A method for the preparation of a bioactive artificial ligament

Abstract

The application discloses a preparation method of a bioactive artificial ligament, and the bioactive artificial ligament is prepared from collagen aggregates extracted from animal tissues as raw materials, and collagen filaments are formed after electrospinning treatment; the collagen filaments are subjected to heat setting treatment under the conditions of pre-treatment, directional stretching and radial rotation; and collagen fibers are obtained; the artificial ligament is prepared from the collagen fibers after further weaving; the bioactive artificial ligament has a fibrous microstructure, can promote the migration, differentiation and proliferation of bone marrow stem cells, fibroblasts and ligament cells, has excellent biocompatibility, and has great application value since the mechanical properties of the product are far superior to those of other products in the prior art.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and more particularly to a method for preparing a bioactive artificial ligament for the treatment of ligament or tendon injuries. Background Technology

[0002] The main components of ligaments are collagen fibers and elastic fibers. Collagen fibers give ligaments strength and stiffness, while elastic fibers endow them with the ability to stretch under load. Most ligament fibers are arranged in parallel, so their function is relatively specialized, often bearing loads in only one direction. Because ligaments are very tough, they enhance bone stability, fix them in their normal position, and limit their range of motion. Major ligaments in the human body include the cruciate ligaments of the knee joint, the inguinal ligament, the coracoacromial ligament, and the radial and ulnar collateral ligaments of the elbow joint.

[0003] Ligaments can be injured by force. Mild injuries include ligament sprains, caused by overstretching or partial fiber rupture, presenting as pain, tenderness, varying degrees of swelling, and limited range of motion. Severe injuries involve ligament ruptures, sometimes accompanied by joint sprains, avulsion fractures, or joint dislocations, presenting as pain, significant swelling, bruising, joint effusion or hematoma, and significantly limited range of motion. X-rays show uneven joint cavity width. The knee joint is one of the most easily injured joints in the human body. Multiple ligament injuries of the knee generally refer to damage to two or more of the four main ligaments of the knee joint, leading to severe knee instability and functional impairment. These injuries are often accompanied by vascular and nerve damage, making clinical treatment relatively complex, increasing the likelihood of cartilage damage, and also increasing the risk of knee osteoarthritis. Severe ligament injuries, such as complete ruptures, rarely heal on their own and generally require surgical treatment, using biomaterials for ligament reconstruction.

[0004] Ligament repair and reconstruction mainly involves the use of materials, and there are currently three methods. The most common method is autologous tendon, but this method has inherent drawbacks; it's essentially "robbing Peter to pay Paul," which can cause damage to the tendon harvesting site. The second method is allogeneic tendon, which, due to its ancestral origin, is currently extremely scarce. The third method is artificial ligaments, including those made of synthetic and biological materials. Currently used PET polyester synthetic fiber artificial ligaments cannot fuse with bone and can only rely on mechanical fixation, leading to a rising failure rate year by year.

[0005] The latest ligament treatment concept is called "super-strong reconstruction," which means that after ligament repair, the new ligament is stronger and more stable than the original one. Achieving this goal requires advancements in materials. High-strength, bioactive artificial ligaments that can induce ligament regeneration have elasticity, toughness, and rigidity similar to human ligaments. They can effectively distribute the body's weight, and their microstructure is identical to human tissue, supporting cell growth and being absorbed by the body without rejection. Ultimately, they form entirely new autologous ligaments, achieving the most ideal tissue reconstruction.

[0006] Patent CN201410324893.5 discloses an antigen-free collagen aggregate and its preparation method. It mainly mentions using animal tissue as raw material, which is subjected to degreasing, repeated ultrasonic low-temperature washing, acid inactivation, centrifugation washing, and sterilization to obtain an aggregate containing collagen fibers or collagen fiber bundles. It has certain mechanical properties. The product in this document is ultimately used as a biomedical material, which needs to have certain mechanical properties. However, when used for the repair and treatment of ligament or tendon injuries, the collagen fiber material produced by this method does not meet the high mechanical properties required. Therefore, it is necessary to further develop bio-collagen fiber materials with high mechanical properties. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to provide a method for preparing a bioactive artificial ligament for the treatment of ligament or tendon injuries.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a method for preparing a bioactive artificial ligament, wherein the bioactive artificial ligament is made from collagen aggregates extracted from animal tissue as raw material, which are electrospinned to form collagen filaments, pretreated under heat and moisture conditions, and then subjected to radial rotation while being stretched in a directional manner to complete the heat setting treatment, thereby obtaining collagen fibers; the collagen fibers are further woven to form an artificial ligament.

[0009] In the preparation method of this invention, the processing method of collagen filaments is as follows: 1) Pretreatment: The collagen filaments are treated at a temperature of 35-50℃ and a humidity of RH of 75-99% for 8-48 hours; 2) Oriented rotational stretching: The pretreated collagen filaments are subjected to directional rotational stretching at a temperature of 40-75℃ and a humidity of RH of 75-90%, with a stretching rate of 0.5-2 mm / min and a rotational rate of 0.1-5° / min. 3) Heat setting treatment: After directional rotation and stretching, the temperature is raised to 60-80℃ and maintained for 3-8 hours to obtain collagen fibers.

[0010] The temperature for directional rotational stretching in this invention is 45–65°C; the rotational speed is 0.5–1.5° / min.

[0011] The animal tissues used in this invention are selected from mammals, including cattle, pigs, horses, sheep, donkeys, and deer; the animal tissues are selected from skin, submucosal basal tissue of the intestinal tract, blood vessels, bladder, tendons, and ligaments. Cattle are preferred as the mammals used in this invention, and tendons and ligaments are preferred as the animal tissues used.

[0012] In the process of weaving collagen fibers according to the present invention, other materials are incorporated, including natural polymer materials, synthetic polymer materials, and medical metal materials. Natural polymer materials are biological polysaccharides and structural protein materials, including one or more of chitosan, cellulose and its derivatives, starch and its derivatives, sericin, and fibroin.

[0013] The synthetic polymer materials of this invention include biodegradable polymer materials and non-biodegradable polymer materials; the biodegradable polymer materials include one or more of polylactic acid, polyglycolic acid, polylactic-co-hydroxyacetic acid copolymer, polycaprolactone, polyhydroxyalkanoates, polybutylene succinate, polydioxanone, polytrimethylene carbonate, polyanhydride, polyglutamic acid, polylysine, and polyphosphazene; the non-biodegradable polymer materials include one or more of polyethylene terephthalate, polypropylene, polyurethane, polyethylene, ultra-high molecular weight polyethylene, polytetrafluoroethylene, polyetheretherketone, and polymethacrylates such as polymethyl methacrylate, polyethyl methacrylate, polypropyl methacrylate, and polybutyl methacrylate.

[0014] The medical metal materials of the present invention include one or more of the following: pure titanium, nickel-titanium alloy, Ti-6Al-4V titanium alloy, Ti-6Al-7Nb titanium alloy, β titanium alloy, 316L stainless steel, high-nitrogen stainless steel, cobalt-chromium-molybdenum alloy, and cobalt-chromium-tungsten-nickel alloy.

[0015] The woven artificial ligament of the present invention undergoes a cross-linking operation, and the cross-linking agent is an ethanol solution of 2-5% glutaraldehyde, polyglycidyl ether and carbodiimide.

[0016] The woven artificial ligaments of the present invention are dyed using dyes including melanin, gentian violet, crystal violet, D&C violet, bright green, D&C green, trypan blue, D&C blue, acridine orange, proflavin, and alizarin yellow.

[0017] The woven artificial ligaments of the present invention are subjected to chemical, irradiation or supercritical carbon dioxide sterilization, and then packaged in an airtight sterile barrier for storage.

[0018] The advantages of this invention are as follows: Using collagen, which is identical in composition to human ligaments, and through specific rotational stretching and strengthening treatments, the mechanical strength of the bioactive artificial ligament is significantly improved, meeting the ligament repair requirements of different parts of the body. After implantation, its rotating collagen fiber structure guides the migration, colonization, and differentiation of ligament cells and fibroblasts, secreting extracellular matrix. Simultaneously, the implanted collagen fibers degrade and are absorbed, ultimately growing into entirely new autologous ligaments, truly achieving biological reconstruction of ligaments.

[0019] This invention relates to a bioactive artificial ligament, a cell-free tissue-engineered scaffold derived from animal tissue. Through extraction, spinning, and targeted reinforcement, it exhibits excellent biocompatibility and biomechanical properties, providing an ideal guiding scaffold for cell regeneration and ligament regeneration. Human fibroblasts and ligament cells are recolonized within the fibrous structure of the bioactive artificial ligament. After in vivo remodeling, it fully integrates into the patient's own tissue, becoming an active component of the body. This achieves functional repair identical to the original ligament tissue, resolving biocompatibility and long-term complications associated with currently used allogeneic transplants (such as immunogenic reactions or potential disease transmission risks), autologous transplants (secondary trauma), and synthetic materials. Furthermore, the material source is unrestricted, meeting diverse clinical needs for ligament repair and enabling ligament and tendon regeneration in various parts of the body. It is the optimal alternative to autologous grafts, allogeneic grafts, and synthetic materials.

[0020] The bioactive artificial ligament produced by this invention has the following overall mechanical properties: tensile strength ≥1730N; stiffness 150~220 N / mm; and elongation at break 10~30%. From the perspective of mechanical properties, it far surpasses other products in the prior art, demonstrating excellent performance and significant application value. Attached Figure Description

[0021] Figure 1 The collagen fibers after reinforcement treatment obtained in Example 3; Figure 2 This is a cross-sectional view of the woven artificial ligament in Example 4; Figure 3 The bioactive artificial ligament sample after tensile reinforcement obtained in Example 7; Figure 4 The mixed silk fibroin / collagen fibers obtained in Example 13; Figure 5 The cross-section of the mixed silk fibroin / collagen fiber obtained in Example 13; Figure 6 The polylactic acid composite collagen fiber in Example 14; Figure 7 The polyethylene terephthalate composite collagen fiber in Example 15; Figure 8 The cross-section of the composite collagen fiber with nickel-titanium shape memory alloy wire as the core in Example 16 is shown. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0023] Example 1: Fresh Achilles tendons from 24-48 month old cattle were processed in a laminar flow hood, where muscle and fat were removed, and bone fragments, hair, and other impurities were removed. The tendons were then immersed in a 0.1% peracetic acid (PAA) solution for 30 minutes, followed by immersion in acetone solution, preferably 99% (v / v). The tendons were submerged and stirred for 3-5 hours. After immersion, the tendons were rinsed with pure water and then added to a sufficient volume of ultrapure water at a weight ratio of 1:10. The tendons were stored at 4°C for 12-24 hours. The process was repeated three times, with fresh ultrapure water added. The tendons were then removed and stored at -80°C for 24 hours. Immediately after removal, the tendons were immersed in a sufficient volume of fresh ultrapure water and stirred slowly for 1 hour. This freeze-thaw cycle was repeated three times. The tendons were then sonicated in ultrapure water for 30 minutes, maintaining a temperature not exceeding 30°C. The tendons were washed twice with sufficient ultrapure water. Finally, the tendons were immersed in a DNase I 10.0 μg / ml EDTA solution and stored overnight at 4°C.

[0024] Example 2: The extracted collagen tissue was repeatedly dehydrated three times with acetone solution at a ratio of 1:10, each time for five hours. After drying under vacuum at 30°C, it was weighed and dissolved in hexafluoroisopropanol solution to a concentration of 3%. After complete dissolution, electrospinning was performed with a positive voltage of 10kV, a negative voltage of -10kV, a flow rate of 0.5mL / h, and a receiving distance of 10cm. The resulting collagen fibers were then fixed at 95% humidity and 60°C.

[0025] Example 3: Collagen filaments were left to stand at 40°C and 95% RH for 24 hours, then subjected to directional stretching at 0.6 mm / min at 61°C and 75% RH, while simultaneously undergoing radial rotation at a rate of 1.2° / min. After rotational stretching, the temperature was raised to 65°C and set for 8 hours to obtain reinforced collagen fibers; specific results are as follows. Figure 1 As shown.

[0026] Example 4: The shaped collagen fibers or mixed fibers were bundled into bundles of 5 filaments each, and 3 bundles were twisted together into a single strand. Using the mixed collagen fiber as the core, 4 strands of collagen fiber were then twisted into a thread. The collagen fiber thread was then braided into artificial ligaments of different sizes according to the required dimensions. The results are as follows... Figure 2 As shown.

[0027] Example 5: The two ends of the above-mentioned artificial ligament were fixed on stainless steel clamps to keep it straight, cross-linked in 2% glutaraldehyde ethanol solution for 6 hours, cleaned with alcohol 3 times, vacuum dried and packaged in paper-plastic bags, and further sealed in aluminum foil bags, and sterilized by irradiation with a dose of 25 kGy.

[0028] Example 6: The mechanical properties and related technical indicators of the artificial ligaments obtained in the above examples are as follows: Appearance: White fibrous, free of impurities visible to the naked eye; Fiber length: 10-50cm; Tensile strength: ≥1730N; Stiffness: 150~180 N / mm; Elongation at break: 20-30%; Moisture content: ≤5% (wt); Heavy metal content: ≤10μg / g; Hydroxyproline content: not less than 10% (w / w) of total protein content; Cytotoxicity: Cytotoxic reaction not greater than grade 1; Sensitization test: No delayed hypersensitivity reaction; Intradermal reaction test: Primary irritation index (PII) < 0.4 Endotoxin content: ≤20 EU Sterility test: Sterile.

[0029] Example 7: The collagen filaments involved in this invention were subjected to directional stretching at a stretching rate of 0.6 mm / min under conditions of 40°C, RH 95%, and a standing temperature of 40°C and RH 95% for 24 hours, while simultaneously undergoing radial rotation at a rotation rate of 1.2° / min. After rotational stretching, the temperature was raised to 65°C and set for 8 hours to obtain reinforced collagen fibers; the results are as follows. Figure 3 As shown.

[0030] Comparative Example 1: The collagen filaments involved in this invention require a pretreatment step before reinforcement treatment. When the pretreatment is insufficient, some fibers may break during the rotation and stretching process.

[0031] With other reaction conditions remaining unchanged, a comparative experiment was conducted using the reaction conditions in Example 3: 1) When the pretreatment humidity is below 60% and the pretreatment time is less than 7 hours, the proportion of broken collagen fibers is 3%. When the treatment temperature and humidity are further reduced and the pretreatment time is further shortened, the proportion of broken collagen fibers increases, which seriously affects the tensile strength of the bioactive artificial ligament.

[0032] 2) When the pretreatment temperature is 25℃ and the humidity is 50% (RH), the tensile strength of the prepared product decreases to 450N after 4 hours of treatment, which does not meet the requirements for clinical use.

[0033] Comparative Example 2: The collagen filaments involved in this invention, after reinforcement treatment, can promote the directional alignment of collagen molecules after spinning, increasing the hydrogen bond check and van der Waals forces between collagen molecular chains. This stretching treatment needs to be carried out under specific temperature, humidity, and stretching rate conditions. When the temperature and relative humidity are too low, or the stretching rate is too fast, collagen fiber breakage is likely to occur.

[0034] With other reaction conditions remaining unchanged, a comparative experiment was conducted using the reaction conditions in Example 3: 1) Under conditions of 40℃ temperature and RH < 70%, and with a stretching rate of 1.0 mm / min, the proportion of collagen fiber breakage reached 5%. As the temperature and humidity further decreased, and the stretching rate further increased, the proportion of collagen fiber breakage increased. 2) Under the conditions of stretching at 35℃ and relative humidity RH 50%, with a stretching rate of 1.5mm / min, the proportion of collagen fiber breakage reached 8%, and the tensile strength of the prepared product was only 700N, which could not meet the requirements for clinical use.

[0035] Comparative Example 3: Studies have shown that applying radial rotation simultaneously during the stretching process can significantly improve the reinforcing effect of collagen filaments.

[0036] With other reaction conditions remaining unchanged, a comparative experiment was conducted using the reaction conditions in Example 3: 1) The product was subjected to directional stretching at a stretching rate of 0.6 mm / min after standing for 24 hours at 40℃ and RH 95% for 60℃ and RH 75%. The tensile strength of the resulting product reached 980 N.

[0037] 2) Using the same directional stretching conditions, a further radial rotation of 1.2° / min was applied, resulting in a tensile strength of 1800N. When the rotation speed was increased to 3° / min, the tensile strength decreased to 1500N.

[0038] Comparative Example 4: Studies have shown that the direction of radial rotation does not affect the reinforcement effect; relative rotation to the left or right can enhance tensile strength and stiffness.

[0039] With other reaction conditions remaining unchanged, a comparative experiment was conducted using the reaction conditions in Example 3: 1) Using the same tensile conditions, a radial rotation of 1.3° / min is applied to the left, and the tensile strength of the resulting product reaches 1780N.

[0040] 2) Using the same tensile conditions, a radial rotation of 1.3° / min is applied to the right, and the tensile strength of the resulting product reaches 1810N.

[0041] Example 12: The tensile strength of the enhanced bioactive artificial ligament from Example 7 was tested according to YY / T096—2025 "Specific Requirements for Artificial Ligaments in Sports Medicine Implant Devices".

[0042] The results showed that, under the same experimental conditions, the mechanical properties of the bioactive artificial ligaments were significantly enhanced after stretching and shaping treatment compared to the untreated samples. The data are as follows:

[0043] Example 13: The above-mentioned collagen fibers were uniformly mixed with natural biomaterial silk fibroin fibers in a 5:1 ratio, and then bundled into bundles of 6 fibers each. Three bundles were twisted together, and then four strands of collagen fibers were twisted into a thread, with the mixed collagen fibers as the core. The collagen fiber threads were then braided into artificial ligaments of different sizes according to the required dimensions. Specific results are as follows... Figure 4 and 5 As shown. The mechanical properties of the obtained composite artificial ligament are as follows: tensile strength: 4500 N; stiffness: 230 N / mm; elongation at break: 15%.

[0044] Example 14: Under unchanged conditions, biodegradable polylactic acid (PLA) with a molecular weight (Mw) of 200,000-800,000 was purified by dissolution-precipitation and dried. It was then added at a ratio of 0.1-10% to the collagen solution obtained in Example 2 above. After complete dissolution, electrostatic blending was performed, with a positive pressure of 25 kV, a negative pressure of -10 kV, a flow rate of 0.4 mL / h, and a receiving distance of 50 cm. The resulting mixed collagen fibers were then set at 90% humidity and 75°C. The set composite fibers were then woven into composite artificial ligaments of different sizes. The results are as follows... Figure 6 As shown. The mechanical properties of the obtained composite artificial ligament are as follows: tensile strength: 2300 N; stiffness: 170 N / mm; elongation at break: 22%.

[0045] Example 15: Under otherwise unchanged conditions, polyethylene terephthalate (PET) with a molecular weight (Mw) of 70,000 to 130,000 was dissolved in hexafluoroisopropanol solution and then mixed with the above collagen solution at a ratio of 1:2. After complete dissolution, electrostatic blending was performed, with a positive voltage of 28 kV, a negative voltage of -12 kV, a flow rate of 0.6 mL / h, and a receiving distance of 40 cm. The resulting mixed collagen fibers were then set at 95% humidity and 80°C. The set composite fibers were then woven into composite artificial ligaments of different sizes; the results are as follows. Figure 7 As shown. The mechanical properties of the obtained composite artificial ligament are as follows: tensile strength: 5100 N; stiffness: 190 N / mm; elongation at break: 28%.

[0046] Example 16: A φ0.1mm nickel-titanium shape memory alloy wire, after heat setting, was used as the core and twisted with the collagen fiber bundles obtained in Example 7 to form threads. These alloy collagen fiber threads were then braided into artificial ligaments of different diameters. The results are as follows: Figure 8 As shown. The mechanical properties of the obtained composite artificial ligament are as follows: tensile strength: 6000 N; stiffness: 250 N / mm; elongation at break: 12%.

[0047] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any combination or equivalent transformation made based on the above embodiments shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing a bioactive artificial ligament, characterized in that, The bioactive artificial ligament uses collagen aggregates extracted from animal tissue as raw material. After electrospinning, collagen filaments are formed. After pretreatment under heat and moisture conditions, the filaments are directionally stretched and radially rotated to complete the heat setting process, resulting in collagen fibers. The collagen fibers are further woven to form artificial ligaments.

2. The preparation method according to claim 1, characterized in that, The preparation method described above includes the following treatment of the collagen filaments: 1) Pretreatment: The collagen filaments are treated at a temperature of 35-50℃ and a humidity of RH of 75-99% for 8-48 hours; 2) Oriented rotational stretching: The pretreated collagen filaments are subjected to directional rotational stretching at a temperature of 40-75℃ and a humidity of RH of 75-90%, with a stretching rate of 0.5-2 mm / min and a rotational rate of 0.1-5° / min. 3) Heat setting treatment: After directional rotation and stretching, the temperature is raised to 60-80℃ and maintained for 3-8 hours to obtain collagen fibers.

3. The preparation method according to claim 2, characterized in that, The temperature of the directional rotational stretching is 45–65°C; the rotational speed is 0.5–1.5° / min.

4. The preparation method according to claim 1 or 2, characterized in that, The animal tissues are selected from mammals, including cattle, pigs, horses, sheep, donkeys and deer; the animal tissues are selected from skin, submucosal basal tissue of the intestinal tract, blood vessels, bladder, tendons and ligaments.

5. The preparation method according to claim 1 or 2, characterized in that, Other materials are incorporated into the collagen fiber weaving process, including natural polymer materials, synthetic polymer materials, and medical metal materials.

6. The preparation method according to claim 5, characterized in that, The natural polymeric materials mentioned are biological polysaccharides and structural protein materials, including one or more of chitosan, cellulose and its derivatives, starch and its derivatives, sericin and fibroin.

7. The preparation method according to claim 6, characterized in that, The synthetic polymer materials include biodegradable polymer materials and non-biodegradable polymer materials; Biodegradable polymer materials include one or more of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, polyhydroxy fatty acid ester, polybutylene succinate, polydioxanone, polytrimethylene carbonate, polyanhydride, polyglutamic acid, polylysine, and polyphosphazene. Non-degradable polymeric materials include one or more of polyethylene terephthalate, polypropylene, polyurethane, polyethylene, ultra-high molecular weight polyethylene, polytetrafluoroethylene, polyetheretherketone, and polymethacrylates such as polymethyl methacrylate, polyethyl methacrylate, polypropyl methacrylate, and polybutyl methacrylate.

8. The preparation method according to claim 5, characterized in that, The medical metal materials mentioned include one or more of the following: pure titanium, nickel-titanium alloy, Ti-6Al-4V titanium alloy, Ti-6Al-7Nb titanium alloy, β titanium alloy, 316L stainless steel, high-nitrogen stainless steel, cobalt-chromium-molybdenum alloy, and cobalt-chromium-tungsten-nickel alloy.

9. The preparation method according to claim 1 or 2, characterized in that, The artificial ligament is cross-linked using a cross-linking agent consisting of a 2-5% glutaraldehyde, polyglycidyl ether, and carbodiimide ethanol solution.

10. The preparation method according to claim 1 or 2, characterized in that, The artificial ligament is stained with dyes including melanin, gentian violet, crystal violet, D&C violet, bright green, D&C green, trypan blue, D&C blue, acridine orange, proflavin, and alizarin yellow.

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