Preparation method and application of anterior cruciate ligament bridging material

Abstract

The invention provides a preparation method and application of an anterior cruciate ligament bridging material. The preparation method comprises the following steps: treating a bovine-derived collagen raw material, and carrying out pure water extraction treatment after flaking the bovine-derived collagen raw material; treating the thin sheet through an acid solution and an alkaline solution; then collagen fibers are dried and then added into an acid solution to be treated into collagen fiber slurry, then an alkaline solution is added to enable the collagen fibers to be separated out, and the separated-out collagen fibers have flowability and are convenient for further finishing; further finishing the collagenous fiber into a directional / non-directional shape, dehydrating and freeze-drying to obtain an uncrosslinked collagenous fiber scaffold; and finally carrying out high-temperature physical crosslinking. The bridging material is applied to an anterior cruciate ligament repair operation material. According to the method, a good ACL repairing effect can be achieved, and compared with a conventional repairing method, the postoperative exercise ability better than that of a conventional repairing method is achieved; and the material is selected from animal collagen substances, so that the material has relatively good biocompatibility, and tissue repair is promoted to a greater extent.

Description

Technical Field

[0001] The invention relates to the field of medical consumables, and in particular to a preparation method and application of an anterior cruciate ligament bridging material. Background Art

[0002] Anterior cruciate ligament (ACL) injury is the most common knee injury and its healing requires surgery.

[0003] Conventional treatment methods are mainly autologous tendon transplantation and reconstruction and artificial ligament transplantation and replacement: Problems with autologous tendon transplantation and reconstruction: 1. The problem of ligament maturity of the transplanted tendon; 2. Problems related to the donor site, such as muscle weakness and limited availability of grafts; Problems with artificial ligament transplantation and replacement: higher incidence of osteoarthritis, higher re-rupture rate, and problems such as ligament formation process depression and knee joint laxity and hyperplasia, which affect the widespread application of such grafts in clinical practice.

[0004] There is a new anterior cruciate ligament repair method, BEAR (Bridge Enhanced ACL Repair), which is different from traditional ACL reconstruction. It is a less invasive surgery; it can restore the natural anatomy and function of the knee joint. Studies have reported that patients are more satisfied with pain, symptoms and preparation. This technology does not require a second wound site to remove the healthy tendon and does not involve a donor (allogeneic reconstruction).

[0005] The specific method of enhanced ACL repair is to fill and fix a "bridging" material between the torn ends of the patient's ACL. The material is filled with autologous blood to form a clot. The blood clot can serve as a natural scaffold for medial collateral ligament repair, thereby promoting ligament repair.

[0006] The "bridging" material currently used in BEAR surgery is a collagen-based scaffold material, which mainly has problems such as rapid degradation in the body, insufficient support for tissues, and substandard mechanical properties. While synthetic materials solve these problems, they are accompanied by defects such as poor biocompatibility and lack of biological activity. Appropriate functionalization and modification of collagen-based materials can often achieve improvements in material performance. Summary of the invention

[0007] The purpose of the present invention is to provide a preparation method and application of an anterior cruciate ligament bridging material.

[0008] The present invention is achieved through the following technical solution: a method for preparing anterior cruciate ligament bridging material, comprising the following steps:

[0009] Step 1: Remove the surface fascia and fat from bovine collagen material;

[0010] Step 2, cutting the bovine collagen material processed in step 1 into thin slices, and subjecting the slices to pure water extraction;

[0011] Step 3, treating the slices obtained in step 2 with an acidic solution and an alkaline solution; here, the bovine Achilles tendon is extracted with an acidic aqueous solution to purify the collagen fibers to a certain extent and disperse them in the aqueous solution to a certain extent, and the alkaline aqueous solution is used to preliminarily inactivate the virus;

[0012] Step 4, drying the collagen fibers and adding them into an acidic solution to form a collagen fiber slurry, and then adding an alkaline solution to precipitate the collagen fibers, wherein the precipitated collagen fibers have fluidity and are convenient for further finishing;

[0013] Step 5, manually or by equipment, arranging the collagen fibers into oriented / non-oriented shapes and removing a certain amount of water; freeze-drying the dehydrated collagen fibers to obtain uncross-linked collagen fiber scaffolds;

[0014] Step 6, subjecting the uncrosslinked collagen fiber scaffold obtained in step 5 to high-temperature physical crosslinking.

[0015] The bridging material prepared by the preparation method is used in anterior cruciate ligament repair surgery materials.

[0016] Compared with the previous technology, the beneficial effects of the present invention are:

[0017] 1. The present invention can achieve a good repair effect on ACL and bring better postoperative exercise ability than conventional repair methods. This is because the present invention can cooperate with the patient's own PRP when used, and release and activate multiple growth factors, promote the growth and proliferation of fibroblasts and the production of collagen, and further accelerate the production of blood vessels and neuron bodies to achieve the repair of ruptured ACL.

[0018] 2. The present invention designs an appropriate collagen solid content and forms micropores inside the sample through freeze-drying, which brings good water absorption performance and a faster water absorption rate, ensures the penetration of PRP inside the material and the smooth exchange between the material itself and the patient's body fluids, helps the exchange of nutrients and thus accelerates tissue reconstruction; the present invention adopts a physical cross-linking method, and the material itself is taken from animal collagen, has good biocompatibility, and promotes tissue repair to a greater extent.

[0019] 3. The present invention imparts good mechanical properties to the material through freeze drying and vacuum high temperature cross-linking, ensuring that the material maintains a good shape and is not destroyed during surgery, and maintains a good shape when used with surgical sutures and syringes. Secondly, after implantation in the body, the material is required to have good mechanical retention until it is completely degraded.

[0020] 4. The present invention adjusts the parameters of physical cross-linking to design a degradation rate that matches ACL repair, and completely degrades within 8 weeks, thus safeguarding tissue repair.

[0021] 5. If necessary, the invented product may be designed to have a directional effect, which can further accelerate the repair and reconstruction of tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of collagen fiber scaffold;

[0023] Figure 2 This is a scanning electron microscope image of the cut surface of the collagen fiber scaffold;

[0024] Figure 3 Schematic diagram of surgical sutures passing through the collagen fiber scaffold;

[0025] Figure 4 Thermal stability of collagen fiber scaffold. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with the accompanying drawings:

[0027] The collagen repair material of the present invention is taken from bovine collagen material. The present invention specifically uses the Achilles tendon of the main rod of the hind hoof of a bovine. After removing the surface fascia and fat, the slices are cut into thin slices of 0.5 to 1 mm manually or by a slicing device. The slices are immersed in pure water to remove impurities. After the pure water is poured out, an acidic substance aqueous solution is added for extraction to dissociate the collagen fibers in the bovine Achilles tendon. After the acid solution is poured out, it is washed and an alkaline substance aqueous solution is added for preliminary virus inactivation. The alkaline solution is poured out and washed. The washed bovine Achilles tendon is placed in an oven for drying. The dried bovine Achilles tendon is added with an acid aqueous solution and processed into a collagen fiber slurry by a homogenizer. An alkaline aqueous solution is dripped into the slurry to realize the precipitation of collagen fibers. A certain amount of water is removed from the precipitated collagen fibers, and the collagen fibers are arranged into a directional / non-directional form. The arranged collagen fibers are placed in a mold and enter a freeze dryer for freeze drying. The dried product is physically cross-linked by high-temperature equipment such as a vacuum drying oven to prepare a collagen fiber scaffold.

[0028] The specific preparation method and parameters are as follows: A method for preparing anterior cruciate ligament bridging material, characterized in that: comprising the following steps

[0029] Step 1: Remove the surface fascia and fat from bovine collagen material;

[0030] Step 2, cutting the bovine collagen material processed in step 1 into thin slices, and subjecting the slices to pure water extraction;

[0031] Step 3, treating the slices in step 2 with an acidic solution and an alkaline solution;

[0032] Step 4, drying the collagen fibers and adding them into an acidic solution to prepare a collagen fiber slurry, and then adding an alkaline solution to precipitate the collagen fibers;

[0033] Step 5, manually or by equipment, arranging the collagen fibers into oriented / non-oriented shapes, removing water, and freeze-drying the dehydrated collagen fibers to obtain uncross-linked collagen fiber scaffolds;

[0034] Step 6, subjecting the uncrosslinked collagen fiber scaffold obtained in step 5 to high-temperature physical crosslinking.

[0035] Wherein, the thickness of the slice in step 2 is 0.5-1 mm, and the time of pure water extraction treatment is 15-30 hours.

[0036] The specific process of treating the slice with an acid solution and an alkaline solution in step 3 is as follows:

[0037] Process 1, soaking the slice treated in step 3 in an acid solution for 15-30 hours, and then washing with pure water or saline solution to remove the acid;

[0038] Process 2, soaking the slices treated in process 1 in an alkaline solution for 15-30 hours, and then washing and removing the alkali with pure water or saline solution; the saline solution is sodium sulfate solution or sodium chloride solution.

[0039] The pH range of the acidic solution in process 1 is 0-2, and the acidic solution is one of a hydrochloric acid solution and a sulfuric acid solution; the brine solution is one of a sodium sulfate solution and a sodium chloride solution or a mixture thereof;

[0040] In process 2, the pH value of the alkaline solution is 12-14, the alkaline solution is a mixed solution of sodium hydroxide and salt, the salt is sodium sulfate or sodium chloride; the saline solution is one of sodium sulfate solution and sodium chloride solution or a mixture thereof.

[0041] Wherein, in step 4, the collagen fibers are dried and added into an acidic solution, and placed into a homogenizer for processing to form a collagen fiber slurry; the speed of the homogenizer is 2500rpm to 6000rpm, and the homogenization time is 5 to 15 minutes.

[0042] In step 4, the pH of the acidic solution used in preparing the collagen fiber slurry is 2-4, and the acidic solution is selected from one of hydrochloric acid, sulfuric acid solution, acetic acid solution, and lactic acid solution.

[0043] In step 4, an alkaline solution is added to the collagen fiber slurry to re-precipitate the collagen fibers, wherein the pH of the alkaline solution is 10-12, and the alkaline solution is one of sodium hydroxide solution, potassium hydroxide solution, ammonia solution or copper hydroxide solution, and then the collagen fibers are dehydrated by physical pressing.

[0044] In step 5, the collagen fibers of step 4 are freeze-dried to obtain an uncross-linked collagen fiber scaffold. The parameters of the freeze-drying treatment are: in an environment with a vacuum degree of less than 200 Pa, the temperature is changed in sequence. The temperature and time changes are as follows: -50°C is maintained for 3 hours, then the temperature is adjusted to -30°C and maintained for 3 hours, then the temperature is adjusted to -20°C and maintained for 4 hours, then the temperature is adjusted to -10°C and maintained for 3 hours, then the temperature is adjusted to 0°C and maintained for 3 hours, then the temperature is adjusted to 10°C and maintained for 3 hours, and finally the temperature is adjusted to 25°C and maintained for 3 hours.

[0045] In step 6, the uncrosslinked collagen fiber scaffold obtained in step 5 is subjected to high-temperature physical crosslinking in a vacuum at 100 to 150 degrees. The time of the high-temperature physical crosslinking is 12 to 48 hours, and finally a collagen fiber scaffold is prepared.

[0046] A method for preparing an anterior cruciate ligament bridging material and application of the bridging material prepared in anterior cruciate ligament repair surgery materials.

[0047] Based on the above steps, this application defines a specific production process to obtain an actual product, and the steps of the specific production process are as follows:

[0048] Step 1: Remove the surface fascia and fat from bovine collagen material;

[0049] Step 2, cutting the bovine collagen material processed in step 1 into 0.7-0.8 mm slices, and extracting with pure water for 24 hours;

[0050] Step 3, soaking the slice treated in step 2 in a hydrochloric acid solution with a pH value of 2 for 24 hours, and then washing with pure water for multiple times to remove the acid;

[0051] Step 4, after drying the collagen fiber slices in step 3, mix with a hydrochloric acid solution with a pH value of 2, and place it in a homogenizer at 5000 rpm for 10 minutes to form a collagen fiber slurry; then put the collagen fiber slurry into a sodium hydroxide solution with a pH value of 12 to precipitate the collagen fibers, and finally squeeze and dehydrate.

[0052] It should be noted that the drying process in step 4 is to dry the slices at a temperature of 45° C. for 6 hours to ensure that the dehydration rate is 65%±3%.

[0053] In step 5, the collagen fibers in step 4 are manually or by equipment arranged into a directional / non-directional shape, and a certain amount of water is removed to form a collagen fiber scaffold; the dehydrated collagen fiber scaffold is freeze-dried; the parameters of the freeze-drying process are, in an environment where the vacuum degree is less than 200Pa, the temperature is changed in sequence, and the temperature and time changes are as follows: -50°C is maintained for 3 hours, then the temperature is adjusted to -30°C and maintained for 3 hours, then the temperature is adjusted to -20°C and maintained for 4 hours, then the temperature is adjusted to -10°C and maintained for 3 hours, then the temperature is adjusted to 0°C and maintained for 3 hours, then the temperature is adjusted to 10°C and maintained for 3 hours, and finally the temperature is adjusted to 25°C and maintained for 3 hours.

[0054] In step 6, the uncrosslinked collagen fiber scaffold obtained in step 5 is subjected to high-temperature physical crosslinking in an environment of vacuum degree less than 200 Pa and 130°C for 24 hours to finally prepare a collagen fiber scaffold.

[0055] The specific use method is as follows: the above collagen fiber scaffold is arranged as a bridging material into a cylindrical shape (see Figure 1 ), or any other specifications and shapes. The collagen fibers in the material are designed to be directional or non-directional, and the material has good hydrophilicity. The material preparation process includes acid-base treatment, homogenization, freeze-drying and other process steps. The material has no chemical cross-linking and is sterilized by electron beam irradiation. The total protein content of the material exceeds 80%, the thermal transition temperature is greater than 60°C, and it has good mechanical properties. It can pass through surgical needles and sutures and has good stability in use.

[0056] When in use, it is activated by adding the patient's own platelet-rich plasma (PRP). It is completely absorbed within 8 weeks after implantation into the human body and replaced by fibrovascular repair tissue.

[0057] The product of the present invention can ensure that the medial collateral ligament can form a blood clot at the injured part, and this blood clot can serve as a natural scaffold for the repair of the medial collateral ligament, thereby promoting the repair of the ligament. Specifically, the collagen fiber scaffold of the present invention can be designed to have a directional effect (it is stipulated that more than 50% of the collagen fibers in the same baseline direction under the microscope are in the same direction), and this design can further accelerate the repair and reconstruction of the tissue; because the tissue cells including ligaments and tendons in the human body are arranged and grown in a directional manner, the scaffolds and other materials used in tissue repair will be placed at the injury site to play the role of cell scaffolds, and the cells move with the body fluid and adhere to the fibers for proliferation. More parallel fibers can be conducive to the migration of cells, and the opposite vertical fibers will bring greater migration resistance (guiding cells); the result of fiber orientation will increase the space utilization rate in the scaffold, provide more places for cell proliferation and differentiation, and the fiber network structure is more conducive to the entry and growth of cells. The fiber orientation and multi-layer assembly can realize the appearance of more network structures, providing a favorable place for cell growth and proliferation and tissue reconstruction.

[0058] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing anterior cruciate ligament bridging material, characterized in that: The following steps are included Step 1: Remove the surface fascia and fat from bovine collagen material; Step 2, cutting the bovine collagen material processed in step 1 into thin slices, and subjecting the slices to pure water extraction; Step 3, treating the slices from step 2 with an acidic solution and an alkaline solution; Step 4, drying the collagen fibers and adding them into an acidic solution to prepare a collagen fiber slurry, and then adding an alkaline solution to precipitate the collagen fibers; Step 5, manually or by equipment, arranging the collagen fibers into oriented / non-oriented shapes, removing water, and freeze-drying the dehydrated collagen fibers to obtain uncross-linked collagen fiber scaffolds; Step 6, subjecting the uncrosslinked collagen fiber scaffold obtained in step 5 to high-temperature physical crosslinking.

2. The method for preparing an anterior cruciate ligament bridging material according to claim 1, characterized in that: In step 2, the thickness of the slice is 0.5-1 mm, and the time of pure water extraction treatment is 15-30 hours.

3. The method for preparing an anterior cruciate ligament bridging material according to claim 1, characterized in that: The specific process of treating the slice with an acid solution and an alkaline solution in step 3 is as follows: Process 1, soaking the slice treated in step 3 in an acid solution for 15-30 hours, and then washing with pure water or saline solution to remove the acid; Process 2: soak the slices processed in process 1 in an alkaline solution for 15-30 hours, and then use pure water or saline solution to wash and remove the alkali.

4. The method for preparing anterior cruciate ligament bridging material according to claim 3, characterized in that: The pH range of the acidic solution in process 1 is 0-2, and the acidic solution is one of a hydrochloric acid solution and a sulfuric acid solution; the brine solution is one of a sodium sulfate solution and a sodium chloride solution or a mixture thereof; In process 2, the pH value of the alkaline solution is 12-14, the alkaline solution is a mixed solution of sodium hydroxide and salt, the salt is sodium sulfate or sodium chloride; the saline solution is one of sodium sulfate solution and sodium chloride solution or a mixture thereof.

5. The method for preparing anterior cruciate ligament bridging material according to claim 1, characterized in that: In step 4, the dried collagen fibers are added into an acidic solution and placed in a homogenizer to form a collagen fiber slurry; the speed of the homogenizer is 2500 rpm to 6000 rpm, and the homogenization time is 5 to 15 minutes.

6. The method for preparing anterior cruciate ligament bridging material according to claim 5, characterized in that: In step 4, the pH of the acidic solution used in preparing the collagen fiber slurry is 2-4, and the acidic solution is selected from one of hydrochloric acid, sulfuric acid solution, sulfuric acid solution, and lactic acid solution.

7. The method for preparing anterior cruciate ligament bridging material according to claim 1, characterized in that: In step 4, an alkaline solution is added to the collagen fiber slurry to re-precipitate the collagen fibers, wherein the pH of the alkaline solution is 10-12, and the alkaline solution is one of sodium hydroxide solution, potassium hydroxide solution, ammonia solution or copper hydroxide solution, and then the collagen fibers are dehydrated by physical pressing.

8. The method for preparing anterior cruciate ligament bridging material according to claim 1, characterized in that: In step 5, the collagen fibers of step 4 are freeze-dried to obtain an uncross-linked collagen fiber scaffold. The parameters of the freeze-drying treatment are: in an environment with a vacuum degree of less than 200 Pa, the temperature is changed in sequence. The temperature and time changes are as follows: -50°C is maintained for 3 hours, then the temperature is adjusted to -30°C and maintained for 3 hours, then the temperature is adjusted to -20°C and maintained for 4 hours, then the temperature is adjusted to -10°C and maintained for 3 hours, then the temperature is adjusted to 0°C and maintained for 3 hours, then the temperature is adjusted to 10°C and maintained for 3 hours, and finally the temperature is adjusted to 25°C and maintained for 3 hours.

9. The method for preparing anterior cruciate ligament bridging material according to claim 1, characterized in that: In step 6, the uncrosslinked collagen fiber scaffold obtained in step 5 is subjected to high-temperature physical crosslinking in vacuum at 100-150° C. The time for the high-temperature physical crosslinking is 12-48 hours, and finally a collagen fiber scaffold is prepared.

10. Use of a bridging material prepared according to the preparation method according to any one of claims 1 to 9 in anterior cruciate ligament repair surgery.

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