SECM-SIS biological membrane as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to an sECM-SIS biological membrane as well as a preparation method and application thereof. According to the present invention, an extracellular matrix (sECM) derived from stem cells is integrated to a small intestine submucosa (SIS) to prepare the sECM-SIS biological membrane; after the sECM-SIS biological membrane is implanted into a full-thickness skin defect, wound healing is remarkably accelerated, and the sECM-SIS biological membrane is characterized by rapid wound reepithelization, strong angiogenesis, approximate normal epidermis thickness recovery, mature collagen deposition, skin appendage regeneration and macrophage polarization adjustment. According to the results, the sECM-SIS has huge potential in the aspect of skin wound repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a sECM-SIS biomembrane and a preparation method and use thereof. BACKGROUND

[0002] As the largest protective organ of the human body, the integrity of the barrier function of the skin is crucial for resisting pathogen invasion and maintaining tissue homeostasis. However, skin defects caused by trauma, chronic diseases (such as diabetes and vascular diseases) or the aging process often cause delayed or abnormal wound healing, which can lead to complications including infection, tissue necrosis and systemic problems. These complications seriously threaten the quality of life of patients. Promoting wound healing can reduce hospitalization time, reduce the risk of infection and alleviate the socio-economic burden. Traditional dressings often fail to regulate the complex wound microenvironment due to the lack of biological activity, which can lead to frequent occurrence of scarring or dysfunction. Therefore, the development of new biomaterials that can promote wound healing (especially epithelial regeneration and angiogenesis) has become an urgent need to overcome the current treatment limitations.

[0003] Cell-derived extracellular matrix (ECM) not only retains a variety of bioactive factors that can promote wound healing, such as vascular endothelial growth factor, fibroblast growth factor and transforming growth factor-beta, but also reduces the risk of immune rejection and pathogen transmission. Among them, stem cell-derived extracellular matrix (sECM) has advantages in treating skin wounds, such as richness in bioactive factors. However, it is worth noting that the mechanical properties of cell-derived ECM prepared by decellularization of cell sheets are not ideal, and the effect of decellularized ECM on promoting wound healing is greatly affected by the decellularization method. Therefore, it is necessary to choose a suitable decellularization method or integrate sECM with other scaffold materials to enhance its suitability for skin wound repair.

[0004] Small intestinal submucosa (SIS) is an extracellular matrix (ECM) scaffold extracted from the membranous tissue of the small intestinal submucosa. Due to its excellent biocompatibility, biodegradability and inherent bioactive ingredients, it has received extensive attention in the field of tissue engineering. SIS has been applied to various tissue repairs, including bone regeneration, valve replacement and tympanic membrane repair.

[0005] Therefore, how to construct a sECM composite material suitable for skin wound repair is a problem faced by the field. SUMMARY

[0006] In view of the defects of the prior art, the present application provides a sECM-SIS biomembrane and a preparation method and use thereof.

[0007] The present application provides an sECM-SIS biomembrane, which is characterized by being prepared by decellularization of a complex of stem cells and SIS membrane, wherein the stem cells are selected from the group consisting of urinary stem cells and bone marrow-derived mesenchymal stem cells.

[0008] Preferably, the stem cells are selected from bone marrow-derived mesenchymal stem cells.

[0009] Preferably, the complex of stem cells and SIS membrane is prepared by seeding stem cells onto the surface of the SIS membrane and culturing.

[0010] Preferably, the amount of stem cells seeded onto the surface of the SIS membrane is (1-10) × 10 4 / cm 2 .

[0011] Preferably, the culturing process comprises culturing in α-MEM or DMEM complete medium for 5-10 days, and then culturing in α-MEM or DMEM complete medium containing 25-100 ng / ml vitamin C for 10-20 days.

[0012] Preferably, the step of decellularization comprises surfactant A treatment and DNAase treatment.

[0013] Preferably, the surfactant A is selected from the group consisting of sodium dodecyl sulfate, triton X-100, Tween-100, sodium deoxycholate, tributyl phosphate; and / or, the surfactant A treatment is performed by adding ammonia water, wherein the amount of ammonia water is 10-50 mM; and / or, the DNAase is selected from DNase I.

[0014] and / or, the amount of surfactant A is 0.25-1% by volume fraction; and / or, the surfactant A treatment is performed at a temperature of 37±1℃ and a rotation speed of 50-100 rpm for 15-60 min.

[0015] and / or, the amount of DNAase is 50-200 U / ml; and / or, the DNAase treatment is performed at a temperature of 37±1℃ for 1-3 hours at a rotation speed of 50-100 rpm.

[0016] Preferably, the SIS membrane is prepared by the following steps:

[0017] The animal jejunum is mechanically treated to retain the submucosal layer, and then subjected to defatting, protease treatment and surfactant B treatment to obtain the SIS membrane.

[0018] Preferably, the defatting is performed using a mixed solution of methanol and chloroform at a volume ratio of 0.8-1.2:0.8-1.2; and / or, the defatting is performed for 8-16 hours.

[0019] and / or, the protease is selected from trypsin, papain, pepsin; and / or, the protease is used in a mass concentration of 0.2-0.5%, the protease treatment is carried out at a temperature of 2-8℃, and the protease treatment is carried out for 8-16 hours;

[0020] and / or, the surfactant B is selected from sodium dodecyl sulfate, triton X-100, Tween-100 ethylenediaminetetraacetic acid; and / or, the surfactant B is used in a mass concentration of 0.25-1%, and the surfactant B treatment is carried out for 2-8 hours;

[0021] and / or, after each of the steps of defatting, protease treatment, and surfactant B treatment is completed, the submucosal tissue is washed with water;

[0022] and / or, after the steps of defatting, protease treatment, and surfactant B treatment are completed, the submucosal tissue is subjected to lyophilization treatment.

[0023] The present application provides a method for preparing the biological membrane as described in any of the above, which comprises: preparing a decellularized product from a composite of stem cells and SIS membrane, the stem cells being selected from urinary-derived stem cells and bone marrow-derived mesenchymal stem cells.

[0024] Preferably, the biological membrane is used for preparing an implantable medical material for treating skin defects.

[0025] The present application prepares an sECM-SIS biological membrane material that significantly promotes skin wound healing by screening the preparation process and ECM of stem cells from different sources. The sECM-SIS biological membrane material exhibits excellent cell compatibility, significantly promotes the activity and proliferation behavior of fibroblasts, promotes fibroblast migration and enhances the ability of endothelial cell tubular formation. After the sECM-SIS biological membrane material is implanted into a full-thickness skin wound, the results show that the wound healing is significantly accelerated, which is characterized by rapid re-epithelialization of the wound, strong angiogenesis, near-normal recovery of epidermal thickness, deposition of mature collagen, regeneration of skin appendages, and regulation of macrophage polarization. In combination with these findings, it is shown that sECM-SIS has great potential in skin wound repair.

[0026] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the present application.

[0027] The above summary of the application will be further elaborated in the following detailed description in the form of examples. However, it should not be understood that the above summary of the subject matter of the application is limited to the following examples. Any technology realized based on the above summary of the application falls within the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Macroscopic appearance of bECM-SIS;

[0029] Figure 2 (a) is a graph of DNA content determination results of bone marrow mesenchymal stem cells / pig small intestinal submucosa (BM-MSCs / SIS) composite material before and after decellularization treatment (n=4 in each group); Figure 2 (b) is a DAPI staining graph of BM-MSCs / SIS composite material before and after decellularization treatment, scale: 100 microns;

[0030] Figure 3 is a graph of live / dead cell staining results of pig small intestinal submucosa (SIS) and bECM-SIS co-cultured with NIH3T3 cells; green fluorescence: live cells; red fluorescence: dead cells; scale: 100 microns;

[0031] Figure 4 (a) is a scanning electron microscope (SEM) graph of SIS and bECM-SIS, including graphs of the material alone and in co-culture with NIH3T3 cells, scale: 50 microns; and Figure 4 (b) is a graph of the proliferation of NIH3T3 cells on bECM-SIS (n=3 in each group), *p<0.05;

[0032] Figure 5 is a representative image of a cell scratch experiment, scale: 200 microns;

[0033] Figure 6 is a graph of the migration ratio obtained by statistical analysis of the cell scratch experiment (n=6 in each group);

[0034] Figure 7 is a representative image of a tube formation experiment, scale: 200 microns;

[0035] Figure 8 is a graph of the statistical data analysis results of the tube formation experiment (n=3 in each group), *p<0.05; **p<0.01; ***p<0.001;

[0036] Figure 9 is a macroscopic graph of the wound in the animal experiment in Example 4;

[0037] Figure 10 (a) is a schematic graph of the wound area in each group in Example 4;Figure 10 (b) is the wound healing rate result graph in Experimental Example 4 (n = 6 per group), *p < 0.05; #p < 0.01;

[0038] Figure 11 is the result of a-SMA immunohistochemical staining; scale: 100 microns;

[0039] Figure 12 is the quantitative analysis of vascular density (n = 4 per group); *p < 0.05;

[0040] Figure 13 is the hematoxylin-eosin (HE) staining result of the wound tissue on postoperative days 3 and 7, the scales are 2 microns, 100 microns and 100 microns respectively, corresponding to the upper, middle and lower three rows, the upper row shows the entire wound area, the middle row shows the central area of the wound, and the lower row shows the edge area of the wound;

[0041] Figure 14 is the HE staining result of the wound tissue on postoperative days 14 and 21, the scales are 2 microns, 100 microns and 100 microns respectively, corresponding to the upper, middle and lower three rows, the upper row shows the entire wound area, the middle row shows the central area of the wound, and the lower row shows the edge area of the wound;

[0042] Figure 15 (a) is the epidermal thickness result graph (n = 4 per group); Figure 15 (b) is the wound re-epithelialization rate result graph (n = 4 per group), *p < 0.05;

[0043] Figure 16 (a) is the Masson staining result in Experimental Example 4, scale: 100 microns; Figure 16 (b) is the quantitative analysis result graph of the wound collagen deposition rate in Experimental Example 4 (n = 4 per group), *p < 0.05, **p < 0.01;

[0044] Figure 17 (a) is the result of Sirius red staining, scale: 100 microns; Figure 17 (b) is the result graph of the ratio of type I collagen to type III collagen in the wound counted by the Sirius red staining method (n = 4 per group), *p < 0.05, **p < 0.01;

[0045] Figure 18 (a) is the result of immunofluorescence staining of CD163 (green fluorescence) and iNOS (red fluorescence), scale: 50 microns; Figure 18 (b) is the result graph of the ratio of M2-type to M1-type macrophages in the wound (n = 4 per group), *p < 0.05, **p < 0.01;

[0046] Figure 19(a) is the HE staining result of wound tissue on the 21st day after surgery, scale bar: 200 microns; Figure 19 (b) is the result of quantitative analysis of wound skin appendages (n = 4 per group);

[0047] Figure 20 is the wound general observation chart of the animal experiment in Experimental Example 5;

[0048] Figure 21 (a) is a schematic diagram of the wound area in each group in Experimental Example 5; Figure 21 (b) is the result chart of wound healing rate in Experimental Example 5 (n = 6 per group), *p < 0.05;

[0049] Figure 22 is the Masson staining result in Experimental Example 5, scale bar: 100 microns;

[0050] Figure 23 is the result chart of quantitative analysis of wound collagen deposition rate in Experimental Example 5 (n = 4 per group), *p < 0.05, **p < 0.01. DETAILED DESCRIPTION

[0051] In the following examples and experimental examples, reagents and materials not specifically stated are commercially available.

[0052] Example 1b ECM-SIS biological membrane and its use in treating skin defects

[0053] First, the bECM-SIS biological membrane of the present embodiment is prepared by the following method:

[0054] 1. Preparation of SIS

[0055] (1) Fresh jejunum of pigs with death time within 3 hours is obtained from a slaughterhouse.

[0056] (2) The jejunum is cut into segments of about 15 cm in length and opened longitudinally, and the segments are thoroughly washed with PBS solution.

[0057] (3) The serosa layer, muscle layer and mucosa layer of the segments are mechanically scraped off, and the submucosa tissue is retained.

[0058] (4) The submucosa tissue is soaked in a mixture of methanol and chloroform (volume ratio 1:1) overnight to remove fat.

[0059] (5) After defatting, the segments are rinsed with deionized water 8 times, and then soaked in a 0.25% trypsin solution at 4°C overnight.

[0060] (6) The segments are rinsed with deionized water 3 times, and then soaked in a 0.5% sodium dodecyl sulfate solution overnight.

[0061] (7) Deionized water rinsing 10 times, and freeze-drying for 48 hours.

[0062] (8) After freeze-drying, ethylene oxide sterilization for standby.

[0063] In other embodiments, the length of the intestinal segment in step (2) can also be adjusted in the range of 10-20 cm; the number of deionized water rinsing in step (5) can also be adjusted in the range of 5-10 times; the number of deionized water rinsing in step (6) can also be adjusted in the range of 3-5 times, and the soaking time in 0.5% sodium dodecyl sulfate solution can also be adjusted in the range of 2-8 hours; the number of deionized water rinsing in step (7) can also be adjusted in the range of 10-15 times, and the freeze-drying time can also be adjusted in the range of 24-48 hours.

[0064] 2. Compound culture of bone marrow-derived MSCs and SIS

[0065] (1) Cut SIS into a round piece with a diameter of 1.5 cm, and sterilize by ethylene oxide at 37°C for standby.

[0066] (2) Place the SIS round piece on a six-well plate with a sterile forceps, so that the SIS round piece is tightly attached to the bottom of the well.

[0067] (3) Take 50 microliters of serum and carefully coat the surface of SIS, and stand for 30 minutes.

[0068] (4) Take P3 generation of bone marrow-derived MSCs (BM-MSCs), and inoculate on the surface of SIS at a density of 5×10 4 / cm 2 .

[0069] (5) Culture in a 37°C, 5% CO2 incubator, and replace the culture medium every 2 days.

[0070] (7) After 5 days of culture, replace the culture medium with complete culture medium containing vitamin C (50 μg / ml), and continue to culture for 10 days to prepare a BM-MSCs / SIS composite.

[0071] In other embodiments, the diameter of the round piece in step (1) can also be adjusted in the range of 1.5-2 cm; and the BM-MSCs in step (4) can also be P3-P5 generation.

[0072] 4. Preparation of bECM-SIS biomembrane

[0073] (1) Prepare working solution A: add concentrated ammonia water to a solution of Triton X-100 to make the concentration of ammonia water 20 mM, and the volume concentration of Triton X-100 0.5%.

[0074] (2) The BM-MSCs / SIS complex was placed in a six-well plate, one piece of BM-MSCs / SIS complex per well, and then 2 ml of working solution A was added to each well, and the plate was placed in a 37°C shaking bed (60 rpm) for 30 minutes, and then washed with deionized water.

[0075] (3) The BM-MSCs / SIS complex was further treated with 100 U / mL of deoxyribonuclease solution at 37°C for 3 hours, and then washed with PBS.

[0076] (4) The treated BM-MSCs / SIS complex was subjected to overnight freeze-drying in a vacuum freeze dryer to prepare a bECM-SIS biological membrane.

[0077] II. Use of bECM-SIS biological membrane for skin defects

[0078] 1. Establishment of animal model

[0079] A full-thickness skin defect model was established in 12-week-old male Sprague-Dawley rats. The rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg), and a circular full-thickness skin defect with a diameter of 1 cm was made on the back of each rat.

[0080] 2. Material treatment

[0081] The bECM-SIS membrane was applied to the wound and fixed with sutures, and one bECM-SIS biological membrane was used for each wound. After 21 days, the skin wound was completely healed.

[0082] Example 2 uECM-SIS biological membrane and its use in treating skin defects

[0083] The uECM-SIS biological membrane was prepared according to the method of Example 1 and used for skin defects, with the difference that BM-MSCs were replaced by urine-derived stem cells.

[0084] The technical solutions of the present application are further illustrated by experiments. The samples SIS, BM-MSCs / SIS complex and bECM-SIS biological membrane detected in the following experimental examples were prepared by the method of Example 1, and the uECM-SIS biological membrane was prepared by the method of Example 2.

[0085] Experimental Example 1 Decellularization effect determination

[0086] I. Experimental method

[0087] The decellularization effect of bECM-SIS biomembrane was evaluated by DAPI staining and DNA content determination. To detect the residual nuclei after decellularization, the bECM-SIS sample was stained with DAPI staining solution (dilution ratio 1:1000) in the dark for 10 minutes, and then analyzed using a fluorescence microscope. The residual DNA content in bECM-SIS was quantified by extracting total DNA using a DNA extraction kit and measuring it with a microspectrophotometer (NanoDrop, Thermo, USA). The DNA content was expressed in nanograms per milligram of sample (ng / mg). The BM-MSCs / SIS complex before decellularization served as a control group.

[0088] II. Experimental results

[0089] The overall morphology of bECM-SIS biomembrane was very similar to SIS ( Figure 1 ), presenting a film-like scaffold with a white surface. Scanning electron microscopy (SEM) analysis showed that bECM-SIS exhibited a rough surface compared to the relatively smooth surface of SIS, due to the presence of cell-derived extracellular matrix (ECM) Figure 4 a). During the preparation of bECM-SIS, the decellularization process of the BM-MSCs / SIS complex was evaluated by DNA quantification and DAPI staining. As shown in Figure 2 a, the residual DNA content in bECM-SIS was 47.39 ± 2.51 ng / mg, which was lower than the safety threshold for decellularized scaffolds (50 ng / mg). In addition, after decellularization of the BM-MSCs / SIS complex, no identifiable nuclei were found by DAPI staining Figure 2 b). Integrating these findings confirmed the successful decellularization of bECM-SIS prepared by the present application, as well as the retention of cell-derived ECM on the surface.

[0090] This experimental example demonstrated the successful preparation of bECM-SIS, as well as good decellularization effect.

[0091] Experimental Example 2 Cell compatibility of bECM-SIS biomembrane

[0092] I. Experimental methods

[0093] 1. Cell viability and proliferation activity determination experiment

[0094] Fibroblasts (NIH3T3) were seeded at 1 × 10 4 / cm 2NIH3T3 cells were seeded on SIS or bECM-SIS surfaces at a density of 1 x 10 4 / cm 2 and cultured in complete medium. Cell viability was assessed using live / dead staining technique at 1, 3 and 5 days after seeding. Specifically, the culture medium was aspirated and then cells were stained with Calcein-AM and propidium iodide (PI) double staining kit for 30 minutes at 37°C. Subsequently, the samples were observed using a laser scanning confocal microscope.

[0095] NIH3T3 cell proliferation activity on SIS or bECM-SIS was determined using Cell Counting Kit-8 (CCK-8). At 1, 3 and 5 days after seeding, CCK-8 reagent was diluted in DMEM medium at a ratio of 1:10, and then 220 μL of diluted CCK-8 solution was added to each sample (n=3 for each group) and incubated at 37°C for 2 hours. Subsequently, 100 μL of supernatant from each sample was transferred to a 96-well plate, and the absorbance was measured at 450 nm using a spectrophotometer.

[0096] 2. Scanning electron microscope analysis

[0097] NIH3T3 cells were seeded on SIS or bECM-SIS surfaces at a density of 1 x 10 4 / cm 2 and cultured in complete medium for 5 days. SEM was used to observe the surface morphology of the samples. For SEM analysis, the cell / scaffold composites went through a series of steps: light washing with PBS three times, fixation with 2.5% glutaraldehyde, drying under vacuum, gold spraying, and finally observation using a scanning electron microscope. In this experiment, SIS and bECM-SIS were used as control groups.

[0098] II. Experimental results

[0099] Live / dead staining showed that cell viability on SIS or bECM-SIS was good at 1, 3 and 5 days after culture Figure 3 ). At 5 days, SEM analysis of the cell / scaffold composites showed that NIH3T3 cells spread well on the surface of both SIS and bECM-SIS, forming multilayer cell sheets with normal morphology Figure 4 a). In addition, CCK-8 results showed that NIH3T3 in both groups showed strong growth Figure 4 b), with increasing optical density (OD) values during the culture period. At 3 days, the OD value of the bECM-SIS group was significantly higher than that of the SIS group (p<0.05), indicating that bECM-SIS enhanced the proliferation activity of fibroblasts.

[0100] The results of the present experiment show that bECM-SIS has good cell compatibility, can promote the proliferation of fibroblasts, and the proliferation-promoting effect of the bECM-SIS biological membrane is better than that of SIS.

[0101] Experimental Example 3 Influence of bECM-SIS biological membrane on skin repair cells in vitro

[0102] I. Experimental Methods

[0103] 1. Cell scratch test

[0104] NIH3T3 fibroblasts were seeded into a 12-well plate at a density of 2 x 10 5 / well. When the cell confluence reached 100%, a straight-line scratch was made using a 200-μL tip. Subsequently, the cells were washed with PBS for 3 times, and then cultured in serum-free DMEM medium (control group), SIS conditioned medium or bECM-SIS conditioned medium. The conditioned medium was prepared by incubating SIS or bECM-SIS biological membrane in serum-free DMEM medium at a ratio of 1 cm 2 of membrane per 6 mL of medium at 37°C for 72 hours, after which the supernatant was collected. At 0, 6, 12 and 24 hours after scratching, the cell migration was observed and recorded using an inverted microscope. The migration rate was calculated according to the formula: migration rate = (migrated area / initial scratch area) x 100%.

[0105] 2. Matrigel tube formation test

[0106] Matrigel (Corning, USA) and DMEM medium were mixed at a volume ratio of 1:1 and added to a 24-well plate, and incubated at 37°C for 45 minutes to gel. After gel formation, human umbilical vein endothelial cells (HUVECs) were seeded onto the gel surface at a density of 8 x 10 4 / well. Subsequently, 500 μL of different medium was added to each well, with serum-free DMEM as the control group. In the SIS and bECM-SIS groups, SIS conditioned medium or bECM-SIS conditioned medium was used, respectively. After incubation at 37°C for 6 hours, the formation of tubular structures was observed and recorded using an inverted microscope. ImageJ software was used for quantitative analysis, and the parameters evaluated included total tube length, mesh number and branch points.

[0107] II. Experimental Results

[0108] To evaluate the effect of bECM-SIS on fibroblast migration, a NIH3T3 cell scratch test was performed using the conditioned medium of SIS or bECM-SIS. Cell migration was evaluated at 6, 12 and 24 hours, as shown in Figure 5As shown, bECM-SIS exhibited the fastest fibroblast migration. Statistically significant differences were observed between the control and bECM-SIS groups at all time points. Furthermore, after 6 hours of culture, the migration rate of bECM-SIS was higher than that of SIS (…). Figure 6 (p<0.05). These results indicate that bECM-SIS has a more significant effect on fibroblast migration compared to SIS.

[0109] To investigate the effects of bECM-SIS on vascular endothelial cells, HUVECs were cultured in conditioned media derived from SIS or bECM-SIS, and Matrigel tube formation assays were performed. After 6 hours of co-culture, significant differences were observed among the experimental groups. Figure 7 Compared to the control group and the SIS group, the bECM-SIS group had a larger number of grids. Figure 8 a&c; p<0.05). Similarly, the bECM-SIS group also had the longest total tube length ( Figure 8 (b) These findings suggest that bECM-SIS has a stronger tubular formation capacity compared to SIS, which is beneficial for promoting angiogenesis in wound healing.

[0110] The results of this experiment show that, compared with SIS, bECM-SIS biomembrane significantly promotes the migration ability of fibroblasts and the tube-forming ability of vascular endothelial cells.

[0111] Experimental Example 4: Application of the ECM-SIS biomembrane in a full-thickness skin defect model

[0112] I. Experimental Methods

[0113] 1. Animal models

[0114] A full-thickness skin defect model was established using 12-week-old male Sprague-Dawley rats. The rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg), and a circular full-thickness skin defect with a diameter of 1 cm was created on the back of the rats. The rats were then randomly assigned to the following three groups: 1) control group, receiving no treatment; 2) SIS group, with the wound covered with SIS and fixed with sutures; 3) bECM-SIS group, with the wound covered with a bECM-SIS biofilm and fixed with sutures.

[0115] 2. Wound healing rate

[0116] The wound was assessed on postoperative days 3, 7, 14, and 21. ImageJ software was used to quantify the wound area. The healing rate was calculated using the formula [(S0-St) / S0]×100%, where S0 is the initial wound area, St is the healed ... t : Wound area at various time points.

[0117] 3. Histological analysis

[0118] On days 3, 7, 14 and 21 post-surgery, rats were euthanized and tissue samples were obtained from the wound site, fixed in 4% paraformaldehyde solution and submitted to histological staining, including hematoxylin-eosin (HE) staining, Masson staining and Sirius red staining.

[0119] HE staining was used to analyze: 1) epidermis thickness; 2) number of skin appendages; 3) degree of re-epithelialization of the wound, calculated by the formula [(Lo - L t ) / Lo] x 100%, where Lo represents the initial wound length and L t represents the wound length at each time point. Masson staining was used to examine collagen deposition within the wound area. Sirius red staining was used to assess the deposition of type I and III collagens in the wound area. All histological analyses were performed using ImageJ software.

[0120] 4. Immunohistochemistry and immunofluorescence staining

[0121] Tissue sections were submitted to antigen retrieval in citrate buffer. For immunohistochemistry staining, endogenous peroxidase activity was inhibited using 3% hydrogen peroxide, followed by treatment with 0.2% Triton X-100 for 10 minutes and then blocking with a protein-free quick block for 30 minutes. Sections were treated with primary antibody (a-SMA; dilution 1:1500) and incubated overnight at 4°C. The next day, sections were incubated with secondary antibody (dilution 1:500) for 1 hour, followed by DAB staining and hematoxylin counterstaining. After dehydration, sections were mounted with neutral resin, scanned using the VS200 system and analyzed using ImageJ software.

[0122] For immunofluorescence staining, sections were incubated overnight at 4°C with primary antibodies for iNOS (dilution 1:200) and CD206 (dilution 1:200). Subsequently, sections were incubated with fluorescent secondary antibodies (dilution 1:400) for 1 hour and the nuclei were stained with DAPI. Fluorescence was observed using a confocal microscope and ImageJ software was used for quantitative analysis of M1 and M2 macrophages in the wound area.

[0123] 6. Statistical analysis

[0124] Data are presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism software. For multiple comparisons, one-way ANOVA followed by Tukey's post-hoc test was used. For comparison between two groups, the unpaired Student's t-test was used. P values less than 0.05 were considered statistically significant.

[0125] II. Experimental results

[0126] 1. Wound healing

[0127] Wounds were grossly observed, quantified, and histologically analyzed at days 3, 7, 14, and 21 postoperatively. The wound area was reduced in the SIS group and the bECM-SIS group at days 3, 7, and 14 compared to the control group Figure 9 Figure 10 a). Importantly, the wound area was significantly smaller in the bECM-SIS group than in the control group at days 3 and 7 Figure 10 b; p < 0.01). At day 7, the wound area was significantly smaller in the bECM-SIS group than in the SIS group Figure 10 b; p < 0.01). At day 21, all wounds were completely healed Figure 9

[0128] 2. bECM-SIS promotes wound angiogenesis

[0129] To evaluate the wound angiogenesis, a-smooth muscle actin (a-SMA) immunohistochemical staining was performed Figure 11 At day 3, the bECM-SIS group had a significantly higher vessel density than the control group Figure 12 ; p < 0.05). By day 7, the bECM-SIS group still had a higher vessel density than the other groups. As the newly formed tissue matured, the vessel density began to approach the level of healthy skin tissue. At day 21, the bECM-SIS group had a vessel density that was lower than the control group Figure 12 ; p < 0.05) and very close to the vessel density of normal rat skin tissue (11.06 ± 4.87 / cm 2 ). These results indicate that bECM-SIS enhances angiogenesis during the early stages of wound healing.

[0130] 3. bECM-SIS promotes re-epithelialization of wounds

[0131] As shown in Figure 13-14 , the bECM-SIS group had superior re-epithelialization of the wound compared to the other groups. At day 14, the bECM-SIS group had a re-epithelialization rate of 84.29 ± 16.74%, which was significantly higher than that of the control group (52.74 ± 2.59%; Figure 15 b; p < 0.05). At days 3, 7, and 14, the epidermis thickness was slightly greater in the SIS group and the bECM-SIS group than in the control group Figure 15 a).

[0132] 4. bECM-SIS improves wound collagen deposition

[0133] ​​Masson staining was used to assess collagen deposition at the wound site. The results showed that at days 7, 14 and 21, there was a large amount of collagen present at the wound site in all groups Figure 16 a). The bECM-SIS group resulted in more collagen deposition at days 3 and 7 compared to the control group Figure 16 b).

[0134] It is known that type III collagen is more prevalent than type I collagen during the early stages of wound healing. Conversely, type I collagen becomes the predominant component during the later stages of wound healing. In this experiment, Sirius red staining was used to assess the deposition of type I and type III collagen in the wound tissue, where type I collagen appears red / orange and type III collagen appears green. The results showed that the bECM-SIS group had a significant increase in type I collagen deposition compared to the other groups Figure 17 a). The bECM-SIS group had a higher ratio of type I to type III collagen at all time points Figure 17 b; p<0.05).

[0135] 5. bECM-SIS promotes M2 macrophage polarization at the wound site

[0136] Macrophages are a key factor in skin wound healing and their transition between the pro-inflammatory Ml phenotype and the anti-inflammatory M2 phenotype is crucial for healing. Failure to transition from the Ml phenotype to the M2 phenotype can result in persistent inflammation and delayed healing. In this experiment, immunofluorescence staining for iNOS (Ml macrophage marker) and CD163 (M2 macrophage marker) was used to assess macrophage polarization at the wound site. As shown in Figure 18 a, the bECM-SIS group had a decrease in the number of Ml macrophages at days 3 and 7 and an increase in the number of M2 macrophages at days 14 and 21 compared to the control and SIS groups.

[0137] The bECM-SIS group had a significantly higher M2 / Ml ratio of macrophages at days 3 and 7 compared to the control and SIS groups. Similarly, the bECM-SIS group had a higher M2 / Ml ratio at day 14 compared to the control group Figure 18 b; p<0.05). However, at day 21, no significant differences were detected between the groups as the regenerative tissue matured Figure 18 . These findings suggest that bECM-SIS has immunomodulatory capabilities at the wound site, aiding in the polarization of macrophages towards an anti-inflammatory phenotype.

[0138] 6. bECM-SIS promotes adnexal regeneration at the wound site

[0139] Wound skin appendage regeneration is essential for restoring normal skin function. In this experiment, HE staining was used to evaluate the regeneration of skin appendages on the wound surface. The results showed that the number of skin appendages on the wound surface was very small in all groups on days 3, 7, and 14 Figure 19 ). Notably, by day 21, the bECM-SIS group showed significantly more skin appendages (13.08 ± 12.78 per field), while the control and SIS groups were 3.50 ± 1.29 and 7.92 ± 4.76 per field, respectively Figure 19 b). These results indicate that ECM from BM-MSCs creates a more favorable microenvironment for skin appendage regeneration.

[0140] The results of this experimental example show that the bECM-SIS biomembrane exhibits superior wound repair capacity compared to SIS. First, bECM-SIS exhibits superior re-epithelialization capacity, both in terms of re-epithelialization rate and new epidermis thickness, which is superior to the control group. In terms of collagen deposition, enhanced collagen deposition and maturation were observed in the bECM-SIS group compared to the SIS group. Polarization of macrophages to the M2 phenotype can significantly improve tissue repair. In the early stage of wound healing, the bECM-SIS group exhibited the highest ratio of M2 / M1 type macrophages, confirming the immunomodulatory effect of the bECM-SIS biomembrane.

[0141] Experimental Example 5 Therapeutic effect of ECM-SIS prepared from stem cells of different sources

[0142] I. Experimental Methods

[0143] The animal model was established according to the experimental method of Experimental Example 4, and the wound healing rate was determined, and Masson staining was used to evaluate the collagen deposition in the wound area. The difference is that this experimental example is divided into four groups: control group, SIS group, uECM-SIS, bECM-SIS; among them, the control group, SIS group, bECM-SIS group are treated the same as Experimental Example 4, the uECM-SIS group is to cover the wound with uECM-SIS and fix it with sutures, and the uECM-SIS is prepared according to the preparation method of Example 2.

[0144] II. Experimental Results

[0145] 1. Wound healing

[0146] The results of wound healing are shown in Figure 20 . Gross observation found that the stem cell ECM modified SIS membrane material uECM-SIS and bECM-SIS both significantly promoted wound healing. In particular, the effect of bECM-SIS biomembrane in promoting wound healing was significantly better than uECM-SIS Figure 21a). Quantitative results of wound area further indicate that the bECM-SIS biofilm is superior to uECM-SIS in promoting wound healing, especially in the early stages of wound healing ( Figure 21 (b) This indicates that bECM-SIS is more effective at promoting the healing of full-thickness skin wounds compared to uECM-SIS.

[0147] 2. Collagen deposition in the wound

[0148] like Figure 22 As shown, in the early stage of wound healing, such as day 3, bECM-SIS promoted collagen deposition to a significantly higher degree than uECM-SIS, which may be related to the higher wound healing efficiency of the bECM-SIS group; in the later stage of wound healing, the degree to which bECM-SIS promoted collagen deposition was still significantly higher than that of uECM-SIS. Figure 23 The results indicate that the bECM-SIS group has higher wound healing efficiency and better outcomes.

[0149] The comparative experiment in this case shows that ECM-SIS materials prepared from ECM stem cells of different origins have different effects on promoting skin wound healing, with bECM-SIS showing better results.

[0150] As can be seen from the above embodiments and experimental examples, this invention, through screening preparation processes and ECMs from stem cells of different sources, prepares sECM-SIS biomembrane materials that significantly promote skin wound healing. This sECM-SIS biomembrane material exhibits excellent cell compatibility, which is confirmed by the high activity and enhanced proliferation capacity of fibroblasts in in vitro experiments. Scratch assays and tubular formation assays further confirm that sECM-SIS promotes fibroblast migration and enhances the tubular formation ability of vascular endothelial cells. After implantation of this sECM-SIS biomembrane material into full-thickness skin defects, the results showed significantly accelerated wound healing, characterized by rapid re-epithelialization, robust angiogenesis, near-normal epidermal thickness recovery, mature collagen deposition, regeneration of skin appendages, and regulation of macrophage polarization. These findings collectively indicate that sECM-SIS biomembranes have great potential in skin defect repair.

Claims

1. An sECM-SIS biomembrane, characterized in that: The decellularization treatment is prepared by a complex of stem cells and SIS membrane, and the stem cells are selected from urinary stem cells.

2. The biofilm according to claim 1, characterized in that: The complex of stem cells and SIS membrane is prepared by seeding stem cells on the surface of SIS membrane and culturing.

3. The biofilm according to claim 2, characterized in that: The amount of stem cells inoculated on the surface of the SIS membrane is (1-10) x 10 4 / cm 2 .

4. The biofilm according to claim 2, characterized in that: The culturing process comprises culturing in α-MEM or DMEM complete medium for 5-10 days, and then culturing in α-MEM or DMEM complete medium containing 25-100 ng / ml vitamin C for 10-20 days.

5. The biofilm according to claim 1, characterized in that, The decellularization treatment comprises surfactant A treatment and DNA enzyme treatment.

6. The biofilm according to claim 5, characterized in that, The surfactant A is selected from sodium dodecyl sulfate, triton X-100, Tween-100, sodium deoxycholate and tributyl phosphate; and / or, the surfactant A treatment is added with ammonia water, and the amount of the ammonia water is 10-50 mM; and / or, the DNA enzyme is selected from DNase I; And / or, the amount of the surfactant A is 0.25-1% by volume fraction; and / or, the surfactant A treatment is performed at a temperature of 37±1℃ and a rotation speed of 50-100 rpm for 15-60 min; And / or, the amount of the DNA enzyme is 50-200 U / ml; and / or, the DNA enzyme treatment is performed at a temperature of 37±1℃ and a rotation speed of 50-100 rpm for 1-3 hours.

7. The biofilm according to claim 1, characterized in that, The SIS membrane is prepared by the following steps: The animal jejunum is mechanically treated to reserve submucosal tissue, and then defatted, treated with protease and treated with surfactant B to obtain the SIS membrane.

8. The biofilm according to claim 7, characterized in that: The defatting is performed by a mixed solution of methanol and chloroform in a volume ratio of 0.8-1.2:0.8-1.2; and / or, the defatting is performed for 8-16 hours; And / or, the protease is selected from trypsin, papain and pepsin; and / or, the amount of the protease is 0.2-0.5% by mass concentration, the protease treatment is performed at a temperature of 2-8℃, and the protease treatment is performed for 8-16 hours; And / or, the surfactant B is selected from sodium dodecyl sulfate, triton X-100, Tween-100 and ethylenediaminetetraacetic acid; and / or, the amount of the surfactant B is 0.25-1% by mass concentration, and the surfactant B treatment is performed for 2-8 hours; And / or, after each of the defatting, protease treatment and surfactant B treatment is completed, the submucosal tissue is washed with water; And / or, after the defatting, protease treatment and surfactant B treatment are completed, the submucosal tissue is freeze-dried.

9. The method of producing a biofilm according to any one of claims 1 to 8, characterized in that, It comprises: The decellularization treatment is prepared by a complex of stem cells and SIS membrane, and the stem cells are selected from urinary stem cells, bone marrow-derived mesenchymal stem cells.

10. Use of the biological membrane according to any one of claims 1-8 for the preparation of an implantable medical material for the treatment of skin defects.

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