Injectable osteochondral repair material and method of making same

Abstract

The application provides an injectable osteochondral repair material and a preparation method thereof, and the injectable osteochondral repair material comprises a cartilage repair solution, a bone repair solution and a neutral ion trigger solution; the cartilage repair solution comprises a first collagen, a phosphoserine modified magnesium-based microsphere and a first acid solvent; the bone repair solution comprises a second collagen, a phosphoserine modified magnesium-based mineralized microsphere and a second acid solvent, the phosphoserine modified magnesium-based mineralized microsphere comprises a magnesium-based core, a phosphoserine interface coupling layer and a calcium-phosphorus mineralized precursor coating, and the phosphoserine interface coupling layer couples the magnesium-based core and the calcium-phosphorus mineralized precursor coating. By using the injectable osteochondral repair material, synchronous repair of cartilage and subchondral bone and continuous reconstruction of an osteochondral interface can be realized.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, and in particular to injectable osteochondral repair materials and their preparation methods. Background Technology

[0002] Osteoarthritis (OA) is a chronic degenerative disease characterized by degeneration of articular cartilage, subchondral bone remodeling, synovial inflammation, and joint dysfunction. It is one of the leading causes of chronic pain, limited mobility, and long-term disability in adults. Osteoarthritis most commonly affects synovial joints such as the knee, hip, and hand.

[0003] Synovial joints are composed of articular cartilage, calcified cartilage, subchondral bone plates, subchondral cancellous bone, synovium, menisci, ligaments, and joint capsule. Among these, articular cartilage and subchondral bone together form the osteochondral unit, which is the core structure maintaining low-friction joint movement, load transmission, and mechanical cushioning. Articular cartilage primarily performs low-friction sliding and compression cushioning functions; while subchondral bone provides mechanical support, load distribution, and nutritional regulation of deeper cartilage.

[0004] Osteoarthritis is not simply a "cartilage wear disease," but a degenerative disease involving multiple tissues, including articular cartilage, calcified cartilage, subchondral bone, synovium, meniscus, and the immune microenvironment. In the early stages of osteoarthritis, pathological processes such as abnormal subchondral bone remodeling, bone marrow lesions, vascular and nerve invasion, and changes in the osteocartilage interface may occur before obvious cartilage degeneration. These processes continuously affect chondrocyte metabolism and extracellular matrix homeostasis through bone-cartilage crosstalk, ultimately leading to increased cartilage damage and joint dysfunction. Therefore, repair strategies that solely target cartilage defects often fail to achieve long-term stable therapeutic effects. Achieving simultaneous repair of cartilage and subchondral bone, as well as continuous reconstruction of the osteocartilage interface, is a pressing issue that needs to be addressed. Summary of the Invention

[0005] Based on this, this application provides an injectable osteochondral repair material and its preparation method to achieve synchronous repair of cartilage and subchondral bone and continuous reconstruction of the osteochondral interface.

[0006] The first aspect of this application provides an injectable osteocartilage repair material, comprising: a cartilage repair solution, a bone repair solution, and a neutral ion triggering solution; the cartilage repair solution comprises a first collagen, phosphoserine-modified magnesium-based microspheres, and a first acidic solvent; the bone repair solution comprises a second collagen, phosphoserine-modified magnesium-based mineralized microspheres, and a second acidic solvent, wherein the phosphoserine-modified magnesium-based mineralized microspheres comprise a magnesium-based core, a phosphoserine interface coupling layer, and a calcium phosphate mineralization precursor coating, the phosphoserine interface coupling layer coupling the magnesium-based core and the calcium phosphate mineralization precursor coating.

[0007] In some embodiments, the mass percentage of the phosphoserine-modified magnesium microspheres relative to the total mass of the first collagen and the first acidic solvent is 0.05wt%-5wt%.

[0008] In some embodiments, the mass percentage of the phosphoserine-modified magnesium-based mineralized microspheres relative to the total mass of the second collagen and the second acidic solvent is 0.1 wt% to 20 wt%.

[0009] In some embodiments, the mass-volume concentration of the first collagen in the cartilage repair solution is 4 mg / mL to 8 mg / mL.

[0010] In some embodiments, the mass-volume concentration of the second collagen in the bone repair solution is 4 mg / mL to 8 mg / mL.

[0011] In some embodiments, the cartilage repair solution further comprises a first additive, the first additive including one or more of hyaluronic acid, chondroitin sulfate and oxidized chondroitin sulfate.

[0012] In some embodiments, the mass-volume concentration of the first additive in the cartilage repair solution is 0.1 mg / mL to 5 mg / mL.

[0013] In some embodiments, the bone repair solution further comprises a second additive, the second additive including one or more of hyaluronic acid, chondroitin sulfate, and oxidized chondroitin sulfate.

[0014] In some embodiments, the mass-volume concentration of the second additive in the bone repair solution is 0.1 mg / mL to 5 mg / mL.

[0015] In some embodiments, the pH value of the neutral ion triggering solution is 7.2-8.2, optionally 7.4-7.8.

[0016] In some embodiments, the neutral ion triggering solution includes a buffer system, an ion regulating component, and a physiological salt system.

[0017] In some embodiments, the buffer system includes one or more of PBS buffer, HEPES buffer, Tris buffer, and NaHCO3 buffer.

[0018] In some embodiments, the ion-regulating component includes one or more of a calcium-containing soluble salt, a magnesium-containing soluble salt, and a potassium-containing soluble salt.

[0019] In some embodiments, the ion-regulating component includes one or more of calcium chloride, magnesium chloride, and potassium chloride.

[0020] In some embodiments, the physiological salt system is sodium chloride.

[0021] A second aspect of this application provides a method for preparing an injectable osteocartilage repair material, comprising the following steps:

[0022] Prepare a first acidic collagen solution containing a first collagen and a first acidic solvent, and a second acidic collagen solution containing a second collagen and a second acidic solvent;

[0023] The first acidic collagen solution was mixed with phosphoserine-modified magnesium microspheres to prepare a cartilage repair solution.

[0024] The second acidic collagen solution is mixed with phosphoserine-modified magnesium-based mineralized microspheres to prepare a bone repair solution; the phosphoserine-modified magnesium-based mineralized microspheres include a magnesium-based core, a phosphoserine interface coupling layer and a calcium phosphate mineralization precursor coating, wherein the phosphoserine interface coupling layer couples the magnesium-based core and the calcium phosphate mineralization precursor coating.

[0025] Prepare the neutral ion trigger solution.

[0026] In some embodiments, the mass-volume concentration of the first collagen in the first acidic collagen solution is 4 mg / mL to 8 mg / mL.

[0027] In some embodiments, the mass-volume concentration of the second collagen in the second acidic collagen solution is 4 mg / mL to 8 mg / mL.

[0028] In some embodiments, the mass percentage of the phosphoserine-modified magnesium microspheres relative to the mass of the first acidic collagen solution is 0.05wt%-5wt%.

[0029] In some embodiments, the mass percentage of the phosphoserine-modified magnesium-based mineralized microspheres relative to the mass of the second acidic collagen solution is 0.1 wt% to 20 wt%.

[0030] In some embodiments, the first acidic collagen solution is mixed with phosphoserine-modified magnesium microspheres at 2°C-8°C.

[0031] In some embodiments, the second acidic collagen solution is mixed with phosphoserine-modified magnesium-based mineralized microspheres at 2°C-8°C.

[0032] In some embodiments, the first acidic solvent is an aqueous solution of acetic acid, wherein the molar concentration of acetic acid in the aqueous solution is 0.01 mol / L to 0.05 mol / L.

[0033] In some embodiments, the second acidic solvent is an aqueous solution of acetic acid, wherein the molar concentration of acetic acid in the aqueous solution is 0.01 mol / L to 0.05 mol / L.

[0034] In some embodiments, the first acidic collagen solution further contains a first additive, which includes one or more of hyaluronic acid, chondroitin sulfate, and oxidized chondroitin sulfate.

[0035] In some embodiments, the mass-volume concentration of the first additive in the first acidic collagen solution is 0.1 mg / mL to 5 mg / mL.

[0036] In some embodiments, the second acidic collagen solution further contains a second additive, the second additive including one or more of hyaluronic acid, chondroitin sulfate and oxidized chondroitin sulfate.

[0037] In some embodiments, the mass-volume concentration of the second additive in the second acidic collagen solution is 0.1 mg / mL to 5 mg / mL.

[0038] In some embodiments, the method for preparing the phosphoserine-modified magnesium microspheres includes: mixing a first magnesium microsphere with a first phosphoserine solution, adjusting the pH of the mixture to 6-7, and then washing and drying the first magnesium microsphere to prepare the phosphoserine-modified magnesium microspheres.

[0039] In some embodiments, the molar concentration of phosphoserine in the first phosphoserine solution is 0.5 mmol / L to 20 mmol / L.

[0040] In some embodiments, the mass-to-volume ratio of the first magnesium-based microspheres to the first phosphoserine solution is (1-50) mg:1 mL.

[0041] In some embodiments, the Dv50 of the first magnesium-based microspheres is 15 μm-60 μm.

[0042] In some embodiments, the method for preparing the phosphoserine-modified magnesium-based mineralized microspheres includes:

[0043] The second magnesium-based microspheres were mixed with a second phosphoserine solution, the pH of the mixture was adjusted to 6-7, and then the second magnesium-based microspheres were washed and dried to prepare a phosphoserine-modified magnesium-based core.

[0044] The phosphoserine-modified magnesium-based core was mixed with a mineralization solution and then incubated to prepare the phosphoserine-modified magnesium-based mineralized microspheres; wherein the mineralization solution contained calcium ions and phosphate ions.

[0045] In some embodiments, the mineralization solution contains a calcium ion source, which includes one or more of calcium chloride, calcium nitrate, and calcium acetate.

[0046] In some embodiments, the mineralization solution contains a phosphate ion source, which includes one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium phosphate.

[0047] In some embodiments, the molar ratio of calcium ions to phosphate ions in the mineralization solution is (1.3-1.67):1.

[0048] In some embodiments, the molar concentration of calcium ions in the mineralization solution is 1 mmol / L to 20 mmol / L.

[0049] In some embodiments, the molar concentration of phosphate ions in the mineralization solution is 0.5 mmol / L to 10 mmol / L.

[0050] In some embodiments, the pH of the mineralization solution is 6.5-7.5.

[0051] In some embodiments, the incubation temperature is 20°C-37°C, optionally 25°C-37°C.

[0052] In some embodiments, the incubation time is 4h-48h, optionally 10h-24h.

[0053] In some embodiments, the molar concentration of phosphoserine in the second phosphoserine solution is 0.5 mmol / L to 20 mmol / L.

[0054] In some embodiments, the mass-to-volume ratio of the second magnesium-based microspheres to the second phosphoserine solution is (1-50) mg:1 mL.

[0055] In some embodiments, the Dv50 of the second magnesium-based microspheres is 15 μm-60 μm.

[0056] In some embodiments, the pH value of the neutral ion triggering solution is 7.2-8.2, optionally 7.4-7.8.

[0057] In some embodiments, the preparation method of the neutral ion triggering solution includes: mixing a buffer system, an ion regulating component, and a physiological salt system.

[0058] In some embodiments, the buffer system includes one or more of PBS buffer, HEPES buffer, Tris buffer, and NaHCO3 buffer.

[0059] In some embodiments, the ion-regulating component includes one or more of a calcium-containing soluble salt, a magnesium-containing soluble salt, and a potassium-containing soluble salt.

[0060] In some embodiments, the ion-regulating component includes one or more of calcium chloride, magnesium chloride, and potassium chloride.

[0061] In some embodiments, the physiological salt system is sodium chloride.

[0062] The aforementioned injectable osteocartilage repair materials have at least the following beneficial effects:

[0063] (1) The injectable osteocartilage repair material adopts a three-component synergistic system of cartilage repair solution, bone repair solution and neutral ion triggering liquid. The cartilage repair solution constructs a biomimetic extracellular matrix microenvironment for cartilage cells, and the bone repair solution constructs an organic-inorganic composite mineralization system with continuous mineralization ability. The two layers of materials gel in situ under the action of neutral ion triggering liquid and form a continuous and combined osteocartilage repair structure. After injection, a continuous and combined double-layer repair structure can be formed in the defect area. There is no need for prefabricated scaffolds or regular trimming of the defect area. It can adapt to osteocartilage defects of different shapes and sizes, and improve the matching and interfacial bonding ability between the material and the host tissue.

[0064] (2) The cartilage repair solution is mainly composed of collagen, with magnesium-based microspheres modified with phosphoserine. Collagen undergoes fiber self-assembly to form a three-dimensional network under neutral environment and body temperature conditions. Phosphoserine binds through hydrogen bonds and Mg... 2+ Mediated dynamic ion bridging enhances collagen network stability and continuously releases appropriate amounts of Mg. 2+ It regulates the cartilage microenvironment, promotes chondrocyte adhesion, extracellular matrix secretion, and hyaline cartilage regeneration.

[0065] (3) The bone repair solution uses collagen as an organic template and phosphoserine-modified magnesium-based mineralized microspheres as an inorganic mineralization core. Phosphoserine, as a mineralization interface coupling molecule, enhances the binding stability between the magnesium-based core and the calcium-phosphorus mineralization precursor coating, and provides continuous nucleation sites for subsequent mineralization. After the phosphoserine-modified magnesium-based mineralized microspheres are dispersed in the collagen network, the calcium-phosphorus mineralization precursor coating further migrates into the interior and surface of the collagen fibers, undergoing continuous mineralization under the induction of the collagen template, forming a hierarchical organic-inorganic composite mineralization network, realizing subchondral bone repair and continuous repair of the osteochondral interface.

[0066] (4) In the phosphoserine-modified magnesium-based mineralized microspheres of the bone repair solution, phosphoserine constructs a stable interface between the magnesium-based core and the calcium-phosphine mineralization precursor coating, effectively reducing the initial rapid corrosion and hydrogen release of the magnesium-based material. Simultaneously, it continuously releases Ca through the calcium-phosphine mineralization precursor coating. 2+ PO4 3- and Mg 2+ This achieves synergistic regulation of ion slow release and continuous mineralization, providing a stable ion microenvironment for subchondral bone repair and bone tissue regeneration.

[0067] (5) The injectable osteocartilage repair material adopts a dual-layer functional partition design. The cartilage repair solution mainly constructs a collagen-rich, low-mineralized cartilage biomimetic microenvironment to promote chondrocyte adhesion, proliferation and hyaline cartilage matrix formation. The bone repair solution mainly constructs a bone repair layer with continuous mineralization capacity to promote subchondral bone reconstruction and osteocartilage interface mineralization repair, realize the synergistic regeneration of cartilage layer and bone layer, and reduce the risk of ectopic mineralization of cartilage layer.

[0068] (6) Compared with pre-formed block osteocartilage scaffolds, the injectable osteocartilage repair material of this application does not require regular pore enlargement or excessive trimming of the defect area, can adapt to complex and irregular defect morphologies, and reduces additional damage to residual healthy cartilage, calcified cartilage layer, and subchondral bone tissue. Simultaneously, the cured double-layer porous scaffold provides a spatial structural basis for cell migration, nutrient exchange, blood vessel ingrowth, and new tissue formation, and through Mg... 2+ The release and calcium-phosphorus mineralization process promotes subchondral bone repair and osteochondral interface reconstruction.

[0069] (7) Injectable osteocartilage repair materials have good injectability and can be implanted into osteocartilage defect areas through arthroscopic minimally invasive methods; the materials quickly gel in situ in vivo without the need for complex fixation devices, which can reduce surgical trauma and operation time, and have good clinical application value and application prospects. Attached Figure Description

[0070] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0071] Figure 1 The expression levels of cartilage differentiation-related genes SOX9 and COL II in Examples 1-2, Example 8 and Comparative Example 1 are shown.

[0072] Figure 2 Gross observations and histological results of animal experiments on osteochondral repair.

[0073] Figure 3 Immunohistochemical results of animal experiments on osteochondral repair.

[0074] Figure 4 Micro-CT results of animal experiments on osteochondral repair.

[0075] Figure 5 This is a schematic diagram illustrating the use of injectable in-situ double-layer osteochondral repair material. Detailed Implementation

[0076] To facilitate understanding of this application, a more complete description of the application will be provided below with reference to relevant embodiments. Preferred embodiments of the application are given below. However, the application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0078] As used herein, the terms "and / or," "or / and," and "and / or" encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that, in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR."

[0079] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0080] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0081] This document only specifically discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, each individually disclosed point or single value can itself serve as a lower or upper limit and be combined with any other point or single value or with other lower or upper limits to form an unspecified range.

[0082] Unless otherwise specified, the temperature parameters in this application may be either constant temperature processing or processing within a certain temperature range. The constant temperature processing allows temperature fluctuations within the precision range controlled by the instrument, such as ±5°C, ±4°C, ±3°C, ±2°C, or ±1°C.

[0083] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0084] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0085] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0086] In the description of the application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0087] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions. Unless otherwise specified, all technical features and optional technical features of this application can be combined to form new technical solutions.

[0088] Unless otherwise specified, all steps of this application may be performed sequentially or randomly, but sequentially is preferred.

[0089] It should be noted that in this article, terms such as "first collagen," "second collagen," "first acidic solvent," "second acidic solvent," "first additive," "second additive," "first acidic solvent," "second acidic solvent," "first acidic collagen solution," and "second acidic collagen solution" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Furthermore, terms such as "first" and "second" serve only as a non-exhaustive enumeration and should be understood as not constituting a closed-ended limitation on quantity.

[0090] Osteoarthritis is not simply a "cartilage wear disease," but a degenerative disease involving multiple tissues, including articular cartilage, calcified cartilage, subchondral bone, synovium, meniscus, and the immune microenvironment. In the early stages of osteoarthritis, pathological processes such as abnormal subchondral bone remodeling, bone marrow lesions, vascular and nerve invasion, and changes in the osteocartilage interface may occur before obvious cartilage degeneration. These processes continuously affect chondrocyte metabolism and extracellular matrix homeostasis through bone-cartilage crosstalk, ultimately leading to increased cartilage damage and joint dysfunction. Therefore, repair strategies that solely target cartilage defects often fail to achieve long-term stable therapeutic effects. Achieving simultaneous repair of cartilage and subchondral bone, as well as continuous reconstruction of the osteocartilage interface, is a pressing issue that needs to be addressed.

[0091] Currently, the cartilage repair techniques used in clinical practice mainly include microfracture surgery, autologous osteochondral transplantation, autologous chondrocyte transplantation (ACI), matrix-induced autologous chondrocyte transplantation (MACI), and tissue engineering scaffold repair, etc., which generally have the following problems: (1) It is difficult to simultaneously repair cartilage and subchondral bone, as well as continuously reconstruct the osteochondral interface; (2) Pre-formed block or membrane structures require defect shaping, open or semi-open implantation, which is difficult to adapt to complex and irregular defect morphologies and may cause additional iatrogenic damage to normal cartilage and subchondral bone tissue; (3) The adaptability of minimally invasive operation is limited, especially for deep, complex morphology and weight-bearing area defects, the implantation operation is complicated and the operator's technical requirements are high; (4) There is a lack of active regulation of the pathological microenvironment such as inflammation, abnormal bone remodeling and osteochondral crosstalk.

[0092] To address the aforementioned issues, this application provides an injectable osteocartilage repair material, comprising a cartilage repair solution, a bone repair solution, and a neutral ion triggering solution. The cartilage repair solution contains a first collagen protein, phosphoserine-modified magnesium microspheres, and a first acidic solvent. After injection, under neutral ion conditions and body temperature, the collagen protein undergoes fiber self-assembly to form a three-dimensional network, and the phosphoserine protein, through hydrogen bonds and magnesium... 2+ Ion bridging enhances the stability of the collagen network, forming a biomimetic microenvironment for cartilage with good bioactivity, thereby promoting chondrocyte adhesion, proliferation, and hyaline cartilage regeneration. The bone repair solution contains a second collagen protein, phosphoserine-modified magnesium-based mineralized microspheres, and a second acidic solvent. The phosphoserine-modified magnesium-based mineralized microspheres include a magnesium-based core, a phosphoserine interface coupling layer, and a calcium-phosphine mineralization precursor coating. Phosphoserine, as a mineralization interface regulating molecule, forms a stable bond with the magnesium-based core surface through its phosphate groups, further promoting the uniform deposition of calcium-phosphine mineralization precursors on the magnesium-based microparticle surface. After the mineralized microspheres are dispersed in the collagen solution, they further induce the deposition of mineralization precursors into the collagen interior and surface using collagen fibers as templates, resulting in further biomimetic mineralization of collagen. A neutral ion triggering solution is used to neutralize the acidic collagen solution and trigger collagen fiber self-assembly, dynamic ion bridging, and in-situ solidification of the collagen network in a body fluid environment, thereby forming a continuous, integrated bilayer repair structure between the cartilage and bone layers.

[0093] The first aspect of this application provides an injectable osteocartilage repair material, comprising: a cartilage repair solution, a bone repair solution, and a neutral ion triggering solution; the cartilage repair solution comprises a first collagen, phosphoserine-modified magnesium-based microspheres, and a first acidic solvent; the bone repair solution comprises a second collagen, phosphoserine-modified magnesium-based mineralized microspheres, and a second acidic solvent, wherein the phosphoserine-modified magnesium-based mineralized microspheres comprise a magnesium-based core, a phosphoserine interface coupling layer, and a calcium phosphate mineralization precursor coating, and the phosphoserine interface coupling layer couples the magnesium-based core and the calcium phosphate mineralization precursor coating.

[0094] Understandably, the injectable osteocartilage repair material of this application has at least the following beneficial effects:

[0095] (1) The injectable osteocartilage repair material adopts a three-component synergistic system of cartilage repair solution, bone repair solution and neutral ion triggering liquid. The cartilage repair solution constructs a biomimetic extracellular matrix microenvironment for cartilage cells, and the bone repair solution constructs an organic-inorganic composite mineralization system with continuous mineralization ability. The two layers of materials gel in situ under the action of neutral ion triggering liquid and form a continuous and combined osteocartilage repair structure. After injection, a continuous and combined double-layer repair structure can be formed in the defect area. There is no need for prefabricated scaffolds or regular trimming of the defect area. It can adapt to osteocartilage defects of different shapes and sizes, and improve the matching and interfacial bonding ability between the material and the host tissue.

[0096] (2) The cartilage repair solution is mainly composed of collagen, with magnesium-based microspheres modified with phosphoserine. Collagen undergoes fiber self-assembly to form a three-dimensional network under neutral environment and body temperature conditions. Phosphoserine binds through hydrogen bonds and Mg... 2+ Mediated dynamic ion bridging enhances collagen network stability and continuously releases appropriate amounts of Mg. 2+ Regulating the cartilage microenvironment promotes chondrocyte adhesion, extracellular matrix secretion, and hyaline cartilage regeneration.

[0097] (3) The bone repair solution uses collagen as an organic template and phosphoserine-modified magnesium-based mineralized microspheres as an inorganic mineralization core. Phosphoserine, as a mineralization interface coupling molecule, enhances the binding stability between the magnesium-based core and the calcium-phosphorus mineralization precursor coating, and provides continuous nucleation sites for subsequent mineralization. After the phosphoserine-modified magnesium-based mineralized microspheres are dispersed in the collagen network, the calcium-phosphorus mineralization precursor coating further migrates into the interior and surface of the collagen fibers, undergoing continuous mineralization under the induction of the collagen template, forming an organic-inorganic composite mineralization network with layers and structure, realizing subchondral bone repair and continuous repair of the osteochondral interface.

[0098] (4) In the phosphoserine-modified magnesium-based mineralized microspheres of the bone repair solution, phosphoserine constructs a stable interface between the magnesium-based core and the calcium-phosphine mineralization precursor coating, effectively reducing the initial rapid corrosion and hydrogen release of the magnesium-based material. Simultaneously, it continuously releases Ca through the calcium-phosphine mineralization precursor coating. 2+ PO4 3- and Mg 2+ This achieves synergistic regulation of ion slow release and continuous mineralization, providing a stable ion microenvironment for subchondral bone repair and bone tissue regeneration.

[0099] (5) The injectable osteocartilage repair material adopts a dual-layer functional partition design. The cartilage repair solution mainly constructs a collagen-rich, low-mineralized cartilage biomimetic microenvironment to promote chondrocyte adhesion, proliferation and hyaline cartilage matrix formation. The bone repair solution mainly constructs a bone repair layer with continuous mineralization capacity to promote subchondral bone reconstruction and osteocartilage interface mineralization repair, realize the synergistic regeneration of cartilage layer and bone layer, and reduce the risk of ectopic mineralization of cartilage layer.

[0100] (6) Compared with pre-formed block osteocartilage scaffolds, the injectable osteocartilage repair material of this application does not require regular pore enlargement or excessive trimming of the defect area, can adapt to complex and irregular defect morphologies, and reduces additional damage to residual healthy cartilage, calcified cartilage layer, and subchondral bone tissue. Simultaneously, the cured double-layer porous scaffold provides a spatial structural basis for cell migration, nutrient exchange, blood vessel ingrowth, and new tissue formation, and through Mg... 2+ The release and calcium-phosphorus mineralization process promotes subchondral bone repair and osteochondral interface reconstruction.

[0101] (7) Injectable osteocartilage repair materials have good injectability and can be implanted into osteocartilage defect areas through arthroscopic minimally invasive methods; the materials quickly gel in situ in vivo without the need for complex fixation devices, which can reduce surgical trauma and operation time, and have good clinical application value and application prospects.

[0102] During use, the cartilage repair solution and bone repair solution are mixed separately with a neutral ion triggering solution, and the pH of the system is rapidly adjusted from acidic to neutral. Under body temperature conditions, collagen fibers undergo self-assembly to form a three-dimensional network; simultaneously, phosphoserine mediates Ca2+... 2+ Mg 2+ Dynamic ion bridging is formed, which promotes the migration of calcium phosphate mineralization precursors on the surface of mineralized microspheres to the surface and interior of collagen fibers, and further mineralization is induced by collagen templates, thereby realizing in-situ gelation, continuous interfacial bonding and organic-inorganic synergistic regeneration in osteochondral defect areas.

[0103] In some embodiments, the mass percentage of the phosphoseserine-modified magnesium-based microspheres relative to the total mass of the first collagen and the first acidic solvent is 0.05wt%-5wt%; for example, it can be, but is not limited to, 0.05wt%, 0.1wt%, 0.2wt%, 0.4wt%, 0.6wt%, 0.8wt%, 1wt%, 1wt%, 0.05wt%, 1wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 2wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, 3wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, 4wt%, 4.2wt%, 4.4wt%, 4.6wt%, 4.8wt%, 5wt%, or any range between any two of the above values. Therefore, the appropriate amount of phosphoserine-modified magnesium microspheres is beneficial for the product to have suitable compressive strength. At the same time, the appropriate proportion of inorganic particles in the cartilage repair solution system is beneficial for maintaining the composition and mechanical microenvironment of the extracellular matrix of the cartilage layer, enhancing the interaction between cells and collagen networks, and maintaining the cartilage phenotype.

[0104] As one possible implementation, the mass percentage of the phosphoserine-modified magnesium-based mineralized microspheres relative to the total mass of the second collagen and the second acidic solvent is 0.1 wt% to 20 wt%; for example, it can be, but is not limited to, 0.1 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt%, 8.5 wt%, 9 wt%, 9.5 wt%, 10 wt%, 10.5 wt%, 11 wt%, 11.5 wt%, 12 wt%, 12.5 wt%, 13 wt%, 13.5 wt%, 14 wt%, 14.5 wt%, 15 wt%, 15.5 wt%, 16 wt%, 16.5 wt%, 17 wt%, 17.5 wt%, 18 wt%, 18.5 wt%, 19 wt%, 19.5 wt%, 20 wt%, or any range between any two of the above values. Therefore, when the amount of phosphoserine-modified magnesium-based mineralized microspheres is appropriate, the phosphoserine-modified magnesium-based mineralized microspheres can be uniformly dispersed in the collagen network and enhance the organic-inorganic composite structure through interfacial interaction, thereby improving the mechanical properties of the product.

[0105] In some embodiments, the mass-volume concentration of the first collagen in the cartilage repair solution is 4 mg / mL to 8 mg / mL; for example, it can be, but is not limited to, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, or any range between two of the above concentrations. Therefore, a collagen concentration within a suitable range is beneficial for forming a stable supporting structure during use, and also facilitates the uniform dispersion of phosphoserine-modified magnesium-based microspheres in the collagen network, thereby improving the overall strength of the gel network formed during use.

[0106] As one possible implementation, the mass-volume concentration of the second collagen in the bone repair solution is 4 mg / mL to 8 mg / mL; for example, it can be, but is not limited to, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, or any range between two of the above concentrations. Therefore, a collagen concentration within a suitable range is beneficial for forming a stable supporting structure during use, and also facilitates the uniform dispersion of phosphoserine-modified magnesium-based mineralized microspheres in the collagen network, thereby improving the overall strength of the gel network formed during use.

[0107] In some embodiments, the cartilage repair solution further comprises a first additive, which includes one or more of hyaluronic acid, chondroitin sulfate, and oxidized chondroitin sulfate. Thus, introducing an appropriate amount of phosphoserine-modified magnesium-based microspheres into the collagen system, and combining them with additives such as hyaluronic acid, chondroitin sulfate, and / or oxidized chondroitin sulfate to construct a biomimetic extracellular matrix, can significantly promote the differentiation of mesenchymal stem cells into chondrocytes.

[0108] In some alternative embodiments, the mass-volume concentration of the first additive in the cartilage repair solution is 0.1 mg / mL to 5 mg / mL; for example, it can be, but is not limited to, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, or any range between two of the above concentrations.

[0109] In some embodiments, the bone repair solution further comprises a second additive, which includes one or more of hyaluronic acid, chondroitin sulfate, and oxidized chondroitin sulfate. Thus, introducing an appropriate amount of phosphoserine-modified magnesium-based mineralized microspheres into the collagen system, and combining them with additives such as hyaluronic acid, chondroitin sulfate, and / or oxidized chondroitin sulfate to construct a biomimetic extracellular matrix, can significantly promote the differentiation of mesenchymal stem cells into chondrocytes.

[0110] As one possible implementation, the mass-volume concentration of the second additive in the bone repair solution is 0.1 mg / mL to 5 mg / mL; for example, it can be, but is not limited to, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, or any range between two of the above concentrations.

[0111] In some embodiments, the pH value of the neutral ion triggering solution is 7.2-8.2; for example, it can be, but is not limited to, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2 or any two of the above pH values.

[0112] In some alternative implementations, the pH of the neutral ion triggering solution is 7.4-7.8.

[0113] In some implementations, the neutral ion triggering solution includes a buffer system, an ion-regulating component, and a physiological salt system.

[0114] As one possible implementation, the buffer system includes one or more of PBS buffer, HEPES buffer, Tris buffer, and NaHCO3 buffer.

[0115] In some alternative embodiments, the ion-regulating component includes one or more of a calcium-containing soluble salt, a magnesium-containing soluble salt, and a potassium-containing soluble salt.

[0116] In some alternative embodiments, the ion-regulating component includes one or more of calcium chloride, magnesium chloride, and potassium chloride.

[0117] In some embodiments, the physiological saline system is sodium chloride. The physiological saline system is used to adjust the ionic strength and osmotic pressure of the neutral ion trigger solution.

[0118] A second aspect of this application provides a method for preparing an injectable osteocartilage repair material, comprising the following steps:

[0119] A first acidic collagen solution containing a first collagen protein and a first acidic solvent, and a second acidic collagen solution containing a second collagen protein and a second acidic solvent are prepared. The first acidic collagen solution is mixed with phosphoserine-modified magnesium-based microspheres to prepare a cartilage repair solution. The second acidic collagen solution is mixed with phosphoserine-modified magnesium-based mineralized microspheres to prepare a bone repair solution. The phosphoserine-modified magnesium-based mineralized microspheres include a magnesium-based core, a phosphoserine interface coupling layer, and a calcium phosphate mineralization precursor coating. The phosphoserine interface coupling layer couples the magnesium-based core and the calcium phosphate mineralization precursor coating. A neutral ion triggering solution is prepared.

[0120] After mixing the second acidic collagen solution with phosphoserine-modified magnesium-based mineralized microspheres, the phosphoserine-modified magnesium-based mineralized microspheres are dispersed in the second acidic collagen solution. The calcium phosphate mineralization precursor coating remains active in the acidic environment and interacts with collagen molecules through adsorption, coordination and hydrogen bonding to form a stable and dispersed organic-inorganic mineralization precursor system.

[0121] It should be noted that the injectable osteocartilage repair material prepared by this method has similar beneficial effects to the aforementioned injectable osteocartilage repair material, and will not be elaborated further here.

[0122] In some embodiments, the mass-volume concentration of the first collagen in the first acidic collagen solution is 4 mg / mL to 8 mg / mL; for example, it can be, but is not limited to, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, or any range between two of the above concentrations. Therefore, a collagen concentration within a suitable range is beneficial for forming a stable supporting structure during use, and also facilitates the uniform dispersion of phosphoserine-modified magnesium-based microspheres in the collagen network, thereby improving the overall strength of the gel network formed during use.

[0123] As one possible implementation, the mass-volume concentration of the second collagen in the second acidic collagen solution is 4 mg / mL to 8 mg / mL; for example, it can be, but is not limited to, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, or any range between two of the above concentrations. Therefore, a collagen concentration within a suitable range is beneficial for forming a stable supporting structure during use, and also facilitates the uniform dispersion of phosphoserine-modified magnesium-based mineralized microspheres in the collagen network, thereby improving the overall strength of the gel network formed during use.

[0124] In some embodiments, the mass percentage of the phosphoserine-modified magnesium microspheres relative to the mass of the first acidic collagen solution is 0.05wt%-5wt%; for example, it can be, but is not limited to, 0.05wt%, 0.1wt%, 0.2wt%, 0.4wt%, 0.6wt%, 0.8wt%, 1wt%, 1wt%, 0.05wt%, 1wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 2wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, 3wt%, 3.2wt%, 3.4wt%, 3.6wt%, 3.8wt%, 4wt%, 4.2wt%, 4.4wt%, 4.6wt%, 4.8wt%, 5wt%, or any range between any two of the above values. Therefore, the appropriate amount of phosphoserine-modified magnesium microspheres is beneficial for the product to have suitable compressive strength. At the same time, the appropriate proportion of inorganic particles in the cartilage repair solution system is beneficial for maintaining the composition and mechanical microenvironment of the extracellular matrix of the cartilage layer, enhancing the interaction between cells and collagen networks, and maintaining the cartilage phenotype.

[0125] As one possible implementation, the mass percentage of phosphoseserine-modified magnesium-based mineralized microspheres relative to the mass of the second acidic collagen solution is 0.1 wt% to 20 wt%; for example, it can be, but is not limited to, 0.1 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, 3.5 wt%, 4 wt%, 4.5 wt%, 5 wt%, 5.5 wt%, 6 wt%, 6.5 wt%, 7 wt%, 7.5 wt%, 8 wt%, 8.5 wt%, 9 wt%, 9.5 wt%, 10 wt%, 10.5 wt%, 11 wt%, 11.5 wt%, 12 wt%, 12.5 wt%, 13 wt%, 13.5 wt%, 14 wt%, 14.5 wt%, 15 wt%, 15.5 wt%, 16 wt%, 16.5 wt%, 17 wt%, 17.5 wt%, 18 wt%, 18.5 wt%, 19 wt%, 19.5 wt%, 20 wt%, or any range between any two of the above values. Therefore, when the amount of phosphoserine-modified magnesium-based mineralized microspheres is appropriate, the phosphoserine-modified magnesium-based mineralized microspheres can be uniformly dispersed in the collagen network and enhance the organic-inorganic composite structure through interfacial interaction, thereby improving the mechanical properties of the product.

[0126] In some embodiments, the first acidic collagen solution is mixed with phosphoserine-modified magnesium microspheres at 2°C-8°C. This reduces the self-assembly rate of collagen molecules during mixing, minimizes premature gelation or local aggregation caused by local temperature increases, facilitates uniform dispersion of phosphoserine-modified magnesium microspheres in the acidic collagen system, improves the stability and component distribution uniformity of the mixed system, and provides sufficient operating time for subsequent mixing with neutral ion triggering solution and in-situ gelation.

[0127] As one possible implementation method, a second acidic collagen solution is mixed with phosphoserine-modified magnesium-based mineralized microspheres at 2℃-8℃. This facilitates the uniform dispersion of the mineralized microspheres in the continuous collagen phase and reduces the impact of local enrichment of mineralized particles on the flowability and injectability of the system, thereby improving the uniformity, operability, and consistency of the subsequent gel structure of the obtained precursor system.

[0128] In some embodiments, the first acidic solvent is an aqueous solution of acetic acid, wherein the molar concentration of acetic acid in the aqueous solution is 0.01 mol / L to 0.05 mol / L; for example, it can be, but is not limited to, 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, or any range between any two of the above molar concentrations.

[0129] As one possible implementation, the second acidic solvent is an aqueous solution of acetic acid, wherein the molar concentration of acetic acid in the aqueous solution is 0.01 mol / L to 0.05 mol / L; for example, it can be, but is not limited to, 0.01 mol / L, 0.02 mol / L, 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, or any range between any two of the above molar concentrations.

[0130] In some embodiments, the first acidic collagen solution further contains a first additive, which includes one or more of hyaluronic acid, chondroitin sulfate, and oxidized chondroitin sulfate. Thus, introducing an appropriate amount of phosphoserine-modified magnesium microspheres into the collagen system, and combining them with additives such as hyaluronic acid, chondroitin sulfate, and / or oxidized chondroitin sulfate to construct a biomimetic extracellular matrix, can significantly promote the differentiation of mesenchymal stem cells into chondrocytes.

[0131] In some alternative embodiments, the mass-volume concentration of the first additive in the first acidic collagen solution is 0.1 mg / mL to 5 mg / mL; for example, it can be, but is not limited to, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, or any range between two of the above concentrations.

[0132] In some embodiments, the second acidic collagen solution also contains a second additive, which includes one or more of hyaluronic acid, chondroitin sulfate, and oxidized chondroitin sulfate. Thus, by introducing an appropriate amount of phosphoserine-modified magnesium-based mineralized microspheres into the collagen system and combining them with additives such as hyaluronic acid, chondroitin sulfate, and / or oxidized chondroitin sulfate to construct a biomimetic extracellular matrix, mesenchymal stem cells can be significantly promoted to differentiate into chondrocytes.

[0133] In some optional embodiments, the mass-volume concentration of the second additive in the second acidic collagen solution is 0.1 mg / mL to 5 mg / mL; for example, it can be, but is not limited to, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, or any range between two of the above concentrations.

[0134] In some embodiments, the preparation method of phosphoserine-modified magnesium microspheres includes: mixing a first magnesium microsphere with a first phosphoserine solution, adjusting the pH of the mixture to 6-7, and then washing and drying the first magnesium microsphere to prepare phosphoserine-modified magnesium microspheres.

[0135] Adjusting the pH of the mixing system to 6-7 allows the functional groups such as phosphate and carboxyl groups in the phosphoserine molecule to be in a suitable ionized state, which is conducive to their interfacial bonding with the magnesium-based microspheres and the formation of a relatively uniform phosphoserine modification layer. At the same time, it can avoid the magnesium-based microspheres from corroding too quickly under excessively low pH conditions and the local precipitation or particle agglomeration under excessively high pH conditions, thereby improving the surface modification uniformity and stability of the phosphoserine-modified magnesium-based microspheres.

[0136] In some alternative implementations, the pH of the mixture is adjusted to 6-7 using a sodium hydroxide buffer solution.

[0137] In some optional embodiments, the molar concentration of phosphoserine in the first phosphoserine solution is 0.5 mmol / L to 20 mmol / L; for example, it can be, but is not limited to, 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, or any range between two of the above molar concentrations.

[0138] In some optional embodiments, the molar concentration of phosphoserine in the first phosphoserine solution is 0.5 mmol / L-20 mmol / L, and the mass-to-volume ratio of the first magnesium-based microspheres to the first phosphoserine solution is (1-50) mg:1 mL; for example, but not limited to 1 mg:1 mL, 5 mg:1 mL, 10 mg:1 mL, 15 mg:1 mL, 20 mg:1 mL, 25 mg:1 mL, 30 mg:1 mL, 35 mg:1 mL, 40 mg:1 mL, 45 mg:1 mL, 50 mg:1 mL, or any range between two of the above mass-to-volume ratios. Therefore, a mass-to-volume ratio of magnesium-based microspheres to phosphoserine solution within this range helps the magnesium-based microspheres to be fully dispersed and contacted in the phosphoserine solution, promoting the uniform binding of phosphoserine on the surface of the magnesium-based microspheres, reducing particle aggregation and localized uneven modification, thereby improving the uniformity and stability of the phosphoserine modification on the surface of the magnesium-based microspheres.

[0139] As one possible implementation, the Dv50 of the first magnesium-based microsphere is 15μm-60μm; for example, it can be, but is not limited to, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm or any range between two of the above Dv50.

[0140] As an example, after adjusting the pH value, the magnesium-based microspheres were washed with purified water 2-3 times, and then placed in a low-temperature vacuum drying oven to dry for 1-4 hours.

[0141] In some embodiments, the preparation method of phosphoserine-modified magnesium-based mineralized microspheres includes: mixing a second magnesium-based microsphere with a second phosphoserine solution, adjusting the pH of the mixture to 6-7, then washing and drying the second magnesium-based microsphere to prepare a phosphoserine-modified magnesium-based core; mixing the phosphoserine-modified magnesium-based core with a mineralization solution and then incubating the mixture to prepare phosphoserine-modified magnesium-based mineralized microspheres; wherein the mineralization solution contains calcium ions and phosphate ions.

[0142] During the incubation process, the phosphate and carboxyl groups on the surface of phosphoserine react with Ca... 2+ Coordination occurs, further inducing crystal nucleus formation and continuous growth, and a uniform and dense calcium phosphate mineralization precursor coating is constructed in situ on the surface of the magnesium-based core, resulting in phosphose-modified magnesium-based mineralized microspheres.

[0143] In some alternative embodiments, the mineralization solution contains a calcium ion source, which includes one or more of calcium chloride, calcium nitrate, and calcium acetate.

[0144] As one possible implementation, the mineralization solution contains a phosphate ion source, which includes one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium phosphate.

[0145] In some alternative embodiments, the molar ratio of calcium ions to phosphate ions in the mineralization solution is (1.3-1.67):1; for example, it can be, but is not limited to, 1.3:1, 1.35:1, 1.4:1, 1.45:1, 1.5:1, 1.55:1, 1.6:1, 1.65:1, 1.67:1, or any range between any two of the above molar ratios.

[0146] In some embodiments, the molar concentration of calcium ions in the mineralization solution is 1 mmol / L to 20 mmol / L; for example, it can be, but is not limited to, 1 mmol / L, 2 mmol / L, 4 mmol / L, 6 mmol / L, 8 mmol / L, 10 mmol / L, 12 mmol / L, 14 mmol / L, 16 mmol / L, 18 mmol / L, 20 mmol / L, or any range between two of the above molar concentrations.

[0147] In some optional embodiments, the molar concentration of phosphate ions in the mineralization solution is 0.5 mmol / L to 10 mmol / L; for example, it can be, but is not limited to, 0.5 mmol / L, 1 mmol / L, 1.5 mmol / L, 2 mmol / L, 2.5 mmol / L, 3 mmol / L, 3.5 mmol / L, 4 mmol / L, 4.5 mmol / L, 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, 7.5 mmol / L, 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, 10 mmol / L, or any range between two of the above molar concentrations.

[0148] In some alternative embodiments, the pH of the mineralization solution is 6.5-7.5; for example, it can be, but is not limited to, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5 or any range between two of the above pH values.

[0149] In some embodiments, the incubation temperature is 20°C-37°C; for example, it can be, but is not limited to, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 37°C, or any two of the above temperatures.

[0150] In some alternative implementations, the incubation temperature is 25°C-37°C.

[0151] As one possible implementation, the incubation time is 4h-48h; for example, it can be, but is not limited to, 4h, 5h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 45h, 48h or any range between two of the above times.

[0152] In some optional implementations, the incubation period is 10h-24h. For example, it can be, but is not limited to, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, or any range between two of the above times.

[0153] In some embodiments, the molar concentration of phosphoserine in the second phosphoserine solution is 0.5 mmol / L to 20 mmol / L; for example, it can be, but is not limited to, 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 3 mmol / L, 4 mmol / L, 5 mmol / L, 6 mmol / L, 7 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L, 20 mmol / L, or any range between two of the above molar concentrations.

[0154] In some optional embodiments, the molar concentration of phosphoserine in the second phosphoserine solution is 0.5 mmol / L-20 mmol / L, and the mass-to-volume ratio of the second magnesium-based microspheres to the second phosphoserine solution is (1-50) mg:1 mL; for example, it can be, but is not limited to, 1 mg:1 mL, 5 mg:1 mL, 10 mg:1 mL, 15 mg:1 mL, 20 mg:1 mL, 25 mg:1 mL, 30 mg:1 mL, 35 mg:1 mL, 40 mg:1 mL, 45 mg:1 mL, 50 mg:1 mL, or any range between the above two mass-to-volume ratios. This facilitates the full dispersion and contact of the second magnesium-based microspheres in the phosphoserine solution, promotes the uniform binding of phosphoserine on the surface of the second magnesium-based microspheres, forms a relatively uniform phosphoserine-modified interface, and provides functional sites for the enrichment of calcium ions and the nucleation and deposition of calcium-phosphoserine mineralization precursors during subsequent calcium-phosphoserine mineralization processes, thereby facilitating the acquisition of magnesium-based mineralized microspheres with relatively uniform surface mineralization.

[0155] As one possible implementation, the Dv50 of the second magnesium-based microsphere is 15μm-60μm; for example, it can be, but is not limited to, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm or any range between two of the above Dv50.

[0156] In some embodiments, after the incubation process is completed, the product is washed 2 to 5 times with purified water to remove unbound ions and residual mineralization solution, and then dried under freeze-drying or vacuum drying conditions to obtain phosphoserine-modified magnesium-based mineralized microspheres for later use.

[0157] In some embodiments, the pH value of the neutral ion triggering solution is 7.2-8.2; for example, it can be, but is not limited to, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2 or any two of the above pH values.

[0158] In some alternative implementations, the pH of the neutral ion triggering solution is 7.4-7.8.

[0159] In some embodiments, the preparation method of the neutral ion triggering solution includes mixing a buffer system, an ion regulating component, and a physiological salt system.

[0160] As one possible implementation, the buffer system includes one or more of PBS buffer, HEPES buffer, Tris buffer, and NaHCO3 buffer.

[0161] In some alternative embodiments, the ion-regulating component includes one or more of a calcium-containing soluble salt, a magnesium-containing soluble salt, and a potassium-containing soluble salt.

[0162] In some alternative embodiments, the ion-regulating component includes one or more of calcium chloride, magnesium chloride, and potassium chloride.

[0163] In some embodiments, the physiological saline system is sodium chloride. The physiological saline system is used to adjust the ionic strength and osmotic pressure of the neutral ion trigger solution.

[0164] As an example, the preparation method of acidic collagen solution includes: removing the fat layer of animal-derived collagen tissue, cutting the raw material into slices, and repeatedly rinsing with PBS or purified water to remove blood and tissue residues; then performing defatting and decellularization treatment to obtain purified collagen tissue; adding the purified collagen tissue to a 0.01 mol / L-0.05 mol / L acetic acid solution, stirring to fully dissolve the collagen tissue to form a homogeneous acidic collagen solution, centrifuging and dialysis to remove acid, salt, and small molecule impurities, and then freeze-drying to prepare collagen powder; and then using a 0.01 mol / L-0.05 mol / L acetic acid solution to prepare an acidic collagen solution with a concentration of 4 mg / mL-8 mg / mL.

[0165] In some alternative implementations, animal-derived collagen tissue includes one or more of bovine pericardium, porcine pericardium, porcine peritoneum, and porcine skin.

[0166] The technical solutions of this application will be described in detail below with reference to specific embodiments. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the field, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the field.

[0167] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0168] Unless otherwise specified, all raw materials in the following examples and comparative examples are commercially available or prepared using conventional methods.

[0169] I. Preparation of Injectable Osteocartilage Repair Materials

[0170] Example 1

[0171] Step 1: Preparation of Acidic Collagen Solution: The fat layer of bovine pericardial tissue containing animal collagen was removed using a thinner, and the raw material was cut into 1x1cm slices. The slices were repeatedly rinsed with purified water to remove blood and tissue residue. Then, the animal-derived material underwent defatting and decellularization to obtain purified collagen tissue. The purified collagen tissue was added to a 0.01 mol / L acetic acid solution and continuously stirred to fully dissolve the collagen and form a homogeneous acidic collagen solution. This solution was then centrifuged and dialyzed to remove acid, salt, and small molecule impurities before lyophilization to prepare collagen powder. An acidic collagen solution with a collagen concentration of 8 mg / mL was prepared using a 0.01 mol / L acetic acid solution. Hyaluronic acid with a molecular weight of 80 kDa was added to the acidic collagen solution, resulting in a hyaluronic acid concentration of 1 mg / mL. A portion of the acidic collagen solution was used for the subsequent preparation of a cartilage repair solution, and the remainder was used for the subsequent preparation of a bone repair solution.

[0172] Step 2: Preparation of phosphoserine-modified magnesium microspheres: Magnesium microspheres with a Dv50 of 15 μm were washed and dried sequentially with ethanol and purified water. Then, a 20 mmol / L phosphoserine solution was added (mass-volume ratio of magnesium microspheres to phosphoserine solution: 10 mg: 1 mL), and the mixture was stirred for 2 h. The pH was adjusted to 6 with sodium hydroxide buffer. The treated magnesium microspheres were then rapidly washed with purified water three times, and finally dried in a low-temperature vacuum drying oven for 4 h to obtain phosphoserine-modified magnesium microspheres. A portion of the obtained phosphoserine-modified magnesium microspheres were used in the subsequent preparation of phosphoserine-modified magnesium mineralization microspheres.

[0173] Step 3, Cartilage Repair Solution: A portion of the phosphoserine-modified magnesium microspheres obtained in Step 2 was added to a portion of the acidic collagen solution obtained in Step 1. The mixture was stirred at 50 rpm at 2°C until homogeneous. The amount of phosphoserine-modified magnesium microspheres added was 1 wt% of the mass of the acidic collagen solution. The homogeneous cartilage repair solution was aseptically filled into 5 mL pre-filled syringes, sealed, and stored as a precursor solution for cartilage repair.

[0174] Step 4: Preparation of phosphoserine-modified magnesium-based mineralized microspheres: A portion of the phosphoserine-modified magnesium-based microspheres obtained in Step 2 were added to a mineralization solution and incubated with slow shaking at 30°C for 12 hours. The mineralization solution consisted of calcium chloride (calcium ion source) and disodium hydrogen phosphate (phosphate ion source), with a molar concentration of 9 mmol / L for calcium chloride, 6 mmol / L for disodium hydrogen phosphate, a Ca / P molar ratio of 1.5, a pH of 7, an incubation temperature of 30°C, and an incubation time of 4 hours. After incubation, the microspheres were washed five times with purified water to remove unbound ions and residual mineralization solution, and then dried under vacuum to obtain phosphoserine-modified magnesium-based mineralized microspheres.

[0175] Step 5: Preparation of bone repair solution: The phosphoserine-modified magnesium-based mineralized microspheres obtained in Step 4 are added to a portion of the acidic collagen solution prepared in Step 1. The mixture is slowly stirred at 2°C to ensure uniform dispersion of the mineralized microspheres in the acidic collagen solution, thus obtaining the bone repair solution. The amount of phosphoserine-modified magnesium-based mineralized microspheres added is 5 wt% of the total mass of the acidic collagen solution, and the collagen concentration in the acidic collagen solution is 4 mg / mL.

[0176] Step 6: Preparation of neutral ion trigger solution: Using PBS buffer as the base solution, add 2 mmol / L CaCl2, 1 mmol / L MgCl2, and 4 mmol / L KCl to adjust the pH of the buffer to 7.5. After aseptic mixing and filtration, the above components are filled into 5 mL pre-filled syringes for later use.

[0177] Example 2

[0178] Step 1: Preparation of Acidic Collagen Solution: The fat layer of the animal-derived collagen porcine skin tissue was removed using a thinning machine. The raw material was cut into 1x1cm slices and repeatedly rinsed with purified water to remove blood and tissue residue. Then, the animal-derived material underwent defatting and decellularization to obtain purified collagen tissue. The purified collagen tissue was added to a 0.05 mol / L acetic acid solution and continuously stirred to fully dissolve the collagen and form a homogeneous acidic collagen solution. This solution was then centrifuged and dialyzed to remove acid, salt, and small molecule impurities before freeze-drying to prepare collagen powder. An acidic collagen solution with a collagen concentration of 8 mg / mL was prepared using a 0.05 mol / L acetic acid solution. 40 kDa medical-grade chondroitin sulfate was added to the acidic collagen solution, with a concentration of 1 mg / mL. A portion of the acidic collagen solution was used for the subsequent preparation of a cartilage repair solution, and the remainder was used for the subsequent preparation of a bone repair solution.

[0179] Step 2: Preparation of phosphoserine-modified magnesium microspheres: Magnesium microspheres with a Dv50 of 15 μm were washed and dried sequentially with ethanol and purified water. Then, a 20 mmol / L phosphoserine solution was added (mass-volume ratio of magnesium microspheres to phosphoserine solution: 10 mg: 1 mL), and the mixture was stirred for 2 h. The pH was adjusted to 6 with sodium hydroxide buffer. The treated magnesium microspheres were then rapidly washed with purified water three times, and finally dried in a low-temperature vacuum drying oven for 4 h to obtain phosphoserine-modified magnesium microspheres. A portion of the obtained phosphoserine-modified magnesium microspheres were used in the subsequent preparation of phosphoserine-modified magnesium mineralization microspheres.

[0180] Step 3, Cartilage Repair Solution: A portion of the phosphoserine-modified magnesium microspheres obtained in Step 2 was added to a portion of the acidic collagen solution obtained in Step 1. The mixture was stirred at 50 rpm at 2°C until homogeneous. The amount of phosphoserine-modified magnesium microspheres added was 1 wt% of the mass of the acidic collagen solution. The homogeneous cartilage repair solution was aseptically filled into 5 mL pre-filled syringes, sealed, and stored as a precursor solution for cartilage repair.

[0181] Step 4: Preparation of phosphoserine-modified magnesium-based mineralized microspheres: A portion of the phosphoserine-modified magnesium-based microspheres obtained in Step 2 were added to a mineralization solution and incubated with slow shaking at 30°C for 12 hours. The mineralization solution consisted of calcium chloride (calcium ion source) and disodium hydrogen phosphate (phosphate ion source), with a molar concentration of 9 mmol / L for calcium chloride, 6 mmol / L for disodium hydrogen phosphate, a Ca / P molar ratio of 1.5, a pH of 7, an incubation temperature of 30°C, and an incubation time of 4 hours. After incubation, the microspheres were washed five times with purified water to remove unbound ions and residual mineralization solution, and then dried under vacuum to obtain phosphoserine-modified magnesium-based mineralized microspheres.

[0182] Step 5: Preparation of bone repair solution: The phosphoserine-modified magnesium-based mineralized microspheres obtained in Step 4 are added to a portion of the acidic collagen solution prepared in Step 1. The mixture is slowly stirred at 2°C to ensure uniform dispersion of the mineralized microspheres in the acidic collagen solution, thus obtaining the bone repair solution. The amount of phosphoserine-modified magnesium-based mineralized microspheres added is 5 wt% of the total mass of the acidic collagen solution, and the collagen concentration in the acidic collagen solution is 4 mg / mL.

[0183] Step 6: Preparation of neutral ion trigger solution: Using PBS buffer as the base solution, add 2 mmol / L CaCl2, 1 mmol / L MgCl2, and 4 mmol / L KCl to adjust the pH of the buffer to 7.5. After aseptic mixing and filtration, the above components are filled into 5 mL pre-filled syringes for later use.

[0184] Example 3

[0185] The preparation method of Example 3 is similar to that of Example 1, except that: in step 3 of Example 3, the concentration of collagen in the acidic collagen solution used to prepare the cartilage repair solution is 4 mg / mL, and in step 5 of Example 3, the concentration of collagen in the acidic collagen solution used to prepare the bone repair solution is 8 mg / mL; all other aspects are the same.

[0186] Example 4

[0187] The preparation method of Example 4 is similar to that of Example 1, except that the concentration of collagen in the acidic collagen solution used in step 3 of Example 4 when preparing the cartilage repair solution and in step 5 when preparing the bone repair solution is 10 mg / mL; all other aspects are the same.

[0188] Example 5

[0189] The preparation method of Example 5 is similar to that of Example 1, except that the concentration of collagen in the acidic collagen solution used in the preparation of cartilage repair solution in step 3 and bone repair solution in step 5 of Example 5 is 2 mg / mL; all other aspects are the same.

[0190] Example 6

[0191] The preparation method of Example 6 is similar to that of Example 1, except that: in step 3 of Example 6, the amount of phosphoserine-modified magnesium microspheres added when preparing the cartilage repair solution is 0.05 wt% of the mass of the acidic collagen solution; all other aspects are the same.

[0192] Example 7

[0193] The preparation method of Example 7 is similar to that of Example 1, except that: in step 3 of Example 7, the amount of phosphoserine-modified magnesium microspheres added when preparing the cartilage repair solution is 5 wt% of the mass of the acidic collagen solution; all other aspects are the same.

[0194] Example 8

[0195] The preparation method of Example 8 is similar to that of Example 1, except that: in step 3 of Example 8, the amount of phosphoserine-modified magnesium microspheres added when preparing the cartilage repair solution is 7 wt% of the mass of the acidic collagen solution; all other aspects are the same.

[0196] Example 9

[0197] The preparation method of Example 9 is similar to that of Example 1, except that: in step 5 of Example 9, the amount of phosphoserine-modified magnesium-based mineralized microspheres added when preparing the bone repair solution is 0.1 wt% of the mass of the acidic collagen solution; all other aspects are the same.

[0198] Example 10

[0199] The preparation method of Example 10 is similar to that of Example 1, except that: in step 5 of Example 10, the amount of phosphoserine-modified magnesium-based mineralized microspheres added when preparing the bone repair solution is 20 wt% of the mass of the acidic collagen solution; all other aspects are the same.

[0200] Example 11

[0201] The preparation method of Example 11 is similar to that of Example 1, except that: in step 5 of Example 11, the amount of phosphoserine-modified magnesium-based mineralized microspheres added when preparing the bone repair solution is 22 wt% of the mass of the acidic collagen solution; all other aspects are the same.

[0202] Comparative Example 1

[0203] Step 1: Preparation of Acidic Collagen Solution: The fat layer of bovine pericardial tissue containing animal collagen was removed using a thinner, and the raw material was cut into 1x1cm slices. The slices were repeatedly rinsed with purified water to remove blood and tissue residue. Then, the animal-derived material underwent defatting and decellularization to obtain purified collagen tissue. The purified collagen tissue was added to a 0.01 mol / L acetic acid solution and continuously stirred to fully dissolve the collagen and form a homogeneous acidic collagen solution. This solution was then centrifuged and dialyzed to remove acid, salt, and small molecule impurities, and the collagen concentration was adjusted to 8 mg / mL to obtain the acidic collagen solution. Subsequently, hyaluronic acid with a molecular weight of 80 kDa was added to the acidic collagen solution at a concentration of 1 mg / mL. After thorough mixing, a homogeneous monophase collagen solution was obtained.

[0204] Step 2, Preparation of Neutralization Buffer: Using PBS buffer as the base solution, add 2 mmol / L CaCl2, 1 mmol / L MgCl2, and 4 mmol / L KCl to adjust the pH of the buffer to 7.5. After aseptic mixing and filtration, the above components are filled into 5 mL pre-filled syringes for later use. In use, load the monophase collagen solution and neutralization buffer into separate dual syringes, mix them using a mixer, and then inject them into the defect area to form a homogeneous collagen hydrogel under physiological conditions.

[0205] Comparative Example 2

[0206] The preparation method of Comparative Example 2 is similar to that of Example 1, except that the preparation of phosphoserine-modified magnesium-based mineralized microspheres in step 4 was not performed in Comparative Example 2. Instead, a portion of the phosphoserine-modified magnesium-based microspheres prepared in step 2 were directly added to an acidic collagen solution to prepare a bone repair solution; all other steps are the same.

[0207] When using the cartilage repair solution, bone repair solution, and neutral ion triggering solution, the mixing method, injection method, and in-situ gelation process are all the same as in Example 1.

[0208] Comparative Example 3

[0209] The preparation method of Comparative Example 3 is similar to that of Example 1, except that: when preparing the cartilage repair solution in Comparative Example 3, magnesium-based microspheres were used directly, and phosphoserine-modified magnesium-based microspheres were not prepared; when preparing the bone repair solution, magnesium-based microspheres were directly incubated to mineralize them, and then the mineralization product was used to prepare the bone repair solution; all other aspects are the same.

[0210] The main differences between the above embodiments and comparative examples are as follows:

[0211] Table 1

[0212]

[0213] In Table 1, collagen concentration 1 refers to the concentration of collagen in the acidic collagen solution used in preparing the cartilage repair solution; collagen concentration 2 refers to the concentration of collagen in the acidic collagen solution used in preparing the bone repair solution. Mass percentage 1 refers to the percentage of the mass of phosphoserine-modified magnesium microspheres added in preparing the cartilage repair solution relative to the mass of the acidic collagen solution; mass percentage 2 refers to the percentage of the mass of phosphoserine-modified magnesium mineralized microspheres added in preparing the bone repair solution relative to the mass of the acidic collagen solution. "Whether phosphoserine-modified magnesium microspheres are used" refers to whether the magnesium microspheres used in preparing the cartilage and bone repair solutions are phosphoserine-modified; "Whether the magnesium microspheres are incubated for mineralization" refers to whether the magnesium microspheres used in preparing the cartilage and bone repair solutions are incubated to achieve mineralization. "Whether it is a two-component material solution" refers to whether the cartilage repair solution and bone repair solution are prepared separately.

[0214] The differences between Example 2 and Example 1 also include: in step 1 of Example 2, the concentration of the acetic acid solution used in preparing the acidic collagen solution is 0.05 mol / L, and the chondroitin sulfate with a molecular weight of 40 kDa, which is medical grade, is added to the acidic collagen solution.

[0215] II. Performance Testing

[0216] The performance of the repair materials prepared in the above embodiments and comparative examples was tested as follows. It should be noted that in the following description, component A represents the cartilage repair solution and component B represents the bone repair solution.

[0217] 1. Mechanical property testing

[0218] Gel samples obtained from Examples 1-11 (gels of components A and B, respectively), Comparative Examples 1, 2 (gel of component B), and 3 (gels of components A and B, respectively) were prepared. Each sample was mixed with the corresponding neutral ion triggering solution according to the proportions used in the examples and allowed to stand at 37 °C for 10 min to allow for complete in-situ gelation. The gels were then prepared into cylindrical specimens with a diameter of 8 mm and a height of 5 mm, with at least five parallel samples per group. Uniaxial compression tests were performed on each gel group using a universal testing machine with a 100 N load cell and a compression rate set to 1 mm / min, compressing to 50% of the original height of the sample. The stress-strain curves during compression were recorded in real time. The compressive modulus of the sample within the strain range of 10%–20% was calculated based on the stress-strain curves. Experimental results are expressed as mean ± standard deviation (Mean ± SD).

[0219] The apparent viscosity of the precursor liquids (component A and component B) prepared in each example and comparative example was measured using a rotational rheometer at a specified temperature. During testing, the sample was placed on the rheometer platform, and a parallel plate clamp was used. The testing temperature was controlled at 25 °C, and the shear rate was set to 10 s⁻¹. - ¹, After the sample reaches a stable shear state, its apparent viscosity value is recorded. Each group of samples is tested in triplicate, and the results are expressed as mean ± standard deviation.

[0220] The test results are shown in Table 2:

[0221] Table 2

[0222]

[0223] Wherein, compression modulus 1 refers to the compression modulus of component A gel, and compression modulus 2 refers to the compression modulus of component B gel. Viscosity 1 refers to the viscosity of component A gel, and viscosity 2 refers to the viscosity of component B gel.

[0224] The results above show that the monolayer collagen gel in Comparative Example 1 has the lowest compressive modulus, at only 12.4 ± 0.9 kPa, indicating that the mechanical support capacity of the simple collagen system is limited. In Example 1, the compressive modulus of component A gel increased to 24.8 ± 1.6 kPa due to the introduction of phosphoserine-modified magnesium microspheres, indicating that functionally modified microparticles can enhance the structural stability of the collagen network.

[0225] Collagen concentration has a significant impact on the mechanical and rheological properties of gels. When the collagen concentration in both the first and second acidic collagen solutions was reduced to 2 mg / mL, the compressive modulus of component A gel obtained in Example 5 was only 8.3 ± 1.2 kPa, and the compressive modulus of component B gel was 21.4 ± 3.2 kPa, both significantly lower than that in Example 1. This indicates that the continuous collagen network formed when the collagen concentration is too low is insufficient, making it difficult to provide stable structural support for the material. Conversely, when the collagen concentration was increased to 10 mg / mL, the compressive modulus of both component A and component B gels obtained in Example 4 further increased, but the viscosity of the corresponding precursor system also increased significantly, leading to increased resistance to material mixing and injection, which is not conducive to the uniform dispersion of functional microspheres in the collagen system and the uniform filling of the material in the defect area. Therefore, a higher collagen concentration is not necessarily better. Considering the mechanical support performance, mixing uniformity, and injectability of the material, the preferred collagen concentration is 4 mg / mL to 8 mg / mL.

[0226] Although component B of Comparative Example 2 contained phosphose-modified magnesium-based microspheres, it did not undergo further calcium-phosphine mineralization precursor construction, and its compressive modulus was 28.9 ± 2.1 kPa. After further mineralization treatment, the compressive modulus of component B in Example 1 increased to 72.6 ± 4.8 kPa. These results indicate that further construction of a calcium-phosphine mineralization precursor on the surface of phosphose-modified magnesium-based microspheres is beneficial for the formation of a composite structure between the inorganic phase and the collagen continuous phase, thereby improving the gel's compressive strength and defect space maintenance ability, and providing more stable structural support for the subchondral bone region.

[0227] The content of functional microspheres in component A also affects the material properties. When the amount of phosphoserine-modified magnesium-based microspheres added was increased to 7 wt%, the compressive modulus of the component A gel obtained in Example 8 increased to 38.2 ± 3.3 kPa, which was higher than 24.8 ± 1.6 kPa in Example 1, indicating that increasing the content of functional microspheres can further enhance the compressive strength of component A gel. However, component A is mainly used to construct the regenerative microenvironment of the cartilage layer, and its performance requirements are not simply to pursue high mechanical strength, but also to take into account material flexibility and the supporting role of mesenchymal stem cell cartilage differentiation.

[0228] For component B, the compressive strength of the gel generally increased with the increase of phosphoserine-modified magnesium-based mineralized microspheres. In Example 1, the compressive modulus of the component B gel was 72.6 ± 4.8 kPa; when the concentration of phosphoserine-modified magnesium-based mineralized microspheres in Example 11 was increased to 22 wt%, its compressive modulus further increased to 97.2 ± 1.5 kPa. This indicates that increasing the content of mineralized microspheres can increase the proportion of inorganic phase in component B and enhance the organic-inorganic composite structure formed by the mineralized microspheres and collagen network, thereby improving the compressive strength of the gel.

[0229] However, as an injectable repair material for subchondral bone regions, the performance of component B is not simply about achieving a high compressive modulus; the dispersion uniformity, flowability, and injectability of the precursor system must also be considered. When the concentration of mineralized microspheres increases to 22 wt%, although the compressive modulus of the resulting gel further improves, the viscosity of the precursor system increases significantly, leading to increased mixing and injection resistance. This is detrimental to the uniform dispersion of the mineralized microspheres in the collagen system and the uniform injection and filling of the material within the defect area. Therefore, considering the material's mechanical support properties, system uniformity, and injectability, the optimal concentration of phosphoserine-modified magnesium-based mineralized microspheres in component B is 0.1 wt%–20 wt%.

[0230] 2. Evaluation of the effect of materials on chondrogenesis

[0231] Mesenchymal stem cells were harvested at a ratio of approximately 2 × 10⁻⁶ cells per well. 4Cells were seeded at densities on the surfaces of the gels containing component A in Example 1, the gel containing component A in Example 2, the monolayer collagen gel in Comparative Example 1, and the gel containing component A in Example 8, and cultured at 37°C and 5% CO2. After cell attachment, the culture medium was replaced with chondrogenic medium and cultured for 7, 14, and 21 days, respectively.

[0232] Samples were taken at various time points, and the expression levels of chondrocyte differentiation-related genes SOX9 and COL II were detected by RT-qPCR. The results are as follows: Figure 1 As shown. By Figure 1 It was observed that the expression levels of SOX9 and COL II genes gradually increased in each group, but significant differences existed between the groups. Example 1 showed the highest SOX9 and COL II expression levels at all culture time points, followed by Example 2, then Example 8, while Comparative Example 1 showed the lowest. The results indicate that introducing an appropriate amount of phosphoserine-modified magnesium microspheres into the collagen system, combined with hyaluronic acid or chondroitin sulfate to construct a biomimetic extracellular matrix, can significantly promote the differentiation of mesenchymal stem cells into chondrocytes. Comparative Example 1, a monolayer homogeneous collagen system, did not contain any phosphoserine-modified magnesium microspheres or inorganic mineralization components, only providing basic cell adhesion support and lacking continuous biological activity stimulation; therefore, it exhibited the lowest SOX9 and COL II expression levels. Although Example 8 introduced magnesium-based microspheres modified with phosphoserine, its chondrogenic differentiation ability was better than that of Comparative Example 1. However, because the amount of microspheres added in component A was increased to 7 wt%, which exceeded the preferred range, the proportion of inorganic particles in the system was too high. This changed the composition of the extracellular matrix and the mechanical microenvironment of the cartilage layer, weakened the interaction between cells and the collagen network, and was not conducive to maintaining the cartilage phenotype. Therefore, the expression of SOX9 and COL II was lower than that in Examples 1 and 2.

[0233] 3. Animal experiments on osteochondral defect models

[0234] Eighteen healthy adult New Zealand white rabbits, weighing 2.5 kg–3.0 kg, were randomly divided into an experimental group and a control group, with nine rabbits in each group. After general anesthesia, a rabbit knee joint osteochondral defect model was established using aseptic surgery. The skin and joint capsule were incised layer by layer along the medial side of the knee joint to expose the weight-bearing area of ​​the trochlear groove of the femur. A cylindrical osteochondral defect of approximately 4 mm depth was created using a 4 mm diameter trephine, penetrating the articular cartilage, calcified cartilage layer, and extending into the subchondral bone, forming a standardized osteochondral defect model. Microfracture holes, approximately 2 mm–3 mm deep and spaced approximately 2 mm apart, were evenly created at the base of the defect using a microfracture cone to promote the recruitment of bone marrow-derived mesenchymal stem cells. Subsequently, the bone repair solution of component A in Example 1 and Comparative Example 2 was thoroughly mixed with the neutral ion triggering solution of component C at a 1:1 volume ratio. This mixture was then injected into the subchondral bone defect area using a dual-injector and allowed to stand for approximately 2-3 minutes to allow for in-situ gelation. Next, the cartilage repair solution of component A was mixed with component C at a 1:1 volume ratio and injected onto the surface of the bone repair layer. This mixture was allowed to stand for another 3-5 minutes to allow the upper and lower layers to form a continuous, integrated bilayer gel structure. Animals were sacrificed at 3, 6, and 12 months post-surgery, and knee joint specimens were collected for gross observation, histological evaluation, immunohistochemical analysis, and Micro-CT analysis.

[0235] Gross observation and histological results as follows Figure 2 As shown, by Figure 2 As can be seen, in Example 1, the defect area was gradually completely filled by newly formed repair tissue. The surface of the repair tissue was smooth, the color was close to that of normal articular cartilage, and it formed good continuous integration with the surrounding normal osteochondral tissue. As the repair time increased, the defect boundary gradually disappeared, and by 12 months postoperatively, the normal joint surface morphology was basically restored. In contrast, in Comparative Example 2, the defect area still showed obvious depressions, and there was a clear boundary between the repair tissue and the surrounding normal tissue, with poor tissue continuity. In Example 1, the repair area formed continuous hyaline cartilage-like tissue, with significantly enhanced proteoglycan staining. The chondrocytes were round or oval, arranged relatively regularly, and formed a continuous osteochondral interface with the surrounding normal cartilage. In contrast, in Comparative Example 2, there was less proteoglycan deposition in the repair tissue, the cells were arranged disorderly, and it mainly showed fibrocartilage-like repair tissue. The tissue structure integrity was significantly lower than that of Example 1. Further quantitative analysis of the glycosaminoglycan (GAG) content and type II collagen (Col II) expression level of the repair tissue was performed, and the results are as follows: Figure 3 As shown. By Figure 3It was found that the GAG ​​content in Example 1 group was significantly higher than that in Comparative Example 2 group at 3, 6, and 12 months post-operation, and continued to increase with the extension of repair time, indicating that the injectable osteochondral repair material of this application can continuously promote the deposition of proteoglycans in the extracellular matrix of chondrocytes. At the same time, the expression level of type II collagen (Col II) in Example 1 group was significantly higher than that in Comparative Example 2 group at all time points, indicating that the material of this invention can promote the formation of hyaline cartilage-specific extracellular matrix and maintain chondrocyte phenotype.

[0236] Micro-CT 3D reconstruction results are as follows Figure 4 As shown. By Figure 4 It was observed that in Example 1 group, the amount of newly formed bone tissue in the defect area gradually increased, the subchondral bone structure recovered its continuity, and the trabecular bone arrangement gradually became regular, forming a dense and complete bone tissue structure with the extension of repair time. In contrast, in Comparative Example 2 group, the formation of new bone was relatively less, the trabecular bone structure was sparser, and some bone tissue loss still existed in the defect area. Quantitative analysis of bone volume (BV / TV) showed that the bone volume fractions in Example 1 group at 3, 6, and 12 months postoperatively were 28.6±2.3%, 41.8±2.8%, and 55.2±3.1%, respectively, all significantly higher than those in Comparative Example 2 group (18.9±2.0%, 29.7±2.5%, and 37.5±2.9%), indicating that Example 1 group could significantly promote new bone formation in the defect area and increase subchondral bone mass. Simultaneously, bone mineral density (BMD) measurements showed that in Example 1 group, the levels reached 420±20 mgHA / cm³ at 3, 6, and 12 months postoperatively. 3 560±24 mgHA / cm 3 and 690±28 mgHA / cm 3 The concentration was significantly higher than that of control group 2 (330±18 mgHA / cm). 3 430±20 mgHA / cm 3 and 520±25 mgHA / cm 3 This indicates that the materials in this application can promote the maturation of new bone tissue and improve the mineralization of subchondral bone and the quality of bone tissue.

[0237] The above results indicate that the calcium phosphate mineralization precursor induced by phosphoserine in this application can significantly promote the regeneration and mineralization reconstruction of subchondral bone, increase bone volume fraction and bone density, improve the local microenvironment of subchondral bone, provide stable structural support and favorable biological microenvironment for the regeneration of upper cartilage tissue, thereby promoting the continuous reconstruction of the osteochondral interface and improving the overall repair effect of osteochondral defects.

[0238] 4. Material Usage Instructions

[0239] like Figure 5As shown, the osteochondral defect area was fully exposed arthroscopically. Fibrous and necrotic tissue on the surface of the diseased subchondral bone was thoroughly removed, and the defect area was thoroughly rinsed with saline to remove debris and blood, keeping the surgical area clean. Microfractures were performed on the subchondral bone plate using a microfracture cone or micro-drill. The depth of the microfracture holes was 2mm-5mm, and the spacing between adjacent holes was 2mm-4mm. The micro-holes were evenly distributed throughout the defect area, avoiding communication between holes to maintain the overall stability of the subchondral bone plate. After completing the microfractures, the fluid in the joint cavity was fully drained, and the defect area was kept relatively dry using sterile gauze or a suction device.

[0240] Component B bone repair solution and component C neutral ion triggering solution were connected in a dual-syringe at a 1:1 volume ratio to form a homogeneous bone repair solution. This mixture was then slowly injected into the subchondral bone defect area, ensuring it fully filled the microfracture holes and bone defect spaces. After standing for 2-3 minutes, collagen fibers underwent self-assembly, while phosphoserine-modified mineralized magnesium microspheres induced organic-inorganic composite mineralization in a neutral ion environment, forming a bone repair layer with initial mechanical support. Subsequently, component A cartilage repair solution and component C neutral ion triggering solution were thoroughly mixed at a 1:1 volume ratio and slowly injected over the bone repair layer, filling the entire cartilage defect area and adhering closely to the surrounding normal cartilage edges. After standing for 3-5 minutes at body temperature, collagen fibers further completed self-assembly, forming a three-dimensional network.

[0241] As the gel solidifies, collagen fibers at the interface between components A and B undergo interpenetration of chains and molecular entanglement. Simultaneously, phosphoserine molecules bind with Ca through their phosphate and carboxyl groups. 2+ Mg 2+ Dynamic ion bridging is formed, creating a continuous, integrated interface between the upper and lower layers. The implanted material further adsorbs and fixes bone marrow clots and mesenchymal stem cells released from microfractures, continuously releasing Mg... 2+ Ca 2+ It contains phosphate ions, which promote subchondral bone mineralization and repair, and induce extracellular matrix deposition in chondrocytes, thus achieving integrated osteochondral regeneration.

[0242] After the material has completely solidified, the knee joint is flexed and extended several times to confirm that there is no obvious dislodgement, displacement or loss of the implanted material before the surgery is completed.

[0243] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0244] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. An injectable osteocartilage repair material, characterized in that, include: Cartilage repair solution, bone repair solution, and neutral ion triggering solution; The cartilage repair solution comprises a first collagen, phosphoserine-modified magnesium microspheres, and a first acidic solvent; The bone repair solution comprises a second collagen, phosphoserine-modified magnesium-based mineralized microspheres, and a second acidic solvent. The phosphoserine-modified magnesium-based mineralized microspheres comprise a magnesium-based core, a phosphoserine interface coupling layer, and a calcium phosphate mineralization precursor coating. The phosphoserine interface coupling layer couples the magnesium-based core and the calcium phosphate mineralization precursor coating.

2. The injectable osteocartilage repair material as described in claim 1, characterized in that, The mass percentage of the phosphoserine-modified magnesium microspheres relative to the total mass of the first collagen and the first acidic solvent is 0.05 wt%-5 wt%; and / or, The mass percentage of the phosphoserine-modified magnesium-based mineralized microspheres relative to the total mass of the second collagen and the second acidic solvent is 0.1 wt% to 20 wt%.

3. The injectable osteocartilage repair material as described in claim 1, characterized in that, The cartilage repair solution contains a first collagen protein with a mass-to-volume concentration of 4 mg / mL to 8 mg / mL; and / or, The mass-volume concentration of the second collagen in the bone repair solution is 4 mg / mL to 8 mg / mL.

4. The injectable osteocartilage repair material as described in claim 1, characterized in that, The cartilage repair solution further comprises a first additive, which includes one or more of hyaluronic acid, chondroitin sulfate, and oxidized chondroitin sulfate; optionally, the mass-volume concentration of the first additive in the cartilage repair solution is 0.1 mg / mL-5 mg / mL; and / or, The bone repair solution further comprises a second additive, which includes one or more of hyaluronic acid, chondroitin sulfate, and oxidized chondroitin sulfate; optionally, the mass-volume concentration of the second additive in the bone repair solution is 0.1 mg / mL to 5 mg / mL.

5. The injectable osteocartilage repair material as described in claim 1, characterized in that, The pH value of the neutral ion triggering solution is 7.2-8.2, optionally 7.4-7.8; and / or, The neutral ion triggering solution includes a buffer system, ion regulating components, and a physiological salt system. Optionally, the buffer system includes one or more of PBS buffer, HEPES buffer, Tris buffer, and NaHCO3 buffer; Optionally, the ion regulating component includes one or more of a soluble salt containing calcium ions, a soluble salt containing magnesium ions, and a soluble salt containing potassium ions; further optionally, the ion regulating component includes one or more of calcium chloride, magnesium chloride, and potassium chloride. Optionally, the physiological salt system is sodium chloride.

6. A method for preparing an injectable osteocartilage repair material, characterized in that, Includes the following steps: Prepare a first acidic collagen solution containing a first collagen and a first acidic solvent, and a second acidic collagen solution containing a second collagen and a second acidic solvent; The first acidic collagen solution was mixed with phosphoserine-modified magnesium microspheres to prepare a cartilage repair solution. The second acidic collagen solution was mixed with phosphoserine-modified magnesium-based mineralized microspheres to prepare a bone repair solution; The phosphoserine-modified magnesium-based mineralized microspheres include a magnesium-based core, a phosphoserine interface coupling layer, and a calcium phosphate mineralization precursor coating, wherein the phosphoserine interface coupling layer couples the magnesium-based core and the calcium phosphate mineralization precursor coating. Prepare the neutral ion trigger solution.

7. The method for preparing the injectable osteocartilage repair material as described in claim 6, characterized in that, Includes one or more of the following conditions: (1) The mass-volume concentration of the first collagen in the first acidic collagen solution is 4 mg / mL-8 mg / mL; (2) The mass-volume concentration of the second collagen in the second acidic collagen solution is 4 mg / mL-8 mg / mL; (3) The mass percentage of the phosphoserine-modified magnesium microspheres relative to the mass of the first acidic collagen solution is 0.05wt%-5wt%; (4) The mass percentage of the phosphoserine-modified magnesium-based mineralized microspheres relative to the mass of the second acidic collagen solution is 0.1wt%-20wt%; (5) The first acidic collagen solution was mixed with phosphoserine-modified magnesium microspheres at 2℃-8℃; (6) The second acidic collagen solution was mixed with phosphoserine-modified magnesium-based mineralized microspheres at 2℃-8℃; (7) The first acidic solvent is an aqueous solution of acetic acid, wherein the molar concentration of acetic acid in the aqueous solution of acetic acid is 0.01 mol / L-0.05 mol / L; (8) The second acidic solvent is an aqueous solution of acetic acid, wherein the molar concentration of acetic acid in the aqueous solution is 0.01 mol / L-0.05 mol / L; (9) The first acidic collagen solution further contains a first additive, which includes one or more of hyaluronic acid, chondroitin sulfate and oxidized chondroitin sulfate; optionally, the mass-volume concentration of the first additive in the first acidic collagen solution is 0.1 mg / mL to 5 mg / mL. (10) The second acidic collagen solution also contains a second additive, which includes one or more of hyaluronic acid, chondroitin sulfate and oxidized chondroitin sulfate; optionally, the mass-volume concentration of the second additive in the second acidic collagen solution is 0.1 mg / mL to 5 mg / mL.

8. The method for preparing the injectable osteocartilage repair material as described in claim 6, characterized in that, The preparation method of the phosphoserine-modified magnesium-based microspheres includes: The first magnesium-based microspheres were mixed with the first phosphoserine solution, the pH of the mixture was adjusted to 6-7, and then the first magnesium-based microspheres were washed and dried to prepare the phosphoserine-modified magnesium-based microspheres. Optionally, the molar concentration of phosphoserine in the first phosphoserine solution is 0.5 mmol / L-20 mmol / L; further optionally, the mass-to-volume ratio of the first magnesium-based microspheres to the first phosphoserine solution is (1-50) mg:1 mL. Optionally, the Dv50 of the first magnesium-based microsphere is 15μm-60μm.

9. The method for preparing the injectable osteocartilage repair material as described in claim 6, characterized in that, The preparation method of the phosphoserine-modified magnesium-based mineralized microspheres includes: The second magnesium-based microspheres were mixed with a second phosphoserine solution, the pH of the mixture was adjusted to 6-7, and then the second magnesium-based microspheres were washed and dried to prepare a phosphoserine-modified magnesium-based core. The phosphoserine-modified magnesium-based core was mixed with a mineralization solution and then incubated to prepare the phosphoserine-modified magnesium-based mineralized microspheres; wherein the mineralization solution contained calcium ions and phosphate ions; Optionally, the mineralization solution contains a calcium ion source, which includes one or more of calcium chloride, calcium nitrate, and calcium acetate. Optionally, the mineralization solution contains a phosphate ion source, which includes one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium phosphate. Optionally, the molar ratio of calcium ions to phosphate ions in the mineralization solution is (1.3-1.67):1; Optionally, the molar concentration of calcium ions in the mineralization solution is 1 mmol / L to 20 mmol / L; Optionally, the molar concentration of phosphate ions in the mineralization solution is 0.5 mmol / L to 10 mmol / L; Optionally, the pH value of the mineralization solution is 6.5-7.5; Optionally, the incubation temperature is 20℃-37℃, or 25℃-37℃; the incubation time is 4h-48h, or 10h-24h. Optionally, the molar concentration of phosphoserine in the second phosphoserine solution is 0.5 mmol / L-20 mmol / L; further optionally, the mass-to-volume ratio of the second magnesium-based microspheres to the second phosphoserine solution is (1-50) mg:1 mL; Optionally, the Dv50 of the second magnesium-based microsphere is 15 μm-60 μm.

10. The method for preparing the injectable osteocartilage repair material according to any one of claims 6 to 9, characterized in that, The pH value of the neutral ion triggering solution is 7.2-8.2, and can be selected as 7.4-7.8; And / or, The preparation method of the neutral ion triggering solution includes: mixing the buffer system, ion regulating component and physiological salt system; Optionally, the buffer system includes one or more of PBS buffer, HEPES buffer, Tris buffer, and NaHCO3 buffer; Optionally, the ion regulating component includes one or more of a soluble salt containing calcium ions, a soluble salt containing magnesium ions, and a soluble salt containing potassium ions; further optionally, the ion regulating component includes one or more of calcium chloride, magnesium chloride, and potassium chloride. Optionally, the physiological salt system is sodium chloride.

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