Preparation method of directional osteochondral scaffold for in situ deposition of drugs and genes

Abstract

The present invention discloses a method for preparing a directional osteochondrocyte scaffold for in situ deposition of drugs and genes. The method involves pouring a hydrogel precursor solution into a mold to form a patterned hydrogel membrane comprising upper and lower grooves. A nanoparticle suspension containing a cartilage repair and regeneration drug and a catechol-containing polyphenol compound / buffer solution are transferred into the upper grooves, whereupon the cartilage repair drug and catechol-containing polyphenol compound nanoparticles are synthesized in situ on the hydrogel membrane. A solution containing a bone regeneration gene drug and CaCl2 and Na2HPO4 is transferred into the lower grooves, whereupon amorphous calcium phosphate nanoparticles are synthesized in situ on the hydrogel membrane. Based on the structure of the Haversian canals of natural bone units, the patterned hydrogel membrane is rolled into a cylindrical shape using a simple and efficient roll-to-roll method to form a directional osteochondrocyte scaffold. The preparation method provided by the present invention achieves the spatial integration of multiple factors, simultaneously enabling the repair and regeneration of cartilage and bone, and the integration of newly formed cartilage and subchondral bone.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and particularly relates to a method for preparing a directional osteochondrocyte scaffold for in-situ deposition of drugs and genes. Background Art

[0002] Clinically, vertical integration of newly formed cartilage with subchondral bone and subsequent subchondral bone regeneration are crucial for mitigating tissue deterioration. Simultaneous regeneration of bone and cartilage is challenging due to the inherent heterogeneity of bone and cartilage, making simultaneous osteochondral regeneration a promising research direction. However, many studies in the literature focus primarily on cartilage scaffolds in osteochondral tissue engineering, often overlooking the need for subchondral bone regeneration.

[0003] The key to achieving high-quality osteochondral repair and regeneration lies in constructing an osteochondral scaffold with a native tissue biomimetic hierarchical structure. At present, there are many technologies to manufacture double-layer or multi-layer osteochondral scaffolds, such as hydrogel precursor solution layered preparation method, 3D printing, electrospinning, microfluidics, magnetron method and buoyancy drive method. However, most of the existing methods involve complex multi-step manufacturing processes, which hinders their practical application. For example, the Chinese patent with publication number CN114681677A discloses a cartilage scaffold material and its application in cartilage scaffold construction, wherein the cartilage scaffold material includes hydrogel, cartilage fragments and chondrocyte spheres. The present application prints a mixture of cartilage fragments and chondrocyte spheres, and the two complement each other. And the Chinese patent with publication number CN110368529A discloses a full-layer repair scaffold for cartilage or subchondral bone and its preparation method, which includes: preparing a gradient soft skeleton, preparing a double-layer scaffold, and preparing a full-layer repair scaffold finished product. The application uses buoyancy-driven pore-making technology to construct a porous structure with gradient density in the cartilage layer.

[0004] Hydrogels, the most typical polymer materials with three-dimensional network structures, have attracted widespread attention due to their outstanding characteristics, including high water content, tunable mechanical properties, excellent biodegradability, and biocompatibility. Advances in hydrogel material design have revolutionized the way biomedical problems are addressed, including osteochondral tissue engineering. Due to their similarities to the natural extracellular matrix, hydrogels are one of the most attractive materials for osteochondral tissue engineering.

[0005] Due to the significant heterogeneity of cartilage and bone, different drugs or growth factors need to be loaded onto the scaffold in a gradient and released in a continuous response to simultaneously repair bone and cartilage. However, most scaffolds currently rely on a single bioactive factor to repair osteochondral cartilage, failing to achieve simultaneous repair and integration of bone and cartilage.

[0006] There are many methods for loading bioactive agents onto stents, but physical adsorption alone is not robust enough, significantly reducing the drug's efficacy during use. Furthermore, the more tenacious covalent chemical interactions can affect the release of bioactive agents, increasing the uncontrollability of drug release. Due to its dynamic reversibility, physical interaction is a more efficient and controllable drug loading method than physical adsorption and covalent crosslinking.

[0007] Therefore, how to simplify the preparation method of osteochondral scaffolds and integrate multiple factors to simultaneously achieve the repair and integration of cartilage and bone is currently a research hotspot in this field. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for preparing a directional osteochondral scaffold for in situ deposition of drugs and genes. The preparation method provided by the present invention is simple and rapid, and the prepared directional osteochondral scaffold has stable mechanical properties, self-healing ability, reactive oxygen capture ability and drug controlled release ability. In addition, the preparation method provided by the present invention realizes the spatial integration of multiple factors, and simultaneously realizes the repair and regeneration of cartilage and bone and the integration of new cartilage and subchondral bone.

[0009] The present invention provides the following technical solutions:

[0010] A method for preparing a directional osteochondral scaffold for in-situ deposition of drugs and genes, the method comprising the following steps:

[0011] (1) pouring a hydrogel precursor solution into a customized mold to prepare a patterned hydrogel film, wherein the patterned hydrogel film includes an upper groove and a lower groove;

[0012] (2) transferring the nanoparticle suspension containing the cartilage repair and regeneration drug and the catechol-containing polyphenol compound / buffer solution into the upper groove of the patterned hydrogel film, and in situ synthesizing the cartilage repair drug@catechol-containing polyphenol compound nanoparticles on the hydrogel film;

[0013] (3) The solution containing bone regeneration gene drug / CaCl2 and Na2HPO4 was transferred into the groove of the lower layer of the patterned hydrogel film, and the bone regeneration gene drug@amorphous calcium phosphate nanoparticles were synthesized in situ on the hydrogel film;

[0014] (4) The patterned hydrogel film was rolled into a cylindrical shape using the roll film method to form a directional osteochondral scaffold.

[0015] Among them, the grooves are designed on the patterned hydrogel film to provide spatial locations for the in situ deposition of nanomedicines.

[0016] The present invention designs a patterned hydrogel with a directional double-layer structure, on which grooves for depositing nanoparticles are provided. The dynamic responsiveness of metal ions and their ligands is then utilized to in situ deposit them on a patterned hydrogel film rich in hydroxyl and carboxyl groups, and a directional hydrogel osteochondrocyte scaffold is obtained by a roll-to-roll method.

[0017] In step (1), the hydrogel precursor solution is a polymer / metal ion gel containing carboxyl and hydroxyl groups.

[0018] The polymer containing carboxyl and hydroxyl groups is gelatin and its derivatives, alginate and its derivatives, carboxymethyl cellulose or carboxymethyl chitosan; the metal ion is selected from Ag + Mg 2+ 、Zn 2+ 、Sr 2+ 、Mn 2+ 、Cu 2+ 、Mo 2+ 、Fe 2+ 、、Fe 3+ 、Al 3+ and Ti 4+ .

[0019] Among them, gelatin and its derivatives are selected from methacrylic anhydride gelatin, 2-ureido-4[lH]-pyrimidinone modified gelatin and 3-amino-4-methoxybenzoic acid modified gelatin.

[0020] In step (2), the cartilage repair and regeneration drug is selected from bone morphogenetic protein (BMP), transforming growth factor (TGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), Wnt5a protein, Kartogenin (KGN), curcumin, andrographolide, icariin and platelet-rich plasma (RPR), containing catechol The polyphenolic compounds are all compounds containing catechol, selected from dopamine (DA), tannic acid (TA), tea polyphenols (flavonoids), gallic acid (GA), propyl gallate, pyrogallol (PG), catechin gallate (ECG), catechin ((2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-benzopyran-3,5,7-triol), epigallocatechin (EGC) and epigallocatechin gallate (EGCG)).

[0021] In step (3), the bone regeneration gene drug is selected from miR-26a, miR-146a, miR-542a, miR-27a, miR-23a, miR-24-2, miR-138, miR-34a, miR-22, miR-199a-5P, miR-1260a, miR-140-3p or miR-144-5.

[0022] Preferably, the length ratio of the upper groove to the lower groove is 0.5-1:2.5-2, and the width of the upper groove to the lower groove is 0.5mm-12mm. Preferably, the length ratio of the upper groove to the lower groove is 1:2.

[0023] The shapes of the upper groove and the lower groove are selected from cylindrical, rectangular or triangular.

[0024] The directional osteochondral scaffold for in situ deposition of drugs and genes has a diameter of 3-5 mm and a length of 3-5 mm. Preferably, the directional osteochondral scaffold has a diameter of 4 mm and a length of 3 mm.

[0025] Compared with the prior art, the technical effect of the present invention is that, based on the Haversian canal structure of natural bone units, the hydrogel film is rolled into a cylinder using the film rolling method to obtain a directional osteochondral scaffold. The method of the present invention is simple and rapid, avoids complex manufacturing processes, and can be widely promoted. The osteochondral scaffold prepared by the present invention has stable mechanical properties, self-healing ability, active oxygen capture ability and drug controlled release ability. The present invention realizes the spatial integration of multiple bioactive factors to obtain a directional osteochondral scaffold. Cartilage repair and regeneration drugs @ polyphenol compound nanoparticles containing catechol and bone regeneration gene drugs @ calcium phosphate nanoparticles promote the chondrogenic differentiation and migration of mesenchymal stem cells and osteogenic differentiation and migration respectively. The simultaneous repair of multiple factors can achieve the directional osteochondral scaffold to show optimal subchondral bone and cartilage regeneration and integration of new cartilage with subchondral bone in vivo, and the accumulation of glycosaminoglycans and collagen in the relevant areas is significantly increased. This multi-factor nanobiomaterial-guided osteochondral repair will become a new approach for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the in situ deposition of catechol-containing polyphenol compounds on patterned hydrogel films for cartilage repair and regeneration drugs (using iron ions as an example).

[0027] Figure 2 Schematic diagram of in situ deposition of calcium phosphate gene drug for bone repair on patterned hydrogel membranes.

[0028] Figure 3A, B, and C in the figure are Micro-CT images of the patterned hydrogel film, the patterned hydrogel film with in situ deposition of two nanodrugs, and the oriented osteochondral scaffold, respectively.

[0029] Figure 4 This is a self-healing photo of GTU-Fe hydrogel in Example 1.

[0030] Figure 5 This is the stress-strain curve of the hydrogel in Example 1 before and after self-healing.

[0031] Figure 6 This is the stress-strain curve of the directional osteochondral scaffold with in situ deposition of drugs and genes under compression in Example 1.

[0032] Figure 7 This is the stress curve of the directional osteochondral scaffold with in situ deposition of drugs and genes in Example 1 under cyclic compression at 50% constant strain.

[0033] Figure 8 The active oxygen scavenging ability of the directional osteochondrocyte scaffold with in situ deposition of drugs and genes in Example 1. DETAILED DESCRIPTION

[0034] The technical solutions of the present invention are further described below with reference to specific embodiments, but these embodiments are not intended to limit the present invention.

[0035] The method for preparing a directional osteochondrocyte scaffold for in-situ deposition of drugs and genes provided by the present invention comprises the following steps:

[0036] (1) Prepare a patterned hydrogel film by pouring the hydrogel precursor solution into a customized mold, letting it stand for 24 hours, and removing the mold to obtain a patterned hydrogel film including upper and lower grooves.

[0037] (2) 6 mg of catechol-containing polyphenol compound was added to a buffer solution (pH = 8.5), and the cartilage repair and regeneration drug nanoparticle suspension was added to the solution and quickly transferred to the upper groove of the patterned hydrogel. The cartilage repair drug @ catechol-containing polyphenol compound nanoparticles were in situ synthesized on the patterned hydrogel film. The reaction mechanism is as follows Figure 1 As shown, the catechol groups on the catechol-containing polyphenolic compound can further complex with metal ions. Therefore, the cartilage repair and regeneration drug @ catechol-containing polyphenolic compound can be combined with the hydrogel film and in situ deposited on the hydrogel film.

[0038] (3) The bone regeneration gene drug / CaCl2 solution and Na2HPO4 solution are mixed and quickly transferred to the lower groove of the patterned hydrogel film, and the bone regeneration gene drug@calcium phosphate nanoparticles are prepared in situ on the patterned hydrogel film. The reaction mechanism is as follows Figure 2As shown, the interaction between calcium ions and the hydroxyl and carboxyl groups on the hydrogel film enables the bone repair gene drug @calcium phosphate to be deposited in situ on the patterned hydrogel film, thereby forming a mineralized hydrogel for bone regeneration.

[0039] (4) The hydrogel patch was rolled into a cylindrical shape using the film rolling method to form a directional osteochondral scaffold (4 mm in diameter and 3 mm in height).

[0040] Example 1

[0041] 2-Urea-4[1H]-pyrimidinone-modified gelatin was used as the substrate for patterned hydrogel films. 3+ As metal ions, KGN@PDA and mi-26a@CaP were used as in situ deposited nanoparticles as examples to illustrate the preparation of directional osteochondral scaffolds.

[0042] (1) Synthesis of 2-ureido-4[lH]-pyrimidinone-modified gelatin. Under nitrogen atmosphere and reaction conditions of 80°C, 5g of gelatin was dissolved in 50mL of anhydrous DMSO. After the gelatin was completely dissolved and cooled to room temperature, 0.25g, 0.5g and 0.75g of 2-ureido-4[lH]-pyrimidinone (UPy-NCO) were added dropwise to the gelatin solution. The reactants were stirred overnight at 25°C under nitrogen. The reactants were precipitated in ethanol three times, filtered to obtain the precipitate, and dialyzed with deionized water for three days. The dialysis water was changed every day, and the water was changed 3-4 times a day. The dialyzed product was freeze-dried to obtain a white foamy product UPy-gelatin (GTU). Too high a GTU grafting rate is not conducive to dissolution, so the optimal feed amount of UPy-NCO is 0.5g.

[0043] (2) GTU solution (20% wt%) was mixed with FeCl3 solution (12 mM), and then vortexed at 3000 rpm for 10 seconds to obtain GTU-Fe hydrogel.

[0044] (3) Preparation of patterned GTU-Fe membranes: The obtained hydrogel precursor solution was poured into a custom mold, allowed to stand for 24 hours, and then removed from the mold to obtain a patterned hydrogel membrane. The length ratio of the upper groove to the lower groove of the patterned hydrogel membrane was 1:2.

[0045] (4) 6 mg of dopamine hydrochloride was added to a buffer solution (pH = 8.5), and the KGN nanoparticle suspension was added to the solution and quickly transferred into the grooves of the patterned hydrogel. KGN@PDA nanoparticles were synthesized in situ on the patterned hydrogel film.

[0046] (5.) The mi-RNA26a / CaCl2 solution and Na2HPO4 solution were mixed and quickly transferred into the grooves of the patterned hydrogel film to in situ prepare mi-26a@CaP nanoparticles on the patterned hydrogel film.

[0047] (6) The hydrogel patch was rolled into a cylindrical shape using the film rolling method to form a directional osteochondral plug (4 mm in diameter and 3 mm in height).

[0048] Example 2

[0049] 2-ureido-4[1H]-pyrimidinone-modified gelatin was used as the patterned substrate. 3+ As metal ions, KGN@PTA and mi-26a@CaP were used as examples of in situ deposited nanoparticles to illustrate the preparation of directional osteochondral scaffolds.

[0050] (1) Synthesis of 2-ureido-4[lH]-pyrimidinone-modified gelatin. Under nitrogen atmosphere and reaction conditions of 80°C, 5g of gelatin was dissolved in 50mL of anhydrous DMSO. After the gelatin was completely dissolved and cooled to room temperature, 0.25g, 0.5g and 0.75g of 2-ureido-4[lH]-pyrimidinone (UPy-NCO) were added dropwise to the gelatin solution. The reactants were stirred overnight at 25°C under nitrogen. The reactants were precipitated in ethanol three times, filtered to obtain the precipitate, and dialyzed with deionized water for three days. The dialysis water was changed every day, and the water was changed 3-4 times a day. The dialyzed product was freeze-dried to obtain a white foamy product UPy-gelatin (GTU). Too high a GTU grafting rate is not conducive to dissolution, so the optimal feed amount of UPy-NCO is 0.5g.

[0051] (2) GTU solution (20% wt%) was mixed with FeCl3 solution (12 mM), and then vortexed at 3000 rpm for 10 seconds to obtain GTU-Fe hydrogel.

[0052] (3) Prepare a patterned GTU-Fe membrane. The resulting hydrogel precursor solution was poured into a custom mold, allowed to stand for 24 hours, and then removed from the mold to obtain a patterned hydrated hydrogel membrane. The length ratio of the upper groove to the lower groove of the patterned hydrogel membrane was 1:2.

[0053] (4) 6 mg of tannic acid was added to a buffer solution (pH = 8.5), and the KGN nanoparticle suspension was added to the solution and quickly transferred into the grooves of the patterned hydrogel. KGN@PTA nanoparticles were synthesized in situ on the patterned hydrogel film.

[0054] (5.) The mi-RNA26a / CaCl2 solution and Na2HPO4 solution were mixed and quickly transferred into the grooves of the patterned hydrogel film to in situ prepare mi-26a@CaP nanoparticles on the patterned hydrogel film.

[0055] (6) The hydrogel patch was rolled into a cylindrical shape using the film rolling method to form a directional osteochondral plug (4 mm in diameter and 3 mm in height).

[0056] Example 3

[0057] 2-ureido-4[1H]-pyrimidinone-modified gelatin was used as the patterned substrate. 3+ As metal ions, KGN@PDA and mi-26a@CaP were used as in situ deposited nanoparticles as examples to illustrate the preparation of directional osteochondral scaffolds.

[0058] (1) Synthesis of 2-ureido-4[lH]-pyrimidinone-modified gelatin. Under nitrogen atmosphere and reaction conditions of 80°C, 5g of gelatin was dissolved in 50mL of anhydrous DMSO. After the gelatin was completely dissolved and cooled to room temperature, 0.25g, 0.5g and 0.75g of 2-ureido-4[lH]-pyrimidinone (UPy-NCO) were added dropwise to the gelatin solution. The reactants were stirred overnight at 25°C under nitrogen. The reactants were precipitated in ethanol three times, filtered to obtain the precipitate, and dialyzed with deionized water for three days. The dialysis water was changed every day, and the water was changed 3-4 times a day. The dialyzed product was freeze-dried to obtain a white foamy product UPy-gelatin (GTU). Too high a GTU grafting rate is not conducive to dissolution, so the optimal feed amount of UPy-NCO is 0.5g.

[0059] (2) GTU solution (20% wt%) was mixed with FeCl3 solution (12 mM), and then vortexed at 3000 rpm for 10 seconds to obtain GTU-Fe hydrogel.

[0060] (3) Prepare a patterned GTU-Fe membrane. The resulting hydrogel precursor solution was poured into a custom mold, allowed to stand for 24 hours, and then removed from the mold to obtain a patterned hydrated hydrogel membrane. The length ratio of the upper groove to the lower groove of the patterned hydrogel membrane was 1:2.

[0061] (4) 6 mg of tannic acid was added to a buffer solution (pH = 8.5), and the KGN nanoparticle suspension was added to the solution and quickly transferred into the grooves of the patterned hydrogel. KGN@PTA nanoparticles were synthesized in situ on the patterned hydrogel film.

[0062] (5.) The mi-RNA26a / CaCl2 solution and Na2HPO4 solution were mixed and quickly transferred into the grooves of the patterned hydrogel film to in situ prepare mi-26a@CaP nanoparticles on the patterned hydrogel film.

[0063] (6) The hydrogel patch was rolled into a cylindrical shape using the film rolling method to form a directional osteochondral plug (4 mm in diameter and 3 mm in height).

[0064] Example 4

[0065] Methacrylated gelatin (GelMA) was used as the patterned substrate. 3+ As metal ions, KGN@PDA and mi-RNA26a@CaP were used as in situ deposited nanoparticles as examples to illustrate the preparation of directional osteochondral scaffolds.

[0066] (1) Gelatin was added to phosphate buffered saline (PBS) and stirred at 50°C with a magnetic stirrer at a speed of 500 rpm until the gelatin was completely dissolved. Then, methacrylic anhydride (MA) was added dropwise to the dissolved gelatin solution at a rate of 0.2 mL / min using a microinjection pump. The mixed solution was reacted at 50°C with a magnetic stirrer at a speed of 500 rpm for 2 h. The reactants were precipitated in ethanol three times, filtered to obtain the precipitate, and dialyzed with deionized water for three days. The dialysis water was changed every day, and the water was changed 3-4 times a day. The dialyzed product was freeze-dried to obtain a white foamy product.

[0067] (2) GelMA solution (20% wt%), FeCl3 solution (12 mM) and photoinitiator 2959 were mixed, then vortexed at 3000 rpm for 10 seconds and cured under UV for 5 minutes to obtain GTU-Fe hydrogel.

[0068] (3) Preparation of a patterned GelMA-Fe membrane: The resulting hydrogel precursor solution was poured into a custom mold, cured under UV light for 5 min, allowed to stand for 24 h, and then removed from the mold to obtain a patterned hydrogel membrane. The length ratio of the upper groove to the lower groove of the patterned hydrogel membrane was 1:2.

[0069] (4) 6 mg of dopamine hydrochloride was added to a buffer solution (pH = 8.5), and the KGN nanoparticle suspension was added to the solution and quickly transferred into the grooves of the patterned hydrogel. KGN@PDA nanoparticles were synthesized in situ on the patterned hydrogel film.

[0070] (5.) The mi-RNA26a / CaCl2 solution and Na2HPO4 solution were mixed and quickly transferred into the grooves of the patterned hydrogel film to in situ prepare mi-26a@CaP nanoparticles on the patterned hydrogel film.

[0071] (6) The hydrogel patch was rolled into a cylindrical shape using the film rolling method to form a directional osteochondral plug (4 mm in diameter and 3 mm in height).

[0072] like Figure 3 As shown, Figure 3 Figures A, B, and C show Micro-CT images of a patterned GTU-Fe hydrogel film, a patterned GTU-Fe hydrogel film with in situ deposition of two nanomedicines, and a directional osteochondral scaffold, respectively. The patterned hydrogel film includes upper and lower grooves designed to provide spatial locations for in situ deposition of nanomedicines.

[0073] like Figure 4 As shown, the GTU-Fe hydrogel has self-healing properties and can be spliced ​​together after being cut into two halves, and the crack disappears.

[0074] like Figure 5 As shown in the figure, the self-healing performance of GTU-Fe hydrogel can be proved by tensile experiments. The tensile strength of the hydrogel before and after self-healing is basically the same, indicating that the tensile strength of the hydrogel after self-healing can be restored.

[0075] like Figure 6 As shown in the figure, the GTU-Fe / KGN@PDA / miRNA@CaP oriented hydrogel scaffold has compression resistance and a compression strength of up to 2.59 MPa.

[0076] like Figure 7 As shown in Figure 3, the GTU-Fe / KGN@PDA / miRNA@CaP oriented hydrogel scaffold exhibited excellent fatigue resistance in cyclic compression tests.

[0077] like Figure 8 As shown in the results, the GTU-Fe / KGN@PDA / miRNA@CaP oriented hydrogel scaffold can capture 91% of DPPH within 60 minutes, 39% of ABTS within 6 minutes, and 16% of PTIO. The introduction of PDA enhances the free radical scavenging ability of the GTU-Fe / KGN@PDA / miRNA@CaP oriented hydrogel scaffold.

[0078] The above is an exemplary description of the present invention. It should be noted that, without departing from the core of the present invention, any simple deformation, modification or other equivalent replacement that can be made by other skilled in the art without expending creative labor falls within the scope of protection of the present invention.

Claims

1. A method for preparing a directional osteochondral scaffold for in situ deposition of drugs and genes, characterized in that: The preparation method comprises the following steps: (1) pouring a hydrogel precursor solution into a customized mold to prepare a patterned hydrogel film, wherein the patterned hydrogel film includes an upper groove and a lower groove; (2) transferring the nanoparticle suspension containing the cartilage repair and regeneration drug and the catechol-containing polyphenol compound / buffer solution into the upper groove of the patterned hydrogel film, and in situ synthesizing the cartilage repair drug@catechol-containing polyphenol compound nanoparticles on the hydrogel film; (3) The solution containing bone regeneration gene drug / CaCl2 and Na2HPO4 was transferred into the groove of the lower layer of the patterned hydrogel film, and the bone regeneration gene drug@amorphous calcium phosphate nanoparticles were synthesized in situ on the hydrogel film; (4) The patterned hydrogel film was rolled into a cylindrical shape using the roll film method to form a directional osteochondral scaffold.

2. The method for preparing a directional osteochondrocyte scaffold for in situ deposition of drugs and genes according to claim 1, characterized in that: In step (1), the hydrogel precursor solution is a hydrogel comprising a polymer containing carboxyl and hydroxyl groups and metal ions.

3. The method for preparing a directional osteochondrocyte scaffold for in situ deposition of drugs and genes according to claim 2, characterized in that: The polymer containing carboxyl and hydroxyl groups is gelatin and its derivatives, alginate and its derivatives, carboxymethyl cellulose or carboxymethyl chitosan; the metal ion is selected from Ag + Mg 2+ 、Zn 2+ 、Sr 2+ 、Mn 2+ 、Cu 2+ 、Mo 2+ 、Fe 2+ 、Fe 3+ 、Al 3+ and Ti 4+ .

4. The method for preparing a directional osteochondrocyte scaffold for in situ deposition of drugs and genes according to claim 1, characterized in that: In step (2), the cartilage repair and regeneration drug is selected from bone morphogenetic protein, transferring growth factor, insulin-like growth factor, platelet-derived growth factor, Wnt5a protein, Kartogenin, curcumin, andrographolide, icariin or platelet-rich plasma, and the polyphenol compound containing catechol is selected from dopamine, tannic acid, tea polyphenols, gallic acid, propyl gallate, pyrogallol, catechin gallate, catechin, epigallocatechin or epigallocatechin gallate.

5. The method for preparing a directional osteochondrocyte scaffold for in situ deposition of drugs and genes according to claim 1, characterized in that: In step (3), the bone regeneration gene drug is selected from miR-26a, miR-146a, miR-542a, miR-27a, miR-23a, miR-24-2, miR-138, miR-34a, miR-22, miR-199a-5P, miR-1260a, miR-140-3p or miR-144-5.

6. The method for preparing a directional osteochondrocyte scaffold for in situ deposition of drugs and genes according to claim 1, characterized in that: The length ratio of the upper groove to the lower groove is 0.5-1:2.5-2, and the width of the upper groove to the lower groove is 0.5mm-12mm.

7. The method for preparing a directional osteochondrocyte scaffold for in situ deposition of drugs and genes according to claim 1, characterized in that: The shapes of the upper groove and the lower groove are selected from cylindrical, rectangular or triangular.

8. The method for preparing a directional osteochondrocyte scaffold for in situ deposition of drugs and genes according to claim 1, characterized in that: The directional osteochondral scaffold for in-situ deposition of drugs and genes has a diameter of 3-5 mm and a length of 3-5 mm.

Images