An immunomodulatory scaffold for tendon-bone integration repair and a preparation method and application thereof

Abstract

The application discloses an immune-regulation scaffold for tendon-bone integrated repair, which is a three-dimensional framework with a layered structure, wherein the layered structure comprises a sublayer X with bone marrow mesenchymal stem cells and a sublayer Y with tendon stem cells; the bone marrow mesenchymal stem cells in the sublayer X are uniformly distributed in a high-molecular framework body containing a functional manganese component, and the tendon stem cells in the sublayer Y are uniformly distributed in a high-molecular framework body containing a functional manganese component; and the sublayer X and the sublayer Y are stacked to obtain the immune-regulation scaffold for tendon-bone integrated repair. The immune-regulation scaffold for tendon-bone integrated repair promotes tendon-bone integration through immune regulation by means of release of inorganic bioactive ions and delivery of tissue cells.

Description

Technical Field

[0001] This invention relates to an immunomodulatory scaffold for integrated tendon-bone repair, its preparation method, and its application, belonging to the field of biotechnology. Background Technology

[0002] Tendon-bone interface injuries are common in sports medicine, occurring in multiple locations including the rotator cuff, Achilles tendon, and anterior cruciate ligament. They have a high incidence rate and significantly impact patients' quality of life. Commonly used clinical treatments such as sports rehabilitation, hormone injections, and surgical interventions all suffer from high failure rates. This is due to the gradient characteristics from tendon to bone, such as increased mineral content, decreased collagen fiber tissue, and thickened matrix tissue. At the cellular level, the tendon-bone interface exhibits type changes from fibroblasts to fibrochondrocytes and then to osteocytes. During the healing process, M1 macrophages are the main driver of early inflammatory processes in the healing tendon, while the M2 macrophage phenotype plays a role in fibroblast proliferation and stimulating new tissue deposition. Tendon-bone regeneration is closely related to the immune microenvironment. These challenges hinder complete regeneration of the tendon-bone interface, preventing it from restoring to its natural structure and morphology. Therefore, there is an urgent need to develop a tissue-engineered product with immunomodulatory tendon-bone repair functions. Summary of the Invention

[0003] The technical objective of this invention is to provide an immunomodulatory scaffold for integrated tendon-bone repair, its preparation method, and its application. This immunomodulatory scaffold for integrated tendon-bone repair promotes tendon-bone integration through immune regulation, utilizing the release of inorganic bioactive ions and delivery by tissue cells.

[0004] In a first aspect, the present invention provides an immunomodulatory scaffold for integrated tendon-bone repair. The immunomodulatory scaffold is a three-dimensional framework with a layered structure, comprising a sublayer X containing bone marrow mesenchymal stem cells and a sublayer Y containing tendon stem cells; in sublayer X, bone marrow mesenchymal stem cells are uniformly distributed within a polymeric framework containing functional manganese components, and in sublayer Y, tendon stem cells are uniformly distributed within a polymeric framework containing functional manganese components; sublayers X and Y are stacked layer by layer to obtain the immunomodulatory scaffold for integrated tendon-bone repair.

[0005] Preferably, the precursor slurry used for printing sublayer X comprises a polymer matrix, a functional manganese component, and bone marrow mesenchymal stem cells; wherein the polymer matrix has a mass concentration of 4-8% (w / v), the functional manganese component accounts for 2-15% of the polymer matrix mass, and the cell concentration is (4-6)×10⁻⁶. 6 per mL.

[0006] Preferably, the precursor slurry used for printing sublayer Y comprises a polymer matrix, a functional manganese component, and tendon stem cells; wherein the polymer matrix has a mass concentration of 4-8% (w / v), the functional manganese component accounts for 2-15% of the polymer matrix mass, and the cell concentration is (4-6)×10⁻⁶. 6 per mL.

[0007] Preferably, the polymer matrix includes at least one of hyaluronic acid, methacrylated gelatin, methacrylated hyaluronic acid, and sodium alginate; more preferably, the polymer matrix is ​​methacrylated gelatin.

[0008] Preferably, the functional manganese component is a manganese-containing silicate active material; more preferably, the functional manganese component is manganese silicate.

[0009] Preferably, the sublayer X containing bone marrow mesenchymal stem cells has 3-5 layers; the sublayer Y containing tendon stem cells has 3-5 layers.

[0010] Secondly, the present invention provides a method for preparing an immunomodulatory scaffold for integrated tendon-bone repair. The method includes: preparing precursor slurries for printing sublayers X and Y; sequentially depositing sublayers X and Y according to a specified procedure; and obtaining an immunomodulatory scaffold for integrated tendon-bone repair with bone marrow mesenchymal stem cells and tendon stem cells spatially distributed vertically through layer-by-layer stacking and photocrosslinking.

[0011] Preferably, the method for preparing the precursor slurry for printing sublayers X and Y includes: dissolving a polymer matrix in a solvent and mixing it uniformly to obtain a polymer solution; mixing a functional manganese component, the polymer solution, and a photoinitiator uniformly to obtain a mixture; and adding appropriate cells to the mixture to obtain the precursor slurry for printing.

[0012] Thirdly, the present invention provides the application of the immunomodulatory scaffold for integrated tendon-bone repair as described in any of the above claims in promoting tendon-bone interface repair. Attached Figure Description

[0013] Figure 1 The cell proliferation activity of scaffolds containing bone marrow mesenchymal stem cells and tendon stem cells was characterized. The bar charts from left to right are GelMA, GelMA-2MS, GelMA-5MS, GelMA-10MS, and GelMA-15MS.

[0014] Figure 2The biomechanical and internal structure of the manganese silicate and scaffold used in this invention are characterized. Among them, (a) is a transmission electron microscope image of manganese silicate; (b) is a scanning electron microscope image of the manganese silicate-containing scaffold; (c) is the stress-strain curve, and the curves from bottom to top are GelMA, GelMA-2MS, GelMA-5MS, and GelMA-10MS; (d) is the elastic modulus.

[0015] Figure 3 The survival and distribution of bone marrow mesenchymal stem cells and tendon stem cells in the scaffold of this invention were characterized by live-dead staining. (a) Live-dead staining images of the two types of cells in the scaffold on day 1 and day 21. (b) The arrangement of the two types of cells in the scaffold on days 1, 4, 7, and 14.

[0016] Figure 4 The ALP activity of the cell scaffolds in this invention was characterized after 7 and 14 days of culture. (a) is an ALP staining image, with circles being optical photographs and a scale bar of 1 cm; rectangles are optical photographs with a scale bar of 500 μm; (b) is the ALP activity statistics.

[0017] Figure 5 The ARS activity of the cell culture scaffold of the present invention was characterized after 14 and 21 days of culture. (a) is an ARS staining image, with circles being optical photographs and a scale bar of 0.5 cm; rectangles are optical photographs with a scale bar of 500 μm; (b) is the ARS activity statistics.

[0018] Figure 6 Immunofluorescence staining characterizes the cell scaffold cultured for 7 days according to the present invention. (a) Immunofluorescence staining of OPN in bone marrow mesenchymal stem cells and TNMD in tendon stem cells; (b) Semi-quantitative results of immunofluorescence staining.

[0019] Figure 7 The immunomodulatory effects of the scaffold of the present invention are characterized. (a) Gene expression of OPN, OCN, BMP2, Runx2, and BSP in bone marrow mesenchymal stem cells. The bars in each group, from left to right, are GelMA-Co, GelMA-Co-5MS, GelMA-Co+RAW, and GelMA-Co-5MS+RAW. (b) Gene expression of TNC, DCN, and BGN in tendon stem cells. The bars in each group, from left to right, are GelMA-Co, GelMA-Co-5MS, GelMA-Co+RAW, and GelMA-Co-5MS+RAW. (c) Gene expression of macrophage phenotypic markers CCR7, iNOS, CD206, Arg-1, and related inflammatory factors IL-6, IL-β, TNF-α, IL-10, and IL-4. The bars in each group, from left to right, are GelMA-Co+RAW and GelMA-Co-5MS+RAW.

[0020] Figure 8 Characterizing the recruitment of immune cells in the rotator cuff injury area after GelMA-cells-MS scaffold implantation. (a) Immunofluorescence staining images of CD4 (T cell marker), CD18 (neutrophil marker), CD21 (B cell marker), and CD68 (macrophage marker) in the wound at 4, 7, and 14 days post-operation. (b) Quantitative analysis of the corresponding markers for T cells, neutrophils, B cells, and macrophages. The bars in each group, from left to right, represent CD4, CD18, CD21, and CD68.

[0021] Figure 9 Characterizing the in vivo immunomodulatory effect of the scaffold of the present invention. (a) CD68 / Arg-1 positive cells 14 days after scaffold implantation; (b) Arg-1 positive cell percentage statistics, with the bars in each group from left to right being GelMA-cells and GelMA-cells-MS.

[0022] Figure 10 Gait characterization data acquisition. (a) is the walking channel device for recording rat footprints; (b) is a schematic diagram of footprint parameters; (c) are representative paw prints from each group 8 weeks post-surgery.

[0023] Figure 11 Characterizing the repair results of cell-containing scaffolds for rat rotator cuff injuries: gait analysis. (a) gait length analysis; (b) gait width analysis; (c) gait length asymmetry index; (d) paw length asymmetry index; (e) toe width asymmetry index in rats 1, 4, and 8 weeks after scaffold implantation. The bar charts from left to right represent Blank, GelMA-cells, GelMA-cells-MS, and Normal (Normal group only included at 8 weeks).

[0024] Figure 12 Characterization of the repair effect of cell-containing scaffolds on rat rotator cuff injuries: biomechanical analysis. (a) Schematic diagram of mechanical testing; (b) Ultimate load of tendon-bone failure at 8 weeks postoperatively; (c) Stiffness of tendon-bone at 8 weeks postoperatively.

[0025] Figure 13 Characterizing the repair effect of cell-containing scaffolds on rabbit rotator cuff injuries: Safranin-Fix-Green staining, toluidine blue staining, and quantitative analysis of the tendon-bone interface at 12 weeks post-surgery.

[0026] Figure 14 Characterizing the repair effect of cell-containing scaffolds on rabbit rotator cuff injuries: H&E staining and Masson staining of the tendon-bone interface at 12 weeks postoperatively.

[0027] Figure 15Characterizing the repair effect of cell-containing scaffolds on rabbit rotator cuff injuries: Micro-computed tomography (Micro-CT) images of bone 12 weeks post-surgery, and quantitative analysis of the volume ratio of new bone to the defect area. Detailed Implementation

[0028] The present invention is further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention. Unless otherwise specified, all percentage contents refer to mass percentage contents.

[0029] The integrated tendon-bone repair immunomodulatory scaffold described in this invention can also be called an integrated tendon-bone repair immunomodulatory scaffold, or a cell scaffold that can immunomodulate and promote tendon-bone regeneration, or an integrated tendon-bone repair immunomodulatory scaffold.

[0030] The immunomodulatory scaffold is a three-dimensional framework with a layered structure, comprising a sublayer X containing bone marrow mesenchymal stem cells and a sublayer Y containing tendon stem cells. In sublayer X, bone marrow mesenchymal stem cells are uniformly distributed within a polymeric framework containing functional manganese components, and in sublayer Y, tendon stem cells are uniformly distributed within a polymeric framework containing functional manganese components. Sublayers X and Y are stacked layer by layer to obtain the immunomodulatory scaffold for integrated tendon-bone repair.

[0031] The precursor slurry used for printing sublayer X comprises a polymer matrix, a functional manganese component, and bone marrow mesenchymal stem cells; wherein the polymer matrix has a mass concentration of 4-8% (w / v), the functional manganese component accounts for 2-15% of the polymer matrix mass, and the cell concentration is (4-6)×10⁻⁶. 6 per mL.

[0032] Similarly, the precursor slurry used to print sublayer Y comprises a polymer matrix, a functional manganese component, and tendon stem cells; wherein the polymer matrix has a mass concentration of 4-8% (w / v), the functional manganese component accounts for 2-15% of the polymer matrix mass, and the cell concentration is (4-6)×10⁻⁶. 6 per mL.

[0033] The polymer matrix (polymer component) should have good biocompatibility and meet the following requirements: 1) It should be able to flow in aqueous solutions and within a cell-friendly temperature range, and be thermosensitive at different temperatures. 2) It should have good molding properties, meaning that the polymer matrix can be smoothly extruded into uniform filamentous strips and its shape fixed under the action of a pneumatic extrusion printer. 3) It should have good mass exchange capacity, with sufficient space inside the scaffold for the transport of nutrients and metabolic products with the culture medium.

[0034] The polymer matrix includes, but is not limited to, at least one of hyaluronic acid, methacrylated gelatin, methacrylated hyaluronic acid, and sodium alginate. Preferably, the polymer matrix is ​​methacrylated gelatin. Methacrylated gelatin is soluble in water at 50-70°C, and cells are then added to the resulting aqueous solution to form a precursor for printing. A photoinitiator may also be added if necessary. This precursor is then frozen, 3D printed, and photocrosslinked to obtain a scaffold of a specific shape.

[0035] In some embodiments, the mass concentration of the polymer matrix is ​​4-8% (w / v). To balance cell compatibility and precursor printing performance, the concentration of the polymer matrix is ​​particularly preferably 6% (w / v).

[0036] The functional manganese component is a manganese-containing silicate active material. Preferably, the functional manganese component is manganese silicate. In some embodiments, the functional manganese component accounts for 2-15% of the polymer matrix mass. Excessive concentration of the functional manganese component may be detrimental to the long-term survival of cells in the scaffold. Preferably, the functional manganese component accounts for 5% of the polymer matrix mass. Nanoscale (approximately 100 nm particle size) manganese silicate can be used.

[0037] The live cells include at least one of bone marrow mesenchymal stem cells and tendon stem cells. As an example, the cells are used in a cell suspension. In some embodiments, the cell concentration in the precursor slurry is (4-6) × 10⁻⁶. 6 Cells / mL, preferably 5×10⁻⁶ 6 per mL.

[0038] The preparation method of the immunomodulatory scaffold for integrated tendon-bone repair is then illustrated by way of example.

[0039] The functional manganese component is pretreated. UV-sterilized manganese silicate can be used as the functional manganese component. In some embodiments, the manganese silicate is pre-sterilized with UV light for 3-5 hours.

[0040] The polymer matrix is ​​dissolved in a solvent and mixed thoroughly to obtain a polymer solution. In some embodiments, methacrylated gelatin is dissolved in water and heated in a water bath at 50-70°C for 30 minutes until completely dissolved, during which the mixture is thoroughly homogenized by shaking. The hot solution is then rapidly filtered using a filter and a syringe.

[0041] A mixture is obtained by mixing the functional components and the polymer solution. In some embodiments, the functional components, photoinitiator, and polymer solution are mixed. For example, the functional components, methacrylated gelatin, and photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphonate) are mixed. In some technical solutions, manganese silicate is mixed into the solution (manganese silicate accounts for 2-15% of the polymer matrix mass), and the mixture is sealed and protected from light in a sterile refrigerator at 4°C. Before use, the mixture in the refrigerator is heated to 37°C.

[0042] A precursor solution for printing is prepared by adding (live) cells. The precursor is thermosensitive and crosslinkable, and is in a gel state after refrigeration before printing. In some embodiments, cells with a confluence of 70-90% are collected after digestion and centrifugation, and resuspended in a mixture to prepare a cell concentration of (4-6)×10⁻⁶. 6 A suspension of cells / mL was prepared. The suspension was transferred to a temperature-controlled container and cooled at 4°C for 10-30 minutes to form a gel. The live cell components were uniformly dispersed in the precursor slurry.

[0043] The precursor slurry is printed into a scaffold using a bio-3D printer and a pre-defined program. In some embodiments, the cell scaffold is obtained by irradiating it with lithium phenyl-2,4,6-trimethylbenzoylphosphonate and blue light after printing.

[0044] In some preferred embodiments, the scaffold is layered and printed from a precursor slurry X containing bone marrow mesenchymal stem cells and a precursor slurry Y containing tendon stem cells. For example, 3-5 layers of precursor slurry X containing bone marrow mesenchymal stem cells are deposited, followed by 3-5 layers of precursor slurry Y containing tendon stem cells, and so on, to form a scaffold containing both bone marrow mesenchymal stem cells and tendon stem cells. This layered structure remains stable during culture, and the cells within the scaffold exhibit good viability. The functional components promote both osteogenic and tendonogenic differentiation; therefore, thanks to the functional release of manganese ions from the scaffold, cells differentiate into osteogenic and tendonogenic groups, respectively.

[0045] This invention utilizes bio-inks containing tendon stem cells and bone marrow mesenchymal stem cells respectively to co-print and construct a cell scaffold mimicking a tendon-bone structure. The two types of cells are spatially distributed vertically within the scaffold, with the lower layer corresponding to bone marrow mesenchymal stem cells and the upper layer corresponding to tendon stem cells, resulting in an immunomodulatory scaffold for integrated tendon-bone repair.

[0046] The aforementioned immunomodulatory scaffold exhibits enhanced effects in the presence of macrophages. For example, the cell scaffold and macrophages were indirectly co-cultured in a Transwell chamber, with the cell scaffold placed in the upper layer and macrophages cultured in the lower layer. In the presence of macrophages, manganese silicate stimulates macrophages, creating a beneficial immune microenvironment that further enhances the differentiation of bone marrow mesenchymal stem cells and tendon stem cells.

[0047] Existing scaffolds containing living cells typically benefit cellular activity through the direct stimulation of tissue cells (endothelial cells, bone marrow mesenchymal stem cells, nerve cells, endothelial cells, fibroblasts, etc.) by the active ions released from inorganic materials. However, the immunomodulatory scaffold described in this invention demonstrates that, in addition to the direct stimulation of tissue cells by active ions, there is also an immune-modulatory pattern in which active ions indirectly stimulate tissue cells by stimulating immune cells (macrophages) to form an immune microenvironment.

[0048] This invention also discloses the application of the aforementioned immunomodulatory scaffold in the repair of rotator cuff injuries. Specifically, it describes the application of the immunomodulatory scaffold in promoting tendon-bone interface repair by regulating the immune microenvironment around the wound after rotator cuff loss. The cell scaffold of this invention has been demonstrated to exhibit beneficial effects of integrated tendon-bone repair in animal models, which is attributed to the scaffold's unique immunomodulatory function. Specifically, a rotator cuff injury model was established in SD rats. After surgical implantation of the cell scaffold, the repair function of the scaffold was verified through gait analysis and biomechanical testing. The delivery of cells and the release of manganese silicate active ions within the scaffold provide an anti-inflammatory immune microenvironment and missing tissue cells to the injury site, facilitating efficient and high-quality repair.

[0049] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0050] Example 1

[0051] Preparation of cell scaffolds

[0052] Manganese silicate was sterilized with UV light for 4 hours beforehand. 6% (w / v) methacrylated gelatin was dissolved in water and heated in a 60°C water bath for 30 minutes until completely dissolved, with agitation to ensure thorough homogenization. The hot solution was rapidly filtered using a filter and syringe. Manganese silicate was then added to the solution in gradient amounts: 2%, 5%, 10%, and 15% of the polymer matrix (methacrylated gelatin) by mass (GelMA-2MS, GelMA-5MS, GelMA-10MS, GelMA-15MS). A solution without manganese silicate served as a control (GelMA). The mixture was sealed and protected from light in a sterile 4°C refrigerator. The mixture in the refrigerator was heated to 37°C for later use. Cells with a confluence of 70-90% were collected after digestion and centrifugation, and resuspended in the mixture to prepare a cell concentration of 5 × 10⁶ cells / mL. 6 A suspension of cells / mL was prepared. The suspension was transferred to a temperature-controlled container and cooled at 4°C for 20 minutes to form a gel. Bone marrow mesenchymal stem cell precursor X and tendon stem cell precursor Y were deposited layer by layer according to a specified procedure, first depositing 4 layers of X, then 4 layers of Y. During the 21-day culture period, proliferation activity was measured by CCK8 assay on days 1, 7, 14, and 21. Cell viability was observed by live / dead staining on days 1 and 21. Cell distribution was observed on days 1, 4, 7, and 14.

[0053] Figure 1 The results of culturing CCK8 cells on cell scaffolds for 21 days showed that, except for GelMA-15MS, all other groups promoted cell proliferation. This indicates that manganese silicate has good biocompatibility. Figure 2 (a) is manganese silicate used in this invention, which has a hollow structure and a particle size of about 100 nm. Figure 2 As can be seen from (b), the scaffold has a large porous structure, which is the basis for the swelling of the hydrogel, and the swelling property is conducive to the exchange of nutrients and metabolic waste. Figure 2 (c) and (d) show that the elastic modulus of the scaffold gradually increases with the increase of manganese silicate content. The elastic modulus of the GelMA-10MS scaffold (4.7 kPa) is 1.7 times that of the GelMA scaffold (2.1 kPa), indicating that the incorporation of manganese silicate enhances the mechanical properties of GelMA hydrogel, which is beneficial to osteogenic differentiation of bone marrow mesenchymal stem cells. Figure 3 (a) indicates that cell viability staining suggests that the scaffold can support long-term cell survival. Figure 3 (b) shows that the two cell types in the scaffold are spatially distributed vertically, with the lower layer corresponding to bone marrow mesenchymal stem cells and the upper layer corresponding to tendon stem cells. During the 14-day culture period, both cell types maintained this distribution, indicating that the prepared cell scaffold conformed to the design and had long-term stability.

[0054] Example 2

[0055] The role of cell scaffolds in tendon and osteogenic differentiation

[0056] Manganese silicate was sterilized with UV light for 4 hours beforehand. 6% (w / v) methacrylated gelatin was dissolved in water and heated in a 60°C water bath for 30 minutes until completely dissolved, with shaking to ensure thorough homogenization. The hot solution was rapidly filtered using a filter and syringe. Manganese silicate was then added to the solution in gradient amounts, at concentrations of 2%, 5%, and 10% of the polymer matrix mass (GelMA-2MS, GelMA-5MS, GelMA-10MS). A solution without manganese silicate served as a control (GelMA). The solution was sealed and protected from light in a sterile 4°C refrigerator. The mixture in the refrigerator was heated to 37°C for later use. Cells with a confluence of 70-90% were collected after digestion and centrifugation, and resuspended in the mixture to prepare a cell concentration of 5 × 10⁶ cells / mL. 6 The suspension was prepared at a concentration of [number] cells / mL. The suspension was transferred to a temperature-controlled container and cooled at 4°C for 20 minutes to form a gel. Bone marrow mesenchymal stem cell precursor X was deposited in 8 layers to obtain a bone marrow mesenchymal stem cell scaffold, and tendon stem cell precursor Y was deposited in 8 layers to obtain a tendon stem cell scaffold. Osteogenic properties of the scaffolds were characterized. At days 7 and 14, early osteogenic marker ALP staining and quantitative analysis were performed, and at days 14 and 21, terminal osteogenic marker ARS staining and quantitative analysis were performed, respectively. On day 7, the osteogenic marker OPN and the tendonogenic marker TNMD were subjected to immunofluorescence staining.

[0057] Figure 4 It can be seen that GelMA-2MS and GelMA-5MS enhanced ALP activity in the cell scaffold on days 7 and 14. GelMA-10MS had no significant effect on ALP activity on day 7, but enhanced it on day 14. This indicates that ALP activity did not increase positively with increasing MS concentration; specific doping concentrations had a more significant effect on enhancing ALP activity. ALP is an early marker of osteogenic differentiation, and calcium nodules are deposited extensively in the terminal stage of osteogenic differentiation. Qualitative analysis of calcium nodules was performed using Alizarin Red staining, and semi-quantitative analysis was performed after dissolving the calcium nodules. Figure 5It can be seen that, compared with the control group, GelMA-2MS, GelMA-5MS, and GelMA-10MS all promoted the formation of calcium nodules on days 14 and 21. On day 14, GelMA-5MS showed the highest calcium nodule formation, while there was no significant difference in calcium nodule formation between GelMA-2MS and GelMA-10MS. On day 21, the calcium nodule formation in all groups increased compared with day 14, with GelMA-5MS and GelMA-10MS showing higher calcium nodule formation than GelMA-2MS, and GelMA-5MS still maintained the highest formation rate. Figure 6 This study presents immunofluorescence staining and semi-quantitative statistical results for OPN in bone marrow mesenchymal stem cells and TNMD in tendon stem cells. OPN is an osteogenic-related protein that plays an important role in bone formation and remodeling. Compared with the GelMA group, OPN expression was increased in the GelMA-MS group, with the highest expression in the GelMA-5MS group. TNMD, a transmembrane glycoprotein marker of tendon cell maturation, showed significantly increased expression in the GelMA-2MS and GelMA-5MS groups compared to the GelMA group. The highest TNMD expression was observed in the GelMA-5MS group, which was also significantly higher than in the GelMA-2MS and GelMA-10MS groups. The combined results of ALP staining, ARS staining, immunofluorescence staining, and semi-quantitative analysis indicate that MS nanoparticles have osteogenic and tendonogenic differentiation effects on bone marrow mesenchymal stem cells and tendon stem cells, respectively.

[0058] Example 3

[0059] In vitro immunomodulatory effects of cell scaffolds

[0060] Manganese silicate was sterilized with UV light for 4 hours beforehand. 6% (w / v) methacrylated gelatin was dissolved in water and heated in a 60°C water bath for 30 minutes until completely dissolved, with shaking to ensure thorough homogenization. The hot solution was rapidly filtered using a filter and syringe. Manganese silicate was then added to the solution, at a concentration of 5% of the polymer matrix. A solution without manganese silicate served as a control. The mixture was sealed and protected from light in a sterile 4°C refrigerator. The mixture in the refrigerator was then heated to 37°C for later use. Cells with a confluence of 70-90% were collected after digestion and centrifugation, and resuspended in the mixture to prepare a cell concentration of 5 × 10⁻⁶ cells / mL. 6A suspension of macrophages was prepared at a concentration of [number] cells / mL. The suspension was transferred to a temperature-controlled container and cooled at 4°C for 20 minutes to form a gel. Bone marrow mesenchymal stem cell precursor X and tendon stem cell precursor Y were deposited layer by layer according to a specified procedure, first depositing 4 layers of X, then 4 layers of Y. Both scaffold compositions were indirectly co-cultured with macrophages in Transwell chambers, with the cell scaffold placed on the upper layer and macrophages cultured on the lower layer, labeled GelMA-Co+RAW and GelMA-Co-5MS+RAW. Scaffolds without macrophages served as controls, labeled GelMA-Co and GelMA-Co-5MS. After 7 days of culture, the expression of osteogenic and tendonogenic genes in the scaffolds was measured. The expression of inflammatory factors in the two macrophage-containing groups was also compared.

[0061] Figure 7 As shown in (a) and (b), the manganese silicate-containing scaffold enhanced the expression of osteogenic and tendonogenic genes, while the presence of macrophages alone could not promote their differentiation. The enhancement effect was more significant when both manganese silicate and macrophages were present. This indicates that manganese silicate and macrophages form an immune microenvironment that promotes tendon-bone differentiation. Figure 7 In contrast to (c), under the influence of manganese silicate, macrophages secreted more anti-inflammatory factors such as IL-4 and IL-10, while the expression of pro-inflammatory factors such as IL-6 and IL-1β decreased. This further confirms the immunomodulatory effect of manganese silicate.

[0062] Example 4

[0063] In vivo immune regulation by cell scaffolds

[0064] A rat rotator cuff injury model was established to evaluate the immunomodulatory effects of multicellular scaffolds in vivo. Forty male Sprague-Dawley rats (6 weeks old) were selected and randomly divided into four groups: normal (no surgery), Blank (no scaffold), GelMA-Cells (no MS scaffold), and GelMA-Cells-MS (MS and cell-containing scaffold). After anesthesia, the rats were placed in a lateral decubitus position. The anterolateral aspect of the shoulder was incised, and the deltoid muscle was cut to expose the supraspinatus tendon. The supraspinatus tendon was then damaged with a scalpel, and a 0.8 mm diameter bone tunnel was drilled at the insertion point. Finally, the scaffold was fixed to the tendon-bone interface, and the tendon and bone were sutured together. The skin incision was then sutured. At predetermined time points (4, 7, and 14 days post-surgery), the cell types and phenotypes of the tissues surrounding the scaffold were detected by immunofluorescence staining.

[0065] Figure 8Immunofluorescence staining and semi-quantitative analysis showed that within 14 days post-surgery, the most abundant cells around the GelMA-Cells-MS scaffold were macrophages (CD68+ cells), while other immune cells such as neutrophils (CD18+ cells), T cells (CD4+ cells) and B cells (CD21+ cells) were relatively few.

[0066] Figure 9 Immunofluorescence staining for Arg-1 / CD68 was used to identify the phenotype of macrophages recruited around the scaffold. Quantitative analysis showed that at 3 days post-surgery, the proportion of Arg-1 positive cells was low in both groups, with no statistically significant difference between the two groups. However, at 7 and 14 days post-surgery, the proportion of M2 macrophages (Arg-1+ / CD68+ cells) in the GelMA-cells-MS group was significantly higher than that in the GelMA-cells group. These results indicate that the MS-based scaffold has significant immunomodulatory function, enabling the recruited macrophages to polarize to the M2 phenotype.

[0067] Example 5

[0068] The effect of cell scaffolds on the repair of rotator cuff injuries in rats

[0069] A rat model of rotator cuff injury was established to evaluate the immunomodulatory effect of multicellular scaffolds in vivo. Male Sprague-Dawley rats (6 weeks old) were selected and randomly divided into four groups: normal (no surgery), Blank (no scaffold), GelMA-Cells (no MS scaffold), and GelMA-Cells-MS (MS and cell-containing scaffold). After anesthesia, the rats were placed in a lateral decubitus position. The anterolateral aspect of the shoulder was incised, and the deltoid muscle was dissected to expose the supraspinatus tendon. The supraspinatus tendon was then damaged with a scalpel, and a 0.8 mm diameter bone tunnel was drilled at the insertion point. Finally, the scaffold was fixed to the tendon-bone interface, and the tendon and bone were sutured together. The skin incision was then sutured. Gait analysis was performed at predetermined time points (1, 4, and 8 weeks post-surgery). The remaining rats were sacrificed 8 weeks post-surgery, and the biomechanical properties of the rotator cuff were evaluated.

[0070] Gait analysis is an effective method for assessing functional recovery after rotator cuff tears, providing quantifiable behavioral data on animal movement. Figure 10 (a) The device is used to collect gait data. Figure 10 (b) and (c) are schematic diagrams of gait parameters and claw print images collected 8 weeks post-surgery, respectively. Figure 11It can be seen that gait abnormalities were observed in all groups of rats one week after surgery, with no statistically significant differences in any parameters. Although all rats showed a tendency to recover over time, stride length and stride width in the GelMA-cells and GelMA-cells-MS groups showed significant recovery at four weeks post-surgery, while the control group only showed partial recovery. Figure 11 (a)-(b)). The asymmetry of other parameters also confirms this difference. Figure 11 (c), (d), and (e)). The differences increased further at 8 weeks post-surgery, with the GelMA-cells-MS group showing advantages in all indicators. Notably, all gait parameters in the GelMA-cells-MS group showed continuous improvement over time.

[0071] The mechanical properties of the tendon-bone junction are an important indicator for evaluating the healing quality and shoulder function recovery after rotator cuff repair. Figure 12 (a) Mechanical testing was performed on rotator cuff samples from each group 8 weeks post-surgery. The ultimate destructive load of the GelMA-cells-MS group (19.93±1.56N) was significantly higher than that of the GelMA-cells and blank groups. Figure 12 (b) indicates that there was no significant difference between the GelMA-cells-MS group and the normal group. Figure 12 (c) indicates that in the stiffness analysis, although the stiffness of the rotator cuff in the GelMA-cells-MS group did not return to normal after surgery, it was still significantly better than that in the blank group.

[0072] In summary, compared to other conditions, rats with rotator cuff injuries exhibited better gait behavior after repair with GelMA-cells-MS, and the biomechanical properties of the newly formed tendon-bone interface were significantly improved. This demonstrates the excellent repair effect of the GelMA-cells-MS scaffold on rotator cuff injuries.

[0073] Example 6

[0074] The effect of cell scaffolds on the repair of rotator cuff injuries in rabbits

[0075] A rabbit rotator cuff injury model was established to evaluate the repair effect of multicellular scaffolds in vivo. Male adult New Zealand white rabbits weighing 3.0±0.5 kg were randomly divided into four groups: Blank group, GelMA-cells scaffold group, MS-cells scaffold group, and MS and two-cells scaffold group (GelMA-cells-MS). The surgical procedure was as follows: The rabbits were fixed in a supine position and anesthetized. The shoulder area was shaved and disinfected. The skin was incised to expose the rotator cuff tissue. The supraspinatus tendon was then dissected at the greater tubercle, and a 4 mm diameter hole was drilled at the greater tubercle. A 4 mm diameter scaffold was inserted into the hole. Finally, the wound was sutured with 3-0 sutures. The rabbits were sacrificed at 12 weeks post-surgery. The healing effect was evaluated using safranin-fast green staining, toluidine blue staining, H&E staining, Masson staining, and micro-computed tomography (Micro-CT).

[0076] Fibrocartilage regeneration is a key indicator for evaluating the repair effect of rotator cuff injuries. Safranin-Fix-Green staining and toluidine blue staining are used to mark the newly formed fibrocartilage at the tendon-bone interface. For example... Figure 13 As shown, compared to the control group, the implanted scaffold group had a higher proportion of newly formed fibrocartilage, with the GelMA-cells-MS group exhibiting the best effect in promoting fibrocartilage regeneration. Next, H&E staining results at 12 weeks post-operation (…) Figure 14 As can be seen, the Blank group contained obvious unhealed gaps, inflammatory infiltration was observed in both the Blank and GelMA-cells groups, while inflammation was significantly reduced in the GelMA-MS group. Furthermore, the GelMA-cells-MS group showed almost no inflammatory infiltration and high orderliness at the interface, with the presence of tidal lines. Masson staining results showed that the implanted scaffold group exhibited more collagen deposition. In summary, MS can inhibit inflammation, and the combined use of MS and cells (i.e., GelMA-cells-MS) significantly enhanced the repair quality of tendon-bone interface injuries in rabbits.

[0077] Furthermore, bone defects in rabbits can be reconstructed using Micro-CT. For example... Figure 15 As shown, the newly formed bone tissue in the Blank group was discontinuous. In contrast, the GelMA-MS and GelMA-cells-MS groups containing MS formed intact cortical bone. Statistical results regarding the volume ratio of newly formed bone to the defect area indicate that the GelMA-cells-MS scaffold has the best bone repair capacity.

[0078] Therefore, the rabbit rotator cuff injury model further confirmed the integrated repair effect of the GelMA-cells-MS scaffold on tendon-bone.

Claims

1. The application of an immunomodulatory scaffold containing manganese silicate nanoparticles in the preparation of materials for restoring motor function after tendon-bone interface injury, characterized in that, The immunomodulatory scaffold is a three-dimensional framework with a layered structure, comprising a sublayer X containing bone marrow mesenchymal stem cells and a sublayer Y containing tendon stem cells; in sublayer X, bone marrow mesenchymal stem cells are uniformly distributed within a polymeric framework containing nanoscale manganese silicate, and in sublayer Y, tendon stem cells are uniformly distributed within a polymeric framework containing nanoscale manganese silicate; the sublayers X and Y are stacked layer by layer to obtain an immunomodulatory scaffold containing manganese silicate nanoparticles. The precursor slurry used to print sublayer X comprises a polymer matrix, nano-sized manganese silicate, bone marrow mesenchymal stem cells, and a photoinitiator; wherein the polymer matrix has a mass concentration of 4%-8% w / v, the nano-sized manganese silicate accounts for 2%-10% of the polymer matrix mass, and the bone marrow mesenchymal stem cell concentration is (4-6)×10 6 cells / mL; The precursor slurry used for printing sublayer Y comprises a polymer matrix, nano-sized manganese silicate, tendon stem cells, and a photoinitiator; wherein the polymer matrix has a mass concentration of 4%-8% w / v, the nano-sized manganese silicate accounts for 2%-10% of the polymer matrix mass, and the tendon stem cell concentration is (4-6)×10 6 cells / mL; The polymer matrix is ​​methacrylated gelatin; The method for preparing the immunomodulatory scaffold containing manganese silicate nanoparticles includes: preparing precursor slurries for printing sublayers X and Y respectively; sequentially depositing sublayers X and Y according to a specified procedure; and obtaining an immunomodulatory scaffold containing manganese silicate nanoparticles with bone marrow mesenchymal stem cells and tendon stem cells spatially distributed vertically through layer-by-layer stacking and photocrosslinking.

2. The application according to claim 1, characterized in that, Sublayer X, containing bone marrow mesenchymal stem cells, consists of 3-5 layers; sublayer Y, containing tendon stem cells, consists of 3-5 layers.

3. The application according to claim 1, characterized in that, The method for preparing the precursor slurry for printing sublayers X and Y includes: dissolving a polymer matrix in a solvent and mixing it evenly to obtain a polymer solution; mixing nano-sized manganese silicate, the polymer solution and a photoinitiator evenly to obtain a mixture; and adding the corresponding cells to the mixture to obtain the precursor slurry for printing.

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