A synthetic dermal underlaying subacromial synovial stem cell composite patch, its preparation method and application

Abstract

The application relates to the technical field of biology, and particularly discloses an artificial synthetic dermis loaded with subacromial synovial mesenchymal stem cell composite patch as well as a preparation method and application thereof. The artificial synthetic dermis loaded with subacromial synovial mesenchymal stem cell composite patch has the advantages of convenient raw material source, no immune rejection, no supply area complications, no disease transmission risk and the like. Moreover, the composite patch provides initial mechanical support, and improves the tendon-bone interface microenvironment through the paracrine effect (including pro-angiogenic related factors) of the subacromial synovial mesenchymal stem cells, promotes host cell infiltration and blood vessel ingrowth, and enhances tissue integration and rotator cuff healing.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a synthetic composite patch of dermal loaded with subacromial stem cells, its preparation method, and its application. Background Technology

[0002] Large rotator cuff tears or chronic degenerative tears are often accompanied by poor tendon tissue quality, tendon retraction, and insufficient local blood supply, leading to difficulties in tendon-bone interface reconstruction, slow healing, and a high rate of re-tears. Clinically, methods such as patch-enhanced repair, bridging repair, and superior bursa reconstruction are commonly used to improve initial mechanical stability and promote tissue integration. Currently, commonly used materials and techniques for repairing rotator cuff injuries mainly include autologous or allogeneic tissue patches, such as latissimus dorsi tendon, biceps brachii long head tendon transposition, or acellular allogeneic dermis, but these have limitations such as limited availability and postoperative pain due to donor site injury.

[0003] Artificial dermis, also known as dermal substitute or dermal scaffold, is a biomaterial that guides tissue regeneration through a biomimetic dermal structure. Its main components are collagen and chondroitin sulfate. Artificial dermis has been widely used in the repair of burns, plastic surgery wounds, and other ulcers, with good therapeutic effects. Some studies have attempted to use human acellular dermal matrix for rotator cuff repair with promising results, but the availability of materials is limited. Porcine acellular dermis, on the other hand, suffers from problems such as foreign body reactions, immune-related inflammation, and overly dense structures leading to limited cell infiltration and blood vessel ingrowth, affecting repair outcomes. Therefore, synthetic dermis with collagen as its main component is an excellent biological patch. Meanwhile, the need for removal of the subacromial membrane in rotator cuff repair remains controversial. Current research is gradually shifting from simple anatomical resection to function preservation and active therapeutic targets. The synovial membrane of the acromion is rich in mesenchymal stem cells with high proliferative potential and paracrine function, which can regulate the local inflammatory microenvironment. In arthroscopic surgery, doctors can now more selectively debride the synovium, remove the thickened and fibrotic inflammatory synovium, preserve healthy synovial tissue, and use its own repair potential to help the rotator cuff heal. Summary of the Invention

[0004] The purpose of this application is to overcome the shortcomings of the prior art and provide a synthetic composite patch of dermal loaded with acromial subacromial stem cells, its preparation method and application.

[0005] To achieve the above objectives, the technical solution adopted in this application is as follows: This application provides a synthetically produced composite patch of dermal stem cells loaded with subacromial stem cells. The composite patch comprises a double-layered artificial dermis and subacromial stem cells; the subacromial stem cells are loaded onto the double-layered artificial dermis. The dual-layer structure of artificial dermis includes a silicone membrane layer and a collagen sponge layer; The number of cells loaded by the acromial subacromial stem cells per square centimeter of composite patch is 1 × 10⁻⁶. 5 ~1×10 6 cells / cm 2 .

[0006] The synthetic dermal graft containing subacromial stem cells provided in this application has the advantages of convenient raw material sourcing, no immune rejection, no donor site complications, and no risk of disease transmission.

[0007] The composite patch using the above-mentioned artificially synthesized dermal loaded with acromial subacromial stem cells is specifically applied to rotator cuff tears. The material integrates with the body's own tissue, which can "repair" the rotator cuff while achieving "regeneration", promoting the healing of the tendon-bone connection point, reducing the risk of re-tear, promoting the reconstruction of the tendon-bone interface, improving the quality of rotator cuff healing and reducing the risk of re-tear.

[0008] Furthermore, the silicone membrane layer serves as a barrier and moisturizing protection, while the collagen sponge layer acts as a three-dimensional scaffold for cell adhesion and infiltration. The silicone membrane layer and the collagen sponge layer together form the artificial dermis with a bilayer structure as described in this application. The paracrine function of the subacromial stem cells (including pro-angiogenic factors) improves the microenvironment of the tendon-bone interface, promotes host cell infiltration and blood vessel ingrowth, and enhances tissue integration and rotator cuff healing.

[0009] Furthermore, using the above-mentioned loading range of subacromial stem cells ensures good cell adhesion and distribution on the surface of the composite patch, thus better achieving rotator cuff repair. Too low a loading of subacromial stem cells hinders sufficient cell adhesion and the formation of a local paracrine microenvironment; too high a loading may lead to excessively dense local distribution, decreased composite uniformity, and even affect nutrient exchange and cell survival.

[0010] In some specific embodiments, the thickness of the silicone film layer is 0.15 mm; the thickness of the collagen sponge layer is 3 mm.

[0011] The use of double-layer artificial dermis of the aforementioned thickness offers the following advantages: First, the appropriate thickness ensures adequate mechanical support while maintaining the material's flexibility and adherence, facilitating better contact with the irregular surface of the rotator cuff area and reducing curling, lifting, or localized suspension. Second, the appropriate thickness promotes cell distribution and attachment within the collagen sponge layer pores, while also facilitating the diffusion and exchange of nutrients, oxygen, and metabolic products within the scaffold. Third, the suitable thickness of the double-layer artificial dermis maintains sufficient cell loading space within the collagen sponge layer while preventing excessive scaffold thickness from slowing early tissue integration, thus promoting the ingrowth of host cells, capillaries, and new matrix into the material. Fourth, from a practical standpoint, the appropriate thickness also facilitates intraoperative cutting, suturing, and matching with the recipient area, balancing ease of operation with supportive effectiveness.

[0012] In a preferred embodiment of the method for preparing the synthetic dermal composite patch loaded with subacromial stem cells as described in this application, the number of subacromial stem cells loaded per square centimeter of composite patch is 5 × 10⁻⁶ cells. 5 cells / cm 2 .

[0013] In the technical solution of this application, the subacromial stem cells of this application are 5×10 5 pcs / cm 2 The amount of cells loaded onto the synthetic dermal scaffold is sufficient to ensure good cell adhesion and distribution on the surface of the composite patch, while also considering cell viability and scaffold support feasibility. This facilitates the tissue repair function of the composite patch and enables better rotator cuff repair.

[0014] In some specific embodiments, the subacromial stem cells are loaded into the pores of the collagen sponge layer. As a preferred embodiment of the synthetic dermal composite patch loaded with subacromial stem cells described in this application, the subacromial stem cells include human subacromial mesenchymal stem cells.

[0015] This application provides a method for preparing the above-mentioned synthetic dermal composite patch loaded with subacromial stem cells, comprising the following steps: 1) Take the subacromial membrane tissue from the surgical site, cut it into small pieces, digest the tissue, centrifuge it, collect the precipitate, resuspend the cells, culture the resuspended cells in a culture vessel, and then passage the cells to obtain a subacromial membrane stem cell suspension. 2) Cell suspensions were prepared by adding 3rd to 6th generation acromion subacromial stem cells to low-glucose DMEM medium; 3) Fix the double-layered artificial dermis to the supraspinatus tendon and the greater tubercle of the humerus respectively, and then drop the cell suspension from step 2) onto the double-layered artificial dermis to form a composite patch.

[0016] This application uses a double-layer artificial dermis to be fixed to the supraspinatus tendon and the greater tubercle of the humerus to achieve enhanced repair. During use, the cell suspension is dropped onto the double-layer artificial dermis to avoid the double-layer artificial dermis becoming gel-like when it comes into contact with aqueous solution, which is not conducive to subsequent operations. On-site addition allows the cell suspension to adhere better to the artificial dermis.

[0017] In a preferred embodiment of the method for preparing the synthetic dermal graft containing subacromial stem cells described in this application, the cell seeding density on the artificial dermis is 1×10⁻⁶. 5 ~1×10 6 cells / cm 2 The volume of the acromion subacromial stem cell suspension added was controlled at 50~100 μl / cm³. 2 .

[0018] In a preferred embodiment of the method for preparing the synthetic dermal graft containing subacromial stem cells described in this application, the cell seeding density on the artificial dermis is 5 × 10⁻⁶ cells / cm². 5 cells / cm 2 The application utilizes the aforementioned cell seeding density and cell suspension volume range to achieve better rotator cuff repair.

[0019] This application used subacromial stem cells from the acromion at the above-mentioned cell seeding density. HE staining showed that the fibers were regularly arranged. Masson staining showed that it better promoted the generation of collagen fibers, which were thicker, more continuous, and arranged more parallel to the direction of stress, indicating a high degree of tissue maturity in the repair process. Immunofluorescence staining showed that a higher CD31 positive area was maintained, and some vascular structures showed a more network-like distribution, indicating more complete and stable vascularization.

[0020] The composite patch prepared by the above method provides initial mechanical support and improves the tendon-bone interface microenvironment through the paracrine effect of acromial subacromial stem cells (including pro-angiogenic factors), promotes host cell infiltration and blood vessel ingrowth, and enhances tissue integration and rotator cuff healing.

[0021] In a preferred embodiment of the method for preparing the synthetic dermal-loaded acromial subacromial stem cell composite patch described in this application, step 1) includes digestion of the shredded tissue using a digestive solution containing trypsin and collagenase.

[0022] In a preferred embodiment of the method for preparing the synthetic dermal-loaded subacromial stem cell composite patch described in this application, step 1) includes digestion of the shredded tissue using a digestive solution containing trypsin and collagenase. The mass concentration of trypsin in the digestive fluid is 0.05%~0.25%; the mass concentration of collagenase in the digestive fluid is 1~4 mg / ml.

[0023] In some specific embodiments, the mass concentration of trypsin in the digestive fluid is 0.1% to 0.25%; the mass concentration of collagenase in the digestive fluid is 2 to 4 mg / ml.

[0024] This application uses trypsin and collagenase at the above-mentioned mass concentrations to better isolate and obtain mesenchymal stem cells derived from the acromion subacromial membrane.

[0025] The mass concentration of trypsin in the digestive fluid is 0.25%; the mass concentration of collagenase in the digestive fluid is 3 mg / ml.

[0026] This application uses 3rd to 6th generation subacromial stem cells, which helps to ensure that the subacromial mesenchymal stem cells used have good adhesion and proliferation ability, phenotypic stability and biological activity, thereby improving their adhesion and survival effect after entering the pores of the artificial dermal collagen sponge layer, and enhancing the consistency and reproducibility of composite patch preparation.

[0027] This application also provides the application of the above-mentioned synthetic dermal composite patch loaded with acromial subacromial stem cells in the preparation of rotator cuff repair products.

[0028] The synthetic dermal graft containing acromial subacromial stem cells provided in this application can be used for rotator cuff enhancement repair, bridging repair, or superior bursa reconstruction.

[0029] Compared with the prior art, this application has the following beneficial effects: This application provides a synthetic dermal composite patch loaded with subacromial stem cells, its preparation method, and its application. The synthetic dermal composite patch provided by this application has advantages such as convenient raw material sourcing, no immune rejection, no donor site complications, and no risk of disease transmission. Furthermore, the composite patch provides initial mechanical support and improves the tendon-bone interface microenvironment through the paracrine effects of subacromial stem cells (including pro-angiogenesis-related factors), promoting host cell infiltration and blood vessel ingrowth, and enhancing tissue integration and rotator cuff healing. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the subacromial membrane tissue during surgery; Figure 240X microscope images of cultured P0 and P3 subacromial stem cells of the acromion; Figure 3 This is a flow cytometry result of acromial subacromial stem cell. Figure 4 This is a diagram showing the verification results of the compatibility between human acromial subacromial mesenchymal stem cells and Pelnac in Example 2; Figure 5 This is a diagram showing the results of the angiogenesis verification experiment in Example 3; Figure 6 This is a diagram showing the in vivo verification results in Example 4; Figure 7 The figure shows the results of the tensile test in Example 5. Detailed Implementation

[0031] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0032] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0033] In the technical solution of this application, a composite patch of artificially synthesized dermis loaded with subacromial stem cells is provided. The composite patch includes a double-layer artificial dermis and subacromial stem cells. The double-layer artificial dermis includes a silicone membrane layer and a collagen sponge layer. The subacromial stem cells are filled in the pores of the collagen sponge layer.

[0034] The double-layer artificial dermis uses Pelnac reinforced material.

[0035] Example 1: A synthetic dermal composite patch loaded with subacromial stem cells and its preparation method. This embodiment provides a method for preparing a synthetic dermal graft containing subacromial stem cells, comprising the following steps: 1) Isolation and culture of subacromial mesenchymal stem cells (hSMSCs): Subacromial tissue was harvested intraoperatively. After removing significantly inflammatory and necrotic tissue, relatively healthy areas were preserved. The attached fascia and fat were carefully dissected, and the tissue was thoroughly minced with sterile scissors. The tissue was then digested for 2 hours in a 1:3 ratio of 0.25% trypsin and 3 mg / ml collagenase (freshly prepared). The digested tissue was filtered through a 100 μm filter and placed in a 50 mL centrifuge tube. After slight shaking for 30 seconds and centrifugation at 2000 rpm for 5 minutes at room temperature, the supernatant was removed. The cells were resuspended in 1 mL of low-glucose DMEM complete medium. The cells were then seeded into 25 mL culture flasks and cultured in a 37°C, 5% CO2 incubator for primary (P0) culture. The medium was changed for the first time after cell attachment (usually 3-5 days), and then every 3 days thereafter with fresh medium. When the cell clones reached a full-grown state, they were passaged: First, the cells were gently washed three times with PBS to remove residual culture medium. Then, an appropriate amount of trypsin was added for digestion. Under a microscope, the cells were observed until most became rounded and detached (approximately 1-2 minutes). Immediately afterward, 1 mL of complete culture medium was added to stop the digestion. The cells were repeatedly pipetted to ensure complete detachment. After centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1 mL of complete culture medium. 20 μL of the cell suspension was used for cell counting, and the cells were reseeded at a density of 3000 cells / cm². When the cells reached passage 3 (P3) and there were few contaminating cells, flow cytometry was performed for identification. Flow cytometry analysis of the expression of hSMSC surface markers showed the expression of CD73, CD90, and CD105, indicating that these cells conformed to the characteristics of mesenchymal stem cells, thus obtaining human acromial subacromial mesenchymal stem cells (hSMSCs).

[0036] 2) Preparation of hSMSC supernatant: After the acromial subacromial stem cells were cultured to about 80% confluence, they were collected and cultured on (acromial subacromial stem cells-CM), centrifuged at 4℃ to remove cell debris (500×g, 5 min), and filtered with 0.22μm for later use.

[0037] 20% hSMSC supernatant: acromial subacromial stem cells-CM diluted to a concentration of 20%.

[0038] The diagram of the subacromial membrane tissue during the operation is shown below. Figure 1 As shown; 40X microscope images of P0 cultured human subacromial mesenchymal stem cells and P3 cultured human subacromial mesenchymal stem cells are shown. Figure 2 As shown.

[0039] The results of flow cytometry analysis of human subacromial mesenchymal stem cells are shown in the figure below. Figure 3 As shown.

[0040] Example 2: Verification experiment on the compatibility of human acromial subacromial mesenchymal stem cells with Pelnac. Human subacromial mesenchymal stem cells (hSMSCs) obtained in step 1) of Example 1 were labeled with pKh67 and seeded at a cell density of 1×10⁻⁶. 5 cells / cm 2 Cells were seeded on the collagen sponge layer (inner / deep side) of Pelnac (3×3cm pieces, adjustable as needed). After 3 days, microscopic observation showed that hSMSCs could grow on Pelnac (staining as live cells) using DAPI and pKh67 staining, indicating that hSMSCs can grow and proliferate on Pelnac, demonstrating the good biocompatibility of Pelnac. Figure 4 As shown.

[0041] Example 3: Angiogenesis Verification Experiment 1) Preparation of collagen membrane extract: Under aseptic conditions, prepare a double-layer artificial dermis (Pelnac). Place each sample (1×1×3 mm) of double-layer artificial dermis (Pelnac) into a sterile centrifuge tube; add serum-free basal culture medium (DMEM) at a ratio of 3 cm² / mL, adding 1.0~1.1 mL per sample; incubate at 37℃ with gentle shaking for 24 h; collect the extract after removing the material, and then filter it through a 0.22 μm filter for sterilization. The filtered stock solution is defined as 100% extract (Pelnac extract). 2) Spread 20 μL of matrix gel into a 24-well plate and incubate at 37°C for 1 hour. The cell density is 1.5 × 10⁻⁶ cells / well. 4 Endothelial cell suspension was added to wells, with 500 μL added to each well. The control group was given serum-free high-glucose medium, while the experimental groups were given 50% Pelnac extract (250 μL of Pelnac extract was added to a 500 μL system to dilute to 50%) and 50% Pelnac extract + 20% hSMSC supernatant, respectively. The results were recorded by taking pictures at 12 h.

[0042] Nb nodes represent the number of branch points (red in the image below). More branch points generally indicate stronger angiogenic activity and are a key indicator of the complexity of the vascular network; Tot.length represents the total length of the vessel (…). Figure 5 (Medium yellow) is a fundamental core indicator for assessing angiogenesis capacity.

[0043] Depend on Figure 5 It can be seen that the Nb nodes and Tot. length of the Pelnac extract group and the Pelnac extract + hSMSC group were better than those of the blank control group, indicating that both Pelnac and hSMSC have the potential to promote angiogenesis, and the Pelnac extract + hSMSC group is better than Pelnac alone.

[0044] Example 4: In vivo validation experiment Twenty-four healthy male SD rats, weighing 250-300 g, were selected and randomly divided into three groups by random number table: blank control group, Pelnac dermal enhancement group, and Pelnac dermal + hSMSC enhancement group, with eight rats in each group.

[0045] Establishing a rotator cuff defect model: A 1.5 cm longitudinal incision was made on the lateral side of the left acromion of the rat. The neck and back muscles were separated layer by layer to fully expose the acromion and supraspinatus tendon. The insertion point of the supraspinatus tendon on the humerus was determined. The tendon was completely severed and part of the tissue was removed to construct a rotator cuff defect model.

[0046] The blank control group underwent direct suturing; The Pelnac dermal reinforcement group consisted of a 1×1cm Pelnac reinforcement repair after direct suturing. The Pelnac dermal + hSMSC enhancement group consisted of: after adding 1×1 cm Pelnac, the concentration was increased to 1×10⁻⁶. 6 / cm 2 50 μL of mlhSMSC suspension was added to Pelnac, and the skin was sutured layer by layer.

[0047] Postoperatively, tissue repair and angiogenesis were assessed using HE staining, Masson staining, and the vascular-related marker CD31.

[0048] The results are as follows Figure 6 As shown.

[0049] HE staining revealed a large number of inflammatory cells clustered around the tendon-bone repair interface in each group, with irregular arrangement of fibrous scar tissue. The fiber arrangement was disordered in the blank control group, relatively regular in the Pelnac dermal enhancement group, and more regular in the Pelnac dermal + hSMSC enhancement group than in the blank control group and the Pelnac dermal enhancement group.

[0050] Masson staining revealed the formation of new collagen fibers in all three repair areas. In the blank control group, the collagen fibers were disordered and loosely structured, while the collagen fibers in the Pelnac dermal enhancement group were increased and more densely structured. The collagen fibers in the Pelnac dermal + hSMSC enhancement group were larger, more continuous, and tended to be arranged parallel to the direction of force, indicating a higher degree of maturity in the repaired tissue.

[0051] Immunofluorescence staining revealed that CD31, a marker of blood formation, was expressed with green fluorescence. In the blank control group, CD31 expression was less and focal. In the Pelnac dermal enhancement group, CD31 was widely distributed. The Pelnac dermal + hSMSC enhancement group maintained a higher CD31 positive area, and some vascular structures showed a more network-like distribution, indicating more complete and stable vascularization.

[0052] Example 5: Tensile Test The supraspinatus tendon-humerus complex (tendon and bone from the rotator cuff defect model constructed in Example 4) was removed, preserving the integrity of the tendon-bone attachment structure and removing excess soft tissue. The specimen was treated under moist conditions (wrapped in saline-soaked gauze) to avoid dehydration and mechanical deviation. Before testing, ensure that the loading axis is aligned with the long axis of the tendon.

[0053] Preload: 0.1–0.5 N for 30 s, tensile rate: 20 mm / min, initial load: continuously applied from 0 N, record load-displacement curve until interface failure or tendon rupture.

[0054] (1) Initial “toe region”: The load slowly increases from 0 N, and the collagen fiber folds in the corresponding tendon-bone complex are gradually straightened; (2) Near-linear increase stage: The load increases nearly linearly with the increase of displacement, reflecting the enhanced load-bearing capacity of the repaired structure and the healing tissue; (3) Peak decline stage: After the curve reaches the maximum load peak (ultimate failure load), it drops rapidly, indicating structural failure (mostly tendon-bone interface tearing, tendon rupture or suture cutting), and then enters the residual load stage.

[0055] Compared with the blank control group, the Pelnac dermal reinforcement group (i.e., the Pelnac group) and the Pelnac dermal + hSMSC reinforcement group (i.e., the hSMSC / Pelnac group) showed higher maximum failure loads at all time points. Furthermore, the load-displacement curve peak was higher and the subsequent decline was relatively delayed when combining Pelnac dermal with hSMSC, suggesting that Pelnac dermal and hSMSC can improve early structural stability and promote subsequent mechanical property recovery. Figure 7 .

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A synthetically produced dermal composite patch loaded with subacromial stem cells, characterized in that, The composite patch comprises a double-layered artificial dermis and subacromial stem cells; the subacromial stem cells are loaded onto the double-layered artificial dermis. The dual-layer structure of artificial dermis includes a silicone membrane layer and a collagen sponge layer; The number of cells loaded by the acromial subacromial stem cells per square centimeter of composite patch is 1 × 10⁻⁶. 5 ~1×10 6 cells / cm 2 .

2. The synthetic dermal graft containing subacromial stem cells as described in claim 1, characterized in that, The number of cells loaded onto the subacromial stem cells was 5 × 10⁻⁶. 5 cells / cm 2 .

3. The synthetic dermal graft containing subacromial stem cells as described in claim 1, characterized in that, The acromion subacromial stem cells are loaded in the pores of the collagen sponge layer.

4. The synthetic dermal graft containing subacromial stem cells as described in claim 1, characterized in that, The acromial subacromial stem cells include human acromial subacromial mesenchymal stem cells.

5. The method for preparing the synthetic dermal-loaded subacromial stem cell composite patch according to any one of claims 1 to 4, characterized in that, Includes the following steps: 1) Take the subacromial membrane tissue from the surgical site, cut it into small pieces, digest the tissue, centrifuge it, collect the precipitate, resuspend the cells, culture the resuspended cells in a culture vessel, and then passage the cells to obtain a subacromial membrane stem cell suspension. 2) Cell suspensions were prepared by adding 3rd to 6th generation acromial subarachnoid stem cells to low-glucose DMEM medium; 3) Fix the double-layered artificial dermis to the supraspinatus tendon and the greater tubercle of the humerus respectively, and then drop the cell suspension from step 2) onto the double-layered artificial dermis to form a composite patch.

6. The method for preparing the synthetic dermal-loaded subacromial stem cell composite patch as described in claim 5, characterized in that, The cell seeding density on the artificial dermis is 1×10⁻⁶. 5 ~1×10 6 cells / cm 2 The volume of the acromion subacromial stem cell suspension added was controlled at 50~100 μl / cm³. 2 .

7. The method for preparing the synthetic dermal-loaded subacromial stem cell composite patch as described in claim 6, characterized in that, The cell seeding density on the artificial dermis is 5 × 10⁻⁶. 5 cells / cm 2 .

8. The method for preparing the synthetic dermal-loaded subacromial stem cell composite patch as described in claim 5, characterized in that, In step 1), digestion includes digesting the shredded tissue with a digestive solution containing trypsin and collagenase. The mass concentration of trypsin in the digestive fluid is 0.05%~0.25%; the mass concentration of collagenase in the digestive fluid is 1~4 mg / ml.

9. The method for preparing the synthetic dermal-loaded subacromial stem cell composite patch as described in claim 8, characterized in that, The mass concentration of trypsin in the digestive fluid is 0.25%; the mass concentration of collagenase in the digestive fluid is 3 mg / ml.

10. The use of the synthetic dermal composite patch loaded with acromial subacromial stem cells as described in any one of claims 1 to 4 in the preparation of rotator cuff repair products.

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