CN116650729A - Preparation method of novel cartilage matrix bionic material - Google Patents

Abstract

The invention discloses a chemically reinforced decellularized living transparent cartilage graft (eHCMG), which uses a double emulsion method to prepare gelatin microspheres and screens the gelatin microspheres; preparing sodium alginate hydrogel prepolymer liquid, pretreating the screened gelatin microspheres, adding the pretreated gelatin microspheres into the sodium alginate hydrogel prepolymer liquid, culturing cartilage cells of pigs by using a microporous hydrogel three-dimensional culture system (PTCC), secreting cartilage matrixes, and forming a matrix interpenetrating network system; culturing the constructed matrix interpenetrating network system in vitro by using a CC culture medium, and decomposing alginic acid by using sodium citrate to obtain a cell-carrying living transparent cartilage graft (HCLG); obtaining a decellularized hyaline cartilage graft (HCMG) by using a decellularization method; the use of Chondroitin Sulfate (CS) crosslinked with collagen fibers in the decellularized hyaline cartilage graft resulted in a reinforced decellularized hyaline cartilage graft (eHCMG). Brings a new therapeutic strategy and a reference system for the cartilage repair field.

Description

Preparation method of a new cartilage matrix biomimetic material

technical field

The invention relates to the fields of medicine and biomedical engineering, in particular to a preparation method of a novel cartilage matrix bionic material.

Background technique

Articular cartilage, a connective tissue covering the epiphyseal surface of joint bones, is highly hydrated, has blood and lymphatic vessels, and has only a single cell type, chondrocytes. Continuous exposure of articular cartilage to mechanical stress can easily cause cartilage damage or cartilage degeneration, which in turn leads to osteoarthritis. Once a cartilage defect occurs, it is almost impossible for articular cartilage to repair and regenerate spontaneously. Even, due to the vicious circle existing between cartilage defect and osteoarthritis, it will lead to aggravated cartilage destruction and long-term development of osteoarthritis. Therefore, an effective approach to the treatment of osteoarthritis relies on successful orthotopic implantation of cartilage grafts.

In the field of tissue engineering, extracellular matrix (ECM), regarded as an ideal biological scaffold, is widely used in tissue repair and regenerative medicine, including cartilage repair. ECM is secreted by cells around tissues and has tissue-specific three-dimensional microstructure and functional bioactive molecules. The extracellular matrix is decellularized to remove immunogenic cellular components, and can be made into a decellularized extracellular matrix (dECM) scaffold. Scaffolds can provide sufficient mechanical support for regenerating tissues, release biological signals, guide cell proliferation and tissue remodeling, and ultimately complete the repair and replacement of damaged tissues. However, traditional natural cartilage-derived dECM scaffolds have limitations: the high cell mass makes it difficult to completely remove the decellularized reagents and easily damages the ECM; the hard tissue that concentrates the ECM makes integration with the surrounding cartilage problematic.

Contents of the invention

The present invention aims at the problem of impaired extracellular matrix (ECM) function and poor integration with surrounding cartilage tissue existing in natural cartilage-derived acellular cartilage grafts in the prior art, and provides a new type of bionic cartilage matrix The method of preparation of the material.

The object of the present invention is achieved in that a kind of MWCNT/PCN/Co3O4 preparation method of composite nanomaterial comprises the following steps:

(1) Preparation of gelatin microsphere pore-forming system: gelatin microspheres were prepared by the double emulsion method, and gelatin microspheres with a diameter of 150-180 μm were screened out to be evenly dispersed in a PBS solution containing 10% PS for sterilization. use;

(2) Construct matrix interpenetrating network: configure sodium alginate hydrogel prepolymerization liquid, pretreat gelatin microspheres screened in step (1), add sodium alginate hydrogel prepolymerization liquid, use microporous water Gel three-dimensional culture system (PTCC) cultivates pig chondrocytes, secretes cartilage matrix, and forms a matrix interpenetrating network system;

(3) Preparation of cartilage base material: The matrix interpenetrating network system constructed in step (2) was cultured in vitro for more than 30-35 days in CC medium, and the alginic acid component was dissociated with sodium citrate to obtain a transparent cell-loaded living body cartilage graft (HCLG);

(4) Culture the hyaline cartilage graft prepared in step (3) for another 10-15 days, and use the decellularization method to obtain the decellularized hyaline cartilage graft (HCMG);

(5) Using chondroitin sulfate (CS) to cross-link the collagen fibers in the acellular hyaline cartilage graft prepared in step (4), to obtain a reinforced acellular hyaline cartilage graft (eHCMG).

Further, the preparation of gelatin microspheres by the double emulsion method described in step (1) specifically includes the following steps:

(1.1) Mix ethyl acetate and gelatin solution, and stir at 600-800 rpm for 1-3 minutes to obtain a gelatin solution/ethyl acetate mixture, namely mixed emulsion 1;

(1.2) Mix mixed emulsion 1 and soybean oil, and stir at 600-800 rpm for 1-3 min to obtain mixed emulsion 2;

(1.3) Drop the temperature of mixed emulsion 2 to below 10 ℃, and continue to stir for 12-18 min to obtain mixed emulsion 3; filter mixed emulsion 3, remove the oil phase, and put the filtered gelatin microspheres at -15 ℃ to Stir rapidly in ethanol at -22 ℃; remove the ethanol on the upper layer, wash the gelatin microspheres on the lower layer once with dioxane and acetone, then wash three times with absolute ethanol, and dry them in an oven overnight to obtain gelatin microspheres. ball particles.

Further, the concentration of the gelatin solution is 0.08-0.12 g/mL, and the volume ratio of the gelatin solution to ethyl acetate is 1:2.2-2.8; in step (1.2), the volume of the mixed emulsion 1 and soybean oil The ratio is 1:1.3-1.6.

Further, the pretreatment of the gelatin microspheres is specifically: heating the gelatin microsphere particles at 120-130°C for 1.5-2.5 hours, and then rapidly cooling them at -15 to -23°C for 0.5-0.8h to obtain the quenched gelatin microspheres; add penicillin/streptomycin (PS) lotion to the quenched gelatin microspheres, disperse the gelatin microspheres, and let stand overnight at 4°C to obtain matrix interpenetrating network system .

Further, the PS lotion is prepared by mixing penicillin/streptomycin with phosphate buffer (10 mM phosphate, 150 mM sodium chloride, pH 7.3 to 7.5) and filtering; the solution contains 400-600 units/mL penicillin and 400-600 µg/mL streptomycin.

Further, the configuration of the alginic acid-based hydrogel prepolymer solution in step (2) is as follows: the concentration of sodium alginate solution is 1.4-1.7% (wt); the concentration of chondrocytes is 0.5-1.5*10 ⁶ cells/mL , the concentration of the gelatin microsphere pore-forming system is 0.2-0.4g/mL.

Further, in step (3), the in vitro culture conditions are 30-35 days at 37°C and 5% CO ₂ environment, using sodium citrate (50-58 mM concentration) to dissociate calcium alginate 8- After 12 minutes, the hyaline cartilage graft (HCLG) loaded with cells was obtained.

Further, in step (3), the CC medium includes high-sugar DMEM as the basal medium, 20% fetal bovine serum, 1 mmol/L HEPES, 1 mmol/L sodium pyruvate, 0.4 mmol/L proline , 0.1 g/mL vitamin C.

Further, in step (4), the decellularization method can be prepared by one of the following methods:

Method 1. Use Triton X-100 (Triton X-100) method to decellularize the prepared cell-loaded living hyaline cartilage graft (HCLG). The specific steps include:

a) Soak the prepared HCLG in a hypotonic solution (0.1-0.3% EDTA in 10-15 mM Tris-HCl buffer, pH=8-9) for 24-36 hours;

b) use 3-5% Triton X-100 solution to soak for 24-36 hours;

c) Wash 3-4 times with 1×PBS (50-60 mL) solution, 3-4 hours each time;

d) Add DNase solution (0.5-0.75 mg/mL) for 3-4 hours;

e) Wash with 1×PBS (50-60 mL) solution overnight, and then wash with deionized water 3-4 times, each time for 3-4 hours.

Method 2. The prepared cell-loaded living hyaline cartilage graft (HCLG) was decellularized by using Triton sodium hydroxide (NaOH) + Triton X-100 (Triton X-100) method. Specific steps include:

a) Soak the prepared HCLG into 0.4-0.6% NaOH solution for 45-60 minutes (2-8°C);

b) Treat with a hypotonic solution containing 1% Triton X-100 for 48-60 hours;

c) Immerse in PBS solution and wash 3-4 times, each time for 3-4 hours;

d) Add DNase solution (0.5-0.75 mg/mL) for 3-4 hours;

e) Wash with PBS solution overnight, and then wash with deionized water 3-4 times, 3-4 hours each time.

Method 3. The prepared cell-loaded living hyaline cartilage graft (HCLG) was decellularized by using Triton X-100 (Triton X-100) + sodium hydroxide (NaOH) method. Specific steps include:

Soak the prepared HCLG into a hypotonic solution containing 1-1.2% Triton X-100 for 48-60 hours;

Then use 0.4-0.6% NaOH solution to soak for 45-60 minutes (2-8°C);

Wash 3-4 times with 1× PBS (50-60 mL) solution, 3-4 hours each time;

Add DNase solution (0.5-0.75 mg/mL) for 3-4 hours;

Wash with 1×PBS (50-60 mL) solution overnight, and then wash with deionized water 3-4 times, each time for 3-4 hours.

Further, the chemical reinforcement method in step (5) includes the following steps:

(5.1) Weigh 50-60 mg of decellularized hyaline cartilage graft (HCMG), and place it in 20-30 mL of 50-60 mM tetramorpholineethanesulfonic acid (MES) buffer containing 40-50% ethanol (pH = 5.5-6.5) pretreatment for 30-60 minutes;

(5.2) Take out the decellularized hyaline cartilage graft (HCMG) and place it in 20-30 mL of 40-50% ethanol solution containing 50-60 mM MES, 1-ethyl-(3-dimethylaminopropyl The molar ratio of carbodiimide (EDC) to N-hydroxysuccinimide (NHS) is 1:1-1:1.2, and the molar concentrations of EDC are 5-6 mM, 10-12 mM, 30 -35 mM, 50-55 mM;

(5.3) Add 2-3 % (w/v) chondroitin sulfate (CS) to the reaction system, react at room temperature for 4-5 hours, and obtain enhanced acellular hyaline cartilage grafts under the conditions of 4 different cross-linking agent concentrations substance (eHCMG);

(5.4) All four materials were washed 6-7 times with PBS, 2-3 hours each time, and then washed 3-4 times with deionized water, 2-3 hours each time, to wash away the cross-linking agent residue.

The present invention uses the double emulsion method to prepare and screen out the required gelatin microspheres, and encapsulates the prepared gelatin microspheres and porcine chondrocytes in sodium alginate hydrogel, and the gel containing gelatin microspheres and soft cells at 37 ℃, 5% CO ₂ for 35 days, the medium is liquid DEME (Dulbecco's Modified Eagle Medium) medium containing 20% fetal bovine serum (FBS), and the living hyaline cartilage graft (HCLG) loaded with cells is obtained. Tissue residue will cause the defect that the biological activity of the prepared hyaline cartilage graft is reduced. The decellularized hyaline cartilage graft (HCMG) is obtained by using the decellularization method. To solve the problem of poor mechanical properties of some living hyaline cartilage grafts, the present invention prepares The decellularized living hyaline cartilage graft (HCMG) was chemically reinforced using chondroitin sulfate (CS) to cross-link the collagen fibers in the decellularized hyaline cartilage graft to reinforce the material and compensate for the loss of polysaccharides during the process, resulting in Enhanced decellularized hyaline cartilage graft (eHCMG).

Description of drawings

Figure 1 is the cross-sectional SEM images of HCMG samples prepared by five different decellularization methods.

Figure 2 is a schematic diagram of the preparation of chondroitin sulfate-reinforced decellularized hyaline cartilage graft (eHCMG).

Fig. 3 is a scanning electron microscope cross-sectional view of acellular hyaline cartilage graft (eHCMG) reinforced with different concentrations of chondroitin sulfate.

Figure 4 shows the cross-linking statistics of different concentrations of chondroitin sulfate reinforced decellularized hyaline cartilage graft (eHCMG).

Figure 5 shows the statistics of the swelling rate of the acellular hyaline cartilage graft and the acellular hyaline cartilage graft (eHCMG) reinforced with different concentrations of chondroitin sulfate.

Figure 6 A is the stress-strain curves of five groups of scaffold materials (HCMG is the control group); B is the compressive modulus of five groups of scaffold materials (40% strain).

Fig. 7 is the change trend diagram of the elastic modulus and dissipation modulus of the stress-strain curves (HCMG is the control group) of the five groups of scaffold materials.

Figure 8 shows the DNA residues of different decellularization methods.

Figure 9 shows the statistics of cellular glycosaminoglycan (GAG) and collagen (Collagen) content of acellular hyaline cartilage grafts and different concentrations of chondroitin sulfate-reinforced acellular hyaline cartilage grafts (eHCMG).

Figure 10 shows the distribution statistics of collagen (Collagen) types in the acellular hyaline cartilage grafts and the acellular hyaline cartilage grafts (eHCMG) reinforced with different concentrations of chondroitin sulfate.

Fig. 11 Safranin (Saf-O) staining and Mason (MT) staining of decellularized hyaline cartilage grafts and decellularized hyaline cartilage grafts reinforced with different concentrations of chondroitin sulfate (eHCMG).

Fig. 12 Live/dead cell staining (A) and cytotoxicity statistics (B) of decellularized hyaline cartilage grafts and acellular hyaline cartilage grafts reinforced with different concentrations of chondroitin sulfate (eHCMG).

Implementation

Example 1: Preparation of living decellularized hyaline cartilage graft (eHCMG):

(1) Preparation of living cell-loaded hyaline cartilage graft (HCLG):

a) Extraction of pig-derived chondrocytes: Fresh domestic pig hind leg knee joints were obtained from a local fresh supermarket, excess tissue was removed with surgical instruments, cartilage tissue was taken, and sterilized with 75% for use. The stripped cartilage tissue was washed three times with 10% PS solution, and then washed with PBS. The washed tissues were transferred to modified DMEM medium containing 12% fetal bovine serum (FBS). The primary porcine articular chondrocytes were obtained by type II collagenase digestion, and cultured in a modified DMEM medium containing 12% FBS;

b) Preparation of HCLG: firstly, gelatin microspheres were prepared by the double emulsion method, and the microspheres with a diameter of 165 μm were screened out and uniformly dispersed in a PBS solution containing 10% PS for sterilization. The obtained porcine chondrocytes (1.0×10 ⁶ cell/mL) and gelatin microspheres (0.35 g/mL) were co-encapsulated in sodium alginate hydrogel, and then the constructed hydrogel was cultured in a non-adherent culture cultured in special DMEM medium containing 17% FBS. The culture was carried out on an orbital shaker in a humidified incubator at 37 °C and 5% CO ₂ . After 34 days of culture, the alginate component was washed away with 56 mM sodium citrate solution to obtain HCLG. HCLG continued to culture for 7 days in preparation for decellularization and further use.

(2) Preparation of living decellularized hyaline cartilage graft (HCMG):

a) The living hyaline cartilage graft containing cells prepared by the Triton X-100 method was decellularized. Specific steps include:

i) Soak the prepared HCLG in a hypotonic solution (0.2% EDTA in 13 mM Tris-HCl buffer, pH=8.5) for 24 hours;

ii) use 4% Triton X-100 solution to soak for 24 hours;

iii) Wash with 1×PBS (55 mL) solution 3 times, 3 hours each time;

iv) Add DNase solution (0.6 mg/mL) for 3 hours;

v) Wash with 1×PBS (55 mL) solution overnight, and then wash with deionized water 3 times, 3 hours each time.

(3) Preparation of living decellularized hyaline cartilage graft (eHCMG):

Weigh 55 mg of HCMG and pretreat it in 25 mL of 50 mM tetramorpholineethanesulfonic acid (MES) buffer (pH=5.5) containing 45% ethanol for 30 minutes;

The HCMG was taken out and placed in 25 mL of 45% ethanol solution containing 55 mM MES, 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide The molar ratio of amine (NHS) is 1:1.05, and the molar concentrations of EDC are 5 mM, 10 mM, 30 mM, 50 mM;

Add 2.5 % (w/v) chondroitin sulfate (CS) to the reaction system, react at room temperature for 4 hours, and obtain enhanced decellularized hyaline cartilage grafts (eHCMG) under 4 different cross-linking agent concentrations;

All four materials were washed 6 times with PBS, 2 hours each time, and then washed 3 times with deionized water, 2 hours each time, to wash away the cross-linking agent residue;

Example 2: The difference between this example and Example 1 lies in the different decellularization methods in the preparation of the living decellularized hyaline cartilage graft (eHCMG).

(1) The living hyaline cartilage graft containing cells was decellularized by using Triton sodium hydroxide (NaOH) + Triton X-100 (Triton X-100) method. Specific steps include:

a) Soak the prepared HCLG in 0.4% NaOH solution for 45 minutes (6°C);

b) treated with a hypotonic solution containing 1% Triton X-100 for 48 hours;

c) Immerse in PBS solution and wash 3 times, 3 hours each time;

d) Add DNase solution (0.6 mg/mL) for 3 hours;

e) Wash with PBS solution overnight, and then wash with deionized water 3 times, 3 hours each time.

Example 3: The difference between this example and Example 1 lies in the different decellularization methods in the preparation of decellularized living hyaline cartilage graft (eHCMG):

(2) The hyaline cartilage grafts loaded with cells were decellularized by using Triton X-100 (Triton X-100) + sodium hydroxide (NaOH) method. Specific steps include:

The prepared HCLG was soaked into a hypotonic solution containing 1% Triton X-100 for 48 hours;

Soak in 0.5% NaOH solution for 45 minutes (2-8°C);

Wash 3 times with 1× PBS (55 mL) solution, 3 hours each time;

Add DNase solution (0.6 mg/mL) for 3 hours;

Wash with 1×PBS (55 mL) solution overnight, and then wash with deionized water 3 times, 3 hours each time.

The cross-sectional structure of HCMG samples prepared by five different decellularization methods was studied by scanning electron microscopy (SEM), and the results are shown in Figure 1. It can be seen from the figure that the collagen fibers formed a porous natural fiber network structure inside the HCLG. Compared with the control group, the HCMG prepared by different decellularization methods retained the network structure of the HCLG, but in different degrees, appeared hollow change. That is, some larger holes appeared in the fibers in the middle of the section, and the collagen membrane tended to be divided into two layers.

The schematic diagram of chemical reinforcement is shown in Fig. 2. The cross-sectional microstructures of four eHCMGs prepared with different concentrations of NHS/EDC crosslinker were photographed by scanning electron microscopy (SEM). The results are shown in Figure 3. It can be seen that compared with the control group, the cross-linked eHCMG still retains a loose and porous fiber network structure. However, as the concentration of the crosslinking agent increases, it can be found that the pores of the material become smaller, and the overall structure becomes more compact and orderly, presenting a highly porous structure that is interconnected and arranged in parallel.

Test Example 1: Determination of the degree of chemical cross-linking of enhanced decellularized hyaline cartilage grafts (eHCMG):

Prepare 4% sodium bicarbonate as a reaction buffer solution, accurately weigh HCMG and four kinds of eHCMG samples to be about 1 mg, add 0.5 mL of sodium bicarbonate solution to each sample, and let stand for 30 minutes;

Prepare a fresh 0.05% trinitrobenzenesulfonic acid (TNBS) solution, add 0.5 mL of TNBS solution to the EP tube where each sample is located, and incubate for 4 hours in a water bath at 40 °C;

Prepare 6 M hydrochloric acid, add 1.5 mL hydrochloric acid to each sample, and place in a water bath at 60 °C for 90 minutes;

Measure the absorbance at 345 nm using a microplate reader;

The degree of cross-linking is expressed by the loss rate of free amino groups after cross-linking, and the calculation formula is as follows:

Among them, ABS _c represents the absorbance of the cross-linked group, ABS _nc represents the absorbance of the uncross-linked group (HCMG), m _c and m _nc are the masses of the two, and the free amino groups in the collagen are determined by the trinitrobenzenesulfonic acid (TNBS) TNBS method content to determine the degree of cross-linking of eHCMG.

Figure 4 shows the degree of cross-linking of eHCMG prepared with four different concentrations of NHS/EDC cross-linking agent compared with control HCMG. It can be seen from the figure that NHS/EDC has a fairly good cross-linking efficiency, and a high degree of cross-linking can be achieved in a short cross-linking time. With the increase of the concentration of cross-linking agent, the degree of cross-linking increased rapidly at the beginning, but when the concentration of cross-linking agent reached 10 mM, and then increased its concentration, the degree of cross-linking increased slowly. This may be due to the short cross-linking time, and the free amino groups on the surface of HCMG have basically reacted completely, while the free amino groups inside HCMG are difficult to fully react with the cross-linking agent.

Test Example 2: Swellability Test of Enhanced Decellularized Hyaline Cartilage Graft (eHCMG):

The samples were cut into discs with a diameter of 8 mm with a trephine cutter, and the samples were placed in a freeze dryer overnight to remove moisture. After weighing, record it as W ₁ . After immersing the sample in deionized water for 24 hours, take out the sample, absorb the moisture on the surface of the sample with a dust-free paper, and weigh it as W ₂ . The swelling rate is calculated by the following formula:

Figure 5 shows the changes in swelling properties of eHCMG prepared with four different concentrations of NHS/EDC cross-linking agent compared with the control group HCMG. It can be seen from the figure that the swelling rate of the 5 mM group and the 10 mM group did not increase significantly compared with HCMG. When the cross-linking agent concentration reached 30 mM, the swelling rate of eHCMG increased significantly, and the swelling rate of the 50 mM group and the 30 mM group remained basically unchanged. This is due to the formation of amide bonds between the free amino groups on the collagen in HCMG and the carboxyl groups in chondroitin sulfate, and the introduction of chondroitin sulfate. affinity.

(n=3, ns: no statistical difference, **p<0.01)

Test Example 3: Static Compression Test of Enhanced Decellularized Hyaline Cartilage Graft (eHCMG):

The samples were cut into 1 mm diameter discs with a trephine cutter and loaded on the rheometer. Adjust the distance between the indenter and the sample so that the indenter just touches the surface of the sample without pressure. Set the indenter speed to 0.01 mm/min for static compression test, and stop the test when the deformation is 80%. Record the data of compressive load and deformation, and make the stress-strain curve.

Five groups of stress-strain curves (HCMG is the control group) are given in Fig. 6A. It can be seen from the figure that all the stress-strain curves are upward concave curves, showing nonlinear characteristics, but showing elastic characteristics in the initial stage of strain. The compressive modulus of the material was obtained by linear fitting to the elastic deformation region (40% strain) of the material, as shown in Figure 6B. From the figure, we can find that the compressive modulus of eHCMG increases with the increase of crosslinker concentration.

Test Example 4: Dynamic Frequency Sweep Test of Enhanced Decellularized Hyaline Cartilage Graft (eHCMG):

The dynamic viscoelastic behavior of four different eHCMGs was tested by a DHR rotational rheometer. Dynamic frequency sweeps (storage modulus G´ and loss modulus G") were performed at a fixed strain in the frequency range of 0.1-10 Hz, the strain was fixed at 5%, and the stress was controlled between 0.001 N-0.2 N.

The dynamic mechanical viscoelasticity of eHCMG with different cross-linking agent concentrations was tested, and the results are shown in Figure 7. It can be seen from the figure that after adding CS for cross-linking, the storage modulus G' of the material is improved. With the increase of the cross-linking agent concentration, the G' of eHCMG gradually increases. When the concentration reaches 50 mM, G 'Reaches the maximum value. The change of the same dissipation modulus G" has a similar trend. When the concentration of the cross-linking agent is 5 mM, it is close to the control group, but it increases significantly when the cross-linking agent reaches 10 mM. At 50 mM, the dissipation modulus G" reaches its maximum value.

Test example 5: DNA residue test in living decellularized hyaline cartilage graft (HCMG):

Prepare 1 mg/mL Hochest 33258 ^® fluorescent dye and store at 4°C in the dark. Prepare 10×TNE buffer solution: 12.11 g tris(hydroxymethyl)aminomethane, 3.72 g EDTA, 116.89 g sodium chloride dissolved in ultrapure water, 1 M HCl to adjust the pH of the solution to 7.4, and then add ultrapure water to volume to 1 L. Take 50 mL of 1× TNE buffer and add 5 μL of Hochest33258 fluorescent dye to make Hoechst 33258 ^® working solution. Store at room temperature, ready to use now. Prepare calf thymus DNA standard solution: prepare 1 μg/mL calf thymus DNA solution with 1×TNE buffer, and serially dilute to 500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.25 ng /mL, 15.625 ng/mL, 7.8125 ng/mL and 0 ng/mL. Take 10 μL sample digestion solution and add 1 mL Hoechst 33258 ^® working solution. Incubate for 5 min in the dark. A microplate reader measures the absorbance at an absorption wavelength of 350 nm and an emission wavelength of 450 nm. The DNA standard solution is the same. Absorbance was converted to DNA content by a standard curve.

Figure 8 shows the DNA residues in HCMG prepared by different decellularization methods. As shown in the figure, according to the measured DNA residues, it was found that the DNA content in all HCMGs was reduced to an acceptable range.

(n=3, ****p<0.0001)

Test Example 6: Cell Glycosaminoglycan (GAG) Content Test in Enhanced Decellularized Hyaline Cartilage Graft (eHCMG):

Prepare DMMB chromogenic solution: Take 50 mL of glycine/sodium chloride solution, add 0.8 mg of 1,0-dimethylene blue-zinc chloride double salt dye (DMMB) to dissolve, and store at 4 °C in the dark for use. The glycine/sodium chloride solution was prepared as follows: Weigh 3.04 g glycine and 2.37 g sodium chloride, add 95 mL 0.1 M HCl solution to adjust the pH, and dilute to 1 L with deionized water. Prepare a standard solution of chondroitin sulfate (1 mg/mL) and serially dilute it so that the concentration of chondroitin sulfate solution is 0 µg/mL, 3.125 µg/mL, 6.25 µg/mL, 12.5 µg/mL, 25 µg/mL, 50 µg/mL and 100 µg/mL. Add 0.1 mL of each sample digestion solution to 1 mL of DMMB reagent, mix well, and immediately measure the absorbance of the mixture with a microplate reader at 530 nm (this operation should be carried out within 3 minutes after mixing). The standard solution is the same, and the absorbance is converted into GAG content through the standard curve.

Test Example 7: Test of total collagen content in enhanced decellularized hyaline cartilage graft (eHCMG):

Take 500 µL of the papain digestion solution of the sample to be tested, add 5 mL of 6M hydrochloric acid to the pressure bottle, seal the tube with nitrogen gas, hydrolyze at 105 °C for 22 hours, wait to cool to room temperature, and adjust the pH to 6-8 with 6 M NaOH solution , and dilute to 10 mL with primary water. Dilute hydroxyproline standards to 7.5 µg/mL, 3.75 µg/mL, 1.875 µg/mL, 0.938 µg/mL, 0.469 µg/mL, 0.234 µg/mL, 0.117 µg/mL, 0 µg/mL. According to the operation of the hydroxyproline kit, after adding the relevant reagents to the hydrolyzed sample, measure the absorbance value at 560 nm, and the hydroxyproline standard solution is the same. Absorbance was converted to hydroxyproline content by drawing a standard curve. The hydroxyproline content was converted to total collagen content according to the ratio of hydroxyproline in the articular cartilage collagen.

The biochemical analysis of the glycosaminoglycan components and collagen components in the four eHCMGs with different cross-linking agent concentrations is shown in FIG. 9 . The results showed that compared with the control group HCMG, the GAG content of all eHCMGs was significantly increased, while the collagen content was decreased. And with the increase of cross-linking agent concentration, the content of GAG increased correspondingly, while the content of collagen decreased in turn. This shows that with the increase of the concentration of cross-linking agent, the content of cross-linked CS in eHCMG gradually increases within the same time period, and at the same time the proportion of collagen in the material naturally decreases accordingly. (n=3,, *p<0.05, **p<0.01)

Test Example 8: Immunofluorescent staining of collagen expression patterns in enhanced decellularized hyaline cartilage grafts (eHCMG):

Immunofluorescent staining was performed on four eHCMGs to determine the type of collagen (collagen I and collagen II) in the material. Graft samples were fixed overnight with 4% paraformaldehyde. After washing with PBS solution, after equilibrating with sucrose in gradient sucrose solution, they were embedded with embedding agent, and sliced into 7 μm sections by cryostat. Frozen sections were immersed in water and washed twice for 3 minutes each time. Use an immunohistochemical pen to draw a closed hydrophobic circle around the sample. Block with 10% goat serum for 2 hours. Prepare the primary antibody solution, incubate with the sample for 12 hours (4°C) and pour off the blocking serum. Wash 3 times with PBS, 5 minutes each time. Incubate with secondary antibody solution at room temperature for 2 hours in the dark. Wash 3 times with PBS, 5 minutes each time. Prepare 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (dilution ratio 1:3000), incubate at room temperature for 25 minutes, and wash 3 times with PBS. Mount the slides with an anti-fluorescence quenching medium.

Immunofluorescence staining in Figure 10 also shows that cross-linked eHCMG has no change in collagen typing compared with HCMG (by comparison of fluorescence intensity), and still maintains a hyaline cartilage phenotype with high expression of type II collagen and weak expression of type I collagen .

Test Example 9: Enhanced Safranin O (Saf-O) Staining of Decellularized Hyaline Cartilage Grafts (eHCMG):

The prepared graft sample slices were immersed in water and washed twice for 3 minutes each time. The safranin O staining solution was dropped on the sample and treated for 10 minutes. Dump the dye solution and rinse with running water. Immerse the slices in 95% ethanol for 10 seconds and absolute ethanol for 1 minute. Dip the slices into xylene clear twice for 2 min each. Mount the slides with neutral resin. Photographs were taken using an optical microscope.

Test Example 10: Enhanced Mason's Trichrome (MT) Staining of Decellularized Hyaline Cartilage Graft (eHCMG):

The prepared graft sample slices were immersed in water and washed twice for 3 minutes each time. The slices were dipped in mordant solution and left overnight at room temperature or in an incubator at 60°C for 1 hour. Lapis lazuli dyed for 2 minutes, washed twice with water for 15 seconds each time. Mayer's hematoxylin was stained for 2 minutes, washed twice with water for 15 seconds each time. Use acidic ethanol differentiation solution for differentiation for a few seconds, wash with water to stop the differentiation, and wash with water for 10 minutes. Ponceau hong dyed for 10 minutes, washed 2 times, 15 seconds each time. Phosphomolybdic acid treatment for 8 minutes. The liquid was poured off, the sections were not washed with water, and stained with aniline blue for 5 minutes. Use weak acid to wash away the aniline blue solution, and continue to add weak acid dropwise for 2 minutes. Use 95% ethanol to dehydrate for 30 seconds, and 100% ethanol to dehydrate twice, each time for 1 minute. Clarify 2 times with 5 mL of xylene for 2 min each. Mount with neutral resin. Photographs were taken using an optical microscope.

The four eHCMGs were stained with Safranin O and Mason's trichrome, and the results are shown in Figure 11. The staining of safranin O showed that with the increase of cross-linking agent concentration, the positive degree of eHCMG safranin O staining became stronger. Mason's trichrome staining showed that the collagen structure of eHCMG did not change significantly compared with HCMG.

Test Example 11: Cytotoxicity of enhanced decellularized hyaline cartilage graft (eHCMG) extract:

Preparation of HCMG extract: put HCMG in the culture medium at a material-to-medium ratio of 0.1 g/mL for 24 hours. Digest mouse fibroblast L929 cells, plant the cells in a 24-well plate with 10,000 cells per well, and culture overnight to allow the cells to attach. The medium was sucked off, and the extract was added dropwise to culture the cells, and the medium was used as the control group. The Cell-Counting Kit-8 ^® kit was used to detect cell proliferation on the first day and the fourth day respectively.

Test Example 12: Enhanced Viability Staining of Extract from Decellularized Hyaline Cartilage Graft (eHCMG):

As previously described, on the fifth day after the cells were cultured with the extract, the cells in the 24-well plate were stained for live death, and the cells cultured in the normal medium were used as the control group. Dye solution preparation: Dilute Calcein AM ^® (Calcein AM) and propidium iodide ^® (PI) staining agent to 1×, mix 1:1 evenly and set aside. Aspirate the extract and medium, wash with PBS, then aspirate the PBS, add the prepared dye solution, and incubate in the incubator for 30 minutes. Aspirate the dye solution, add 250 mL of buffer solution to each well, then take pictures with a fluorescence microscope, and count the ratio of the luminescence intensities of the two dyes to determine the cell death/survival ratio.

Fig. 12A is a live-death staining image of L929 cells cultured with eHCMG extract on day 5. The results showed that compared with the control group, there were no obvious apoptotic cells in the 5mM and 10 mM groups, and the number of living cells was close. In the 30mM and 50mM groups, there were certain apoptotic cells, and the number of living cells was less. The cell proliferation toxicity of L929 cells cultured with eHCMG extract is shown in Figure 12B. Likewise, on the 4th and 7th day, the high concentration group (30 mM and 50 mM) showed some inhibition of cell proliferation, while the low concentration group (5 mM and 10 mM) had no obvious proliferation inhibition phenomenon. (n=3, ns: no statistical difference, *p<0.05, **p<0.01, ***p<0.001).

Claims (10)

1. The preparation method of the novel cartilage matrix bionic material comprises the following steps:

(1) Preparing a gelatin microsphere pore-forming system: preparing gelatin microsphere by double emulsion method, screening out gelatin microsphere with diameter of 150-180 μm, uniformly dispersing in PBS solution containing 10% PS, and sterilizing;

(2) Building a matrix interpenetrating network: preparing sodium alginate hydrogel prepolymer liquid, pretreating the gelatin microspheres screened in the step (1), adding the pretreated gelatin microspheres into the sodium alginate hydrogel prepolymer liquid, and culturing cartilage cells of pigs by using a microporous hydrogel three-dimensional culture system (PTCC), secreting cartilage matrixes and forming a matrix interpenetrating network system;

(3) Preparation of cartilage base material: culturing the matrix interpenetrating network system constructed in the step (2) in vitro for more than 30-35 days by using a CC culture medium, and dissociating alginic acid components by using sodium citrate to obtain a cell-carrying living transparent cartilage graft (HCLG);

(4) Culturing the hyaline cartilage graft prepared in the step (3) for 10-15 days, and obtaining a decellularized hyaline cartilage graft (HCMG) by using a decellularization method;

(5) Using Chondroitin Sulfate (CS) to crosslink with collagen fibers in the decellularized hyaline cartilage graft prepared in step (4) to obtain a reinforced decellularized hyaline cartilage graft (eHCMG).

2. The method according to claim 1, wherein the preparation of gelatin microspheres by the double emulsion method in step (1) comprises the steps of:

(1.1) mixing ethyl acetate with gelatin solution, stirring for 1-3 min at 600-800 rpm to obtain gelatin solution/ethyl acetate mixed solution, namely mixed emulsion 1;

(1.2) mixing the mixed emulsion 1 with soybean oil, and stirring for 1-3 min at 600-800 rpm to obtain mixed emulsion 2;

(1.3) suddenly reducing the temperature of the mixed emulsion 2 to below 10 ℃, and continuously stirring for 12-18 min to obtain mixed emulsion 3; filtering the mixed emulsion 3, removing the oil phase, and putting the filtered gelatin microspheres into ethanol with the temperature of between 15 ℃ below zero and 22 ℃ below zero for rapid stirring; removing the upper ethanol layer, cleaning the gelatin microsphere at the lower layer with dioxane and acetone once, cleaning with absolute ethanol three times, and oven drying overnight to obtain gelatin microsphere particles.

3. The method of manufacture as claimed in claim 2, wherein in step (1.1), the gelatin solution concentration is 0.08-0.12 g/mL and the volume ratio of gelatin solution to ethyl acetate is 1:2.2-2.8; in the step (1.2), the volume ratio of the mixed emulsion 1 to the soybean oil is 1:1.3-1.6.

4. The preparation method according to claim 1, wherein in the step (2), the pretreatment of the gelatin microsphere is specifically: heating gelatin microsphere particles at 120-130 ℃ for 1.5-2.5-h, and then rapidly cooling at-15-23 ℃ for 0.5-0.8h to obtain quenched gelatin microsphere particles; the penicillin/streptomycin (PS) wash solution was added to the quenched gelatin microsphere particles and the gelatin microsphere particles were dispersed and allowed to stand overnight at 4 ℃ to give a matrix interpenetrating network system.

5. The method according to claim 4, wherein the PS washing solution is prepared by mixing penicillin/streptomycin with phosphate buffer (10 mM phosphate, 150 mM sodium chloride, pH 7.3 to 7.5) and filtering; the solution contains 400-600 units/mL penicillin and 400-600 mug/mL streptomycin.

6. The method according to claim 1, wherein the preparation of the alginic acid based hydrogel prepolymer in step (2) is carried out according to a concentration of sodium alginate solution of 1.4-1.7% (wt); chondrocyte concentration is 0.5-1.5 x 10 ⁶ The concentration of the gelatin microsphere pore-forming system is 0.2-0.4g/mL.

7. The method according to claim 1, wherein in step (3), the in vitro culture conditions are 37℃and 5% CO ₂ Culturing for 30-35 days under environment, and dissociating calcium alginate with sodium citrate (concentration of 50-58 mM) for 8-12 min to obtain cell-loaded living transparent cartilage graft (HCLG).

8. The method according to claim 1, wherein in the step (3), the CC medium comprises a high glucose DMEM-based medium, 20% fetal bovine serum, 1 mmol/L HEPES,1 mmol/L sodium pyruvate, 0.4 mmol/L proline, 0.1. 0.1 g/mL vitamin C.

9. The method of claim 1, wherein in step (4), the decellularized process is performed by one of the following methods:

method one, carrying out decellularization operation on the prepared cell-loaded living transparent cartilage graft (HCLG) by using a Triton X-100 (Triton X-100) method, comprising the following specific steps:

a) Soaking the prepared HCLG into hypotonic solution (10-15 mM Tris-HCl buffer solution with 0.1-0.3% EDTA, pH=8-9) for 24-36 hours;

b) Soaking in 3-5% Triton X-100 solution for 24-36 hr;

c) Washing 3-4 times with 1 XPBS (50-60 mL) solution for 3-4 hours each;

d) Adding DNase solution (0.5-0.75 mg/mL) for 3-4 hours;

e) Washing with 1 XPBS (50-60 mL) solution overnight, and washing with deionized water for 3-4 times, each for 3-4 hours;

and secondly, performing decellularization operation on the prepared cell-loaded living transparent cartilage graft (HCLG) by using a Triton sodium hydroxide (NaOH) +triton X-100 (Triton X-100) method. The method comprises the following specific steps:

a) Soaking the prepared HCLG into 0.4-0.6% NaOH solution for 45-60 min (2-8 ℃);

b) Treating with hypotonic solution containing 1% Triton X-100 for 48-60 hr;

c) Immersing in PBS solution for washing for 3-4 times, each time for 3-4 hours;

d) Adding DNase solution (0.5-0.75 mg/mL) for 3-4 hours;

e) Washing the PBS solution overnight, and then washing the PBS solution with deionized water for 3-4 times, each time for 3-4 hours;

method three, the prepared cell-loaded living transparent cartilage graft (HCLG) was subjected to decellularization using the Triton X-100 (Triton X-100) +sodium hydroxide (NaOH) method. The method comprises the following specific steps:

soaking the prepared HCLG into hypotonic solution containing 1-1.2% Triton X-100 for 48-60 hours;

soaking in 0.4-0.6% NaOH solution for 45-60 min (2-8deg.C);

washing 3-4 times with 1 XPBS (50-60 mL) solution for 3-4 hours each;

adding DNase solution (0.5-0.75 mg/mL) for 3-4 hours;

the solution was washed overnight with 1 XPBS (50-60 mL) and then 3-4 times with deionized water for 3-4 hours each.

10. The method of claim 1, wherein the chemical strengthening in step (5) comprises the steps of:

(5.1) weighing 50-60 mg decellularized hyaline cartilage graft (HCMG), and pre-treating in 50-60 mM tetramorpholinoethanesulfonic acid (MES) buffer (ph=5.5-6.5) containing 40-50% ethanol for 30-60 min at 20-30 mL;

(5.2) the decellularized hyaline cartilage graft (HCMG) was removed and placed in 20-30 mL of 40-50% ethanol solution containing 50-60 mM MES, 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) to N-hydroxysuccinimide (NHS) in a molar ratio of 1:1-1:1.2 molar concentration of EDC of 5-6 mM,10-12 mM,30-35 mM,50-55 mM, respectively;

(5.3) adding 2-3% (w/v) Chondroitin Sulfate (CS) into the reaction system, and reacting for 4-5 hours at room temperature to obtain 4 reinforced decellularized hyaline cartilage grafts (eHCMG) under the condition of different cross-linking agent concentrations;

(5.4) four materials were washed with PBS 6-7 times, each for 2-3 hours, and then with deionized water 3-4 times, each for 2-3 hours, to wash off crosslinker residue.