling is crucial for normal skin formation and scar prevention. Type III collagen mainly forms in early scar tissue and gradually transforms into type I collagen as the wound matures. To investigate the effects of different treatments on scarring, immunohistochemical staining was performed on wound samples, and the results are as follows: Figure 11 As shown in g, the HC-exos@Org group exhibited high expression of type I collagen after healing, while type III collagen expression was relatively lower than in other groups. These results indicate that HC-exos@Org accelerates wound healing during the proliferative phase and participates in scar remodeling in the later stages, playing a significant role.

[0090] To investigate the expression of key proteins required for skin regeneration, researchers performed immunofluorescence staining on tissue samples. Differentiation cluster 31 (CD31) is a transmembrane protein expressed during angiogenesis in the early stages of wound healing, responsible for promoting local perfusion. The results showed that the fluorescence intensity of the HC-exos@Org group was significantly enhanced, indicating a significant pro-angiogenic effect on day 7. Figure 12 a, f). Similarly, on day 14, α-smooth muscle actin (α-SMA) expression was significantly higher in the HC-exos@Org group, while other groups showed no significant difference compared to the blank control group. Figure 12 c, e). These findings confirm that HC-exos@Org effectively promotes angiogenesis not only in the early stages of proliferation but also in the later stages. Furthermore, data showed that the expression level of the nuclear protein Kiel 67 antigen (KI67)—a protein crucial for cell proliferation and expressed only in dividing cells—was significantly increased in both the HC-exos@Org and HC-exos@GECM groups. Figure 12 b, g), demonstrating its ability to effectively promote cell proliferation within wounds, which is crucial for tissue repair and angiogenesis. The staining results for cytokeratin-10 (CK-10)—a marker of keratinocyte differentiation and a key factor in epidermal maturation—showed a similar trend, further confirming that HC-exos@Org contributes to epidermal regeneration. Figure 12 d, i). Selenoprotein staining also showed a similar pattern: the HC-exos@Org group exhibited the highest fluorescence intensity in the epidermal and dermal regions, as observed in quantitative results ( Figure 12From the perspective of e,j), this further confirms that HC-exos@Org can enhance the expression of antioxidant proteins in vivo.

[0091] 4.7. Study on the mechanism of HC-exos@Org promoting skin regeneration in vivo

[0092] To further elucidate the molecular mechanism by which HC-exos@Org promotes wound healing in vivo, transcriptomic analysis was performed on wound tissue samples from the control and treatment groups. Figure 13 a and volcano Figure 13 b shows that differentially expressed genes were significant in the HC-exos@Org group, with 659 genes upregulated and 2061 genes downregulated. Principal component analysis (PCA) was performed. Figure 13 c shows that the gene distributions in the blank group and the HC-exos@Org group are significantly different. Gene Ontology (GO) enrichment analysis. Figure 13 The results showed a significant enrichment of oxidoreductase activity during biological processes, confirming that HC-exos@Org can activate redox-related genes and proteins, consistent with wound tissue staining results. (Kyoto Encyclopedia of Genetics and Genomes (KEGG) analysis) Figure 13 The study showed a significant enrichment of pathways essential for tissue regeneration, including the AMPK (adenosine monophosphate-activated protein kinase) signaling pathway, cytokine-cytokine receptor interaction, PPAR signaling pathway, and sphingolipid signaling pathway. Literature reports that the sphingolipid signaling pathway is crucial for cellular functions such as cell motility, signaling protein synthesis, and transmission. Simultaneously, the HC-exos@Org group downregulated multiple pro-inflammatory pathways, such as nuclear factor κB (NF-κB), Toll-like receptor, and NOD-like receptor signaling pathways, which inhibit the healing process. The reaction set plots showed downregulation of pathways such as neutrophil degranulation, collagen degradation, and ECM degradation, which are beneficial for wound healing and prevention of fibrotic scars. Gene set enrichment analysis (GSEA) was also performed. Figure 13 gh further confirmed that the AMPK-related gene set was moderately upregulated and the inflammation-related gene set was downregulated in the HC-exos@Org group. In conclusion, hepatocyte-derived exosomes can effectively enhance the function of skin organoids and accelerate the healing of complex full-thickness wounds.

[0093] The histological analysis involved in the above experiments was as follows: skin samples were collected, fixed in 4% paraformaldehyde for 24 hours, dehydrated with alcohol series, and then embedded in paraffin, with a section thickness of 5 μm. After dewaxing, the skin sections were stained with HE and Masson's trichrome, and scanned using a Slideview VS200 (Olympus Corporation, Japan). The sections were then stained with specific antibodies for immunohistochemical staining and observed under bright-field and fluorescence microscopy using a BX45 fluorescence microscope (Olympus Corporation, Japan).

[0094] Statistical analysis in the above experiments was performed using GraphPad Prism 8.0 software. All experiments were repeated at least three times. Two-tailed t-tests were used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. *p<0.05, **p<0.01, and ***p<0.001 were considered statistically significant. All data are expressed as mean ± standard deviation.

[0095] The primer sequences involved in the above experiments are as follows:

[0096]

[0098] The antibody information involved in the above experiments is as follows:

[0099]

[0101] The effect of this embodiment:

[0102] This study demonstrates that the secretome of healthy hepatocytes and HC-exos have significant regenerative effects on skin cells, primarily through promoting proliferation, gene regulation, anti-oxidation, and angiogenesis. Based on their potent regenerative effects on skin cells, this study designed and fabricated a 3D bioprinted skin organoid scaffold, encapsulating keratinocytes, fibroblasts, and endothelial cells within an HC-exos-containing GelMa-ECM hydrogel to mimic the structure of natural skin. With the addition of HC-exos, this construct exerts important functions such as anti-oxidation, upregulation of regenerative cell processes, and regulation of signaling pathways, achieving efficient tissue regeneration within the wound healing microenvironment, providing a promising solution for the treatment of large-area, full-thickness skin injuries. This construct exhibits excellent biocompatibility and efficacy, showing broad prospects for clinical translation.

[0103] Studies have confirmed that HC-exos is a rich source of organic SELENOP, which can upregulate various glutathione-related genes and proteins, exhibits low immunogenicity and high bioavailability, and its supplementation is crucial for tissue regeneration. This study demonstrates that HC-exos can improve angiogenesis and enhance skin cell regeneration by upregulating cell mitotic activity, migration capacity, and the expression of antioxidant molecules such as GPX-4 and SELENOP; these are essential for promoting wound healing. The findings could provide insights for future exosome-mediated "liver-skin axis" therapy for various skin diseases. Simultaneously, HC-exos-containing bioconstructs can promote collagen transformation, ultimately preventing scar formation. This research provides potential directions for future synergistic treatment of hair-related diseases using HC-exos in conjunction with other innovative platforms. For example, combining microneedles with hepatocyte exosomes can target specific skin layers, providing a minimally invasive and convenient treatment for refractory diseases related to anti-oxidation, hair follicle regeneration, and scar remodeling. Furthermore, this micro-tissue can upregulate the AMPK signaling pathway. Moderate activation of AMPK in keratinocytes is beneficial for normal wound healing and regeneration of aging or diabetic wounds, opening up exciting possibilities for future treatment of dermatological and other systemic inflammatory pathologies.

Claims

1. Application of hepatocyte conditioned medium in the preparation of skin regeneration / repair / healing products.

2. Application of hepatocyte conditioned medium in the preparation of products that promote angiogenesis.

3. Application of hepatocyte-derived exosomes in the preparation of skin regeneration / repair / healing products.

4. Application of hepatocyte-derived exosomes in the preparation of products that promote angiogenesis.

5. Application of hepatocyte-derived exosomes in the preparation of scar remodeling products.

6. Application of hepatocyte-derived exosomes in the preparation of products for preventing scar formation.

7. Application of hepatocyte-derived exosomes in the preparation of products for the regeneration of aging wounds.

8. Application of hepatocyte-derived exosomes in the preparation of products for diabetic wound regeneration.

9. Application of hepatocyte-derived exosome-assisted 3D bioprinting of skin organoids in wound regeneration.

10. A method for preparing 3D bioprinted skin organoids assisted by hepatocyte-derived exosomes, characterized in that: Keratinocytes, fibroblasts, and endothelial cells were encapsulated in GelMa-ECM hydrogel containing hepatocyte-derived exosomes, and skin organoids were 3D bioprinted to simulate the structure of natural skin. The bottom layer contains endothelial cells, the middle layer contains fibroblasts, and the top layer contains keratinocytes.

Images