CN113577391A

CN113577391A - Preparation method of epiphyseal cartilage combined bone acellular material from natural tissue source

Info

Publication number: CN113577391A
Application number: CN202110962367.1A
Authority: CN
Inventors: 林贤丰; 张琪; 范顺武
Original assignee: Affiliated Sir Run Run Shaw Hospital of School of Medicine Zhejiang University
Current assignee: Affiliated Sir Run Run Shaw Hospital of School of Medicine Zhejiang University
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2021-11-02

Abstract

The invention discloses a preparation method of an epiphyseal cartilage combined bone acellular material from a natural tissue source, and belongs to the technical field of biological materials for repairing and regenerating tissues or organs. The preparation method comprises the steps of rinsing, repeatedly freezing and thawing the epiphyseal cartilage combined bone at the distal end of the femur of the young large white pig, and treating with PBS buffer solution containing TritonX-100, PBS buffer solution containing SLES, PBS buffer solution containing HTHOPS, PBS buffer solution containing DNase I and physiological saline to obtain the acellular epiphyseal cartilage combined bone material. The invention carries out complete decellularization treatment on the epiphyseal cartilage combined bone material, completely removes cells with immunogenicity and cell content components, simultaneously furthest retains the integrity of the epiphyseal cartilage combined bone ECM, has the capability of repairing osteochondral defect, and can be used for treating orthopedic diseases caused by osteochondral defect.

Description

Preparation method of epiphyseal cartilage combined bone acellular material from natural tissue source

Technical Field

The invention relates to the technical field of biomaterials for tissue or organ repair and regeneration, in particular to a preparation method of an epiphyseal cartilage combined bone acellular material from a natural tissue source.

Background

At present, as China gradually enters an aging society, osteoarthritis and diseases such as cartilage damage and cartilage defect caused by severe activities increasingly affect human health, heavy economic and physical and psychological burdens are caused on individuals, heavy social burdens are caused, and the problem to be solved in orthopedics clinic is solved urgently. In order to solve the above problems, the conventional solutions at present include arthroscopic repair surgery, bone joint replacement surgery, etc., which still have many problems, such as poor repair effect, huge surgical trauma, etc. In recent years, the use of biomaterials to repair cartilage tissue defects has received much attention, but the current biomaterials have problems of poor biocompatibility and insufficient cartilage repair and regeneration, and the goal of repair is focused only on simple hyaline cartilage. As is well known, osteoarthritis and cartilage damage involve not only hyaline cartilage itself but also subchondral bone, and therefore, in the repair and treatment of osteoarthritis and cartilage defects, a comprehensive repair of hyaline cartilage and subchondral bone is required. The development of biomaterials with the effect of promoting cartilage and bone regeneration becomes the bottleneck of urgent need for breakthrough in the current treatment of osteoarthritis and cartilage defects.

Epiphyseal cartilage is at both ends of the long bone, where secondary ossification centers occur. The intermediate layer of epiphyseal cartilage, which surrounds the secondary ossification centre, in normal organisms eventually differentiates into bone tissue so that the epiphyseal is closed. However, research shows that epiphyseal cartilage stores a layer of chondroblast cell band and has the potential of differentiating into cartilage, so that epiphyseal cartilage combined bone has the capacity of repairing cartilage combined bone defect.

The extracellular matrix derived from natural tissues can promote cell adhesion, growth, migration and guide cell differentiation to bone tissues, and is the focus of bone tissue engineering research in recent years. The natural tissue acellular material has unique biocompatibility due to its structure and composition being highly similar to those of living organisms, thus exhibiting excellent tissue repair effects. The preparation of the extracellular scaffold by using the decellularization technology provides a feasible idea for the preparation of tissue repair and regeneration materials. At present, few biological materials which can be used for repairing cartilage combined bones are reported, and epiphyseal cartilage combined bones are not reported for repairing osteoarthritis and cartilage defects.

Therefore, it is an urgent problem to provide a method for preparing a natural tissue-derived epiphyseal cartilage combined bone decellularized material.

Disclosure of Invention

In view of the above, the invention provides a preparation method of an epiphyseal cartilage combined bone acellular material from a natural tissue source, and the prepared epiphyseal cartilage combined bone acellular material is a biological material with good material characteristics, excellent biocompatibility and excellent bone cartilage repair effect, and provides a new material and a new way for repairing cartilage defects and treating osteoarthritis.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of epiphyseal cartilage combined bone acellular material from natural tissue sources comprises the following specific steps:

(1) repeatedly rinsing the distal epiphyseal cartilage joint bone tissue of the femur of a fresh 3-4 month old white pig (female and male without limitation) with sterile PBS for 5 times at a low temperature of 4 ℃ for 10min each time; removing blood and tissue fluid on the material;

(2) placing in an organic solvent, and shaking and degreasing in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for 2-6 h; then washing with sterile PBS for 12 h; the organic solvent is an ethanol solution with the mass concentration of 50-70% or an acetone solution with the mass concentration of 20-40%;

(3) placing in liquid nitrogen for 30min, quickly taking out, and placing in 37 deg.C water bath for 30 min; repeating the above process for 3 times, and finally rinsing with sterile PBS for 2 times, each time for 5 min;

(4) adding the mixed antibacterial solution into 1-2% volume concentration PBS buffer solution containing Triton X-100, and shaking in a constant temperature shaking table with constant temperature of 25 deg.C and rotation speed of 100rpm for 24-72 h; then washing with sterile PBS for 12 h;

(5) adding mixed antibacterial solution into PBS buffer solution containing sodium dodecyl sulfate sulfonate (SLES) with mass concentration of 2-5%, and shaking in constant temperature shaking table with constant temperature of 25 deg.C and rotation speed of 100rpm for 12-48 h; then washing with sterile PBS for 12 h;

(6) adding mixed antibacterial solution into PBS buffer solution of 3-allyloxy-2-hydroxy-1-propane sulfonic acid sodium salt (HT HOPS) with mass concentration of 1-2%, and shaking in constant temperature shaking table with constant temperature of 25 deg.C and rotation speed of 100rpm for 6-12 h; then washing with sterile PBS for 12 h;

(7) adding the mixed antibacterial solution into PBS buffer solution containing 0.01mg/ml DNase I, and carrying out thermostatic water bath at 37 ℃ for 6-12 h;

(8) placing the mixture in 50-70% ethanol, and shaking in a constant temperature shaking table with constant temperature of 25 deg.C and rotation speed of 120rpm for 24-48 h;

(9) adding the mixed antibacterial solution into sterile physiological saline, placing in a constant temperature shaking table with the temperature of 37 ℃ and the rotation speed of 120rpm, shaking for 12h, and repeating for 3 times; obtaining epiphyseal cartilage combined bone acellular material from natural tissue;

in the steps (4) to (7), the concentrations of penicillin and streptomycin in the mixed antibacterial solution are respectively 100U/ml and 100 mu g/ml; the volume ratio of the penicillin to the streptomycin is 1: 1; the volume ratio of the PBS buffer solution to the mixed antibacterial solution is 10: 1;

in the step (9), the concentrations of penicillin and streptomycin in the mixed antibacterial solution are respectively 100U/ml and 100 mu g/ml; the volume ratio of the penicillin to the streptomycin is 1: 1; the volume ratio of the sterile normal saline to the mixed antibacterial solution is 10: 1.

The above steps are all kept as relatively sterile as possible.

Aiming at the problems of the existing preparation method for cartilage-combined bone repair and regeneration materials, the invention establishes a preparation method for epiphyseal cartilage-combined bone acellular materials from natural tissue sources, and the method comprises the steps of repeatedly rinsing, degreasing, repeatedly freezing and thawing the distal epiphyseal cartilage-combined bone tissue of young large white pig femur, and obtaining the epiphyseal cartilage-combined bone acellular materials after detoxification treatment of PBS buffer solution containing Triton X-100, PBS buffer solution containing SLES, PBS buffer solution containing HT HOPS, PBS buffer solution containing DNase I and physiological saline.

According to the technical scheme, compared with the prior art, the invention discloses a preparation method of the epiphyseal cartilage combined bone decellularized material from natural tissue sources, and the preparation method has the following beneficial effects:

(1) the invention realizes the preparation of the epiphyseal cartilage combined bone acellular material for the first time, adopts SLES and HT HOPS to replace Sodium Dodecyl Sulfate (SDS), and adopts SLES and HT HOPS as detergents, but is milder compared with SDS and has considerable acellular effect, and can completely remove cells and cell contents in the material, simultaneously retain the original natural ECM components and structural integrity to a greater extent, and the structure of the epiphyseal cartilage combined bone is very similar to that of normal cartilage combined bone, and can simulate the components and structure of normal cartilage combined bone tissues to a maximum extent.

(2) After the material is subjected to cell removal and then is repeatedly rinsed by ethanol and sterile physiological saline for detoxification, the material has no cytotoxicity and immunogenicity, and has good biocompatibility and excellent cartilage combined bone defect repair effect.

(3) The material of the invention is from thighbone of young large white pig, has wide raw material source and can be used for batch production.

(4) The invention can be an individualized epiphyseal cartilage combined bone biomaterial with customizable size and dimension so as to meet the clinical complex and diversified needs for repairing cartilage combined bones.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic view of the epiphyseal cartilage joint bone site of the present invention;

wherein, the left figure is a general figure of the distal femur epiphysis cartilage plate, and the square frame is a material drawing part; the right image shows HE staining of the material-drawing part, showing that the delta region is epiphyseal cartilage region;

FIG. 2 is a drawing showing the quantitative DNA detection of each region of the epiphyseal cartilage combined bone decellularized material before and after decellularization according to the invention; ***: p is less than 0.001;

FIG. 3 is a diagram showing the evaluation of the decellularization efficiency of the epiphyseal cartilage and bone decellularization material before and after decellularization according to the invention; wherein, the 2 nd column diagram is a macro diagram of the 3 rd, 4 th and 5 th column diagrams; the scale size is 100 μm;

FIG. 4 is a diagram showing qualitative evaluation of main component retention of epiphyseal cartilage combined bone decellularized material before and after decellularization according to the present invention; wherein, the 1 st column diagram is a macro diagram of the 2 nd, 3 rd and 4 th column diagrams; the scale size is 100 μm;

FIG. 5 is a diagram showing quantitative evaluation of main component retention of epiphyseal cartilage combined bone decellularized material before and after decellularization according to the present invention; wherein, the left graph is a collagen quantitative evaluation bar graph; the right panel is a glycosaminoglycan (GAG) quantitative assessment bar chart; ns: p is more than 0.05; *: p is less than 0.05;

FIG. 6 is a diagram showing the evaluation of the preservation of the three-dimensional structure of the epiphyseal cartilage associated bone decellularized material before and after decellularization according to the present invention;

FIG. 7 is a graph of uniaxial compression curves for a normal set of the invention;

FIG. 8 is a graph showing uniaxial compression of a decellularized group according to the invention;

FIG. 9 is a graph showing the elastic modulus statistics of the present invention; ns: p is more than 0.05;

FIG. 10 is a graph showing the relative elastic limit statistics of the present invention; *: p is less than 0.05;

FIG. 11 is a graph showing the cytotoxicity evaluation of decellularized epiphyseal cartilage in combination with bone decellularized material in accordance with the present invention; ns: p is more than 0.05;

FIG. 12 is a graph showing the evaluation of cytotoxicity and cell adhesion of a decellularized epiphyseal cartilage combined bone decellularized material in accordance with the invention; wherein a, b, c represent cells in the epiphyseal cartilage, the junction region and the cancellous bone region, respectively, to indicate the adherability of the scaffold and to promote proliferative migration of the target cells; the image in the square frame is a low power mirror image, and the image outside the square frame is a high power mirror image for selecting a specific target from the low power mirrors; the scale size is 60 μm;

FIG. 13 is a graph showing the immunogenicity assessment of a decellularized epiphyseal cartilage in combination with a bone decellularized material according to the invention;

wherein the first column "generally" is an embedded overview picture,

columns

2 and 3 are enlarged views of the epiphyseal cartilage and bone, respectively, of column 1; the scale size is 100 μm;

FIG. 14 is a drawing showing in vitro osteogenesis and chondrogenesis induction evaluation of a decellularized epiphyseal cartilage combined bone decellularized material of the invention; ns: p is more than 0.05; *: p is less than 0.05; **: p is less than 0.01; the left side of each group of column graphs is a blank group, and the right side is an experimental group;

FIG. 15 is a drawing showing the in vivo osteogenic and chondrogenic tissue evaluation of a decellularized epiphyseal cartilage associated osteocytic material of the invention; the scale size is 1 mm;

FIG. 16 is a drawing showing the ratio of cartilage in the cartilage region of the present invention; *: p is less than 0.05; ***: p is less than 0.001;

FIG. 17 is a drawing showing the ratio of cartilage in the subchondral bone region of the present invention; ns: p is more than 0.05; ***: p is less than 0.001;

FIG. 18 is a drawing showing the proportion of bone mass in the subchondral bone region of the present invention; ***: p is less than 0.001; .

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(1) material taking: rinsing the distal epiphyseal cartilage combined bone of the femur of a fresh 3-month-old young white pig for 5 times and 10min each time in a sterile PBS buffer solution at the low temperature of 4 ℃, and removing impurities such as hair, muscle, fascia, blood, tissue fluid and the like on the surface; the size of the obtained materials is determined according to actual requirements, and cylinders with the diameter of 5mm and the height of 5mm are generally selected as the sizes;

(2) a cell removing process:

placing the mixture in 500ml of organic solvent solution (20% acetone solution) in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for shaking and degreasing for 4 hours, and then washing the mixture for 12 hours by using sterile PBS (phosphate buffer solution);

secondly, wiping the materials with sterile gauze, placing the materials in liquid nitrogen for 30min, then quickly taking the materials out, and placing the materials in water bath at 37 ℃ for 30 min; repeating for 3 times; finally rinsing with sterile PBS for 5min for 2 times;

③ adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and 100 mu g/ml into 500ml of PBS buffer solution containing 1 percent of Triton-X100 by volume concentration, and placing the mixture in a constant temperature shaking table with the temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 48 hours; then washing with sterile PBS for 12 h;

fourthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and the concentration of 100 mu g/ml into 500ml of PBS buffer solution containing the SLES with the mass concentration of 2%, and placing the mixed antibacterial solution in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 36 hours; then washing with sterile PBS for 12 h;

fifthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and 100 mug/ml into 500ml of PBS buffer solution containing 1% by mass of 3-allyloxy-2-hydroxy-1-propanesulfonic acid sodium salt (HT HOPS), and placing the mixture in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm to vibrate for 6 hours; then washing with sterile PBS for 12 h;

sixthly, adding 10ml of mixed antibacterial solution of 100U/ml and 100 mu g/ml of penicillin and streptomycin into 100ml of PBS buffer solution containing DNase I with the concentration of 0.01mg/ml, and placing the mixed antibacterial solution in a constant-temperature 37 ℃ water bath box for 12 hours;

seventhly, placing the mixture in 500ml of ethanol with the mass concentration of 70% in a constant-temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 120rpm to shake for 24 hours;

adding 50ml of mixed antibacterial solution of 100U/ml penicillin and streptomycin with the concentration of 100 mu g/ml and the concentration of 100 mu g/ml into 500ml of sterile physiological saline, placing the mixture into a constant-temperature shaking table with the temperature of 37 ℃ and the rotating speed of 120rpm, shaking for 12 hours, and repeating the shaking for 3 times to obtain the epiphyseal cartilage combined bone acellular material from natural tissue sources.

A series of tests and experiments were carried out using the epiphyseal cartilage associated bone decellularized material prepared in example 1 as a template, as follows:

the schematic drawing of the epiphyseal cartilage joint bone drawing part is shown in figure 1; FIG. 1 shows, on the left, a schematic view of a distal epiphyseal chondroplane of a femur, with material access in boxes; the right panel shows HE staining of the site, showing the Δ region as the epiphyseal cartilage region.

FIG. 2 is a DNA quantitative determination diagram of each region of epiphyseal cartilage associated bone decellularized material before and after decellularization; extracting spongy bone and bone epiphysis region DNA in the epiphyseal cartilage combined bone acellular material before and after acellular by adopting a DNA genome extraction kit, determining corresponding OD values, and determining the DNA content of each region. The result shows that the DNA in the cell of the epiphyseal cartilage combined bone decellularized material after decellularization is reduced by more than 95 percent compared with that of the bracket before decellularization, and no obvious DNA residue exists. (p < 0.001).

FIG. 3 is a diagram showing the evaluation of the decellularization efficiency of the epiphyseal cartilage and bone decellularized material before and after decellularization; the general picture of the epiphyseal cartilage combined bone decellularized material before and after decellularization shows that the bracket changes from pink to milky white after decellularization; the HE staining graph shows that the bracket has no obvious cells and cell content residues after the decellularization; the DAPI staining pattern showed no significant nuclei and fragments remaining after the scaffold was detached. In conclusion, the method shows that the acellular effect of the epiphyseal cartilage combined bone acellular material is good, the scaffold has no obvious cells and no residue in the content.

FIG. 4 is a qualitative evaluation diagram of main component retention of epiphyseal cartilage combined bone decellularized material before and after decellularization; the first and second line images are Masson trichrome staining images, and the results show that the collagen component in the epiphyseal cartilage combined bone decellularized material before and after decellularization is well preserved; the third and fourth panels show fast green staining with safranin O, indicating that the epiphyseal cartilage glycosaminoglycans are more lost after decellularization, while the cancellous bone portion glycosaminoglycans remain substantially intact.

FIG. 5 is a diagram showing quantitative evaluation of the retention of main components of the epiphyseal cartilage associated bone decellularized material before and after decellularization; the left graph is a collagen quantitative evaluation bar graph, and the result shows that the collagen component in the epiphyseal cartilage combined bone decellularized material is well preserved after decellularization; the right graph is a bar chart for quantitative evaluation of glycosaminoglycan (GAG), and the result shows that the content of the GAG in the decellularized epiphyseal cartilage is obviously reduced compared with that in the normal group, and the GAG in the decellularized cancellous bone part is kept intact.

FIG. 6 is a diagram showing the evaluation of the retention of the three-dimensional structure of the epiphyseal cartilage associated bone decellularized material before and after decellularization; the surface three-dimensional structures of the epiphyseal cartilage and bone decellularized material before and after decellularization are evaluated through a Scanning Electron Microscope (SEM), and the results show that the three-dimensional structures of the surfaces of all parts of the scaffold are not obviously damaged, and the porosity of the scaffold after decellularization is obviously increased.

FIGS. 7-10 are graphs showing quantitative assessment of mechanical retention of epiphyseal cartilage in combination with bone decellularized material before and after decellularization; the mechanical properties of the epiphyseal cartilage combined bone decellularized material before and after decellularization are measured by adopting a uniaxial compression test, and the result shows that the mechanical properties of the epiphyseal cartilage combined bone decellularized material after decellularization are well reserved.

FIG. 11 is a graph of cytotoxicity assessment of decellularized epiphyseal cartilage in combination with bone decellularized material; extracting a leaching liquor of the decellularized epiphyseal cartilage combined bone decellularized material by adopting a cck-8 method, diluting by different times, and determining the OD value of the concentration of a cell metabolite, wherein the OD value is compared with the OD value of the concentration of the cell metabolite determined after the cell metabolite is cultured by a common culture medium, and the result shows that the decellularized epiphyseal cartilage combined bone decellularized material has no obvious cytotoxicity.

FIG. 12 is a graph of the assessment of cytotoxicity, cell adhesion of decellularized epiphyseal cartilage in combination with bone decellularized material; after mouse Bone Mesenchymal Stem Cells (BMSCs) are planted on the decellularized epiphyseal cartilage combined bone decellularized material for 1 week, it can be seen that the BMSCs can be normally adhered to the scaffold and can migrate to a spongy bone area in the scaffold (figure C), and the scaffold is free from cytotoxicity and can be used as a medium for allowing cells to normally adhere and migrate.

FIG. 13 is a graph of the immunogenicity assessment of a decellularized epiphyseal cartilage in combination with a bone decellularized material; the acellular epiphyseal cartilage and bone acellular material is embedded in subcutaneous tissues of the skin on the back of the rat for 2 and 4 weeks to evaluate the immunogenicity of the scaffold, and the result shows that a certain degree of inflammatory reaction exists at 2 weeks; but at 4 weeks the inflammatory response was markedly reduced and visible signs of stent degradation.

FIG. 14 is a diagram of in vitro osteogenesis and chondrogenesis induction evaluation of a decellularized epiphyseal cartilage combined bone decellularized material; the mouse bone mesenchymal stem cells are planted on the decellularized epiphyseal cartilage combined bone decellularized material for 3, 7 and 14 days, after the cells are collected and RNA is extracted, PCR test is carried out, and then the relative expression quantity of the mouse bone mesenchymal stem cells and blank groups (the mesenchymal stem cells are simply planted in a culture dish) of specific genes (RUNX2, Collagen I and ALP are osteogenic genes, and SOX9, Collagen II and aggrecan are chondrogenic genes) is measured. The results show that the acellular epiphyseal cartilage combined bone acellular material has the function of obviously promoting the generation of bones and cartilage.

FIG. 15 is a graph of in vivo osteogenic and chondrogenic tissue evaluation of decellularized epiphyseal cartilage in combination with osteocytic material; after the acellular epiphyseal cartilage and the bone are embedded in the distal articular surface defect of the femur of an adult New Zealand white rabbit for 4 weeks and 8 weeks, the experimental part is taken out, and HE staining and safranin fast green staining are carried out to preliminarily judge the bone and cartilage repairing effect. The results show that at week 4, cartilage and subchondral bone are initially repaired as seen in the blank; at week 8, the subchondral bone is basically and completely repaired, and the defects of the cartilage part except the defect junction part are not completely repaired, but the rest parts are basically and completely repaired. In conclusion, the decellularized epiphyseal cartilage combined bone decellularized material can promote the repair and regeneration of osteochondral defects.

FIGS. 16-18 are graphs showing quantitative assessment of in vivo osteogenic and chondrogenic induction of decellularized epiphyseal cartilage in combination with osteocytic material; in FIG. 15, the area under the joint surface of the patient is regarded as the cartilage area, and the area under the joint surface of the patient is regarded as the subchondral bone area, and the areas occupied by the cartilage and the bone in the subchondral bone area are measured by a quantitative method, so as to quantitatively evaluate the repair effect of the cartilage and the bone at the defect part. The result shows that at the 8 th week, the decellularized epiphyseal cartilage combined bone decellularized material has obvious effects of promoting cartilage and osteogenesis.

Example 2

(2) a cell removing process:

placing the mixture in 500ml of organic solvent solution (20% acetone solution) in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for shaking and degreasing for 2 hours, and then washing the mixture for 12 hours by using sterile PBS (phosphate buffer solution);

③ adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and 100 mu g/ml into 500ml of PBS buffer solution containing 2 percent of Triton-X100 by volume concentration, and placing the mixture in a constant temperature shaking table with the temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 48 hours; then washing with sterile PBS for 12 h;

fourthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and the concentration of 100 mu g/ml into 500ml of PBS buffer solution containing 5% SLES, and placing the mixed antibacterial solution in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 36 hours; then washing with sterile PBS for 12 h;

fifthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and 100 mug/ml into 500ml of PBS buffer solution containing 2% by mass of 3-allyloxy-2-hydroxy-1-propanesulfonic acid sodium salt (HT HOPS), and placing the mixture in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm to vibrate for 6 hours; then washing with sterile PBS for 12 h;

sixthly, adding 10ml of mixed antibacterial solution of 100U/ml and 100 mu g/ml of penicillin and streptomycin into 100ml of PBS buffer solution containing DNase I with the concentration of 0.01mg/ml, and placing the mixed antibacterial solution in a constant-temperature 37 ℃ water bath box for 6 hours;

seventhly, placing the mixture in 500ml of 50% ethanol at the constant temperature of 25 ℃ and the rotating speed of 120rpm in a constant-temperature shaking table to shake for 24 hours;

Example 3

(2) a cell removing process:

placing the mixture in 500ml of organic solvent solution (30% acetone solution) in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for shaking and degreasing for 6 hours, and then washing the mixture for 12 hours by using sterile PBS (phosphate buffer solution);

③ adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and 100 mu g/ml into 500ml of PBS buffer solution containing 1 percent of Triton-X100 by volume concentration, and placing the mixture in a constant temperature shaking table with the temperature of 25 ℃ and the rotating speed of 100rpm to shake for 24 hours; then washing with sterile PBS for 12 h;

fourthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and the concentration of 100 mu g/ml into 500ml of PBS buffer solution containing the SLES with the mass concentration of 2%, and placing the mixed antibacterial solution in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 12 hours; then washing with sterile PBS for 12 h;

fifthly, adding 50ml of mixed antibacterial solution of 100U/ml and 100 mu g/ml of penicillin and streptomycin into 500ml of PBS buffer solution containing 1 mass percent of 3-allyloxy-2-hydroxy-1-propanesulfonic acid sodium salt (HT HOPS), and placing the mixture in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm to vibrate for 12 hours; then washing with sterile PBS for 12 h;

sixthly, adding 10ml of mixed antibacterial solution of 100U/ml and 100 mu g/ml of penicillin and streptomycin into 100ml of PBS buffer solution containing DNase I with the concentration of 0.01mg/ml, and placing the mixed antibacterial solution in a constant-temperature 37 ℃ water bath box for 9 hours;

seventhly, placing the mixture in 500ml of 50% ethanol at the constant temperature of 25 ℃ and the rotating speed of 120rpm to shake for 48 hours;

Example 4

(1) Material taking: rinsing the distal epiphyseal cartilage combined bone of the femur of a fresh 4-month-old young white pig for 5 times and 10min each time in a sterile PBS buffer solution at the low temperature of 4 ℃, and removing impurities such as hair, muscle, fascia, blood, tissue fluid and the like on the surface; the size of the obtained materials is determined according to actual requirements, and cylinders with the diameter of 5mm and the height of 5mm are generally selected as the sizes;

(2) a cell removing process:

placing the mixture in 500ml of organic solvent solution (40% acetone solution) in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for shaking and degreasing for 2 hours, and then washing the mixture for 12 hours by using sterile PBS (phosphate buffer solution);

③ adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and 100 mu g/ml into 500ml of PBS buffer solution containing 2 percent of Triton-X100 by volume concentration, and placing the mixture in a constant temperature shaking table with the temperature of 25 ℃ and the rotating speed of 100rpm to shake for 24 hours; then washing with sterile PBS for 12 h;

fourthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and the concentration of 100 mu g/ml into 500ml of PBS buffer solution containing 5% SLES, and placing the mixed antibacterial solution in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 12 hours; then washing with sterile PBS for 12 h;

fifthly, adding 50ml of mixed antibacterial solution of 100U/ml and 100 mu g/ml of penicillin and streptomycin into 500ml of PBS buffer solution containing 2% by mass of 3-allyloxy-2-hydroxy-1-propanesulfonic acid sodium salt (HT HOPS), and placing the mixture in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm to vibrate for 12 hours; then washing with sterile PBS for 12 h;

seventhly, placing the mixture in 500ml of ethanol with the mass concentration of 70% in a constant-temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 120rpm to vibrate for 48 hours;

Example 5

(2) a cell removing process:

placing the mixture in 500ml of organic solvent solution (40% acetone solution) in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for shaking and degreasing for 6 hours, and then washing the mixture for 12 hours by using sterile PBS (phosphate buffer solution);

③ adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and 100 mu g/ml into 500ml of PBS buffer solution containing 1 percent of Triton-X100 by volume concentration, and placing the mixture in a constant temperature shaking table with the temperature of 25 ℃ and the rotating speed of 100rpm to shake for 72 hours; then washing with sterile PBS for 12 h;

fourthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and the concentration of 100 mu g/ml into 500ml of PBS buffer solution containing the SLES with the mass concentration of 2%, and placing the mixed antibacterial solution in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 48 hours; then washing with sterile PBS for 12 h;

fifthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and 100 mug/ml into 500ml of PBS buffer solution containing 1% by mass of 3-allyloxy-2-hydroxy-1-propanesulfonic acid sodium salt (HT HOPS), and placing the mixture in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 9 hours; then washing with sterile PBS for 12 h;

seventhly, placing the mixture in 500ml of 60% ethanol at the constant temperature of 25 ℃ and shaking for 24 hours in a constant-temperature shaking table at the rotating speed of 120 rpm;

Example 6

(2) a cell removing process:

placing the mixture in 500ml of organic solvent solution (50% ethanol solution) in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for shaking and degreasing for 2 hours, and then washing the mixture for 12 hours by using sterile PBS (phosphate buffer solution);

③ adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and 100 mu g/ml into 500ml of PBS buffer solution containing 2 percent of Triton-X100 by volume concentration, and placing the mixture in a constant temperature shaking table with the temperature of 25 ℃ and the rotating speed of 100rpm to shake for 72 hours; then washing with sterile PBS for 12 h;

fourthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and the concentration of 100 mu g/ml into 500ml of PBS buffer solution containing the SLES with the mass concentration of 5%, and placing the mixed antibacterial solution in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 48 hours; then washing with sterile PBS for 12 h;

fifthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and 100 mug/ml into 500ml of PBS buffer solution containing 2% by mass of 3-allyloxy-2-hydroxy-1-propanesulfonic acid sodium salt (HT HOPS), and placing the mixture in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 9 hours; then washing with sterile PBS for 12 h;

seventhly, placing the mixture in 500ml of 60% ethanol at the constant temperature of 25 ℃ and in a constant temperature shaking table with the rotating speed of 120rpm to shake for 48 hours;

Example 7

(2) a cell removing process:

placing the mixture in 500ml of organic solvent solution (50% ethanol solution) in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for shaking and degreasing for 6 hours, and then washing the mixture for 12 hours by using sterile PBS (phosphate buffer solution);

fourthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and the concentration of 100 mu g/ml into 500ml of PBS buffer solution containing 3% SLES, and placing the mixed antibacterial solution in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 36 hours; then washing with sterile PBS for 12 h;

seventhly, placing the mixture in 500ml of 50% ethanol at the constant temperature of 25 ℃ and the rotating speed of 120rpm to shake for 36 hours;

Example 8

(2) a cell removing process:

placing the mixture in 500ml of organic solvent solution (70% ethanol solution) in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for shaking and degreasing for 2 hours, and then washing the mixture for 12 hours by using sterile PBS (phosphate buffer solution);

fourthly, adding 50ml of mixed antibacterial solution of penicillin and streptomycin with the concentration of 100U/ml and the concentration of 100 mu g/ml into 500ml of PBS buffer solution containing the SLES with the mass concentration of 4%, and placing the mixed antibacterial solution in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm for shaking for 36 hours; then washing with sterile PBS for 12 h;

seventhly, placing the mixture in 500ml of 60% ethanol at the constant temperature of 25 ℃ and shaking for 36 hours in a constant-temperature shaking table at the rotating speed of 120 rpm;

Example 9

(2) a cell removing process:

placing the mixture in 500ml of organic solvent solution (70% ethanol solution) in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for shaking and degreasing for 6 hours, and then washing the mixture for 12 hours by using sterile PBS (phosphate buffer solution);

seventhly, placing the mixture in 500ml of ethanol with the mass concentration of 70% in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 120rpm to shake for 36 hours;

Example 10

(2) a cell removing process:

placing the mixture in 500ml of organic solvent solution (60% ethanol solution) in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 60rpm for shaking and degreasing for 4 hours, and then washing the mixture for 12 hours by using sterile PBS (phosphate buffer solution);

Examples 2 to 10 all produced epiphyseal cartilage combined bone decellularized materials having complete decellularization, intact scaffold three-dimensional structure and active ingredients, and certain biological activities (such as osteogenic and chondrogenic activities), and the corresponding results were substantially the same as in example 1.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A preparation method of an epiphyseal cartilage combined bone acellular material from natural tissue sources is characterized by comprising the following specific steps:

(1) repeatedly rinsing the distal epiphyseal cartilage joint bone tissue of the femur of a fresh 3-4 month old white pig for 5 times with sterile PBS at a low temperature of 4 ℃ for 10min each time;

(3) placing in liquid nitrogen for 30min, quickly taking out, and placing in 37 deg.C water bath for 30 min; repeating for 3 times; finally rinsing with sterile PBS for 5min for 2 times;

(5) adding the mixed antibacterial solution into PBS buffer solution containing sodium dodecyl sulfate sulfonic acid with the mass concentration of 2-5%, and placing in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotation speed of 100rpm to shake for 12-48 h; then washing with sterile PBS for 12 h;

(6) adding the mixed antibacterial solution into PBS buffer solution of 3-allyloxy-2-hydroxy-1-propane sulfonic acid sodium salt with the mass concentration of 1-2%, and placing in a constant temperature shaking table with the constant temperature of 25 ℃ and the rotating speed of 100rpm to shake for 6-12 h; then washing with sterile PBS for 12 h;

(7) adding the mixed antibacterial solution into PBS buffer solution with the concentration of 0.01mg/ml DNase I, and carrying out constant-temperature water bath at 37 ℃ for 6-12 h;