CN1297658C - Tissue engineering cartilage construction method using bone matrix gelatin - Google Patents

Abstract

A method for constructing tissue-engineered cartilage with bone matrix gel. First, separate and culture chondrocytes or stromal stem cells to induce chondrocytes to obtain seed cells, and then take New Zealand rabbit long bones and metaphyseal cancellous bone to construct bone matrix gel. Glue BMG, inoculate the seed cells harvested by separation and culture on cortical bone or cancellous bone BMG at a density of 4-100,000/ ^mm2 for in vitro culture, and the medium is calf serum containing 15% volume ratio and P/S each 100 units/ml of DMEM culture medium, just change the medium every 3 days. Because the bone matrix gelatin used in the present invention has the advantages of good biocompatibility, degradability, and strong induction of bone and cartilage growth and differentiation, it overcomes the problems of too fast absorption and insufficient mechanical strength after implantation; it can Good results in obtaining normal or near normal structure and function of osteochondral tissue.

Description

A method for constructing tissue engineered cartilage with bone matrix gel

technical field

The invention belongs to the reconstruction of cartilage tissue in vitro, in particular to a method for constructing tissue engineered cartilage with bone matrix gel.

Background technique

At present, the progress of tissue engineering research at home and abroad has opened up a new way for the research and treatment of cartilage diseases, and it is one of the important means for the transformation of future medicine from "replacement" to "reconstruction". Its development prospect is encouraging. With the rapid development of molecular biology and cell biology, the basic theoretical research on various diseases has not only been done at the level of whole animals, isolated organs and tissues, but people have increasingly realized that from the molecular and cellular levels, especially from the living The study of cell morphology and function from the perspective of tissue cells is of great significance to the study of the etiology, pathogenesis, pathological changes and treatment of various diseases. Tissue engineering is the product of the cross-integration of biomedicine and material science. Its meaning is to apply the principles and methods of life science and engineering to understand the relationship between the structure and function of mammalian normal and pathological tissues, and to research and develop biological substitutes to repair, maintain or Improve the form and function of human tissues and organs. At present, there are individual reports at home and abroad that implanting bone matrix gelatin (BMG) into muscle tissue can induce bone and cartilage formation. However, there are still many problems to be solved in the current research: 1). Seed cells: the prerequisite for tissue engineering is a sufficient number of seed cells with regeneration ability and function. The main source of cartilage seed cells in cartilage tissue engineering is cartilage and mesenchymal stem cells (mainly bone marrow); at present, human or animal cartilage is used at home and abroad, digested with enzymes, separated, and cultured to obtain a high survival rate , high purity, sufficient number of chondrocytes, the method is relatively mature. However, chondrocytes are prone to dedifferentiation after long-term culture and passage, and gradually become flat fibroblast-like cells, losing the differentiated phenotype of chondrocytes. Bone morphogenetic protein (BMP), insulin-like growth factor (IGF), transforming growth factor (TGF) and other cytokines can promote the differentiation of chondrocytes and maintain their phenotype. Bone marrow mesenchymal stem cells (also useful adipose stromal stem cells, embryonic stem cells, etc.) as the source of cartilage seed cells have received increasing attention. The isolated bone marrow mesenchymal stem cells are cultured with TGF and fibroblast growth factor (FGF), and can be differentiated into Chondrocytes that synthesize and secrete cartilage-specific markers such as type II collagen have achieved good results in repairing articular cartilage. And because bone marrow has the advantages of convenient extraction and less damage to the donor, its application prospect is more promising. However, bone marrow stromal stem cells are composed of a variety of cells, which need to be further purified, and appropriate conditions must be selected to induce their differentiation into chondrocytes. Therefore, how to regulate the proliferation and differentiation of chondrocytes in vitro and in vivo, and maintain their phenotypic characteristics are issues that need further study. 2). Three-dimensional cell scaffold: the scaffold for planting chondrocytes is also the key to the success of cartilage tissue engineering. Scaffold materials should have good biocompatibility, no toxic side effects, porous three-dimensional structure, biodegradability, good cell attachment interface, plasticity and certain mechanical strength. At present, there are many biomaterials studied at home and abroad, such as polylactic acid (PLA), polyglycolic acid (PGA), bone matrix gelatin (BMG), collagen sponge, alginate, hyaluronic acid, etc. question. For example, PGA and PLA, which are more optimistic at home and abroad and are the most widely used, are artificially synthesized polymers with good biocompatibility, degradability and plasticity, but both are hydrophobic substances, which are harmful to cells. The adsorption force is weak, and the degradation product is acidic, which is not good for cell growth. Although many improvement measures have been taken at present, as a synthetic material, there are still shortcomings such as lack of cell recognition signals and high cost. BMG is prepared from natural bone materials, has good biocompatibility and degradability, and because it contains BMP, it has a strong effect of inducing bone and cartilage growth, differentiation and maintaining its phenotype, but also There are disadvantages such as inhomogeneous material pores, rapid absorption after implantation, insufficient mechanical strength, and insufficient source of human bone materials. These are problems that need further research and solution. 3). Formation of tissue-engineered cartilage: the stable fusion of cartilage seed cells and bone matrix gelatin to construct tissue-engineered cartilage is the ultimate problem to be solved. Subcutaneous implantation of chondrocyte-scaffold complexes into athymic nude mice is a common method for generating tissue-engineered cartilage. Cao Yilin, a Chinese scholar, inoculated chondrocytes on ear-shaped PGA scaffolds, cultured them in vitro and implanted them subcutaneously in the back of nude mice, successfully recreating human ear-shaped cartilage. At present, human nasal cartilage, bronchial cartilage, meniscal cartilage and articular cartilage have also been studied. Autologous or allogeneic chondrocyte-scaffold complexes have also been successfully transplanted in immunocompetent animals to form cartilage tissue or repair articular cartilage defects. In the early stage of implantation, there is a mild local inflammatory reaction or lymphocyte infiltration, which gradually subsides later, and generally does not affect the formation of cartilage. 4).Evaluation and clinical application of tissue-engineered cartilage: Tissue-engineered cartilage must pass strict biological and mechanical evaluation and animal experiment verification before it is applied clinically. Through gross observation, histomorphology, histochemistry, immunohistochemistry and in situ hybridization techniques, the appearance characteristics of tissue engineered cartilage, the morphology, distribution and activity of chondrocytes, the composition and content ratio of cartilage matrix, etc. were analyzed. By comparing and testing the compression modulus, surface permeability, and aggregation modulus of tissue-engineered cartilage, understand its mechanical properties. Through various animal experiments, the effectiveness, safety and immune reactivity of tissue engineered cartilage transplantation into experimental animal models are studied. The currently developed tissue engineered cartilage can only replace part of the function of the defective tissue, and the maintenance time is relatively short, and there is still a certain distance from permanent functional reconstruction. For human clinical application, in addition to solving the above-mentioned problems, it is necessary to conduct in-depth research and research on how to establish an evaluation system, ensure the quality of tissue engineered cartilage, and the long-term effect and safety of grafts on the body. solve. In terms of bioscaffold research, there are currently many types of materials used at home and abroad. They have good biocompatibility, no side effects, porous three-dimensional structure, biodegradability, good cell attachment interface, plasticity and certain mechanical properties. Strength and other elements have their own strengths and some problems that need to be improved.

Contents of the invention

The object of the present invention is to provide a method for constructing tissue engineered cartilage with bone matrix gel, and the tissue engineered cartilage constructed by the present invention can obtain osteochondral tissue with normal or close to normal structure and function.

For achieving the above object, the preparation method that the present invention adopts is:

1) Seed cell acquisition

Isolation and culture of chondrocytes

①Materials: 1 New Zealand dead rabbit about 4 weeks old, depilated, washed, soaked in 0.1% bromogeramine for 20-30 minutes, cut off the proximal humerus, proximal and distal femur and proximal tibia by aseptic operation, and put them in In the D-Hanks' solution containing 100 units/ml of P/S, remove all soft tissues, cut off the articular cartilage in pieces, and put it into D-Hanks' solution containing 100 units/ml of P/S ' solution container, then suck out the liquid and discard it, digest it with 10ml of hyaluronidase with a mass percent concentration of 0.05% at room temperature for 3 minutes, discard the enzyme solution, and use D- After washing with Hanks' solution, cut the cartilage into ^1mm3 pellets;

②Cell separation: Transfer the cartilage granules into the digestion chamber, add 5ml of trypsin with a mass percentage concentration of 0.2%, digest at 37°C for 30 minutes under magnetic stirring, then suck out the enzyme solution and discard it, then add 5ml of trypsin with a mass percentage concentration of Digest with 0.2% collagenase at 37°C for 60 minutes under magnetic stirring, suck out the enzyme solution containing cells, and place in half of the DMEM containing 15% volume ratio of calf serum and 100 units/ml of P/S for culture centrifuge at 1500rpm for 3 minutes, discard the supernatant to collect the cells, repeat the digestion once, combine the collected cells twice, and use D-Hanks' solution containing 100 units/ml of P/S or containing 15 Wash 1-2 times with the calf serum of % volume ratio and the DMEM culture fluid of 100 units/ml of P/S;

③Primary culture: 400,000 cells/bottle were inoculated into a 5cm×5cm×3cm culture flask, and the medium was cultured in DMEM containing 15% volume ratio of calf serum and 100 units/ml of P/S Cultured in a CO ₂ incubator at 37°C with 4ml of liquid solution, the interval between changing the liquid for the first time was 72 hours, and changing the liquid every other day thereafter until a monolayer of cells was formed;

④ Subculture: Discard the culture medium in each bottle that has formed a cell monolayer in the primary culture, wash the cells 1-2 times with D-Hanks' solution containing 100 units/ml of P/S each, and add 4ml Digest the digestive enzyme solution with a percentage concentration of 0.25% trypsin+0.02% EDTA at 37°C for 10 minutes, pour the cell-containing enzyme solution into a 50ml centrifuge tube, and then use D-Hanks' containing 100 units/ml of P/S solution or 2ml of DMEM culture solution containing 15% volume ratio of calf serum and P/S of 100 units/ml, washed 1-2 times to wash all the cells in the bottle, centrifuged at 1500rpm for 3 minutes, discarded Obtain chondrocytes after clearing;

2) Preparation of bone matrix gel BMG:

①The long bones and metaphyseal spongy bones of New Zealand rabbits were taken under relatively aseptic conditions, and all soft tissues and bone marrow were removed and washed with distilled water;

②Rinse with 3mol/L sodium azide for 2-3 times and then wash with distilled water; use 1:1 chloroform/methanol to degrease overnight under magnetic stirring at room temperature, then wash with distilled water;

③Decalcify with 0.6mol/L hydrochloric acid at 4°C with magnetic stirring, during which time the liquid is changed every 4 hours until the bone becomes soft and has a certain degree of elasticity, then decalcification is terminated, and then washed with distilled water until neutral;

④ Under magnetic stirring at 4°C: treat with 2mol/L calcium chloride for 1 hour, wash with distilled water; treat with 0.5mol/L EDTA for 1 hour, wash with distilled water; treat with 8mol/L lithium chloride for 1 hour, wash with distilled water;

⑤ Treat with sterile distilled water at 55°C for 1 hour;

⑥ Trim the processed BMG to the size required for the experimental design, freeze-dry, sterilize with 60 cobalt source and store in the refrigerator at 0℃～-20℃ for later use;

3) Construction of tissue engineered cartilage:

Inoculate the seed cells harvested by separation and culture on cortical bone or cancellous bone BMG at a density of 4-100,000/ ^mm2 for in vitro culture, and the medium is 100 units each of calf serum and P/S containing 15% volume ratio /ml of DMEM culture medium, and change the medium every 3 days.

The seed cells of the present invention can also be induced into chondrocytes by separation and culture of stromal stem cells, and bone marrow mesenchymal stem cells are obtained by using density gradient centrifugation and adhesion separation methods: taking about four months old New Zealand purebred rabbits or adults for bone marrow puncture, extracting bone marrow by The volume ratio of 1:1 was slowly injected on the surface of 70% Percoll cell separation medium, centrifuged at 1500 rpm for 20 minutes, the liquid layer containing bone marrow mesenchymal stem cells between the upper layer and the middle layer was sucked, and a small volume ratio of 15% was used. Bone marrow mesenchymal stem cells were made into a single cell suspension with bovine serum and P/S of 100 units/ml each in DMEM culture medium, counted nucleated cells and inoculated them in culture flasks for monolayer culture, the first 5 days, Afterwards, replace the DMEM medium containing 15% calf serum by volume and 100 units/ml of P/S every 3 days, and subculture after about 2 weeks to form a cell monolayer; the primary culture has formed a cell monolayer Discard the culture fluid in each bottle, wash the cells 1-2 times with D-Hanks' solution containing 100 units/ml of P/S, and add 4ml of digestive enzyme with mass percent concentration of 0.25% trypsin+0.02% EDTA solution, digested at 37°C for 10 minutes, pour the cell-containing enzyme solution into a centrifuge tube, and then use D-Hanks' solution containing 100 units/ml of P/S or calf serum containing 15% volume ratio and P/S Wash with 2ml of DMEM culture medium with 100 units/ml of S each to elute all the cells in the bottle, centrifuge at 1500rpm for 3 minutes, discard the supernatant, and use 100 units each of calf serum containing 15% volume ratio and P/S /ml of DMEM culture medium to make cell suspension, count the cells and inoculate for subculture. At this time, add inducing factors that induce the transformation of bone marrow mesenchymal stem cells into chondrocytes: 10ng/ml transforming growth factor β1, 10ng/ml insulin-like growth factors and 50 μg/ml vitamin C to induce the transformation of bone marrow mesenchymal stem cells into chondrocytes.

In the present invention, 5 μg/ml fibronectin can be pre-coated on the bottom of the culture bottle to promote the adhesion of the bone marrow mesenchymal stem cells when the nucleated cells are counted and inoculated in the culture bottle for monolayer culture.

Since the present invention adopts bone matrix gelatin, it has better biocompatibility, degradability, and stronger advantages of inducing bone and cartilage growth and differentiation, and overcomes the problems of too fast absorption and insufficient mechanical strength after implantation. Problem: Carry out experimental research on the use of heterogeneous bone matrix gelatin to overcome problems such as insufficient sources of human bone materials, and will achieve good results that the tissue engineered cartilage constructed by the present invention can obtain normal or close to normal structure and function of osteochondral tissue.

Description of drawings

Fig. 1 is the slice HE staining figure that chondrocyte composite cortical bone BMG of the present invention is cultivated for 6 days, 10X;

Fig. 2 is the slice HE staining figure of chondrocyte composite cancellous bone BMG of the present invention cultured for 6 days, 10X;

Fig. 3 is the slice HE staining figure of chondrocyte composite cortical bone BMG of the present invention cultured 12 days; , 10X;

Fig. 4 is the slice PG staining picture of chondrocyte composite cancellous bone BMG of the present invention cultured for 12 days, 10X;

Fig. 5 is the slice HE staining picture of chondrocyte composite cortical bone BMG of the present invention cultured for 18 days, 10X;

Fig. 6 is the slice HE staining figure of chondrocyte composite cancellous bone BMG of the present invention cultured for 18 days, 10X;

Fig. 7 is the section PG staining picture of chondrocyte composite cortical bone BMG of the present invention cultured for 18 days, 10X;

Fig. 8 is the slice PG staining picture of the chondrocyte composite cancellous bone BMG of the present invention cultured for 18 days, 20X;

Fig. 9 is the slice HE staining picture of chondrocyte composite cortical bone BMG of the present invention cultured for 24 days;, 10X;

Fig. 10 is the section collagen staining figure of chondrocyte composite cancellous bone BMG of the present invention cultured for 24 days, 10X;

Detailed ways

Example 1:

1) Seed cell acquisition

Isolation and culture of chondrocytes

①Materials: 1 New Zealand dead rabbit about 4 weeks old was born, depilated with 8% sodium sulfide by mass percentage, washed with running water, soaked in 0.1% bromogeramine for 20-30 minutes; cut off the proximal humerus by aseptic operation, The proximal and distal ends of the femur and the proximal end of the tibia were placed in a container containing D-Hanks' solution containing 100 units/ml of P/S each, and all soft tissues were removed, and the cartilage of each articular surface was sliced off, and placed in a container containing P/S each of 100 units/ml of D-Hanks' solution, then suck out the liquid and discard it, digest it with 10ml hyaluronidase with a mass percentage concentration of 0.05% at room temperature for 3 minutes, discard the enzyme solution, and use After washing with D-Hanks' solution containing 100 units/ml of P/S, the cartilage slices were cut into small cartilage granules with a size of ¹ mm;

②Cell separation: Transfer the cartilage granules into the digestion chamber, add 5ml of trypsin with a mass percentage concentration of 0.2%, digest at 37°C for 30 minutes under magnetic stirring, then suck out the enzyme solution and discard it, then add 5ml of trypsin with a mass percentage concentration of Digest with 0.2% collagenase at 37°C for 60 minutes under magnetic stirring, suck out the enzyme solution containing cells, and place in half of the DMEM containing 15% volume ratio of calf serum and 100 units/ml of P/S for culture centrifuge at 1500rpm for 3 minutes, discard the supernatant, collect the cells, repeat the digestion once, combine the collected cells twice, and wash 1-2 times with D-Hanks' solution;

③Primary culture: 400,000 cells/bottle were inoculated into a 5cm×5cm×3cm culture flask, and the medium was cultured in DMEM containing 15% volume ratio of calf serum and 100 units/ml of P/S cultured in a CO ₂ incubator at 37°C, and observed the cell growth under an inverted microscope every day during the culture. Generally, the cells in the primary culture adhered to the wall within 24 hours, and the cells began to protrude within 48-72 hours. Start to divide and proliferate, and the rabbit chondrocytes can form a single layer in about 10 days;

④ Subculture: Discard the culture medium in each bottle that has formed a cell monolayer in the primary culture, wash the cells 1-2 times with D-Hanks' solution containing 100 units/ml of P/S each, and add 4ml The digestive enzyme solution with a percentage of 0.25% trypsin + 0.02% EDTA was digested at 37°C for 10 minutes. After observing the detachment of the cells under an inverted microscope, pour the enzyme solution containing the cells into a 50ml centrifuge tube, and then use 100% P/S each Wash 1-2 times with 2 ml of D-Hanks' solution of 1 unit/ml to elute all the cells in the bottle, centrifuge at 1500 rpm for 3 minutes, discard the supernatant to obtain the cells;

This method consumes less animals and digestive enzymes, the matrix is completely digested, and the amount of cells obtained is larger. One rabbit can obtain more than 10 million chondrocytes. However, due to the strong enzyme action and long digestion time, the cells are more damaged during separation, and the death rate after the primary inoculation is relatively large. In order to make the experimental results more reliable and to increase the number of cells, generally more than the first generation cells for experimental research. Passage cells can basically survive, and the number of cells obtained is large (up to hundreds of millions), the experiment repeatability is good, and the results are relatively stable.

2) Preparation of bone matrix gel (BMG):

①The long bones and metaphyseal spongy bones of New Zealand rabbits were taken under relatively aseptic conditions, and all soft tissues and bone marrow were removed and washed with distilled water;

② Rinse with 3mol/L sodium azide 2-3 times, wash with distilled water; degrease overnight with 1:1 chloroform/methanol under magnetic stirring at room temperature, and wash with distilled water;

③Decalcify with 0.6mol/L hydrochloric acid at 4°C with magnetic stirring, during which time the liquid is changed every 4 hours until the bone becomes soft and has a certain degree of elasticity, then decalcification is terminated, and then washed with distilled water until neutral;

④Treat with 2mol/L calcium chloride for 1 hour under magnetic stirring at 4°C, wash with distilled water; treat with 0.5mol/L EDTA for 1 hour, wash with distilled water; treat with 8mol/L lithium chloride for 1 hour, wash with distilled water;

⑤ Treat with sterile distilled water at 55°C for 1 hour;

⑥ Trim the processed BMG into the size required for the experimental design, freeze-dry, sterilize with ⁶⁰ cobalt source and store in the refrigerator at 0℃～-20℃ for later use;

3) Construction of tissue engineered cartilage:

Inoculate the seed cells harvested by separation and culture on cortical bone or cancellous bone BMG at a density of 4-100,000/ ^mm2 for in vitro culture, and the medium is 100 units each of calf serum and P/S containing 15% volume ratio /ml of DMEM culture medium, and change the medium every 3 days.

Example 2:

1) Seed cell acquisition

Isolation and culture of chondrocytes

Separation and culture of stromal stem cells to induce chondrocytes:

①Using density gradient centrifugation and adhesion separation to obtain bone marrow mesenchymal stem cells: take New Zealand purebred rabbits about four months old, extract bone marrow and slowly inject it on the surface of 70% Percoll cell separation medium at a volume ratio of 1:1, at 1500 Spin centrifuge for 30 minutes, absorb the liquid layer containing bone marrow mesenchymal stem cells between the upper layer and the middle layer, and fill the bone marrow mesenchymal stem cells with DMEM culture solution containing 15% volume ratio of calf serum and P/S 100 units/ml each. Stem cells were made into a single cell suspension, and the nucleated cells were counted and inoculated in culture flasks for monolayer culture. The first 5 days were replaced every 3 days with calf serum containing 15% volume ratio and 100 P/S each. 1 unit/ml of DMEM culture medium once, after about 2 weeks to form a monolayer of cells, then subculture;

② Subculture to induce transformation into chondrocytes: Discard the culture medium in each bottle that has formed a monolayer of cells in the primary culture, and wash the cells with D-Hanks' solution containing 100 units/ml of P/S each for 1-2 First, add 4ml of digestive enzyme solution with a mass percentage of 0.25% trypsin + 0.02% EDTA, digest at 37°C for 10 minutes, pour the cell-containing enzyme solution into a centrifuge tube, observe the detachment of the cells under an inverted microscope, and then add the cell-containing enzyme Pour the solution into a centrifuge tube, wash with 2ml of D-Hanks' solution containing 100 units/ml of P/S each, so that all the cells in the bottle are eluted, centrifuge at 1500rpm for 3 minutes, discard the supernatant, and wash with 15% volume Ratio calf serum and 100 units/ml of P/S DMEM culture fluid were used to make cell suspension, count the cells and inoculate for subculture, at this time, add inducing factors that induce bone marrow mesenchymal stem cells to transform into chondrocytes: 10ng/ml transforming growth factor β1, 10ng/ml insulin-like growth factor and 50μg/ml vitamin C to induce bone marrow mesenchymal stem cells to transform into chondrocytes;

③Others such as adipose stem cells can be obtained by referring to the separation method of bone marrow mesenchymal stem cells.

2) Preparation of bone matrix gel (BMG):

①The long bones and metaphyseal spongy bones of New Zealand rabbits were taken under relatively aseptic conditions, and all soft tissues and bone marrow were removed and washed with distilled water;

② Rinse with 3mol/L sodium azide 2-3 times, wash with distilled water; degrease overnight with 1:1 chloroform/methanol under magnetic stirring at room temperature, and wash with distilled water;

③Decalcify with 0.6mol/L hydrochloric acid at 4°C with magnetic stirring, during which time the liquid is changed every 4 hours until the bone becomes soft and has a certain degree of elasticity, then decalcification is terminated, and then washed with distilled water until neutral;

④Treat with 2mol/L calcium chloride for 1 hour under magnetic stirring at 4°C, wash with distilled water; treat with 0.5mol/L EDTA for 1 hour, wash with distilled water; treat with 8mol/L lithium chloride for 1 hour, wash with distilled water;

⑤ Treat with sterile distilled water at 55°C for 1 hour;

⑥ Trim the processed BMG into the size required for the experimental design, freeze-dry, sterilize with ⁶⁰ cobalt source and store in the refrigerator at 0℃～-20℃ for later use;

3) Construction of tissue engineered cartilage:

The seed cells harvested by separation and culture were inoculated on cortical bone and cancellous bone BMG at a density of 4-100,000/ ^mm2 for in vitro culture, and the medium was calf serum containing 15% volume ratio and 100 P/S each unit/ml of DMEM culture medium, and change the medium every 3 days.

Example 3:

1) Seed cell acquisition

Isolation and culture of chondrocytes

Separation and culture of stromal stem cells to induce chondrocytes:

①Using density gradient centrifugation and adhesion separation to obtain bone marrow mesenchymal stem cells: Bone marrow puncture was performed on adults, and the bone marrow was extracted and injected slowly on the surface of 70% Percoll cell separation medium at a volume ratio of 1:1, and centrifuged at 1500 rpm for 30 minutes , absorb the liquid layer containing bone marrow mesenchymal stem cells between the upper layer and the middle layer, and prepare the bone marrow mesenchymal stem cells with DMEM culture medium containing 15% volume ratio of calf serum and 100 units/ml of P/S Single cell suspension, count the nucleated cells and inoculate in a culture bottle for monolayer culture, the first 5 days, then every 3 days, replace with calf serum containing 15% volume ratio and P/S 100 units/ml each DMEM culture medium once, and subculture after about 2 weeks to form a cell monolayer;

② Subculture to induce transformation into chondrocytes: Discard the culture medium in each bottle that has formed a monolayer of cells in the primary culture, and wash the cells with D-Hanks' solution containing 100 units/ml of P/S each for 1-2 First, add 4ml of digestive enzyme solution with a mass percentage of 0.25% trypsin + 0.02% EDTA, digest at 37°C for 10 minutes, pour the cell-containing enzyme solution into a centrifuge tube, observe the detachment of the cells under an inverted microscope, and then add the cell-containing enzyme Pour the solution into a centrifuge tube, wash with 2ml of D-Hanks' solution containing 100 units/ml of P/S each, so that all the cells in the bottle are eluted, centrifuge at 1500rpm for 3 minutes, discard the supernatant, and wash with 15% volume Ratio calf serum and 100 units/ml of P/S DMEM culture fluid were used to make cell suspension, count the cells and inoculate for subculture, at this time, add inducing factors that induce bone marrow mesenchymal stem cells to transform into chondrocytes: 10ng/ml transforming growth factor β1, 10ng/ml insulin-like growth factor and 50μg/ml vitamin C to induce bone marrow mesenchymal stem cells to transform into chondrocytes;

③Others such as adipose stem cells can be obtained by referring to the separation method of bone marrow mesenchymal stem cells.

2) Preparation of bone matrix gel (BMG):

①The long bones and metaphyseal spongy bones of New Zealand rabbits were taken under relatively aseptic conditions, and all soft tissues and bone marrow were removed and washed with distilled water;

② Rinse with 3mol/L sodium azide 2-3 times, wash with distilled water; degrease overnight with 1:1 chloroform/methanol under magnetic stirring at room temperature, and wash with distilled water;

③Decalcify with 0.6mol/L hydrochloric acid at 4°C with magnetic stirring, during which time the liquid is changed every 4 hours until the bone becomes soft and has a certain degree of elasticity, then decalcification is terminated, and then washed with distilled water until neutral;

④Treat with 2mol/L calcium chloride for 1 hour under magnetic stirring at 4°C, wash with distilled water; treat with 0.5mol/L EDTA for 1 hour, wash with distilled water; treat with 8mol/L lithium chloride for 1 hour, wash with distilled water;

⑤ Treat with sterile distilled water at 55°C for 1 hour;

⑥ Trim the processed BMG into the size required for the experimental design, freeze-dry, sterilize with ⁶⁰ cobalt source and store in the refrigerator at 0℃～-20℃ for later use;

3) Construction of tissue engineered cartilage:

The seed cells harvested by separation and culture were inoculated on cortical bone and cancellous bone BMG at a density of 4-100,000/ ^mm2 for in vitro culture, and the medium was calf serum containing 15% volume ratio and 100 P/S each unit/ml of DMEM culture medium, and change the medium every 3 days.

According to the engineered cartilage prepared by the preparation method of the present invention, see Figures 1 and 2, the chondrocytes planted in the BMG are cultured for 6 days to form a thin layer of cells; see Figures 3 and 4, the cartilage-like tissue with 8 to 10 layers of cells is formed after being cultivated for 12 days, The staining of amino polysaccharides around the cells is obvious; see Figures 5, 6, 7, and 8, cartilage-like tissue with 12 to 15 layers of cells is formed after 18 days of culture, and the staining of amino polysaccharides around the cells is more obvious and collagen components appear; see Figures 9 and 10, cultured to From 24d to 42d, cartilage-like tissue with more than 20 layers of cells was formed. The difference is that the cartilage-like tissue cultured on cortical bone BMG surrounds the BMG surface, similar to articular surface cartilage; the cartilage-like tissue cultured on cancellous bone BMG has cells distributed in the mesh of cancellous bone BMG to form a seamless body.

I. Composite cortical bone BMG tissue engineered cartilage toxicity test:

Using cell culture method, the cultured New Zealand purebred young rabbit chondrocytes were planted on the autologous cortical bone matrix gelatin (BMG), and the cartilage tissue model was rebuilt in vitro. Compatibility group design was adopted, and different concentrations of suspected KBD pathogenic factor T-2 toxin, nivalenol (NIV), deoxynivalenol (DON) and protective factor selenium were added to rebuild cartilage in vitro Tissues to establish a cartilage degeneration and necrosis model. The results showed that there were multiple layers of chondrocytes on the BMG surface of the control group. With the increase of T-2, NIV and DON toxin concentrations among the groups, the cell density decreased, the damage was aggravated, and necrosis appeared; the trend remained unchanged after adding selenium, and the damage was alleviated compared with the corresponding group without selenium.

II. Composite cancellous bone BMG tissue engineered cartilage transplantation to repair articular cartilage defects:

The cancellous bone BMG-chondrocyte complex formed in vitro cultured for 12 days was implanted in the osteochondral defect (diameter 4mm) of the allogeneic rabbit knee joint, and the functional evaluation was performed at 2, 4, 8, 12, and 24 weeks after operation And dissecting microscope, histology, electron microscope and other dynamic observation. The results showed that all experimental rabbits resumed normal activities within 10 days after operation; 2 weeks after operation, chondrocytes split and proliferated between cancellous bone BMG scaffolds to form cartilage clumps; The chondrocyte clumps formed between the two groups gradually increased, and the BMG was gradually absorbed. At 8 weeks, the BMG was completely absorbed and formed cartilage tissue; at 24 weeks after operation, the osteochondral defect was repaired with cartilage tissue. Cancellous bone BMG has good histocompatibility with host tissue in vivo and is degradable. Combined with the results of in vitro experiments, it is considered that cancellous bone BMG is a suitable cell scaffold, and chondrocytes are loaded on cancellous bone BMG It can repair the osteochondral defects of rabbit joints.

III. Conclusion:

Chondrocytes are planted in bone matrix gelatin and cultured in vitro for 1 to 6 weeks to obtain cartilage-like tissue with the function of secreting aminopolysaccharide and collagen; cartilage-like tissue formed in cortical bone BMG is more suitable for in vitro functional metabolism and injury experiments; Cartilage-like tissue cultured in cancellous bone BMG is more suitable for transplantation and repair of osteochondral defects.

In the present invention, chondrocytes isolated and cultured in vitro are planted on bone matrix gelatin (BMG) to construct tissue engineered cartilage, and osteochondral tissue similar to normal structure and function has been obtained, which is used for in vitro tissue cell function and metabolism research and Used for transplantation to repair osteochondral defects.

Claims (1)

1. A method for constructing tissue engineered cartilage with bone matrix gel, characterized in that: 1) Seed cell acquisition Isolation and culture of chondrocytes ①Materials: 1 New Zealand dead rabbit about 4 weeks old, depilated, washed, soaked in 0.1% bromogeramine for 20-30 minutes, cut off the proximal humerus, proximal and distal femur and proximal tibia by aseptic operation, and put them in In the D-Hanks' solution containing 100 units/ml of P/S, remove all soft tissues, cut off the articular cartilage in pieces, and put it into D-Hanks' solution containing 100 units/ml of P/S ' solution container, then suck out the liquid and discard it, digest it with 10ml of hyaluronidase with a mass percent concentration of 0.05% at room temperature for 3 minutes, discard the enzyme solution, and use D- After washing with Hanks' solution, cut the cartilage into ^1mm3 pellets; ②Cell separation: Transfer the cartilage granules into the digestion chamber, add 5ml of trypsin with a mass percentage concentration of 0.2%, digest at 37°C for 30 minutes under magnetic stirring, then suck out the enzyme solution and discard it, then add 5ml of trypsin with a mass percentage concentration of Digest with 0.2% collagenase at 37°C for 60 minutes under magnetic stirring, suck out the enzyme solution containing cells, and place in half of the DMEM containing 15% volume ratio of calf serum and 100 units/ml of P/S for culture centrifuge at 1500rpm for 3 minutes, discard the supernatant to collect the cells, repeat the digestion once, combine the collected cells twice, and use D-Hanks' solution containing 100 units/ml of P/S or containing 15 Wash 1-2 times with the calf serum of % volume ratio and the DMEM culture fluid of 100 units/ml of P/S; ③Primary culture: 400,000 cells/bottle were inoculated into a 5cm×5cm×3cm culture flask, and the medium was cultured in DMEM containing 15% volume ratio of calf serum and 100 units/ml of P/S Cultured in a CO ₂ incubator at 37°C with 4ml of liquid solution, the interval between changing the liquid for the first time was 72 hours, and changing the liquid every other day thereafter until a monolayer of cells was formed; ④ Subculture: Discard the culture medium in each bottle that has formed a cell monolayer in the primary culture, wash the cells 1-2 times with D-Hanks' solution containing 100 units/ml of P/S each, and add 4ml Digest the digestive enzyme solution with a percentage concentration of 0.25% trypsin+0.02% EDTA at 37°C for 10 minutes, pour the cell-containing enzyme solution into a 50ml centrifuge tube, and then use D-Hanks' containing 100 units/ml of P/S solution or 2ml of DMEM culture solution containing 15% volume ratio of calf serum and P/S of 100 units/ml, washed 1-2 times to wash all the cells in the bottle, centrifuged at 1500rpm for 3 minutes, discarded Obtain chondrocytes after clearing; 2) Preparation of bone matrix gel BMG: ①The long bones and metaphyseal spongy bones of New Zealand rabbits were taken under relatively aseptic conditions, and all soft tissues and bone marrow were removed and washed with distilled water; ②Rinse with 3mol/L sodium azide for 2-3 times and then wash with distilled water; use 1:1 chloroform/methanol to degrease overnight under magnetic stirring at room temperature, then wash with distilled water; ③Decalcify with 0.6mol/L hydrochloric acid at 4°C with magnetic stirring, during which time the liquid is changed every 4 hours until the bone becomes soft and has a certain degree of elasticity, then decalcification is terminated, and then washed with distilled water until neutral; ④ Under magnetic stirring at 4°C: treat with 2mol/L calcium chloride for 1 hour, wash with distilled water; treat with 0.5mol/L EDTA for 1 hour, wash with distilled water; treat with 8mol/L lithium chloride for 1 hour, wash with distilled water; ⑤ Treat with sterile distilled water at 55°C for 1 hour; ⑥ Trim the processed BMG into the size required for the experimental design, freeze-dry, sterilize with ⁶⁰ cobalt source and store in the refrigerator at 0℃～-20℃ for later use; 3) Construction of tissue engineered cartilage: Inoculate the seed cells harvested by separation and culture on cortical bone or cancellous bone BMG at a density of 4-100,000/ ^mm2 for in vitro culture, and the medium is 100 units each of calf serum and P/S containing 15% volume ratio /ml of DMEM culture medium, and change the medium every 3 days.

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