CN118236551A

CN118236551A - Preparation method of type II collagen extracellular matrix cartilage graft

Info

Publication number: CN118236551A
Application number: CN202410323063.4A
Authority: CN
Inventors: 姚航; 王东安; 张雷雷; 张嘉露; 崔金兰; 王迟润
Original assignee: Yantai Dinghao Biotechnology Co ltd
Current assignee: Yantai Dinghao Biotechnology Co ltd
Priority date: 2024-03-21
Filing date: 2024-03-21
Publication date: 2024-06-25

Abstract

The invention discloses a preparation method of a cartilage graft of type II collagen extracellular matrix, which comprises the steps of extracting primary cartilage cells of a knee joint of a pig, carrying out in vitro screening culture to obtain cartilage matrix production cells, simultaneously utilizing alginic acid polysaccharide, gelatin particles with a certain size and the cartilage matrix production cells to construct a microporous hydrogel cartilage matrix secretion system through blending and solidification, incubating the cartilage matrix secretion system in a culture medium, greatly secreting extracellular matrix with hyaline cartilage property by the cartilage matrix production cells, then removing an alginate gel part from the cartilage matrix production system by utilizing citric acid, enabling the cells to continuously secrete the matrix in a large amount, finally removing relevant components of the cells by a mild cell removing method, removing glycosaminoglycan and other proteins in the cells, and retaining porous materials mainly taking type II collagen extracellular matrix, thereby having good application prospects for clinical treatment of cartilage defects in traumatic, degenerative and early arthritic environments and the like.

Description

Preparation method of type II collagen extracellular matrix cartilage graft

Technical Field

The invention relates to the field of medical science, biomedical engineering and tissue engineering material preparation, in particular to a preparation method of a type II collagen extracellular matrix cartilage graft.

Background

Osteoarthritis (OA) refers to inflammation caused by strain and improper treatment of articular cartilage and surrounding tissues. At present, the OA of patients in China exceeds 1.3 hundred million, and the OA causes tissue deterioration and arthralgia and seriously affects the life quality of the patients. Current treatments for osteoarthritis mainly include physical therapy and drug therapy, and patients with severe conditions need cartilage repair by surgery (two-wire therapy) or artificial joint replacement (three-wire therapy). Because no ideal two-line treatment scheme exists in the clinic at present, physical treatment and drug treatment only play a role in relieving the illness state, and finally, the method is developed to the use of a large destructive artificial joint replacement operation. In fact, the root cause of the poor effect of two-wire therapy is that there is no good active biological material to guide the migration of chondrocytes to the tissue defect site, achieving cartilage repair, in particular efficient hyaline cartilage repair.

According to the clinical situation of cartilage defect repair and the biological characteristics of no nerve and no blood vessel of cartilage tissue, the cartilage defect with supercritical size can not be repaired or only fibrous scar tissue can be formed without guiding of a stent material. Based on the structural characteristics of cartilage matrix, the preparation of the bionic extracellular matrix material is a main solution idea. Biological materials currently under study for cartilage repair are mainly polysaccharides or soluble collagen type one extracted naturally, and the materials are obtained by chemical modification and re-crosslinking. Although the method can solve the problems at a certain level, the cartilage repair effect is poor or unstable, because the biological activity of the purified polysaccharide molecules or collagen is obviously reduced, the complex structure in the natural tissue is difficult to simulate, and the re-crosslinking mode is manually added, and the complex structure can be quickly degraded and reconstructed by enzymes in the body, so that the degradation rate is difficult to match with the tissue regeneration, and in addition, the existing component bionic material does not have a matrix bionic material consistent with the natural articular cartilage, namely type II collagen, so that the phenotype can be changed in the cartilage migration and regeneration process. Therefore, a technique is needed to obtain a pure type II collagen scaffold which does not contain type I collagen or polysaccharide molecules.

Disclosure of Invention

The invention aims to provide a preparation scheme of a type II collagen extracellular matrix cartilage implant, which abandons the traditional method of combining component selection with artificial crosslinking of the traditional cartilage repair material, directly constructs a controllable removed hydrogel frame with a micropore structure, selects production cells which are suitable for secreting cartilage matrixes, directly uses the cell secretion matrixes to form in-vitro hyaline cartilage tissues, and finally sequentially removes the hydrogel frame and cytoplasmic components through gradient elution to obtain the type II collagen extracellular matrix implant of the bionic hyaline cartilage with pure natural, material-free, cell-free and component structure.

In order to achieve the above purpose, the present invention is realized by the following specific technical scheme: a preparation scheme of a type II collagen extracellular matrix cartilage graft is characterized in that an alginate-based hydrogel-coated porcine cartilage cell is constructed, the cartilage cell secretes an extracellular matrix to form a network structure, a hydrogel framework, porcine cell components and polysaccharide components are removed, and a blocky porous material taking type II collagen as a component is reserved, so that the type II collagen extracellular matrix cartilage graft is obtained.

As a further description of the above technical solution, the preparation method is carried out according to the following steps:

Step one: harvesting chondrocytes

1.1, Selecting pig hind leg knee joint cartilage, and shearing to obtain cartilage tissue;

1.2 preparing a solution of type II collagenase at a concentration of 0.8-1.2 mg/mL: adding type II collagenase with effective activity of 100-300unit/mL into high-sugar DMEM medium containing 10-15% fetal calf serum to obtain type II collagenase solution;

1.3 cartilage tissue digestion: performing gradient enzymolysis on 1g of cartilage tissue by using 10-30mL type II collagenase solution at 37 ℃ in a 5% CO ₂ incubator, performing 3 cycles in the digestion process of the cartilage tissue, and replacing new type II collagenase digestion solution in each cycle, wherein the digestion time of the first cycle is 10-16 hours, and the digestion time of the second cycle is 4-8 hours; the third digestion cycle is 2-4 hours, after each cycle is finished, the supernatant of the digestion solution is collected and mixed, and the primary chondrocyte is obtained after centrifugal counting;

step two: obtaining extracellular matrix-producing cells

2.1 Plate amplification culture: inoculating the chondrocytes obtained in the first step into a cell culture dish according to 6000-12000 living cells/cm ², wherein the culture medium is a high-sugar DMEM culture medium containing 10-15% fetal bovine serum, and carrying out stationary plate amplification culture in a 5% CO ₂ incubator at 37 ℃ for 3-7 days;

2.2 cell screening: qPCR detection is carried out on cells on the 3 rd day of flat-plate amplification culture, the internal reference gene is RPL4, the expression condition of mRNA of key markers of the cells, namely collagen II, collagen I and collagen X is detected, and the cells with the differences of the reaction cycle numbers of the collagen II, the collagen I and the collagen X and the internal reference standard being respectively more than or equal to 10, less than or equal to-2 and less than or equal to-4 are screened out;

2.3 obtaining cells: observing the growth state of cells on the 3 rd to 7 th days of plate expansion culture, when the occupied area of the cell wall reaches 40% -70% of the total area of the culture dish, digesting the screened cells for 5-12 minutes by using pancreatin with the concentration of 0.25%, then detecting the activity rate of the cells, and screening out the cells with the activity rate of more than 90% to obtain extracellular matrix production cells;

Step three: preparation of first-order extracellular matrix secretion System

3.1 Preparing sodium alginate solution: preparing sodium alginate solution with concentration of 0.5-3% wt. with 12-18g/L physiological saline and alginic acid;

3.2 gelatin granules, extracellular matrix-producing cells were immersed overnight in sodium alginate solution, phosphate Buffer (PBS) at 4 ℃ in 1mL:0.5-1.2 x 10≡6: mixing 0.2-0.45g at room temperature, crosslinking with 80-140mM calcium chloride aqueous solution, and constructing a first-stage extracellular matrix secretion system;

Step four: programmed culture

4.1 First-stage extracellular cartilage matrix secretion system is incubated and cultured for 20-50 days in a 5% CO ₂ incubator at 37 ℃, wherein the culture medium in the system is a high-sugar DMEM culture medium of 10-30% fetal calf serum, 0.05mg/mL ascorbic acid and 0.4mM proline, and the liquid exchange frequency of the culture medium is as follows: 1 time of liquid change every 3-5 days in 1-15 days, 1 time of liquid change every 2-4 days in 16 days and later, and the culture process is in a shaking state of a shaking table at 50 rpm;

4.2 desalginic acid: soaking the first-stage extracellular matrix secretion system with 50-65mM sterile citric acid solution for 3-6 min, removing supernatant, adding sterile citric acid solution, repeatedly soaking the first-stage extracellular matrix secretion system for 3-6 min, repeating for one time, and placing insoluble substances back into the culture medium, wherein the insoluble substances are the second-stage extracellular matrix secretion system;

4.3 incubation culture of the second-stage extracellular cartilage matrix secretion system: continuously culturing the second-stage extracellular matrix secretion system for 5-20 days, wherein the culture medium is a high-sugar DMEM culture medium of 10-30% fetal bovine serum, 0.05mg/mL ascorbic acid and 0.4mM proline, and the liquid exchange frequency of the culture medium is that liquid exchange is carried out once every 3 days;

Step five: elution of cellular Components

5.1 Placing the second-stage extracellular matrix secretion system in PBS, rinsing for 5-20min, placing in 0.5-2% (Wt) Trion-X aqueous solution, rinsing for 20-50 hr, and using horizontal circumferential shaker with rotation speed of 120-160rpm at normal temperature;

5.2 rinsing the second-stage extracellular matrix secretion system after the rinsing step by using PBS for 3-5 times, wherein each time for 4-16 hours, adding 0.3-0.4% (Wt) sodium hydroxide solution to react for 20-70min at 4 ℃ for alkali treatment, and rinsing and vibrating for 15 seconds every 10 min;

5.3 rinsing the second-stage extracellular matrix secretion system after the steps by using PBS for 5-10 times, each time for 4-16 hours, packaging by using a sterile bottle, and irradiating by using cobalt 60 as a radioactive source with the irradiation amount of 25KGY to obtain the type II collagen extracellular matrix implant.

As a further description of the above technical solution, in step one, the source of cartilage tissue is a domestic pig breed: pig breeds which are common in long white, large white, ternary and other markets are raised for a month of between 5 and 9 months.

As a further description of the above technical solution, in the first step, the cartilage tissue is chopped into a granular tissue having a particle size of not more than 4 mm.

As a further description of the above technical scheme, the source of alginic acid used in step three is brown algae, the standard viscosity range is 10-30cP,1% in H ₂ O,25 ℃.

As a further description of the above technical scheme, in the third step, the gelatin particles are derived from pigskin or cow leather, are spherical or amorphous, and have a radius of gyration of 80-120 μm in dry state.

As a further description of the above technical scheme, in step four, the second extracellular cartilage matrix secretion system is LHCG.

As a further description of the technical scheme, the alkali treatment in the step five is a key step of removing swine cells, glycosaminoglycans and other proteins from materials, wherein the alkali treatment time is 20-70min, and different impurity treatment effects are brought by different treatment times.

The invention has the following effects:

1. The invention overcomes the defect of cartilage defect repair materials, prepares the type II collagen extracellular matrix material which has very similar components and structures to the natural transparent cartilage tissue, and ensures that the phenotype of the cartilage in the migration and regeneration process does not change.

2. The type II collagen extracellular matrix implant prepared by the method has good biocompatibility and stable cartilage repair effect, and along with the repair, the tissue of a cartilage repair part is more and more similar to natural hyaline cartilage.

Drawings

FIG. 1 shows the preparation of a type II collagen matrix graft (DLHCG) and the macroscopic morphology during the process;

FIG. 2 shows histological and immunohistological staining of type II collagen matrix grafts (DLHCG);

FIG. 3 is a scanning electron microscope picture of a type II collagen matrix graft (DLHCG);

FIG. 4 shows the condition of live and dead cell staining of chondrocytes, mesenchymal cells and dermal fibroblasts by type II collagen matrix grafts (DLHCG);

FIG. 5 is a macroscopic, histological and immunohistological staining of type II collagen matrix grafts (DLHCG) for repair of primary cartilage defects in rabbit knee joints;

FIG. 6 shows macroscopic appearance of a type II collagen matrix graft (DLHCG) produced using cells of varying lunar age porcine origin;

FIG. 7 is a Mersen stain of type II collagen matrix grafts (DLHCG) produced by swine-derived cells of different ages;

FIG. 8 shows the mechanical properties of type II collagen matrix grafts (DLHCG) produced by swine-derived cells of different ages;

Figure 9 is the effect of gelatin particle pore formers on extracellular matrix cartilage secretion system and implant construction.

Detailed Description

The invention is further described below with reference to examples.

The test methods used in the following examples are conventional methods unless otherwise specified.

The material reagents and the like used in the following examples are commercially available unless otherwise specified.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Examples:

step one: extraction of primary chondrocytes

Selecting a white breed boar with 6 months of age; dissecting the femoral component of the hind leg, and obtaining transparent cartilage of the knee joint surface (medial and lateral condyles and the scooter surface) by using a surgical knife and forceps; after washing with 1X Phosphate Buffer (PBS), removing non-cartilage miscellaneous tissues of the subchondral bone surface with a scalpel, and cutting the cartilage tissues into 2X 2mm particles with a scalpel; 15mL type II collagenase solution (1 mg/mL, solvent 10% in high-sugar DMEM solution) was used to digest cartilage particles according to 1.5g on a shaker at 45 rpm; after 14 hours of digestion in the first stage, the supernatant is sucked away, new digestion liquid is replaced for digestion in the second stage for 5 hours, and digestion in the third stage is carried out for 3 hours; all supernatants were centrifuged to obtain cells.

Step two: screening of cartilage matrix producing cells

Cells obtained by digestion of cartilage tissue were treated at 8000 cells/cm ²; inoculation was performed with medium composition high-sugar DMEM medium containing 12.5% fetal bovine serum. Cells were changed on the first day; the third day, the rare-phase cells are taken for detecting the specific markers of the cartilage matrix production cells, and the first-pass markers of the samples are as follows: qPCR detection is carried out on the collagen II mRNA, and cell samples with the cycle difference less than 10 are discarded; the fourth day of digestion is performed to obtain cells, 0.25% of pancreatin 8mL is used for digesting the chondrocytes in a 15cm culture dish, and meanwhile, the activity rate of the cells is detected, and the cells with the activity rate reaching more than 90% can be used as cartilage matrix production cells.

Step three: construction of type II collagen extracellular matrix secretion System

Gelatin substrate preparation: prepared aqueous calcium chloride (105 mM) was mixed with 15g of gelatin per 100mL, and autoclaved for use: a polytetrafluoroethylene 24mm diameter four-hole mold was used: adding 8mL gelatin substrate into a 10cm cell culture dish, rotating to uniformly spread, carefully placing into a mold, and cooling in a refrigerator at 4 ℃ for standby after solidification;

Based on the cell count, 0.4g of gelatin particles per 8×10≡6 living cells. Gelatin particles soaked in DMEM were weighed: peeling and weighing 50ml of flat-bottom centrifuge tubes in advance, sucking a sufficient amount of gelatin particles in a natural sedimentation state by using a pipette, adding the gelatin particles into the peeled and weighed centrifuge tubes for standing, sucking and discarding upper-layer liquid after obvious layering occurs in the centrifuge tubes, tilting a tube orifice downwards for 45 degrees, weighing after no water precipitation, and placing the centrifuge tubes into a refrigerator at 4 ℃ for standby; sucking sodium alginate solution (1 mL of every 8×10≡6 living cells), re-suspending the centrifugal cells, and uniformly mixing with gelatin particles to obtain a three-dimensional culture system preparation solution; taking out the gelatin substrate mould, using a shearing gun head, sucking the mixed solution with the transport capacity, adding the mixed solution into the mould, rotating uniformly, placing the tray filled with the mould into a refrigerator with the temperature of 4 ℃ for 4min, spraying CaCl ₂ solution on the cell suspension on each tray for 25 times, and placing the tray into the refrigerator for 4min; a first order extracellular matrix secretion system is obtained.

Step four: programmed culture

Adding 20mL of culture medium into each dish to protect a first-stage extracellular matrix secretion system, clamping a die out by using forceps with hooks, gently shaking the dishes to separate the first-stage extracellular matrix secretion system from a gelatin substrate, carefully transferring the dishes into high-sugar DMEM containing a proper amount of culture medium (20% fetal bovine serum, 0.05mg/mL of ascorbic acid and 0.4mM of proline) and placing the dishes in a carbon dioxide incubator (37 ℃ C., 5% CO ₂) for shake culture, wherein the macroscopic appearance of the first-stage extracellular matrix secretion system is as shown in FIG. 1B;

the programmed culture was performed for 35 days, and the frequency schedule of medium exchange in the system was as follows: changing liquid 1 time every 5 days 1-15 days; changing liquid 1 time every 4 days after 16 days; the culture process is in a shaking state of 50rpm of a shaking table; after 35 days, the alginate in the system was eluted and cultured continuously with citric acid, and the specific operations were: immersing the first order extracellular matrix secretion system with a sterile citric acid solution having a concentration of 55mM for 5 minutes; repeating the steps once after removing the supernatant; placing insoluble substance (second extracellular matrix secretion system LhCG) back into culture medium, changing liquid every 3 days, and culturing for 12 days; the macroscopic appearance of the second-stage extracellular matrix secretion system is shown in FIG. 1B;

Step five: decellularized acquisition of type II collagen extracellular matrix cartilage graft

LhCG was rinsed in 1 XPBS for 10min; then placing the mixture into 0.1% (wt) Trion-X aqueous solution to rinse for 40 hours, wherein the rinsing time is 150rpm of a horizontal circumferential table; the temperature is 25 ℃; lhCG after the above steps was rinsed 3 with PBS for 10 hours each; then adding 0.4% (wt) sodium hydroxide solution to react for 20-70min at 4 ℃ and rinsing and vibrating every 10min; rinsing LhCG times with PBS for 5 hours, packaging with sterile bottle, and performing cobalt 60; irradiation of 25 KGY; i.e. obtaining type ii collagen extracellular matrix cartilage graft (DLHCG) test example 1: histological and immunohistological section staining characterization of type II collagen extracellular matrix grafts

Obtaining a graft sample (LHCG for a control group and DLHCG for an experimental group), cutting a wafer with the diameter of 8mm by using a rotary cutter, slicing by using a frozen slicing step, specifically, sequentially soaking the wafer for more than 5 hours by using 10 wt%, 30 wt% and 50wt% of sucrose solution for balancing, and then embedding the wafer by using a frozen slice embedding agent, wherein the slice thickness is 7 mu m; the sections were left at room temperature for 24 hours and then stored at-20 ℃. Carrying out safranin O staining, meissen trichromatic staining, collagen II type, collagen I type, aggrecan and fibronectin immunofluorescence staining on a sample group, and analyzing the histological morphology of the sample and the expression condition of key active markers;

Safranin O staining and merson staining respectively demonstrate the persistence of glycosaminoglycans and total collagen in the tissue. It can be seen in fig. 2A that decellularization resulted in a significant loss of glycosaminoglycans, wherein the glycosaminoglycan content was further reduced by the use of different alkali treatment times until undetectable, while the collagen content was not greatly changed, and the tissue morphology remained well except for the removal of cells throughout the graft, with classical cartilage crypt structures still present.

Immunofluorescent staining primarily indicated the persistence of key cartilage proteins in the graft; the hyaline cartilage tissue is characterized in that collagen II type protein is highly expressed, collagen I type protein is basically not expressed, aggrecan is not expressed, and fibronectin is expressed; as is evident from fig. 2B, the expression profile of hyaline cartilage was essentially unchanged before and after decellularization, demonstrating that the decellularization process does not affect the morphology of the tissue and key active protein markers.

Test example 2: characterization of dry Structure and Biochemical Components of type II collagen extracellular matrix grafts

A type ii collagen extracellular matrix graft sample (control LHCG) was taken and discs 8mm in diameter were cut using a rotary knife; freeze-drying and weighing dry weight; observing the section of the sample by using a scanning electron microscope; testing the DNA residual quantity, the glycosaminoglycan content and the total collagen content in the sample;

DNA content test: 1mg/mL Hoechst 33258 fluorescent dye is prepared and stored in a dark place at 4 ℃; preparing 10XTNE buffer solution: 12.11g of tris (hydroxymethyl) aminomethane, 3.72g EDTA,116.89g sodium chloride is dissolved in ultrapure water, the pH of the solution is regulated to 7.4 by 1M HCl, and then the ultrapure water is added to a constant volume of 1L; 50mL of 1 XTNE buffer was added to 5. Mu.L of Hoechst 33258 fluorescent dye as Hoechst working solution. Placing at room temperature, and preparing at present; preparing calf thymus DNA standard solution: preparing 1 mu g/mL calf thymus DNA solution by 1 XTNE buffer solution, and gradually diluting to 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.625ng/mL, 7.8125ng/mL and 0ng/mL; 10. Mu.L of the sample digest was added to 1mLHoechst of the working solution. Incubate in the dark for 5min. The microplate reader measures the absorbance at an absorption wavelength of 350nm and an emission wavelength of 450 nm. The DNA standard solution was the same. Absorbance was converted to DNA content by a standard curve.

Glycosaminoglycan (GAG) content assay: preparing DMMB color development liquid: 50mL glycine/sodium chloride solution is taken, added with 0.8mgDMMB dye for dissolution, and placed at 4 ℃ for standby in a dark place. The glycine/sodium chloride solution was prepared as follows: weighing 3.04g of glycine, 2.37g of sodium chloride, adding 95mL of 0.1M HCl solution to adjust pH, and fixing the volume to 1L with deionized water; standard solutions of chondroitin sulfate (1 mg/mL) were prepared and diluted in a gradient so that the chondroitin sulfate solution concentrations were 0. Mu.g/mL, 3.125. Mu.g/mL, 6.25. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL and 100. Mu.g/mL, respectively; 0.1mL of each sample digest was added to 1mLDMMB reagent, and after mixing well, the absorbance of the mixture was measured immediately with an enzyme-labeled instrument at 530nm (this operation was performed within 3 minutes after mixing). The absorbance was converted to GAG content by standard curve, similarly to standard solution.

Total collagen assay: taking 500 mu L of papain digestive juice of a sample to be detected, adding 5mL of 6M hydrochloric acid into a pressure bottle, filling nitrogen into a tube for sealing, hydrolyzing at 105 ℃ for 22 hours, cooling to room temperature, adjusting pH to 6-8 by using 6M NaOH solution, and fixing the volume of primary water to 10mL for later use; the hydroxyproline standard was diluted to 7.5. Mu.g/mL, 3.75. Mu.g/mL, 1.875. Mu.g/mL, 0.938. Mu.g/mL, 0.469. Mu.g/mL, 0.234. Mu.g/mL, 0.117. Mu.g/mL, 0. Mu.g/mL; the method is operated according to a hydroxyproline kit, and after a hydrolysis sample is added into relevant reagents, the absorbance value is measured at 560nm, and the same is true of a hydroxyproline standard solution. Converting the absorbance into hydroxyproline content by drawing a standard curve; the hydroxyproline content was converted to total collagen content according to the ratio of hydroxyproline in articular cartilage collagen.

By scanning electron microscope analysis of the dry section of the sample, as shown in fig. 3, the whole structure of the sample after cell removal is not greatly changed, and the internal cavity is more loose and is the space left after cell removal. As shown in Table 1, the DNA content of the type II collagen extracellular matrix grafts, the glycosaminoglycan content and the total collagen content were compared, and the DNA content had been reduced to below 10ng/mg to an acceptable range for immune response. The glycosaminoglycan content test showed that the decellularization process resulted in a significant loss of glycosaminoglycan, similar to the results in safranin O staining (test example 1), collagen was less affected by decellularization, and the proportional content of collagen was greatly increased to over 99% due to the substantial reduction of glycosaminoglycan and cellular components, eventually the graft scaffold became a pure decellularized scaffold with collagen as the main component.

Table 1 shows the variation of the content of glycosaminoglycans and hydroxyproline with different alkali treatments compared to the sample conditions

Table 1 shows that after various times of alkali treatment, the glycosaminoglycan content in the product gradually decreased until undetected, and the DNA content further decreased, but when reaching around 5, the decrease in the value was not significant. At the same time, the alkali treatment time is increased, and the product form is damaged. Thus, the alkali treatment is critical to the removal of impurities from the product and can be extended appropriately, but not indefinitely. Finally, selecting an optimal solution with the alkali treatment time of 45-60 min. The analysis reasons may be that the structure of the product is loose, and the polysaccharide component is sensitive to alkali liquor, so that the increase of the alkali treatment time can obtain good effects, and the excessive alkali treatment can cause the damage of the structure of the product.

Test example 3: characterization of biocompatibility of type II collagen extracellular matrix grafts

And (3) performing a cell compatibility experiment on the sample by using a leaching solution method, selecting chondrocytes, mesenchymal cells and skin fibroblasts, detecting, and observing whether the leaching solution of the sample has an effect on the growth and proliferation of the cells. dLhCG was placed in medium at a material to medium ratio of 0.1g/mL for 24 hours; digesting cells, planting cells in 24 pore plates, planting 1 ten thousand cells per pore, and culturing overnight to attach cells; sucking the culture medium, dripping the leaching solution to culture cells, and taking the culture medium as a control group; as described above, on the fifth day of cell culture with the leaching solution, cells in the 24-well plate were subjected to live-dead staining, and cells cultured in the normal medium were used as a control group; calceinAM and PI staining were diluted to 1X,1:1, uniformly mixing for standby; sucking the leaching solution and the culture medium, cleaning with PBS, sucking the PBS, adding the prepared dye solution, and incubating in an incubator for 30 minutes; the dye was aspirated, 250mL buffer was added to each well, and photographs were taken with a fluorescence microscope.

The results of dead and alive staining of chondrocytes, mesenchymal cells and dermal fibroblasts using the extract are shown in fig. 4, and it can be found that the sample DLHCG extract group was not significantly different from the control group of normal cells, indicating that the biocompatibility of the material was good.

Test example 4: evaluation of ability of type II collagen extracellular matrix graft to repair cartilage damage in knee joint of animals

Animal experiments prove that the cartilage repair effect of DLHCG is achieved by constructing a full-layer cartilage defect model at the back leg knee joint pulley part of a New Zealand white rabbit. The modeling non-repaired group served as a negative control.

Experimental animal modeling and graft surgical implantation: the method comprises the steps of intravenous injection of anesthetic into the ear margin of an experimental animal, preparation of skin at the femoral part on the right side, fixation on an operating table, respiratory anesthesia, iodophor disinfection in an operating area, alcohol deiodination and sterile hole towel spreading. The skin and subcutaneous tissue is cut to expose the operation area, and a medical bone drill is used for washing bone fragments and tissues and the like by using physiological saline to flush the bone fragments and tissues with the diameter of 4mm on the lower pulley surface of the right leg patella. After hemostasis and irrigation of the wound, cartilage grafts DLHCG were separately implanted in parallel in animal models as test specimens. Untreated defect areas served as negative controls. After implantation, the incision skin is sutured and the wound is disinfected.

Sample acquisition: the rabbits of the experimental group were sacrificed at two time points of 50 days and 120 days of repair, the knee joints of the rabbits were opened, macroscopic photographing was performed on the repaired parts, and then the knee joints of the rabbits were excised for further histological and immunohistological characterization.

Macroscopic situation of repair site at two time points of 50 days and 120 days is shown in fig. 5. The repair photo of 50 days shows that the joint surface of the non-repair group is proliferated at a plurality of positions, the cartilage defect part is not repaired completely, and the edge of the defect part is provided with a proliferation tissue. Group DLHCG defect sites were essentially repaired, except that the edge sites were not fully integrated with the surrounding native cartilage. The 120-day repair photo shows that the joint cavity of the non-repair group for molding has good appearance, no obvious swelling, no obvious inflammation and no effusion after the joint cavity is opened. The defect at the defect part still exists obviously, part of subchondral bone is exposed, and cartilage-like tissue repair is carried out at the edge of the defect. DLHCG has obvious repairing effect, smooth tissue at the repairing part, and is level with and well integrated with surrounding natural cartilage.

Safranin O staining showed that the cartilage matrix glycosaminoglycan at DLHCG cartilage repair sites was strongly positive, whereas the non-repair groups were negative or weakly positive. According to H & E staining and Meissen staining, the cartilage repair part of the modeling unrepaired group is uneven, the cartilage cell morphology of the defect repair part is similar to that of fibroblast, and the fibrous tissue after the remodeling of subchondral bone is judged to be repaired, so that the overall repair effect is poor. The thickness of articular cartilage at DLHCG bone defect part is recovered, the surface is reconstructed smoothly, transparent cartilage is restored, and the cartilage is well integrated with surrounding cartilage. Mersen staining was slightly general in staining, but compared with surrounding natural cartilage (positive control area) at the site of defect repair, collagen secretion and distribution were similar. In addition, cartilage cells and their fossa size, morphology and arrangement are normal at the defect repair site. The subchondral tide line is basically clear and complete, and is locally seen as infiltration of subchondral bone into cartilage, and the subchondral bone is normal in morphology.

Immunohistochemical staining for 50 days and 120 days shows that the natural cartilage part around the modeling non-repair group has normal phenotype, col I is negative, col II is positive, the matrix and the cell part Col I of the cartilage defect repair part are positive, and the extracellular matrix of Col II is negative, which indicates that the repair part is fibrocartilage repair. The result of the DLHCG groups on 50 days shows that Col I is negative, col II is positive or weak positive, which indicates that the repair part is regenerated cartilage tissue and has a corresponding hyaline cartilage phenotype. The 120-day immunohistochemical result shows that DLHCG groups of Col I are negative and Col II are strong positive. It is demonstrated that as repair proceeds, the tissue of the cartilage repair site becomes more and more similar to natural hyaline cartilage.

Comparative example 1: preparation of type II collagen extracellular matrix cartilage grafts using different month-old pigs

Aiming at the month age of a pig source of cell sources in a type II collagen secretion system, separately selecting pig species of 5, 6 and 6.5 months of age for comparison, and preparing a type II collagen extracellular matrix implant (DLHCG) according to the method of the step 1-5; observing the product difference caused by the change of the pig age of the cells of the comparative example; the method specifically comprises macroscopic characterization, and mersen staining and mechanical property characterization of cross section sections; the mechanical property characterization method comprises the following steps: taking DLHCG implant samples, cutting out wafers with the diameter of 8mm by using a rotary cutter, and carrying out static compression test and dynamic torsion test; the compressive stress strain curve, the elastic modulus at 20% deformation, and the dynamic modulus under dynamic torsion of the sample were measured, respectively.

FIG. 6 is a macroscopic physical image of DLHCG prepared at 5, 7.5 and 9 months of age in the comparative example, and it can be seen that all DLHCG stents were relatively complete in their entirety, slightly broken at 9 months of age, slightly cracked at the surface of the 7.5 month-old sample, and the 5 month-old sample was the most full. Collagen staining in combination with cross section can be seen (as in fig. 7), and DLHCG whole matrices prepared from 5 month old and 7.5 month old swine-derived cells are uniformly distributed; slight delamination inside 9 months old; since different swine-derived cells have a certain change in the state of secreting matrix for the same culture system. The cartilage biological microstructure (e.g., cartilage pits) matrix secretion is normal for all month old samples. The mechanical property test can find that from the elastic modulus value layer, the values of the static modulus and the dynamic modulus are not greatly different, and the 5 month age is slightly better than 7.5 month age and is slightly better than 9 month age; the trend of the stress strain curve and dynamic modulus also demonstrates that the mechanical properties are consistent for different ages of the month, and also reflects that the internal structure is similar under compression. According to this, the age of the pig is selected to be suitable for 5-7.5 months, and the 9-month-old pig is unsuitable.

Comparative example 2: construction of type II collagen extracellular matrix cartilage grafts using different size gelatin particle pore formers

Aiming at the working characteristics of a type II collagen matrix secretion system, the specific size of the gelatin particles used as pore-forming agents and different mixing modes are compared; firstly, the size of gelatin particles is subdivided into large, medium and small sizes (the average radius of gyration in a dry state is 80 mu m, 100 mu m and 120 mu m); the method is carried out in three modes; the control group adopts pore-forming agent with gelatin particle diameter of 100 micrometers; the mixing group is a mixed gradient group of 100-80-micrometer pore-forming agent, when the matrix secretion system is prepared, the mixture is poured in layers, the pore-forming agent is 120 micrometers in the outer layer, the pore-forming agent is 100-80 micrometers in the inner layer, the method of the comparative example is shown in fig. 9A, and the electron microscope observation of different gelatin pore-forming agent particles is shown in fig. 9B; the other methods of sample construction in comparative example 2 were performed according to invention steps 1-5 to obtain LHCG comparative example samples, and pathological sections were directly sectioned and safranin O-staining and meisen-staining were performed.

The pathological staining results are shown in fig. 9C, and can be visualized in all safranin O staining and mersen staining of the comparative examples, with normal glycosaminoglycan secretion and collagen secretion, compounding the standard features of LHCG; the cartilage structure within the comparative sample is clearly visible. The observation of the internal microstructure can show that the mixed gradient group can significantly improve the internal layering phenomenon of the whole LHCG, the internal structure of LHCG has better continuity, the layering phenomenon of the contrast component is obvious, the mixture is enough to have a certain degree of life, and the layering phenomenon is still visible. From the comparative example, the structural continuity and integrity of the interior of the type II collagen matrix can be improved by refining the distribution and modification of the internal gelatin pore former.

Claims

1. A method for preparing a type II collagen extracellular matrix cartilage graft, which is characterized by comprising the following steps: the pig-source chondrocyte coated with the alginic acid-based hydrogel is constructed, the chondrocyte secretes extracellular matrix to form a network structure, a hydrogel frame and pig-source cell components are removed, and a blocky porous material taking the type II collagenase extracellular matrix as a component is reserved, so that the type II collagenase extracellular matrix cartilage graft is obtained.

2. The method of preparing a type ii collagenase extracellular matrix cartilage graft according to claim 1, comprising the steps of:

Step one: harvesting chondrocytes

step two: obtaining extracellular matrix-producing cells

Step three: preparation of first-order extracellular matrix secretion System

Step four: programmed culture

Step five: elution of cellular Components

5.2 rinsing the second-stage extracellular matrix secretion system after the rinsing step by using PBS for 3-5 times, 4-16 hours each time, adding 0.3-0.4% (Wt) sodium hydroxide solution to react for 20-70min at 2-8 ℃ for alkali treatment, rinsing and vibrating for 15 seconds every 10 min;

3. A method of preparing a type ii collagen extracellular matrix cartilage graft according to claim 2, wherein: in the first step, the cartilage tissue source is a domestic pig variety: pig breeds which are common in long white, large white, ternary and other markets are raised for 5-9 months.

4. A method of preparing a type ii collagen extracellular matrix cartilage graft according to claim 2, wherein: in the first step, the cartilage tissue is chopped into granular tissue with the particle size not more than 4 mm.

5. A method of preparing a type ii collagen extracellular matrix cartilage graft according to claim 2, wherein: the alginic acid used in the third step is brown algae with standard viscosity ranging from 10-30cP,1% in H ₂ O,25 ℃.

6. A method of preparing a type ii collagen extracellular matrix cartilage graft according to claim 2, wherein: in the third step, the gelatin particles are derived from pigskin or cow leather, are spherical or amorphous, and have a radius of gyration of 80-120 μm.