Mineralized collagen cartilage repair scaffold, preparation method and application thereof

Abstract

The application provides a mineralized collagen cartilage repair scaffold and a preparation method and application thereof, relates to the technical field of biomedical materials, and comprises the following steps: injecting a mineralized collagen solution into a mold, and obtaining a mineralized collagen layer through deep freezing and primary freeze drying; taking the mineralized collagen layer as a bottom layer, injecting an un-mineralized collagen solution, and obtaining a freeze-dried scaffold through deep freezing and first freeze drying; and obtaining the mineralized collagen cartilage repair scaffold through compression after cross-linking and second freeze drying of the freeze-dried scaffold. Necessary biological active interfaces, continuous interpenetrating structures of collagen fiber networks, and cross-linking treatment are provided for bone integration, and layer separation is avoided; bidirectional regeneration of bone and cartilage is realized, and the complexity and risk of clinical application are reduced; good tailorability and sufficient stiffness are simultaneously provided in a wet state, and stable mechanical support is provided; and the technical problems of systematic insufficiency of scaffold materials in multi-scale structure bionics and biological safety of interface bonding agents in the prior art are solved.

Description

A mineralized collagen cartilage repair scaffold, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a mineralized collagen cartilage repair scaffold, its preparation method, and its application. Background Technology

[0002] In the clinical treatment of articular cartilage injuries, tissue-engineered scaffolds utilize biomimetic materials to create a microenvironment conducive to cell adhesion, proliferation, and differentiation, thereby promoting cartilage regeneration. However, existing cartilage repair scaffolds suffer from several key technical deficiencies in terms of osseointegration performance. For example, traditional homogeneous collagen scaffolds (such as commercially available membrane products) lack bioactive mineral phases when repairing full-thickness cartilage defects. This results in an inability to effectively stimulate osteoblast responses in the underlying bone environment, leading to the scaffold-host bone interface forming only a fibrous tissue isolation zone with poor mechanical properties, failing to achieve true osseointegration. Existing collagen / hydroxyapatite composite materials prepared using physical blending methods suffer from uneven distribution of mineral phases in the form of micron-sized aggregates, leading to stress concentration and potentially triggering inflammatory reactions due to their rough surfaces. Existing bilayer scaffolds generally employ a process of separate preparation followed by bonding, resulting in weak interlayer bonding and a risk of in vivo delamination. Furthermore, existing enhancement strategies relying on exogenous growth factors suffer from high costs and uncontrollable safety issues.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] One of the objectives of this invention is to provide a method for preparing a mineralized collagen cartilage repair scaffold, in order to solve the technical problems of insufficient scaffold materials in terms of multi-scale structural biomimicry and the biosafety of interfacial binders in the prior art.

[0005] The second objective of this invention is to provide a mineralized collagen cartilage repair scaffold.

[0006] The third objective of this invention is to provide the mineralized collagen cartilage repair scaffold prepared by the above-mentioned preparation method, or the application of the above-mentioned mineralized collagen cartilage repair scaffold in cartilage tissue regeneration or cartilage defect repair materials.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0008] In a first aspect, the present invention provides a method for preparing a mineralized collagen cartilage repair scaffold, comprising the following steps:

[0009] A. The mineralized collagen solution was injected into a mold and then subjected to deep freezing and primary freeze-drying to obtain a mineralized collagen layer.

[0010] B. Using the mineralized collagen layer from step A as the bottom layer, inject an unmineralized collagen solution to form a non-mineralized collagen layer, and obtain a freeze-dried scaffold by deep freezing and a first freeze-drying.

[0011] C. After cross-linking and a second freeze-drying, the freeze-dried scaffold from step B is compressed to obtain a mineralized collagen cartilage repair scaffold.

[0012] Furthermore, the concentration of the collagen solution is 3~20 mg / mL, preferably 8~15 mg / mL;

[0013] Preferably, the collagen includes type I collagen.

[0014] Furthermore, the mineralization process includes simultaneously and slowly adding calcium salt and phosphate to the collagen solution while stirring to prepare the mineralization reaction.

[0015] Preferably, the stirring speed is 200~400 rpm;

[0016] Preferably, the slow dripping rate is 0.5~2 ml / min;

[0017] Preferably, the temperature of the collagen solution during the dripping process is 2~8℃, more preferably 4℃;

[0018] Preferably, the mineralization reaction includes heating to 37°C and reacting for 12-48 hours;

[0019] Preferably, the heating rate is 0.5~1℃ / min;

[0020] Preferably, the mineralization treatment is followed by centrifugation at 2000-3000 rpm for 5-10 minutes.

[0021] Furthermore, the Ca / P molar ratio is 1.5~2, preferably 1.67;

[0022] Preferably, the concentration of the calcium salt is 50-200 mM, more preferably 100-150 mM;

[0023] Preferably, the concentration of the phosphate is 30-120 mM, more preferably 50-100 mM;

[0024] Preferably, the calcium salt comprises CaCl2;

[0025] Preferably, the phosphate includes Na2HPO4.

[0026] Furthermore, the deep-freezing temperature is -60 to -80°C, and the deep-freezing time is ≥6 hours;

[0027] Preferably, the temperature for the primary freeze-drying is -50 to -60°C, more preferably -50°C;

[0028] Preferably, the vacuum degree of the primary freeze-drying is 7~10 Pa, more preferably 10 Pa;

[0029] Preferably, the primary freeze-drying time is 24-48 hours.

[0030] Preferably, the conditions for the first freeze-drying are the same as those for the second freeze-drying.

[0031] Furthermore, the crosslinking includes immersing the lyophilized scaffold in a crosslinking solution for crosslinking;

[0032] Preferably, the crosslinking solution comprises 10-50 mM carbodiimide and 5-20 mM N-hydroxysuccinimide, and the solvent is a 70%-90% aqueous ethanol solution;

[0033] Preferably, the concentration of the carbodiimide is 20-30 mM;

[0034] Preferably, the concentration of the N-hydroxysuccinimide is 10~15mM;

[0035] Preferably, the crosslinking time is 6~24h;

[0036] Preferably, the crosslinking process further includes washing to remove crosslinking byproducts.

[0037] Furthermore, the compression includes applying mechanical pressure to compress the thickness to 2.0 ± 0.3 mm;

[0038] Preferably, the conditions for applying mechanical pressure include continuous pressurization of 0.5-5 MPa for 1-2 hours;

[0039] Preferably, before compression, the thickness of the mineralized collagen layer is 1-2 mm, and the thickness of the non-mineralized collagen layer is 1-2 mm.

[0040] Secondly, the present invention provides a mineralized collagen cartilage repair scaffold, which is prepared by the above-mentioned preparation method;

[0041] The mineralized collagen cartilage repair scaffold includes a mineralized collagen layer and a non-mineralized collagen layer covering the mineralized collagen layer, wherein the mineralized collagen layer is formed by the interpenetration of mineralized collagen fibers and non-mineralized collagen fibers.

[0042] Furthermore, the porosity is >90%;

[0043] Preferably, the average pore size of the non-mineralized collagen layer is 100~300μm;

[0044] Preferably, the average pore size of the mineralized collagen layer is 50~250μm.

[0045] Thirdly, the present invention provides the application of the mineralized collagen cartilage repair scaffold prepared by the above-mentioned preparation method or the above-mentioned mineralized collagen cartilage repair scaffold in cartilage tissue regeneration or cartilage defect repair materials.

[0046] The method for preparing a mineralized collagen cartilage repair scaffold provided by this invention provides a necessary bioactive interface for bone integration through a mineralized collagen layer. An unmineralized collagen solution is injected onto the mineralized collagen layer to form a non-mineralized collagen layer, which is then integrally formed with the mineralized collagen layer. This creates a continuous interpenetrating structure of collagen fiber networks at the interface between the two layers. A unified cross-linked covalent "suture" then forms a stable whole without a clear interface, significantly enhancing the interlayer bonding strength, improving the mechanical properties and stability of the scaffold, and avoiding the risk of interlayer separation. The method utilizes the bioactivity of collagen itself and the gradient structure formed during preparation to achieve bidirectional regeneration of bone and cartilage, reducing the complexity and risk of clinical applications. Compression shaping improves the density and compressive modulus of the scaffold, giving it both good cutability and sufficient "rigidity" in a wet state, greatly facilitating clamping, trimming, and suturing operations during surgery, while providing stable mechanical support for cartilage regeneration. This method solves the systemic technical problems of insufficient multi-scale structural biomimicry and the lack of biocompatibility of interfacial binders in existing scaffold materials. Attached Figure Description

[0047] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0048] Figure 1 is a scanning electron microscope image of the mineralized collagen cartilage repair scaffold provided in Embodiment 1 of the present invention;

[0049] Figure 2 is a scanning electron microscope image of the mineralized collagen cartilage repair scaffold provided in Embodiment 2 of the present invention;

[0050] Figure 3 is a scanning electron microscope image of the mineralized collagen cartilage repair scaffold provided in Comparative Example 3 of the present invention.

[0051] Figure 4 is a scanning electron microscope image of the mineralized collagen cartilage repair scaffold provided in Comparative Example 4 of the present invention.

[0052] Figure 5 is a scanning electron microscope image of the mineralized collagen cartilage repair scaffold provided in Comparative Example 5 of the present invention. Detailed Implementation

[0053] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0054] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein.

[0055] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0056] This invention provides a method for preparing a mineralized collagen cartilage repair scaffold, comprising the following steps:

[0057] A. The mineralized collagen solution was injected into a mold and then subjected to deep freezing and primary freeze-drying to obtain a mineralized collagen layer.

[0058] B. Using the mineralized collagen layer from step A as the bottom layer, inject an unmineralized collagen solution to form a non-mineralized collagen layer, and obtain a freeze-dried scaffold by deep freezing and a first freeze-drying.

[0059] C. After cross-linking and a second freeze-drying, the freeze-dried scaffold from step B is compressed to obtain a mineralized collagen cartilage repair scaffold.

[0060] The mineralized collagen layer provides the necessary bioactive interface for bone integration. An unmineralized collagen solution is injected into the mineralized collagen layer to form a non-mineralized collagen layer, which is then combined with the mineralized collagen layer to form a single, integral structure. This creates a continuous, interpenetrating collagen fiber network at the interface, and through unified cross-linking and covalent "suturing," a stable whole without a clear interface is formed, significantly enhancing interlayer bonding strength, improving the mechanical properties and stability of the scaffold, and avoiding the risk of interlayer separation. The bioactivity of collagen itself and the gradient structure formed during preparation enable bidirectional regeneration of osteochondrium, reducing the complexity and risk of clinical applications. Compression shaping improves the scaffold's density and compressive modulus, giving it both good cutability and sufficient "rigidity" in a wet state, greatly facilitating clamping, trimming, and suturing operations during surgery, while providing stable mechanical support for cartilage regeneration. This addresses the systemic shortcomings of existing scaffold materials in multi-scale structural biomimicry and the biosafety of interfacial binders.

[0061] The core materials are collagen and hydroxyapatite, without introducing any exogenous toxic materials. In some specific embodiments, the concentration of the collagen solution is 3-20 mg / mL, preferably 8-15 mg / mL. In some specific embodiments, the collagen includes type I collagen.

[0062] In some specific embodiments, the mineralization treatment includes simultaneously and slowly adding calcium salt and phosphate to a collagen solution while stirring to induce a mineralization reaction. This slow-release mineralization process, achieved through simultaneous stirring and slow addition, enables uniform deposition of minerals at the molecular level, forming a nanocomposite structure that more closely resembles natural bone tissue and exhibits excellent osteogenic activity. To further improve the mineralization effect, in some specific embodiments, the temperature of the collagen solution during the addition process is 2-8°C, preferably 4°C. In some specific embodiments, the stirring speed is 200-400 rpm; in some specific embodiments, the slow addition rate is 0.5-2 ml / min.

[0063] In some specific embodiments, the mineralization reaction includes heating to 37°C and reacting for 12-48 hours; in some specific embodiments, the heating rate is 0.5-1°C / min. Through "low-temperature slow drop-addition + slow heating," nanoscale, uniformly distributed hydroxyapatite is formed, which efficiently adsorbs osteogenic proteins and activates cell signaling pathways, thereby actively inducing osteoblast differentiation and bone tissue ingrowth.

[0064] In some specific embodiments, the mineralization treatment is followed by centrifugation at 2000-3000 rpm for 5-10 minutes.

[0065] In some specific embodiments, the Ca / P molar ratio is 1.5 to 2, preferably 1.67; in some specific embodiments, the concentration of the calcium salt is 50 to 200 mM, preferably 100 to 150 mM; in some specific embodiments, the concentration of the phosphate is 30 to 120 mM, preferably 50 to 100 mM.

[0066] The calcium salt includes CaCl2; the phosphate includes Na2HPO4.

[0067] In some specific embodiments, the deep-freezing temperature is -60 to -80°C, and the deep-freezing time is ≥6 hours.

[0068] In some specific embodiments, the temperature of the primary freeze-drying is -50~-60℃, preferably -50℃; in some specific embodiments, the vacuum degree of the primary freeze-drying is 7~10Pa, preferably 10Pa; in some specific embodiments, the time of the primary freeze-drying is 24~48h.

[0069] In some specific embodiments, the conditions for the first freeze-drying are the same as those for the second freeze-drying.

[0070] In some specific embodiments, the crosslinking includes immersing the lyophilized scaffold in a crosslinking solution for crosslinking; in some specific embodiments, the crosslinking solution includes 10-50 mM carbodiimide and 5-20 mM N-hydroxysuccinimide, and the solvent is a 70%-90% aqueous ethanol solution; in some specific embodiments, the concentration of the carbodiimide is 20-30 mM; in some specific embodiments, the concentration of the N-hydroxysuccinimide is 10-15 mM; in some specific embodiments, the crosslinking time is 6-24 h.

[0071] In some specific embodiments, the crosslinking process further includes washing to remove crosslinking byproducts.

[0072] In some specific embodiments, the compression includes applying mechanical pressure to compress the thickness to 2.0 ± 0.3 mm. In some specific embodiments, the conditions for applying the mechanical pressure include continuous pressurization of 0.5-5 MPa for 1-2 hours.

[0073] In some specific implementations, the thickness of the mineralized collagen layer is 1-2 mm before compression, and the thickness of the non-mineralized collagen layer is 1-2 mm.

[0074] According to another aspect of the present invention, a mineralized collagen cartilage repair scaffold is also provided, which is prepared by the above-described preparation method;

[0075] The mineralized collagen cartilage repair scaffold includes a mineralized collagen layer and a non-mineralized collagen layer covering the mineralized collagen layer, wherein the mineralized collagen layer is formed by the interpenetration of mineralized collagen fibers and non-mineralized collagen fibers.

[0076] It adopts a functional gradient design, and constructs a precise double-layer structure with an upper layer of non-mineralized collagen and a lower layer of biomimetic mineralized collagen. The composition truly reproduces the biological gradient of the natural cartilage-bone interface, so that the two layers form a continuous interpenetrating collagen fiber network at the interface.

[0077] In some specific implementations, the porosity is >90%;

[0078] In some specific embodiments, the average pore size of the non-mineralized collagen layer is 100~300μm;

[0079] In some specific embodiments, the average pore size of the mineralized collagen layer is 50~250μm.

[0080] According to another aspect of the present invention, the application of the mineralized collagen cartilage repair scaffold prepared by the above-described preparation method or the above-described mineralized collagen cartilage repair scaffold in cartilage tissue regeneration or cartilage defect repair materials is also provided.

[0081] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0082] Example 1

[0083] A mineralized collagen cartilage repair scaffold is prepared according to the following steps:

[0084] 1. Preparation of collagen slurry

[0085] Type I collagen extracted from bovine hide was dissolved in a dilute acetic acid solution (0.5 mol / L); the solution was slowly stirred at 4°C for 36 hours to prevent collagen molecule denaturation and form a homogeneous collagen suspension.

[0086] Adjust the collagen concentration to 8 mg / mL. Slowly add 0.1 M NaOH solution to the solution to neutralize the pH to 7.0-7.4 (physiological pH), and stir until homogeneous.

[0087] 2. Preparation of mineralized collagen layer

[0088] A type I collagen solution with a concentration of 8 mg / mL was pre-cooled at 4°C. Separate solutions of 125 mM CaCl2 and 75 mM Na2HPO4 (with a Ca / P molar ratio of 1.67) were prepared and pre-cooled to 4°C.

[0089] Under low temperature and low speed stirring (400 rpm) conditions at 4℃, CaCl2 solution and Na2HPO4 solution were simultaneously but separately and slowly added dropwise to the collagen solution at a rate of 1 mL / min using a precision syringe pump or peristaltic pump. After the addition was complete, the mixed reaction system was slowly heated to 37℃ at a heating rate of 1℃ / min and the reaction was continued at 37℃ for 24 h to complete the in-situ biomimetic deposition of hydroxyapatite on collagen fibers.

[0090] The reacted mineralized collagen slurry was centrifuged to remove air bubbles (3000 rpm, 10 min). The slurry was poured into a mold, controlling its thickness to 2 mm, and then rapidly deep-frozen in an ultra-low temperature freezer at -80℃ or in liquid nitrogen for more than 6 hours. The deep-frozen sample was transferred to a freeze dryer and initially freeze-dried for 48 hours at a temperature below -50℃ and a vacuum degree below 10 Pa to obtain a porous mineralized collagen sponge.

[0091] 3. Integrated construction and cross-linking of composite scaffolds

[0092] The mineralized collagen sponge prepared above was used as the bottom layer and precisely placed in a new mold. A non-mineralized collagen solution with a concentration of 8 mg / mL was slowly injected into the mold, completely covering the mineralized collagen layer, and the final thickness of the non-mineralized layer was controlled to be 1 mm. The entire composite system mold was directly placed at -80℃ for rapid deep freezing for 24 hours, allowing the upper and lower layers to form a unified ice crystal template. The deep-frozen composite system was then subjected to integrated freeze-drying to form an integrated porous scaffold with interpenetrating collagen fibers between the layers.

[0093] A 75% ethanol aqueous solution was prepared as the crosslinking agent solvent, with a carbodiimide (EDC) concentration of 25 mM and an N-hydroxysuccinimide (NHS) concentration of 12.5 mM. The integrated lyophilized scaffold was completely immersed in the crosslinking solution and crosslinked at room temperature for 12 hours. After crosslinking, it was thoroughly washed with a large amount of ultrapure water and ethanol mixture to completely remove crosslinking byproducts.

[0094] 4. Post-processing and finalization

[0095] The washed stent undergoes a second freeze-drying process to remove solvent and fix the cross-linked structure. The dried composite stent is then placed in a controlled humid and heat environment (e.g., 37℃, >75% relative humidity) and subjected to a mechanical pressure of 3 MPa for 6 hours, ultimately compressing the overall stent thickness to 2.0 ± 0.3 mm and achieving the required mechanical strength and density. The shaped stent is then aseptically cut, packaged, and finally sterilized using ethylene oxide. After sealing, it becomes the finished product.

[0096] Example 2

[0097] Unlike Example 1, in the preparation of the mineralized collagen layer, the mineralization reaction was carried out continuously at 37°C for 48 hours.

[0098] Example 3

[0099] Unlike Example 1, in the preparation of the mineralized collagen layer, the mineralization reaction was carried out continuously at 37°C for 12 hours.

[0100] Example 4

[0101] Unlike Example 1, the concentration of carbodiimide (EDC) in the crosslinking solution was 15 mM and the concentration of N-hydroxysuccinimide (NHS) was 7.5 mM.

[0102] Comparative Example 1

[0103] Unlike Example 1, the integrated porous scaffold was not immersed in the crosslinking solution; post-processing and shaping were performed directly.

[0104] Comparative Example 2

[0105] Unlike Example 1, no non-mineralized collagen layer is provided:

[0106] 3. Crosslinking

[0107] A 75% ethanol aqueous solution was prepared as the crosslinking agent solvent, with carbodiimide (EDC) concentration of 25 mM and N-hydroxysuccinimide (NHS) concentration of 12.5 mM. The prepared mineralized collagen sponge was completely immersed in the crosslinking solution and crosslinked at room temperature for 12 hours. After crosslinking, it was thoroughly washed with a large amount of ultrapure water and ethanol mixture to completely remove crosslinking byproducts.

[0108] Comparative Example 3

[0109] Unlike Example 1, deep freezing is not performed in steps 2 and 3.

[0110] Comparative Example 4

[0111] Unlike Example 1, in step 2, the CaCl2 solution and Na2HPO4 solution are added to the collagen solution simultaneously in one step.

[0112] Comparative Example 5

[0113] Unlike Example 1, in step 4, post-processing and shaping, the washed stent undergoes a second freeze-drying process to remove solvent and fix the cross-linked structure, resulting in a stent with an overall thickness of 3 mm and achieving the required mechanical strength and density. The shaped stent is then cut and packaged under aseptic conditions, and finally sterilized using ethylene oxide. After sealing and packaging, it becomes the finished product.

[0114] Comparative Example 6

[0115] Unlike Example 1, in step 4, post-processing and shaping, the washed stent undergoes a second freeze-drying process to remove solvent and fix the cross-linked structure. The dried composite stent is then placed in a controlled humid and hot environment (e.g., 37°C, >75% relative humidity) and subjected to a mechanical pressure of 5 MPa for 12 hours, ultimately compressing the overall stent thickness to 1-1.5 mm and achieving the required mechanical strength and density. The shaped stent is then aseptically cut, packaged, and finally sterilized using ethylene oxide. After sealing and packaging, it becomes the finished product.

[0116] Comparative Example 7

[0117] To prepare a pure collagen scaffold, follow these steps:

[0118] 1. Preparation of collagen slurry

[0119] Type I collagen extracted from bovine hide was dissolved in a dilute acetic acid solution (0.5 mol / L); the solution was slowly stirred at 4°C for 36 hours to prevent collagen molecule denaturation and form a homogeneous collagen suspension.

[0120] Adjust the collagen concentration to 8 mg / mL. Slowly add 0.1 M NaOH solution to the solution to neutralize the pH to 7.0-7.4 (physiological pH), and stir until homogeneous.

[0121] 2. Preparation of collagen basal layer

[0122] A type I collagen solution with a concentration of 8 mg / mL was pre-cooled at 4°C. The slurry was injected into a mold, controlling its thickness to 2 mm, and then rapidly deep-frozen in an ultra-low temperature freezer at -80°C or in liquid nitrogen for at least 6 hours. The deep-frozen sample was transferred to a freeze dryer and subjected to primary freeze-drying at -50°C or below and a vacuum degree of less than 10 Pa for 48 hours to obtain a porous collagen sponge.

[0123] 3. Integrated construction and cross-linking of composite scaffolds

[0124] The collagen sponge prepared above was used as the bottom layer and precisely placed in a new mold. A non-mineralized collagen solution with a concentration of 8 mg / mL was slowly injected into the mold, completely covering the mineralized collagen layer, and the final thickness of the non-mineralized layer was controlled to be 1 mm. The entire composite system mold was directly placed at -80℃ for rapid deep freezing for 24 hours, allowing the upper and lower layers to form a unified ice crystal template. The deep-frozen composite system was then subjected to integrated freeze-drying to form an integrated porous scaffold with interpenetrating collagen fibers between the layers.

[0125] A 75% ethanol aqueous solution was prepared as the crosslinking agent solvent, with a carbodiimide (EDC) concentration of 25 mM and an N-hydroxysuccinimide (NHS) concentration of 12.5 mM. The integrated lyophilized scaffold was completely immersed in the crosslinking solution and crosslinked at room temperature for 12 hours. After crosslinking, it was thoroughly washed with a large amount of ultrapure water and ethanol mixture to completely remove crosslinking byproducts.

[0126] 4. Post-processing and finalization

[0127] The washed stent undergoes a second freeze-drying process to remove solvent and fix the cross-linked structure. The dried composite stent is then placed in a controlled humid and heat environment (e.g., 37℃, >75% relative humidity) and subjected to a mechanical pressure of 3 MPa for 6 hours, ultimately compressing the overall stent thickness to 2.0 ± 0.3 mm and achieving the required mechanical strength and density. The shaped stent is then aseptically cut, packaged, and finally sterilized using ethylene oxide. After sealing, it becomes the finished product.

[0128] Test 1 Hygroscopicity

[0129] The mineralized collagen cartilage repair scaffolds from the above examples and comparative examples were selected as samples and measured according to the liquid absorption method in section 6.3 of YY / T1511-2017 Collagen Sponge. The results are shown in Table 1.

[0130] Table 1

[0131]

[0132] Data shows that the examples maintained good toughness and did not separate after absorbing moisture, while the comparative examples showed varying degrees of decreased toughness or separation. Comparative example 6, being too thin, did not separate, but its dilution factor decreased.

[0133] Experiment 2

[0134] The porosity of the scaffold was tested according to GB / T1966, with an optimal porosity ≥90%, and the results are shown in Table 2. SEM images of the samples from Examples 1, 2, and Comparative Examples 3-5 are shown in Figures 1-5. The scale bars for Figures 1 and 3-5 are 200 μm, and the scale bar for Figure 2 is 500 μm.

[0135] Experiment 3

[0136] The cytotoxicity of the scaffold material was detected using the MTT assay. First, an extract of the scaffold material was prepared. 2.0 g of the sterile double-layer osteochondral repair scaffold material prepared in the above-described embodiments of the present invention, sterilized by electron beam radiation, was added to 20 mL of LDM complete culture medium and extracted at 37°C for 72 ± 2 h to obtain a scaffold extract of 100 mg / mL. hBMSC cells were cultured at 37°C with a CO2 concentration of 5.0%. The MTT assay was performed according to GB / T16886.5, and the absorbance was measured at 450 nm using a microplate reader. The relative proliferation rate of hBMSC cells was calculated, and the results are shown in Table 2.

[0137] Table 2

[0138]

[0139] Data shows that, compared with Comparative Example 7, Examples 1-4 maintained a relatively stable range of porosity and showed no significant difference in cell growth rate, indicating that none of the examples and comparative examples were cytotoxic.

[0140] Experiment 4

[0141] The mineralized collagen cartilage repair scaffolds from the above embodiments and comparative examples were selected as samples for cell adhesion testing, and the specific procedures were as follows:

[0142] According to 1.5×10 5 Cells were seeded into 24-well plates at a density of cells / well, with four parallel experiments for each sample. After culturing the cells in an incubator for 12 hours, the waste liquid was aspirated, and the pre-prepared drug solution was added, followed by another 12 hours of culturing. After aspirating the waste liquid, the cells were washed three times with PBS buffer (15 vortex cycles per wash). Cells that were fully digested with trypsin were counted and statistically analyzed. The results are shown in Table 3.

[0143] Table 3

[0144]

[0145] Initial cell count: 34 ± 3 (×10⁻⁶) 4 Experiments showed that, compared with Comparative Example 2, Examples 1-4 effectively improved cell adhesion, reaching or even exceeding the cell adhesion of the pure collagen scaffold in Comparative Example 7. This indicates that setting a mineralized collagen layer is beneficial for cell adhesion and promotes cartilage repair and regeneration. Compared with Example 1, the cell adhesion effects of Comparative Examples 1 and 4-6 were all reduced, indicating that the different preparation steps in this scheme can synergistically improve the cell adhesion effect of the scaffold.

[0146] Experiment 5 Osteointegration

[0147] According to YY / T 1447-2016, the scaffold samples were immersed in simulated body fluid (SBF) and cultured at 37℃ for different time points (e.g., 7, 14, and 21 days). The calcium-to-phosphorus ratio was analyzed by energy dispersive spectroscopy (EDS), and the results are shown in Table 4.

[0148] Table 4

[0149]

[0150] Experiment 6 In vitro degradation

[0151] In vitro degradation performance tests were conducted. 1 g of sample was weighed and soaked in PBS buffer (pH=7.4) at 37℃ in a 0.1 g / mL system. The degradation of the sample was observed periodically until it was no longer visible to the naked eye. The corresponding time was recorded. The results are shown in Table 4.

[0152] Data shows that the scaffolds prepared by the processes in Examples 1-4 degraded in 8-16 weeks, close to the cartilage repair time (8-16 weeks), indicating that they did not degrade too quickly. Furthermore, their calcium-to-phosphorus ratio remained consistently between 1.5 and 2, close to the composition of natural bone minerals. Compared to Example 1, Comparative Examples 1 and 5 maintained a calcium-to-phosphorus ratio between 1.5 and 2, but degraded too quickly. Comparative Examples 2 and 4 showed little difference in calcium-to-phosphorus ratio and degradation rate, but Comparative Example 2 had low porosity and poor cell growth and adhesion, which was still unfavorable for cartilage repair and regeneration during application. Comparative Example 4 had its calcium and phosphate added all at once, resulting in disordered interlayer structure, uneven pore distribution, poor growth and adhesion, and delamination during use, affecting repair and regeneration. Comparative Example 3 was not subjected to deep freezing treatment, affecting the initial release of calcium and phosphorus elements. Comparative Example 6 had a low initial calcium-to-phosphorus ratio; although it was pressed thinner, its degradation rate was close to the maximum optimal degradation time, and its porosity was substandard, resulting in poor cell adhesion.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a mineralized collagen cartilage repair scaffold, characterized in that, Includes the following steps: A. Inject the mineralized collagen solution into the mold, and then deep freeze and primary freeze-dry to obtain a mineralized collagen layer; B. Using the mineralized collagen layer from step A as the bottom layer, inject an unmineralized collagen solution to form a non-mineralized collagen layer, and then deep freeze and a first freeze-dry to obtain a freeze-dried scaffold. C. After cross-linking and a second freeze-drying, the freeze-dried scaffold from step B is compressed to obtain a mineralized collagen cartilage repair scaffold. The mineralization process includes simultaneously and slowly adding calcium salt and phosphate to the collagen solution while stirring for mineralization reaction. The stirring speed is 200~400 rpm. The slow addition rate is 0.5~2 ml / min. The deep freezing temperature is -60~-80℃, and the deep freezing time is 6~8 h.

2. The preparation method according to claim 1, characterized in that, The concentration of the collagen solution is 3~20 mg / mL; the collagen includes type I collagen.

3. The preparation method according to claim 2, characterized in that, The temperature of the collagen solution during the dropwise addition process is 2~8℃; the mineralization reaction includes heating to 37℃ and reacting for 12~48h; the heating rate is 0.5~1℃ / min; the mineralization treatment is followed by centrifugation at 2000~3000rpm for 5~10min.

4. The preparation method according to claim 3, characterized in that, The Ca / P molar ratio is 1.5~2; the concentration of the calcium salt is 50~200 mM; the concentration of the phosphate is 30~120 mM; the calcium salt includes CaCl2; the phosphate includes Na2HPO4.

5. The preparation method according to claim 1, characterized in that, The temperature of the primary freeze-drying is -50 to -60°C; the vacuum degree of the primary freeze-drying is 7 to 10 Pa; the time of the primary freeze-drying is 24 to 48 hours; the conditions for the first freeze-drying and the second freeze-drying are the same.

6. The preparation method according to claim 1, characterized in that, The crosslinking process includes immersing the lyophilized scaffold in a crosslinking solution for crosslinking; the crosslinking solution includes 10-50 mM carbodiimide and 5-20 mM N-hydroxysuccinimide, and the solvent is a 70%-90% aqueous ethanol solution; the crosslinking time is 6-24 h; the crosslinking process also includes washing to remove crosslinking byproducts.

7. The preparation method according to claim 1, characterized in that, The compression includes applying mechanical pressure to compress the thickness to 2.0±0.3mm; the conditions for applying the mechanical pressure include continuous pressure of 0.5-5MPa for 1-2 hours; before compression, the thickness of the mineralized collagen layer is 1-2mm and the thickness of the non-mineralized collagen layer is 1-2mm.

8. A mineralized collagen cartilage repair scaffold, characterized in that, The mineralized collagen cartilage repair scaffold is prepared by any one of claims 1 to 7; the mineralized collagen cartilage repair scaffold comprises a mineralized collagen layer and a non-mineralized collagen layer covering the mineralized collagen layer, wherein the mineralized collagen layer is formed by the interpenetration of mineralized collagen fibers and non-mineralized collagen fibers.

9. The mineralized collagen cartilage repair scaffold according to claim 8, characterized in that, Porosity > 90%; the average pore size of the non-mineralized collagen layer is 100~300μm; the average pore size of the mineralized collagen layer is 50~250μm.

10. The application of the mineralized collagen cartilage repair scaffold prepared by the preparation method according to any one of claims 1 to 7, or the mineralized collagen cartilage repair scaffold according to claim 8 or 9, in the preparation of cartilage defect repair materials.

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