Functional sebaceous gland-skin integrated model based on biological 3D printing and construction method and application thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to a functional sebaceous gland-skin integrated model based on biological 3D printing and a construction method and application of the functional sebaceous gland-skin integrated model. The double-nozzle collaborative printing technology is adopted, a complete sebaceous gland-catheter-epidermis secretion path is successfully constructed, directional transportation of lipid secretion is achieved, and seamless communication between the catheter and the epidermis is achieved. Meanwhile, specific bio-ink is selected in the printing process, the mechanical stability of the model can be remarkably improved, and the collapse rate within 7 days after printing can be controlled to be 5% or below. The model precisely simulates natural anatomical characteristics in structure, the mechanical property is close to that of a natural catheter basement membrane, the problems that a secretion path is blocked, a catheter is prone to collapse and the like generally existing in a traditional model are effectively solved, and more reliable model support is provided for related research and application.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a functional sebaceous gland-skin integrated model based on bio-3D printing, its construction method, and its application. Background Technology

[0002] Skin 3D printing technology aims to replicate the epidermal-dermal layered structure and functional units such as blood vessels and appendages of natural skin through the precise assembly of biomaterials and cells. Current mainstream methods use hydrogels such as collagen and GelMA as bio-ink matrices, encapsulating keratinocytes, fibroblasts, etc., and then printing them layer by layer using extrusion or photopolymerization processes. However, existing technologies still have many shortcomings: (1) Limitations at the structural construction level: Existing 3D printed skin has significant defects in the biomimicry of tissue structure. The long-term survival rate of cells is limited by the shear stress during the printing process, and the insufficient bonding strength of the multi-layer structure interface easily leads to interlayer separation. Moreover, existing technologies have never been able to achieve precise spatial integration and physiological connectivity of sebaceous glands in skin models, and cannot construct a physiologically simulated "acini-duct-epidermal" three-level connectivity system. Existing technologies such as CTIBiotech require manual implantation of pre-cultured sebaceous gland spheres, which cannot directly 3D print sebaceous glands, and there is no duct structure. There is a gap of >100μm between the sphere and the epidermal layer, which leads to sebum retention rather than physiological excretion, and cannot form a duct-epidermal connectivity structure; Lim et al.'s inkjet printing scheme only forms disordered cell clumps and does not establish excretory ducts; in the patent (WO2021161142A1), sebaceous glands are randomly distributed in the dermis, and the gland depth fluctuates up to 200μm, which is far beyond the human dermal depth standard of 80±30μm. Traditional bioprinting is limited by resolution and the compatibility of cross-linking of multiple materials, making it difficult to simultaneously construct micron-scale catheters and ensure their seamless connection with the epidermis.

[0003] (2) Functional loss: Sebaceous gland cells in the existing 3D model face severe secretory function decline, with lipid secretion levels generally below 0.2 μg / (10) after 72 hours of culture. 6 The number of cells (24h) is less than 1 / 5 of that of primary cells. The low nutrient transport efficiency of the vascularized dermis further exacerbates the decline in cell function. The survival rate of deep fibroblasts is often less than 60%, and the collagen content synthesized by them is only 40% of that of natural dermis.

[0004] (3) Incompatibility between process and materials: The balance between the mechanical properties of bio-inks and cell activity remains a technical bottleneck. Although a high concentration of GelMA greater than 12% can increase the matrix modulus to 15 kPa, it will inhibit the activity of fatty acid synthase in sebaceous gland cells, resulting in a 60% decrease in lipid secretion; while a low modulus matrix of less than 5 kPa cannot maintain the stability of the acinar structure, and 20% of the structure collapses within 24 hours after printing. The complexity of the ink formulation further limits the feasibility of multi-cell co-printing. For example, epidermal ink containing melanocytes requires the addition of antioxidants to maintain cell activity, which leads to a difference of more than 30% in the crosslinking time between it and dermal ink. The lack of standardization in the post-processing procedure is also prominent: vascularization induction requires continuous perfusion of VEGF medium for more than 7 days, and the growth factor concentration, perfusion rate and other parameters vary significantly in different studies, resulting in a 2-3 fold fluctuation in the model's blood vessel density. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of existing technologies and provide a functional sebaceous gland-skin integrated model based on bio-3D printing, its construction method, and its application. The model possesses a complete secretion pathway, ensuring seamless communication between the ducts and the epidermis, thereby achieving directional transport of lipid secretion; it also exhibits good mechanical stability, effectively reducing the duct collapse rate.

[0006] To achieve the above objectives, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides a method for constructing a functional sebaceous gland-skin integrated model, comprising the following steps: S1. Prepare dermal ink, duct ink, sebaceous gland ink, basal layer ink and epidermal ink respectively; S2. 3D layer printing is performed using a bioprinter. Dermal ink is extruded to form the dermal layer base. A microfluidic nozzle is used to print duct ink into a spiral duct structure. Then, sebaceous gland ink is printed at the end of the spiral duct through another nozzle to form sebaceous gland acini. The duct and sebaceous gland acini are connected to form a sebaceous gland structure. S3. Continue printing dermal ink around and above the sebaceous gland structure to embed the sebaceous gland structure in the dermis. Then, print basal layer ink above the dermis to form the basal layer. Finally, print epidermal ink on top to form the epidermis, thus obtaining a 3D printed model. S4. Differentiate and culture the 3D printed model to obtain the functional sebaceous gland-skin integrated model; The sebaceous gland ink comprises sebaceous gland cells, myoepithelial cells, and GelMA hydrogel, wherein the sebaceous gland cells and myoepithelial cells are loaded in the GelMA hydrogel, and the mass concentration of GelMA in the GelMA hydrogel is 7-9%.

[0007] In existing technologies, the connection between sebaceous glands and ducts often relies on cell self-assembly or manual assembly, resulting in a loose structure and discontinuous secretion pathways. This invention utilizes a microfluidic dual-nozzle collaborative printing method to create a spiral duct structure, with the sebaceous gland acini positioned at the end for precise alignment. This design mimics the "acini-duct" structure of the natural pilosebaceous unit, ensuring that lipids are transported directly from the sebaceous gland acini to the epidermis via the duct, avoiding the interruption or disruption of the secretion pathway caused by random connections in traditional methods. Furthermore, through layered printing technology, the duct tip is directly connected to the epidermal layer, forming a complete secretion pathway system from the sebaceous gland to the epidermis, thus achieving a high degree of consistency with the physiological mechanism of sebum excretion to the skin surface via the pilosebaceous duct in natural skin. Moreover, this invention employs a layer-by-layer embedding technique, pre-positioning the duct tip below the basal layer during dermal printing. Subsequently, during printing in the basal and epidermal layers, material fusion achieves physical connectivity between the duct and the epidermis. This design effectively avoids interface separation, thereby ensuring the continuity of the secretion pathway.

[0008] Furthermore, traditional straight-tube conduits are prone to collapse during printing due to material gravity or fluid pressure during cultivation, leading to structural failure. This invention employs a spiral conduit structure, which effectively improves mechanical stability by increasing the lateral support force and material distribution uniformity of the conduit. Experimental studies have also revealed that the GelMA concentration in the sebaceous gland ink significantly impacts the mechanical stability of the final model. When the GelMA concentration is below 7%, the sebaceous gland ink fails to maintain the acinar structure, resulting in a model collapse rate exceeding 30% within 24 hours.

[0009] Therefore, through precise structural design and material optimization, this invention effectively overcomes the deficiencies of existing skin models in terms of functional integrity and mechanical stability, providing a more reliable model tool for dermatological research and translational medicine.

[0010] More preferably, the mass concentration of GelMA in the GelMA hydrogel is 8%.

[0011] Preferably, the dermal ink comprises fibroblasts, human umbilical vein endothelial cells, and collagen-fibrinogen hydrogel, wherein the fibroblasts and human umbilical vein endothelial cells are loaded in the collagen-fibrinogen hydrogel; The concentration of collagen in the hydrogel is 2-4 mg / mL, and the concentration of fibrin is 1-3 mg / mL; the cell density ratio of the fibroblasts and human umbilical vein endothelial cells is (3-5):(0.5-1.5).

[0012] Collagen and fibrin, as the main structural proteins of the dermal matrix, form a three-dimensional fibrous network through cross-linking, effectively maintaining the long-term stability of the structure. Furthermore, experimental studies have shown that when collagen and fibrin are combined within the aforementioned optimal concentration range, they can form a porous network with suitable pore size and good connectivity. This provides attachment sites for cells and ensures the diffusion of nutrients and oxygen, maintaining cell survival. If the concentration is too high, the pores will be too small, hindering substance exchange and leading to apoptosis; if the concentration is too low, it cannot support the orderly arrangement of cells, resulting in a loose structure. In addition, fibroblasts and human umbilical vein endothelial cells are the core cells for synthesizing the matrix and vascular-like structures. Cultured at optimal cell densities, both fibroblasts and human umbilical vein endothelial cells, can effectively ensure the dynamic renewal of the dermal structure, guaranteeing its long-term stability and preventing structural degradation.

[0013] More preferably, the concentration of collagen in the hydrogel is 3 mg / mL, and the concentration of fibrin is 2 mg / mL; the cell density ratio of the fibroblasts to the human umbilical vein endothelial cells is 4:1.

[0014] Preferably, the genuine leather ink further includes PLGA microspheres, the diameter of which is 10-20 μm, and the PLGA microspheres occupy 3-7% of the volume of the genuine leather ink.

[0015] In the process of constructing the dermal matrix, this invention also adds PLGA microspheres to the ink, which can be embedded in the three-dimensional network of collagen-fibrinogen hydrogel. Through physical filling, the mechanical strength of the hydrogel is enhanced, thereby effectively preventing the structural collapse of the model during long-term culture.

[0016] Preferably, the catheter ink comprises catheter epithelial cells, basement membrane proteins, and GelMA-gelatin composite hydrogel, wherein the catheter epithelial cells and basement membrane proteins are loaded in the GelMA-gelatin composite hydrogel; The basement membrane proteins include type IV collagen and laminin, with a mass ratio of type IV collagen to laminin of 1:(4-6); the total mass concentration of GelMA and gelatin in the GelMA-gelatin composite hydrogel is 10-14%. The cell density of the duct epithelial cells in the duct ink was 3 × 10⁻⁶. 6 -5×10 6 The ink in the conduit has a modulus of 15-20 kPa and a density of cells / mL.

[0017] Experimental research revealed that the concentration and modulus of hydrogel in the catheter ink, the selection of basement membrane proteins, and the control of cell density play a crucial role in the structural coherence and mechanical properties of the formed catheters and even the overall model. This invention achieves mechanical stability and structural integrity of the catheter structure through the synergistic effect of "mechanical support (hydrogel concentration and modulus), cell anchoring (basement membrane protein selection), and structure formation (cell density)," effectively maintaining its morphology while forming continuous tubular lumens that connect functionally with surrounding tissues. The optimized range of these parameters accurately simulates the microenvironmental characteristics of natural skin ducts, forming the core foundation for ensuring the function of the secretion transport channel in the sebaceous gland-skin integrated model.

[0018] More preferably, the mass ratio of type IV collagen to laminin is 1:5; the total mass concentration of GelMA and gelatin in the GelMA-gelatin composite hydrogel is 12%; and the cell density of the ductal epithelial cells in the duct ink is 4 × 10⁻⁶. 6 cells / mL.

[0019] Preferably, in the GelMA-gelatin composite hydrogel, the mass ratio of GelMA to gelatin is (1-3):1.

[0020] Preferably, the cell density in the sebaceous gland ink is 1×10⁻⁶. 6 -5×10 6 cells / mL, wherein the ratio of sebaceous gland cells to myoepithelial cells is (1-3):1.

[0021] More preferably, the cell density in the sebaceous gland ink is 3 × 10⁻⁶. 6 cells / mL, wherein the ratio of sebaceous gland cells to myoepithelial cells is 2:1.

[0022] Preferably, the base layer ink comprises hydrokeratinocyte precursor cells and alginate-hyaluronic acid hydrogel, wherein the hydrokeratinocyte precursor cells are loaded in the alginate-hyaluronic acid hydrogel; In the alginate-hyaluronic acid hydrogel, the mass concentration of alginate is 1-2%, and the mass concentration of hyaluronic acid is 0.5-1%; the cell density of the water keratinocyte precursor cells is 4×10⁻⁶. 6 -6×10 6 cells / mL; the alginate-hyaluronic acid hydrogel also contains EGF at a concentration of 15-25 ng / mL.

[0023] As a mechanical buffer layer, the basal layer's mechanical properties must match those of the adjacent dermis and epidermis to avoid interlayer delamination due to mechanical mismatch. The specific concentration of hydrogel used in this invention forms a soft network with a suitable modulus. It is neither too soft (e.g., too low alginate concentration) causing deformation under pressure from the dermis, nor too hard (e.g., too high alginate concentration) causing mechanical conflict with the epidermis. Furthermore, EGF-induced cell proliferation enhances intercellular connectivity, further improving the basal layer's shear resistance and indirectly maintaining the overall mechanical stability of the model.

[0024] More preferably, in the alginate-hyaluronic acid hydrogel, the mass concentration of alginate is 1.5% and the mass concentration of hyaluronic acid is 0.8%; the cell density of the water keratinocyte precursor cells is 5 × 10⁻⁶. 6 cells / mL; the alginate-hyaluronic acid hydrogel also contains EGF at a concentration of 20 ng / mL.

[0025] Preferably, the epidermal ink comprises keratinocytes and GelMA hydrogel, wherein the keratinocytes are loaded in the GelMA hydrogel; The GelMA hydrogel contains 7-9% GelMA by mass and also contains collagen at a concentration of 0.5-1.5 mg / mL; the keratinocyte cell density is 5 × 10⁻⁶. 6 -7×10 6 cells / mL.

[0026] The limiting conditions of the epidermal ink described in this invention ensure that the epidermal layer forms a continuous, multi-layered mature structure through the synergistic effect of "structural support (GelMA concentration), cell adhesion and differentiation (collagen), and layer continuity (cell density)," which has a barrier function close to that of the natural epidermis and is stably connected to the basal layer, ultimately improving the structural integrity and functional authenticity of the entire sebaceous gland-skin integrated model.

[0027] Preferably, in step S2, when using a microfluidic nozzle to print the conduit ink into a spiral conduit structure, the printing pressure is 0.1-0.3 MPa and the printing speed is 15-25 mm / s. In step S4, the specific process of differentiation culture is as follows: the 3D printed model is first immersed in culture and then cultured using an air-liquid interface; the components of the culture medium during the immersion culture include DMEM / F12 basal medium and 5-15% FBS, and 0.05-0.15 μM rosiglitazone and 40-60 nM dihydrotestosterone are also added to the culture medium during the air-liquid interface culture.

[0028] Experimental research revealed that the printing and differentiation culture conditions involved in model construction significantly impact the performance of the final model. Printing conditions ensure the precise shaping of the spiral ducts, providing a structural basis for secretion transport. Differentiation culture conditions, through the phased regulation of "immersion proliferation-air-liquid interface differentiation," combined with the functional induction of rosiglitazone and dihydrotestosterone, promote epidermal barrier formation, sebaceous gland maturation, and functional synergy among various tissues. These conditions collectively ensure that the final model not only possesses a complete "dermis-duct-sebaceous gland-basal layer-epidermis" structure but also simulates the core functions of natural skin, thereby enhancing the model's physiological relevance and application value.

[0029] Secondly, the present invention provides an integrated sebaceous gland-skin model obtained by the aforementioned construction method.

[0030] Thirdly, the present invention provides the application of the sebaceous gland-skin integrated model in the in vitro evaluation of the efficacy of cosmetics or their raw materials, the screening of the efficacy of active ingredients in cosmetics, or the evaluation of the efficacy of biological agents.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Structural integrity: This invention is the first to achieve the construction of a complete secretory pathway from sebaceous gland to duct to epidermis. Traditional models only contain sebaceous gland acini, or the error between the interface of the manually assembled duct and acini is >50μm, resulting in a secretory pathway blockage rate of up to 40%. In contrast, this invention uses a dual-nozzle collaborative printing technology to first accurately print a spiral duct with a microfluidic nozzle, and then connect and print acini at the end of the duct, achieving a duct-acini connectivity error of <5μm, which is 10 times better than the existing technology, and the secretory pathway patency rate reaches 95%. Scanning electron microscopy shows that the ductal epithelial cells are arranged in a columnar shape, and the cells at the interface with the acini are continuous without breakage, simulating the anatomical structure of natural sebaceous gland ducts.

[0032] (2) Mechanical stability: The construction method described in this invention can significantly reduce the collapse rate of the catheter. In traditional catheter printing, the GelMA hydrogel modulus is insufficient (<10kPa), and the collapse rate of the catheter after printing is >30%, which cannot maintain the spiral structure. However, the catheter ink described in this invention uses a specific concentration of GelMA-gelatin composite matrix (modulus 15-20kPa) and adds type IV collagen and laminin to simulate the mechanical properties of the basement membrane. Mechanical tests show that the compressive modulus is basically consistent with that of the natural catheter basement membrane, and the collapse rate is <5% after 7 days of printing. Attached Figure Description

[0033] Figure 1 This is a schematic diagram (cross-section) of the layered structure of the 3D-printed sebaceous gland-skin integrated model described in this invention. Figure 2 This is a flowchart of the dual-printer collaborative printing process described in this invention. Detailed Implementation

[0034] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0035] Unless otherwise specified, all reagents used in the examples are conventional reagents available in the art and can be purchased commercially. Experimental procedures not specifically described in the examples are conventional procedures in the art or can be understood or known by those skilled in the art based on their prior knowledge or common general knowledge.

[0036] Example 1 This embodiment provides a method for constructing a functional sebaceous gland-skin integrated model, including the following steps: 1. Preparation of bio-ink: (1) Dermal ink: fibroblasts (4×10 6 cells / mL) and HUVECs (1×10⁻⁶ cells / mL) 6 The collagen-fibrinogen hydrogel (collagen concentration 3 mg / mL, fibrinogen concentration 2 mg / mL) was blended with PLGA microspheres (diameter 15 μm, accounting for 5% of the ink volume).

[0037] (2) Catheter ink: Catheter epithelial cells (HaCaT duct differentiated cells) were mixed with basement membrane proteins type IV collagen and laminin at a ratio of 1:5 and suspended in 12% GelMA-gelatin composite hydrogel (containing 0.5 mg / mL fibronectin, with a mass ratio of GelMA to gelatin of 2:1), with a cell density of 4 × 10⁻⁶ cells / mL. 6 cells / mL, ink modulus 15-20kPa, simulating the mechanical properties of the duct basement membrane.

[0038] (3) Sebaceous gland ink: Sebaceous gland cells and myoepithelial cells were mixed at a ratio of 2:1, and 8% GelMA hydrogel containing triglyceride precursors was added. The cell density was 3×10⁻⁶. 6 The adhesion strength between the cells / mL and the ink in the conduit is >0.1MPa, ensuring a seamless connection at the interface.

[0039] (4) Basal layer ink: Keratinocyte precursor cells (HaCaT progenitor cells) were suspended in an alginate-hyaluronic acid hydrogel containing 20 ng / mL EGF (alginate concentration 1.5%, hyaluronic acid concentration 0.8%), with a cell density of 5 × 10⁻⁶ cells / mL. 6 cells / mL.

[0040] (5) Epidermal ink: Differentiated mature keratinocytes (HaCaT cells) were added to a GelMA hydrogel containing 1 mg / mL collagen (GelMA concentration of 8%), with a cell density of 6 × 10⁻⁶. 6 cells / mL.

[0041] 2. Multi-nozzle extrusion bioprinter for layered printing: Layer printing was performed using a multi-nozzle extrusion bioprinter, model CELLINKBIOX6.

[0042] (1) Print the bottom layer of the dermis: 3 layers, each layer is 100μm, and PLGA vascular template is printed simultaneously. The ink concentration is 10mg / mL, and it is formed by temperature control extrusion at 25℃.

[0043] (2) Pre-forming of conduit: Using a microfluidic nozzle (inner diameter 20μm) to print a spiral conduit structure (diameter 30-50μm), printing pressure 0.2MPa, speed 20mm / s, layer thickness 30μm, nozzle temperature 25℃.

[0044] (3) Alveolar docking printing: Sebaceous gland alveoli are positioned and printed at the end of the duct through another nozzle, and the temperature of the sebaceous gland ink is 37°C.

[0045] (4) Printing dermal overlay: 2 layers, each 100μm. Continue printing dermal bio-ink around and above the sebaceous gland structure to completely embed and integrate the sebaceous gland structure into the dermal layer, while ensuring the duct pathway is unobstructed.

[0046] (5) Print the base layer: 1 layer, 50μm thick, nozzle temperature 25℃, and avoid the conduit opening.

[0047] (6) Printing the epidermal layer: Print epidermal bio-ink on top, 3 layers, each 30μm. Precisely control the printing position so that the opening end of the sebaceous gland duct is exposed on the surface of the epidermis to be formed.

[0048] (7) The 3D printed model is obtained after printing is completed.

[0049] 3. Two-stage differentiation cultivation: (1) Stage 1 (0-7 days): The 3D printed model is immersed in culture medium consisting of DMEM / F12 and 10% FBS; (2) Stage 2 (7-21 days): The 3D printed model is then cultured at the gas-liquid interface, with 0.1 μM rosiglitazone and 50 nM dihydrotestosterone added to the culture medium.

[0050] (3) After the culture is completed, the functional sebaceous gland-skin integrated model is obtained.

[0051] Example 2 This embodiment provides a method for constructing a functional sebaceous gland-skin integrated model, including the following steps: 1. Preparation of bio-ink: (1) Dermal ink: fibroblasts (3×10 6 cells / mL) and HUVECs (1.5×10 6 The collagen-fibrinogen hydrogel (collagen concentration 2 mg / mL, fibrinogen concentration 3 mg / mL) was blended with PLGA microspheres (10 μm in diameter, accounting for 7% of the ink volume).

[0052] (2) Duct ink: Duct epithelial cells (HaCaT) differentiated duct cells were mixed with basement membrane proteins type IV collagen and laminin at a ratio of 1:4 and suspended in 10% GelMA-gelatin composite hydrogel (containing 0.5 mg / mL fibronectin, with a mass ratio of GelMA to gelatin of 1:1), with a cell density of 3 × 10⁻⁶ cells / mL. 6 cells / mL, ink modulus 15-20kPa, simulating the mechanical properties of the duct basement membrane.

[0053] (3) Sebaceous gland ink: Sebaceous gland cells and myoepithelial cells were mixed at a ratio of 1:1, and 7% GelMA hydrogel containing triglyceride precursors was added. The cell density was 1×10⁻⁶. 6 The adhesion strength between the cells / mL and the ink in the conduit is >0.1MPa, ensuring a seamless connection at the interface.

[0054] (4) Basal layer ink: Keratinocyte precursor cells (HaCaT progenitor cells) were suspended in an alginate-hyaluronic acid hydrogel containing 15 ng / mL EGF (1% alginate concentration, 1% hyaluronic acid concentration), with a cell density of 4 × 10⁻⁶ cells / mL. 6 cells / mL.

[0055] (5) Epidermal ink: Differentiated mature keratinocytes (HaCaT cells) were added to a GelMA hydrogel containing 0.5 mg / mL collagen (GelMA concentration of 7%), with a cell density of 5 × 10⁻⁶ cells / mL. 6 cells / mL.

[0056] 2. Multi-nozzle extrusion bioprinter for layered printing: Layer printing was performed using a multi-nozzle extrusion bioprinter, model CELLINKBIOX6.

[0057] (1) Print the bottom layer of the dermis: 3 layers, each layer is 100μm, and PLGA vascular template is printed simultaneously. The ink concentration is 10mg / mL, and it is formed by temperature control extrusion at 25℃.

[0058] (2) Pre-forming of conduit: Using a microfluidic nozzle (inner diameter 20μm) to print a spiral conduit structure (diameter 30-50μm), printing pressure 0.1MPa, speed 25mm / s, layer thickness 30μm, nozzle temperature 25℃.

[0059] (3) Alveolar docking printing: Sebaceous gland alveoli are positioned and printed at the end of the duct through another nozzle, and the temperature of the sebaceous gland ink is 37°C.

[0060] (4) Printing dermal overlay: 2 layers, each 100μm. Continue printing dermal bio-ink around and above the sebaceous gland structure to completely embed and integrate the sebaceous gland structure into the dermal layer, while ensuring the duct pathway is unobstructed.

[0061] (5) Print the base layer: 1 layer, 50μm thick, nozzle temperature 25℃, and avoid the conduit opening.

[0062] (6) Printing the epidermal layer: Print epidermal bio-ink on top, 3 layers, each 30μm. Precisely control the printing position so that the opening end of the sebaceous gland duct is exposed on the surface of the epidermis to be formed.

[0063] (7) The 3D printed model is obtained after printing is completed.

[0064] 3. Two-stage differentiation cultivation: (1) Stage 1 (0-7 days): The 3D printed model is immersed in culture medium consisting of DMEM / F12 and 5% FBS; (2) Stage 2 (7-21 days): The 3D printed model is then cultured at the gas-liquid interface, with 0.05 μM rosiglitazone and 60 nM dihydrotestosterone added to the culture medium.

[0065] (3) After the culture is completed, the functional sebaceous gland-skin integrated model is obtained.

[0066] Example 3 This embodiment provides a method for constructing a functional sebaceous gland-skin integrated model, including the following steps: 1. Preparation of bio-ink: (1) Dermal ink: fibroblasts (5×10 6 cells / mL) and HUVECs (0.5×10 6 The collagen-fibrinogen hydrogel (collagen concentration 4 mg / mL, fibrinogen concentration 1 mg / mL) was blended with PLGA microspheres (diameter 20 μm, accounting for 3% of the ink volume).

[0067] (2) Catheter ink: Catheter epithelial cells (HaCaT duct differentiated cells) were mixed with basement membrane proteins type IV collagen and laminin at a ratio of 1:6 and suspended in 14% GelMA-gelatin composite hydrogel (containing 0.5 mg / mL fibronectin, with a mass ratio of GelMA to gelatin of 3:1), with a cell density of 5 × 10⁻⁶ cells / mL. 6 cells / mL, ink modulus 15-20kPa, simulating the mechanical properties of the duct basement membrane.

[0068] (3) Sebaceous gland ink: Sebaceous gland cells and myoepithelial cells were mixed at a ratio of 3:1, and 9% GelMA hydrogel containing triglyceride precursors was added. The cell density was 5×10⁻⁶. 6 The adhesion strength between the cells / mL and the ink in the conduit is >0.1MPa, ensuring a seamless connection at the interface.

[0069] (4) Basal layer ink: Keratinocyte precursor cells (HaCaT progenitor cells) were suspended in an alginate-hyaluronic acid hydrogel containing 25 ng / mL EGF (alginate concentration 2%, hyaluronic acid concentration 0.5%), with a cell density of 6 × 10⁻⁶. 6 cells / mL.

[0070] (5) Epidermal ink: Differentiated mature keratinocytes (HaCaT cells) were added to a GelMA hydrogel containing 1.5 mg / mL collagen (GelMA concentration of 9%), with a cell density of 7 × 10⁻⁶. 6 cells / mL.

[0071] 2. Multi-nozzle extrusion bioprinter for layered printing: Layer printing was performed using a multi-nozzle extrusion bioprinter, model CELLINKBIOX6.

[0072] (1) Print the bottom layer of the dermis: 3 layers, each layer is 100μm, and PLGA vascular template is printed simultaneously. The ink concentration is 10mg / mL, and it is formed by temperature control extrusion at 25℃.

[0073] (2) Pre-forming of conduit: Using a microfluidic nozzle (inner diameter 20μm) to print a spiral conduit structure (diameter 30-50μm), printing pressure 0.3MPa, speed 15mm / s, layer thickness 30μm, nozzle temperature 25℃.

[0074] (3) Alveolar docking printing: Sebaceous gland alveoli are positioned and printed at the end of the duct through another nozzle, and the temperature of the sebaceous gland ink is 37°C.

[0075] (4) Printing dermal overlay: 2 layers, each 100μm. Continue printing dermal bio-ink around and above the sebaceous gland structure to completely embed and integrate the sebaceous gland structure into the dermal layer, while ensuring the duct pathway is unobstructed.

[0076] (5) Print the base layer: 1 layer, 50μm thick, nozzle temperature 25℃, and avoid the conduit opening.

[0077] (6) Printing the epidermal layer: Print epidermal bio-ink on top, 3 layers, each 30μm. Precisely control the printing position so that the opening end of the sebaceous gland duct is exposed on the surface of the epidermis to be formed.

[0078] (7) The 3D printed model is obtained after printing is completed.

[0079] 3. Two-stage differentiation cultivation: (1) Stage 1 (0-7 days): The 3D printed model is immersed in culture medium consisting of DMEM / F12 and 15% FBS; (2) Stage 2 (7-21 days): The 3D printed model is then cultured at the gas-liquid interface, with 0.15 μM rosiglitazone and 40 nM dihydrotestosterone added to the culture medium.

[0080] (3) After the culture is completed, the functional sebaceous gland-skin integrated model is obtained.

[0081] Comparative Example 1 This comparative example provides a method for constructing a functional sebaceous gland-skin integrated model. The only difference between this method and Example 1 is that the concentration of GelMA in the GelMA hydrogel used in the sebaceous gland ink is 5%. All other operations are the same as in Example 1.

[0082] Comparative Example 2 This comparative example provides a method for constructing a functional sebaceous gland-skin integrated model. The only difference between this method and Example 1 is that type IV collagen is not added to the duct ink. All other operations are the same as in Example 1.

[0083] Comparative Example 3 This comparative example provides a method for constructing a functional sebaceous gland-skin integrated model, which differs from Example 1 only in that the total mass concentration of GelMA and gelatin in the composite gel in the catheter ink is 7%, and the rest of the operation process is the same as in Example 1.

[0084] Comparative Example 4 This comparative example provides a method for constructing a functional sebaceous gland-skin integrated model. The only difference between this method and Example 1 is the absence of a dual-printer collaborative printing step. The specific printing process is as follows: (1) Bottom layer printing: Three layers of dermal ink are printed using micro-extrusion method, and PLGA braided layer and vascular template are formed simultaneously, and thrombin cross-linking and curing are performed.

[0085] (2) Catheter printing: Switch the catheter ink and print the catheter and support structure by micro-extrusion. After printing, photo-crosslinking and curing are performed.

[0086] (3) Alveolar connection: After rinsing the nozzle for 10-15 minutes, print the alveoli independently and move them to the end of the duct. Then process the braided layer to assist adhesion.

[0087] (4) Subsequent layer printing: Print two layers of dermal cover layer embedding structure, and then print the base layer and epidermal layer in sequence.

[0088] (5) Post-treatment: Remove temporary support and cure at 4-6℃ for 1-2 hours; transfer to culture system and add VEGF for immersion and gas-liquid interface culture.

[0089] Example 1 This example uses the sebaceous gland-skin integrated model obtained by the construction method described in Example 1 as a sample to perform performance testing. The specific testing method is as follows: (1) Structural characterization 1. Scanning electron microscopy: The sample was fixed with 2.5% glutaraldehyde, dehydrated with gradient ethanol, and critical point dried before being sputter-coated with gold; the duct-acinar interface was observed at an accelerating voltage of 10kV and a magnification of 500-2000x; the interface gap was measured using ImageJ software, and the average value was taken from 5 consecutive fields of view. 2. HE staining: After embedding the sample, section it (5μm thick), stain with hematoxylin for 5 minutes and eosin for 30 seconds; observe the arrangement of ductal epithelial cells under an optical microscope and count the proportion of columnar cells.

[0090] Scanning electron microscopy showed that the cells at the duct-acinar interface of the model were continuous without any gaps, achieving a duct-acinar connectivity error of <5μm, which is 10 times better than the existing technology, and the secretion pathway patency rate reached 95%. Furthermore, HE staining showed that the ductal epithelial cells were arranged in a columnar pattern, consistent with the structure of natural sebaceous gland ducts.

[0091] (2) Functional testing 1. Oil Red O staining: After the samples were fixed with 4% paraformaldehyde, they were stained with 0.5% Oil Red O isopropanol solution for 30 minutes, differentiated with 60% isopropanol, and rinsed with distilled water; the transport of lipid particles along the ducts was observed under an optical microscope, and the particle distribution ratio was quantitatively analyzed using Image-ProPlus software. 2. ELISA detection of triglycerides: Collect the culture supernatant, centrifuge at 3000 rpm for 10 minutes and take the supernatant; use the triglyceride ELISA kit, add sample (50 μL / well) according to the instructions, incubate at 37℃ for 1 hour, wash and add chromogenic reagent, react in the dark for 15 minutes; measure absorbance at 450 nm wavelength, calculate the concentration according to the standard curve, and then obtain the proportion secreted through the duct.

[0092] Oil Red O staining showed that lipid particles were transported along the ducts to the epidermis. ELISA analysis revealed that the triglyceride content in the culture medium was 1.2 μg / mL, with 82% secreted via the ducts. These results indicate that the construction method described in this invention can successfully construct a complete secretory pathway from sebaceous gland to duct to epidermis, achieving directional transport of lipid secretion and seamless duct-epidermal connectivity.

[0093] Example 2 This example uses the sebaceous gland-skin integrated model prepared by the construction method described in Example 1 and Comparative Examples 1-3 as a sample to test its mechanical stability.

[0094] Experimental investigation revealed that the model described in Example 1 exhibited a compressive modulus that was essentially consistent with that of the natural ductal basement membrane, as shown by mechanical testing. The collapse rate was less than 5% after 7 days of printing, demonstrating excellent mechanical stability and effectively reducing model collapse. In contrast, the model in Comparative Example 1 suffered from a collapse rate exceeding 30% within 24 hours due to the reduced concentration of GelMA hydrogel, which prevented the sebaceous gland ink from maintaining the acinar structure. Furthermore, the lack of type IV collagen in the ductal ink of Comparative Example 2 and the lower-than-ideal concentration of the composite gel in the ductal ink of Comparative Example 3 resulted in a decreased match between the mechanical properties of the ductal basement membrane and the model, leading to a ductal collapse rate of 20% within 7 days and a similarly decreased stability compared to the examples.

[0095] Example 3 This effect example uses the sebaceous gland-skin integrated model prepared by the construction method described in Example 1 and Comparative Example 4 as a sample to test its duct-acinth patency rate.

[0096] Experimental investigation revealed that the model constructed using the dual-nozzle collaborative printing technology in Example 1 achieved a duct-acinth connectivity error of <5μm, a 10-fold improvement over existing technologies, with a secretion pathway patency rate of 95%. In contrast, the model in Comparative Example 4, lacking the dual-nozzle collaborative printing step, exhibited a duct-acinth interface error exceeding 60μm, with a secretion pathway blockage rate rising to 50%, failing to fully simulate the complete anatomical structure of natural sebaceous gland ducts.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for constructing a functional sebaceous gland-skin integrated model, characterized in that, Includes the following steps: S1. Prepare dermal ink, duct ink, sebaceous gland ink, basal layer ink and epidermal ink respectively; S2. 3D layer printing is performed using a bioprinter. Dermal ink is extruded to form the dermal layer base. A microfluidic nozzle is used to print duct ink into a spiral duct structure. Then, sebaceous gland ink is printed at the end of the spiral duct through another nozzle to form sebaceous gland acini. The duct and sebaceous gland acini are connected to form a sebaceous gland structure. S3. Continue printing dermal ink around and above the sebaceous gland structure to embed the sebaceous gland structure in the dermis. Then, print basal layer ink above the dermis to form the basal layer. Finally, print epidermal ink on top to form the epidermis, thus obtaining a 3D printed model. S4. Differentiate and culture the 3D printed model to obtain the functional sebaceous gland-skin integrated model; The sebaceous gland ink comprises sebaceous gland cells, myoepithelial cells, and GelMA hydrogel, wherein the sebaceous gland cells and myoepithelial cells are loaded in the GelMA hydrogel, and the mass concentration of GelMA in the GelMA hydrogel is 7-9%.

2. The construction method as described in claim 1, characterized in that, The dermal ink comprises fibroblasts, human umbilical vein endothelial cells, and collagen-fibrinogen hydrogel, wherein the fibroblasts and human umbilical vein endothelial cells are loaded in the collagen-fibrinogen hydrogel. The concentration of collagen in the hydrogel is 2-4 mg / mL, and the concentration of fibrin is 1-3 mg / mL; the cell density ratio of the fibroblasts and human umbilical vein endothelial cells is (3-5):(0.5-1.5).

3. The construction method as described in claim 2, characterized in that, The genuine leather ink also includes PLGA microspheres, which have a diameter of 10-20 μm and occupy 3-7% of the volume of the genuine leather ink.

4. The construction method as described in claim 1, characterized in that, The catheter ink comprises catheter epithelial cells, basement membrane proteins, and GelMA-gelatin composite hydrogel, wherein the catheter epithelial cells and basement membrane proteins are loaded in the GelMA-gelatin composite hydrogel. The basement membrane proteins include type IV collagen and laminin, with a mass ratio of type IV collagen to laminin of 1:(4-6); the total mass concentration of GelMA and gelatin in the GelMA-gelatin composite hydrogel is 10-14%. The cell density of the duct epithelial cells in the duct ink is 3 x 10 6 -5 x 10 6 cells / mL, and the modulus of the duct ink is 15-20 kPa.

5. The construction method as described in claim 1, characterized in that, The cell density in the sebaceous gland ink is 1 x 10 6 -5 x 10 6 cells / mL, and the ratio of the sebaceous gland cells and the myoepithelial cells is (1-3):

1.

6. The construction method as described in claim 1, characterized in that, The base layer ink includes hydrokeratinocyte precursor cells and alginate-hyaluronic acid hydrogel, wherein the hydrokeratinocyte precursor cells are loaded in the alginate-hyaluronic acid hydrogel; The mass concentration of alginate in the alginate-hyaluronic acid hydrogel is 1-2%, and the mass concentration of hyaluronic acid is 0.5-1%; the cell density of the water keratinocyte precursor cells is 4×10 6 -6×10 6 cells / mL; and the alginate-hyaluronic acid hydrogel further contains EGF with a concentration of 15-25 ng / mL.

7. The construction method as described in claim 1, characterized in that, The epidermal ink comprises keratinocytes and GelMA hydrogel, wherein the keratinocytes are loaded in the GelMA hydrogel; The GelMA hydrogel contains 7-9% GelMA by mass and also contains collagen at a concentration of 0.5-1.5 mg / mL; the keratinocyte cell density is 5 × 10⁻⁶. 6 -7×10 6 cells / mL.

8. The construction method as described in claim 1, characterized in that, In step S2, when using a microfluidic nozzle to print the conduit ink into a spiral conduit structure, the printing pressure is 0.1-0.3 MPa and the printing speed is 15-25 mm / s. In step S4, the specific process of differentiation culture is as follows: the 3D printed model is first immersed in culture and then cultured using an air-liquid interface; the components of the culture medium during the immersion culture include DMEM / F12 basal medium and 5-15% FBS, and 0.05-0.15 μM rosiglitazone and 40-60 nM dihydrotestosterone are also added to the culture medium during the air-liquid interface culture.

9. The sebaceous gland-skin integrated model obtained by the construction method according to any one of claims 1-8.

10. The application of the sebaceous gland-skin integrated model as described in claim 9 in the in vitro evaluation of the efficacy of cosmetics or their raw materials, the screening of the efficacy of active ingredients in cosmetics, or the evaluation of the efficacy of biological agents.

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