A bme-sis biofilm and methods of making and using the same

Abstract

The application belongs to the technical field of medicine, and particularly relates to a BME-SIS biological membrane and a preparation method and application thereof. The BME-SIS biological membrane is prepared by screening a preparation process, based on SIS material, and through composite culture with BMSCs and decellularization treatment. The BME-SIS biological membrane has good decellularization effect, and avoids the problem of insecurity caused by xeno / allograft transplantation. Compared with SIS material, the BME-SIS biological membrane has the effect of significantly promoting cell adhesion, proliferation and differentiation. When applied to a rat rotator cuff tear model, the BME-SIS biological membrane of the application is confirmed to have the effect of significantly promoting rotator cuff tear healing from multiple aspects of molecular, histological, motility, mechanical and imaging, is beneficial to reducing the risk of postoperative re-tear of the rotator cuff, and has stability in the treatment effect. The BME-SIS biological membrane of the application can be used for biological repair patches, and has good prospects in the aspect of rotator cuff tear healing.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a BME-SIS biomembrane, its preparation method, and its uses. Background Technology

[0002] The rotator cuff is a crucial structure for shoulder joint movement. Rotator cuff injuries are the most common cause of shoulder pain, severely impacting patients' quality of life. Surgical treatment is recommended for rotator cuff tears that do not respond well to conservative treatment and for those with significant symptoms, as it can significantly alleviate symptoms and improve shoulder function. However, a considerable number of patients experience poor rotator cuff healing or even re-tears after surgery. Studies have reported that the re-tear rate after rotator cuff repair surgery can reach as high as 94%. Improving rotator cuff healing remains a pressing challenge in shoulder surgery.

[0003] Animal studies have shown that the submucosal layer of the small intestine (SIS) can repair rotator cuff tears. SIS can promote host tissue growth and has demonstrated therapeutic potential in rotator cuff defect models. Clinicians have attempted to use commercially available SIS products to repair human rotator cuff tears, but the results have been less than ideal. A review of previous clinical studies reveals that the limitations of SIS in rotator cuff repair are mainly due to two factors: firstly, SIS is associated with a higher rate of adverse reactions after rotator cuff surgery; and secondly, SIS does not significantly reduce the rate of re-tears after rotator cuff surgery.

[0004] Adverse reactions to xenogeneic decellularized biomaterials are related to DNA residue. Improving the material preparation process and ensuring complete decellularization can mitigate these adverse reactions. Clinical studies have found that SIS failed to significantly reduce the rotator cuff re-tear rate, which may be related to the following reasons: First, SIS is rarely modified or altered, and is often used only for repairing rotator cuff tears. Second, most studies use SIS to fill tendon defects in large, irreparable rotator cuff tears, or to cover the rotator cuff surface to enhance the repair of repairable rotator cuff tears; there are few reports of SIS being placed between the tendon and bone. SIS modification and its specific application can both affect the repair outcome.

[0005] In recent years, there have been increasing reports on the use of bone marrow-derived mesenchymal stem cells (BMSCs) to improve tendon-bone healing, but the effects have varied. In a rat Achilles tendon insertion reconstruction model, local injection of BMSCs formed a typical structure similar to the normal tendon-bone interface. However, in a rat rotator cuff tear model, transplanted BMSCs did not promote tendon-bone interface formation, nor did it improve biomechanical strength (Am J Sports Med, 2009, 37(11): 2126-33). While BMSCs have some potential in promoting rotator cuff healing and reducing re-tear rates, the problem of unstable therapeutic effects needs to be overcome. In particular, transplanted BMSCs carry risks such as low cell survival and tendon calcification. How to utilize the healing-promoting capacity of BMSCs while reducing transplantation risks and overcoming the problem of therapeutic instability is key to clinical application.

[0006] Therefore, how to construct a BMSC-modified SIS material that can safely and stably improve the healing of rotator cuff tears is a challenge in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a BME-SIS biofilm, its preparation method, and its applications.

[0008] This invention provides a BME-SIS biomembrane, which is prepared by a method comprising the following steps:

[0009] Step 1: Bone marrow-derived mesenchymal stem cells were seeded onto the surface of the SIS membrane and cultured to obtain the BMSCs-SIS complex;

[0010] Step 2: The BMSCs-SIS complex is obtained by decellularization.

[0011] The ratio of the SIS membrane to bone marrow-derived mesenchymal stem cells used was 1 cm. 2 : 2×10 4 -15×10 4 indivual.

[0012] Preferably, the culture includes: first culturing in α-MEM complete medium for 3-10 days, and then culturing in α-MEM complete medium containing 50-200 μg / ml vitamin C for 5-10 days.

[0013] Preferably, the SIS membrane is prepared by the following steps:

[0014] Animal jejunum is taken, mechanically processed while preserving the submucosa, and then defatted, treated with protease, and treated with surfactant to obtain the final product.

[0015] Preferably, the degreasing is carried out by a mixed solution of methanol and chloroform in a volume ratio of 0.8-1.2:0.8-1.2; and / or, the degreasing time is 10-16 hours;

[0016] And / or, the protease is selected from trypsin, papain, and pepsin; and / or, the amount of the protease used is 0.1-0.5% by mass, the temperature of the protease treatment is 3-8°C, and the treatment time is 4-18 hours;

[0017] And / or, the surfactant is selected from sodium dodecyl sulfate, Triton X-100, Tween-100, and ethylenediaminetetraacetic acid; and / or, the amount of the surfactant is 0.25-1% by mass, and the treatment time of the surfactant is 6-24 hours;

[0018] And / or, after each step of the defatting, protease treatment, and surfactant treatment is completed, the submucosal tissue is washed with water 5-15 times;

[0019] And / or, after the defatting, protease treatment, and surfactant treatment steps are completed, freeze-drying is performed.

[0020] Preferably, the decellularization process includes: surfactant treatment and DNase treatment.

[0021] Preferably, the surfactant is selected from sodium dodecyl sulfate, Triton X-100, Tween-100, and sodium deoxycholate; and / or, concentrated ammonia is added during the surfactant treatment, and the volume ratio of the surfactant to concentrated ammonia is 50-500:200; and / or, the DNase is selected from DNase I.

[0022] Preferably, the amount of surfactant used is 0.25-1% by volume; and / or, the surfactant treatment time is 15-60 min, the temperature is 25-37℃, and the rotation speed is 50-100 rpm.

[0023] And / or, the amount of DNase used is 50-250 U / ml; and / or, the temperature of the DNase treatment is 25-37℃, the time is 1.5-6 hours, and the rotation speed is 50-100 rpm.

[0024] Preferably, the culture medium is changed every 1-2 days during the culture process; and / or, the culture conditions include incubation at 37±1℃ in a 5% CO2 cell culture incubator; and / or, the SIS membrane is a circular disc with a diameter of 1.5-10cm; and / or, after decellularization, the membrane is washed with an aqueous solution; and / or, the bone marrow-derived mesenchymal stem cells are P3-7 generation bone marrow-derived mesenchymal stem cells.

[0025] This invention provides a method for preparing the BME-SIS biofilm according to any one of the above claims, comprising:

[0026] Step 1: Bone marrow-derived mesenchymal stem cells were seeded onto the surface of the SIS membrane and cultured to obtain the BMSCs-SIS complex;

[0027] Step 2: The BMSCs-SIS complex is obtained by decellularization.

[0028] The ratio of the SIS membrane to bone marrow-derived mesenchymal stem cells used was 1 cm. 2 : 2×10 4 -15×10 4 indivual.

[0029] The present invention provides the use of the BME-SIS biomembrane described in any of the above claims in the preparation of implantable medical materials for the treatment of rotator cuff tears.

[0030] This invention, through screening raw materials and preparation processes, provides a BME-SIS biomembrane based on SIS materials, after co-culturing with BMSCs and decellularization. This BME-SIS biomembrane exhibits excellent decellularization, avoiding the safety issues associated with xenotransplantation. The BME-SIS biomembrane possesses improved surface properties and mechanical properties. Compared to SIS materials, it significantly promotes cell adhesion, proliferation, and differentiation. Applied to a rat rotator cuff tear model, multiple aspects including molecular, histological, motor function, mechanical, and imaging studies have confirmed that the BME-SIS biomembrane of this invention significantly promotes rotator cuff tear healing, improves rat motor function, and helps reduce the risk of postoperative rotator cuff re-tear, with stable therapeutic effects. The BME-SIS biomembrane of this invention can be used as a biological repair patch, showing promising potential in rotator cuff tear healing.

[0031] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0032] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0033] Figure 1 Here are bright-field images of BMSCs under a microscope, in which... Figure 1 A is a microscopic image of P0 generation BMSCs. Figure 1B is a microscopic image of P1 generation BMSCs. Figure 1 C is a microscope image of P2 generation BMSCs. Figure 1 D is a microscopic image of P3 generation BMSCs;

[0034] Figure 2 This is a comparative observation of the entire process of co-culturing BMSCs and SIS, in which... Figure 2 A shows a gross observation of BMSCs and SIS co-cultured on day 0, in which... Figure 2 B is a gross observation image taken on day 14 of co-culture of BMSCs and SIS.

[0035] Figure 3 This image shows the results of live and dead staining of the BMSCs-SIS complex. Figure 3 A is a graph showing cell viability. Figure 3 B is a diagram showing cell nuclear staining.

[0036] Figure 4 This is a general observational comparison of BME-SIS and SIS, in which... Figure 4 A is a general observation diagram of SIS. Figure 4 B is a macroscopic view of BME-SIS;

[0037] Figure 5 The graph shows the results of DNA quantification detection of SIS, BMSCs-SIS complex, and BME-SIS.

[0038] Figure 6 These are scanning electron microscope images, in which... Figure 6 A is a scanning electron microscope image of SIS. Figure 6 B is a scanning electron microscope image of BME-SIS; the pentagram indicates that the surface of the BME-SIS fiber is coated with a uniform ECM.

[0039] Figure 7 The image shows the surface roughness results for BME-SIS and SIS. Figure 7 A shows representative atomic force microscope images of BME-SIS and SIS. Figure 7 B represents the results of the quantitative roughness analysis of BME-SIS and SIS.

[0040] Figure 8 The following are the water contact angle results for BME-SIS and SIS, where... Figure 8 A shows representative images of the water contact angles of BME-SIS and SIS. Figure 8 Figure B shows the quantitative analysis results of water contact angle for BME-SIS and SIS.

[0041] Figure 9The graph shows the mechanical property test results for BME-SIS and SIS. Figure 9 Figure A shows the quantitative analysis results of the elastic modulus of BME-SIS and SIS. Figure 9 Figure B shows the quantitative analysis results of tensile strength of BME-SIS and SIS;

[0042] Figure 10 The image shows the ELISA detection results for BME-SIS and SIS extracts. Figure 10 A shows the results of FGF-1 content detection. Figure 10 B is the graph showing the TGF-β1 content detection results. Figure 10 C represents the results of VEGF content detection;

[0043] Figure 11 Images showing the live / dead staining results of BMSCs cultured on different materials, among which... Figure 11 A shows the live / dead staining results of the control group on day 1. Figure 11 B shows the live / dead staining results of the control group on day 3. Figure 11 C shows the live / dead staining results of the control group on day 5. Figure 11 D is the live / dead staining result image of the SIS group on day 1. Figure 11 E shows the live / dead staining results of the SIS group on day 3. Figure 11 F shows the live / dead staining results of the SIS group on day 5. Figure 11 G is the live / dead staining result image of the BME-SIS group on day 1. Figure 11 H represents the live / dead staining results of the BME-SIS group on day 3. Figure 11 Image I shows the live / dead staining results of the BME-SIS group on day 5. In each image, the small image on the upper left is a green fluorescence channel image, the small image on the lower left is a red fluorescence channel image, and the large image on the right is a mixed fluorescence channel image. Red fluorescence indicates dead cells, and green fluorescence indicates live cells.

[0044] Figure 12 Experimental results of CCK-8 after treating BMSCs with different materials;

[0045] Figure 13 Figure 1 shows the results of cell scratch assays after treating BMSCs with different materials. Figure 13 A shows representative images of cell scratches at 0 h and 24 h for each group. Figure 13 B shows the results of quantitative analysis of cell migration rate in each group over 24 hours.

[0046] Figure 14 Each component of the bone differentiation gene is a bone differentiation gene, among which, Figure 14 A represents the constituent osteodifferentiation genes. RUNX2 Expression level results graph Figure 14B consists of various bone differentiation genes OSX Graph of expression level results;

[0047] Figure 15 This is a graph showing the expression levels of genes involved in chondrogenesis. Figure 15 A represents the cartilage differentiation genes of each component. ACAN Expression level results graph Figure 15 B represents the cartilage differentiation genes of each component. COMP Graph of expression level results;

[0048] Figure 16 This is a graph showing the expression levels of genes involved in tendon differentiation. Figure 16 A represents the genes responsible for tendon differentiation. TNC Expression level results graph Figure 16 B represents the genes responsible for tendon differentiation. DCN Graph of expression level results;

[0049] Figure 17 Representative gait diagrams of rats in each group at 4 and 8 weeks post-surgery;

[0050] Figure 18 The graph shows the quantitative analysis results of walking speed in each group of rats. Figure 18 Figure A shows the quantitative analysis results of walking speed in each group of rats 4 weeks after surgery. Figure 18 B shows the quantitative analysis results of walking speed in each group of rats 8 weeks after surgery;

[0051] Figure 19 The image shows the results of quantitative analysis of the maximum intensity of the right forefoot of rats in each group. Figure 19 Figure A shows the quantitative analysis results of the maximum strength of the right forefoot of rats in each group 4 weeks after surgery. Figure 19 B shows the quantitative analysis results of the maximum strength of the right forefoot of rats in each group 8 weeks after surgery;

[0052] Figure 20 The graph shows the quantitative analysis results of the maximum gripping force of the forelimbs of rats in each group. Figure 20 Figure A shows the quantitative analysis results of the maximum gripping force of the forelimbs of rats in each group 4 weeks after surgery. Figure 20 B shows the quantitative analysis results of the maximum gripping force of the forelimbs of rats in each group 8 weeks after surgery;

[0053] Figure 21 These are representative Micro-CT images of the greater tubercle footprint area of ​​rats in each group at 4 and 8 weeks post-surgery. The dashed boxes represent the tendon-bone junction of the greater tubercle footprint area.

[0054] Figure 22 The image shows the results of quantitative analysis of BV / TV at the tendon-bone junction in the greater tubercle footprint area of ​​rats in each group. Figure 22Figure A shows the quantitative analysis results of BV / TV at the tendon-bone junction in the greater tubercle footprint area of ​​rats in each group 4 weeks after surgery. Figure 22 Figure B shows the quantitative analysis results of BV / TV at the tendon-bone junction of the greater tubercle footprint area in each group of rats 8 weeks after surgery.

[0055] Figure 23 Representative MRI images of the shoulder joints of rats in each group at 4 and 8 weeks post-surgery, where arrows indicate the supraspinatus tendon;

[0056] Figure 24 The images show representative gross images of the humeral-supraspinatus complex in rats at 4 and 8 weeks post-surgery, where dashed circles indicate tendon-bone junctions.

[0057] Figure 25 The image shows the results of quantitative analysis of the ultimate load on the humeral-supraspinatus complex in each group of rats. Figure 25 Figure A shows the quantitative analysis results of the humeral-supraspinatus complex limit load in each group of rats 4 weeks after surgery. Figure 25 Figure B shows the results of quantitative analysis of the ultimate load on the humeral-supraspinatus complex in each group of rats 8 weeks after surgery.

[0058] Figure 26 The image shows the quantitative analysis results of the tensile strength of the humerus-supraspinatus muscle complex in each group of rats. Figure 26 Figure A shows the quantitative analysis results of the tensile strength of the humeral-supraspinatus muscle complex in each group of rats 4 weeks after surgery. Figure 26 Figure B shows the quantitative analysis results of the tensile strength of the humerus-supraspinatus complex in each group of rats 8 weeks after surgery.

[0059] Figure 27 The images show representative HE-stained images of the tendon-bone interface of rats in each group at 4 and 8 weeks post-surgery. The images in the second and fourth rows are magnified views of the dashed boxes in the first and third rows, respectively. B represents bone, I represents interface, and T represents tendon.

[0060] Figure 28 The graph shows the maturity score of the supraspinatus tendon in each group of rats. Figure 28 Figure A shows the results of the supraspinatus tendon maturity scores of rats in each group 4 weeks after surgery. Figure 28 B shows the results of supraspinatus tendon maturity scores in each group of rats 8 weeks after surgery.

[0061] Figure 29The images shown are representative Masson staining images of the tendon-bone interface in rats. The first row of images shows representative Masson staining images of the tendon-bone interface in rats from each group 4 weeks after surgery. The second row of images shows magnified views of the dashed box area in the first row of images. The third row of images shows representative Masson staining images of the tendon-bone interface in rats from each group 8 weeks after surgery. The fourth row of images shows magnified views of the dashed box area in the third row of images. B represents bone, I represents interface, and T represents tendon.

[0062] Figure 30 The images are representative COL-II immunohistochemical staining images of the tendon-bone interface in rats. The first row of images shows representative COL-II immunohistochemical staining images of the tendon-bone interface in rats from each group 4 weeks after surgery, and the second row shows representative COL-II immunohistochemical staining images of the tendon-bone interface in rats from each group 8 weeks after surgery. B represents bone, I represents interface, and T represents tendon.

[0063] Figure 31 The image shows the quantitative analysis results of COL-II immunohistochemical staining at the tendon-bone interface in each group of rats. Figure 31 Figure A shows the quantitative analysis results of COL-II immunohistochemical staining at the tendon-bone interface in rats of each group 4 weeks after surgery. Figure 31 B represents the quantitative analysis of COL-II immunohistochemical staining at the tendon-bone interface in rats from each group after 8 weeks. Detailed Implementation

[0064] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0065] Example 1: A BME-SIS biomembrane and its preparation method

[0066] The BME-SIS biofilm in this embodiment was prepared by the following method:

[0067] 1. Isolation and culture of BMSCs

[0068] The specific procedure for isolating BMSCs from the rat femur is as follows:

[0069] (1) Sacrifice 2-week-old SD rats and immerse them in a beaker containing povidone-iodine for 5 minutes for disinfection.

[0070] (2) Cut off both hind limbs of the rat at the hip joint with surgical scissors, disinfect in 75% ethanol for 10 minutes, and then transfer to a clean bench.

[0071] (3) Use sterile forceps to remove the lower limb, place it in sterile PBS containing 2% double antibody, remove the skin and soft tissues such as muscles of the lower limb, and free the femur for later use.

[0072] (4) Use ophthalmic scissors to cut off both ends of the femur to fully expose the medullary cavity. Draw α-MEM complete culture medium with a 1 ml syringe. Insert the needle into one end of the medullary cavity and slowly flush the medullary cavity into a 10 ml culture dish.

[0073] (5) Collect the rinsing solution, gently blow it several times, transfer the rinsing solution to a T25 culture flask, and incubate it overnight in a cell culture incubator at 37°C and 5% CO2.

[0074] (6) Replace half of the α-MEM complete medium after 24 hours. Thereafter, change the medium every two days, and passage the cells after they have reached 80% confluence.

[0075] (7) BMSCs were successfully isolated from the rat femur by microscopy. Figure 1 BMSCs from generation P0 to P3 all adhered to the culture wall and grew, which is consistent with the growth characteristics of stem cells.

[0076] 2. Preparation of SIS

[0077] (1) Obtain fresh jejunum from pigs that died within 3 hours of slaughter.

[0078] (2) Cut the jejunum into segments about 15 cm long and cut them longitudinally, then wash them thoroughly with PBS.

[0079] (3) Mechanically scrape away the serosa, muscle and mucosa of the intestinal segment, while preserving the submucosa.

[0080] (4) Soak the submucosal tissue in a mixture of methanol and chloroform (volume ratio 1:1) overnight for defatting.

[0081] (5) Rinse three times with deionized water and soak in 0.25% trypsin solution overnight at 4°C.

[0082] (6) Rinse three times with deionized water and soak it overnight in a 0.5% sodium dodecyl sulfate solution.

[0083] (7) Rinse with deionized water 3 times and freeze dry for 24 hours.

[0084] The obtained SIS materials, such as Figure 3 As shown, it is a white, semi-transparent film visible to the naked eye.

[0085] In other embodiments, deionized water may be used to rinse 4-5 times.

[0086] 3. Co-culture of BMSCs and SIS

[0087] (1) Cut the SIS into round pieces with a diameter of 1.5 cm, sterilize with ethylene oxide at 37°C and set aside.

[0088] (2) Use sterile forceps to place the SIS disc in the center of the six-well plate so that the SIS disc is in close contact with the bottom of the well.

[0089] (3) Take healthy P3 generation BMSCs and feed them with 5 × 10⁻⁶ cells / mL. 4 pcs / cm 2 The concentration was uniformly inoculated onto the SIS surface.

[0090] (4) Transfer the six-well plate to a 37°C, 5% CO2 cell culture incubator and incubate it as day 0. Add 2 ml of complete culture medium to each well and change the culture medium every 2 days.

[0091] (5) On day 6 of culture, the culture medium was replaced with a complete culture medium containing vitamin C (50 μg / ml). The medium was then changed every 2 days thereafter, and the culture was continued for 8 days to obtain the BMSCs-SIS complex.

[0092] BMSCs were inoculated onto the surface of SIS and cultured. On day 0, SIS was observed spreading at the bottom of the well plate. Figure 2 A). After 14 days of culture, SIS showed signs of shrinkage ( Figure 2 B), which is related to the large-scale growth of BMSCs on SIS. After 14 days of culture, live / dead staining showed that a large number of BMSCs survived on SIS, with only a very small number of dead cells (BMSCs). Figure 3 The above results demonstrate that the BMSCs-SIS complex was successfully prepared in this embodiment.

[0093] 4. Preparation of BME-SIS

[0094] (1) Preparation of working solution A: Add 200 μL of concentrated ammonia to every 50 ml of 0.5% Triton X-100 solution, mix thoroughly and set aside.

[0095] (2) Place the BMSCs-SIS complex in a six-well plate, one plate per well, add 2 ml of working solution A per well, and treat in a shaker at 37°C (60 rpm) for 30 min.

[0096] (3) Remove working solution A, add 2 ml of deionized water to each well, place on a shaker at 37°C (60 rpm) and wash for 5 minutes. Repeat washing 10 times.

[0097] (4) Prepare DNase I working solution: Dissolve DNase I in calcium and magnesium PBS to a concentration of 100 U / ml for later use.

[0098] (5) Remove deionized water, slowly add 1.5 ml of DNase I working solution to each well, and treat in a shaker at 37°C (60 rpm) for 3 hours.

[0099] (6) Remove the DNase I working solution, add 2 ml PBS to each well, and wash at 37°C on a shaker (60 rpm) for 5 minutes. Repeat the washing 10 times.

[0100] (7) Remove the PBS solution and store the six-well plate in a -80°C freezer for 24 hours.

[0101] (8) Take it out of the -80℃ freezer, put it into the freeze dryer, freeze dry for 24 hours to obtain BME-SIS.

[0102] Observations show that, compared to SIS, BME-SIS has an additional layer of white powdery substance on its surface. Figure 4 ).

[0103] The technical solution of the present invention will be further illustrated by the following experiments. The SIS, BMSCs-SIS and BME-SIS samples tested in the following experimental examples were all prepared by the method of Example 1.

[0104] Experimental Example 1: Material Characterization

[0105] I. Experimental Methods

[0106] 1. DNA content determination

[0107] This experiment used a commercially available genome extraction kit to extract total DNA from the samples. The procedure is as follows:

[0108] (1) Take SIS, BMSCs-SIS or BME-SIS and measure the sample weight with a balance;

[0109] (2) Load the samples into EP tubes, add 200 μL of GTL to each tube, and shake thoroughly;

[0110] (3) Add 20 μL Proteinase K and 200 μL Buffer GL to each tube, vortex to mix, and incubate in a water bath at 56°C for 10 minutes;

[0111] The extracted DNA solution was then collected according to the instructions; the DNA concentration was detected using a Nanodrop spectrophotometer, and the DNA content per milligram of sample was calculated.

[0112] 2. Surface morphology and surface roughness

[0113] The surface morphology of the samples was examined using scanning electron microscopy (SEM). The samples were sputter-coated with gold for 300 seconds using a double electron scanning mode with an accelerating voltage of 30 kV. Multiple image acquisitions were performed on different areas of the samples at a magnification of 800x. The surface roughness of the samples was then measured using atomic force microscopy (AFM). A contact scanning mode was used, with a scanning range of 5 μm × 5 μm. Three randomly selected, far apart areas on the sample surface were measured, taking care to avoid the sample edges. The acquired images were processed using the AFM software to calculate the average roughness (Ra).

[0114] 3. Surface hydrophilicity

[0115] SIS and BME-SIS samples were cut into 1cm×1cm pieces. 2 μL of deionized water was automatically dropped onto the sample surface. The droplet was released from a height of 5 mm above the sample surface. The hydrophilicity of the sample surface was detected using an optical contact angle meter. The droplet contour image was captured by a side-view optical system. The static contact angle was calculated using the circle fitting method with the software provided with the equipment.

[0116] II. Experimental Results

[0117] 1. DNA content detection results

[0118] The DNA content of SIS was 29.30 ± 2.25 ng / mg ( Figure 5 The DNA content of the BMSCs-SIS complex was 176.27 ± 3.75 ng / mg, significantly higher than that of the SIS complex (50 ng / mg), indicating that the SIS decellularization effect was good. P <0.0001, Figure 5 This indicates that the present invention successfully prepared a SIS loaded with a large amount of BMSCs, namely the BMSCs-SIS complex. The DNA content of the BME-SIS was 16.12 ± 0.48 ng / mg, which is lower than the international standard (50 ng / mg) and significantly lower than that of the BMSCs-SIS complex. P <0.0001, Figure 5 This further demonstrates that BME-SIS has a good decellularization effect.

[0119] 2. Surface morphology

[0120] Scanning electron microscopy results ( Figure 6 The results showed that no cell residues were observed in either SIS or BME-SIS, and both exhibited a three-dimensional network structure with interwoven and overlapping fibers. The difference was that the surface of SIS fibers was smooth, while the surface of BME-SIS fibers formed a coating-like structure; specifically, in sparse areas of BME-SIS fibers, a uniform layer of ECM (indicated by yellow pentagrams) was visible on the fiber surface.

[0121] 3. Surface roughness

[0122] Atomic force microscopy results ( Figure 7 The results show that the arithmetic mean roughness (Ra) of BME-SIS is significantly higher than that of SIS. P <0.05).

[0123] 4. Surface hydrophilicity

[0124] The results showed that the water contact angles of BME-SIS and SIS were 54.90±4.40° and 67.63±3.45°, respectively, with the water contact angle of BME-SIS being significantly smaller than that of SIS. P <0.01, Figure 8 This indicates that the hydrophilicity of BME-SIS is significantly improved compared to SIS.

[0125] Good decellularization effect helps reduce adverse reactions of xenogeneic / allogeneic decellularized biomaterials in vivo; changes in the surface morphology, roughness and hydrophilicity of BME-modified SIS materials will affect cell biological functions.

[0126] Experimental Example 2: Mechanical Property Analysis

[0127] I. Experimental Methods

[0128] This experiment uses an electronic universal testing machine to test the mechanical properties of the samples, including the elastic modulus and tensile strength. The specific procedure is as follows:

[0129] (1) Cut SIS and BME-SIS into 1cm×2cm samples, and use vernier calipers to measure the width and thickness of the samples to calculate the cross-sectional area;

[0130] (2) Use transparent tape to fix the sample to the clamp so that the tension is transmitted to the inside of the sample and to prevent slippage;

[0131] (3) Perform uniaxial tensile tests at a rate of 10 mm / min until the sample breaks. The elastic modulus and tensile strength are directly exported from the software.

[0132] II. Experimental Results

[0133] The test results showed that the elastic modulus of BME-SIS was 52.97±14.40 MPa, and the tensile strength was 10.89±2.81 MPa; the elastic modulus of SIS was 141.35±16.74 MPa, and the tensile strength was 12.56±2.69 MPa. The elastic modulus of BME-SIS was significantly lower than that of SIS. P <0.001, Figure 9 The tensile strength of BME-SIS tends to decrease compared to SIS.

[0134] Compared to SIS, the changes in the mechanical properties of BME-SIS materials will significantly affect the material's biological functions in cells, such as cell differentiation.

[0135] Experimental Example 3: Bioactive Components of BME-SIS

[0136] TGF-β1 (transforming growth factor-β1), FGF-1 (fibroblast growth factor-1), and VEGF (vascular endothelial growth factor) are all important cytokines that play a key role in cell growth, differentiation, migration, and tissue repair.

[0137] I. Experimental Methods

[0138] This experiment used enzyme-linked immunosorbent assay (ELISA) to detect the contents of TGF-β1, FGF-1, and VEGF in the material extract. The specific procedure is as follows:

[0139] (1) Preparation of extract: Take 4 portions of SIS and 4 portions of BME-SIS, and soak them in α-MEM medium respectively (material surface area: medium volume = 6 cm³). 2 (1 ml), shaker at 37℃ and 60 rpm for 72 hours.

[0140] (2) Diluting the standard: Dilute the standard with diluent to the following concentrations: 0 pg / mL, 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL.

[0141] (3) Sample addition: Set up blank control wells, standard wells and sample wells. The blank control wells are the same as those without sample, biotin-labeled antibody and enzyme-labeled reagent. Add 50 μL of different concentrations of standard to the standard wells in sequence; add 40 μL of sample to the sample wells first, and then add 10 μL of biotin-labeled antibody.

[0142] (4) Enzyme addition and color development: Except for the blank control wells, 50 μL of enzyme-labeled reagent was added to each well. After sealing with sealing film, the plate was incubated at 37°C for 30 minutes. After washing with washing buffer, 50 μL of colorimetric reagent A and 50 μL of colorimetric reagent B were added to each well. The plates were gently shaken to mix and incubated at 37°C in the dark for 20 minutes. The reaction was terminated by adding stop solution. The absorbance (OD value) of each well was measured at 450 nm using the blank control well as the zeroing point.

[0143] (5) Calculation of experimental results: The quadratic regression equation of the standard curve is calculated with the OD value of the standard as the x-axis and the concentration as the y-axis. The OD value of the sample is substituted into the equation to calculate the actual concentration of the sample.

[0144] II. Experimental Results

[0145] ELISA results showed that the contents of FGF-1 and TGF-β1 in the BME-SIS extract were significantly higher than those in the SIS extract. Figure 10 A, P <0.01; Figure 10 B, P <0.05; The VEGF content of BME-SIS extract tended to increase compared with SIS extract, but the difference was not statistically significant. Figure 10 C, P >0.05). This indicates that BME-SIS significantly increases the release of cytokines such as FGF-1 and TGF-β1.

[0146] Experiment Example 4: Effects of BME-SIS biomembrane on cell proliferation and migration

[0147] I. Experimental Methods

[0148] 1. Experimental Grouping

[0149] This experiment consisted of three groups: a control group, a SIS group, and a BME-SIS group. The SIS group and the BME-SIS group used extracts of SIS and BME-SIS biofilms, respectively, while the control group used α-MEM complete culture medium.

[0150] 2. Preparation of extracts for each group

[0151] Control group: α-MEM complete medium (containing 1% antibiotics and 10% FBS) was placed in a shaker at 37℃ and 60 rpm for 72 hours. SIS group and BME-SIS group: SIS and BME-SIS samples were immersed in α-MEM complete medium (material surface area: medium volume = 6 cm²). 2 (1 ml), and incubate at 37°C and 60 rpm for 72 hours. Filter each extract separately for later use. In the following cell scratch assays, the α-MEM complete medium was replaced with α-MEM low-serum complete medium.

[0152] 3. Live and dead staining

[0153] To evaluate the effect of BME-SIS on BMSC proliferation, a live / dead staining experiment was conducted. This experiment included three time points: Day 1, Day 3, and Day 5. The details are as follows:

[0154] Take healthy P3 generation BMSCs, resuspend the cells in α-MEM complete medium, and adjust the cell concentration to 5 × 10⁻⁶. 4Cells / ml. Three 24-well plates were seeded at 5000 cells / well and incubated at 37°C with 5% CO2 for 24 hours. The old culture medium for each group was aspirated, and 2 ml of the corresponding extraction buffer was added to each well for intervention. The plates were then incubated at 37°C with 5% CO2 for 24 hours. For Day 3 and Day 5 groups, the extraction buffer was changed every two days, continuing until Day 3 and Day 5, respectively. Cells were stained with Calcein-AM and PI, and observed under an inverted fluorescence microscope.

[0155] 4. CCK-8 Experiment

[0156] To evaluate the effect of BME-SIS on BMSC proliferation, a CCK-8 assay was conducted, with three time points: Day 1, Day 3, and Day 5. Details are as follows:

[0157] Take healthy P3 generation BMSCs, resuspend the cells in α-MEM complete medium, and adjust the cell concentration to 2×10⁻⁶. 4 Cells / ml. Add 100 μL of cell suspension to each well of a 96-well plate and incubate at 37°C with 5% CO2 for 24 hours. Discard the old culture medium and add 200 μL of extraction buffer to each well for intervention. Incubate at 37°C with 5% CO2 for 24 hours. Co-incubate with CCK-8 solution at 37°C with 5% CO2 for 1 hour. Measure the absorbance (OD) at 450 nm using a multi-mode microplate reader. Use the mean OD value of the blank control group as the calibration value. The actual OD value of each well = OD value of each well - calibration value.

[0158] 5. Cell scratch test

[0159] To evaluate the effect of BME-SIS on the migration ability of BMSCs, a cell scratch assay was performed. This experiment included two time points: 0 h and 24 h. Details are as follows:

[0160] Take healthy P3 generation BMSCs, resuspend them in α-MEM complete medium, and adjust the cell concentration to 5 × 10⁻⁶. 5 Cells / ml. Add 1 ml of α-MEM complete culture medium to each well, followed by 1 ml of cell suspension. Incubate the six-well plate at 37°C with 5% CO2 for 24 hours. When cell confluence reaches 100%, perform a scratch test using a 200 μL sterile pipette tip, and wash twice with PBS to remove any detached cells. Add 2 ml of the appropriate extraction solution to each well for intervention, and immediately acquire scratch images at the 0 h time point under a microscope. After image acquisition, incubate the six-well plate at 37°C with 5% CO2 for 24 hours, and acquire scratch images at the 24 h time point under a microscope. Analyze the scratch area and calculate the migration rate using ImageJ software.

[0161] II. Experimental Results

[0162] 1. Effects on cell proliferation

[0163] Live and dead cell staining results showed that the number of live cells in the control group, SIS group, and BME-SIS group all increased over time; at 1 day of culture, there was no significant difference in the number of live cells among the three groups; at 3 and 5 days of culture, the number of live cells in the BME-SIS group was significantly higher than that in the SIS group and the Control group, and the number of live cells in the SIS group was significantly higher than that in the Control group; at 5 days of culture, there were no obvious dead cells in any of the three groups. Figure 11 The above results indicate that SIS and BME-SIS cells have good cell compatibility and both promote the proliferation of BMSCs.

[0164] The CCK-8 experiment results were consistent with the live-dead staining results: the absorbance values ​​of the control group, SIS group, and BME-SIS group all increased over time; at day 1 of culture, there was no statistically significant difference in absorbance among the three groups; at days 3 and 5 of culture, the absorbance of the BME-SIS group was significantly higher than that of the SIS group and the Control group, and the absorbance value of the SIS group was significantly higher than that of the Control group. Figure 12 The results showed that BME-SIS significantly enhanced the proliferation-promoting effect compared to SIS.

[0165] 2. Effects on cell migration

[0166] Cell scratch assay results showed that the scratch area in the control group, SIS group, and BME-SIS group was smaller than the initial scratch area after 24 h. Figure 13 A); Quantitative analysis of cell migration rate showed that the BME-SIS group was significantly higher than the SIS group and the control group, and the SIS group was significantly higher than the control group (A); Figure 13 B). These results indicate that both SIS and BME-SIS can promote BMSC migration, with BME-SIS showing a significantly greater promoting effect than SIS.

[0167] The results of this experiment show that BME-SIS can significantly promote the proliferation and migration of BMSCs, and its effect on promoting proliferation and migration is significantly higher than that of SIS.

[0168] Experimental Example 5: Effect of BME-SIS on BMSC Differentiation

[0169] I. Experimental Methods

[0170] 1. Experimental Grouping

[0171] This experiment was divided into three groups: control group, SIS group, and BME-SIS group.

[0172] 2. RT-PCR

[0173] Take healthy P3 generation BMSCs, resuspend the cells in α-MEM complete medium, and adjust the concentration to 5 × 10⁻⁶. 4 Cells / ml. 2 ml of cell suspension was added to the surface of each group of materials. Cells were cultured at 37℃ in a 5% CO2 incubator for 7 days, with the culture medium changed every 2 days. After 7 days of culture, RT-PCR was performed to detect osteogenic (…). RUNX2 , OSX ), cartilage ( ACAN , COMP ) and tendon ( TNC , DCN The relative expression levels of relevant genes were determined. RNA was extracted from each group using the Total RNA Isolation Kit, reverse transcription was performed using HiScript III All-in-one RT SuperMix Perfect for qPCR, and PCR was performed using Taq Pro Universal SYBR qPCR Master Mix. Each gene in each group was tested in triplicate, and the relative expression levels were calculated based on the Ct value. -△△Ct The value is used to determine the relative expression level of mRNA.

[0174] II. Experimental Results

[0175] 1. Effects of BME-SIS on osteogenic differentiation of BMSCs

[0176] about RUNX2 The expression level in the BME-SIS group was significantly higher than that in the SIS group and the control group. Figure 14 A); Regarding OSX The expression levels in both the SIS group and the BME-SIS group were significantly higher than those in the control group. Figure 14 B).

[0177] 2. Effects of BME-SIS on chondrogenic differentiation of BMSCs

[0178] about ACAN and COMP Expression levels were significantly higher in the BME-SIS group than in the SIS group and the control group, with the SIS group showing a significantly higher level than the control group. Figure 15 ).

[0179] 3. Effects of BME-SIS on tendinogenic differentiation of BMSCs

[0180] about TNC The expression level in the BME-SIS group was significantly higher than that in the SIS group and the control group, and the expression level in the SIS group was significantly higher than that in the control group. Figure 16 A); Regarding DCNThe expression level in the SIS group was significantly higher than that in the control group and the BME-SIS group, and the BME-SIS group was significantly higher than that in the control group. Figure 16 B).

[0181] The above results indicate that BME-SIS significantly promotes osteogenic, chondrogenic, and tendonogenic differentiation of BMSCs, and its effect is superior to that of SIS.

[0182] Experimental Example 6: Application of BME-SIS biomembrane in in vivo repair of rotator cuff tears

[0183] I. Experimental Methods

[0184] 1. Animal experimental grouping

[0185] This experiment was approved by the Laboratory Animal Ethics Committee of West China Hospital, Sichuan University, and all experimental procedures strictly adhered to animal ethics regulations. A total of 52 eight-week-old male SD rats (104 shoulders) were randomly divided into four groups: Sham group (sham surgery group; only the supraspinatus tendon was exposed without severing), Control group (supraspinatus tendon severing followed by simple repair surgery), SIS group (supraspinatus tendon severing followed by repair surgery + SIS implantation), and BME-SIS group (supraspinatus tendon severing followed by repair surgery + BME-SIS implantation). Gait, grip strength, imaging, biomechanics, and histological examinations were performed at 4 and 8 weeks post-surgery. 2. Animal Model Establishment

[0186] The procedure for establishing a rat model of bilateral acute rotator cuff tear is as follows:

[0187] (1) Preoperative preparation: Rats were anesthetized by inhalation of isoflurane. After satisfactory anesthesia, they were placed in a supine position, the skin was prepared, and the rats were disinfected with povidone-iodine three times and draped. The sterile SIS / BME-SIS disc was divided in half along the diameter (for implantation in both shoulders). The semi-circular material was folded twice along the radius to form a fan shape (with the ECM loading side facing outward) for subsequent implantation between the tendon and bone on one side.

[0188] (2) Starting from the proximal end of the humerus, make an incision of about 2 cm in length along the supraspinatus muscle on the anterolateral side of the shoulder joint, and cut the skin and subcutaneous tissue layer by layer, bluntly dissecting the muscle. Slightly adduct, extend and internally rotate the rat's shoulder joint to facilitate exposure of the supraspinatus tendon, and carefully identify its attachment point on the greater tubercle of the humerus. After suturing the tissue layer by layer in the Sham group, disinfect the incision and proceed to step (6). The other three groups performed all subsequent steps.

[0189] (3) Gently lift the supraspinatus tendon with a bone aspiration needle, and then fix the supraspinatus tendon with 5-0 suture. Completely transection the supraspinatus tendon at the insertion point of the greater tubercle. Carefully clean the remaining supraspinatus tendon and fibrocartilage in the footprint area of ​​the humeral head with a burr, and perform mild decorticization.

[0190] (4) Using a 1 mm drill perpendicular to the direction of the supraspinatus muscle, create a bone tunnel in the humeral head from anterior to posterior. Pass a 5-0 absorbable suture through the bone tunnel and then through the end of the supraspinatus tendon (for the SIS / BME-SIS group, it is also necessary to pass through a fan-shaped SIS / BME-SIS). Tighten the suture to reposition the supraspinatus tendon insertion point to the footprint area, and then tie a knot while maintaining the tension of the supraspinatus muscle to securely suture the supraspinatus tendon to the footprint area (for the SIS / BME-SIS group, ensure that the material is accurately placed between the tendon and bone).

[0191] (5) Remove excess sutures, suture the tissue layer by layer, and disinfect the incision.

[0192] (6) After the rats regained consciousness, they were given an intramuscular injection of 400,000 units of penicillin to prevent infection, once a day for three days. After the operation, the rats were allowed to move freely and eat and drink, and the local wound and overall activity were closely observed.

[0193] 3. Gait analysis

[0194] This experiment used the VisuGait animal gait analysis system and its accompanying software. A high-speed camera captured the spontaneous movement trajectory of rats within a closed glass track, simultaneously recording information such as plantar pressure distribution. Qualitative analysis of the rats' gait sequence was performed, and quantitative analysis of walking speed and maximum intensity in the right forefoot was conducted. The procedure is as follows:

[0195] (1) Before the formal test, the rats were subjected to track adaptation training for 3 consecutive days (twice a day, 5 minutes each time) to eliminate environmental stress interference and enable them to quickly pass through the entire glass track.

[0196] (2) During the formal test, each rat completed 3 effective walking tests (walking speed difference <15%), and the average value of each index was used for statistical analysis. Note that the test should be conducted in a quiet and dark environment to eliminate external interference.

[0197] 4. Forelimb grip strength test

[0198] This experiment used a rat and mouse grip force measuring instrument to detect forelimb grip force. This instrument is equipped with a rat-specific horizontal grip bar and a high-precision force sensor, which can record peak grip force data in real time.

[0199] (1) Adaptation training: Three days before the formal test, the rats were trained to grasp the horizontal bar naturally with their forelimbs for 5 minutes each time. The rats were trained to use their forelimbs to grasp the bar naturally, avoiding interference from the hindlimbs.

[0200] (2) Testing environment: The testing is conducted in a quiet room, and the testing time is fixed from 9:00 am to 12:00 pm.

[0201] (3) Instrument calibration: Calibrate with a 50 g standard weight before formal testing.

[0202] (4) During the formal test, each rat completed 3 effective grasps with a 2-minute rest interval. The main indicator was the maximum grasping force, defined as the peak grasping force recorded by the sensor during the grasping process.

[0203] 5. Micro-CT Experiment

[0204] This experiment used micro-CT scans of small animals to examine the bone quality of the greater tuberosity footprint area of ​​the rat humerus, with the main indicator being bone volume fraction (BV / TV). The specific procedure is as follows:

[0205] (1) Rats were anesthetized by inhalation of isoflurane. After satisfactory anesthesia, the rats were placed in a prone position in the scanning chamber.

[0206] (2) Scanned with Cruiser software, pixel size 0.050 mm, slice thickness 0.058 mm.

[0207] (3) Reconmd software is used for three-dimensional reconstruction of the image, with FDK algorithm and resolution of 2k×2k.

[0208] (4) Randomly select a cylinder with a bottom diameter of 1 mm and a height of 1 mm as the region of interest (ROI) in the footprint area of ​​the greater tubercle of the humerus, and measure the BV / TV.

[0209] 6. MRI Experiment

[0210] This experiment used a small animal-specific MRI system to evaluate the tissue integrity of the supraspinatus tendon in rats. Before the formal scan, rats were euthanized, and their forelimbs were wrapped with tape to maintain slight adduction and internal rotation of both shoulder joints. The rats were placed in a prone position, head first, on a dedicated MRI animal bed. During the formal scan, coronal T2-weighted imaging was used, and images were processed using RadiantViewer 2023 software.

[0211] 7. Obtain materials

[0212] In this experiment, the rat humerus-supraspinatus complex was subjected to biomechanical and histological examinations. The complete upper arm-scapula complex was isolated, and only the humerus-supraspinatus complex was retained.

[0213] 8. Biomechanical testing

[0214] This experiment used an electronic universal testing machine to test the mechanical properties of the humerus-supraspinatus complex to evaluate the strength of tendon-bone healing. The procedure was as follows: the thickness and width of the supraspinatus tendon insertion point were measured using vernier calipers, and the cross-sectional area was calculated; the humeral shaft and supraspinatus muscle were placed in the instrument fixtures, keeping the long axis of the humerus and the long axis of the supraspinatus muscle in the same straight line; a tension of 0.1 N was pre-loaded on the specimen, followed by a uniaxial tensile test at a rate of 10 mm / min until the humerus-supraspinatus complex fractured at the insertion point; the ultimate load and tensile strength of the humerus-supraspinatus complex in each group were recorded and statistically analyzed.

[0215] 9. Tissue staining

[0216] Fresh humeral-supraspinatus complex was placed in 4% paraformaldehyde and fixed at room temperature for 48 hours. After decalcification and trimming, paraffin sections were prepared for HE, Masson and immunohistochemical staining.

[0217] 10. Histological score

[0218] In this experiment, a histologist scored the HE staining results using a modified supraspinatus tendon maturity scoring system (Table 1), without informing the patient of the section grouping and time points.

[0219] Table 1. Supraspinatus Tendon Maturity Scoring System

[0220]

[0221] Note: C: Continuity; R: Regularity; F: Fibrocartilage; T: Tidal line; +: Positive.

[0222] 11. Statistical Analysis

[0223] In this invention, all experiments were blinded by the statisticians, who were unaware of the experimental groups and time points. SPSS 25.0 software was used for data analysis. If the measurement data conformed to a normal distribution, it was expressed as mean ± standard deviation; if it did not conform to a normal distribution, it was expressed as median and interquartile range. If two groups of measurement data conformed to a normal distribution and had homogeneous variances, a two-sample t-test was used for comparison between the two groups; if they did not conform to a normal distribution or had unequal variances, a Wilcoxon rank-sum test was used for comparison between the two groups. Comparisons of multiple groups of measurement data were first performed using one-way ANOVA (CCK-8 used two-way ANOVA), followed by LSD-t test for multiple comparisons. P <0.05 indicates a statistically significant difference; * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001, **** indicates P<0.0001.

[0224] II. Experimental Results

[0225] 1. Step sequence analysis

[0226] Through step sequence diagram ( Figure 17 It can be clearly seen that: 4 weeks after the operation, the rats in the sham-operated group had no abnormal gait; the rats in the control group and SIS group had more abnormal gait, and the overall gait stability and regularity were poor, indicating poor motor coordination; the rats in the BME-SIS group had fewer abnormal gait, indicating that the motor coordination of the rats in the BME-SIS group was better than that of the control group and SIS group, but worse than that of the sham-operated group 4 weeks after the operation.

[0227] Eight weeks post-surgery, the gait pattern of rats in the sham-operated group was similar to that at four weeks post-surgery, with good overall motor coordination. The gait patterns of the other three groups improved at eight weeks post-surgery compared to four weeks post-surgery: the control group showed fewer abnormal gaits, but its overall gait pattern stability and regularity were poor, indicating continued poor motor coordination; the SIS group showed no abnormal gaits, but its overall gait pattern stability and regularity were poor, indicating that at eight weeks post-surgery, the SIS group's motor coordination was better than the control group, but worse than the sham-operated group and the BME-SIS group; the BME-SIS group showed no abnormal gaits, and its overall gait pattern stability and regularity were good, indicating that at eight weeks post-surgery, the BME-SIS group's motor coordination was no significantly different from the sham-operated group.

[0228] 2. Quantitative Gait Analysis

[0229] (1) Walking speed

[0230] Walking speed refers to the distance a rat walks per unit time, and its value is positively correlated with the overall motor ability of the rat. Results showed that 4 weeks post-surgery, the walking speed of the sham-operated group was significantly higher than that of the control group, the SIS group, and the BME-SIS group, while the differences among the other three groups were not significant. Figure 18 A); At 8 weeks post-operation, walking speed increased in all groups compared to 4 weeks post-operation. The sham surgery group was still significantly faster than the other three groups. The BME-SIS group was significantly faster than the control group and the SIS group. There was no statistically significant difference between the control group and the SIS group. Figure 18 B).

[0231] (2) Maximum strength of the forefoot

[0232] Maximum forefoot strength refers to the maximum pressure generated by the forefoot during landing, and its value is positively correlated with forefoot motor function and weight-bearing capacity. Results showed that at 4 weeks post-surgery, the maximum forefoot strength in the control group was significantly lower than that in the sham-operated group. The SIS and BME-SIS groups showed a decreasing trend compared to the sham-operated group, but the differences were not statistically significant. There were no statistically significant differences among the control group, SIS group, and BME-SIS group. Figure 19A). At 8 weeks post-operation, the maximum strength of the right forefoot in all groups increased compared to 4 weeks post-operation, with the control group showing a significant increase compared to the sham-operated group, SIS group, and BME-SIS group. There were no statistically significant differences among the other three groups. Figure 19 B).

[0233] This experiment used walking speed and forefoot maximum strength to quantitatively evaluate overall movement, gait posture, and limb control in rats, which were positively correlated with shoulder joint function. Gait analysis results showed that at 4 and 8 weeks post-surgery, SIS did not significantly improve walking speed and forefoot stride length; only at 8 weeks post-surgery did SIS significantly improve forefoot maximum strength. In contrast, at 8 weeks post-surgery, BME-SIS significantly improved walking speed and forefoot maximum strength in rats. Overall, this experiment found that BME-SIS has a greater advantage in improving gait in rats compared to SIS.

[0234] 3. Forelimb grip strength test

[0235] Maximum forelimb grip strength in rats was positively correlated with shoulder joint function. Results showed that at 4 weeks post-surgery, the maximum forelimb grip strength in the control group was significantly lower than that in the sham-operated group and the BME-SIS group, and slightly lower than that in the SIS group, but the difference was not statistically significant. The SIS group was significantly lower than that in the sham-operated group, and showed a decreasing trend compared to the BME-SIS group. There was no statistically significant difference between the BME-SIS group and the sham-operated group. Figure 20 A). At 8 weeks post-surgery, the maximum forelimb grip strength in all groups was higher than at 4 weeks post-surgery, but the trend of differences among the four groups did not change significantly compared to 4 weeks post-surgery. Figure 20 B).

[0236] 4. Imaging examination

[0237] (1) Micro-CT

[0238] Results showed that 4 weeks post-surgery, the tendon-bone junction of the greater tuberosity footprint area in the sham surgery group ( Figure 21 The bone cortex (shown in the yellow dashed box) showed good continuity, thicker cortical bone, and denser subchondral bone. In the control group, SIS group, and BME-SIS group, the bone cortex at the tendon-bone junction was damaged to varying degrees, exhibited poor continuity, thinner cortical bone, and varying degrees of osteoporosis in the subchondral bone.

[0239] At 8 weeks post-operation, there was no significant difference in bone quality between the sham surgery group and the 4 weeks post-operation. The other three groups showed improvement compared to 4 weeks post-operation, characterized by restoration of cortical continuity, cortical bone thickening, and subchondral bone regeneration. However, the subchondral bone quality in the control group and the SIS group was more porous than that in the BME-SIS group.

[0240] Bone volume analysis results showed that at 4 weeks post-surgery, the bone volume fraction (BV / TV) at the tendon-bone junction in the greater tuberosity footprint area was significantly lower in the control group than in the SIS and BME-SIS groups. The BME-SIS group showed a trend towards higher values ​​compared to the SIS group. Figure 22 A). At 8 weeks post-operation, the bone-tendon junction volume (BV) / tube size (TV) at all groups was higher than at 4 weeks post-operation. The sham surgery group remained significantly higher than other groups, while the control group remained significantly lower than the SIS and BME-SIS groups. The BME-SIS group was significantly higher than the SIS group. Figure 22 B).

[0241] Bone volume fraction (BV / TV) is defined as the proportion of skeletal tissue in a specific volume of bone tissue sample, and it is positively correlated with bone strength and load-bearing capacity. Our results showed that at 4 and 8 weeks post-surgery, both SIS and BME-SIS significantly increased the BV / TV in the humeral head footprint region of rats, with BME-SIS showing a superior effect compared to SIS, indicating that BME-SIS can improve bone quality in the humeral head footprint region.

[0242] (2) MRI

[0243] The results showed that at 4 weeks post-operation, the supraspinatus tendon in the sham-operated group exhibited uniform low signal intensity, regular cord-like shape, clear and distinguishable boundaries, and excellent continuity. The supraspinatus tendons in the control and SIS groups showed internal high signal intensity, irregular shape, and less clear boundaries, with some tendons exhibiting poor continuity. The supraspinatus tendon in the BME-SIS group showed increased thickness, internal mixed high signal intensity, relatively regular shape, clearer boundaries, and acceptable continuity. At 8 weeks post-operation, there was no significant difference in the supraspinatus tendon quality in the sham-operated group compared to 4 weeks post-operation. The tendon quality in the other three groups improved compared to 4 weeks post-operation. Specifically, the supraspinatus tendon in the control group showed improved signal intensity and continuity, but the tendon shape was thin and the boundaries were still unclear. The supraspinatus tendon in the SIS group showed improved continuity, increased thickness, and clearer boundaries, but internal mixed high signal intensity, still significantly different from the control group. The supraspinatus tendon in the BME-SIS group showed further improved continuity, uniform low signal intensity internally, further shaped shape, and clear boundaries, and was generally similar to the tendon in the control group. Figure 23 ).

[0244] MRI results at 8 weeks post-operation showed that: in the sham-operated group, the supraspinatus tendon was classified as type I, representing normal supraspinatus tendon quality; in the BME-SIS group, the supraspinatus tendon was classified as type II, representing near-normal supraspinatus tendon quality; and in the control group and SIS group, the supraspinatus tendon was classified as type III to IV, representing poor supraspinatus tendon quality.

[0245] Imaging studies in this study suggest that BME-SIS can significantly promote the healing of rotator cuff tears.

[0246] 5. Results of material collection

[0247] In this experiment, samples were taken at 4 and 8 weeks post-surgery for relevant testing. Prior to euthanasia, no significant limitation of shoulder joint movement due to tissue adhesion was observed in any group of rats. During sample collection, no significant infection or heterotopic ossification was observed at the surgical site in any group of rats. No implant residue was observed in either the SIS or BME-SIS groups, indicating that the implants had biodegraded.

[0248] The gross differences in the humeral-supraspinatus complex among the groups of rats are as follows: At 4 weeks post-operation, the supraspinatus tendon in the sham-operated group was silvery-white, dense, and had a smaller cross-sectional area. There were no significant differences in the supraspinatus tendons of the control, SIS, and BME-SIS groups; their texture resembled adipose tissue, and their cross-sectional areas were all larger than those of the sham-operated group. At 8 weeks post-operation, the supraspinatus tendon in the sham-operated group showed no significant difference compared to 4 weeks post-operation. The supraspinatus tendons in the control and SIS groups showed no significant improvement compared to 4 weeks post-operation. The supraspinatus tendon in the BME-SIS group showed significant improvement compared to 4 weeks post-operation, exhibiting further reshaping and an appearance similar to the sham-operated group. Figure 24 ).

[0249] 6. Biomechanical test results

[0250] (1) Limit load of humeral-supraspinatus complex

[0251] The results showed that at 4 weeks post-operation, the sham surgery group had a significantly higher maximum workload than the control group and the SIS group, but no statistically significant difference compared to the BME-SIS group. The control group had significantly lower maximum workloads than the SIS group and the BME-SIS group, while there was no statistically significant difference between the SIS group and the BME-SIS group. Figure 25 A). At 8 weeks post-operation, the maximum workload in all groups increased compared to 4 weeks post-operation. The sham surgery group remained significantly higher than the control group and the SIS group, but there was no statistically significant difference between the sham surgery group and the BME-SIS group. The BME-SIS group was significantly higher than the SIS group ( Figure 25 B).

[0252] (2) Tensile strength of the humerus-supraspinatus complex

[0253] The results showed that at 4 weeks post-operation, the tensile strength of the humeral-supraspinatus complex in the sham-operated group was significantly higher than that in the control group, the SIS group, and the BME-SIS group. The control group was significantly lower than that in the SIS group and the BME-SIS group, while there was no statistically significant difference between the SIS group and the BME-SIS group. Figure 26 A). At 8 weeks post-operation, the tensile strength of all groups increased compared to 4 weeks post-operation. The sham-operated group still showed significantly higher tensile strength than the other three groups. The BME-SIS group showed significantly higher tensile strength than the control group and the SIS group. Figure 26 B).

[0254] These results indicate that at 8 weeks post-surgery, BME-SIS significantly improves the ultimate load and tensile strength of the humeral-supraspinatus complex compared to SIS. This directly suggests that BME-SIS provides better rotator cuff healing strength and may significantly reduce the risk of postoperative rotator cuff re-tear.

[0255] 7. Histological staining

[0256] (1) HE staining

[0257] The results showed that at 4 weeks post-operation, the sham surgery group performed best overall, with an intact tendon-bone interface, clearly distinguishable four-layer transitional structure (tendon-uncalcified fibrocartilage-calcified fibrocartilage-bone), natural transition, and no inflammatory cell infiltration. The control group performed worst overall, with a completely disordered tendon-bone interface structure, disappearance of the four-layer transitional structure, highly irregular cell arrangement, and significant inflammatory cell infiltration. The SIS group performed better overall than the control group, but worse than the sham surgery group and the BME-SIS group, exhibiting a relatively disordered tendon-bone interface structure, only faintly discernible four-layer transitional structure, relatively irregular cell arrangement, and milder inflammatory cell infiltration. The BME-SIS group was second only to the sham surgery group, exhibiting a relatively intact tendon-bone interface structure, relatively clearly distinguishable four-layer transitional structure, relatively regular cell arrangement, and no significant inflammatory cell infiltration. Figure 27 ).

[0258] Eight weeks post-surgery, the sham surgery group remained the best overall, with no significant difference in the tendon-bone interface compared to four weeks post-surgery. The other three groups all showed varying degrees of improvement compared to four weeks post-surgery. The control group remained the worst overall, with a disordered tendon-bone interface structure and the four transitional layers still not visible, although cell arrangement was more regular and inflammatory cell infiltration was reduced. The SIS group remained better than the control group, but worse than the sham surgery group and the BME-SIS group, showing improved tendon-bone interface structure, clearer four transitional layers, more regular cell arrangement, and reduced inflammatory cell infiltration. The BME-SIS group was second only to the sham surgery group, with a tendon-bone interface structure similar to the sham surgery group, clearly distinguishable four transitional layers with a more natural transition, tendon insertion into bone tissue, further regular cell arrangement, and no significant inflammatory cell infiltration.

[0259] (2) Histological score

[0260] The results showed that at 4 weeks post-operation, the supraspinatus tendon maturity in the control group was significantly lower than that in the other three groups, the SIS group was significantly lower than that in the BME-SIS group, and the BME-SIS group was significantly lower than that in the sham surgery group. Figure 28 A). At 8 weeks post-operation, the maturity of the supraspinatus tendon in the sham-operated group was not significantly different from that at 4 weeks post-operation, while the other three groups all showed improvement compared to 4 weeks post-operation, but the trend of differences among the four groups was similar to that at 4 weeks post-operation. Figure 28 B).

[0261] (3) Masson staining

[0262] The results showed that at 4 weeks post-surgery, the sham surgery group performed best overall, exhibiting uniform and regularly arranged collagen fibers. The control group performed worst overall, with almost no collagen fibers, replaced by a large amount of granulation tissue. There was no significant difference between the SIS group and the BME-SIS group; both groups showed a high content of collagen fibers, but their arrangement was disordered and disorganized. Figure 29 At 8 weeks post-surgery, there was no significant difference in collagen fiber content in the sham surgery group compared to 4 weeks post-surgery. The content and arrangement of collagen fibers in the other three groups showed improvement compared to 4 weeks post-surgery. The control group remained the worst overall, showing some recovery in collagen fiber content, but with disordered and disorganized arrangement. The SIS group was better overall than the control group, but worse than the sham surgery group and the BME-SIS group, showing a further increase in collagen fiber content, but the arrangement was still somewhat disordered, and the direction was still faintly discernible. The BME-SIS group was second only to the sham surgery group, showing a more regular and orderly arrangement of collagen fibers, with the direction still faintly discernible. Figure 29 ).

[0263] (4) COL-II immunohistochemical staining and quantitative analysis

[0264] The results showed that at 4 weeks post-operation, the cartilage layer structure at the tendon-bone interface was intact in the sham-operated group, and COL-II expression was significantly higher than in the other three groups. In the control group, the cartilage layer at the tendon-bone interface almost disappeared, and COL-II expression was significantly lower than in the SIS and BME-SIS groups. In the SIS group, the cartilage layer at the tendon-bone interface partially recovered, but COL-II distribution was more localized, and its expression was significantly lower than in the BME-SIS group. In the BME-SIS group, the cartilage layer at the tendon-bone interface partially recovered, and COL-II distribution was more widespread; its expression was second only to the sham-operated group. Figure 30 , Figure 31 A).

[0265] At 8 weeks post-operation, there was no significant difference in the sham surgery group compared to 4 weeks post-operation. The other three groups showed improvement compared to 4 weeks post-operation. Specifically, in the control group, the cartilage layer at the tendon-bone interface partially recovered, but COL-II distribution was relatively limited, and its expression remained significantly lower than in the sham surgery group, SIS group, and BME-SIS group. In the SIS group, the cartilage layer at the tendon-bone interface further recovered, and COL-II distribution was more widespread, but its expression remained significantly lower than in the sham surgery group. In the BME-SIS group, the cartilage layer at the tendon-bone interface recovered to a level close to that of the sham surgery group, and COL-II distribution became more widespread; its expression was not statistically different from that of the sham surgery group. Figure 30 , Figure 31 B).

[0266] 8. In vivo biosafety assessment

[0267] The rats in all groups were in good condition after surgery, with normal food and water intake and activity. There were no infections or deaths, and no pathological abnormalities were observed in vital organs, indicating that SIS and BME-SIS have good biocompatibility.

[0268] In this experimental case, a comprehensive series of tests, including gait, forelimb grip strength, imaging, biomechanics, and histology, were conducted at 4 and 8 weeks postoperatively. The results showed that the BME-SIS biomembrane applied to the tendon-bone space in a rat rotator cuff tear model significantly promoted rotator cuff tear healing and significantly improved the rats' motor coordination. The BME-SIS biomembrane can provide better rotator cuff healing strength, which is beneficial in reducing the risk of rotator cuff re-tear after surgery, and also demonstrates the stability of the therapeutic effect of BME-SIS biomembrane.

[0269] As can be seen from the above embodiments and experimental examples, this invention provides a BME-SIS biomembrane through screening and preparation processes, based on SIS materials, and after co-culturing and decellularization with BMSCs. This BME-SIS biomembrane exhibits good decellularization effects, avoiding the safety issues associated with xenotransplantation. The BME-SIS biomembrane possesses improved surface properties and mechanical properties. Compared to SIS, it significantly promotes cell adhesion, proliferation, and differentiation. Applied to a rat rotator cuff tear model, multiple aspects, including molecular, histological, motor, mechanical, and imaging studies, have confirmed that the BME-SIS biomembrane of this invention significantly promotes rotator cuff tear healing, improves rat motor function, and helps reduce the risk of postoperative rotator cuff re-tear, with stable therapeutic effects. The BME-SIS biomembrane of this invention can be used as a biological repair patch and shows great promise in rotator cuff tear healing.

Claims

1. A BME-SIS biofilm, characterized in that: It is prepared by a method including the following steps: Step 1: Bone marrow-derived mesenchymal stem cells were seeded onto the surface of the SIS membrane and cultured to obtain the BMSCs-SIS complex; Step 2: The BMSCs-SIS complex is obtained by decellularization. The ratio of the SIS membrane to bone marrow-derived mesenchymal stem cells used was 1 cm. 2 : 2×10 4 -15×10 4 The culture includes: first culturing in α-MEM complete medium for 3-10 days, and then culturing in α-MEM complete medium containing 50-200 μg / mL vitamin C for 5-10 days.

2. The BME-SIS biofilm according to claim 1, characterized in that, The SIS membrane is prepared by the following steps: Animal jejunum is taken, mechanically processed while preserving the submucosa, and then defatted, treated with protease, and treated with surfactant to obtain the final product.

3. The BME-SIS biofilm according to claim 2, characterized in that, The degreasing is carried out by a mixed solution of methanol and chloroform in a volume ratio of 0.8-1.2:0.8-1.2; and / or the degreasing time is 10-16 hours; And / or, the protease is selected from trypsin, papain, and pepsin; and / or, the amount of the protease used is 0.1-0.5% by mass, the temperature of the protease treatment is 3-8°C, and the treatment time is 4-18 hours; And / or, the surfactant is selected from sodium dodecyl sulfate, Triton X-100, Tween-100, and ethylenediaminetetraacetic acid; and / or, the amount of the surfactant is 0.25-1% by mass, and the treatment time of the surfactant is 6-24 hours; And / or, after each step of the defatting, protease treatment, and surfactant treatment is completed, the submucosal tissue is washed with water 5-15 times; And / or, after the defatting, protease treatment, and surfactant treatment steps are completed, freeze-drying is performed.

4. The BME-SIS biofilm according to claim 1, characterized in that, The decellularization process includes: surfactant treatment and DNase treatment.

5. The BME-SIS biofilm according to claim 4, characterized in that, The surfactant is selected from sodium dodecyl sulfate, Triton X-100, Tween-100, and sodium deoxycholate; and / or, concentrated ammonia is added during the surfactant treatment, and the volume ratio of the surfactant to concentrated ammonia is 50-500:200; and / or, the DNase is selected from DNase I.

6. The BME-SIS biofilm according to claim 4, characterized in that, The amount of surfactant used is 0.25-1% by volume; and / or the surfactant treatment time is 15-60 min, the temperature is 25-37℃, and the rotation speed is 50-100 rpm. And / or, the amount of DNase used is 50-250 U / mL; and / or, the temperature of the DNase treatment is 25-37℃, the time is 1.5-6 hours, and the rotation speed is 50-100 rpm.

7. The BME-SIS biomembrane according to claim 1, characterized in that: During the culture process, the culture medium is changed every 1-2 days; and / or, the culture conditions include incubation at 37±1℃ in a 5% CO2 cell culture incubator; and / or, the SIS membrane is a circular disc with a diameter of 1.5-10cm; and / or, after the decellularization treatment, it is washed with an aqueous solution; and / or, the bone marrow-derived mesenchymal stem cells are P3-7 generation bone marrow-derived mesenchymal stem cells.

8. The method for preparing the BME-SIS biofilm according to any one of claims 1-7, characterized in that, It includes: Step 1: Bone marrow-derived mesenchymal stem cells were seeded onto the surface of the SIS membrane and cultured to obtain the BMSCs-SIS complex; Step 2: The BMSCs-SIS complex is obtained by decellularization. The ratio of the SIS membrane to bone marrow-derived mesenchymal stem cells used was 1 cm. 2 : 2×10 4 -15×10 4 indivual.

9. Use of the BME-SIS biomembrane according to any one of claims 1-7 in the preparation of implantable medical materials for treating rotator cuff tears.

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