A method for constructing rapid tissue-engineered skin, rapid tissue-engineered skin and application

Abstract

The application discloses a kind of construction methods of quick tissue engineering skin, quick tissue engineering skin and application, the construction method includes the following steps: obtaining biological tissue sample (EpiSCs) containing epidermal stem cell;Epidermal stem cells in part the donor skin flap are separated, and epidermal stem cell suspension (EpiSCs) is obtained;Obtain three-dimensional support;The epidermal stem cell suspension is mixed with the three-dimensional support, and the remaining donor skin flap is transplanted, and the functional tissue engineering skin is obtained.The application completely gives up the in-vitro culture link of traditional tissue engineering for 2-3 weeks, and the entire preparation process is compressed to 30-50 minutes, so that the quick tissue engineering skin closer to normal skin in texture, thickness, elasticity and flexibility, etc.

Description

Technical Field

[0001] This invention relates to the field of skin construction technology, and in particular to a method for constructing rapid tissue-engineered skin, rapid tissue-engineered skin, and its applications. Background Technology

[0002] Skin defect repair, especially full-thickness skin damage caused by trauma, burns, or surgical excision, remains a major challenge in regenerative medicine. Such damage not only disrupts the skin barrier but also frequently leads to functional impairment and cosmetic defects. Currently, autologous skin grafting is the most commonly used clinical treatment strategy, but various traditional grafting methods have significant limitations: split-thickness grafts, while having high survival rates and fewer donor site complications, are prone to contracture; intermediate-thickness and full-thickness grafts, while superior in durability and appearance, come at the cost of increased donor site trauma; composite grafts (such as those combining artificial dermal matrix) can reduce the amount of donor skin, but still face problems such as delayed vascularization and low initial survival rates. Overall, existing technologies struggle to achieve an ideal balance between donor site protection and repair quality (including function and aesthetics), necessitating a novel treatment strategy that minimizes donor site damage while more realistically reconstructing the structure and function of natural skin.

[0003] Currently, skin substitutes and regeneration technologies used clinically to repair full-thickness skin defects caused by large-area burns, trauma, or chronic ulcers are mainly divided into the following three categories. Despite their respective advantages, each faces significant technical bottlenecks in clinical application: 1. In vitro constructed composite tissue-engineered skin (represented by Epicel®, Apligraf®): Features: Contains seed cells and a biological scaffold, formed through 2-3 weeks of in vitro expansion culture in a strictly GMP laboratory environment. Limitations: Long preparation cycle: The long culture cycle of several weeks makes it unsuitable for routine surgical applications. High cost and barriers to entry: Relies on expensive GMP facilities and cold chain transportation; due to the long time cells are out of the human environment, their viability and dryness are easily damaged, and it is difficult to promote in primary hospitals. 2. Acellular dermal matrix / artificial dermis (represented by Integra®, Pelnac®): Features: Only provides a three-dimensional physical scaffold ("soil") such as collagen, without containing active cells ("seeds"). Existing limitations: Slow vascularization: Due to the lack of cell release of vascular endothelial growth factor (VEGF) and other signals, it relies entirely on ingrowth into host blood vessels, and establishing blood supply usually takes 2-3 weeks. Staged surgery required: Clinically, a "two-step" approach is often used: first, a scaffold is implanted, and after granulation tissue fills the scaffold pores, a second-stage skin graft is performed, significantly prolonging the treatment cycle and increasing the risk of infection. 3. Autologous cell suspension spraying technology (represented by ReCell®) Technical characteristics: Autologous cells are separated during the operation and sprayed directly onto the wound, providing only scattered seed cells. Existing limitations: Lack of three-dimensional structure: Cells lack the support and guidance of a dermal scaffold, making it impossible to reconstruct a thick dermal layer. Limited healing quality: For full-thickness skin defects, simple spraying easily leads to wound contracture, and scar contracture and pigmentation are very likely to occur after healing. In summary, existing technologies either have structure but no cells (slow growth), or have cells but no structure (prone to contracture), or both but are too slow and too expensive. Summary of the Invention

[0004] The main objective of this invention is to propose a method for constructing rapid tissue-engineered skin, rapid tissue-engineered skin, and its application, aiming to solve the problems of long in vitro culture cycles, slow vascularization of simple scaffolds, and lack of dermal support in simple cell spraying of existing tissue-engineered skin.

[0005] To achieve the above objectives, this invention proposes a method for rapidly constructing tissue-engineered skin, the method comprising the following steps: S10. Obtain a biological tissue sample containing epidermal stem cells; S20. Take a portion of the biological tissue sample and separate the epidermal stem cells to obtain an epidermal stem cell suspension. S30, Obtain the three-dimensional scaffold; S40. The epidermal stem cell suspension is mixed with the three-dimensional scaffold and cultured in vitro with the remaining biological tissue sample to obtain the tissue-engineered skin.

[0006] This technical approach involves, firstly, obtaining a biological tissue sample containing epidermal stem cells during the procedure. This sample is then divided into two parts proportionally by area: one part, with an area roughly equivalent to the wound to be repaired and kept intact, serves as the upper covering skin graft; the remaining part acts as a "seed source," preparing an epidermal stem cell suspension. This strategy eliminates the need to expand the biological tissue sample area, diverting only a small amount of tissue on top of routine sampling, significantly reducing donor site damage. Next, when the obtained epidermal stem cell suspension is combined with a three-dimensional scaffold, the highly active epidermal stem cells overcome surface tension and uniformly engraft into the deep pores of the scaffold, rather than merely adhering to the surface. This structure continuously releases exosomes and various cytokines, effectively inducing rapid vertical ingrowth of blood vessels into the scaffold within 7–14 days post-procedure. This avoids the need for a second-stage skin graft required in traditional artificial dermal transplantation, significantly shortening the healing period. Finally, a skin graft, with an area equivalent to the wound, was directly placed over the surface of the cell-loaded scaffold. Intraoperatively, a biomimetic "sandwich" three-layer composite structure was constructed. The upper layer consisted of autologous split-thickness skin, providing an immediate physical barrier to prevent scaffold drying and cell loss, and inhibiting scar contracture and promoting tissue remodeling through moderate compression and moisturizing. The middle layer was a three-dimensional scaffold functionalized with stem cells, serving as the regenerative core to drive vascularization and orderly matrix reconstruction. The lower layer was the wound base, receiving new tissue for integration. The resulting regenerated skin was closer to normal skin in texture, thickness, elasticity, and suppleness, with a VSS (Vancouver Scar Scale) score significantly better than that of simple cell therapy or traditional autologous skin transplantation.

[0007] Crucially, if the construction method of this invention is applied clinically, in vitro culture can be eliminated, thus completely eliminating the 2-3 week in vitro culture step in traditional tissue engineering. The entire preparation process is compressed to within 30-50 minutes, enabling real-time intraoperative construction without in vitro modification and culture. This truly achieves rapid, efficient, and low-trauma skin regeneration and repair during surgery, allowing for "immediate collection, immediate preparation, and immediate use."

[0008] In one embodiment, in step S10, the source of the biological tissue sample containing epidermal stem cells includes autologous split-thickness skin grafts.

[0009] It should be noted that a split-thickness skin graft refers to a thin layer of skin, consisting only of the epidermis and part of the papillary dermis, harvested from the donor site using a scalpel (such as an electric scalpel). Its thickness is typically 0.2–0.4 mm. Split-thickness skin grafts contain the complete epidermal basal layer, where epidermal stem cells are primarily located. Compared to full-thickness skin grafts, split-thickness grafts are less invasive and allow for faster donor site recovery. They are suitable for harvesting additional skin for a small number of cells, providing immediate biological dressings. Furthermore, they can seal the wound, reduce moisture evaporation, and prevent infection, offering excellent protection. Split-thickness epidermal grafts have strong adaptability, and their degradation rate is synchronized with the tissue regeneration process. They can guide stem cell proliferation and differentiation, promote angiogenesis, and prevent wound contracture, providing structural support and a growth microenvironment for rapidly tissue-engineered skin.

[0010] Specifically, the steps for obtaining biological tissue samples containing epidermal stem cells include: setting the total skin harvesting area: To simultaneously meet the dual requirements of "scaffold surface coverage" and "epidermal stem cell preparation," the total skin harvesting area is specifically set to approximately 1.025 times the area of ​​the wound to be treated (i.e., 1 + 1 / 40). Skin graft segmentation and usage allocation: The obtained autologous blade-thickness skin grafts are divided into two parts according to their area ratio: The first part (covering skin graft): accounting for the vast majority of the total area (approximately 40 / 41), its area is consistent with the area of ​​the wound to be treated (and the surface area of ​​the three-dimensional scaffold). This part of the skin graft remains intact and is used to cover the surface of the cell-loaded three-dimensional scaffold as the epidermal layer of tissue-engineered skin; The second part (cell-derived skin graft): accounting for a small portion of the total area (approximately 1 / 41), its area is in a 1:40 ratio to the area of ​​the wound to be treated. This part of the skin graft is cut off and specifically used for enzymatic digestion to prepare a high-concentration epidermal stem cell suspension.

[0011] In one embodiment, step S20 includes: separating epidermal stem cells from a portion of the biological tissue sample using Myseed® patented technology to obtain an epidermal stem cell suspension.

[0012] Specifically, the Myseed® patented technology processing steps include: Step 1: Start the dissociation apparatus and set the temperature to 37°C. Add 10 ml of dissociation reagent to the dissociation dish and 5 ml of fibronectin (FN) at a concentration of 20 μg / ml to the culture flask, spreading them evenly. Place the dissociation dish and culture flask on the apparatus's incubation platform.

[0013] Step 2: Tissue dissociation and preliminary cell acquisition. Take 1-10 cm² skin flaps (0.15-0.2 mm thick) and place them in a dissociation dish, incubate at a constant temperature for 10-20 minutes. After digestion, add lactated Ringer's solution or PBS solution to terminate dissociation and wash the skin flaps. Separate the epidermis and dermis, and scrape epidermal cells from the basal layer of the epidermis using a scalpel. After collecting the cells, dilute them with lactated Ringer's solution or PBS solution at a ratio of 1 ml / cm², filter through a filter screen, and collect the initial epidermal cell suspension in the filter groove.

[0014] Step 3: After the culture flask with FN evenly spread has been left to stand at 37°C for 30 minutes, excess liquid in the culture flask is aspirated. The initial epidermal cell suspension is slowly injected into the culture flask and evenly spread to allow for rapid FN adsorption. The flask is then left to stand at 37°C for 20-30 minutes.

[0015] Step 4: Use physical shaking to allow the cell membranes or clumps adsorbed by FN at the bottom of the culture flask to detach naturally. Use a 10ml syringe and a flat-tipped needle to aspirate the detached intact cell membranes or clumps, add physiological saline to the aspirated cell clumps, and mix mechanically to form a uniform epidermal stem cell suspension for later use.

[0016] In the technical solution of the present invention, the cell concentration of the epidermal stem cell suspension is ≥1×106 cells / mL. Using physiological saline as the solvent medium, after appropriate pipetting, the dispersion is good and there is no obvious agglomeration. The epidermal stem cell suspension is prepared by a standardized enzymatic digestion process, and its cell activity is maintained above 90%.

[0017] In one embodiment, the concentration range of the epidermal stem cell suspension is ≥1×10⁻⁶. 6 cells / mL.

[0018] In one embodiment, in step S30: The three-dimensional scaffold includes acellular dermal matrix or tissue-engineered porous scaffold with a three-dimensional mesh structure.

[0019] Specifically, dermal matrix or tissue-engineered scaffolds are selected as three-dimensional scaffold materials to provide support for skin-like tissue structures. Dermal matrix (such as acellular allogeneic dermis) or synthetic / natural tissue-engineered scaffolds are selected as three-dimensional scaffold materials and pre-cut to the size that matches the wound surface for later use.

[0020] Specifically, Lando's dual-layer tissue engineering scaffold (BAS-C) can be used. It is specially designed to carry autologous epidermal stem cells and adapt to intraoperative construction, possessing excellent biocompatibility and cell adaptability. The dual-layer tissue-engineered scaffold employs a biomimetic structural design. The upper layer is an epidermal replacement layer made of implantable medical-grade silicone, while the lower layer is a dermal simulation layer composed of collagen and glycosaminoglycans. This three-dimensional porous structure efficiently adsorbs and loads autologous epidermal stem cells obtained during the procedure, providing stable anchoring points. It exhibits excellent biocompatibility, showing strong compatibility with stem cells and thick epidermis. Its degradation rate is synchronized with the tissue regeneration process, guiding stem cell proliferation and differentiation, promoting angiogenesis, and preventing wound contracture, thus providing structural support and a growth microenvironment for rapidly tissue-engineered skin.

[0021] In one embodiment, step S40 includes: mixing the epidermal stem cell suspension with the three-dimensional scaffold to form a complex; The remaining biological tissue sample is applied onto the complex to obtain functional tissue-engineered skin.

[0022] The epidermal stem cell suspension is sprayed onto a three-dimensional scaffold to form a preliminary "cell-scaffold" complex. The remaining donor skin graft is then placed on top of the cell-scaffold complex to form a "double-layer repair structure": the upper layer is a temporary covering skin graft, and the lower layer is a stem cell-driven regenerating dermal-epidermal complex, which takes into account both short-term protection and long-term regeneration.

[0023] The specific application steps include the following: 1. Preparation: Place the purchased 3D scaffold (pre-wetted or dry) flat in a sterile petri dish or stainless steel tray.

[0024] 2. Initial Spray: Load the epidermal stem cell suspension into a sterile syringe or spray device equipped with an atomizing nozzle. At a distance of 5-10 cm from the scaffold surface, spray approximately half the volume of the cell suspension evenly onto the front of the scaffold in a zigzag pattern. At this time, some of the cell suspension will seep into the scaffold pores, while some may flow through the scaffold to the bottom of the culture dish.

[0025] 3. Flipping and Respraying (to solve uniformity issues): Use sterile forceps to hold the edge of the scaffold and flip it 180 degrees. Spray the remaining 1 / 2 of the cell suspension evenly onto the reverse side of the scaffold in the same manner.

[0026] 4. Infiltration and reabsorption (solving the problem of effluent): After spraying, if there is effluent cell suspension at the bottom of the culture dish, let it stand for 1-2 minutes. Utilize the hydrophilicity and capillary effect of the scaffold material to allow the scaffold to automatically absorb the suspension at the bottom. Alternatively, use a pipette to collect the effluent at the bottom and add it again to the surface of the scaffold until the entire scaffold is uniformly moistened, semi-transparent, and saturated, and there is no obvious effluent residue at the bottom of the container.

[0027] 5. Skin covering: After confirming that the complex has been prepared, place it on the wound and cover it with the remaining spare donor skin grafts.

[0028] Furthermore, the donor skin graft is an autologous cut-thickness skin graft with a thickness of approximately 0.25 mm, and the area of ​​the donor skin graft is approximately 1:1 to the area of ​​the wound.

[0029] In one embodiment, the mixing method includes spraying.

[0030] The specific steps include: placing the three-dimensional scaffold in a sterile container, spraying the epidermal stem cell suspension onto the three-dimensional scaffold in multiple times and from multiple angles (or alternating between the front and back sides), and using the scaffold's adsorption properties to absorb the suspension flowing out of the container until the three-dimensional scaffold is in a saturated adsorption state.

[0031] The mixing ratio is such that 1 mL of epidermal stem cell suspension is loaded onto the three-dimensional scaffold with a surface area of ​​35-45 cm².

[0032] Specifically, 1 mL of epidermal stem cell suspension (cell count ≥ 1 × 10⁻⁶) is obtained per 1 cm² of donor skin. 6 A standard preparation suspension (cells / mL) was prepared, and 1 mL of the epidermal stem cell suspension was loaded onto the three-dimensional scaffold with a surface area of ​​35-45 cm²; wherein, preferably, the surface area of ​​the three-dimensional scaffold corresponds to the area of ​​the skin wound to be treated, so as to achieve a treatment expansion ratio of 1:40 between the donor skin and the recipient wound.

[0033] This invention provides a rapid tissue-engineered skin, which is constructed using the rapid tissue-engineered skin construction method described in any one of the above claims.

[0034] This invention provides a method for constructing an in vitro skin tissue model, comprising the following steps: Obtain isolated biological tissue samples and isolate epidermal stem cell suspensions from them; A three-dimensional scaffold is provided, and the epidermal stem cell suspension is loaded onto the three-dimensional scaffold to obtain a cell-scaffold complex. The cell-scaffold complex was placed in an in vitro culture system for induction culture to induce epidermal stem cells to differentiate and form a multilayered skin tissue structure, thereby obtaining the in vitro skin tissue model.

[0035] Tissue-engineered skin prepared using the above methods can be applied in the medical field. These methods can also serve as a platform for constructing in vitro skin tissue models to simulate the physiological structure and pathological changes of human skin. Furthermore, they can be used as a drug permeability testing carrier for evaluating the safety and efficacy of cosmetics or cosmeceuticals. For example, simulating physiological structures and pathological changes can be used in new product experiments for skincare products.

[0036] The present invention also provides an application of the rapidly tissue-engineered skin described above in skin injury repair, the application comprising: transplanting functional tissue-engineered skin together with the remainder of a donor skin graft to the skin defect site.

[0037] This technique involves the following steps during surgery: First, a donor skin graft is obtained and divided into two parts proportional to its area. One part, with an area roughly equivalent to the wound surface and kept intact, serves as the upper covering skin graft. The remaining part acts as a "seed source," preparing an epidermal stem cell suspension. This strategy eliminates the need to expand the donor area, diverting only a small amount of tissue on top of the routine skin harvest, significantly reducing donor site damage. Next, when the obtained epidermal stem cell suspension is combined with a three-dimensional scaffold, the highly active epidermal stem cells overcome surface tension and uniformly engraft into the deep pores of the scaffold, rather than merely adhering to the surface. This structure continuously releases exosomes and various cytokines, effectively inducing rapid vertical ingrowth of blood vessels into the scaffold within 7–14 days post-surgery. This avoids the need for a second-stage skin grafting procedure required in traditional artificial dermal transplantation, significantly shortening the healing period. Finally, a skin graft, with an area equivalent to the wound, was directly placed over the surface of the cell-loaded scaffold. Intraoperatively, a biomimetic "sandwich" three-layer composite structure was constructed. The upper layer consisted of autologous split-thickness skin, providing an immediate physical barrier to prevent scaffold drying and cell loss, and inhibiting scar contracture and promoting tissue remodeling through moderate compression and moisturizing. The middle layer was a three-dimensional scaffold functionalized with stem cells, serving as the regenerative core to drive vascularization and orderly matrix reconstruction. The lower layer was the wound base, receiving new tissue for integration. The resulting regenerated skin was closer to normal skin in texture, thickness, elasticity, and suppleness, with a VSS (Vancouver Scar Scale) score significantly better than that of simple cell therapy or traditional autologous skin transplantation. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0039] Figure 1 This is a schematic diagram illustrating the detection of cell microstructure and molecular biology in Example 1 of the present invention. Figure 2 This is a schematic diagram illustrating the physicochemical properties and cell compatibility testing of the scaffold in Embodiment 1 of the present invention; Figure 3 This is a schematic diagram of the gross morphology and healing dynamics assessment of the wound in Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of histological and immunofluorescence detection in Example 1 of the present invention; Figure 5 This is a schematic diagram illustrating the design concept of Embodiment 2 of the present invention.

[0040] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. 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.

[0042] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0043] Experimental materials: The three-dimensional scaffolds used in this study included: JAYYA® acellular dermal matrix (ADM), purchased from Beijing JAYYA® Biotechnology Co., Ltd. (Beijing, China), which is an acellular allogeneic dermal three-dimensional scaffold (model: J-1A, medical device registration certificate number: 20153130864); and a double-layer tissue engineering scaffold (through-type, model: BAS-C-0707, 7 × 7 mm, medical device registration certificate number: 20173131356), purchased from Lando Biotechnology.

[0044] Example 1 verifies the repair effect (feasibility and effectiveness) of "rapidly tissue-engineered skin" in an animal model of full-thickness skin defects. This embodiment uses SD rats to construct a full-thickness skin defect model. Twenty-four healthy SD rats were included in the study and randomly divided into three groups: the STSG group (n=8), the CS group (n=8), and the TES group (n=8). The specific steps are as follows: I. Common Preoperative Preparation and Model Establishment Rats in each group underwent back preparation and routine disinfection under anesthesia. Using a scalpel and scissors, full-thickness skin (reaching the fascia layer) was removed from the back of each rat, creating a circular full-thickness skin defect with a diameter of 2.5 cm. The wound area (denoted as Awound) was measured and recorded.

[0045] II. The experimental group is divided into three groups, and the specific operating steps are as follows. 1. Operating procedures for the thick-bladed leather skid plate assembly (STSG assembly): This group adopted the "simple autologous split-thickness skin graft" protocol, and the specific steps are as follows: Step S101 (Donor Acquisition): Set the skin graft area A_graft = Awound. Use a micro scalpel to obtain an autologous blade-thickness skin graft from the rat donor area.

[0046] Step S102 (Transplantation): The obtained blade-thickness skin graft is laid flat directly over the full-thickness wound base. The edges are sutured and fixed, and pressure bandaged.

[0047] 2. Procedure for the Composite Skin Group (CS Group): Without the addition of stem cells, this group served as a control group. The procedure involved "acellular dermal matrix (ADM) + autologous skin grafting," and the specific steps are as follows: Step S201 (Donor Acquisition): Same as STSG group, set the skin grafting area A_graft = Awound, and obtain the autologous blade-thickness skin for later use.

[0048] Step S202 (Stent Preparation): Prepare an acellular dermal matrix (ADM) of the same size as the wound area (Ascaffold = Awound, diameter 2.5cm). Soak it in sterile saline to rehydrate it for later use. The acellular dermal matrix (ADM) is also known as JAYYA® acellular dermal matrix (ADM) or a three-dimensional (3D) ADM scaffold.

[0049] Step S203 (Transplantation): Apply the rehydrated ADM directly to the base of the wound, cover the ADM surface with the blade-thickness skin graft obtained in step S10, fix it, and apply pressure bandage.

[0050] 3. Procedure for the Rapid Tissue Engineered Skin Treatment Group (TES Group): This group adopted the construction method described in this invention, namely the "in situ cell loading + ADM + skin graft" sandwich structure, to achieve one-step intraoperative construction. The specific operation steps are as follows: Step S301 (Donor Acquisition): Select the wound area Awound, set the total skin graft area Attotal = Awound + Awound / 40, and use a micro-skin grafting scalpel to obtain an autologous skin graft of blade thickness. Usage allocation: Part A (for covering): Area equal to Awound, used for subsequent surface covering. Part B (for cells): Area approximately Awound / 40 (i.e., a 1:40 ratio), used for intraoperative preparation of cell suspension.

[0051] Step S302 (Intraoperative rapid preparation of epidermal stem cell suspension) includes the following steps: (1) Start the dissociation apparatus and set the temperature to 37°C. Add dissociation reagent to the dissociation dish and add fibronectin (FN) at a concentration of 20 μg / ml to the culture flask and spread it evenly. Place the apparatus on the incubator to preheat.

[0052] (2) Tissue dissociation and preliminary cell acquisition: A portion of the B skin flaps were placed in a dissociation dish and incubated at a constant temperature. After dissociation, buffer solution was added to terminate digestion and the skin flaps were washed. The epidermis and dermis were separated, and epidermal cells were scraped off. After collecting the cells, they were diluted with buffer solution and filtered through a filter to obtain the initial epidermal cell suspension (EBCs).

[0053] (3) Rapid intraoperative targeted sorting: The initial epidermal cell suspension was slowly injected into the culture flask of the FN plate and spread evenly. It was placed in a 37°C environment for a short period of static incubation, and the epidermal stem cells (ESCs) were rapidly adsorbed by the specificity of FN.

[0054] (4) Use physical shaking to allow the cell membrane sheets or clumps adsorbed by FN at the bottom of the culture flask to detach naturally. Aspirate the cell clumps and add physiological saline for mechanical mixing to form a highly active epidermal stem cell (ESC) suspension for later use. (Flow cytometry verification showed that the viability of ESCs rapidly extracted during this procedure reached 83.7%, with a yield of 1.03 × 10⁻⁶ cells.) 5 ESCs / cm², and high expression of CD44, CD90, and CD106 markers.

[0055] Step S303 (scaffold preparation): Prepare an Awound (2.5 cm in diameter) decellularized dermal matrix (ADM) and pre-wet it in a sterile container to obtain the hydrated decellularized dermal matrix (ADM).

[0056] Step S304 (Mixing and Rapid Construction): According to the area ratio of 1:40, the epidermal stem cell suspension obtained in step S20 is evenly added dropwise or sprayed onto the ADM surface in step S30. Allow it to stand for 1-2 minutes for saturation adsorption, allowing the cell suspension to penetrate into the internal pores of the ADM to form an "ESCs-ADM complex".

[0057] Final in situ transplantation: The ESCs-ADM complex is applied to the full-thickness wound base, and then the portion of skin graft A (for covering) retained in step S301 is precisely covered on the surface of the complex to complete the one-step tissue-engineered skin (TES) construction. Suture fixation and pressure bandage are applied.

[0058] III. Effectiveness Verification Methods To verify the repair effect of the "rapid tissue-engineered skin" described in this invention in a full-thickness skin defect model, the following tests were performed on the cell suspension, scaffold material, and postoperative recovery of rats in each group described in Example 1: 1. Microscopic morphology of cells at different stages, used for direct comparison of cell state before and after separation. For the initial epidermal cell suspension (EBCs) obtained in step S302 (2) of the embodiment, about 50 μL of cell suspension was dropped onto a glass slide, allowed to stand for about 4 minutes, covered with a coverslip, and observed and photographed under a 4x objective lens using a regular optical microscope (bright field mode) to obtain... Figure 1 Figure A in the figure shows that the cells in the initial epidermal cell suspension are distributed in clumps or mixed patterns, with many incompletely dispersed cell clusters and uneven cell distribution, indicating that the initial cell population still has a certain degree of heterogeneity.

[0059] Correspondingly, approximately 50 μL of the epidermal stem cell suspension (ESCs) obtained in step S302 (4) of the embodiment was added to a glass slide and allowed to settle naturally for 8 min. The slide was then stained with 1 μg / mL DAPI staining solution and incubated at room temperature in the dark for 10 min. After washing with PBS buffer, a coverslip was applied, and the slide was observed and photographed under a fluorescence microscope with a 4x objective lens to obtain... Figure 1 Figure B in the diagram shows the blue fluorescent dots representing DAPI-stained cell nuclei. The results indicate that the cell nuclei in the ESCs cell suspension were relatively evenly distributed, with significantly reduced aggregation, suggesting that the cell suspension obtained after FN adsorption enrichment had a better dispersion state and was more suitable for subsequent tissue-engineered skin construction.

[0060] 2. Molecular biological detection For the initial epidermal cell suspension (EBCs) obtained in step S302 (2), approximately 1×10^6 cells were taken and set up CD106, CD90, and CD44 detection groups, respectively. Each group of cells was resuspended in PBS buffer to approximately 100 μL, and then FITC-labeled anti-CD106 antibody, FITC-labeled anti-CD90 antibody, and FITC-labeled anti-CD44 antibody working solutions prepared at a 1:100 dilution were added. The cells were incubated at 4 ℃ in the dark for 30 min. After incubation, the cells were washed twice with PBS buffer to remove unbound antibodies, centrifuged, the supernatant was discarded, and the cells were resuspended in PBS buffer. Gating was performed using corresponding isotype controls, and the proportion of cells positive for each marker was detected by flow cytometry to obtain the desired cell count. Figure 1 Figure C in the diagram shows that the CD106 positivity rate in EBCs was approximately 18.1%, while the CD90 and CD44 positivity rates were approximately 3.4% and 4.6%, respectively. This indicates that in the unenriched initial cell population, the proportion of CD90 and CD44 positive cells was low, and the proportion of the target stem cell-related cell subset was limited.

[0061] For the epidermal stem cell suspension (ESCs) obtained in step S302 (4), antibody staining and flow cytometry were performed using the same method as above to obtain... Figure 1 Figure D in the diagram shows that the positivity rates of CD106, CD90, and CD44 in ESCs were approximately 98.0%, 98.4%, and 98.2%, respectively. Figure 1 Compared with EBCs in C, the positive expression rates of the above three surface markers were significantly increased, indicating that the FN adsorption method can effectively enrich cell populations with epidermal stem cell-related phenotypic characteristics.

[0062] Further, for the initial epidermal cell suspension (EBCs) obtained in step S302 (2), approximately 1 × 10^6 cells were taken and resuspended in PBS buffer to approximately 100 μL. Then, FITC-labeled anti-CD90 antibody working solution and APC-labeled anti-CD106 antibody working solution were added for mixed staining. The antibody working solutions were prepared by diluting the corresponding fluorescently labeled antibodies at a ratio of 1:100 and incubated at 4 ℃ in the dark for 30 min. After incubation, the cells were washed twice with PBS buffer to remove unbound antibodies, centrifuged, the supernatant was discarded, and the cells were resuspended in PBS buffer. Compensation and gating were performed using corresponding monochrome controls and blank controls. Dual-parameter flow cytometry was used to detect the cells. Figure 1 The figure shows E, where the horizontal and vertical axes represent the intensities of two different fluorescence channels, and the four quadrants Q1-Q4 represent different cell subpopulations. As can be seen from the figure, cells are distributed in all quadrants of the initial cell population, with the proportions in quadrants Q1, Q2, Q3, and Q4 being approximately 16.4%, 4.48%, 1.44%, and 77.7%, respectively, indicating a relatively complex and heterogeneous initial cell population composition.

[0063] For the epidermal stem cell suspension (ESCs) obtained in step S302 (4), the same method was used. Figure 1 The same processing method as E in the image is used for two-color flow cytometry detection to obtain... Figure 1 The results, as shown in the F-plot, indicate that the proportions of cells in quadrants Q1, Q2, Q3, and Q4 in ESCs were approximately 13.9%, 1.01%, 1.38%, and 83.7%, respectively. Compared to EBCs, the cell population distribution of ESCs changed, with the main cell population becoming more concentrated in the two-parameter space, suggesting that the cell population composition tended to be more consistent after FN adsorption and enrichment.

[0064] In summary, the positive expression rates of CD44, CD90, and CD106 in the ESCs suspension obtained in step S302 of Example 1 were approximately 98.2%, 98.4%, and 98.0%, respectively, which were significantly higher than those in the pre-purified EBCs (4.6%, 3.4%, and 18.1%). Furthermore, the ESCs acquisition efficiency was approximately 8.62%, and the acquisition amount was approximately 1.03 × 10^5 cells / cm², which is sufficient to meet the needs of subsequent tissue-engineered skin construction.

[0065] Beneficial effects: This invention utilizes the principle of specific adsorption of extracellular matrix to rapidly enrich and obtain high-purity, highly active autologous epidermal stem cells from the initial epidermal cell population in a short period of time. This avoids the shortcomings of traditional tissue-engineered skin, which requires long-term in vitro expansion and culture. It helps to simplify clinical operation procedures and provides a reliable source of seed cells for the rapid construction of tissue-engineered skin during surgery.

[0066] 3. Scaffold physicochemical properties and cell compatibility testing Completeness of decellularization and morphological observation: The decellularized dermal matrix (ADM) obtained and hydrated in step S303 of Example 1 (Rapid Tissue-Engineered Skin Treatment Group, TES Group) was observed for its macroscopic morphology, thickness, and microstructure, and histological examination was performed. The results are as follows: Figure 2 As shown.

[0067] in, Figure 2 As shown in Figure A, the hydrated ADM appears as a soft, milky-white film with a thickness of approximately 0.4 ± 0.17 mm. Further observation using a scanning electron microscope revealed... Figure 2 B. Before observation, the ADM sample was fixed and dehydrated, and then observed using a scanning electron microscope. It can be seen that ADM retains the natural collagen fiber structure and has a relatively obvious porous structure.

[0068] Continue with HE staining and Masson staining of ADM to obtain... Figure 2Figures C and D in the diagram illustrate the process. Specifically, the prepared ADM tissue was fixed in 4% paraformaldehyde fixative for 24 h, rinsed with running water, and then sequentially dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and cut into continuous tissue sections with a thickness of approximately 4 μm. For HE staining, the tissue sections were dewaxed, rehydrated, and then routinely stained with hematoxylin and eosin, followed by dehydration, clearing, and mounting. The tissue morphology was observed under an optical microscope. For Masson staining, the tissue sections were dewaxed, rehydrated, and then routinely stained using the Masson trichrome staining kit, followed by dehydration, clearing, and mounting. The distribution of collagen fibers was observed under an optical microscope. The results showed that the HE staining results ( Figure 2 (Figure C) No obvious intact cells or nuclear structures were observed, indicating that the ADM decellularization treatment was relatively thorough; Masson staining results ( Figure 2 Figure D in the diagram shows that the collagen fibers are arranged intact and the collagen matrix structure is well preserved, indicating that the cellular components were effectively removed during the decellularization process, while the natural extracellular matrix structure was well preserved.

[0069] The ESCs obtained in step S302 (4) of the example were uniformly dropped or sprayed onto the surface of the ADM, with a cell seeding density of approximately 1×10^5 to 5×10^5 cells / cm². The cells were allowed to stand for 15 min to promote cell adsorption and allow the cell suspension to penetrate the pore structure of the ADM. They were then placed in DMEM / F12 medium containing 10% fetal bovine serum and cultured for 24 h at 37°C in a 5% CO2 incubator. After culture, the composite scaffold was fixed and observed using a scanning electron microscope to obtain… Figure 2 As shown in the figure, ESCs can adhere to the surface of ADM collagen fibers and are distributed along the scaffold surface and pore structure, suggesting that ADM can provide a good three-dimensional adhesion microenvironment for ESCs.

[0070] In summary, the thickness of the hydrated ADM remained at 0.4 ± 0.17 mm, preserving the natural porous collagen network and exhibiting relatively thorough decellularization. Combined with subsequent testing results, ADM demonstrates good biocompatibility and is suitable as a material for three-dimensional scaffolds in rapidly tissue-engineered skin.

[0071] Beneficial effects: Acellular dermal matrix not only retains the collagen scaffold and porous structure of natural dermis, but also provides a suitable three-dimensional survival environment for loaded ESCs, thus providing good physical support and cell carrier for wound repair.

[0072] 4. Cell compatibility and survival evaluation The ESCs suspension obtained in step S302 (4) of Example 1 was seeded onto two-dimensional (2D) culture dishes and three-dimensional (3D) ADM scaffolds, respectively, with a cell seeding density of approximately 1×10^5 cells / cm², and placed in DMEM / F12 medium containing 10% fetal bovine serum for culture at 37°C and 5% CO2.

[0073] (1) Evaluation of cell adhesion rate (corresponding to) Figure 2 (F in the middle) Twenty-four hours after inoculation, suspended cells were collected from both the two-dimensional culture dish group and the three-dimensional ADM scaffold group. For the ADM group, the scaffold surface was gently rinsed with PBS buffer, and unattached cells in the rinse solution were collected and combined with suspended cells in the culture supernatant. The percentage of non-adhered cells (%) was then calculated using a hemocytometer. The ADM group represents the experimental group where ESCs were seeded on a three-dimensional decellularized dermal matrix scaffold; the Dish group represents the control group where ESCs were seeded in a conventional two-dimensional culture dish. The results showed no significant difference in the percentage of non-adhered cells between the ADM and Dish groups (P > 0.05), indicating that the early attachment ability of ESCs on the ADM scaffold was comparable to that under conventional two-dimensional culture conditions, suggesting that the ADM scaffold has good cell compatibility.

[0074] (2) Evaluation of cell proliferation activity (corresponding to) Figure 2 (G in) On days 1, 4, and 7 after inoculation, MTT solution was added to the Dish+ESCs group and the ADM+ESCs group to a final concentration of approximately 0.5 mg / mL, and incubation was continued at 37°C for 4 h. After incubation, the culture medium was removed, and DMSO or other formazan crystal dissolving solutions were added to fully dissolve the formazan crystals formed in the cells. For the ADM+ESCs group, the shaking dissolution time was appropriately extended to promote the full release of formazan products within the scaffold. The absorbance of the dissolved solution was then measured at 490 nm. A blank ADM scaffold without inoculated cells was used as a background control to eliminate the possible influence of absorbance caused by the scaffold material itself. The Dish+ESCs group represents the control group where ESCs were inoculated in a traditional two-dimensional culture dish; the ADM+ESCs group represents the experimental group where ESCs were inoculated on a three-dimensional ADM scaffold. The results showed that the absorbance values ​​of both groups of cells gradually increased with the extension of culture time, indicating that ESCs maintained a certain degree of proliferative activity in both culture systems. On day 7 of culture, the absorbance value of the ADM+ESCs group was significantly higher than that of the Dish+ESCs group (P<0.05), suggesting that the three-dimensional scaffold environment of ADM is more conducive to the long-term proliferation and activity maintenance of ESCs.

[0075] Beneficial effects: The ADM scaffold does not affect the early adhesion of ESCs and is more conducive to maintaining their proliferation activity during long-term culture, indicating that the scaffold has good cell compatibility and can provide a stable three-dimensional culture environment for rapid tissue-engineered skin.

[0076] 5. Assessment of wound gross morphology and healing dynamics Macroscopic observation and necrosis rate recording: On postoperative days 4, 7, 14, and 21, the survival of grafts on the back wounds of rats in each group (TES group, CS group, STSG group), the appearance of the wounds, and the healing status were observed and recorded using a digital camera. The results are as follows: Figure 3 As shown in the figure. The results showed that one case of early graft necrosis occurred in the TES group on postoperative day 4, three cases of necrosis occurred in the CS group (two cases on postoperative day 4 and one case on postoperative day 7), while no early graft necrosis was observed in the STSG group. By postoperative day 21, the wound in the TES group had achieved almost complete epithelialization, with a relatively smooth wound surface and texture and color close to normal skin; healing in the CS group was significantly delayed, with eschar and incompletely epithelialized areas still visible on postoperative day 21; although the STSG group achieved wound closure earlier, significant wound contraction was observed in the later stages.

[0077] Calculation of wound healing rate and contracture rate: Wound photographs were taken at the above-mentioned time points. The area of ​​the non-epithelialized region and the wound edge area were measured using image processing software. The wound healing rate and wound contracture rate were calculated respectively. The formula for calculating the wound contracture rate is as follows: Wound contracture rate = (initial wound area - wound edge area at each time point) / initial wound area × 100%, the result is as follows Figure 3 B and Figure 3 As shown in Figure C, the wound healing speed in the TES group was significantly faster than that in the CS group; at the same time, the wound contracture rate in the TES group was significantly lower than that in the STSG group. Quantitative analysis showed that on postoperative day 21, the wound contracture rate in the TES group was approximately 20.85%, significantly lower than the 39.48% in the STSG group (P < 0.001). This indicates that the TES group can effectively reduce wound contracture while promoting wound healing.

[0078] In summary, the necrosis rate was high in the traditional composite skin group (CS group) in the early postoperative period (3 / 8), while only 1 / 8 of the TES group of this invention showed early necrosis, indicating better graft survival. By day 21 postoperatively, the TES group achieved better wound epithelialization and exhibited a lower wound contracture rate and better cosmetic repair.

[0079] Beneficial effects: The rapid tissue-engineered skin constructed by this invention not only improves the early survival rate of grafts, but also retains the good anti-contraction properties of the dermal scaffold. It can promote rapid wound healing while reducing the obvious contracture and deformity problems common in traditional skin grafting.

[0080] 6. Histological and immunofluorescence detection Tissue section preparation: Rats were sacrificed on postoperative day 21, and tissue from the healed wound was excised. The tissue was fixed in 4% paraformaldehyde for 24 h, followed by routine dehydration, paraffin embedding, and sectioning. The section thickness was approximately 5 μm. Subsequent HE staining, Masson staining, and immunofluorescence detection were performed. The results are as follows: Figure 4 As shown.

[0081] Morphological and thickness measurements (HE / Masson staining): HE staining was used to observe the integrity of the epidermal structure and the morphology of the dermal tissue after wound repair. The results are shown in the figure. Figure 4 The results showed that the TES group grafts exhibited a more continuous epidermal structure, closely resembling normal skin; the CS group showed incomplete local structures and a reduced number of skin appendages; and the STSG group showed a thinner dermis. Further quantitative analysis of skin thickness was performed, and the results are shown in [Figure A]. Figure 4 The D-values ​​in the data show that the new dermal thickness in both the TES and CS groups was better than that in the STSG group, and was closer to the level of normal skin.

[0082] Masson's trichrome staining was used to observe collagen deposition and distribution. The results are shown in [Figure number missing]. Figure 4 B in the figure, and further calculate the collagen volume fraction (CVF), the results are shown in Figure 4 E. The results showed that the collagen deposits in the TES and CS groups were more abundant and the collagen distribution was more continuous than that in the STSG group, indicating that the ADM-based repair method is beneficial to the reconstruction of the dermal collagen matrix.

[0083] Cell proliferation and differentiation assessment (immunofluorescence): Tissue sections were subjected to double immunofluorescence staining with keratin 10 (K10) and Ki67 to assess local epidermal differentiation and cell proliferation. Specifically, after dewaxing, rehydration, and antigen retrieval, tissue sections were blocked with blocking solution, followed by co-incubation with K10 and Ki67 primary antibodies at a dilution of 1:100, at 4 °C overnight. After washing with PBS buffer, fluorescently labeled secondary antibodies corresponding to the primary antibodies were added at a dilution of 1:500, and incubated in the dark. After washing again, cell nuclei were counterstained with DAPI, and the sections were mounted and observed and imaged under a fluorescence microscope. The results are shown in the figure below. Figure 4Figures C and F in the diagram show that K10 reflects the epidermal differentiation state, and Ki67 reflects cell proliferation activity. The results showed that the TES group exhibited a relatively continuous K10-positive epidermal layer in the repaired area, along with a higher number of Ki67-positive cells, indicating better epidermal differentiation and active local cell proliferation. Further quantitative analysis showed that the proportion of Ki67-positive cells in both the TES and CS groups was higher than that in the STSG group, with the TES group showing better overall performance.

[0084] In summary, HE and Masson staining results confirmed that on postoperative day 21, the grafts in the TES group formed relatively complete epidermis and thick dermal-like tissue with abundant collagen deposition; immunofluorescence detection further indicated that the local cell proliferation activity in the TES group was high and the tissue reconstruction quality was good.

[0085] Beneficial effects: The above results demonstrate that rapidly tissue-engineered skin loaded with ESCs can significantly improve the microscopic reconstruction quality and collagen distribution of wounds, not only promoting epidermal regeneration but also promoting physiological remodeling of the dermis, ultimately achieving single-stage high-quality full-thickness repair that is close to normal skin structure.

[0086] Example 2 verifies the safety and efficacy of "rapid tissue-engineered skin treatment" compared to "standard treatment" in repairing complex wounds. This study included 232 patients with deep burns or chronic, difficult-to-heal wounds who met the criteria. They were randomly assigned to either the standard treatment group (Control Group, n=112) or the rapid tissue-engineered skin treatment group (Experimental Group, n=120). The specific steps are as follows: I. Common Preoperative Preparation: Both groups of patients underwent debridement under general or local anesthesia to remove necrotic tissue until healthy granulation tissue or a freshly bleeding base was exposed. The wound area after debridement was measured (denoted as Awound).

[0087] II. The specific experimental group's steps are as follows; Standard treatment group (control group) procedure: This group adopted the currently clinically accepted "dermal scaffold + autologous skin graft" combined treatment regimen, and the specific steps are as follows: Step S10 (Donor Acquisition): Set the graft area A_graft = Awound. Use an electric dermatome to harvest a 0.25 mm (0.010 inch) thick autologous skin graft from the donor site on the patient's thigh. Purpose: This skin graft will be used entirely to directly cover the wound.

[0088] Step S20 (Stent Preparation): Prepare a three-dimensional scaffold (Lando Biotechnology's double-layer tissue engineering scaffold) that is the same size as the wound area (Ascaffold = Awound), and soak it in sterile saline for 5-10 minutes for rehydration.

[0089] Step S30 (Transplantation): The three-dimensional scaffold is directly applied to the wound base. The split-thickness skin graft obtained in step S10 is then directly placed on the scaffold surface at a 1:1 ratio. The area is then sutured and fixed, and pressure bandaged.

[0090] Rapid tissue-engineered skin treatment group (experimental group) operation steps: This group adopts the construction method described in this invention, namely the sandwich structure of "in situ cell loading + scaffold + skin graft", and the specific operation steps are as follows: Step S10 (Donor Acquisition): Select the wound area Awound, and set the total skin graft area Attotal = Awound + Awound / 40. Use an electric dermatome to obtain an autologous skin graft with a thickness of 0.25 mm. Allocation of uses: Part A (for covering): Area equal to Awound, used for subsequent covering (same as the control group). Part B (for cells): Area approximately Awound / 40 (i.e., a 1:40 ratio), used for preparing cell suspension.

[0091] Step S20 (preparation of epidermal stem cell suspension) includes the following steps: (1) Start the dissociation apparatus and set the temperature to 37°C. Add 10 ml of dissociation reagent to the dissociation dish and 5 ml of fibronectin (FN) at a concentration of 20 μg / ml to the culture flask and spread it evenly. Place the dissociation dish and culture flask on the apparatus's incubation platform. (2) Tissue dissociation and preliminary cell acquisition: Place 1-10 cm² skin flaps (0.15-0.2 mm thick) into the dissociation dish and incubate at a constant temperature for 15 minutes. After dissociation, add lactated Ringer's solution or PBS solution to stop digestion and wash the skin flaps. Separate the epidermis and dermis, and scrape epidermal cells from the basal layer of the epidermis using a scalpel. After collecting the cells, dilute them with lactated Ringer's solution or PBS solution at a ratio of 1 ml / cm², filter through a filter screen, and collect the initial epidermal cell suspension in the filter trough. (3) After the culture flask with FN evenly spread is left to stand at 37°C for 30 minutes, excess liquid in the culture flask is aspirated. The initial epidermal cell suspension is slowly injected into the culture flask and evenly spread to allow for rapid FN adsorption. The flask is then left to stand at 37°C for 25 minutes. (4) The cell membranes or clumps that have been adsorbed by FN at the bottom of the culture flask are naturally detached by physical shaking. The detached intact cell membranes or clumps are aspirated using a 10ml syringe and a flat-tipped needle. Physiological saline is added to the aspirated cell clumps, and after mechanical mixing, a uniform epidermal stem cell suspension is finally formed for later use.

[0092] Step S30 (Stent Preparation): Same as the control group, prepare a three-dimensional scaffold with an area of ​​Awound (Lando Biotechnology's double-layer tissue engineering scaffold) and pre-wet it in a sterile container.

[0093] Step S40 (Mixing and Construction): Based on a 1:40 area ratio, where 1 corresponds to skin and 40 corresponds to the scaffold, the epidermal stem cell suspension obtained in Step S20 is sprayed evenly and at multiple angles onto the surface of the three-dimensional scaffold from Step S30 using a sterile spray device. After saturation and allow to stand for 1-2 minutes, the cell suspension is fully absorbed and penetrates the scaffold's pores through capillary action until saturation is achieved, forming a "cell-scaffold complex." Final Construction: This complex is applied to the wound base, and then the portion of skin graft A (for covering) retained in Step S10 is precisely placed over the complex surface. The area is then sutured and fixed, and pressure bandaged.

[0094] Postoperative care was standardized, with the first dressing change performed on the 6th postoperative day. Subsequent dressing changes were performed daily or every other day depending on the healing progress. Follow-up continued for 6 months, with key assessment points at 2 weeks, 3 months, and 6 months postoperatively. The treatment plan was as follows: Figure 5 As shown. Figure 5This study demonstrates a clinical trial design for wound repair using tissue-engineered skin rapidly constructed from an autologous cell suspension rich in epidermal stem cells. The design encompasses the preparation of the cell suspension, the construction process of the tissue-engineered skin, and the clinical steps for its application in wound repair. Simultaneously, Figure 5 The study timeline, procedures, and tests for subjects randomly assigned to the cell group or control group are also presented. All subjects underwent wound assessment two weeks post-surgery and were followed up for wound recurrence and scar formation at 3 and 6 months post-treatment. The test results are shown in Table 1. Table 1

[0095] Table 1 shows that the gender distribution was identical in both groups, with males accounting for 62.5% and females for 37.5% in both the standard treatment group and the rapid tissue-engineered skin treatment group. This indicates a good gender balance between the two groups (p=1.000), with no statistically significant difference. Patients were divided into three age groups: under 18 years old, 18-60 years old, and over 60 years old. In the standard treatment group, 50% of patients were under 18 years old, 43.8% were between 18 and 60 years old, and 6.2% were over 60 years old. In the rapid tissue-engineered skin treatment group, 44.2% of patients were under 18 years old, 43.3% were between 18 and 60 years old, and 12.5% ​​were over 60 years old. Although the proportion of patients over 60 years old was slightly higher in the rapid tissue-engineered skin treatment group, the difference was not statistically significant (p=0.246). Wound causes included scarring, ulceration, and burns. In the standard treatment group, 45.5% of wounds were caused by scarring, 25.9% by ulceration, and 28.6% by burns. In the rapid tissue-engineered skin treatment group, 40% of wounds were caused by scarring, 38.3% by ulceration, and 21.7% by burns. Although the proportion of ulcers was higher in the rapid tissue-engineered skin treatment group, this difference was not statistically significant (p=0.117). Wounds were categorized by location: upper limbs, lower limbs, trunk, and head and neck. In the standard treatment group, 42.9% of wounds were located on the upper limbs, 46.4% on the lower limbs, 8.9% on the trunk, and 1.8% on the head and neck. In the rapid tissue-engineered skin treatment group, 32.5% of wounds were located on the upper limbs, 50.8% on the lower limbs, 9.2% on the trunk, and 7.5% on the head and neck. The proportion of head and neck wounds was higher in the rapid tissue-engineered skin treatment group, but the difference was not statistically significant (p=0.118). Wound areas were categorized as less than 30 cm², 30-50 cm², 50-100 cm², and greater than 100 cm². In the standard treatment group, 44.6% of wounds were less than 30 cm², 24.1% were 30-50 cm², 10.7% were 50-100 cm², and 20.5% were greater than 100 cm². In the rapid tissue-engineered skin treatment group, 45.8% of wounds were less than 30 cm², 18.3% were 30-50 cm², 22.5% were 50-100 cm², and 13.3% were greater than 100 cm². The proportion of larger wounds (50-100 cm²) showed an increasing trend in the rapid tissue-engineered skin treatment group (p=0.057), suggesting a potentially clinically significant difference. Baseline characteristics were generally comparable between the standard treatment and rapid tissue-engineered skin treatment groups, with no statistically significant differences in gender, age, wound cause, wound location, or wound area. However, some trends were observed in the distribution of wound cause, location, and size that may require further consideration when interpreting treatment outcomes. These findings suggest that any observed differences in efficacy are likely attributable to the intervention itself rather than to baseline differences between groups.

[0096] It should be noted that there were no statistically significant differences between the two groups in terms of gender (both males accounted for 62.5%, p=1.000), age distribution (p=0.246), wound cause (scarring / ulceration / burn, p=0.117), location (upper limb / lower limb / trunk / head and neck, p=0.118), and area (<30 to >100 cm², p=0.057), indicating that randomization was successful and the groups were comparable. Although there were slight trends in wound size and type, the overall balance was good, suggesting that any subsequent differences in efficacy could be reasonably attributed to the intervention itself rather than confounding factors.

[0097] Example 3: The effect of rapid tissue-engineered skin treatment on wound healing Test Procedure: This Example 3 included 232 patients with wounds treated with single-stage surgery, who were randomly assigned to either the standard treatment group (n=112) or the rapid tissue-engineered skin treatment group (n=120). All patients received standardized postoperative care and were followed up until week 2 to assess whether complete wound healing was achieved. Univariate and multivariate logistic regression analyses were used to compare the effects of the two treatment methods on early healing, and the association between potential factors such as gender, age, wound cause, location, and area and healing outcome was further investigated. The test results are shown in Table 2. Table 2

[0098] Table 2 shows that 141 patients achieved complete wound healing within two weeks, while 91 did not (Table 2). Patients were divided into two groups based on treatment method: the standard treatment group (n=112) and the rapid tissue-engineered skin treatment group (n=120). The complete wound healing rate in the rapid tissue-engineered skin treatment group (56.7%) was higher than that in the standard treatment group (43.3%). Univariate analysis showed that patients receiving tissue-engineered skin treatment had an increased likelihood of achieving complete wound healing, with an odds ratio (OR) of 1.67 (95% CI: 0.98–2.84, p=0.058). This association reached statistical significance in multivariate analysis, with an adjusted OR of 1.87 (95% CI: 1.06–3.28, p=0.029). Sex was a significant predictor of wound healing. Women were more likely to achieve complete wound healing compared to men. In univariate analysis, the odds ratio (OR) for females was 1.91 (95% CI: 1.08–3.35, p = 0.025). This finding remained significant in multivariate analysis, with an OR of 1.83 (95% CI: 1.02–3.27, p = 0.042). Age had no significant effect on wound healing outcomes. The healing rate was slightly higher in patients under 18 years of age (51.8%) than in patients aged 18–60 years (39%) and patients over 60 years of age (9.2%). Multivariate analysis did not show significant differences between age groups: the OR for patients aged 18–60 years was 0.67 (95% CI: 0.36–1.26, p = 0.216), and the OR for patients over 60 years of age was 0.76 (95% CI: 0.26–2.19, p = 0.607). Wound etiology (scarring, ulceration, or burn) did not significantly affect healing outcomes. The healing rate of ulcer patients (29.8%) was slightly lower than that of scar patients (46.1%) and burn patients (24.1%). Multivariate analysis showed an odds ratio (OR) of 0.78 for ulcers (95% CI: 0.35–1.70, p=0.529) and 0.80 for burns (95% CI: 0.39–1.62, p=0.529), indicating no significant difference. Wound location also did not significantly affect the healing process. The healing rate was highest for upper limb wounds (41.8%), followed by lower limb wounds (45.4%), trunk wounds (8.5%), and head and neck wounds (4.3%). Multivariate analysis confirmed a lack of significant association; the OR for lower limb wounds was 0.73 (95% CI: 0.37–1.43, p=0.357) compared to upper limb wounds. Wound area was not a significant factor determining healing outcome. The healing rate for wounds smaller than 30 cm² was 46.8%, while the healing rates for wounds with areas of 30-50 cm², 50-100 cm², and larger than 100 cm² were 18.4%, 18.4%, and 16.3%, respectively.No significant differences were observed in the multivariate analysis: the odds ratio (OR) for wounds of 30–50 cm² was 0.67 (95% CI: 0.34–1.33, p = 0.249), the OR for wounds of 50–100 cm² was 1.18 (95% CI: 0.54–2.56, p = 0.672), and the OR for wounds larger than 100 cm² was 0.85 (95% CI: 0.40–1.80, p = 0.670).

[0099] In summary, tissue-engineered skin rapidly constructed using EpiSCs showed a more significant positive effect on wound healing outcomes than standard treatment, especially in female patients. Other factors, including age, wound cause, location, and size, were not significant predictors of healing success in this study.

[0100] Example 4: Long-term results of rapidly tissue-engineered skin in wound healing and recurrence. Test Procedure: This study evaluated the long-term efficacy of rapid tissue-engineered skin treatment on wound recurrence in 214 patients who completed a 3-month follow-up. All patients were stratified based on whether they achieved complete healing by week 2 post-surgery, and the recurrence rate was compared between the standard treatment group (n=112) and the rapid tissue-engineered skin treatment group (n=120). Univariate and multivariate logistic regressions were used to assess the association between early healing, treatment method, and other clinical variables (gender, age, wound cause, location, and area) with wound recurrence at 3 months. The results are shown in Table 3. Table 3

[0101] Table 3 shows the long-term effects of tissue-engineered skin rapidly constructed using EpiSCs on wound healing and recurrence rate three months post-treatment (Table 3). A total of 214 patients were evaluated, of whom 196 (91.6%) did not experience wound recurrence, and 18 (8.4%) experienced recurrence. Patients who achieved complete wound healing within the first two weeks had a significantly reduced risk of wound recurrence at three months. Specifically, only 33.3% of patients with complete healing within two weeks experienced recurrence, compared to 66.7% of patients with incomplete healing at two weeks. Univariate analysis showed that early complete healing significantly reduced the likelihood of recurrence (OR: 0.25, 95% CI: 0.09–0.71, p=0.009). This association remained robust in multivariate analysis, with an adjusted OR of 0.26 (95% CI: 0.09–0.75, p=0.012). The study compared the efficacy of standard treatment with rapid tissue-engineered skin treatment. Although the recurrence rate in the rapid tissue-engineered skin treatment group (38.9%) was lower than that in the standard treatment group (61.1%), this difference was not statistically significant (OR: 0.57, 95% CI: 0.21–1.54, p=0.272). This lack of statistical significance may suggest that further research with larger sample sizes is needed, despite the potential benefit of tissue-engineered skin in reducing recurrence. Sex did not significantly affect wound recurrence. In the non-recurrence group, 61.7% were male and 38.3% were female; while in the recurrence group, 55.6% were male and 44.4% were female. Univariate analysis did not show a significant difference in recurrence rates based on sex (OR: 1.29, 95% CI: 0.49–3.42, p=0.607). Age was not a significant predictor of wound recurrence. The recurrence rate was 38.9% in patients under 18 years of age, 44.4% in patients aged 18–60 years, and 16.7% in patients over 60 years of age. Multivariate analysis showed no significant differences between age groups: the odds ratio (OR) was 0.79 (95% CI: 0.23–2.71, p=0.711) for patients aged 18–60 years and 1.23 (95% CI: 0.21–7.36, p=0.821) for patients over 60 years of age. Wound etiology (scarring, ulceration, or burn) did not significantly affect recurrence. Patients with ulcers had the highest recurrence rate (50%), followed by those with scars (27.8%) and burns (22.2%). Although univariate analysis suggested that ulceration increased the risk of recurrence (OR: 2.96, 95% CI: 0.94–9.27, p=0.063), this association was not significant in the multivariate analysis (OR: 2.01, 95% CI: 0.48–8.39, p=0.340). Wound site did not significantly affect the recurrence rate.Lower extremity wounds (66.7%) recurred more frequently than upper extremity wounds (27.8%), but this difference was not statistically significant (OR: 1.68, 95% CI: 0.47–6.05, p=0.428). Wound size was not a significant predictor of recurrence. The recurrence rate was 50% for wounds smaller than 30 cm² and 16.7% for wounds larger than 100 cm². Univariate analysis showed no significant differences among wound size categories: the OR for 30–50 cm² wounds was 0.92 (95% CI: 0.27–3.16, p=0.895), and the OR for wounds larger than 100 cm² was 0.88 (95% CI: 0.22–3.45, p=0.853). Tissue-engineered skin rapidly constructed using EpiSCs significantly reduced the risk of wound recurrence, especially when early wound healing was achieved within the first two weeks. While a trend toward reduced recurrence was observed with tissue-engineered skin treatment, this did not reach statistical significance, indicating the need for further investigation. Other factors such as sex, age, wound cause, location, and size did not significantly affect the recurrence rate in this cohort. These findings highlight the potential of tissue-engineered skin in improving long-term wound healing outcomes and support further research into its clinical application.

[0102] Example 5: Scar Assessment After Rapid Tissue-Engineered Skin Treatment Test Procedure: Six months post-treatment, scar quality was assessed in a blinded manner in 232 patients who completed follow-up (standard group N=103, cell therapy group N=109). The Observer Scar Assessment Scale (EpiSCs) was used to score four dimensions of scars—color, thickness, vascularity, and flexibility—and a total scar score was calculated. Differences in all indicators and total scores were compared between the rapid tissue-engineered skin treatment group and the standard treatment group to assess the impact of EpiSCs on scar formation. The results are shown in Table 4. Table 4

[0103] Note: Lower scores indicate better scar quality (e.g., closer color to normal skin, thinner scar, less vascularization, and greater flexibility). All indicators were significantly better in the cell therapy group than in the standard treatment group.

[0104] Table 4 shows that the effectiveness of tissue-engineered skin using EpiSCs in repairing skin defects was evaluated by comparing scar characteristics at six months post-treatment between the standard treatment group (N=103) and the rapid tissue-engineered skin treatment group (N=109). Four key parameters—color, thickness, vascular distribution, and flexibility—as well as the total scar score, were assessed using the Observer Scar Scores (IQR) (Table 4). The rapid tissue-engineered skin treatment group showed significantly better scar color results, with a median score of 0.0 (IQR: 0.0 to 1.0), compared to a median score of 1.0 (IQR: 0.0 to 2.0) in the standard treatment group. The difference was statistically significant (p=0.001), indicating that patients receiving cell therapy had more desirable scar color. The rapid tissue-engineered skin treatment group also showed significantly reduced scar thickness, with a median score of 1.0 (IQR: 0.0 to 1.0), compared to a median score of 1.0 (IQR: 1.0 to 2.0) in the standard treatment group. Tissue-engineered skin treatment significantly reduced scar thickness (p<0.001). The vascular distribution score was significantly lower in the rapid tissue-engineered skin treatment group, with a median score of 1.0 (IQR: 0.0 to 1.0), compared to 1.0 in the standard treatment group (IQR: 1.0 to 2.0). This difference was statistically significant (p<0.001), indicating that tissue-engineered skin treatment resulted in less vascularized scars. Scar flexibility was significantly improved in the rapid tissue-engineered skin treatment group, with a median score of 1.0 (IQR: 1.0 to 1.0), compared to 2.0 in the standard treatment group (IQR: 1.0 to 2.0). This difference was statistically significant (p<0.001), indicating that the scars in the rapid tissue-engineered skin treatment group were more flexible. In terms of the overall scar score across all four parameters, the median score in the rapid tissue-engineered skin treatment group was 3.0 (IQR: 2.0 to 4.0), significantly lower than the median score of 6.0 (IQR: 4.0 to 8.0) in the standard treatment group (p<0.001). This comprehensive assessment indicates that tissue-engineered skin treatment delivers superior overall scar quality. The results demonstrate that tissue-engineered skin rapidly constructed using EpiSCs significantly improves scar outcomes in patients with skin defects. Compared to standard treatment, tissue-engineered skin treatment results in better scar color, reduced thickness, decreased vascularity, and enhanced flexibility, ultimately significantly improving the overall scar score. These findings highlight the potential of cell-based therapies in improving wound healing and scar formation quality in clinical settings.

[0105] In summary, rapidly tissue-engineered skin constructed using EpiSCs not only promotes healing but also significantly improves post-repair skin quality. Compared to standard treatments, this therapy resulted in scars at 6 months that were closer in color to normal skin, thinner, with less angiogenesis, and better flexibility, with an overall scar score reduction of over 50% (median 3.0 vs 6.0). This demonstrates that the introduction of epidermal stem cells not only accelerates repair but also reduces pathological scar formation by promoting regeneration that more closely resembles physiological structures, thereby improving patient functional recovery and aesthetic satisfaction, highlighting the unique value of cell therapy in high-quality wound repair.

[0106] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for rapidly constructing tissue-engineered skin, characterized in that, The construction method includes the following steps: S10. Obtain a biological tissue sample containing epidermal stem cells; S20. Take a portion of the biological tissue sample and separate the epidermal stem cells to obtain an epidermal stem cell suspension. S30, Obtain the three-dimensional support; S40. The epidermal stem cell suspension is mixed with the three-dimensional scaffold and cultured in vitro with the remaining biological tissue sample to obtain the tissue-engineered skin.

2. The method for constructing rapid tissue-engineered skin as described in claim 1, characterized in that, In step S10, the source of the biological tissue sample containing epidermal stem cells includes autologous split-thickness skin grafts.

3. The method for constructing rapid tissue-engineered skin as described in claim 1, characterized in that, Step S20 includes: Epidermal stem cells from a portion of the biological tissue sample are separated using Myseed® patented technology to obtain an epidermal stem cell suspension.

4. The method for constructing rapid tissue-engineered skin as described in claim 3, characterized in that, The concentration range of the epidermal stem cell suspension is ≥1×10⁻⁶. 6 cells / mL.

5. The method for constructing rapid tissue-engineered skin as described in claim 1, characterized in that, In step S30: The three-dimensional scaffold includes acellular dermal matrix or tissue-engineered porous scaffold with a three-dimensional mesh structure.

6. The method for constructing rapid tissue-engineered skin as described in claim 1, characterized in that, Step S40 includes: mixing the epidermal stem cell suspension with the three-dimensional scaffold to form a complex; The remaining biological tissue sample is applied onto the complex to obtain functional tissue-engineered skin.

7. The method for constructing rapid tissue-engineered skin as described in claim 1, characterized in that, In step S40, the mixing method includes spraying: The mixing ratio is such that 1 mL of epidermal stem cell suspension is loaded onto the three-dimensional scaffold with a surface area of ​​35-45 cm².

8. A rapidly tissue-engineered skin, characterized in that, The rapidly tissue-engineered skin is constructed using the method described in any one of claims 1 to 7.

9. A rapid tissue-engineered skin model for full-thickness wound repair, characterized in that, include: Obtain isolated biological tissue samples and isolate epidermal stem cell suspensions from them; A three-dimensional scaffold is provided, and the epidermal stem cell suspension is loaded onto the three-dimensional scaffold to obtain a cell-scaffold complex. The cell-scaffold complex was placed in an in vitro culture system for induction culture to induce epidermal stem cells to differentiate and form a multilayered skin tissue structure, thereby obtaining the in vitro skin tissue model.

10. The use of the rapidly tissue-engineered skin as described in claim 8 in the preparation of skin repair products.

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