Construction method of composite artificial dermis containing living cells

Abstract

The invention relates to the technical field of biology, and particularly discloses a construction method of composite artificial dermis containing living cells, which comprises the following steps: S1, bracket pretreatment; s2, inoculating fibroblasts; s3, constructing a corium layer; s4, inoculating keratinocytes; s5, carrying out gas-liquid interface culture; s6, performing histological evaluation; according to the constructed dermis, the layers are distinct, the structures are bionic, fibroblasts and keratinocytes are constructed on the same support in a layered mode, the dermis layer and the epidermis layer are mutually coupled, and the morphological structure and the histological structure are similar to those of natural skin; the method has the advantages that cell activity is high, adhesion and proliferation are good, the pretreated hydrated scaffold promotes cell adherence and three-dimensional proliferation, fibroblasts secrete a large amount of extracellular matrixes in the scaffold, keratinocytes are efficiently differentiated under a gas-liquid interface condition, and good proliferation and differentiation activity is maintained.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for constructing a composite artificial dermis containing living cells. Background Technology

[0002] Human skin is composed of the epidermis and dermis, which work together to play important functions in protection, perception, regulation, and repair. The dermis is mainly composed of extracellular matrix such as collagen and elastic fibers secreted by fibroblasts, providing mechanical support and elasticity for the skin. The epidermis, on the other hand, is composed of multiple layers of keratinocytes differentiated from keratinocytes, forming a barrier that directly contacts the body with the external environment. Because various wounds (such as burns, diabetic foot ulcers, and chronic ulcers) are often accompanied by large areas of skin tissue loss, relying solely on autologous skin grafts has limitations such as limited donor sites, secondary trauma, and scar formation.

[0003] Artificial dermis prepared from traditional biomaterials generally only contains the dermal layer structure, lacks an active epidermal layer, or cannot achieve complete skin function reconstruction, making it difficult to meet clinical needs for rapid skin formation, restoration of barrier function, and aesthetics.

[0004] In response to this problem, this application proposes a method for constructing a composite artificial dermis containing living cells. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a composite artificial dermis containing living cells, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for constructing a composite artificial dermis containing living cells, comprising:

[0008] S1. Scaffold pretreatment: The artificial dermal scaffold was washed in sterile phosphate-buffered saline (PBS) and incubated in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum FBS at 37°C and 5% CO2 for 3 hours to obtain the pretreated hydrated scaffold.

[0009] S2. Fibroblast seeding: Human fibroblast suspension (2×105 cells / mL, 500μL) was dropped onto the surface of the pretreated hydrated scaffold and incubated at 37℃ and 5% CO2 for 24h to allow the cells to adhere to the scaffold and obtain a preliminary adherent scaffold.

[0010] S3. Dermal layer construction: The preliminary adherent scaffold was transferred into DMEM containing 10% FBS for routine culture for 7 days, with the medium changed every 2-3 days, to allow fibroblasts to proliferate and secrete extracellular matrix in the scaffold, thus obtaining a composite scaffold containing a dermal layer.

[0011] S4. Keratinocyte seeding: Human keratinocyte suspension (1×106 cells / mL, 500μL) was dropped onto the surface of the composite scaffold containing the dermis and incubated at 37°C and 5% CO2 for 24h to allow the keratinocytes to adhere to the wall, thus obtaining a composite scaffold containing the epidermal precursor layer.

[0012] S5. Gas-liquid interface culture: The composite scaffold containing the epidermal precursor layer is transferred to the gas-liquid interface culture device. Culture medium is added to the bottom of the scaffold in the lower chamber to keep the epidermal layer exposed to air. The culture medium is replaced every 48 hours to promote keratinocyte differentiation and layered construction to obtain mature composite artificial dermis containing live cells.

[0013] S6. Histological evaluation: On days 7, 14, 21 and 28 of culture, the composite artificial dermis was taken for hematoxylin-eosin (HE) staining and immunofluorescence staining to evaluate the histological structure and cell distribution of the epidermis and dermis.

[0014] Preferably, the PBS in step S1 is a calcium- and magnesium-free formulation PBS with a pH of 7.2–7.4.

[0015] Preferably, the DMEM in step S1 is a high-glucose DMEM containing 4.5 g / L glucose, 2 mM glutamate transporter, and 1 mM sodium pyruvate.

[0016] Preferably, the concentration of the fibroblast suspension in step S2 is 1.5 × 10⁻⁶. 5 The inoculum concentration was 2.5 × 10⁵ cells / mL, and the inoculum size was 400–600 μL.

[0017] Preferably, the culture time in step S3 is 7 days ± 1 day, and the culture medium used to replace the culture medium is the same as that in step (1).

[0018] Preferably, the concentration of the keratinocyte suspension in step S4 is 0.8 × 10⁻⁶. 6 Up to 1.2×10 6 The inoculum size is 450–550 μL, with cells / mL.

[0019] Preferably, the gas-liquid interface culture device used in step S5 is Multi-hole support system.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] (1) The dermis constructed in this invention has distinct layers and a biomimetic structure. Fibroblasts and keratinocytes are constructed in layers on the same scaffold, and the dermis and epidermis are coupled to each other. The morphological and histological structures are similar to natural skin. The cells have high activity and good adhesion and proliferation. The pretreated and hydrated scaffold promotes cell adhesion and three-dimensional proliferation, so that fibroblasts secrete a large amount of extracellular matrix inside the scaffold, while keratinocytes differentiate efficiently under air-liquid interface conditions, maintaining good proliferation and differentiation activity.

[0022] (2) The composite dermis constructed in this invention has a complete multilayer keratinocyte structure obtained by gas-liquid interface culture, which can effectively simulate the barrier function of natural epidermis to external physical and chemical stimuli. The preparation process is simple and highly controllable. The conditions of each step (cell density, culture time, frequency of medium change, etc.) can be precisely set. The process does not require complex equipment and is easy to scale up and clinically translate. It can be used for evaluation and customization. Through histological evaluation such as HE staining and immunofluorescence, the construction quality and functional indicators of the composite dermis can be monitored in real time, which is convenient for further optimization and personalized customization. Attached Figure Description

[0023] Figure 1 The image shows the morphology of HFbs observed under an inverted microscope (100×) during fibroblast observation according to the present invention.

[0024] Figure 2 This is a 200× HE staining image of human fibroblasts from the present invention.

[0025] Figure 3 This is a diagram showing the immunofluorescence identification results of the HFbs vimentin protein of the present invention;

[0026] Figure 4 This is a morphological image of HEK isolated and cultured according to the present invention under an inverted microscope;

[0027] Figure 5 This is an immunofluorescence identification image of the HEK keratin K14 isolated and cultured according to the present invention;

[0028] Figure 6 This is a diagram illustrating the cell suspension inoculation process of the present invention;

[0029] Figure 7 This is a diagram of the air-liquid interface (ALI) culture system of the present invention.

[0030] Figure 8 Histological HE (200×) images of normal skin and tissue-engineered skin containing live cell composite material according to the present invention: (a) normal skin; (b) tissue-engineered skin containing live cell composite material; (c) scaffold material alone.

[0031] Figure 9 HE staining images of composite tissue-engineered skin containing live cells and normal skin after different in vitro culture days according to the present invention: (A)(B)(C) Composite tissue-engineered skin containing live cells after 7 days of in vitro culture; (D)(E)(F) Composite tissue-engineered skin containing live cells after 14 days of in vitro culture; (G)(H)(I) Composite tissue-engineered skin containing live cells after 21 days of in vitro culture; (J)(K)(L) Composite tissue-engineered skin containing live cells after 28 days of in vitro culture; (M)(N)(O) Histological structure of normal human skin.

[0032] Figure 10 The image shows the immunofluorescence expression characteristics of K14 and Viminten in normal skin of the present invention (×400).

[0033] Figure 11 Figure 400 shows the expression and distribution of HEK in the live cell composite tissue-engineered skin of the present invention.

[0034] Figure 12 Figure 200 shows the expression and distribution of HFbs in the live cell composite tissue engineered skin of the present invention.

[0035] Figure 13 For the wound covered by the living cell composite tissue-engineered skin transplant of the present invention, (a) marked with methacin; (b) full-thickness skin defect wound; (c) post-transplantation;

[0036] Figure 14 This invention provides a gross observation of the repair of full-thickness skin defects in nude mice.

[0037] Figure 15 The following are examples of scar repair on day 28 for full-thickness skin defects according to the present invention: (a) wound repair on day 28 using composite tissue-engineered skin containing live cells cultured in vitro for 21 days; (b) wound repair on day 28 using composite tissue-engineered skin containing live cells cultured in vitro for 28 days.

[0038] Figure 16 The trend of wound healing rate for each group in this invention;

[0039] Figure 17 HE, representing the skin of nude mice that healed in 28 days according to the present invention;

[0040] Figure 18 This is the normal skin structure of nude mice under HE staining according to the present invention. Detailed Implementation

[0041] 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0042] Example 1:

[0043] A method for constructing a composite artificial dermis containing living cells, comprising:

[0044] Specimen selection: Obtain excess normal skin tissue from healthy adult males aged 20-24 years during surgery.

[0045] Seed cell preparation:

[0046] (1) Isolation, culture and passage of human fibroblasts:

[0047] Approximately 0.5–1 cm of skin tissue from the aforementioned sources was harvested. 2 Place the tissue in a sterile specimen container, add sterile saline containing 50 μg / mL gentamicin, and soak completely for 20 minutes. Immediately transfer to a biosafety cabinet at 4°C, and then transfer to a 60 mm sterile culture dish. Add PBS containing penicillin and streptomycin (100 U / mL) to completely submerge the tissue. Use toothed forceps and ophthalmic scissors to remove as much subcutaneous fat and connective tissue as possible, taking care to prevent the tissue from drying out. After trimming, gently wash the tissue three times repeatedly with disposable pipette tips to remove blood, adhering adipose tissue, and connective tissue. Use fresh PBS and disposable pipette tips for each wash to ensure aseptic technique. Replace with new sterilized instruments and cut the washed skin tissue into pieces approximately 1–1.5 mm in diameter. 2 Small tissue pieces were gently rinsed with PBS containing antibiotics. Using forceps, the tissue pieces were carefully and evenly placed into new 60mm sterile culture dishes, maintaining a 1cm gap between each piece. The dishes were then carefully placed in a 37°C, 5% CO2 saturated humidity incubator for 20 minutes, until the tissue pieces adhered firmly to the bottom of the dish. 4mL of culture medium (DMEM containing antibiotics and 10% fetal bovine serum) was slowly added, and the dishes were then incubated at 37°C, 5% CO2. The culture medium was changed the following day (as above), every 2-3 days. Simultaneously, the growth and morphological changes of cells surrounding the tissue pieces were observed using an inverted microscope. Once the cell density reached 80%, the cells were passaged. All procedures were performed gently to prevent the tissue pieces from detaching from the bottom of the dish or floating.

[0048] When the cell density reaches 80%, passage begins. Discard the culture medium and tissue blocks. Gently rinse the bottom of the culture dish 2-3 times with PBS, then add 1 mL of 0.25% trypsin. Gently agitate the culture dish to ensure the trypsin fully wets the bottom. Incubate at 37°C with 5% CO2 for 3-4 minutes. Under an inverted microscope, observe the cells gradually separating, with increased intercellular spaces and a change in morphology from spindle-shaped to nearly round. Slight agitation causes cells to detach and float. Once completely detached, add 5 mL of 10% FBS medium to stop digestion, and gently pipette any cells still adhering to the bottom of the dish to detach them completely. Collect the cells and transfer them to a 15 mL sterile centrifuge tube. Centrifuge at 1200 rpm for 5 minutes. Cells will settle at the bottom of the centrifuge tube. Discard the supernatant, add culture medium, and gently pipette repeatedly to prepare a cell suspension. Perform trypan blue staining and viable cell counting. Adjust the seeding density of the cell suspension to 1×10⁶ cells / mL. 5 Add / mL to a 60mm culture dish. Incubate at 37℃ with 5% CO2. Change the culture medium (DMEM containing 10% FBS) the next day, repeating every 2-3 days. When the cell density reaches approximately 80%, passage the cells at a 1:3 ratio. The first three generations of cells are collected for subsequent experiments.

[0049] (2) Isolation, culture and identification of keratinocytes:

[0050] Modified skin graft culture method: Under sterile operating room conditions, a graft of approximately 0.5–1 cm is taken. 2 Normal skin tissue was placed in a sterile specimen container. Sterile physiological saline containing 50 μg / mL gentamicin was added, and the tissue was completely immersed for 20 minutes. It was then rapidly transferred to a biosafety cabinet at 4°C. Inside the biosafety cabinet, following the procedure for extracting fibroblast specimens, subcutaneous fat and connective tissue were removed, and the tissue was cut into small skin pieces or strips approximately 3 mm wide. The trimmed tissue pieces were placed in culture dishes pre-coated with type IV collagen and incubated at 37°C with 5% CO2 for 15 minutes. Once the tissue pieces adhered firmly to the bottom of the culture dish, medium containing 10% FBS was slowly added.

[0051] Observe cell growth around the tissue block daily under an inverted microscope. When sheet-like polygonal, irregularly shaped HEK cells appear, replace with EpiLife medium containing 1% HKGS. Thereafter, depending on cell growth, replace the EpiLife medium containing 1% HKGS every 2-3 days. When the cell density reaches approximately 80%, proceed with the next passage.

[0052] like Figures 1-5As shown, the HEK passage procedure is basically the same as the HDF passage, the difference being the reagents used and the pre-coated culture dishes. After the cultured HEK cells have grown into a monolayer, wash twice with calcium- and magnesium-free PBS. Add 4 mL of TrypLE Select per 100 mm culture dish and incubate at 37°C for 5–10 minutes, gently tapping the dish to help cells detach. Then, add 4 mL of 20% PBS to stop the digestion, and gently pipette the cells still adhering to the bottom of the dish. Collect the cells and transfer them to a 15 mL sterile centrifuge tube. Centrifuge at 1200 rpm for 5 minutes, discard the supernatant, and resuspend the cells in EpiLife medium. Gently pipette the cells repeatedly to prepare a cell suspension. Adjust the seeding density of the cell suspension to 2 × 10⁶ cells / mL. 5 / mL, seeded into IV collagen-coated culture dishes. The culture dishes were placed in an incubator at 37°C and 5% CO2, and the medium was replaced with 6mL LEpiLife medium the following day. Thereafter, the medium was changed every 2-3 days depending on cell growth. When the cell density reached approximately 80%, the cells were passaged again at a 1:3 ratio. The first three generations of cells were collected for subsequent experiments.

[0053] Example 2:

[0054] Composite construction:

[0055] Scaffold materials are one of the three core elements in tissue engineering research (seed cells, scaffold materials, and microenvironment). Currently, artificial dermal materials are commonly used in clinical practice. It is a sponge-like scaffold structure mainly composed of porcine collagen. Its pore size facilitates the invasion and growth of blood vessels and cells, and it possesses good mechanical strength, tissue compatibility, biodegradability, and manipulation, often used as a scaffold material for constructing composite tissue-engineered skin. This invention utilizes previously obtained HEK and HFbs to... As a scaffold material, a composite tissue-engineered skin containing living cells was constructed by stepwise culture using an air-liquid interface (ALI) culture system, and its structural characteristics were preliminarily identified.

[0056] (1) Scaffold materials for tissue-engineered skin construction:

[0057] This invention uses reinforced double-layer artificial dermis ( PN-F82060, manufactured by Kori Shiki Co., Ltd., Japan, hereinafter referred to as... It serves as a scaffold material for constructing tissue-engineered skin. It has a double-layer structure. The upper layer is a silicone membrane, which mainly protects the wound and prevents water evaporation. The lower layer is a sponge-like scaffold structure made of porcine collagen, with a pore size of about 70 to 110 μm.

[0058] (2) Construction method:

[0059] 1. Support material Preprocessing:

[0060] After gently washing with sterile PBS, soak the scaffold in a 37°C, 5% CO2 incubator for three hours to ensure complete hydration. Once hydration is complete, discard the PBS, add 5 mL of DMEM medium containing 10% FBS, and incubate the six-well plate in a 37°C, 5% CO2 incubator for another two hours.

[0061] 2. Preparation of cell suspension:

[0062] Third-generation HFbs and HEK cells cultured using the method described in Example 1 were used when the cell growth density reached approximately 80%. The HFbs concentration was adjusted to 2 × 10⁻⁶ cells / cells. 5 / mL of cell suspension, HEK concentration adjusted to 1×10 6 / mL of cell suspension. The cell suspension was tested with trypan blue staining to ensure cell viability was greater than 95%.

[0063] Cell suspension inoculation:

[0064] like Figure 6 As shown, a 1.0cm × 1.0cm pretreated sample... Placed in the chambers of a 6-well cell culture plate. Subsequently, Gently place a sterile medical-grade stainless steel ring (0.9 cm inner diameter, 0.5 cm height) on top, ensuring a tight fit between the ring and the scaffold surface. Accurately add 500 μL of the prepared HFbs or HEK suspension into the stainless steel ring using a pipette, confining the cell suspension within the ring. Take care to prevent the cell suspension from drying out and to prevent culture medium from overflowing into the ring and diluting the cell suspension. Finally, incubate the seeded culture plate at 37°C in a 5% CO2 incubator for 24 hours to allow the cells to fully adhere to the scaffold surface. After 24 hours, gently remove the stainless steel ring using sterile forceps. Avoid changing the medium and minimize movement of the cell culture plate during the initial 48 hours of seeding to ensure successful cell adhesion to the scaffold. Change the medium every 2–3 days, handling gently.

[0065] Co-culture of human fibroblasts and human keratinocytes:

[0066] like Figure 7 As shown. This invention utilizes HFbs, HEK, and Using an air-liquid interface (ALI) culture system, engineered skin containing living cells was constructed through stepwise culture.

[0067] 1. After completing the above After pretreatment and cell suspension preparation, Transfer the cells to the insert chamber of a 6-well cell culture plate, and using the aforementioned cell seeding method, uniformly add 500 μL of HFbs suspension at a concentration of 2 × 10⁵ / mL. On the surface, the inoculated culture plate was placed in a 37°C, 5% CO2 incubator for 24 hours to allow the cells to fully adhere to the scaffold surface. After 24 hours, the stainless steel ring was gently removed using sterile forceps, followed by routine culture.

[0068] 2. After 7 days of culture, when HFbs have proliferated sufficiently and secreted extracellular matrix to form a preliminary dermal structure, HEK cells are inoculated on their surface at a rate of 1×10⁶ cells using the same inoculation method as HFbs.

[0069] 3. Subsequently, adjust the culture conditions and proceed to the ALI culture stage: add a specific co-culture medium to the lower chamber, ensuring the liquid surface is just touching the culture medium. At the bottom, ensure the epidermis is exposed to air and the dermis remains moist. Under ALI culture system conditions, change the culture medium every 48 hours. Samples were taken at 7, 14, 21, and 28 days of culture for HE staining and immunofluorescence analysis to assess the status of the live-cell composite tissue-engineered skin at different in vitro culture times.

[0070] Tissue-engineered skin assessment:

[0071] (2) HE--Histological morphology comparison between normal skin and tissue-engineered skin:

[0072] like Figure 8 As shown, normal human skin ( Figure 8 a): The epidermis shows approximately 4-6 layers of regularly arranged HEK; the dermis is rich in collagen fiber bundles, arranged in an orderly manner, with visible HFbs and a small number of capillaries (200×); containing live cell composite tissue-engineered skin ( Figure 8 b): It exhibits a bilayered structure similar to natural skin, with tightly packed cells in the epidermis (approximately 3-5 layers) and a visible collagen matrix network and embedded HEK (200×) cells in the dermis. Results showed that using... As a support ( Figure 8 c) HEK and HFbs were cultured stepwise to obtain a tissue-engineered skin structure containing live cells that resembled the natural skin structure.

[0073] like Figure 9The images show HE staining of tissue-engineered skin containing live cells and normal skin after different in vitro culture days: (A)(B)(C) Tissue-engineered skin containing live cells after 7 days of in vitro culture; (D)(E)(F) Tissue-engineered skin containing live cells after 14 days of in vitro culture; (G)(H)(I) Tissue-engineered skin containing live cells after 21 days of in vitro culture; (J)(K)(L) Tissue-engineered skin containing live cells after 28 days of in vitro culture; (M)(N)(O) Histological structure of normal human skin. Figures 10-12 The results are from immunofluorescence.

[0074] Furthermore, the experimental procedure is as follows:

[0075] like Figure 13 As shown, the experimental animals were randomly divided into 6 groups, with 10 animals in each group.

[0076] ① Normal saline (NS): The wound is treated with only normal saline;

[0077] ② Scaffold material (Acellular Dermal Matrix, ADM): for wound treatment Support material coverage;

[0078] ③ Tissue Engineered Skin Group Pre-cultured for 7 Days (TES-7d): The wound is covered with tissue engineered skin containing HEK and HFbs cultured in vitro for 7 days.

[0079] ④ Tissue-engineered skin containing live cells cultured for 14 days (TES-14d): The wound was covered with tissue-engineered skin containing HEK and HFbs cultured in vitro for 14 days;

[0080] ⑤ Tissue-engineered skin containing live cells cultured for 21 days (TES-21d): The wound was covered with tissue-engineered skin containing HEK and HFbs cultured in vitro for 21 days;

[0081] ⑥ Tissue-engineered skin containing live cells cultured for 28 days (TES-28d): The wound was covered with tissue-engineered skin containing HEK and HFbs cultured for 28 days in vitro;

[0082] II. Observation Indicators:

[0083] (1) Take photos on days 0, 7, 14, 21 and 28 after surgery, and observe and record the healing process;

[0084] (2) Calculation of wound healing rate: Photos were taken on postoperative days 7, 14, 21, and 28. The wound area was calculated using ImageJ software, and the wound healing rate was calculated. Wound healing rate (%) = (original wound area - unhealed wound area) / original wound area × 100%.

[0085] Histological examination: On the 28th day after surgery, wound tissue from the experimental group was taken, fixed with 4% paraformaldehyde, routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to observe the regeneration and reconstruction of the epidermis and dermis.

[0086] III. Results:

[0087] (a) Wound healing status:

[0088] Figure 14 This diagram shows the healing process of wounds in each group (NS, ADM, TES-7d, TES-14d, TES-21d, and TES-28d) at 0, 7, 14, 21, and 28 days post-traumatic time. The initial wound model for all groups consisted of a circular full-thickness skin defect with an area of ​​1.0 cm².

[0089] Visual observation of wound healing showed that, over time, all groups of wounds improved to varying degrees.

[0090] On day 7, the wound in the NS group contracted and scabs were visible; in the ADM and TES groups, the wound was covered by a relatively transparent thin film and was moist; the TES-7d and TES-14d groups showed tissue integration, the wound was moist and the edges were red and swollen; the wound in the TES-21d and TES-28d groups showed fresh red granulation tissue and significant healing progress.

[0091] On day 14, the wound in the NS group contracted, the scab fell off, and the surrounding area was red and swollen with visible bright red granulation tissue; healing progress was relatively slow. In the ADM group, the wound contracted and was covered with a scab. The TES group healed faster, with better fusion, reduced redness and swelling at the edges, and gradual proliferation of granulation tissue.

[0092] On day 21, the NS group healed through wound edge contraction, with a shallow scar visible in the central area of ​​the wound. The surface was rough, uneven, and lacked normal skin texture. The ADM group had a flatter scar surface than the NS group, with a clear boundary between the scar and normal tissue. In the TES series groups, the wound gradually healed as the culture time increased. The TES-21d and TES-28d groups had relatively smooth scars, and the transition between the healing area and the surrounding normal skin was natural, approaching the restoration of normal skin morphology.

[0093] On day 28, all wounds in all groups showed basic healing. The NS group healed through wound edge contraction, with superficial scarring visible in the central area; the surface was rough and uneven, lacking normal skin texture. The ADM scar surface was flatter than the NS group, with a clear boundary between the scar and normal tissue. In the TES series groups, wounds gradually healed with prolonged culture time; the TES-21d and TES-28d groups showed relatively smooth and even scarring. Figure 15 It exhibits a more delicate surface texture, with a natural transition between the healing area and the surrounding normal skin, and no obvious areas of pigmentation abnormality, almost completely restoring the morphological characteristics of normal skin.

[0094] (II) Wound healing rate:

[0095] (1) Intergroup comparison: There were significant differences in wound healing rates among different experimental groups (p<0.001).

[0096] Wound healing status on day 21: During the observation period on day 21, the TES-28d group had the highest healing rate (87.85±1.11%), while the ADM group had the lowest (74.97±1.36%). The healing rates of each group were ranked as follows: TES-28d > TES-21d (86.34±1.06%) > TES-14d (82.43±0.87%) > TES-7d (78.88±1.35%) > NS (75.16±1.20%) > ADM.

[0097] Wound healing status on day 28: During the observation period on day 28, the wounds in the TES-28d group were almost completely healed (97.53±0.64%), significantly higher than those in other groups. The healing rates of each group were ranked as follows: TES-28d > TES-21d (93.58±1.10%) > TES-14d (91.33±0.97%) > TES-7d (86.66±0.67%) > NS (83.60±0.64%) > ADM (82.44±1.38%).

[0098] In the TES-21d group, the wound healing rate increased from day 7 (46.34±1.37%) to day 28 (93.58±1.10%). Except for the insignificant difference between day 21 and day 28, all pairwise comparisons at other time points showed significant differences.

[0099] In the TES-28d group, the wound healing rate increased from day 7 (59.10±1.83%) to day 28 (97.53±0.64%). Except for the insignificant differences between day 14 and day 21, and between day 21 and day 28, all other time points showed significant differences.

[0100] (3) Trend chart of wound healing rate in each group Figure 16The in vitro culture time of tissue-engineered skin containing live cells was positively correlated with wound healing efficacy. The wound healing rate increased with longer in vitro culture time, especially in the TES-21d and TES-28d groups, which showed a significant wound healing advantage on days 7 and 14, respectively, shortening the wound healing time. In the TES-28d group, the wound healing rate approached 100% (97.53±0.64%) at the end of the 28-day observation period, demonstrating its significant advantage in promoting wound healing.

[0101] (III) Histological observation of HE:

[0102] like Figure 17 The results of HE staining of tissues 28 days post-surgery are shown. In the NS group: the epidermal stratification was indistinct, the basal layer was irregular, the granular layer and stratum corneum were incompletely developed, the cells were loosely arranged, and normal epidermal structure was lacking. Figure 17 A, B). The collagen fibers in the dermis are irregularly arranged, with thin, moderately dense, and loosely distributed collagen bundles. Their orientation is variable, exhibiting a disordered state and lacking the three-dimensional network structure of normal skin. Figure 17 C). ADM group: The epidermis is thinner, the layering is not obvious, and the connection between the epidermis and dermis is not tight enough, and the epidermal ridges have not fully formed. Figure 17 D, E). Dermal collagen fibers showed some improvement compared to the NS group, exhibiting some directionality, but still lacked the tight tissue structure of normal skin collagen. Figure 17 F). TES-7d group: Epidermal cells were observed, but no obvious layered structure was found, and the epidermal thickness was limited. Figure 17 G,H). Its dermis shows a certain degree of tissue organization, with collagen fibers exhibiting a directional alignment trend and fibroblasts more evenly distributed, displaying preliminary tissue organization characteristics. Figure 17 I). TES-14d group: A significant increase in epidermal thickness was observed, with some degree of stratification. The connection between the epidermis and dermis was tighter, and epidermal ridges began to form. Figure 17 J,K). The dermal structure is further improved, with increased collagen fiber density, exhibiting a parallel and interwoven arrangement. Collagen bundles are more uniform in thickness and more regularly arranged, approaching the early tissue characteristics of normal skin. Figure 17 L). TES-21d group: Epidermal stratification is more obvious, and the basal layer, spinous layer and granular layer structure similar to normal skin can be observed, with a thickness close to normal, and the epidermal ridge structure gradually becomes clearer (L). Figure 17 M, N). The dermal fiber structure is abundant and orderly arranged, exhibiting better epidermal layering and dermal matrix organization (M, N). Figure 17 O). TES-28d group: Its epidermis is well-developed, exhibiting a multi-layered, flattened epithelial structure with moderate thickness, including the basal layer, spinous layer, granular layer, and stratum corneum (O). Figure 17P,Q,). Its collagen bundles are uniform in thickness, clearly directional, and densely thickened (P,Q,). Figure 17 R), which is more similar to the epidermal and dermal structure of normal nude mouse skin. Figure 18 ).

[0103] As can be seen from the above, the dermis constructed by this invention has distinct layers and a biomimetic structure. Fibroblasts and keratinocytes are constructed in layers on the same scaffold, and the dermis and epidermis are coupled to each other. The morphological and histological structures are similar to natural skin. The cells have high activity and good adhesion and proliferation. The pretreated and hydrated scaffold promotes cell adhesion and three-dimensional proliferation, allowing fibroblasts to secrete a large amount of extracellular matrix inside the scaffold, while keratinocytes differentiate efficiently under air-liquid interface conditions, maintaining good proliferation and differentiation activity.

[0104] The composite dermis obtained through gas-liquid interface culture has a complete multilayered keratinocyte structure, which can effectively simulate the barrier function of natural epidermis against external physical and chemical stimuli. The preparation process is simple and highly controllable, and the conditions of each step (cell density, culture time, medium change frequency, etc.) can be precisely set. The process does not require complex equipment and is easy to scale up for production and clinical translation. It can be used for evaluation and customization. Histological evaluation such as HE staining and immunofluorescence can monitor the construction quality and functional indicators of composite dermis in real time, which is convenient for further optimization and personalized customization.

[0105] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0106] The accompanying drawings of the embodiments disclosed in this invention only involve structures relevant to the embodiments disclosed in this invention. Other structures can be referred to with common designs. Unless otherwise specified, the same embodiment and different embodiments of this invention can be combined with each other.

[0107] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for constructing a composite artificial dermis containing living cells, characterized in that, include: S1. Scaffold pretreatment: The artificial dermal scaffold was washed in sterile phosphate-buffered saline (PBS) and incubated in high-glucose DMEM containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 for 3 hours to obtain a pretreated hydrated scaffold. S2. Fibroblast seeding: Human fibroblast suspension is dropped onto the surface of the pretreated hydrated scaffold and incubated at 37°C and 5% CO2 for 24 hours to allow the cells to adhere to the scaffold and obtain a preliminary adherent scaffold. S3. Dermal layer construction: The preliminary adherent scaffold was transferred into DMEM containing 10% FBS for routine culture for 7 days, with the medium changed every 2-3 days, to allow fibroblasts to proliferate and secrete extracellular matrix in the scaffold, thus obtaining a composite scaffold containing a dermal layer. S4. Keratinocyte inoculation: Human keratinocyte suspension is dropped onto the surface of the composite scaffold containing the dermis and incubated at 37°C and 5% CO2 for 24 hours to allow the keratinocytes to adhere to the wall, thus obtaining a composite scaffold containing the epidermal precursor layer. S5. Gas-liquid interface culture: The composite scaffold containing the epidermal precursor layer is transferred to the gas-liquid interface culture device. Culture medium is added to the bottom of the scaffold in the lower chamber to keep the epidermal layer exposed to air. The culture medium is replaced every 48 hours to promote keratinocyte differentiation and layered construction to obtain mature composite artificial dermis containing live cells. S6. Histological evaluation: On days 7, 14, 21 and 28 of culture, the composite artificial dermis was taken for hematoxylin-eosin HE staining and immunofluorescence staining to evaluate the histological structure and cell distribution of the epidermis and dermis.

2. The method for constructing a composite artificial dermis containing living cells according to claim 1, characterized in that, In step S1, the PBS is a calcium- and magnesium-free formulation with a pH of 7.2–7.

4.

3. The method for constructing a composite artificial dermis containing living cells according to claim 1, characterized in that, The DMEM mentioned in step S1 is DMEM containing 4.5 g / L glucose, 2 mM glutamate transporter, and 1 mM sodium pyruvate.

4. The method for constructing a composite artificial dermis containing living cells according to claim 1, characterized in that, In step S2, the concentration of the fibroblast suspension is 1.5 × 10⁻⁶. 5 The inoculum concentration was 2.5 × 10⁵ cells / mL, and the inoculum size was 400–600 μL.

5. The method for constructing a composite artificial dermis containing living cells according to claim 1, characterized in that, The culture time in step S3 is 7 days ± 1 day, and the culture medium used to replace the culture medium is the same as that in step (1).

6. The method for constructing a composite artificial dermis containing living cells according to claim 1, characterized in that, In step S4, the concentration of the keratinocyte suspension is 0.8 × 10⁻⁶. 6 Up to 1.2×10 6 The inoculum size is 450–550 μL, with cells / mL.

7. The method for constructing a composite artificial dermis containing living cells according to claim 1, characterized in that, The gas-liquid interface culture device used in step S5 is Multi-hole support system.

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