CN108096632A - Articular cartilage repair materials and preparation method based on oxidized hyaluronic acid-II Collagen Type VIs and self concentration bone marrow nucleated cell - Google Patents

Abstract

The invention relates to an articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells and a preparation method thereof, which is made of oxidized hyaluronic acid, type II collagen and autologous concentrated bone marrow nucleated cells. The autologous enriched bone marrow nucleated cells in the material have the potential of self-renewal and chondrogenic differentiation, and participate in the repair of cartilage defects, so they have a good repair effect on cartilage damage. The degree of integration, type II collagen content, GAG content, calcified cartilage layer and subchondral bone morphology were significantly better than those of the blank control group, and had the potential for clinical transformation.

Description

Based on the relationship between oxidized hyaluronic acid-type II collagen and autologous enriched bone marrow nucleated cells Articular cartilage repair material and preparation method

technical field

The invention belongs to the field of medical materials, and relates to an articular cartilage repair material based on oxidized hyaluronic acid-II collagen and autologous concentrated bone marrow nucleated cells, and also relates to a preparation method and application of the material.

Background technique

Articular cartilage is a thin layer of dense connective tissue lining the surface of joints. Its surface is smooth, firm and elastic, and it is an important load-bearing tissue for human joints. However, since normal articular cartilage has no blood vessels, nerves, and lymphatic distribution, it is difficult to repair itself after injury, and there is still a lack of effective repair strategies. In clinical work, the incidence of articular cartilage damage is relatively high. Widuchowski retrospectively analyzed 25,124 cases of arthroscopic knee surgery and found that 60% of the cases had cartilage damage. Cartilage damage has become an important cause of joint pain, dysfunction and even physical disability. Currently, commonly used clinical repair strategies for damaged cartilage include: joint cavity debridement, microfracture, cartilage transplantation, chondrocyte transplantation, and osteochondral transplantation. Cartilage tissue engineering technology is an ideal method for the repair of articular cartilage damage, but there are still many problems in the use of tissue engineering technology for cartilage repair at this stage, such as: more complicated procedures; poor mechanical properties of repaired tissues, poor integration with host tissues, and fibrous Cartilage degeneration, etc. Therefore, it is of great significance to explore a simpler and better cartilage tissue engineering strategy.

Contents of the invention

In view of this, one purpose of the present invention is to provide a kind of articular cartilage repair material based on oxidized hyaluronic acid-II collagen and autologous concentrated bone marrow nucleated cells; the second purpose of the present invention is to provide a material based on oxidized hyaluronic acid-type II The preparation method of the articular cartilage repair material of type II collagen and autologous concentrated bone marrow nucleated cells; the third object of the present invention is to provide the application of articular cartilage repair materials based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells .

To achieve the above object, the present invention provides the following technical solutions:

1. Articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells, made of oxidized hyaluronic acid, type II collagen and autologous concentrated bone marrow nucleated cells.

Preferably, the amount of oxidized hyaluronic acid, type II collagen and autologous concentrated bone marrow nucleated cells is added to 5×10 ⁶ autologous concentrated bone marrow nucleated cells, and 100 mg type II collagen is added to mix evenly, vortexed and centrifuged, Remove the air bubbles in the mixed gel, and add 10ul oxidized hyaluronic acid solution to the type II collagen and gel of compound cells.

Preferably, the 100 mg/ml oxidized hyaluronic acid is prepared by adding 1 ml of PBS solution for every 100 mg oxidized hyaluronic acid and dissolving it completely.

More preferably, the type II collagen is prepared by the following method: freeze-dried cartilage powder is suspended in a 4M guanidine hydrochloride solution with a mass-volume ratio of 20:1 (g:L), and placed at 4°C after stirring Digest the mucopolysaccharides in the cartilage powder matrix under the same conditions to a homogenate state; after the digestion is complete, centrifuge the digested product at 4°C and 8000rpm for 20min, discard the supernatant, collect the precipitate, add water to dissolve, and then add equivalent hydrochloric acid Acetic acid 3/50 times the volume of the guanidine solution, adding pepsin equivalent to 0.1 times the weight of the freeze-dried cartilage powder, adding water until the final volume is twice the volume of the guanidine hydrochloride solution, adjusting the pH value between 2.5-3, and then heating at 4°C Digest for 48 hours under the same conditions; after digestion, centrifuge at 8000rpm in a vertical high-speed centrifuge for 20 minutes, discard the precipitate, and collect the supernatant; then slowly pour 1.5M NaOH solution into the supernatant and stir until the pH value is 7.5 Stop pouring, add NaCl to make the final concentration 3M, and centrifuge at 8000rpm for 20min at 4°C to collect the sediment; divide the sediment into dialysis bags and stop the dialysis when the conductivity of the dialysate is close to the conductivity of pure water; Centrifuge the viscous colloidal solution in the dialysis bag at 4°C in a vertical high-speed centrifuge at 8000rpm for 20min, collect the precipitate into a sterile volumetric flask, add 0.15mol/L hydrochloric acid solution, dissolve for 3 days, and then the white precipitated protein is dispersed, Dissolve in gel form, stir evenly, subpackage, freeze-dry, and set aside.

More preferably, the oxidized hyaluronic acid is prepared by the following method: prepare a hyaluronic acid solution with a mass fraction of 2%, then add 0.1 g/ml sodium periodate solution, stir, and completely oxidize under the condition of avoiding light After 6 hours, ethylene glycol was added to terminate the reaction, and then the reaction solution was poured into a dialysis bag until the conductivity was close to that of pure water, and the dialysis was terminated to obtain oxidized hyaluronic acid.

More preferably, the autologous concentrated bone marrow nucleated cells are prepared by the following method: the current animal is anesthetized, and then the bone marrow blood is extracted with a syringe pre-wetted with 1000 U/ml heparin, and then the extracted anticoagulated bone marrow fluid is added to an equal volume of Bacterial PBS solution, mix well, slowly inject the bone marrow fluid along the tube wall into the Percoll cell separation solution containing an equal volume of concentration of 1.073g/ml, keep the interface between the separation solution and the mixed solution intact during the injection process, Then use Percoll density gradient centrifugation to obtain autologous concentrated bone marrow nucleated cells. First, centrifuge the mixed liquid at a speed of 2000rpm for 15min, absorb the nucleated cell layer, add PBS solution to wash the absorbed nucleated cells, and centrifuge and wash the cells at a speed of 1500rpm for 5min. Discard the supernatant. Add PBS again to wash the cells by pipetting, centrifuge at 1000rpm for 5min, discard the supernatant, and add PBS to resuspend.

2. The preparation method of the articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells, the specific steps are as follows: first mix the autologous concentrated bone marrow nucleated cells with type II collagen, and then Oxidized hyaluronic acid is added for cross-linking.

3. The application of the articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells in the preparation of articular cartilage repair materials.

The beneficial effects of the present invention are: the present invention discloses an articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells. The implanted autologous enriched bone marrow nucleated cells have the potential of self-renewal and chondrogenic differentiation, and participate in the repair of cartilage defects. Therefore, the prepared material has a good repair effect on cartilage damage. Tissue surface smoothness, degree of integration with adjacent normal cartilage, type II collagen content, GAG content, calcified cartilage layer and subchondral bone morphology were significantly better than those of the blank control group, and had the potential for clinical transformation.

Description of drawings

In order to make the purpose, technical scheme and beneficial effect of the present invention clearer, the present invention provides the following drawings for illustration:

Figure 1 is an SDS-PAGE electrophoresis image (Lane1, Lane2 and Lane3 respectively indicate different loading amounts).

Figure 2 is a shaking experiment of type II collagen hydrogel.

Fig. 3 is a diagram of bone marrow mesenchymal stem cells (A: adherent mesenchymal stem cells cultured for 2 days; B: mesenchymal stem cells passaged at P3).

Fig. 4 is a diagram showing sufficient mixing of type II collagen and mesenchymal stem cells (A: co-culture induction of type II collagen and cells; B: co-culture of gel and mesenchymal stem cells).

Figure 5 shows the Fourier transform infrared spectra of HA and OHA.

Fig. 6 is the detection result of CCK-8.

Figure 7 shows the gelation results of OHA-COLII-BMSC in the mold.

Figure 8 shows the induction results of three lines of porcine bone marrow mesenchymal stem cells (A: osteogenic induction of mesenchymal stem cells; B: adipogenic induction of mesenchymal stem cells; C: chondrogenic induction of mesenchymal stem cells).

Figure 9 shows the results of gelation of OHA-COLII-BMSC and slices at different times of culture (A: gelation results of OHA-COLII-BMSC; B: slice results after 14 hours of culture; C: slice results after 21 hours of culture; D: Gel slice results before culture; E: gel slice results after 14-hour culture; F: gel slice results after 21-day culture).

Figure 10 is the scoring results of cartilage repair tissue (A: MOCART scoring results; B: O`Driscoll scoring results; C: ICRS scoring results).

Fig. 11 is a schematic diagram of the preparation of oxidized hyaluronic acid and the cross-linking reaction with type II collagen.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Example 1, Preparation of Oxidized Hyaluronic Acid Type II Collagen Biomimetic Gel

(1) Extraction and identification of cartilage type II collagen hydrogel

a. Preparation of cartilage powder: obtain the hyaline cartilage of the knee joint of fresh Guizhou Xiaoxiang pig, freeze it in a -20°C refrigerator for 12 hours, collect the frozen cartilage in a glass dish, place it in a vacuum freeze dryer, and freeze-dry it after 24 hours (the temperature drops to -66°C, pressure down to 1Pa), after freeze-drying, pour the cartilage slices into a liquid nitrogen low-temperature pulverizer, continuously fill with liquid nitrogen, crush the cartilage slices into dry cartilage powder, collect the cartilage powder and store it in a sealed bag for later use;

b. Extraction of Type II Collagen: Weigh 20g of freeze-dried cartilage powder and place it in a beaker, pour 1L of 4M guanidine hydrochloride solution into the suspension, stir continuously with a magnetic stirrer and place in a refrigerator at 4°C, digest the cartilage powder for 24 hours The mucopolysaccharide in the matrix is in a homogenized state; after the digestion is complete, centrifuge the digested product at 4°C at 8000rpm×20min, discard the supernatant, collect the precipitate into a beaker, add water to the 1500ml mark, and then measure 60ml of acetic acid into the beaker Add 2g of pepsin, add water to the mark of 2000ml, adjust the pH value between 2.5-3, and place it in the refrigerator at 4°C for 48h with magnetic stirring; after pepsin digestion, centrifuge in a vertical high-speed centrifuge at 8000rpm ×20min, discard the precipitate, collect the supernatant; then slowly pour 1.5M NaOH solution into the supernatant and stir until the pH value is 7.5, stop pouring, add NaCl to make the final concentration 3M, centrifuge at 4°C 8000rpm×20min, collect the sediment; divide the sediment into dialysis bags (diameter 25mm, molecular weight 3500), tie both ends tightly with nylon thread, dialyze with double distilled water, change the dialysate every 6h for the first 48h, and then every 12h Once, the total dialysis time is about 5 days. The conductivity meter monitors the conductivity of the dialysate in real time. When the conductivity of the dialysate is lower than that of normal saline and close to the conductivity of pure water, the dialysis ends; Centrifuge at 8000rpm×20min in a type high-speed centrifuge, collect the sediment into a sterile volumetric flask, add 0.15mol/L hydrochloric acid solution, and dissolve the white precipitated protein after 3 days. , divided into sterilized glass sealed bottles of different volumes, and stored at 4°C for later use;

Take an appropriate amount of collagen hydrogel for freeze-drying, and calculate type II collagen hydrogel w/v according to the formula (wet weight-volatilized liquid weight)/(wet weight volume). The detected mass fraction of type II collagen was 10% (w/v).

After the extracted type II collagen was subjected to SDS-PAGE gel electrophoresis, the results are shown in Figure 1. The results showed that the extracted protein showed only one band. As the amount of sample increased, the concentration of the band increased gradually. The molecular weight of the band was about 120KD, which was a unique protein band of cartilage type II collagen. Obvious miscellaneous band development indicates that the extracted type II collagen has a high purity.

After the type II collagen hydrogel was neutralized with 2M NaOH, it was placed in LB medium, and the shaking experiment was carried out many times. Although some collagen was dissolved in the bottom of the medium, the medium in the upper part of the shaking tube was transparent and clear. , compared with the positive and negative tubes, both showed negative results without contamination (Figure 2).

After the bone marrow mesenchymal stem is subjected to density gradient centrifugation, the separated cell culture medium is resuspended, and the floating lymphocytes and red blood cells are washed away after being cultured for 2 days. The adherent cells are the obtained bone marrow mesenchymal stem cells, and the cells are polygonal , long fusiform, primary cell divisions are more common (A in Figure 3), and multiple cell colonies are formed after 4-5 days. After subculture, the cells grow more vigorously, and the elongated spindle cells can form 80%-90% cell fusion in the culture flask after 5-7 days, and are arranged in a weaving and swirl shape. (B in Figure 3)

After the type II collagen and mesenchymal stem cells are fully mixed, the gel pieces can be immersed in the medium, and a small amount of gel pieces can be dissolved, but it is not turbid due to pollution. When changing the medium, be careful to replace it, avoid blowing, and the gel The blocks are agglomerated with physical connections, with low strength and loose structure (A in Fig. 4). Photographs under an optical microscope show that the collagen fibers are loosely arranged, silky collagen fibers can be seen on the edge of the gel block, and the length is uniform. (B in Figure 4)

(2) Oxidation of hyaluronic acid

Oxidation of hyaluronic acid: Dissolve 4g of hyaluronic acid in 200ml of pure water, stir on a magnetic stirrer for 6h to dissolve completely, and prepare a hyaluronic acid solution with a mass fraction of 2% for later use; take 2g of sodium periodate and add After dissolving in 20ml of pure water, pour it into the prepared 2% hyaluronic acid solution, keep stirring, wrap it in aluminum foil to avoid light, and completely oxidize it at room temperature (18-25°C) for 6 hours; measure 20ml of ethylene glycol and add React in a beaker for 1 hour to terminate the oxidation reaction, pour the reaction solution into a dialysis bag (diameter 25mm, molecular weight 3500), tie the two ends tightly, dialyze with double distilled water, change the dialysate every 6 hours for the first 48 hours, and then replace it every 12 hours . The conductivity meter monitors the conductivity of the dialysate in real time. When the conductivity of the dialysate is close to the conductivity of pure water, the dialysis is terminated to obtain oxidized hyaluronic acid. The dialysate is poured into a freeze-drying tray and placed in a -20°C refrigerator for pre-freezing After freezing for 12 hours, freeze and dry in a vacuum freeze dryer for 24 hours (the temperature drops to -66°C and the pressure drops to 1 Pa). Finally, the dried oxidized hyaluronic acid is divided into EP tubes in units of 100 mg, and sealed with tape Sealed and sterilized by Co 60 irradiation.

Hyaluronic acid is in a transparent liquid state after being oxidized by sodium periodate, and is in the form of a sponge after freeze-drying. The freeze-dried hyaluronic acid and unoxidized hyaluronic acid are tested by Fourier infrared spectroscopy through potassium bromide tablet method , a characteristic change occurs at a wavelength of 1750, indicating that the hydroxyl group on the hyaluronic acid is specifically oxidized to an aldehyde group by sodium periodate (Figure 5).

The oxidized hyaluronic acid can be completely dissolved in the medium, and the porcine bone marrow mesenchymal stem cells are cultured in the 0.5mg/ml, 1.0mg/ml, 1.5mg/ml oxidized hyaluronic acid culture system for 48 hours, and then CCK- 8 detection, there was no significant difference compared with the blank control group, which proved that it had no obvious cytotoxicity and weakened proliferative activity (Fig. 6).

(3) Preparation of oxidized hyaluronic acid type II collagen biomimetic gel

a. Take 100 mg of oxidized hyaluronic acid sterilized by irradiation, add 1 ml of PBS solution, and dissolve it completely for use.

b. Take 1g type II collagen, add 75μl NaOH (2M), stir the nerve strips evenly, add 100μl dissolved oxidized hyaluronic acid with a pipette gun, and stir well. Fill it into a stainless steel mold hole (diameter 8mm, thickness 3mm), and after 2 minutes, the oxidized hyaluronic acid type II collagen (OHA-COLII) is completely gelled and solidified. After pre-freezing in a -20°C refrigerator for 24 hours, freeze-dry in a vacuum freeze dryer to form a sponge. Sent to the Central Laboratory of the Third Medical University, the ion sputtering machine coated the sample with 10nm gold film, and the section was observed by scanning electron microscope. The rest of the gel pieces were soaked in a petri dish of physiological saline, placed in a 37° C. incubator and incubated for 14 days, and the swelling and dissolution were observed ( FIG. 7 ).

Three-line induction of porcine bone marrow mesenchymal stem cells: subcultured mesenchymal stem cells.

a) Osteogenic induction: when the cell confluence reaches 80-90%, digest with 0.25% trypsin; inoculate the digested stem cells in a six-well plate at a cell density of 2×10 ⁴ cells/cm ² Before inoculation, put 0.1% gelatin-coated slides into the well plate, and add 2 mL of complete medium to each well. When the cell confluency reaches 60-70%, aspirate the complete medium in the well. Add 2 mL of adult bone marrow mesenchymal stem cell osteogenic induction and differentiation complete medium to the six-well plate, change the medium every 3 days, and stain with alizarin red after 3 weeks of induction. The results are shown in A in Figure 8, and the results show that the calcium nodules secreted by bone marrow mesenchymal stem cells were stained with alizarin red after osteogenic induction.

b) Adipogenic induction: the cell inoculation method is the same as above, the degree of cell fusion reaches 100%, the complete medium of mesenchymal stem cells is sucked away, and 2ml of bone marrow mesenchymal stem cell adipogenic induction differentiation medium A is added to the six-well plate. After 3 days of induction, liquid A in the six-well plate was sucked away, and 2 ml of bone marrow mesenchymal stem cell adipogenic differentiation medium liquid B was added. After 24 hours, liquid B was sucked away, and liquid A was replaced for induction. After 4 times (15 days) of alternating action of solution A and solution B, the culture was continued with solution B for 5 days until the lipid droplets became sufficiently large and round, and stained with Oil Red O after fixation. The results are shown in Figure 8 B. The results show that after adipogenic induction, the cell body becomes larger, the cells spread, the nuclei are lightly stained, and a large number of fat particles are produced in the cytoplasm, and the larger fat particles can fuse to form fat droplets. .

c) Chondrogenic induction: the cell inoculation method is the same as above, the degree of cell fusion reaches 100%, the complete medium of mesenchymal stem cells is sucked away, and 2ml of bone marrow mesenchymal stem cell chondrogenic induction differentiation medium is added to the six-well plate, cultured for 21 days, Alcian blue staining after fixation. After staining, take out the glass slide in the six-well plate with tweezers, and take pictures under the optical microscope to observe the induction situation; the results are shown in Figure 8 C, and the results show that after chondrogenic induction, the morphology of mesenchymal stem cells changes, from long spindles to Paving stone-like, secreted GAGs were stained light blue by Alixinlan, and the nuclei were not stained.

Take porcine bone marrow mesenchymal stem cells that have been cultured to the third generation, discard the medium, rinse with PBS twice, digest with 0.25% trypsin for 3 minutes, the cells are spherical, pat the bottle wall cells to fall off and suspend, 2ml of complete medium to stop digestion . After centrifugation at 1000rpm×3min, resuspend in 2ml medium, count the cells on a cell counting board, take 5×10 ⁶ and centrifuge again into a 2ml EP tube. Take 1g of neutralized COLII and add it to the EP tube of the centrifuged cells, add 100μl of dissolved OHA, stir well, fill the gel into a high-temperature sterilized stainless steel mold, and wait for 2 minutes for it to become a gel, that is, OHA -COLII-BMSC. Place in complete medium and culture in well plates. The results showed that OHA-COLII-BMSC could form a gel well in the sterile mold (A in Fig. 9).

After culturing in the well plate for 14 days and 21 days, samples were taken respectively, fixed in 10% formalin for 24 hours, stained with HE, and observed under an optical microscope; when cultured to the 14th day, a sample was taken and fixed in 50% formalin. , 70%, 80%, and 90% ethanol gradient dehydration for 15 minutes each, and then dehydration with 100% ethanol three times, each time for 30 minutes; tert-butanol replacement for 30 minutes each time, a total of three replacements. A freeze dryer dries the sample, sticks the sample on the sample stage with a double-sided adhesive tape, coats the sample with a 10nm gold film by an ion sputtering instrument, and observes the profile of the sample with a scanning electron microscope. The results are shown in Figure 9. The results showed that after 14d and 21d of culture, the integrity of the gel was still maintained in the culture medium (B and C in Figure 9), and the section showed that the mesenchymal cells were evenly distributed in the gel block, without Significantly decreased (D, E and F in Figure 9). After dehydration after fixation, the gel block was condensed into a mass, and the section scanning electron microscope showed that the pyknotic spherical cells were distributed in the gel (A in Figure 9).

Example 2. Experiment of repairing cartilage defect with biomimetic gel combined with autologous enriched bone marrow nucleated cells

In order to explore the effect of biomimetic gel combined with autologous concentrated bone marrow nucleated cells in repairing articular cartilage damage, an animal experiment was designed to repair cartilage defects with a one-step method. The bionic gel combined with autologous concentrated bone marrow nucleated cells was used to repair pig knee articular cartilage defects. Taking Guizhou Xiaoxiang pigs as experimental animals, the bone marrow was first aspirated at the iliac crest under general anesthesia, and the autologous concentrated bone marrow nucleated cells were obtained through separation of bone marrow separation fluid, mixed with type II collagen, and then added with oxidized hyaluronic acid to cross-link. A full-thickness cartilage defect model with a diameter of 8 mm was prepared at the femoral trochlea of the knee joint by surgical means, and a hydrogel of mixed cells was implanted into the cartilage defect site. At 1, 3, and 6 months after operation, MRI, gross observation, stereomicroscope, and pathological sections were used for observation and analysis. Through this part of animal experiments, explore the experimental effect of this strategy in repairing cartilage defects, verify the feasibility of this strategy, and provide a basis for later clinical application.

The specific experimental method is as follows:

According to the random number table method, 40 Guizhou small pigs were randomly divided into two groups, A and B, with 20 knees in each group, a total of 40 knees, to avoid secondary damage to the repaired joints, and only one side of the joint was operated on; group A was the blank control group (simple hyaline cartilage defect group); group B was the experimental group (type II collagen-oxidized hyaluronic acid hydrogel combined with autologous bone marrow enriched nucleated cell repair group); knee joints in each group were treated at 4 time points (1, 3, 6 and 12 months) were collected for observation, detection and evaluation, and 5 knee joint samples were selected for each sampling time point.

Acquisition of autologous enriched bone marrow nucleated cells from Guizhou Xiaoxiang pigs: Intramuscular injection of Lumianning 4ml for basic anesthesia, 3% pentobarbital sodium solution at a dose of 0.2mL/kg ear vein injection to anesthetize Guizhou Xiaoxiang pigs; anesthetize Guizhou Xiaoxiang pigs; Guizhou piglets were placed on the operating table in a lateral position, and the contralateral anterior superior iliac spine area was prepared, disinfected, and draped. Use a 16-gauge bone marrow puncture needle to pierce at the iliac crest, and then insert the needle for about 1 cm after feeling a void, take out the needle core, and draw bone marrow blood with a 20 ml syringe (heparin 2 ml) that has been pre-wetted with heparin (1000 U/ml). Properly adjust the position and depth of the puncture needle according to the amount of aspiration, aspirate about 20ml in total, and send it to the laboratory for separation of autologous concentrated bone marrow nucleated cells; inject the extracted anticoagulated Guizhou Xiaoxiang pig bone marrow fluid into a 50ml centrifuge tube, Then add an equal volume of sterile PBS solution and mix well. Slowly inject the mixed bone marrow fluid into the cell separation solution containing an equal volume of Percoll (1.073g/ml) along the wall of the centrifuge tube. The injection process is carried out slowly to keep the integrity of the interface between the separation solution and the mixed solution. Prevent the bone marrow fluid from mixing with the Percoll cell separation medium due to too fast speed, which will affect the separation effect. Autologous concentrated bone marrow nucleated cells were obtained by Percoll density gradient centrifugation: the mixed liquid was centrifuged at 2000rpm for 15 minutes, and the liquid stratification phenomenon was seen: from top to bottom: plasma layer, nucleated cell layer (including bone marrow mesenchymal stem cells) ), transparent separation fluid layer and red blood cell layer. Carefully draw the nucleated cell layer (the second white cloudy layer) into a new centrifuge tube, add 10ml of PBS solution to wash the absorbed nucleated cells, centrifuge and wash the cells at 1500rpm for 5min, and discard the supernatant. Add 10ml of PBS again to wash the cells by pipetting, centrifuge at 1000rpm for 5min, and discard the supernatant. Add 5ml PBS to resuspend, and use a cell counting plate to count the cells, collect the cells for later use; type II collagen complex bone marrow nucleated cells: take 2× ¹⁰⁶ bone marrow nucleated cells, put them in EP tubes, and centrifuge at 1000rpm After 5 minutes, the supernatant was discarded, and 100mg type II collagen was added to mix evenly, and the air bubbles in the mixed gel were removed by vortexing and centrifugation.

Then carry out animal surgery, the specific steps are as follows:

a. After extracting the bone marrow, cut the hair with a hair clipper on the opposite knee to prepare the skin. The size of the skin preparation is about 40cm×30cm. Use sterile gauze dipped in antibacterial hand sanitizer to repeatedly scrub the operation area until it is clean and free of dirt, and spray the operation area with an iodophor pot . Place the operated animal on the V-shaped groove of the animal operating table, take the side lying position, spray iodine again for disinfection, and lay a disposable sterile towel. Take the parapatellar approach on the lateral side of the knee joint, incise the skin longitudinally about 6 cm long, incise the subcutaneous tissue and joint capsule layer by layer, dislocate the patella medially, and expose the femoral trochlea;

b. Use a self-made cartilage defect sleeve with a diameter of 8 mm to drill a circular cartilage defect in the femoral trochlea, the depth of which is consistent with the thickness of the hyaline cartilage layer. Use ophthalmic scissors to cut the hyaline cartilage in the ring into a mesh shape, scrape off the shredded hyaline cartilage with a small curette, and ensure the integrity of the calcified cartilage layer structure, and ensure that there is no bone marrow blood seeping out of the base, and trim the bottom and edge of the defect. Prepare a full-thickness cartilage defect model with a diameter of 8mm;

c. Rinse the modeling area with a saline pulse gun to remove cartilage fragmentation, and add 10 μl of oxidized hyaluronic acid solution to the type II collagen gel of composite cells. The nerve stripper was stirred evenly and implanted in the cartilage defect site, and shaped to make the gel surface flush with the cartilage surface, scrape off excess gel around it, and let it stand for 2 minutes until the gel complex was fully gelled. Restore the patella and passively move the knee joint several times to see that the gel block is firmly attached. Restore the patella with absorbable suture to reinforce the suture of the medial retinaculum, then pulse flushing, and suture the subcutaneous and skin layer by layer;

d. Immediately after the operation, give Luxingning 1 intramuscular injection to wake up, and veterinary penicillin potassium 4 million units intramuscular injection to prevent infection. The knee joint was not fixed after the operation, and the operation area was disinfected twice a day with povidone iodine, and antibiotics were injected intramuscularly twice (veterinary penicillin potassium 4 million units). The animals were fed with a regular diet, and the basic conditions, wound healing and walking gait of the animals were observed daily. Four Guizhou miniature pigs were dropped out of the study series due to anesthesia-related death (n=2), joint dislocation (n=1) and postoperative infection (n=1). Therefore, a total of 36 Guizhou Xiaoxiang pigs completed all operations and postoperative observation and evaluation. Therefore, this study temporarily terminated the long-term follow-up for 12 months after operation, and only followed up the repair effect at 1, 3 and 6 months after operation. The remaining 36 small fragrant pigs basically returned to normal walking gait 7 days after the operation, and the surgical incisions healed to grade A about 10 days after the operation. In the 1-month follow-up group, the data collection time was 33±3 days after operation; in the 3-month follow-up group, the data collection time was 92±1 days after operation; in the 6-month follow-up group, the data collection time was 183±5 days after operation.

(1) MRI score

The modified magnetic resonance observation of cartilage repair tissue score (Modified magnetic resonance observation of cartilage repair tissue score, MOCART) was used to semi-quantitatively score the repair effect of cartilage defects. The specific scoring criteria are shown in Table 1:

Table 1. Modified cartilage repair tissue MR observation scoring system (MOCART)

The scoring results are shown in Table 2 and A in Figure 10. The results showed that the MOCART scores of the blank control group and the experimental group were 10 ± 0 and 53.5 ± 4.74 (n=10, p< 0.001); 42±4.83 and 89.5±5.99 (n=10, p<0.001); and 64±6.99 and 96±5.16 (n=10, p<0.001).

Table 2. MOCART score of cartilage repair tissue

Values are expressed as mean ± SD, Mann-Whitney’s U-test was used. *p<0.05 (Treat.Vs.Cont.)

^a In the original MOCART, the subchondral bone plate was replaced by a calcified layer.

The results show that using the (II collagen-oxidized hyaluronic acid hydrogel compounded with autologous bone marrow concentrated nucleated cells prepared by the present invention has a better effect on cartilage repair.

(2) Modified O'Driscoll histological score: The modified O'Driscoll histological score was used to semi-quantitatively analyze the cartilage defect repair effect of the experimental animals in the two groups, and the results are shown in Table 3 and B in Figure 10. The modified O'Driscoll histological scores of the control group and the treatment group at 1, 3 and 6 months after operation were: 0 vs 13.3±2.21 (n=10, p<0.001); 6.1±2.02vs 16.8±1.4 (n=10 , p<0.001) and 8±1.49 vs 17.6±1.26 (n=10, p<0.001). The repair effect of damaged cartilage in the surface treatment group was better than that in the control group.

Table 3. Modified O’Driscoll histological score of cartilage repair tissue

Values are expressed as mean ± SD, Mann-Whitney’s U-test was used. *p<0.05 (Treat.Vs.Cont.)

(4) Modified ICRS histological score: The modified ICRS histological score was used to evaluate and analyze the repair effect of cartilage defects, including the degree of defect repair, the integration of new tissue and adjacent cartilage, the color of the repaired tissue, the general appearance, and the mechanical strength of the repaired tissue. The evaluation analysis, the results are shown in Table 4. The modified ICRS histological scores of the control group and the treatment group at 1, 3 and 6 months after operation were: 0 vs 16.6±1.78 (n=10, p<0.001); 11.3±2.71 vs 19.3±1.64 (n=10, p<0.001); <0.001) and 15.1±1.85 vs 19.9±0.32 (n=10, p<0.001).

Table 4. Modified ICRS histological score of cartilage repair tissue

Values were presented as mean±SD, Mann-Whitney’s U-test was used.*p<0.05(Treat.Vs.Cont.)

The above results indicated that the oxidized hyaluronic acid-type II collagen biomimetic gel compounded with autologous enriched bone marrow nucleated cells had a good repair effect on cartilage defects in Guizhou Xiaoxiang pigs. The cartilage defect area is filled with cartilage-like new repair tissue, the surface of the repair tissue is flat, and it is well integrated with the adjacent normal cartilage. The cells in the stroma are similar in shape to normal chondrocytes, with division phenomenon, self-renewal ability, and chondrogenic differentiation of mesenchymal stem cells. The content of type II collagen and GAG in the repaired tissue was significantly higher than that of the blank control group, and its MOCART score, O’Driscoll histological score and ICRS histological score were also significantly better than the blank control group, which has the potential of clinical transformation. The principle is: oxidize hyaluronic acid through sodium periodate to modify hyaluronic acid, oxidize its hydroxyl group into aldehyde group, and react the aldehyde group on hyaluronic acid with the amino group on type II collagen through Schiff's base. Chemical cross-linking occurs to form a gel (as shown in Figure 11), this method overcomes the problem that hyaluronic acid cannot polymerize with type II collagen.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that it can be described in terms of form and Various changes may be made in the details without departing from the scope of the invention defined by the claims.

Claims (8)

1. The articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells, characterized in that it is made of oxidized hyaluronic acid, type II collagen and autologous concentrated bone marrow nucleated cells. 2. The articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells according to claim 1, characterized in that: oxidized hyaluronic acid, type II collagen and autologous concentrated bone marrow nucleated cells The amount of addition is based on the addition of 100 mg of type II collagen gel and 10 μl of oxidized hyaluronic acid solution with a concentration of 100 mg/ml for every 2×10 ⁶ autologous concentrated bone marrow nucleated cells. 3. According to claim 1, the articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells is characterized in that: the oxidized hyaluronic acid of 100mg/ml is added by every 100mg oxidized hyaluronic acid 1ml of PBS solution, prepared by complete dissolution. 4. The articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells according to any one of claims 1 to 3, characterized in that: the type II collagen is prepared by the following method: Stir and suspend the freeze-dried cartilage powder with a 4M guanidine hydrochloride solution at a mass volume ratio of 20:1 (g:L), and place it at 4°C after stirring to digest the mucopolysaccharide in the cartilage powder matrix to a homogenate state ; After the digestion is complete, centrifuge the digested product at 4°C and 8000rpm for 20min, discard the supernatant, collect the precipitate, add water to dissolve it, then add acetic acid equivalent to 3/50 times the volume of the guanidine hydrochloride solution, add an amount equivalent to lyophilized Add pepsin with 0.1 times the weight of cartilage powder, add water until the final volume is twice that of the guanidine hydrochloride solution, adjust the pH value between 2.5-3, and then digest at 4°C for 48 hours; after digestion, place in a vertical high-speed centrifuge Centrifuge at 8000rpm for 20min, discard the precipitate, and collect the supernatant; then slowly pour 1.5M NaOH solution into the supernatant and stir until the pH value is 7.5, stop pouring, add NaCl to make the final concentration 3M, 4°C Centrifuge at 8000rpm for 20min under the same conditions to collect the sediment; divide the sediment into the dialysis bag and stop the dialysis when the conductivity of the dialysate is close to the conductivity of pure water; put the viscous colloidal solution in the dialysis bag at 4°C, immediately Centrifuge at 8000rpm for 20min in a high-speed centrifuge, collect the precipitate into a sterile volumetric flask, add 0.15mol/L hydrochloric acid solution, and dissolve the white precipitated protein after 3 days. Disperse and dissolve into a gel, stir evenly, subpackage, freeze-dry, spare. 5. The articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells according to any one of claims 1 to 3, wherein the oxidized hyaluronic acid is prepared by the following method: Prepare a hyaluronic acid solution with a mass fraction of 2%, then add 0.1g/ml sodium periodate solution, stir, and completely oxidize for 6 hours under dark conditions, then add ethylene glycol to terminate the reaction, and then add the reaction solution Pour it into a dialysis bag until the conductivity is close to pure water and end the dialysis to obtain oxidized hyaluronic acid. 6. The articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells according to any one of claims 1 to 3, characterized in that the autologous concentrated bone marrow nucleated cells are prepared by the following method: Anesthetize the current animal, then use a syringe that has been pre-wetted with 1000U/ml heparin to draw bone marrow blood, then add the extracted anticoagulant bone marrow fluid to an equal volume of sterile PBS solution, mix well, and then pour the bone marrow fluid along the tube The wall was slowly injected into an equal volume of Percoll cell separation liquid with a concentration of 1.073g/ml. During the injection process, the interface between the separation liquid and the mixed liquid was kept intact, and then Percoll density gradient centrifugation was used to obtain autologous concentrated bone marrow nucleated cells. First centrifuge the mixed liquid at 2000rpm for 15min, absorb the nucleated cell layer, add PBS solution to wash the absorbed nucleated cells, centrifuge and wash the cells at 1500rpm for 5min, discard the supernatant; add PBS again to blow and wash the cells, and centrifuge at 1000rpm for 5min , discard the supernatant, and add PBS to resuspend. 7. The preparation method of the articular cartilage repair material based on oxidized hyaluronic acid-type II collagen and autologous concentrated bone marrow nucleated cells according to any one of claims 1 to 6, characterized in that, the specific steps are as follows: the autologous concentrated bone marrow has Nuclear cells are first mixed with type II collagen before adding oxidized hyaluronic acid for cross-linking. 8. The use of the articular cartilage repair material according to any one of claims 1 to 6 in the preparation of articular cartilage repair materials.