Artificial ligament with segmented functional coating as well as preparation method and application of artificial ligament

Abstract

The invention discloses a segmented functionalized coating artificial ligament and a preparation method and application thereof. The segmented functionalized coating artificial ligament comprises an articular cavity section PET artificial ligament coating loaded with glucosamine and a bone tunnel section PET artificial ligament coating mineralized by hydroxyapatite. The method comprises the following steps: modifying a PET artificial ligament base material with a phosphate group, and reacting in an alkali solution to obtain a surface-modified PET artificial ligament; activating the modified PET artificial ligament with an MES buffer solution and an EDC / NHS coupling agent, and soaking the activated PET artificial ligament in a glucosamine solution to obtain a glucosamine-loaded PET artificial ligament; the surface-modified PET artificial ligament is placed in simulated body fluid to be mineralized, and the mineralized PET artificial ligament is obtained. The prepared PET artificial ligament has good anti-inflammatory activity and effectively relieves PTOA progress, the bone tunnel section mineralized PET artificial ligament has good osteogenic differentiation promoting capacity, and a new solution is provided for improving the tendon-bone healing capacity of implants after ACL reconstruction.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a segmented functionalized coating artificial ligament and a preparation method and application thereof. Background Art

[0002] With the global popularity of sports and the increasing demand for exercise, anterior cruciate ligament (ACL) injury has become one of the most common knee sports injuries, with an annual incidence rate of 68.6 / 100,000. Due to the poor self-healing ability of the ACL, ACL reconstruction is required to restore its function. The polyethylene terephthalate (PET) artificial ligament commonly used in clinical practice is a permanent implant, but due to the hydrophobicity and bioinertness of the material itself, clinical problems such as synovitis and bone tunnel enlargement occur after reconstruction. Therefore, improving the surface bioactivity of the material to promote bone integration between the implant and the host bone is an urgent clinical problem to be solved.

[0003] Post-traumatic osteoarthritis (PTOA) of the knee is a degenerative joint disease caused by trauma or acute injury, accounting for approximately 12% of all symptomatic osteoarthritis cases. Mechanical damage resulting from knee trauma (such as ligament tears and meniscus injuries) is the primary cause of PTOA. Altered mechanical loads lead to an imbalance in stress distribution within the joint, accelerating cartilage degeneration. Continuous abnormal mechanical loads can cause microdamage to the subchondral bone, accelerating the progression of osteoarthritis. Nonsteroidal anti-inflammatory drugs (NSAIDs) can effectively relieve pain in the short term, but long-term use may cause gastrointestinal discomfort by reducing the production of protective prostaglandins in the gastric mucosa. Furthermore, long-term or frequent intra-articular injections may accelerate cartilage degeneration, are associated with significant individual variability, are costly, and require multiple injections to maintain efficacy.

[0004] Glucosamine (GS), an amino monosaccharide naturally present in cartilage, is a crucial component of the articular cartilage matrix. As a precursor to cartilage matrix components (such as glycosaminoglycans and proteoglycans), GS promotes the production of new matrix by chondrocytes, maintaining cartilage elasticity and lubricity. By inhibiting the NF-κB pathway, it downregulates the expression of the inflammatory cytokine IL-1β. Therefore, there is an urgent need for artificial ligaments with anti-inflammatory properties in clinical research to provide a favorable immune microenvironment for tendon-bone healing.

[0005] Existing research suggests that PET artificial ligaments have achieved some success in clinical application due to their excellent mechanical properties and biomimetic structure. However, their bioinertness and poor tissue integration severely limit their long-term efficacy after ACLR surgery. Within the joint cavity, the surface properties of PET lead to friction with bone tissue during surgery and long-term implantation, producing particulate debris that activates proinflammatory cells and forms a chronic inflammatory microenvironment, leading to the development of postoperative osteoarthritis (PTOA). Within the bone tunnel, PET's limited integration with host bone increases the risk of postoperative bone tunnel enlargement. Therefore, optimizing the interfacial bioactivity and tissue integration of PET artificial ligaments has become an important approach to improving long-term stability and preventing PTOA after ACLR surgery.

[0006] Currently, the preparation method for artificial ligament surface coating (CN104147642B) is to load nanosilver coating after ultrasonic cleaning and corona treatment to give artificial ligament antibacterial and anti-inflammatory properties, but it lacks segmented functional design, that is, functional coating targeting clinical pain points in different parts to meet the biocompatibility and functional requirements of different parts, improve the overall performance of PET artificial ligament, and thus extend its service life. Summary of the Invention

[0007] The purpose of the present invention is to provide an artificial ligament with a segmented functionalized coating and a preparation method thereof in view of the deficiencies in the prior art.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The invention provides a segmented functionalized coating artificial ligament, which comprises a glucosamine-loaded joint cavity segment PET artificial ligament coating and a hydroxyapatite-mineralized bone tunnel segment PET artificial ligament coating.

[0010] The preparation process of the segmented functionalized coating artificial ligament mentioned above mainly includes: 1. artificial ligament surface modification process; 2. artificial ligament loading glucosamine process; 3. artificial ligament mineralization process. The specific preparation steps are as follows:

[0011] (1) Artificial ligament surface modification process

[0012] Step 1: placing the PET artificial ligament substrate in anhydrous ethanol and ultrasonically treating it for 15 to 30 minutes, washing it with deionized water until the pH of the washed water is neutral, and placing it in a vacuum drying oven until it is completely dried;

[0013] Step 2: placing the PET artificial ligament treated in step 1 in a phosphoric acid solution, stirring to allow the phosphoric acid solution to fully infiltrate the PET artificial ligament, washing with deionized water until the pH of the washed water is neutral, and placing in a vacuum drying oven until completely dried to obtain a phosphate-modified PET artificial ligament;

[0014] Step 3: Place the phosphate-modified PET artificial ligament obtained in step 2 in a NaOH solution. After the reaction, wash it with deionized water until the pH of the washed water is neutral, and place it in a vacuum drying oven until it is completely dried to obtain a surface-modified PET artificial ligament.

[0015] (2) Artificial ligament loading glucosamine process

[0016] Step 4, dissolving glucosamine in deionized water and ultrasonically treating the water to obtain a glucosamine solution;

[0017] Step 5, placing the joint cavity segment of the surface-modified PET artificial ligament obtained in Step 3 in a 2-morpholineethanesulfonic acid (MES) buffer solution and reacting until the pH value on the surface of the PET artificial ligament is less than 7;

[0018] Step 6, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) coupling agent to the reaction container of step 5 to react and activate the carboxyl groups on the surface of the PET artificial ligament;

[0019] Step 7, adding 4-dimethylaminopyridine (DMAP) to the reaction vessel of step 6, and then adding the aminoglucose solution of step 4, and reacting;

[0020] Step 8, washing the PET artificial ligament after the reaction in step 7 with deionized water until the pH of the washed water is neutral, placing it in a freeze dryer for freeze drying, and obtaining a PET artificial ligament loaded with glucosamine.

[0021] (3) Artificial ligament mineralization process

[0022] Step 9, placing the surface-modified bone tunnel segment of the PET artificial ligament obtained in Step 3 in a 2-morpholineethanesulfonic acid buffer solution and reacting until the pH value on the surface of the PET artificial ligament is less than 7;

[0023] Step 10, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS) coupling agent to the reaction vessel of step 9 to react and activate the carboxyl groups on the surface of the PET artificial ligament;

[0024] Step 11, washing the PET artificial ligament after carboxyl group activation in step 10 with deionized water until the pH of the washed water is neutral, washing with phosphate buffer until the pH of the surface of the PET artificial ligament is greater than 7, and then placing it in simulated body fluid and mineralizing it on a shaker, and replacing it with fresh simulated body fluid every other day;

[0025] Step 12: Wash the PET artificial ligament obtained in step 11 with deionized water until the pH of the washed water is neutral, and place it in a vacuum drying oven until it is completely dried to obtain a mineralized PET artificial ligament.

[0026] Furthermore, the PET artificial ligament substrate consists of two parts: a joint cavity segment and a bone tunnel segment. The joint cavity segment is about 2.5 cm long, and the bone tunnel segment (femur and tibia) is about 32.5 cm long (the length is cut by the doctor during clinical use). The material of the joint cavity segment and the bone tunnel segment is PET, and the free fibers of the joint cavity segment are tightly connected with the woven fibers of the bone tunnel segment.

[0027] Furthermore, in step 2, the concentration of the phosphoric acid solution is 1 to 2.5 M, the reaction temperature is controlled at 25 to 37° C., and the reaction time is 1 to 3 hours.

[0028] Furthermore, in step 3, the concentration of the NaOH solution is 1 to 5 M, the reaction temperature is controlled at 25 to 37° C., and the reaction time is 1 to 3 h.

[0029] Furthermore, in step 6, the molar ratio of EDC to NHS in the coupling agent is 5:1 to 2:1, the reaction temperature is controlled at 25 to 37° C., and the reaction time is 12 to 24 hours.

[0030] Furthermore, in step 7, the concentration of 4-dimethylaminopyridine is 0.1M to 0.5M, the reaction temperature is controlled at 25 to 37° C., and the reaction time is 12 to 24 hours.

[0031] Furthermore, in step 7, the concentration of the glucosamine solution is 0.01 to 2 mM, preferably 0.05 to 0.1 mM, the reaction temperature is controlled at 25 to 37° C., and the reaction time is 1 to 24 h.

[0032] Furthermore, in step 11, the components of the simulated body fluid are NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, HCl, CaCl2·2H2O, Na2SO4, and Tris base, with a concentration of 1.2 to 1.5 times that of SBF. The amounts of each component are shown in the following table:

[0033]

[0034] Furthermore, in step 11, the reaction temperature of the mineralization process is 36-38° C., the mineralization time is 7-21 days, and the shaking speed is 60-100 rpm.

[0035] The segmented functionalized coating artificial ligament prepared above has the structural characteristics of an integrated woven substrate + a partitioned surface functional coating.

[0036] The segmented functionalized coated artificial ligament prepared above has the ability of anti-inflammatory osteogenesis and accelerated tissue integration, and has good application prospects in anterior cruciate ligament injury repair, reconstruction and soft tissue replacement.

[0037] The present invention implements a segmented functionalized surface modification strategy by grafting glucosamine on the surface of the joint cavity segment of the PET artificial ligament matrix and depositing hydroxyapatite on the surface of the bone tunnel segment of the PET artificial ligament matrix. Through the synergistic effect of functional zoning, the anti-inflammatory and bone integration capabilities of the PET artificial ligament are improved, which is superior to a single coating and has good clinical application prospects.

[0038] Compared with the prior art, the solution of the present invention has the following technical effects:

[0039] The present invention successfully constructed a PET artificial ligament with a segmented functionalized coating. The experimental results showed that the glucosamine-loaded PET artificial ligament in the joint cavity segment had good anti-inflammatory activity and effectively alleviated the progression of PTOA. The mineralized PET artificial ligament in the bone tunnel segment had good osteogenic differentiation ability, providing a new solution for improving the tendon-bone healing ability of implants after ACL reconstruction surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a picture of the PET artificial ligament substrate.

[0041] Figure 2 Comparison of the surface morphology of PET before and after glucosamine grafting; (A) SEM images of PET, PET-COOH and PET-GS with different concentrations; (B) EDS spectrum of PET-GS.

[0042] Figure 3 Comparison of the surface morphology of PET before and after mineralization; (A) SEM images of PET, PET-COOH and PET-HA at different mineralization times; (B) EDS energy spectrum element distribution diagram of PET-HA.

[0043] Figure 4 RT-qPCR was used to detect the expression of related inflammatory genes after RAW264.7 was cultured with different concentrations of PET-GS extracts for one day.

[0044] Figure 5 RT-qPCR was used to detect the expression of chondrogenic-related genes in BMSCs co-cultured with different concentrations of PET-GS chondrogenic induction medium for 7 days and 14 days.

[0045] Figure 6 RT-qPCR was used to detect the expression of osteogenic genes related to BMSCs cultured with PET-HA osteogenic induction medium extract at different mineralization times for 7 days and 14 days. DETAILED DESCRIPTION

[0046] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are not intended to limit the scope of protection of the present invention:

[0047] In the following examples or comparative examples,

[0048] The PET artificial ligament substrate used was taken from Ligatech ligament (Shanghai Ligatech Biotechnology Co., Ltd.), and the overall picture of the substrate is as follows: Figure 1 As shown, the artificial ligament consists of a bone tunnel segment (femur) - an articular cavity segment - and a bone tunnel segment (tibia). The bone tunnel segment is tightly woven, while the articular cavity segment is free-fiber. The free fibers of the articular cavity segment are tightly connected to the woven fibers of the bone tunnel segment. For experiments, the ligament was cut into 1 cm x 2 cm pieces for later use.

[0049] The simulated body fluid 1.5x SBF (Shanghai Yuanye Biotechnology Co., Ltd.) used is shown in the following table:

[0050]

[0051] After the surface of the PET substrate was alkaline hydrolyzed, it was placed in MES buffer at pH 6.0 for EDC / NHS coupling and then loaded with glucosamine and mineralized.

[0052] Example 1

[0053] The preparation of a segmented functionalized coating artificial ligament comprises the following steps:

[0054] (1) Artificial ligament surface modification process

[0055] The PET substrate was soaked in anhydrous ethanol and ultrasonically treated for 15 minutes, washed with deionized water until the pH of the washed water reached 7, and placed in a vacuum drying oven until completely dried. The dried and clean PET artificial ligament was placed in a 1M phosphoric acid solution, stirred at room temperature for 1 hour to allow the phosphoric acid solution to fully infiltrate the PET artificial ligament, washed with deionized water until the pH of the washed water reached 7, and placed in a vacuum drying oven until completely dried. The phosphate-modified PET artificial ligament was placed in a 2.5M NaOH solution, stirred at room temperature for 1 hour, washed with deionized water until the pH of the washed water reached 7, and placed in a vacuum drying oven until completely dried to obtain a surface-modified PET artificial ligament, which was recorded as the PET-COOH group.

[0056] (2) Artificial ligament loading glucosamine process

[0057] 27.725 mg of glucosamine sulfate was dissolved in 10 mL of deionized water and ultrasonically treated at 100 W for 15 min to obtain a 10 mM glucosamine stock solution. The glucosamine stock solution was diluted in sequence to obtain 2 mM, 0.5 mM, 0.1 mM, and 0.05 mM glucosamine solutions. The articular cavity segment of the PET ligament modified in step (1) was placed in a MES buffer solution at pH 6.0 and reacted for 2 h. 1 M EDC and 0.2 M NHS were added and reacted for 16 h. 0.1 M DMAP was added and then the above glucosamine solution was slowly added dropwise. After stirring at room temperature for 24 h, the ligament was washed with deionized water until the pH of the washing water was 7. The ligament was placed in a freeze dryer and freeze-dried to obtain a PET artificial ligament loaded with glucosamine, which was designated as the PET-GS group. According to the different glucosamine concentrations, they were designated as PET-GS2, PET-GS0.5, PET-GS0.1, and PET-GS0.05.

[0058] (3) Artificial ligament mineralization process

[0059] The bone tunnel segment of the modified PET ligament in step (1) was placed in MES buffer at pH 6.0 for 2 h, 1 M EDC and 0.2 M NHS were added and reacted for 16 h, then washed with deionized water until the pH of the washed water was 7, and washed with phosphate buffer until the pH of the surface of the PET artificial ligament was greater than 7. It was placed in simulated body fluid and mineralized on a shaker at 37°C at 60 rpm for 5 days, 7 days, and 14 days, respectively. Fresh simulated body fluid was replaced every other day. The mineralized PET ligament was washed with deionized water until the pH of the washed water was 7, and placed in a vacuum drying oven until completely dried to obtain mineralized PET artificial ligaments, which were recorded as the PET-HA group. According to different mineralization times, they were recorded as PET-HA5, PET-HA7, and PET-HA14.

[0060] Example 2

[0061] Image analysis was performed on the surface-modified PET, glucosamine-loaded PET, and mineralized PET prepared in Example 1.

[0062] 1. Surface morphology and element distribution tests were conducted on PET, PET-COOH and PET-GS materials (PET-GS2, PET-GS0.1, PET-GS0.05).

[0063] like Figure 2As shown in Figure A, the surface of the unmodified PET substrate is smooth without obvious pores, which is not conducive to cell adhesion; the surface of the PET-COOH after phosphate esterification and alkaline hydrolysis treatment shows obvious roughness, which may be caused by chemical etching, which is conducive to subsequent glucosamine modification and cell adhesion; the surface of the low-concentration glucosamine coating (PET-GS0.05 and PET-GS0.1) presents a dense network and porous structure, and the pores gradually increase with the increase of glucosamine concentration. This surface morphology can provide a larger specific surface area, which is conducive to cell adhesion and proliferation; the surface of the high-concentration glucosamine coating (PET-GS2) shows obvious roughness and even wrinkles. The appearance of this morphology may be due to structural collapse caused by excessive glucosamine deposition. High-concentration modification may lead to excessive surface roughness and high brittleness that affect interfacial bonding.

[0064] like Figure 2 As shown in Figure B, after glucosamine was grafted into the EDS spectrum, nitrogen elements appeared on the surface of PET, indicating that glucosamine was successfully modified onto PET, which is beneficial to improving the biocompatibility of the material.

[0065] 2. Surface morphology and element distribution tests were conducted on PET, PET-COOH and PET-HA materials (PET-HA5, PET-HA7, PET-HA14).

[0066] like Figure 3 As shown in Figure A, the surface of unmodified PET is smooth without obvious roughness or particle deposition, which is not conducive to inducing mineralization and cell adhesion. After phosphate esterification and alkaline hydrolysis, the surface of PET-COOH shows a uniform nanoscale rough structure, which improves the surface hydrophilicity and provides a good surface for Ca 2+ and PO4 3- It provides binding sites, which is beneficial to subsequent mineralization and cell adhesion. With the increase of mineralization time, HA deposition gradually occurs on the PET surface, and evenly distributed small particles are formed on the surface of PET-HA5. The particles of PET-HA7 increase in size and form a micron-sized spherical structure. The HA deposits of PET-HA14 further increase in size, presenting a porous spherical structure. The particles fuse with each other, which is beneficial to the subsequent cell adhesion and proliferation.

[0067] like Figure 3 As shown in Figure B, EDS elemental analysis shows that elements such as calcium and phosphorus appear on the surface of PET after mineralization, confirming the successful deposition of HA, which is beneficial to improving the biocompatibility of the material.

[0068] Example 3

[0069] The glucosamine-loaded PET prepared in Example 1 was tested for its anti-inflammatory activity and chondrogenic differentiation ability, and the mineralized PET was tested for its osteogenic differentiation ability.

[0070] RT-qPCR was used to detect the expression of inflammatory factors in mouse mononuclear macrophages (RAW264.7) after PET-GS extracts of different concentrations were cultured.

[0071] (1) Establishment of inflammation model

[0072] 1. Preparation of complete culture medium: Take 500 μL of penicillin / streptomycin (1%) and 5 mL of fetal bovine serum (10%), add DMEM culture medium to 50 mL to obtain complete culture medium.

[0073] 2. Sterilization of materials: Cut the PET sheet into 1cm 2 Place the disc in the bottom of a 48-well plate, add 1 mL of 75% ethanol solution to each well, and expose to UV light for 4 hours. After sterilization, wash three times with PBS and soak overnight. Then, replace the basal medium and soak for 2 hours before use.

[0074] 3. Extract preparation: After sterilizing PET and PET-GS according to the above conditions, refer to the extraction conditions in GB / T 16886.5 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test" and add DMEM medium at a ratio of 1g / 5mL for extraction. Incubate in a shaker at 37°C and 120 rpm for 24 hours. Filter and sterilize through a 0.22μm filter membrane to obtain a sample extract. Add fetal bovine serum for later use.

[0075] 4. Preparation of lipopolysaccharide (LPS) solution: Weigh an appropriate amount of LPS and dissolve it in DMEM medium to prepare a 0.5 mg / mL stock solution. Filter-sterilize with a 0.22 μm filter membrane. Add 10% fetal bovine serum and 1% penicillin / streptomycin solution, and dilute with complete medium to obtain a 500 ng / mL working solution.

[0076] 5. Cell seeding plate: 2×10 5 Raw264.7 were seeded onto 6-well plates at a density of 10 cells / well and cultured in a cell culture incubator for 24 h. After the cells adhered to the wall, the culture medium was slowly aspirated, 500 ng / mL LPS solution was added, and the plates were cultured in a cell culture incubator for another 24 h to induce the cells to polarize toward M1.

[0077] 6. After 24 hours of induction, observe the cell morphology under a microscope to determine whether it is polarized. After polarization, aspirate the lipopolysaccharide solution, slowly rinse twice with PBS, add the sample extract, and detect the expression of inflammatory factors by RT-qPCR after 24 hours.

[0078] (2) RT-qPCR

[0079] The genes tested included tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS). The primer sequences are shown in Table 1. GAPDH was an internal reference gene, meaning that the expression of all genes was compared with that of the internal reference gene.

[0080] Table 1 Primer sequences of inflammation-related genes

[0081]

[0082] Figure 4 Figure 2 shows the relative expression of inflammatory genes in Raw264.7 cells cultured with different concentrations of PET-GS extract for 24 hours after LPS stimulation. (A) Interleukin-6 (IL-6); (B) Inducible nitric oxide synthase (iNOS); (C) Interleukin-1β (IL-1β); (D) Tumor necrosis factor α (TNF-α). *p < 0.05, **p < 0.01, ***p < 0.001. Figure 4 IL-6 in A is a pro-inflammatory cytokine in M1 macrophages. It stimulates hepatocytes to produce C-reactive protein (CRP) through the JAK-STAT3 signaling pathway, enhancing the acute phase response. IL-6 expression levels were highest in the unmodified PET group, indicating that PET alone failed to significantly inhibit the inflammatory response. Glucosamine grafting reduced IL-6 expression, with significantly lower levels in the PET-GS0.05 and PET-GS0.1 groups, indicating that low-concentration glucosamine has a strong anti-inflammatory effect. Figure 4 iNOS is a proinflammatory cytokine in B. Its high expression promotes NO production. Excessive NO may lead to enhanced oxidative stress, mitochondrial damage, and cell apoptosis. The unmodified PET group showed higher iNOS expression, indicating that PET did not inhibit LPS-induced NO production. iNOS expression was significantly reduced in the PET-GS0.05 and PET-GS0.1 groups, indicating that low concentrations of glucosamine can effectively inhibit iNOS-mediated inflammatory responses. The higher iNOS expression levels in the PET-GS0.5 and PET-GS2 groups indicate that the inhibitory effect of high concentrations of glucosamine on iNOS is weakened, possibly related to the regulation of cellular metabolic adaptations. Figure 4IL-1β in C can enhance immune cell activity, promote T and B cell proliferation, amplify inflammatory responses, and induce matrix metalloproteinases (MMPs), which participate in tissue degradation and lead to chronic diseases such as arthritis and fibrosis. The unmodified PET group had the highest IL-1β expression level, indicating a significant LPS-induced inflammatory response, with a similar trend to IL-6 and iNOS. IL-1β expression was significantly reduced in the PET-GS0.05 and PET-GS0.1 groups, while higher in the PET-GS0.5 and PET-GS2 groups. This suggests that low concentrations of glucosamine have a strong inhibitory effect on inflammation, while high concentrations can affect this inhibitory effect. Figure 4 TNF-α in D activates the NF-κB signaling pathway, inducing the secretion of other inflammatory factors (IL-1β, IL-6, and iNOS). TNF-α expression was highest in the PET group, while decreased in the PET-GS0.05 and PET-GS0.1 groups, further confirming the anti-inflammatory effects of low-concentration PET-GS. TNF-α expression rebounded in the PET-GS0.5 and PET-GS2 groups, but remained lower than in the PET group, suggesting that high glucosamine concentrations weaken the anti-inflammatory effect. Glucosamine at concentrations of 0.05 to 0.1 mM exhibited a potent anti-inflammatory effect.

[0083] The chondrogenic differentiation sample was a PET sheet of the same size as the bottom area of a 6-well plate, and the sterilization procedure was the same as above. Cells were plated at 1×10 5 Cells were seeded onto PET sheets at a density of 10 cells / well. The plates were placed in an incubator and incubated for 24 hours. The complete culture medium was aspirated and chondrogenic differentiation medium was added, marking day 0. Chondrogenic differentiation culture was continued for 14 days, with the chondrogenic differentiation medium replaced every two days. Cells were harvested from the chondrogenic differentiation culture for 7 and 14 days, and the expression of relevant genes was analyzed by RT-qPCR. The genes tested included SRY-box transcription factor 9 (Sox9), type I collagen (COL-I), type II collagen (COL-II), and aggrecan. The primer sequences are shown in Table 2.

[0084] Table 2 Primer sequences of chondrogenic differentiation-related genes

[0085]

[0086] Figure 5 The relative expression levels of genes involved in the chondrogenic differentiation of mBMSCs induced by different concentrations of PET-GS in chondrogenic induction medium are shown. (A) Aggrecan; (B) Type II collagen (COL-II); (C) SRY-box transcription factor 9 (Sox9); (D) Type I collagen (COL-I). *p < 0.05, **p < 0.01, ***p < 0.001. Figure 5Acan is the main protein component of the extracellular matrix of cartilage cells and plays a key role in the process of cartilage formation. On the 7th day, the expression of Acan in the PET-GS0.05 and PET-GS0.1 groups was significantly higher than that in the unmodified PET group. On the 14th day, Acan expression was further upregulated, and the PET-GS0.05 group showed a higher expression level, indicating that low concentrations of glucosamine have the potential to promote the early synthesis of mBMSCs cartilage matrix. COL-II is the main structural protein in cartilage tissue, and together with Acan, it constitutes the main component of the cartilage matrix. Figure 5 In Figure B, the expression levels of COL-II in the PET-GS0.05 and PET-GS0.1 groups were significantly higher than those in the PET group, and the expression effect was more obvious at 14 days. Its high expression indicates the transformation of mBMSCs to mature cartilage phenotype. Sox9 is a key transcription factor for chondrogenesis, which directly regulates the expression of Acan and COL-II and maintains the chondrocyte phenotype. Figure 5 At 7 and 14 days of induction in C, Sox9 expression was significantly higher in the glucosamine-grafted group than in the PET group. However, Sox9 expression was slightly lower on day 14 compared to day 7, likely due to chondrocytes gradually entering the ECM synthesis phase. COL-I is primarily present in fibrocartilage and bone tissue. Its expression is upregulated during cartilage degeneration or fibrosis. Although fibrous cartilage can fill defects, its mechanical and biological properties are far inferior to those of hyaline cartilage. Figure 5 The expression of COL-I in the PET group was higher in D, while the expression levels of COL-I in the PET-GS0.05 group and the PET-GS0.1 group were significantly lower than that in the PET group, indicating that glucosamine can help reduce the tendency of cartilage fibrosis and maintain the hyaline cartilage phenotype by inhibiting the overexpression of COL-I.

[0087] The cells were plated at 1×10 5 Cells were seeded onto 6-well plates at a density of 10 cells / well and incubated in an incubator for 24 hours. After the cells adhered, the complete medium in the plate was aspirated, the cells were gently rinsed with PBS, and the osteogenic induction extracts of PET and PET-HA were added. After addition, the plate was placed in an incubator for osteogenic differentiation, with fresh sample osteogenic induction extracts replaced every two days. After 7 and 14 days of osteogenic differentiation, the expression of osteogenesis-related genes was detected by RT-qPCR. The detected genes included alkaline phosphatase (ALP), type I collagen (COL-I), chondrogenesis-related transcription factor 2 (Runx2), and osteopontin (OPN). The primer sequences are shown in Table 3.

[0088] Table 3 Primer sequences of genes related to osteogenic differentiation

[0089]

[0090] Figure 6Figure 2. Relative expression levels of genes involved in the osteogenic differentiation of mBMSCs induced by PET-HA at different mineralization times in osteogenic induction medium. (A) Osteopontin (OPN); (B) Runx2; (C) Alkaline phosphatase (ALP); (D) Collagen type I (COL-I). *p < 0.05, **p < 0.01, ***p < 0.001. Figure 6 Runx2 in A is a key transcription factor for osteogenic differentiation. It works synergistically with downstream factors such as Osx to directly regulate the differentiation of stem cells into osteoblasts. At 7 and 14 days of induction, the expression of Runx2 in the mineralization group was significantly higher than that in the PET group, indicating that the HA coating effectively activated the cell osteogenic transcriptional regulatory pathway. Figure 6 ALP in B is a marker gene for early osteogenic differentiation. The ALP expression level in the PET-HA group was significantly upregulated compared with the PET group at 7 and 14 days of culture, which once again proved that HA coating can effectively promote early osteogenic differentiation of cells. Figure 6 OPN in C is a non-collagenous bone matrix protein. It is a gene expressed in the middle and late stages of osteoblast differentiation. It can regulate the mineralization rate of bone matrix and prevent disordered deposition of calcium salts. At 14 days of induction, OPN was significantly upregulated in the PET-HA group, indicating that mineralization treatment can effectively promote the expression and deposition of bone matrix proteins. COL-I is the main organic component of synthetic bone matrix, providing a structural platform for the adhesion, extension and arrangement of osteoblasts. Figure 6 The expression levels of COL-I in the PET-HA7 and PET-HA14 groups in D were significantly higher than that in the PET group, indicating that the HA coating promoted the synthesis and secretion of bone matrix proteins.

Claims

1. A segmented functionalized coating artificial ligament, characterized in that: The segmented functionalized coating artificial ligament comprises a glucosamine-loaded joint cavity segment PET artificial ligament coating and a hydroxyapatite-mineralized bone tunnel segment PET artificial ligament coating.

2. The method for preparing the segmented functionalized coating artificial ligament according to claim 1, characterized in that: The preparation process includes artificial ligament surface modification process, artificial ligament glucosamine loading process, and artificial ligament mineralization process. The specific steps are as follows: (1) Artificial ligament surface modification process Step 1: ultrasonically treat the PET artificial ligament substrate in anhydrous ethanol, wash it with deionized water until the pH of the washed water is neutral, and place it in a vacuum drying oven until it is completely dry; Step 2: placing the PET artificial ligament treated in step 1 in a phosphoric acid solution, stirring to allow the phosphoric acid solution to fully infiltrate the PET artificial ligament, washing with deionized water until the pH of the washed water is neutral, and placing in a vacuum drying oven until completely dried to obtain a phosphate-modified PET artificial ligament; Step 3, placing the phosphate-modified PET artificial ligament obtained in Step 2 in a NaOH solution, reacting, washing with deionized water until the pH of the washed water is neutral, and placing in a vacuum drying oven until completely dried to obtain a surface-modified PET artificial ligament; (2) Artificial ligament loading glucosamine process Step 4, dissolving glucosamine in deionized water and ultrasonically treating the water to obtain a glucosamine solution; Step 5, placing the surface-modified articular cavity segment of the PET artificial ligament obtained in Step 3 in MES buffer and reacting until the pH value on the surface of the PET artificial ligament is less than 7; Step 6, adding EDC / NHS coupling agent to the reaction container in step 5 to react and activate the carboxyl groups on the surface of the PET artificial ligament; Step 7, adding DMAP to the reaction vessel of step 6, and then adding the glucosamine solution of step 4, and reacting; Step 8, washing the PET artificial ligament after the reaction in Step 7 with deionized water until the pH of the washed water is neutral, and freeze-drying it in a freeze dryer to obtain a PET artificial ligament loaded with glucosamine; (3) Artificial ligament mineralization process Step 9, placing the surface-modified bone tunnel segment of the PET artificial ligament obtained in Step 3 in MES buffer and reacting until the pH on the surface of the PET artificial ligament is less than 7; Step 10, adding EDC / NHS coupling agent to the reaction container in step 9 to react and activate the carboxyl groups on the surface of the PET artificial ligament; Step 11, washing the PET artificial ligament after carboxyl group activation in step 10 with deionized water until the pH of the washed water is neutral, washing with phosphate buffer until the pH of the surface of the PET artificial ligament is greater than 7, and then placing it in simulated body fluid and mineralizing it on a shaker, and replacing it with fresh simulated body fluid every other day; Step 12: Wash the PET artificial ligament obtained in step 11 with deionized water until the pH of the washed water is neutral, and place it in a vacuum drying oven until it is completely dried to obtain a mineralized PET artificial ligament.

3. The preparation method according to claim 2, wherein In step 2, the concentration of the phosphoric acid solution is 1 to 2.5 M, the reaction temperature is controlled at 25 to 37° C., and the reaction time is 1 to 3 hours.

4. The preparation method according to claim 2, wherein In step 3, the concentration of the NaOH solution is 2.5M to 5M, the reaction temperature is controlled at 25 to 37° C., and the reaction time is 1 to 3 hours.

5. The preparation method according to claim 2, wherein In step 6 and step 10, the molar ratio of EDC to NHS is 5:1 to 2:1, the reaction temperature is controlled at 25 to 37° C., and the reaction time is 12 to 24 hours.

6. The preparation method according to claim 2, wherein In step 7, the concentration of DMAP is 0.1M to 0.5M, the reaction temperature is controlled at 25 to 37° C., and the reaction time is 12 to 24 hours.

7. The preparation method according to claim 2, wherein In step 7, the concentration of the glucosamine solution is 0.01 to 2 mM, the reaction temperature is controlled at 25 to 37° C., and the reaction time is 1 to 24 h.

8. The preparation method according to claim 2, wherein In step 11, the components of the simulated body fluid are NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, HCl, CaCl2·2H2O, Na2SO4, and Tris base, and the concentration is 1.2 to 1.5 times that of SBF.

9. The preparation method according to claim 2, wherein In step 11, the modified PET artificial ligament is mineralized in the simulated body fluid for 7 to 21 days, the temperature is 36 to 38° C., and the shaking speed is 60 to 120 rpm.

10. Use of the segmented functionalized coating artificial ligament according to claim 1 or the segmented functionalized coating artificial ligament obtained by the preparation method according to any one of claims 2 to 9 in the repair, reconstruction and soft tissue replacement of anterior cruciate ligament injuries.

Images