CN112206351B - Composite material for repairing articular cartilage defect and preparation method thereof - Google Patents

Abstract

The invention discloses a composite material for repairing articular cartilage defects, which comprises an allogeneic bone and a gel layer wrapping the allogeneic bone; the gel layer includes chondroitin sulfate, hyaluronic acid, glucosamine, and a gel medium. The invention also provides a preparation method of the composite material for repairing articular cartilage defects. The composite material for repairing articular cartilage defects provided by the invention can be beneficial to reducing inflammatory reaction in the articular cartilage repair process, promoting the repair and reconstruction of cartilage matrixes and accelerating the articular cartilage, and is particularly suitable for repairing large-area defects and particularly suitable for repairing and reconstructing large-area articular cartilage defects.

Description

Composite material for repairing articular cartilage defect and preparation method thereof

Technical Field

The invention relates to the field of medical materials, in particular to a composite material for repairing articular cartilage defects and a preparation method thereof.

Background

Articular cartilage is hyaline cartilage, which is smooth and glossy in surface and is a special connective tissue, and consists of chondrocytes, collagen types II, IX and XI, proteoglycan and small glycoprotein, as well as other specific and nonspecific matrix proteins, water and inorganic salts, wherein the collagen type II accounts for 50% of the total dry weight. Articular cartilage covers the surface of the joint and plays important roles in buffering stress, absorbing shock, lubricating the joint surface, preventing abrasion and the like. The articular cartilage of a human body is a non-vascular tissue without blood supply, and nutrition comes from moistening of peripheral joint synovial fluid, and the articular cartilage is difficult to regenerate once being damaged, so that articular cartilage repair becomes one of the most challenging clinical problems for orthopedists.

The incidence rate of cartilage damage in China reaches 68.5%, and the incidence rate of middle-aged and old people is higher than that of teenagers, while the incidence rate of females in middle-aged and old people is 90.5% and far higher than that of males. Cartilage damage is primarily due to meniscal damage in the joints, resulting in softening, cracking, peeling and loss of articular cartilage. The causes of this phenomenon are mainly of three types, the first being the lack of movement, which leads to degeneration of the articular cartilage; the second is strain caused by excessive joint movement; the third category is damage to articular cartilage caused by acute damage to the joint. The current treatment methods for cartilage damage repair mainly comprise: A. oral administration of the medicament; B. physical therapy; C. intraarticular injection of local anesthetics and corticosteroids; D. washing and cleaning under arthroscopy; E. arthroscopic cartilage planing; F. micro-fracture surgery; G. allograft cartilage transplantation; H. autologous chondrocyte culture transplantation.

However, the above methods have certain disadvantages, such as: A. oral administration of the medicine: can alleviate inflammatory reaction, but has side effects and no effect of directly promoting cartilage repair. B. Physical therapy: the pain symptom of the patient is mostly relieved by subjective feeling, and the long-term effect is unknown. C. Intra-articular injection of local anesthetic and corticoid: can quickly relieve symptoms, but can not promote the repair of articular cartilage and even lead to the degeneration of cartilage tissues, and the long-term prediction is more beneficial than the future prediction. D. Washing and cleaning under arthroscopy: clinical symptoms of patients are relieved by removing inflammatory factors and cartilage fragments in joint cavities, the early clinical curative effect is obvious, but the further development of joint cartilage damage is delayed, and the aim of repairing cartilage defects cannot be achieved. E. Arthroscopic cartilage planing: the curative effect of the single use is questioned. F. Microfracture surgery: the damaged part of the cartilage is removed under the articular lens, a small hole is drilled in the subchondral bone below the cleaned cartilage, and bone marrow containing mesenchymal stem cells seeps out of the hole to form blood clots which become smooth and firm repair tissues to replace the function of the cartilage. G. The autologous chondrocyte culture transplantation is characterized in that cartilage in an autologous non-bearing area is sacrificed, the complication of a supply area is high, in-vitro culture is needed, the operation is complex, the infection probability is high, the risks of cell degeneration and genetic change exist, two operations are needed, and the clinical price is high (6-10 ten thousand).

Chinese patent application No. 201110021494.8 discloses a technical solution for repairing articular cartilage damage by using allogeneic decalcified bone, which has good biocompatibility, no immunological rejection, and good repairing effect because hyaline cartilage is generated, but has slow repairing process and poor repairing effect when used for patients with excessive cartilage defect area or lack of normal cartilage tissue around the damage. The Chinese patent application with the application number of 201910893393.6 discloses a production process optimized based on the patent technology, autologous platelet-rich plasma gel is added, the elastic modulus and hardness of the generated new cartilage are closer to those of normal cartilage, the repair area is complete and the flatness is good, and the problem of repairing large-area defects is still not solved.

Disclosure of Invention

In order to overcome at least one of the defects in the prior art, the invention provides a composite material for repairing articular cartilage defects and a preparation method thereof. The composite material for repairing articular cartilage defect provided by the invention adopts allogeneic bone as raw material, can effectively remove immunogenicity and reduce rejection after a series of processes, is favorable for reducing inflammatory reaction and promoting the repair and reconstruction of cartilage matrix by adding gel, and can be more suitable for repairing patients with large-area defect.

Specifically, the invention provides a composite material for repairing articular cartilage defects, which comprises allogeneic bones and a gel layer wrapping the allogeneic bones; decalcifying the allogeneic bone; the gel layer comprises chondroitin sulfate, hyaluronic acid, glucosamine and a gel medium; the effective components of the gel layer consist of 10-25 parts by weight of chondroitin sulfate, 5-10 parts by weight of hyaluronic acid, 15-25 parts by weight of glucosamine and 20-40 parts by weight of gel medium glue; the gel medium is selected from one of tragacanth, gelatin, starch, cellulose derivative, carboxyvinyl polymer or sodium alginate; gelatin is preferred.

In one embodiment according to the present invention, the gel layer contains 25 parts by weight of chondroitin sulfate, 5 parts by weight of hyaluronic acid, 15 parts by weight of glucosamine, and 30 parts by weight of gelatin.

In another aspect of the present invention, there is provided a method for preparing a composite material for repairing articular cartilage defects, comprising:

1) preparing decalcified allogeneic bone;

2) weighing appropriate amount of chondroitin sulfate, hyaluronic acid, glucosamine and gelatin, dissolving in hot water, mixing, and cooling to room temperature to obtain gel;

3) soaking the allogeneic bone in the gel prepared in the step 2), standing overnight at 2-10 ℃, and properly trimming the thickness of the gel wrapping the bone block;

4) drying and sterilizing by irradiation to obtain the composite material.

In one embodiment according to the present invention, the decalcification treatment of the allogeneic bone in the step 1) is performed by a method comprising the steps of:

soaking the allogeneic bone in chelating solution containing 0.1-0.3M disodium edetate and having pH of 6-10 at 10-37 deg.C until the calcium content of the allogeneic bone is 0.5% -2%.

In one embodiment according to the invention, the allogeneic bone comprises cortical and cancellous bone structures.

In one embodiment according to the invention, the gel thickness outside the allograft bone is 3-10 mm.

In one embodiment according to the present invention, the drying process in step 4) is vacuum freeze-drying.

In one embodiment according to the invention, the radiation sterilization in step 4) is cobalt 60 radiation sterilization.

The invention also provides the application of the composite material in preparing and repairing articular cartilage defects.

Compared with the prior art, the invention has at least one of the following beneficial effects:

the invention adopts allogeneic bone as a framework material, and chondroitin sulfate, glucosamine and hyaluronic acid are added, so that the invention is beneficial to reducing inflammatory reaction and promoting the repair and reconstruction of cartilage matrix, and is particularly suitable for repairing patients with large-area defects. The articular cartilage repair material provided by the invention only needs one operation, is simple to operate and low in cost, and causes less pain to patients.

The invention utilizes 1) the advantages that the low molecular weight chondroitin sulfate (3500-5300) body is easy to absorb and can better promote wound healing and cartilage repair, relieves the inflammatory reaction of arthritis, reduces the release of lysosomal enzyme and has the function of anticoagulation. 2) The glucosamine can stimulate cartilage cells to generate proteoglycan with normal polymer structure, improve the repair capability of the cartilage cells, and promote the repair and reconstruction of cartilage matrixes. 3) Hyaluronic acid can restore the characteristics of joint synovial fluid such as viscoelasticity and physiological homeostasis. The three components are combined with the allogeneic bone scaffold, so that the composition has a remarkable synergistic effect, and compared with the method of simply using the allogeneic bone scaffold product, the composition has the advantages of being more beneficial to treating large-area defect, relieving osteoarthritis, promoting subchondral bone repair and the like.

Drawings

Fig. 1 is a schematic view illustrating a method for preparing a composite material for repairing an articular cartilage defect according to an embodiment of the present invention.

Fig. 2 is a graph comparing the elasticity of articular cartilage repaired with a composite material formed by combining decalcified allogeneic bone with different components according to one embodiment of the present invention.

Fig. 3 is a graph comparing the hardness of articular cartilage after repair with a composite material formed by combining decalcified allogeneic bone with different components according to one embodiment of the present invention.

Fig. 4 is a graph illustrating the effect of a composite material formed by combining different components with decalcified allograft bone to repair an articular cartilage defect, according to one embodiment of the present invention.

Fig. 5 is a schematic comparison of bone tissue microstructures for repairing an articular cartilage defect in a composite material formed by combining different components with decalcified allogeneic bone according to one embodiment of the present invention.

Fig. 6 is a graph illustrating the comparative effect of repairing articular cartilage using a composite material formed by combining a single active ingredient with decalcified allograft bone according to one embodiment of the present invention.

Fig. 7 is a graph comparing hardness test results of a new cartilage tissue in an articular cartilage repaired with a composite material formed by combining different components with decalcified allogeneic bone according to one embodiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The composite material for repairing articular cartilage defect of the invention comprises: allograft bone and a gel layer. The gel layer comprises 10-25 parts of chondroitin sulfate, 5-10 parts of hyaluronic acid, 15-25 parts of glucosamine and 20-40 parts of gelatin.

EXAMPLE 1 preparation of composite Material

1. Preparation of the gel

The gel composition and content are shown in table 1.

TABLE 1 gel Components and amounts

Chondroitin sulfate, hyaluronic acid, glucosamine and gelatin are weighed according to the table 1, dissolved in hot water at 80-100 ℃ and mixed evenly, and when the temperature of the solution is reduced to room temperature, gel solution is obtained for later use.

2. Preparation of composite Material

As shown in fig. 1:

1) selecting an allogeneic bone containing a cortical bone and a cancellous bone structure;

2) the allogeneic bone is soaked in the chelating solution for decalcification treatment. The chelating solution is 0.1M-0.3M disodium edetate, the pH value is 6-10, and the temperature is as follows: 10-37 ℃. The calcium content of the allogeneic bone after chelation and decalcification is 0.5-2%. Meanwhile, an atomic absorption spectrophotometer is used for monitoring the decalcification process, and a universal mechanical testing machine is used for detecting the mechanical property of the product.

3) Cleaning the decalcified allogeneic bone, removing inorganic salt ions, and monitoring the cleaning process by using a conductivity meter and an ultraviolet spectrophotometer;

4) soaking the cleaned allogeneic bone in the gel solution, standing overnight at 2-10 ℃, and trimming the gel outside the bone block to a thickness of 3-10mm to obtain the composite material 1. The gel thickness in this example was 3 mm.

5) And (3) carrying out vacuum freeze drying on the composite material.

6) And (3) performing cobalt 60 irradiation sterilization on the composite material to obtain the composite material suitable for cartilage repair.

EXAMPLE 2 biomechanical Studies of various products

After 24 weeks of implantation of the four products of example 1 in rabbits, the biomechanical properties were determined by taking the materials.

The rabbit joint treatment mode is as follows:

the knee joint is prepared by normal skin preparation, disinfection and single laying, the median incision is taken to enter the joint cavity through the medial approach of the patella, and the medial condyle of the femur is exposed after the patella is dislocated laterally. A full-thickness articular cartilage defect with a diameter of 4mm was made with a trephine. The whole experiment is divided into four groups, four products are implanted at the defect part respectively, and the biomechanical property detection is carried out by taking materials after 24 weeks.

The biomechanical property mainly comprises an elastic modulus and a hardness test. The experimental results are shown in table 2, fig. 2 and fig. 3.

TABLE 2 biomechanical property test results

As shown in Table 2, FIG. 2 and FIG. 3, the product with high content of chondroitin sulfate in the above-mentioned product 4 after 24 weeks in rabbits showed higher elastic modulus, indicating that chondroitin sulfate can increase elastic modulus of neogenetic tissue, but corresponding hardness is slightly decreased, and of the above-mentioned four products, the product 4 is preferred. The following experiment was carried out with product 4 from example 1.

Example 3 Properties of the composite Material

Taking product 4 in example 1 as an example, the performance of the composite material was investigated.

The experimental process comprises the following steps: selecting 4-6 month-old miniature pigs to repair cartilage defects, anaesthetizing, preparing skin, sterilizing, cutting a knee joint to expose a femoral pulley, and making a full-layer articular cartilage defect with the diameter of 9mm on the femoral pulley. The whole experiment was divided into four groups: (1) the group using Chondroitin Sulfate (CS), Hyaluronic Acid (HA), and Glucosamine (GA) alone; (2) the pure allograft bone scaffold (DCCBM) group; (3) CS + HA + GA + Chitosan (CTS) gel group; (4) DCCBM + CS + HA + GA group. The materials are obtained and observed at 6, 12 and 24 weeks after operation, and the experimental result is shown in figure 4; HE of the knee joint specimen after 24 weeks was stained, and the experimental results are shown in fig. 5.

As can be seen from fig. 4, the beneficial factor group (CS + HA + GA) used alone was essentially unfilled, while incomplete repair was also observed in the DCCBM group alone, with uneven surface coverage and thinner repaired tissue than the DCCBM + CS + HA + GA group. Hyaline chondrocytes were seen in the repair area in the DCCBM + CS + HA + GA group, with good integration between the repair tissue and normal cartilage. Compared with the simple DCCBM group, the CTS + CS + HA + GA group HAs poorer repairing effect, thinner repairing tissue and larger unrepaired area, and shows that the gel prepared by combining chitosan with chondroitin sulfate, hyaluronic acid and glucosamine HAs poorer effect of repairing large-area cartilage defects.

As can be seen from fig. 5, the DCCBM + CS + HA + GA group HAs better repairing effect, hyaline-like chondrocytes can be seen in the repairing region of the group, and the repairing tissue and normal cartilage have good integration and obvious tide lines.

EXAMPLE 4 Properties of composite materials of different compositions

To prove the gain effect of Hyaluronic Acid (HA), Chondroitin Sulfate (CS) and Glucosamine (GA), pig cartilage repair tests were performed, respectively. The experiment was carried out by compounding Hyaluronic Acid (HA), Chondroitin Sulfate (CS), Glucosamine (GA) and the allograft bone scaffold (DCCBM), which are single active ingredients, respectively, and the experimental results are shown in fig. 6. The hardness test was performed on 24 weeks of newly-grown cartilage tissue, and the test results are shown in FIG. 7.

As can be seen in fig. 6, the allogenic bone-formed scaffold alone combined with one of the active ingredients (CS, HA or GA) all had poor repair. The repair effect of the three groups is slower than that of DCCBM + CS + HA + GA, and the new cartilage tissue is weaker.

As can be seen from fig. 7, the hardness of the cartilage generated by the DCCBM + CS + HA + GA group is closer to that of the normal cartilage, which indicates that the DCCBM + CS + HA + GA group HAs a synergistic effect in repairing cartilage defects by combining with an allogeneic bone scaffold, especially in improving the hardness of the new cartilage tissue.

In conclusion, the composite material disclosed by the invention is good in biocompatibility, free of immunological rejection, and beneficial to reducing inflammatory reaction and promoting the repair and reconstruction of cartilage matrix; the generated hyaline cartilage has good repairing effect, the elastic modulus and the hardness are closer to those of normal cartilage, the repairing area is complete and has good flatness, and the hyaline cartilage repairing area can be suitable for patients with over-large cartilage defect area (for example, the diameter of the defect reaches 9mm, compared with the prior art that only the defect with the diameter of 4mm can be repaired, the product repairing area provided by the invention is enlarged by 4 times) or the periphery of the defect is lack of normal cartilage tissue; the production process is simple, the materials are convenient to obtain, the cost is low, only one operation is needed when the composite material is used, the operation is simple, and the pain of a patient can be greatly relieved.

The foregoing shows and describes the general principles, essential features, and advantageous features of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (8)

1. A composite material for repairing articular cartilage defects comprising an allogeneic bone and a gel layer encapsulating the allogeneic bone; decalcifying the allogeneic bone; the gel layer comprises chondroitin sulfate, hyaluronic acid, glucosamine and a gel medium; the effective components of the gel layer consist of 10-25 parts by weight of chondroitin sulfate, 5-10 parts by weight of hyaluronic acid, 15-25 parts by weight of glucosamine and 20-40 parts by weight of gel medium; the gel medium is selected from one of tragacanth, gelatin, starch, cellulose derivative, carboxyvinyl polymer or sodium alginate.

2. Composite according to claim 1, characterized in that the gel layer contains 25 parts by weight of chondroitin sulfate, 5 parts by weight of hyaluronic acid, 15 parts by weight of glucosamine and 30 parts by weight of gelatin.

3. The method of preparing a composite material for repairing an articular cartilage defect of claim 1 or 2, comprising:

1) preparing decalcified allogeneic bone;

2) weighing appropriate amount of chondroitin sulfate, hyaluronic acid, glucosamine and gel medium, dissolving in hot water, mixing, and cooling to room temperature to obtain gel;

3) soaking the allogeneic bone in the gel prepared in the step 2), standing overnight at 2-10 ℃, and properly trimming the thickness of the gel wrapping the bone block;

4) drying and sterilizing by irradiation to obtain the composite material.

4. The method for preparing as claimed in claim 3, wherein the decalcification treatment of the allogeneic bone in the step 1) is performed by a method comprising the steps of:

soaking the allogeneic bone in chelating solution containing 0.1-0.3M disodium edetate and having pH of 6-10 at 10-37 deg.C until the calcium content of the allogeneic bone is 0.5% -2%.

5. The method of claim 4, wherein the allogeneic bone comprises cortical and cancellous bone structures.

6. The method of claim 3, wherein the gel thickness outside the allograft bone is 3-10 mm.

7. The method according to claim 3, wherein the drying treatment in step 4) is vacuum freeze-drying.

8. The method according to claim 3, wherein the radiation sterilization in the step 4) is cobalt 60 radiation sterilization.

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