A composite fiber scaffold for treating osteoarthritis and promoting tendon-bone healing and a preparation method thereof

Abstract

The application provides a composite fiber scaffold for treating osteoarthritis and promoting tendon-bone healing and a preparation method thereof, and belongs to the technical field of biological medicine. The composite fiber scaffold of the application is a core-shell structure, the shell layer is based on PLGA, and is loaded with platelet exosomes (PLT-Exos) and curcumin; the core is based on gelatin hydrogel, and is loaded with bone morphogenetic protein-2 (BMP-2) and icariin. The scaffold can realize time-controlled release of drugs by coaxial electrospinning technology: the shell layer drug is rapidly released to resist inflammation and regulate the immune microenvironment, and the core drug is slowly released to long-acting promote tendon-bone regeneration. The application is particularly suitable for treating osteoarthritis caused by dislocation of the distal ulno-radiate joint, and can significantly improve the tendon-bone healing quality after ligament reconstruction, and has the advantages of synergistic effect and good biocompatibility.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a composite fiber scaffold for treating osteoarthritis and promoting tendon-bone healing, and its preparation method. Background Technology

[0002] Distal radioulnar joint dislocation is a common wrist injury, often followed by chronic joint instability due to triangular fibrocartilage complex (TFCC) damage, which can lead to traumatic osteoarthritis (OA). Furthermore, during ligament reconstruction surgery, the quality of "tendon-bone healing" between the transplanted tendon and the bone tunnel is crucial to the success of the procedure. However, tendon-bone interface healing is slow and often results in scar tissue with low mechanical strength, leading to a high risk of re-rupture.

[0003] Current clinical treatments have the following limitations: Drug treatment: Systemic administration (such as nonsteroidal anti-inflammatory drugs) is difficult to achieve effective concentrations at the lesion site, and long-term use has significant side effects; intra-articular injection (such as hyaluronic acid and corticosteroids) mainly relieves symptoms, but cannot promote tissue regeneration and may even inhibit healing.

[0004] Surgical treatment: Although it can restore anatomical structure, it cannot improve the inflammatory microenvironment within the joint that is not conducive to healing. The incidence of secondary osteoarthritis and poor tendon-bone healing after surgery remains high.

[0005] Biological therapy: Growth factor therapies such as platelet-rich plasma (PRP) have problems such as poor stability, uncontrollable release, and easy degradation by the inflammatory environment, resulting in inconsistent efficacy.

[0006] Therefore, existing technologies still need to develop a targeted treatment strategy that can actively regulate the joint microenvironment and promote tissue regeneration in a sequential manner, thereby simultaneously solving the problems of osteoarthritis and tendon-bone healing. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a composite fiber scaffold for treating osteoarthritis and promoting tendon-bone healing, and its preparation method. It aims to resolve the contradiction between the continuous destruction of the inflammatory microenvironment and the difficulty of tendon-bone tissue regeneration after distal radioulnar joint dislocation. Through a biomimetic core-shell structure and time-controlled release technology, it achieves a synergistic therapeutic effect of "anti-inflammatory first, then regeneration".

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: A composite fiber scaffold for treating osteoarthritis and promoting tendon-bone healing, wherein the composite fiber scaffold has a core-shell structure, the shell layer is based on polylactic acid-glycolic acid copolymer, the core layer is based on gelatin hydrogel, and the mass ratio of the shell layer to the core layer is (2-4):1. The shell is loaded with platelet exosomes and curcumin. In each milligram of composite fiber scaffold, the content of platelet exosomes is 0.5-5 μg and the content of curcumin is 1-10 μg. The core-loaded bone morphogenetic protein-2 and icariin, with each milligram of composite fiber scaffold containing 0.1-1 μg of BMP-2 and 5-20 μg of icariin.

[0009] Preferably, the osteoarthritis is chronic unstable osteoarthritis caused by distal radioulnar joint dislocation, the tendon-bone healing is the healing between the transplanted tendon and the bone tunnel after ligament reconstruction surgery, and the bracket is adapted to the wrist anatomy and can be implanted in the tissue space or bone tunnel around the distal radioulnar joint.

[0010] Preferably, the nanofibers of the composite fiber scaffold have a diameter of 100-500 nm and a porosity of 70-85%.

[0011] Preferably, the polylactic acid-glycolic acid copolymer is a copolymer of lactic acid and glycolic acid in a molar ratio of 75:25, with a number-average molecular weight of 50-100 kDa and a degradation cycle of 2-3 weeks.

[0012] Preferably, the gelatin hydrogel is a partially hydrolyzed collagen product derived from bovine bone or pigskin, with a number-average molecular weight of 1-10 kDa. After cross-linking treatment, it is in a liquid state at low temperature of 4-10℃ and in a gel state at body temperature, with a degradation cycle of 1-8 weeks.

[0013] The present invention also provides a method for preparing the above-mentioned composite fiber scaffold, comprising the following steps: S1. Solution preparation: Prepare the shell solution and the core solution separately; S2. Coaxial electrospinning: Using a coaxial needle, the shell solution and the core solution are propelled by independent injection pumps for spinning, and the core-shell structured nanofiber membrane is collected on the receiving plate. S3. Post-processing: The nanofiber membrane is cross-linked in genipin vapor and then aseptically packaged to obtain the composite fiber scaffold.

[0014] Preferably, in step S1, the shell solution is prepared by dissolving polylactic acid-glycolic acid copolymer in hexafluoroisopropanol to prepare a PLGA solution with a concentration of 8-15wt%, adding purified platelet exosomes and curcumin, and then subjecting it to ultrasonic treatment to obtain the shell solution; the ultrasonic power is 100-300W, the time is 5-15min, and the temperature is <25℃.

[0015] Preferably, in step S1, the core solution is prepared by dissolving gelatin in a 1-5 vol% aqueous acetic acid solution, stirring in a water bath at 30-40°C for 30-60 min to prepare a 5-10 wt% gelatin solution, cooling to room temperature, adding bone morphogenetic protein-2 and icariin, and stirring for 10-20 min until completely dissolved to obtain the core solution.

[0016] Preferably, in step S2, the inner diameter of the outer needle of the coaxial needle is 0.8-1.2 mm, the inner diameter of the inner needle is 0.3-0.5 mm, the spinning voltage is 15-25 kV, the receiving distance is 10-20 cm, the shell solution flow rate is 0.5-2 mL / h, and the core solution flow rate is 0.1-0.8 mL / h.

[0017] Preferably, in step S2, the temperature of the spinning environment is 20-25°C and the relative humidity is 30-50%.

[0018] Preferably, in step S3, the genipin vapor crosslinking conditions are: genipin ethanol solution concentration of 0.5-2wt%, crosslinking temperature of 30-40℃, and crosslinking time of 100-140min; after crosslinking, the fiber membrane is dried at 25-30℃ for 8-12h.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention achieves targeted synergistic therapy through a core-shell structure design. The shell layer, a PLGA substrate, provides the initial carrier for the drug, and its properties allow the loaded platelet exosomes and curcumin to be rapidly released through diffusion within 1-3 days, synergistically inhibiting the release of inflammatory factors such as TNF-α and IL-1β, and efficiently clearing the destructive inflammatory microenvironment. The core gelatin hydrogel's degradation cycle covers the entire tendon-bone healing cycle, while the loaded BMP-2 and icariin are slowly and continuously released, synergistically inducing osteogenic differentiation and promoting fibrocartilage formation. This addresses the dual problems of osteoarthritis progression and poor tendon-bone healing at their root, overcoming the limitations of single-treatment methods in existing technologies.

[0020] In this invention, platelet exosomes in the shell regulate the immune microenvironment, forming an "anti-inflammatory synergy" with curcumin's anti-inflammatory and antioxidant effects. In in vitro experiments, its effect in inhibiting inflammatory factors (such as TNF-α) is more than 40% higher than that of curcumin or platelet exosomes alone. In the core, BMP-2 potently induces osteogenic formation, and icariin promotes fibrocartilage formation, forming a "regenerative synergy," increasing the maximum load at the tendon-bone interface to 1.8 times that of the control group at 8 weeks post-surgery. The two groups of active ingredients complement each other and synergistically enhance each other, improving the healing microenvironment and directly promoting tissue regeneration, significantly outperforming the therapeutic effects of existing single-component or non-synergistic preparations.

[0021] This invention provides a stent specifically designed for chronic unstable osteoarthritis caused by distal radioulnar joint dislocation, as well as for tendon-bone healing after ligament reconstruction. It is adapted to the wrist anatomy and can be flexibly implanted into the interstitial space or bone tunnel, thus solving the problem of a lack of targeted clinical treatment devices. Detailed Implementation

[0022] This invention provides a composite fiber scaffold for treating osteoarthritis and promoting tendon-bone healing. It has a core-shell structure, with the shell layer made of polylactic acid-glycolic acid copolymer (PLGA) as the substrate and the core layer made of gelatin hydrogel as the substrate. The mass ratio of the shell layer to the core layer is (2-4):1.

[0023] In this invention, the preferred mass ratio of shell to core is 3:1, balancing the burst release efficiency of the shell with the sustained release duration of the core; further preferred is PLGA as a medical-grade high-purity raw material with a purity ≥99%; further preferred is gelatin hydrogel as a medical-grade raw material with a purity ≥98%. PLGA possesses good biocompatibility, biodegradability, and mechanical support, making it suitable for the rapid release of early anti-inflammatory components; gelatin hydrogel exhibits excellent cell affinity, and its temperature-sensitive properties enable stable encapsulation of active ingredients, making it suitable for the sustained release of later regenerative factors. The two work synergistically to construct a time-release drug delivery carrier.

[0024] In this invention, the shell layer is loaded with platelet exosomes (PLT-Exos) and curcumin. In each milligram of composite fiber scaffold, the content of platelet exosomes is 0.5-5 μg, preferably 1-3 μg, more preferably 2 μg; the content of curcumin is 1-10 μg, preferably 3-6 μg, more preferably 5 μg. PLT-Exos can regulate the immune microenvironment and inhibit the release of inflammatory factors, while curcumin possesses potent anti-inflammatory and antioxidant activities. Both are released rapidly within 1-3 days, quickly clearing chronic inflammation after trauma and removing microenvironmental barriers for tendon and bone healing.

[0025] In this invention, the core load contains bone morphogenetic protein-2 (BMP-2) and icariin. In each milligram of the composite fibrous scaffold, the BMP-2 content is 0.1-1 μg, preferably 0.3-0.6 μg, more preferably 0.5 μg; the icariin content is 5-20 μg, preferably 8-15 μg, more preferably 10 μg. BMP-2 can strongly induce osteogenic differentiation, and icariin can promote fibrocartilage formation and improve tendon-bone integration strength. Both are slowly released over 1-8 weeks, improving the quality of tendon-bone healing. In this invention, the nanofibers of the composite fiber scaffold preferably have a diameter of 100-500 nm and a porosity of 70-85%; more preferably, the fiber diameter is 250 nm and the porosity is preferably 80%. Suitable porosity ensures the circulation of synovial fluid, the penetration of nutrients, and the removal of metabolic waste, preventing the aggravation of inflammation caused by fluid accumulation within the scaffold and promoting tissue ingrowth. In this invention, the preferred polylactic acid-glycolic acid copolymer is a copolymer of lactic acid and glycolic acid in a molar ratio of 75:25, with a number-average molecular weight of 50-100 kDa and a degradation period of 2-3 weeks; more preferably, the number-average molecular weight is 60-80 kDa. The 75:25 molar ratio of PLGA allows for controlled degradation rate, enabling the burst release of active ingredients from the shell within 1-3 days; the suitable molecular weight ensures fiber formability during spinning and provides early postoperative mechanical support for the scaffold, preventing scaffold collapse that could affect drug release and tissue adhesion.

[0026] In this invention, the gelatin hydrogel is preferably a partially hydrolyzed collagen product derived from bovine bone or pigskin, with a number-average molecular weight of 1-10 kDa; more preferably derived from pigskin, with a number-average molecular weight of 3-5 kDa. Collagen-derived gelatin can mimic the natural tissue matrix, enhance cell affinity, and promote the adhesion of fibroblasts and osteoblasts; the suitable molecular weight can balance the stability and degradation rate of the hydrogel, providing protection and a sustained-release carrier for the core active ingredients.

[0027] In this invention, the preferred gelatin hydrogel, after cross-linking treatment, is in a liquid state at a low temperature of 4-10℃ and in a gel state at body temperature, with a degradation cycle of 1-8 weeks; more preferably, the degree of cross-linking is 80-90%, and the degradation cycle is 4-8 weeks.

[0028] The present invention also provides a method for preparing the above-mentioned composite fiber scaffold, comprising the following steps: S1. Solution preparation: Prepare the shell solution and the core solution separately; S2. Coaxial electrospinning: Using a coaxial needle, the shell solution and the core solution are propelled by independent injection pumps for spinning, and the core-shell structured nanofiber membrane is collected on the receiving plate. S3. Post-processing: The nanofiber membrane is cross-linked in genipin vapor and then aseptically packaged to obtain a composite fiber scaffold.

[0029] In this invention, the preferred method for preparing the shell solution in step S1 is as follows: (1) Polylactic acid-glycolic acid copolymer (PLGA) is dissolved in hexafluoroisopropanol to prepare a PLGA solution with a concentration of 8-15 wt%, and more preferably a PLGA solution with a concentration of 10-12 wt%. More preferably, the dissolution process is carried out by magnetic stirring at 20-25°C for 30-40 min at a stirring speed of 200-300 r / min to ensure complete dissolution of PLGA without any particle residue. Fluoroisopropanol is a good solvent for PLGA, with high dissolution efficiency and a low boiling point (59°C), allowing it to evaporate quickly during spinning, thus avoiding solvent residue affecting the biocompatibility of the scaffold. A suitable concentration of PLGA solution provides a stable raw material for coaxial spinning, ensuring the quality of the shell fiber formation. (2) Add purified platelet exosomes and curcumin, and sonicate to obtain a shell solution; the sonication power is 100-300W, the time is 5-15min, and the temperature is <25℃; more preferably, the sonication power is 200W and the time is 10min. Sonication can achieve uniform dispersion of platelet exosomes and curcumin in PLGA solution, while avoiding the inactivation of active ingredients caused by vigorous stirring; low temperature control can protect the bioactivity of active ingredients. In this invention, the preferred method for preparing the core solution in step S1 is as follows: (1) Dissolve gelatin in a 1-5 vol% aqueous acetic acid solution, more preferably a 2-3 vol% aqueous acetic acid solution; stir in a water bath at 30-40°C for 30-60 min to prepare a gelatin solution with a concentration of 5-10 wt%, and cool to room temperature; more preferably, stir in a water bath at 35°C for 45 min to prepare a gelatin solution with a concentration of 8 wt%. Low concentrations of acetic acid can gently dissolve gelatin without damaging its collagen structure and biological activity.

[0030] (2) Add bone morphogenetic protein-2 and icariin, stir for 10-20 min until completely dissolved, and further preferably stir for 15 min at a speed of 100 r / min to obtain the core solution.

[0031] In this invention, in step S2, the outer inner diameter of the coaxial needle is preferably 0.8-1.2 mm and the inner inner diameter is preferably 0.3-0.5 mm; more preferably, the outer inner diameter of the coaxial needle is 1 mm and the inner inner diameter is 0.4 mm; more preferably, the needle material is stainless steel and the needle length is 5-8 cm.

[0032] In this invention, in step S2, the preferred spinning voltage is 15-25kV, more preferably 20kV; the preferred receiving distance is 10-20cm, more preferably 15cm; the preferred shell solution flow rate is 0.5-2mL / h, and the core solution flow rate is 0.1-0.8mL / h, more preferably 1.5mL / h for the shell solution and 0.5mL / h for the core solution. A stable injection pump flow rate ensures consistent fiber diameter; a suitable voltage allows the solution to form a stable Taylor cone, achieving nanofiber formation; a reasonable receiving distance prevents fiber adhesion during flight while ensuring sufficient solvent evaporation, thus improving fiber structural stability.

[0033] In this invention, in step S2, the preferred temperature of the spinning environment is 20-25°C, and the relative humidity is 30-50%; more preferably, the temperature is 22°C, and the relative humidity is 40%. The receiving plate is preferably made of polytetrafluoroethylene (PTFE) or aluminum foil; more preferably, the receiving disc adopts a uniform rotation mode with a rotation speed of 50-200 r / min, and more preferably 100-150 r / min.

[0034] In this invention, in step S3, the preferred conditions for genipin vapor crosslinking are: a genipin ethanol solution concentration of 0.5-2 wt%, more preferably 1 wt%; a preferred crosslinking temperature of 30-40°C and a crosslinking time of 100-140 min, more preferably 35°C and 120 min; and preferably, the fiber membrane is dried at 25-30°C for 8-12 h after crosslinking, more preferably 10 h. Genipin is a natural crosslinking agent with better biocompatibility than chemical crosslinking agents (such as glutaraldehyde). It can improve the stability, mechanical strength, and degradation controllability of hydrogels by crosslinking the amino groups in gelatin molecules, while not damaging the bioactivity of the active ingredients within the scaffold.

[0035] In this invention, step S3 preferably includes the following aseptic packaging process: the dried fiber film is aseptically cut (the size is adapted to the needs of radioulnar joint implantation, including but not limited to 1cm×1cm×0.2cm), sterilized with ethylene oxide, sterilization dose 600-800mg / L, sterilization time 2-4h, sterilization temperature 37℃, and then sealed in an aseptic operating table.

[0036] In this invention, the preferred osteoarthritis is chronic unstable osteoarthritis caused by distal radioulnar joint dislocation. Tendon-bone healing refers to the healing between the transplanted tendon and the bone tunnel after ligament reconstruction surgery. The composite fiber scaffold is adapted to the wrist anatomy and can be implanted in the tissue space or bone tunnel around the distal radioulnar joint.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Example 1 A composite fiber scaffold for treating osteoarthritis and promoting tendon-bone healing is prepared as follows: (1) Solution preparation Shell solution preparation: Medical-grade PLGA (lactic-co-glycolic acid molar ratio 75:25, number-average molecular weight 70 kDa, purity ≥99%) was dissolved in anhydrous hexafluoroisopropanol and magnetically stirred at 250 r / min for 35 min at 23°C to prepare a PLGA solution with a mass concentration of 11 wt%. Purified platelet exosomes (purified by ultracentrifugation, purity ≥95%) and pharmaceutical-grade curcumin (purity ≥98%, pre-dissolved in anhydrous ethanol to 10 mg / mL) were added to the above PLGA solution, controlling the final platelet exosome content to be 2 μg and curcumin content to be 5 μg per milligram of stent. Intermittent ultrasound was performed (power 200W, 30s on / 30s off, total duration 10 min), with the ultrasound temperature controlled at 20°C. After ultrasound, the solution was allowed to stand for 8 min to eliminate air bubbles, resulting in the shell solution.

[0039] Core solution preparation: Pigskin-derived gelatin (number-average molecular weight 4 kDa, purity ≥98%) was dissolved in a 2.5 vol% acetic acid aqueous solution and stirred at 180 r / min for 45 min in a 36℃ water bath to prepare a 7.5 wt% gelatin solution. After cooling to 23℃, recombinant human BMP-2 (biological activity ≥1.5 × 10⁻⁶) was added. 6 The core solution was prepared by mixing BMP-2 (20 mg / mL, pre-dissolved in sterile PBS) and icariin (purity ≥98%, pre-dissolved in anhydrous ethanol to 20 mg / mL), controlling the final BMP-2 content to be 0.4 μg and the icariin content to be 12 μg per milligram of scaffold. The solution was gently stirred at 120 rpm for 15 min until completely dissolved to obtain the core solution.

[0040] (2) Coaxial electrospinning A stainless steel coaxial needle (outer needle inner diameter 1.0 mm, inner needle inner diameter 0.4 mm) was used. The shell solution flow rate was set to 1.2 mL / h, the core solution flow rate to 0.4 mL / h, and the shell to core mass ratio to 3:1. The spinning voltage was set to 20 kV, the receiving distance to 14 cm, the receiving plate to be made of polytetrafluoroethylene, and the rotation speed to 120 r / min. The spinning environment was controlled at 23℃ and 40% relative humidity, resulting in a core-shell structured nanofiber membrane with a thickness of 0.25 mm.

[0041] (3) Post-processing The nanofiber membrane was placed in a sealed container and crosslinked by steam using a 1.2 wt% genipin ethanol solution at 35°C for 120 min. After crosslinking, the surface was wiped with sterile deionized water and dried in a vacuum drying oven at 27°C (vacuum degree -0.09 MPa) for 10 h. It was then aseptically cut into 1 cm × 1 cm × 0.2 cm pieces, sterilized with ethylene oxide (700 mg / L, 37°C, 3 h), and aseptically packaged in aluminum foil bags to obtain the composite fiber scaffold.

[0042] The prepared scaffold nanofibers have a diameter of 250 nm and a porosity of 78%.

[0043] Example 2 A composite fiber scaffold for treating osteoarthritis and promoting tendon-bone healing is prepared as follows: (1) Solution preparation Shell solution preparation: Medical-grade PLGA (lactic-co-glycolic acid molar ratio 75:25, number-average molecular weight 50 kDa, purity ≥99%) was dissolved in anhydrous hexafluoroisopropanol and magnetically stirred at 200 r / min for 30 min at 20℃ to prepare an 8 wt% PLGA solution. Purified platelet exosomes and curcumin were added to control the final platelet exosome content to be 0.5 μg and curcumin content to be 1 μg per milligram of stent. Intermittent ultrasound was used (power 100 W, total duration 5 min) at an ultrasound temperature ≤25℃, and the solution was allowed to stand for 5 min to eliminate air bubbles, thus obtaining the shell solution.

[0044] Core solution preparation: Bovine bone-derived gelatin (number-average molecular weight 1 kDa, purity ≥98%) was dissolved in a 1 vol% acetic acid aqueous solution and stirred at 150 r / min for 30 min in a 30℃ water bath to prepare a 5 wt% gelatin solution. After cooling to room temperature, recombinant human BMP-2 and icariin were added, controlling the final BMP-2 content to be 0.1 μg and the icariin content to be 5 μg per milligram of scaffold. The solution was stirred at 100 r / min for 10 min until dissolved to obtain the core solution.

[0045] (2) Coaxial electrospinning A stainless steel coaxial needle (outer needle inner diameter 0.8 mm, inner needle inner diameter 0.3 mm) was used. The shell solution flow rate was 0.5 mL / h, the core solution flow rate was 0.25 mL / h, and the shell to core mass ratio was 2:1. The spinning voltage was set to 15 kV, the receiving distance to 10 cm, the receiving plate to be made of aluminum foil, and the rotation speed to 50 r / min. The spinning environment temperature was controlled at 20℃ and the relative humidity at 30%, and a nanofiber membrane with a thickness of 0.1 mm was collected.

[0046] (3) Post-processing Crosslinking was performed using a 0.5 wt% genipin ethanol solution via vapor crosslinking at 30°C for 100 min. After crosslinking, the sample was dried in a vacuum oven at 25°C for 8 h. The sample was then aseptically cut, sterilized with ethylene oxide (600 mg / L, 2 h), and packaged.

[0047] The remaining parameters are the same as in Example 1. The obtained scaffold nanofibers have a diameter of 100 nm and a porosity of 70%.

[0048] Example 3 A composite fiber scaffold for treating osteoarthritis and promoting tendon-bone healing is prepared as follows: (1) Solution preparation Shell solution preparation: Medical-grade PLGA (lactic-co-glycolic acid molar ratio 75:25, number-average molecular weight 100 kDa, purity ≥99%) was dissolved in anhydrous hexafluoroisopropanol and magnetically stirred at 300 r / min for 40 min at 25°C to prepare a 15 wt% PLGA solution. Purified platelet exosomes and curcumin were added to control the final platelet exosome content to be 5 μg and curcumin content to be 10 μg per milligram of stent. Intermittent ultrasound was used (power 300 W, total duration 15 min) at an ultrasound temperature ≤25°C. After standing for 10 min to eliminate air bubbles, the shell solution was obtained.

[0049] Core solution preparation: Pigskin-derived gelatin (number-average molecular weight 10 kDa, purity ≥98%) was dissolved in a 5 vol% acetic acid aqueous solution and stirred at 200 rpm for 60 min in a 40°C water bath to prepare a 10 wt% gelatin solution. After cooling to room temperature, recombinant human BMP-2 and icariin were added, controlling the final BMP-2 content to be 1 μg and the icariin content to be 20 μg per milligram of scaffold. The solution was stirred at 150 rpm for 20 min until dissolved, yielding the core solution.

[0050] (2) Coaxial electrospinning A stainless steel coaxial needle (outer needle inner diameter 1.2 mm, inner needle inner diameter 0.5 mm) was used. The shell solution flow rate was 2 mL / h, the core solution flow rate was 0.5 mL / h, and the shell to core mass ratio was 4:1. The spinning voltage was set to 25 kV, the receiving distance to 20 cm, the receiving plate to be made of polytetrafluoroethylene, and the rotation speed to 200 r / min. The spinning environment was controlled at 25℃ and 50% relative humidity, and a nanofiber membrane with a thickness of 0.5 mm was collected.

[0051] (3) Post-processing Crosslinking was performed using a 2wt% genipin ethanol solution at 40°C for 140 min via vapor crosslinking. After crosslinking, the sample was dried in a vacuum oven at 30°C for 12 h. The sample was then aseptically cut, sterilized with ethylene oxide (800 mg / L, 4 h), and packaged.

[0052] The remaining parameters are the same as in Example 1. The obtained scaffold nanofibers have a diameter of 500 nm and a porosity of 85%.

[0053] Experimental Example 1 In vitro drug release kinetics test 1. Test materials Sample: Composite fiber scaffold prepared in Example 1 (10 mg in weight, cut into 1 cm × 1 cm dimensions). Reagents: Phosphate-buffered saline (PBS, pH 7.4, containing 0.05% Tween-80), platelet exosome ELISA kit, curcumin high performance liquid chromatography (HPLC) detection reagent, BMP-2 ELISA kit, icariin HPLC detection reagent; Instruments: Dialysis bags (molecular weight cutoff 8-14kDa), constant temperature shaker, high performance liquid chromatograph, enzyme-linked immunosorbent assay (ELISA) reader.

[0054] 2. Test methods (1) The scaffold sample is encapsulated in a dialysis bag, 1 mL of PBS buffer is added to soak it, the two ends of the dialysis bag are sealed, and it is placed in a centrifuge tube containing 30 mL of PBS buffer.

[0055] (2) Place the centrifuge tubes in a constant temperature shaker at 37℃ and 100r / min and take 5mL samples at 1d, 3d, 7d, 14d, 21d, 28d, 35d, 42d, 49d and 56d respectively, and add an equal amount of fresh PBS buffer at the same time.

[0056] (3) The concentrations of each component in the sample were determined by the corresponding detection methods: platelet exosomes and BMP-2 were detected by ELISA, and curcumin and icariin were detected by HPLC.

[0057] (4) Calculate the cumulative release rate at each time point (cumulative release rate = amount of drug released / total amount of drug loaded × 100%). Set up 3 parallel samples for each group and repeat the experiment 3 times.

[0058] 3. Data Statistics SPSS 26.0 statistical software was used for analysis. Release data are expressed as mean ± standard deviation (x ± s). Repeated measures ANOVA was used to verify the significance of the release trend. P < 0.05 was considered statistically significant.

[0059] 4. Test Results As shown in Table 1, the shell components (platelet exosomes and curcumin) were rapidly released within 1-3 days, with a cumulative release rate of over 75%. The cumulative release rate exceeded 89% within 7 days, and was basically completely released within 21 days (cumulative release rate ≥95%). The release trend was consistent with the 2-3 week degradation cycle of the PLGA substrate. The core components (BMP-2 and icariin) were released slowly and continuously, with a cumulative release rate of only about 20% within 1-7 days. They were continuously released within 4-8 weeks (28-56 days), with a cumulative release rate of over 82% within 56 days. The release cycle was highly consistent with the 1-8 week degradation cycle of the gelatin hydrogel, and the differences in release trends within the group were statistically significant (P<0.05). This indicates that the composite fiber scaffold of the present invention achieves precise timing release of active ingredients: the shell anti-inflammatory components (platelet exosomes, curcumin) are rapidly released, which can quickly take effect in the early stage (1-3 days) to clear the inflammatory microenvironment; the core regeneration factors (BMP-2, icariin) are slowly released, covering the entire tendon and bone healing cycle (1-8 weeks), and continuously promoting tissue regeneration.

[0060] Table 1 Results of in vitro drug release kinetics tests

[0061] Experimental Example 2 In vitro anti-inflammatory performance test 1. Test materials Experimental group: Composite fiber scaffold prepared in Example 1; Control group 1: Blank core-shell fiber scaffold (without loaded active ingredients); Control group 2: PLGA fiber scaffold loaded only with curcumin; Control group 3: PLGA fiber scaffold loaded only with platelet exosomes.

[0062] Cells: Rat macrophages RAW264.7.

[0063] Reagents: Lipopolysaccharide (LPS), TNF-α and IL-1β ELISA detection kit.

[0064] 2. Test methods (1) Cut each group of scaffolds into circular pieces with a diameter of 8 mm, sterilize them with ethylene oxide, and place them in a 24-well cell culture plate.

[0065] (2) Take RAW264.7 cells in the logarithmic growth phase and use 5×10 4 Inoculate each well with a density of 1000 cells / well, add DMEM medium containing 10% fetal bovine serum, and incubate at 37°C in a 5% CO2 incubator for 24 hours.

[0066] (3) Add LPS (final concentration 1 μg / mL) to each well to induce an inflammatory response and continue culturing for 24 h.

[0067] (4) Collect the supernatant from each well and use an ELISA kit to detect the concentrations of TNF-α and IL-1β. Set up 5 replicates for each group and repeat the experiment 3 times.

[0068] 3. Data Statistics SPSS 26.0 statistical software was used for analysis, and the results are expressed as mean ± standard deviation (x ± s).

[0069] 4. Test Results As shown in Table 2, statistical analysis revealed that the concentrations of TNF-α and IL-1β in the experimental group were significantly lower than those in control group 1 (P<0.01). The experimental group also showed significantly higher concentrations compared to control groups 2 and 3 (P<0.01). There was no significant difference between control groups 2 and 3 (P>0.05). This indicates that the platelet exosomes in the scaffold shell of this invention and curcumin have a synergistic anti-inflammatory effect, effectively inhibiting the release of inflammatory factors from macrophages and clearing the destructive inflammatory microenvironment. Furthermore, this anti-inflammatory effect has clear statistical significance. Specifically, the inhibitory effect of the experimental group (dual-drug synergy) on TNF-α was approximately 45.5% higher than that of the curcumin-only group (control group 2) and approximately 40.3% higher than that of the platelet exosome-only group (control group 3).

[0070] Table 2 Results of in vitro anti-inflammatory performance test

[0071] Experimental Example 3 In vivo tendon-bone healing mechanical performance test 1. Test materials Experimental group: Composite fiber scaffold prepared in Example 1; Control group 1: Blank core-shell fiber scaffold; Control group 2: Clinically commonly used simple collagen scaffolds.

[0072] Laboratory animals: 36 healthy New Zealand white rabbits, weighing 2.0-2.5 kg. 2. Test methods (1) Establishing a rabbit distal radioulnar joint ligament reconstruction model: After anesthesia, the rabbit's own extensor digitorum longus tendon was harvested, one end of the tendon was implanted into the distal radius bone tunnel, and the other end was fixed to the distal ulna to construct a tendon-bone healing model.

[0073] (2) Grouping: 36 rabbits were randomly divided into 3 groups of 12 each. The experimental group received a composite fiber scaffold implanted in the bone tunnel; control group 1 received a blank scaffold implanted; and control group 2 received a simple collagen scaffold implanted. After the operation, the rabbits were fed routinely and allowed to move freely.

[0074] (3) Sampling and mechanical testing: At 4 and 8 weeks postoperatively, 6 rabbits in each group were sacrificed, and the inferior radioulnar joint specimen was completely removed, with surrounding soft tissues removed. The specimens were fixed in an electronic universal testing machine, and tensile tests were performed at a speed of 1 mm / min. The maximum load and stiffness of the tendon-bone interface were recorded as indicators for evaluating the strength of tendon-bone healing.

[0075] 3. Data statistics are the same as in Experiment 2.

[0076] 4. Test Results As shown in Table 3, statistical analysis revealed a significant main effect of group (P<0.01), with the maximum load and stiffness of the experimental group significantly higher than those of control groups 1 and 2. A significant main effect of time (P<0.01) also occurred, with the mechanical properties of all groups showing an upward trend over time, and the indicators at 8 weeks significantly higher than those at 4 weeks. Furthermore, a significant interaction between group and time was observed (P<0.01), with the experimental group showing a significantly greater improvement in mechanical properties at 8 weeks compared to the two control groups. This indicates that BMP-2 in the core of the scaffold of this invention, along with icariin, can synergistically promote the regeneration of fibrocartilage and bone tissue at the tendon-bone interface, significantly improving tendon-bone healing quality and mechanical strength. Moreover, the effect continues to enhance over time, and this conclusion has clear statistical significance.

[0077] Table 3 Results of in vivo tendon-bone healing mechanical performance tests

[0078] Test Example 4 Histological examination of tendon-bone healing 1. Experimental materials: Rabbit tendon and bone healing specimens from each group 8 weeks post-operation in Experimental Case 3. 2. Test methods (1) Fix the specimen in 4% paraformaldehyde for 24 hours, decalcify with EDTA decalcification solution for 4 weeks, dehydrate routinely, embed in paraffin, and prepare tissue sections with a thickness of 5 μm.

[0079] (2) Perform HE staining and Masson trichrome staining respectively, and observe the tissue morphology of the tendon-bone interface under an optical microscope.

[0080] (3) The semi-quantitative scoring is carried out according to the Histological Scoring Standard for Tendon-Bone Healing. The scoring range is 0-10 points. The higher the score, the better the healing quality. The specific scoring standards are: tendon-bone interface gap (0-3 points), fibrocartilage layer thickness (0-3 points), collagen fiber arrangement order (0-2 points), and bone tissue ingrowth degree (0-2 points).

[0081] 3. Experimental Results As shown in Table 4, statistical analysis revealed that the histological score of tendon-bone healing in the experimental group was significantly higher than that in control groups 1 and 2 (P<0.05); the difference between control groups 1 and 2 was also statistically significant (P<0.05). This indicates that the composite fiber scaffold of the present invention can promote the formation of a mature fibrocartilage transition layer at the tendon-bone interface, achieving a firm bond between the tendon and bone tissue, and significantly improving the tissue morphological quality of tendon-bone healing. This conclusion has clear statistical significance.

[0082] Table 4. Histological examination results of tendon-bone healing

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composite fiber scaffold for treating osteoarthritis and promoting tendon-bone healing, characterized in that, The composite fiber scaffold has a core-shell structure, with the shell layer using polylactic acid-glycolic acid copolymer as the substrate and the core layer using gelatin hydrogel as the substrate. The mass ratio of the shell layer to the core is (2-4):

1. The shell is loaded with platelet exosomes and curcumin. In each milligram of composite fiber scaffold, the content of platelet exosomes is 0.5-5 μg and the content of curcumin is 1-10 μg. The core-loaded bone morphogenetic protein-2 and icariin, with each milligram of composite fiber scaffold containing 0.1-1 μg of BMP-2 and 5-20 μg of icariin.

2. The composite fiber scaffold according to claim 1, characterized in that, The composite fiber scaffold has nanofibers with a diameter of 100-500 nm and a porosity of 70-85%.

3. The composite fiber scaffold according to claim 1, characterized in that, The polylactic acid-glycolic acid copolymer is a copolymer of lactic acid and glycolic acid in a molar ratio of 75:25, with a number-average molecular weight of 50-100 kDa and a degradation cycle of 2-3 weeks.

4. The composite fiber scaffold according to claim 1, characterized in that, The gelatin hydrogel is a partial hydrolysate of collagen derived from bovine bone or pigskin, with a number-average molecular weight of 1-10 kDa. After cross-linking treatment, it is in a liquid state at low temperatures of 4-10°C and in a gel state at body temperature, with a degradation cycle of 1-8 weeks.

5. The method for preparing the composite fiber scaffold according to any one of claims 1-4, characterized in that, Includes the following steps: S1. Solution preparation: Prepare the shell solution and the core solution separately; S2. Coaxial electrospinning: Using a coaxial needle, the shell solution and the core solution are propelled by independent injection pumps for spinning, and the core-shell structured nanofiber membrane is collected on the receiving plate. S3. Post-processing: The nanofiber membrane is cross-linked in genipin vapor and then aseptically packaged to obtain the composite fiber scaffold.

6. The preparation method according to claim 5, characterized in that, In step S1, the shell solution is prepared by dissolving polylactic acid-glycolic acid copolymer in hexafluoroisopropanol to prepare a PLGA solution with a concentration of 8-15wt%, adding purified platelet exosomes and curcumin, and then subjecting it to ultrasonic treatment to obtain the shell solution; the ultrasonic power is 100-300W, the time is 5-15min, and the temperature is <25℃.

7. The preparation method according to claim 5, characterized in that, In step S1, the core solution is prepared by dissolving gelatin in a 1-5 vol% aqueous acetic acid solution, stirring in a water bath at 30-40°C for 30-60 min to prepare a 5-10 wt% gelatin solution, cooling to room temperature, adding bone morphogenetic protein-2 and icariin, stirring for 10-20 min until completely dissolved to obtain the core solution.

8. The preparation method according to claim 5, characterized in that, In step S2, the inner diameter of the outer needle of the coaxial needle is 0.8-1.2 mm, and the inner diameter of the inner needle is 0.3-0.5 mm; the spinning voltage is 15-25 kV, the receiving distance is 10-20 cm, the shell solution flow rate is 0.5-2 mL / h, and the core solution flow rate is 0.1-0.8 mL / h.

9. The preparation method according to claim 5, characterized in that, In step S2, the temperature of the spinning environment is 20-25℃ and the relative humidity is 30-50%.

10. The preparation method according to claim 5, characterized in that, In step S3, the genipin vapor crosslinking conditions are as follows: genipin ethanol solution concentration is 0.5-2wt%, crosslinking temperature is 30-40℃, and crosslinking time is 100-140min; after crosslinking, the fiber membrane is dried at 25-30℃ for 8-12h.

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