CN113456885A

CN113456885A - Gradient material for promoting repair of cartilage calcified layer and preparation method thereof

Info

Publication number: CN113456885A
Application number: CN202110591079.XA
Authority: CN
Inventors: 苟中入; 刘梦涛; 杨贤燕; 钟成; 高长有; 毛铮伟; 沈淼达; 赵腾飞; 谢利军; 金日龙
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-05-28
Filing date: 2021-05-28
Publication date: 2021-10-01
Anticipated expiration: 2041-05-28
Also published as: CN113456885B

Abstract

The invention discloses a gradient material for promoting the repair of a cartilage calcification layer and a preparation method thereof. The gradient material is formed by a superfine fiber microporous membrane and an organic-inorganic composite fiber microporous membrane which cover the upper surface and the lower surface of the superfine fiber microporous membrane respectively, the upper layer membrane contains a component for promoting cartilage healing, the inorganic component in the lower layer membrane is composed of multifunctional inorganic particles for inhibiting inflammation, preventing and controlling infection and promoting regeneration and repair of a cartilage calcification layer, and the gradient material is prepared by a three-step electrostatic spinning process. The method of the invention has convenient operation, has obvious promotion effect on various articular cartilage injuries, especially the regeneration, repair and reconstruction of calcified layers, can promote the healing of subchondral bone and cartilage tissues, has adjustable degradation rate of gradient materials, and has good regulation effect on various articular pathological inflammation.

Description

Gradient material for promoting repair of cartilage calcified layer and preparation method thereof

Technical Field

The invention belongs to an implantation gradient material and a preparation method thereof in the field of biomedical implantation materials, and particularly relates to a gradient functional material for promoting the restoration and reconstruction of a cartilage calcification layer and a preparation method thereof.

Background

The joints between the bones in the human body play the role of coordinating the movement of limbs. However, arthritic lesions and various bone-cartilage injuries in joint parts, joint fractures and other problems can cause cartilage injuries in joint parts, cause persistent or seasonal pain to patients, and the articular cartilage injuries caused by the articular lesions are difficult to attach importance to the patients at early stage and often involve cartilage to lower bones, so that if the articular cartilage injuries are not treated in time, the basic motor functions of the damaged joint parts of the patients are lost, especially the joint injuries of lower limbs can cause disability results, and various complications further increase the fatality risk. In China, tens of millions of patients with joint diseases and mechanical injuries appear every year, but the current clinical treatment method is still very limited, and the problems of arthritis and the like are mainly solved by medicine intervention for relieving pain; for patients with severe joint damage, surgical treatments, such as joint replacement, are used to improve inflammatory symptoms or increase lubrication by injecting drugs into the joint cavity. The excellent effect of treating both principal and secondary aspects of disease is difficult to produce no matter the treatment is carried out by taking medicines or the treatment is carried out by joint replacement surgery, the focus of infection cannot be eradicated by the medicines, and the replacement surgery brings great trauma and huge economic burden to patients.

In recent years, a large amount of research is carried out on the regeneration and repair treatment of joint injury in the academic communities at home and abroad, deep research and clinical trials are gradually carried out from simple cartilage repair to bone-cartilage integrated regeneration and repair, research heat tide is formed from stem cell treatment, extracellular matrix removal bionic material design and multilayered gradient functional material development, the anatomical structure of the joint is comprehensively analyzed, the multilayered structure of the joint cartilage is realized, and a calcified transition layer is found between the joint cartilage and subchondral bone. The cartilage calcified layer plays an important barrier role in the aspect of the transition of soft tissues to hard tissue anatomical structures, thereby playing a buffering role in the biomechanical conduction of a bone-cartilage interface region. However, no research has been conducted on the reconstruction of the calcified layer, and especially, the existing multifunctional (gradient) material has not been successful in constructing the calcified layer structure at the cartilage-bone interface region in various models or clinical treatments, and the new tissue structure after the repair of articular cartilage injury has a significant difference from the physiological tissue, and the therapeutic consequences are as follows: the 'pathological neogenetic tissue' constructed by artificial repair cannot completely adapt to the biomechanical function requirement of a patient because of not conforming to the physiological structure, and the repaired joint bone-cartilage part can continuously generate the problems of degeneration, pain and the like.

Therefore, the new material for promoting the articular cartilage damage repair needs to give consideration to the repair and reconstruction of the cartilage calcified layer, and can also perform synergistic regulation and promote healing on the damage of subchondral bone and cartilage per se when the goal is achieved, so that the new tissue is effectively improved and the long-term biomechanical requirement is met. However, achieving this goal requires solving three aspects of the problem:

firstly, effectively regulate and control the differentiation of chondrocytes close to a calcified layer to hypertrophic chondrocytes and mineralized transformation, and realize the regeneration and reconstruction of the calcified layer.

And secondly, after the cartilage layer is damaged, the tissues of the upper layer, the middle layer and the lower layer need to be repaired cooperatively, so that once the functional substances in the repairing of the calcification layer regulate and control the healing of the cartilage layer and the subchondral bone, the integrated repairing is favorably realized.

Thirdly, the inflammation reaction in pathological joint injury is lasting, the inflammation generally develops irreversibly under the condition of lacking drug intervention, so when the tissue injury is repaired, the anti-inflammatory function of the artificial material is very beneficial to improving the quality of the new tissue in the injury, and therefore, the anti-inflammatory and anti-infection functional bacteria of the artificial material have very good effect on promoting the repair of the joint injury problem.

Therefore, the repair and treatment of the articular cartilage injury problem cannot only depend on the support of anti-infection and anti-inflammatory drugs after the operation, but also cannot only consider the problem of how to reconstruct the cartilage and the subchondral bone, and the problem of repair and treatment of the articular cartilage injury at the middle and late stages in clinic can be solved by paying attention to the repair of the calcified layer and establishing that multiple tissues can be synchronously repaired and enhanced by certain functional substances.

So far, some inorganic salt compound dissolved ions or inorganic ions released by amorphous glassy materials have regulation and control effects on various tissue regeneration cells, have obvious inhibition and even killing effects on some susceptible pathogenic bacteria, and can regulate immune cell activity, thereby mediating inflammatory reaction to develop towards tissue regeneration and repair. Such as zinc ion, can inhibit and kill harmful bacteria in wounds, and has the effects of promoting the expression of antioxidants to resist oxidative damage and the like (Medicina 2020, 56: 614). Meanwhile, magnesium ions can regulate the expression of inflammatory factors of immune cells, reduce the generation of related neuropeptides, promote the polarization of synovial macrophage M2, reduce the secretion of inflammatory factors, thereby reducing the stimulation to nerve terminals and having anti-inflammatory effect (Bioactive Mater 2021, 6: 1341). In addition, strontium ions can also regulate and control the expression of collagen, polysaccharide and protease by chondrocytes and the expression of inflammatory factors, thereby showing excellent effects of improving the repair and reconstruction of joint injury. Secondly, it was also successively confirmed that some degradable calcium (magnesium) silicate inorganic compound materials can promote bone and cartilage tissue repair, and the applicant of the present invention has found that magnesium doped wollastonite can regulate differentiation of chondrocytes to hypertrophic chondrocytes and regulate the high expression of calcified layer extracellular matrix specific markers and collagen X (Scientific reports.2018, 8: 17911). Therefore, the construction of the artificial material with the functions of resisting inflammation, preventing infection and promoting the repair and reconstruction of the calcified cartilage layer is feasible for solving the difficult problems of joint injury lesion and the like in clinic.

According to the research of the prior art, a multifunctional gradient material which can effectively repair articular cartilage injury in composition, structure, physical and chemical properties and biological effect is urgently needed to be created, the material can actively regulate and control calcified layer regeneration repair and cartilage and bone tissue healing, and meanwhile, the microstructure of the material is favorable for nutrient transmission and prevents osteoblasts of subchondral bone from penetrating to a cartilage area. Therefore, the requirement of repairing the calcified layer of the cartilage can be effectively met only by adopting a multi-element synergistic innovative design.

According to the prior patent technology, research literature reports and clinical application, the design of gradient functional materials capable of promoting the repair and reconstruction of damaged calcified layers of articular cartilage is urgently needed, and the functions and efficacies of the materials need to be achieved include:

1) performing autonomous antibiosis;

2) the sustained anti-inflammation effect is achieved, and the effective inhibition effect on inflammatory reaction is achieved;

3) can effectively mediate regeneration, repair and reconstruction of calcified layer tissues;

4) the healing of subchondral bone and cartilage can be effectively promoted, and the repair of different tissues can be synergistically promoted;

5) has no side effect, has excellent biocompatibility, and basically finishes degradation and absorption when the calcified layer is repaired and rebuilt.

Disclosure of Invention

In order to solve the technical blank of the deficiency in the background technology, the invention provides a gradient material for promoting the repair of a cartilage calcification layer and a preparation method thereof.

The multifunctional inorganic ultrafine particles are integrated into the lower layer membrane of the ultrafine fibrous membrane gradient material, functional substances for promoting the healing of the cartilage are integrated into the upper layer membrane, after the gradient material is implanted into articular cartilage injury, a fiber network in contact with subchondral bone contains the inorganic ultrafine particles, can degrade and release a multi-element inorganic ion composition, causes the effects of local inflammation diminishing, infection resistance and regeneration and repair of calcified layer tissues, has excellent effects of promoting repair and reconstruction of various severe articular injuries, and also promotes the healing of the cartilage layer and regulates and controls inflammatory reaction by the upper layer functional substances, thereby solving the functional requirements in the repair of various layer tissues in the articular injury.

The gradient material for promoting the repair of the cartilage calcified layer not only solves the problems of long-term inflammation diminishing, bacteriostasis, regeneration and repair of the calcified layer, cooperative enhancement of healing of subchondral bone and a cartilage layer and the like, but also solves the problems of simple and convenient operability, complete degradation and absorption and the like, thereby achieving the ideal standard for promoting the repair of articular cartilage injury and providing a superior novel gradient material for promoting the repair of the cartilage calcified layer for solving the clinical problem.

The technical scheme adopted by the invention is as follows:

a gradient material for promoting the repair of a cartilage calcification layer:

the gradient material for promoting the repair of the cartilage calcification layer is a three-layer fiber microporous membrane, the lower layer is composed of a superfine fiber microporous membrane containing calcium-silicon-based inorganic particles, the calcium-silicon-based inorganic particles are calcium-silicon-based bioglass particles or biological ceramic particles, the middle layer is composed of a polymer fiber microporous membrane, the upper layer is mainly composed of a superfine fiber microporous membrane containing a functional substance for promoting the healing of cartilage, the micropore size of the gradient material is smaller than 100 micrometers, and micropores are micropores formed by crossing fibers in the lower layer, the middle layer and the upper layer.

The particle size of the calcium-silicon-based bioglass and the biological ceramic particles is 0.04-15 microns, and the diameter of the superfine fiber is 0.1-8.0 microns.

The calcium-silicon-based bioglass particles are degradable bioglass prepared by a sol-gel method, and contain CaO and SiO₂MgO, ZnO and P₂O₅、SrO、CuO、B₂O₃、Li₂O、Na₂O、K₂O、Mn₂O₃At least one oxide of (a);

the calcium-silicon-based biological ceramic particles are heterogeneous ion-doped wollastonite, akermanite, whitlaite, akermanite and diopside inorganic salt compounds, and the heterogeneous ions are one or a combination of more of strontium, zinc, magnesium, copper, phosphorus, sodium, potassium, boron, manganese and lithium.

The calcium-silicon-based biological ceramic particles comprise the following metal ions and acid radical ions in parts by mole:

SiO₃ ^2-24 to 60 portions of

Ca²⁺20 to 60 portions of

Mg²⁺0 to 40 parts of

Zn²⁺0 to 30 parts of

PO₄ ^3-0 to 15 parts of

BO₃ ^-0 to 15 parts of

Cu²⁺0 to 10 parts of

Mn³⁺0 to 5 parts of

Sr²⁺0 to 12 parts of

Li⁺0 to 8 portions of

Na⁺0 to 8 portions of

K⁺0 to 8 portions of

Remove Ca²⁺、SiO₃ ^2-At least one of the other metal ions or acid radical ions is not 0 at the same time, and the rest is crystal water.

The calcium-silicon-based bioglass particles comprise the following oxides in parts by mole:

SiO₂30 to 78 portions of

16-48 parts of CaO

1 to 30 parts of MgO

P₂O₅0 to 20 parts of

B₂O₃0 to 30 parts of

0.5-10 parts of ZnO

0-5 parts of CuO

0 to 5 parts of MnO

0 to 10 parts of SrO

Li₂0 to 10 parts of O

Na₂0 to 15 parts of O

K₂0 to 10 parts of O

Except CaO and SiO₂At least one of the oxides other than ZnO and MgO is not 0 at the same time.

The functional substance for promoting cartilage healing is one or a combination of chondroitin sulfate, glucosamine, type I collagen, type II collagen, transforming growth factor, vascular endothelial growth factor and bone morphogenetic protein.

The particle size of the calcium-silicon-based biological ceramic particles is 0.04-15.00 microns, the biological ceramic particles are heterogeneous ion-doped wollastonite, akermanite, whitish wollastonite, akermanite and diopside inorganic salt compounds, and the heterogeneous ions are one or a combination of more of strontium, zinc, magnesium, copper, phosphorus, sodium, potassium, boron, manganese and lithium.

Secondly, a preparation method of the gradient material for promoting the repair of the cartilage calcification layer comprises the following steps:

1) adding calcium-silicon-based inorganic particles into a solution containing an electro-spinning polymer, fully stirring, wherein the mass ratio of the calcium-silicon-based inorganic particles to the electro-spinning polymer is 1 (2-80), preparing a compound solution with the particle concentration of 1-400 mg/ml, adding the compound solution into a storage container for high-voltage electrostatic spinning, setting the spinning space to be 8-18 cm, carrying out high-voltage electrostatic spinning at the spinning voltage of 6-20 kV at the speed of 0.10-1.20 ml/h, and forming a superfine fiber microporous membrane on a receiving carrier for electrostatic spinning so that the calcium-silicon-based inorganic particles are embedded and formed in the superfine fiber microporous membrane; when the thickness of the superfine fiber microporous membrane on the receiving carrier reaches 20-300 microns, stopping electrostatic spinning, and discharging electrospinning liquid;

2) adding a solution containing an electrospinning polymer into the storage container for electrospinning in the step 1), modulating electrospinning parameters, continuing to perform high-voltage electrospinning, and depositing the electrospinning on the upper surface of the superfine fiber microporous membrane in the step 1) to enable the functional substance for promoting cartilage healing to be embedded and formed in a new layer of superfine fiber microporous membrane; when the thickness of the newly added superfine fiber microporous membrane on the receiving carrier reaches 20-300 microns, stopping electrostatic spinning, and discharging an electrospinning solution;

3) adding a substance containing a function of promoting cartilage healing into a solution containing an electrospun polymer, fully mixing, wherein the mass ratio of the substance containing the function of promoting cartilage healing to the electrospun polymer is 1 (10-200), obtaining a mixed solution, then adding the mixed solution into a storage container of the high-voltage electrostatic spinning equipment in the step 2), modulating electrospinning parameters, continuing to perform high-voltage electrostatic spinning, and depositing the electrospinning on the upper surface of the superfine fiber microporous membrane prepared in the step 2) to enable the substance containing the function of promoting cartilage healing to be embedded and formed in a new layer of the electrospun microporous membrane; and when the thickness of the newly added electrospinning microporous membrane on the receiving carrier reaches 20-300 microns, stopping electrostatic spinning to obtain the microporous membrane gradient material with the total thickness of 60-900 microns.

The receiving carrier is a porous metal roller, the pore size is 10-2500 micrometers, and the pore form is not strictly limited.

The polymer is one or a compound of more of gelatin, chitosan, polylactic acid-glycolic acid copolymer, polycaprolactone, poly-L-lactide-caprolactone, sodium alginate, hyaluronic acid, polymethacrylic acid, dextran sulfate, sodium carboxymethylcellulose, cellulose, polyacrylic acid, polystyrene sulfonic acid, polyvinyl sulfonic acid and polyvinyl phosphoric acid.

The concentration of the organic polymer in the electrospinning solution is not strictly limited, and the concentration level of the composite solution is determined by taking excellent spinnability as a condition; the solvent of the electrospinning solution is not strictly limited, and a liquid substance in which the organic polymer is completely dissolved is used as the solvent.

The application of the gradient material for promoting the repair of the cartilage calcification layer in the repair of joint injury realizes the autonomous antibiosis, the continuous inflammation diminishing, the regeneration and repair of the cartilage calcification layer and the improvement of the healing of cartilage and subchondral bone.

The application of the gradient material for promoting the repair of the cartilage calcified layer is characterized in that the gradient material is constructed in a multilayered way, a polymer fiber porous grid is used as a carrier to solve the problem of mechanical support, and a superfine fiber microporous membrane containing calcium-silicon-based bioglass or bioceramic particles and a superfine fiber membrane containing the function of promoting the regeneration and healing of cartilage are tightly combined with two surfaces of the superfine polymer fiber membrane, so that different multifunctionalities of interfaces in contact with subchondral bone and cartilage are ensured to be regulated and controlled through different chemical components, the repairing and reconstruction of a calcified layer, a subchondral bone layer and a cartilage layer at an articular cartilage injury part are considered, the inflammatory reaction is effectively regulated, and infection is prevented and treated.

The gradient material for promoting the repair of the cartilage calcified layer has special inorganic ions in the inorganic particles, and is favorable for maintaining and improving the biocompatibility, diminishing inflammation, resisting bacteria and promoting the regeneration and repair of the calcified layer.

The gradient material for promoting the repair of the cartilage calcified layer has no strict limitation on the combination form of the inorganic superfine particles in the fiber, and the particles can be embedded into the fiber body or attached to the surface of the fiber.

The gradient material for promoting the repair of the cartilage calcified layer has no strict limitation on the chemical composition of the organic polymer in the superfine fiber membrane, and each layer can be the same polymer or different polymers and can also be a compound of any two or three polymers.

The preparation process of the gradient material for promoting the repair of the cartilage calcification layer, disclosed by the invention, for compounding the microporous structure can be an electrospinning process, and can also be a phase separation method and a freeze-drying method, and the formed three-layer composite material belongs to the field of the gradient material for promoting the repair of the cartilage calcification layer.

The gradient material for promoting the repair of the calcified cartilage layer has no strict limitation on the application range, and can be applied to promoting the regeneration repair of the damaged calcified layer and the cooperative healing repair of cartilage and subchondral bone tissues in the repair and treatment of joint injury at any joint part of a human body.

The invention has the advantages that:

1) in composition, the bioglass particles and the completely or partially crystalline inorganic mineral particles synthesized by the conventional process contain multiple inorganic functional ions which can be slowly degraded when contacting tissue fluid, and the released inorganic ion composition is a mineral substance required by human joint bone-cartilage physiological metabolism or tissue regeneration and repair, so that no ion component which seriously influences the compatibility of bone and cartilage tissue regeneration cells exists, and meanwhile, certain ions also remarkably show anti-inflammatory and anti-harmful bacteria activities and/or calcified layer regeneration promoting effects, thereby preventing various risks and playing a role in stimulating the activity of tissue repair.

2) In the (micro) structure, the micropores of the porous grid formed by three layers of gradients not only effectively prevent the migration of subchondral bone osteoblasts to a cartilage layer, but also provide a mediation effect for inflammatory reactions in the injury of calcified layers, cartilage and subchondral bone tissues and provide a pore channel for the injury, and meanwhile, the superfine fiber micro-nano structure of the gradient material is also extremely beneficial to the adhesion and growth of related tissue repair cells and provides a good support for accelerating the regeneration and reconstruction of calcified layers.

3) In the aspect of biological effect, the biodegradable gradient material is a biodegradable gradient material which is constructed based on human body mineral substances and cartilage reconstruction functional substances in a synergistic mode, and when the biodegradable gradient material is loaded and contacts with cartilage wounds, good conditions are created for rapid inflammation diminishing, antibiosis and tissue regeneration healing of multiple functional ions and functional molecules which are gradually degraded and released by the biodegradable gradient material, so that regeneration and reconstruction of calcified layers in articular cartilage wounds and cooperative healing and repair of cartilage and subchondral bones are realized.

4) In the aspects of preparation process and clinical application operability, the sheet polymer fiber network membrane can be prepared by applying an electrostatic spinning process, and three layers are sequentially superposed to construct a gradient material for promoting the repair of a cartilage calcification layer, so that the problems of nutrient transfer and self healing and repair of tissues in a contact interface area of cartilage damaged tissues are effectively solved.

Therefore, the gradient material for regenerating and repairing the calcified layer of the cartilage is obviously characterized by simple preparation process, continuous inflammation diminishing and pathogenic infection prevention according to the damage degree of the articular cartilage, strengthening the healing of the cartilage and the subchondral bone and improving the pathological tissue formation of articular cartilage damage repair when the calcified layer is regenerated and repaired.

Drawings

Fig. 1 is a flow chart of a gradient material preparation process, wherein A, B, C represents the bottom layer, middle layer and top layer fiber film electrospinning process respectively.

FIG. 2 is a side electron micrograph of the gradient material.

FIG. 3 is a transmission electron microscope image of a fiber network of an underlayer film of a gradient material, wherein both the image A and the image B show that inorganic mineral particles are contained in the fibers.

FIG. 4 is a general observation of the gradient material implanted in the cartilage calcification layer after 8 weeks in the arthritis lesion model, wherein: FIG. A is a control group, which belongs to the repair situation of directly replanting a cartilage layer after a calcified layer is taken out; panel B is a control group, which belongs to the repair situation after the calcified layer is removed and replaced by pure polymer and the cartilage layer is replanted; FIG. C is the restoration condition after the calcified layer is taken out and replaced by the gradient material of the lower magnesium-zinc co-doped wollastonite particle and the cartilage layer is replanted; panel D shows the repair of the calcified layer after removal replaced by the underlying gradient material containing bioglass particles and the cartilage layer after implantation.

Fig. 5 is a two-dimensional and three-dimensional structural observation of μ CT reconstruction 8 weeks after implantation of a gradient material into a cartilage calcification layer of a arthritis lesion model, wherein: FIG. A is a control group, which belongs to the repair situation of directly replanting a cartilage layer after a calcified layer is taken out; panel B is a control group, which belongs to the repair situation after the calcified layer is removed and replaced by pure polymer and the cartilage layer is replanted; FIG. C is the restoration condition after the calcified layer is taken out and replaced by the gradient material of the lower magnesium-zinc co-doped wollastonite particle and the cartilage layer is replanted; panel D shows the repair of the calcified layer after removal replaced by the underlying gradient material containing bioglass particles and the cartilage layer after implantation.

FIG. 6 is a tissue staining observation after 8 weeks of surgery of implanting a cartilage calcification layer in a arthritis lesion model with a gradient material, wherein: FIG. A is a control group, which belongs to the repair situation of directly replanting a cartilage layer after a calcified layer is taken out; panel B is a control group, which belongs to the repair situation after the calcified layer is removed and replaced by pure polymer and the cartilage layer is replanted; FIG. C is the restoration condition after the calcified layer is taken out and replaced by the gradient material of the lower magnesium-zinc co-doped wollastonite particle and the cartilage layer is replanted; panel D shows the repair of the calcified layer after removal replaced by the underlying gradient material containing bioglass particles and the cartilage layer after implantation.

Detailed Description

The present invention will be further illustrated with reference to the following examples, which are not intended to limit the scope of the present invention, and all the techniques and materials prepared based on the above-mentioned contents of the present invention shall fall within the protection scope of the present invention. The purity of the reagents used in the examples is no lower than the purity of the reagents used in the analytical tests.

The examples of the invention are as follows:

example 1:

1) adding magnesium and zinc co-doped wollastonite powder (the content of magnesium and zinc is 0.95 percent and 0.62 percent respectively) with the granularity distribution of 0.20-1.25 micrometers into hyaluronic acid-gelatin composite aqueous solution with the mass ratio of 3:7, uniformly stirring, preparing organic-inorganic composite solution with the ceramic powder concentration of 12.5mg/ml with the mass ratio of the ceramic powder to the organic matter of 1:8, transferring the organic-inorganic composite solution into a liquid storage tank of high-pressure electrostatic spinning, setting the spinning interval to be 12 centimeters, carrying out electrostatic spinning at the spinning voltage of 14kV at the speed of 0.7ml/h, and stopping electrospinning when the thickness of a fiber film on a porous aluminum foil roller carrier reaches 100 micrometers;

2) transferring a solution containing 6% poly L-lactide-caprolactone (PLCL) into the liquid storage tank of the electrospinning device in the step 1, setting the spinning distance to be 15 cm, and continuing electrostatic spinning at the spinning voltage of 16kV at the speed of 0.4 ml/h, depositing PLCL fibers on the fiber membrane prepared in the step 1), and stopping electrospinning until the thickness of the PLCL fiber membrane layer reaches 50 microns;

3) adding chondroitin sulfate into a carboxylated chitosan-gelatin aqueous solution with the mass ratio of 1:3, uniformly stirring to form a mixed solution with the concentration of carboxylated chitosan and the concentration of chondroitin sulfate being 2% and 0.25%, respectively, then carrying out electrostatic spinning on the composite solution, and depositing superfine fibers on the PLCL membrane prepared in the step 2), wherein the spinning distance is 16 cm, the spinning voltage is 12kV, and the spinning speed is 0.3ml/h, so as to form a three-layer superfine fiber microporous membrane with the total thickness of 400 microns.

The preparation process flow of the steps is shown in figure 1, and a three-layer gradient material with a magnesium-zinc co-doped wollastonite composite hyaluronic acid-gelatin fiber microporous membrane at the lower layer, a poly L-lactide-caprolactone fiber microporous membrane at the middle layer and a carboxylated chitosan fiber microporous membrane containing chondroitin sulfate at the upper layer is formed. FIG. 2 is a scanning electron microscope photograph of a side wall of a three-layer gradient film material, and FIG. 3 is a transmission electron microscope photograph of a lower layer film of magnesium-zinc co-doped wollastonite composite hyaluronic acid-gelatin fiber, which shows that inorganic particles are attached to a fiber body.

Example 2:

1) the bioglass powder (chemical composition is 28CaO-42 SiO) with the granularity of 0.10-0.65 micron₂-12B₂O₃-4P₂O₅-6MgO-1CuO-2ZnO-4Na₂O-1K₂O) is added into an organic polymer solution containing 6 percent of gelatin and 4 percent of polyvinyl sulfonic acid and fully stirred, the mass ratio of inorganic particles to organic polymer is 1:15, organic-inorganic compound solution with the concentration of bioglass particles being 18mg/ml is prepared, then the solution is added into a storage container of high-voltage electrostatic spinning, the spinning distance is set to be 12 cm, the high-voltage electrostatic spinning is carried out at the spinning voltage of 15kV and the speed of 0.8ml/h until the fibers are spunStopping electrospinning when the thickness of the dimensional film reaches 150 micrometers;

2) adding an electrospinning solution containing 8% of polycaprolactone into the storage container of the high-voltage electrostatic spinning equipment in the step 1), setting the spinning distance to be 14 cm, carrying out high-voltage electrostatic spinning at the spinning voltage of 12kV at the speed of 0.40ml/h, depositing the polycaprolactone electrospinning on the upper surface of the lower layer gelatin-polyvinyl sulfonic acid composite film in the step 1), and then stopping electrospinning when the thickness of the polycaprolactone film in the middle layer reaches the level of 100 microns;

3) adding chondroitin sulfate and a transforming growth factor with equal mass into a hyaluronic acid-gelatin composite aqueous solution with a mass ratio of 1:5, fully stirring, wherein the content of gelatin is 4.5%, the mass ratio of the total mass of the chondroitin sulfate-transforming growth factor to the hyaluronic acid-gelatin is 1:80, then adding the solution into a storage container of the high-voltage electrostatic spinning equipment in the step 1), setting the spinning distance to be 13 cm, carrying out high-voltage electrostatic spinning at the spinning voltage of 8kV at the speed of 0.2ml/h, depositing the electrospinning on the upper surface of the middle layer film prepared in the step 2), and stopping electrospinning when the thickness of the upper layer electrospinning film reaches the level of 40 microns to obtain a gradient film material with the thickness of 290 microns.

Example 3:

1) adding zinc and phosphorus co-doped akermanite powder (the content of zinc and phosphorus is 1.2 percent and 3.4 percent respectively) with the granularity of 0.80-4.25 micrometers into a solution containing 6 percent poly-L-lactide-caprolactone (PLCL) and fully stirring, wherein the mass ratio of inorganic particles to the PLCL is 1:5, preparing an organic-inorganic compound solution with the concentration of ceramic powder of 40mg/ml, then adding the organic-inorganic compound solution into a storage container for high-pressure electrostatic spinning, setting the spinning interval to be 14 cm, carrying out high-pressure electrostatic spinning at the spinning voltage of 12kV and the speed of 0.6ml/h, and stopping electrospinning until the thickness of the PLCL fiber film reaches 250 micrometers;

2) adding an electrospinning solution containing 6% of polycaprolactone into the storage container of the high-voltage electrostatic spinning equipment in the step 1), setting the spinning distance to be 12 cm, carrying out high-voltage electrostatic spinning at the spinning voltage of 14kV at the speed of 0.60ml/h, depositing the polycaprolactone electrospinning on the upper surface of the PLCL fiber membrane containing the zinc-phosphorus co-doped akermanite particles in the step 1), and then stopping electrospinning when the thickness of the polycaprolactone membrane in the middle layer reaches the level of 120 microns;

3) adding chondroitin sulfate into a hyaluronic acid-sodium alginate composite solution in a mass ratio of 1:5, fully stirring, wherein the content of hyaluronic acid is 0.4%, and the mass ratio of the chondroitin sulfate to hyaluronic acid-gelatin is 1:50, then adding the solution into a storage container of the high-voltage electrostatic spinning equipment in the step 1), setting the spinning interval to be 10 cm, performing high-voltage electrostatic spinning at a spinning voltage of 10kV at a speed of 0.6ml/h, depositing the electrospinning on the upper surface of the middle layer membrane prepared in the step 2), and stopping electrospinning when the thickness of the upper layer electrospinning membrane reaches a level of 80 microns to obtain a gradient microporous membrane material with a total thickness of 450 microns.

Example 4:

1) synchronously adding equal-mass magnesium-doped wollastonite and phosphorus-doped whitlockite powder (the content of magnesium and phosphorus is 0.85 percent and 4.37 percent respectively) with the granularity of 0.2-2.5 micrometers into a solution containing 6 percent poly-L-lactide-caprolactone (PLCL) and fully stirring, wherein the mass ratio of inorganic particles to organic polymers is 1:8, preparing an organic-inorganic compound solution with the concentration of ceramic powder of 25mg/ml, then adding the organic-inorganic compound solution into a storage container for high-pressure electrostatic spinning, setting the spinning interval to be 16 centimeters, carrying out high-pressure electrostatic spinning at the spinning voltage of 14kV and the speed of 0.6ml/h, and stopping electrospinning until the thickness of a fiber film reaches 120 micrometers;

2) adding an electrospinning solution containing 6% poly L-lactide-caprolactone (PLCL) into a storage container of the high-voltage electrospinning device in the step 1), setting the spinning distance to be 14 cm, carrying out high-voltage electrospinning at a spinning voltage of 12kV at a speed of 0.60ml/h, depositing the poly L-lactide-caprolactone (PLCL) on the upper surface of the lower layer film in the step 1) in an electrospinning way, and then stopping electrospinning when the thickness of the middle layer film reaches a level of 120 microns;

3) adding chondroitin sulfate and glucosamine with equal mass into a hyaluronic acid-gelatin composite aqueous solution with the mass ratio of 1:9, fully stirring, wherein the content of gelatin is 4.5%, the mass ratio of the total mass of the chondroitin sulfate-glucosamine to the hyaluronic acid-gelatin is 1:30, then adding the solution into a storage container of the high-voltage electrostatic spinning equipment in the step 1), setting the spinning distance to be 16 cm, carrying out high-voltage electrostatic spinning at the spinning voltage of 12kV at the speed of 0.6ml/h, depositing the electrospinning on the upper surface of the middle layer membrane prepared in the step 2), and stopping electrospinning when the thickness of the upper layer electrospinning membrane reaches the level of 120 microns to obtain a gradient membrane material with the thickness of 360 microns.

Example 5:

1) the bioglass powder with the particle size of 0.30-2.25 microns and the same mass (the chemical composition is 24CaO-36 SiO)₂-16B₂O₃-6P₂O₅-6MgO-0.5CuO-2.5ZnO-6Na₂O-3Li₂O) and manganese-doped wollastonite powder (with the manganese content of 0.8%) are added into an organic polymer solution containing 3% of sodium carboxymethylcellulose and 4% of polyethylene phosphate and fully stirred, the mass ratio of inorganic particles to organic polymers is 1:10, an organic-inorganic compound solution with the concentration of bioglass particles of 30mg/ml is prepared, then the organic-inorganic compound solution is added into a storage container for high-voltage electrostatic spinning, the spinning distance is set to be 14 cm, the high-voltage electrostatic spinning is carried out at the spinning voltage of 16kV at the speed of 0.6ml/h, and the electric spinning is stopped until the thickness of a fiber film reaches 100 microns;

2) adding an electrospinning solution containing 6% dextran sulfate into the storage container of the high-voltage electrostatic spinning equipment in the step 1), setting the spinning distance to be 14 cm, carrying out high-voltage electrostatic spinning at the spinning voltage of 12kV at the speed of 0.40ml/h, depositing the dextran sulfate electrospinning on the upper surface of the lower layer membrane in the step 1), and then stopping electrospinning when the thickness of the middle layer dextran sulfate membrane reaches the level of 100 microns;

3) adding chondroitin sulfate and bone morphogenetic protein with equal mass into hyaluronic acid-gelatin composite aqueous solution with the mass ratio of 1:5, fully stirring, wherein the content of gelatin is 3.5%, the mass ratio of the total mass of the chondroitin sulfate-bone morphogenetic protein to the mass of hyaluronic acid-gelatin is 1:40, then adding the solution into a storage container of the high-voltage electrostatic spinning equipment in the step 1), setting the spinning interval to be 10 cm, carrying out high-voltage electrostatic spinning at the spinning voltage of 12kV at the speed of 0.6ml/h, depositing the electric spinning on the upper surface of the middle layer membrane prepared in the step 2), and stopping electric spinning when the thickness of the upper layer electric spinning membrane reaches the level of 80 microns to obtain a gradient membrane material with the thickness of 280 microns.

The experiments of the examples verify that:

1. bacteriostasis test

Cutting the gradient membrane materials of the embodiment 1, the embodiment 2 and the embodiment 5 into sheets with the diameter of 15mm, respectively inoculating staphylococcus aureus on the lower surfaces of the sheets, then continuously culturing for 8-24 hours in an anaerobic environment, and observing the bacteriostasis condition. The results show that the surface of the gradient material has obvious inhibition effect on the growth of the bacteria, and the inhibition rate is not lower than 79-96% at 8 and 12 hours.

2. Articular animal knee joint cartilage calcified layer repairing and reconstructing experiment

Taking an in-vivo animal model of an adult New Zealand white rabbit with the body weight of about 2.8 kilograms, injecting sodium pentobarbital intravenously, and fixing the supine surface on an operating table after anesthesia. The femoral knee joint is peeled off, circular cartilage with the diameter of 5mm at the joint part is taken out, then the calcified layer under the cartilage is removed, the disc-shaped fibrous membrane gradient material prepared in the example 1 and the example 2 is implanted, the removed cartilage layer is backfilled again, the suture is sewn, the control group 1 is used for directly backfilling the cartilage suture after the calcified layer is removed, the control group 2 is used for implanting pure poly L-lactide-caprolactone (PLCL) fibrous membrane at the part after the calcified layer is removed, then the cartilage layer is backfilled and sewn. All models were routinely used for wound closure and postoperative management. The test results show that all animals have good vital signs and the survival rate is 100% in the period; as shown in fig. 4-6, the calcified layer of the material repair group of examples 1 and 2 was reconstructed at 8 weeks after the operation, and the cartilage layer was well healed (fig. C, D); control 1 cartilage layer healed, but calcified layer was remodeling (panel a); in control 2, there was an inflammatory reaction in the cartilage layer and no remodeling of the calcified layer (panel B).

The experiment shows that the gradient material has excellent repairing and reconstructing functions on the calcified layer of the articular cartilage, has obvious inhibition effect on inflammatory reaction of the cartilage layer and synergistically promotes the healing of the cartilage layer.

The method of the invention has convenient operation, has obvious promotion effect on various articular cartilage injuries, especially the regeneration, repair and reconstruction of calcified layers, can promote the healing of subchondral bone and cartilage tissues, has adjustable degradation rate of gradient materials, and has good regulation effect on various articular pathological inflammation.

Claims

1. A gradient material for promoting the repair of a calcified cartilage layer, comprising:

the gradient material for promoting the repair of the cartilage calcification layer is a three-layer superfine fiber microporous membrane, the lower layer is composed of a superfine fiber microporous membrane containing calcium-silicon-based inorganic particles, the calcium-silicon-based inorganic particles are calcium-silicon-based bioglass particles or biological ceramic particles, the middle layer is composed of a polymer fiber microporous membrane, the upper layer is mainly composed of a superfine fiber microporous membrane containing a functional substance for promoting the healing of cartilage, and the micropore size of the gradient material is smaller than 100 micrometers.

2. A gradient material for promoting the repair of a cartilage calcification layer as recited in claim 1, wherein: the particle size of the calcium-silicon-based bioglass and the biological ceramic particles is 0.04-15 microns, and the diameter of the superfine fiber is 0.1-8.0 microns.

3. A gradient material for promoting the repair of a cartilage calcification layer as recited in claim 1, wherein: the calcium-silicon-based bioglass particles are degradable bioglass prepared by a sol-gel method, and contain CaO and SiO₂MgO, ZnO and P₂O₅、SrO、CuO、B₂O₃、Li₂O、Na₂O、K₂O、Mn₂O₃At least one oxide of (a);

4. A gradient material for promoting the repair of a cartilage calcification layer as recited in claim 3, wherein: the calcium-silicon-based biological ceramic particles comprise the following metal ions and acid radical ions in parts by mole:

SiO₃ ^2-24 to 60 portions of

Ca²⁺20 to 60 portions of

Mg²⁺0 to 40 parts of

Zn²⁺0 to 30 parts of

PO₄ ^3-0 to 15 parts of

BO₃ ^-0 to 15 parts of

Cu²⁺0 to 10 parts of

Mn³⁺0 to 5 parts of

Sr²⁺0 to 12 parts of

Li⁺0 to 8 portions of

Na⁺0 to 8 portions of

K⁺0 to 8 portions of

5. A gradient material for promoting the repair of a cartilage calcification layer as recited in claim 3, wherein: the calcium-silicon-based bioglass particles comprise the following oxides in parts by mole:

SiO₂30 to 78 portions of

16-48 parts of CaO

1 to 30 parts of MgO

P₂O₅0 to 20 parts of

B₂O₃0 to 30 parts of

0.5-10 parts of ZnO

0-5 parts of CuO

0 to 5 parts of MnO

0 to 10 parts of SrO

Li₂0 to 10 parts of O

Na₂0 to 15 parts of O

K₂0 to 10 parts of O

6. A gradient material for promoting the repair of a cartilage calcification layer as recited in claim 1, wherein: the functional substance for promoting cartilage healing is one or a combination of chondroitin sulfate, glucosamine, type I collagen, type II collagen, transforming growth factor, vascular endothelial growth factor and bone morphogenetic protein.

7. A preparation method of a gradient material for promoting the repair of a cartilage calcification layer is characterized by comprising the following steps:

1) adding calcium-silicon-based inorganic particles into a solution containing an electro-spinning polymer, fully stirring, wherein the mass ratio of the calcium-silicon-based inorganic particles to the electro-spinning polymer is 1 (2-80), preparing a compound solution with the particle concentration of 1-400 mg/ml, adding the compound solution into a storage container for electrostatic spinning, setting the spinning distance to be 8-18 cm, carrying out high-pressure electrostatic spinning at the spinning voltage of 6-20 kV and the speed of 0.10-1.20 ml/h, and forming a superfine fiber microporous membrane on a receiving carrier for electrostatic spinning; when the thickness of the superfine fiber microporous membrane on the receiving carrier reaches 20-300 microns, stopping electrostatic spinning, and discharging electrospinning liquid;

2) adding solution containing electrospun polymer into the storage container for electrostatic spinning in the step 1), modulating electrospinning parameters, and continuing to perform electrostatic spinning, wherein the electrospinning is deposited on the upper surface of the superfine fiber microporous membrane in the step 1); when the thickness of the newly added microporous fiber membrane on the receiving carrier reaches 20-300 microns, stopping electrostatic spinning, and discharging the electrospinning liquid;

3) adding a substance containing a function of promoting cartilage healing into a solution containing an electrospun polymer, fully mixing, wherein the mass ratio of the substance containing the function of promoting cartilage healing to the electrospun polymer is 1 (10-200), obtaining a mixed solution, then adding the mixed solution into a storage container of the high-voltage electrostatic spinning equipment in the step 2), modulating electrospinning parameters, continuing to perform high-voltage electrostatic spinning, and depositing the electrospun fiber on the upper surface of the superfine fiber microporous membrane prepared in the step 2); and when the thickness of the newly added electrospinning microporous membrane on the receiving carrier reaches 20-300 microns, stopping electrostatic spinning to obtain the microporous membrane gradient material with the total thickness of 60-900 microns.

8. The method for preparing a gradient material for promoting the repair of a cartilage calcification layer as claimed in claim 7, wherein: the receiving carrier is a porous metal roller, and the pore size is 10-2500 microns.

9. The method for preparing a gradient material for promoting the repair of a cartilage calcification layer as claimed in claim 7, wherein: the polymer is one or a compound of more of gelatin, chitosan, polylactic acid-glycolic acid copolymer, polycaprolactone, poly-L-lactide-caprolactone, sodium alginate, hyaluronic acid, polymethacrylic acid, dextran sulfate, sodium carboxymethylcellulose, cellulose, polyacrylic acid, polystyrene sulfonic acid, polyvinyl sulfonic acid and polyvinyl phosphoric acid.

10. Use of a gradient material for promoting the repair of a cartilage calcification layer as defined in claims 1 to 6 or a gradient material for promoting the repair of a cartilage calcification layer as prepared by the method as defined in claims 7 to 9, wherein: