CN107537066B - Bionic cartilage based on 3D printing and manufacturing method thereof - Google Patents
Bionic cartilage based on 3D printing and manufacturing method thereof Download PDFInfo
- Publication number
- CN107537066B CN107537066B CN201710695028.5A CN201710695028A CN107537066B CN 107537066 B CN107537066 B CN 107537066B CN 201710695028 A CN201710695028 A CN 201710695028A CN 107537066 B CN107537066 B CN 107537066B
- Authority
- CN
- China
- Prior art keywords
- bionic
- cartilage
- printing
- concentration
- collagen
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The invention belongs to the technical field of biomedical engineering, in particular to the technical field of bionic material preparation, and discloses a bionic cartilage based on 3D printing and a manufacturing method thereof, and the bionic cartilage based on 3D printing and the manufacturing method of the 3D printing bionic cartilage are included. The bionic cartilage based on 3D printing is of a multilayer structure and is prepared from modified type II collagen, modified hyaluronic acid, nano-hydroxyapatite and chondrocytes through gradient bionic 3D printing. The bionic cartilage has good biocompatibility and mechanical property equivalent to that of normal cartilage tissue, and is favorable for repairing cartilage defect parts.
Description
Technical Field
The invention belongs to the technical field of biomedical engineering, and particularly relates to a bionic cartilage based on 3D printing and a manufacturing method thereof.
Background
Cartilage degeneration or injury caused by trauma or disease is common in clinic, but due to the special articular cartilage tissue, blood vessels, lymphatic vessels and nerves are not arranged inside, and after traumatic injury, the cartilage degeneration or injury has only extremely low or almost no effective self-repairing capability, and if the treatment is not timely or proper, serious dysfunction can be caused. Like other tissue repair processes, cartilage repair is the ultimate formation of repair tissue at the site of injury through the formation of a fibrin clot and the subsequent cascade of normal cartilage surrounding the defect, bone marrow beneath the defect, or chondrocytes from synovial tissue. Several existing cartilage repair techniques, such as subchondral drilling, debridement drilling, or microfracture, all involve the removal of cartilage fragments, the formation of worn joints, and the flushing of joints, with defects close to the subchondral blood-supplying tissue and bone marrow. However, in the conventional technology, cartilage defects are repaired by fibrocartilage-like tissues, and the repaired tissues are different from normal cartilage in terms of composition, structure and mechanical properties (normal articular cartilage is mainly composed of hyaline cartilage and serves as a cushion pad to resist external stress), so that the long-term clinical effect of cartilage repair cannot be predicted. Patients treated by traditional means all have a short period of pain relief; however, when the repair tissue is degenerated, the patient may develop progressive symptoms. Repair of defects with appropriate cartilage filler materials is an effective method for rapidly restoring diseased or traumatic cartilage defect tissue. Cartilage tissue engineering provides an ideal treatment approach for the treatment of cartilage injury, but the prior art is difficult to prepare a high-quality cartilage repair material which is similar to the physiological structure and the physiological performance of normal cartilage tissues, and has a plurality of problems to be solved, such as fixation of new cartilage tissues, immune rejection after allogeneic cell transplantation, selection of seed cells and growth factors, how to solve the long-term degeneration of cultured cartilage, whether to bear long-term load and friction, and the like.
In recent years, researchers have proposed 4F guidelines for the design of tissue engineering scaffolds: shape complaints (Form), performance complaints (Function), functional complaints (format), and implantability (visualization). Shape appeal means that the scaffold material must be able to completely fill complex three-dimensional defects and can induce tissue regeneration; the performance appeal refers to the requirement that the mechanical property and the like of the bracket can temporarily play a role of the missing tissue before the missing tissue is repaired; the functional appeal means that the material needs to have corresponding biological activity and can provide a proper environment for cells to promote tissue regeneration; by implantable is meant that the stent can be surgically implanted into the body and perform its intended function. By utilizing bionics, a microstructure suitable for the growth of tissue cells is constructed in vitro, the in vivo environment is simulated, the proliferation, differentiation, migration, apoptosis and the like of different cells are coordinated, and the method is a development direction for treating tissue defects.
The personalized scaffold prepared by the 3D printing technology can accurately simulate a complex three-dimensional microstructure of a natural tissue, the shape of the scaffold is highly matched with that of a defective tissue, the scaffold can be endowed with biological activity by supporting the common printing of growth factors and cells, and the physiological structure of a normal tissue is simulated by a gradient printing method, so that a repair material which is not different from the normal tissue in structure and physiological function is prepared, and the personalized scaffold is a necessary trend for the development of the field of tissue engineering.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a preparation method of a bionic cartilage based on 3D printing.
The invention also aims to provide the bionic cartilage based on 3D printing obtained by the preparation method, which is characterized in that the bionic cartilage is prepared by 20-80 parts of modified type II collagen, 20-80 parts of modified hyaluronic acid, 10-20 parts of nano hydroxyapatite and 0.1-0.5 part of chondrocyte through gradient bionic 3D printing according to parts by weight.
Furthermore, the bionic cartilage is formed by sequentially printing a bionic subchondral bone, a bionic calcification layer, a bionic cartilage deep layer, a bionic intermediate layer and a bionic superficial surface layer through a 3D printer from bottom to top, and the whole bionic cartilage is of a multi-layer interpenetrating network structure.
The bionic cartilage based on 3D printing and the manufacturing method thereof specifically comprise the following steps;
(1) preparation of printing material:
① preparing modified type II collagen, adding type II collagen into PBS phosphate at 50 deg.C to obtain 10% solution, adding methacrylic anhydride at volume ratio of type II collagen PBS phosphate solution V methacrylic anhydride =10:0.75, stirring at 50 deg.C for reaction for 4 hr, dialyzing the obtained solution in dialysis bag with molecular weight cutoff of 10000 for 7 days, freeze drying the obtained dialysate in-80 deg.C freeze dryer for 48 hr to obtain modified type II collagen, storing at room temperature;
② preparing modified hyaluronic acid, dissolving hyaluronic acid in distilled water to obtain hyaluronic acid solution with mass concentration of 2%, adding sodium hydroxide solution with molar concentration of 1 mol/L to adjust pH of the solution to 8.5 to obtain sodium hyaluronate solution, adding dimethylaminoethyl methacrylate according to mass ratio of m sodium hyaluronate to m dimethylaminoethyl methacrylate =1:3.5, reacting at 10 ℃ for 24h, placing the obtained solution in a dialysis bag with molecular weight cutoff of 14000 for flowing water dialysis for 7 days, placing the dialysate in a freeze dryer with temperature of minus 80 ℃ for freeze drying for 48 hours to obtain modified hyaluronic acid, and storing at room temperature for later use;
③ the chondrocytes are digested and added to a chondrocyte medium containing 10% by volume bovine serum to prepare a cell concentration of 1 × 106—5×106mL-1Placing the chondrocyte suspension in a cell culture box with the concentration of carbon dioxide of 5% and the temperature of 37 ℃ for standby; the chondrocytes are normally cultured chondrocytes after 3 generations;
(2) preparing precursors of all bionic layers:
① preparation of the bionic subchondral bone precursor, which is to add Irgacure2959 photoinitiator and the modified type II collagen prepared in the step (1) into distilled water in sequence, add the next substance after the substances added each time are completely dissolved, then add nano-hydroxyapatite, stir until the nano-hydroxyapatite is uniformly dispersed in the solution, and obtain a bionic subchondral bone precursor solution for later use, wherein the concentration of the Irgacure2959 photoinitiator is 0.5wt%, the concentration of the nano-hydroxyapatite is 5-10wt%, and the concentration of the modified type II collagen is 5-10 wt%;
② preparation of a bionic calcification layer precursor, namely adding Irgacure2959 photoinitiator and the modified hyaluronic acid prepared in the step (1) into distilled water in sequence, adding the next substance after the substances added each time are completely dissolved, then adding nano-hydroxyapatite, and stirring until the nano-hydroxyapatite is uniformly dispersed in the solution to obtain a bionic calcification layer precursor solution for later use, wherein the concentration of the Irgacure2959 photoinitiator is 0.5wt%, the concentration of the nano-hydroxyapatite is 1-5wt%, and the concentration of the modified hyaluronic acid is 1-3 wt%;
③ preparation of the bionic cartilage deep precursor, which comprises adding Irgacure2959 photoinitiator, the modified hyaluronic acid prepared in step (1) and the modified type II collagen prepared in step (1) into distilled water in sequence, adding the next substance after the substances added in each time are completely dissolved, adding the chondrocyte suspension prepared in step (1) with the same volume, and stirring gently until the mixture is uniform to obtain the bionic cartilage deep precursor solution for later use, wherein the concentration of the Irgacure2959 photoinitiator is 0.5wt%, the concentration of the modified hyaluronic acid is 1-3 wt%, the concentration of the modified type II collagen is 10-15wt%, and the concentration of the chondrocyte is 5 × 10wt%5—2.5×106mL-1；
④ preparation of a bionic intermediate layer precursor, which is to add Irgacure2959, the modified hyaluronic acid prepared in the step (1) and the modified type II collagen prepared in the step (1) into distilled water in sequence, and add the next substance after the substances added each time are completely dissolved to obtain a bionic intermediate layer precursor solution for later use, wherein the concentration of the Irgacure2959 is 0.5wt%, the concentration of the modified hyaluronic acid is 2-5 wt%, and the concentration of the modified type II collagen is 10-15 wt%;
⑤ preparation of a bionic shallow surface layer precursor, namely adding a photoinitiator Irgacure2959 and the modified type II collagen prepared in the step (1) into distilled water in sequence, and adding the next substance after the substances added each time are completely dissolved to obtain a bionic shallow surface layer precursor solution for later use, wherein the concentration of the photoinitiator Irgacure2959 is 0.5wt%, and the concentration of the modified type II collagen is 10-15 wt%;
(3) bionic printing of the bionic cartilage:
sequentially arranging the precursors of the bionic layers prepared in the step (2) according to the bionic subchondral bone precursor, the bionic calcification layer precursor, the bionic cartilage deep layer precursor, the bionic middle layer precursor and the bionic superficial layer precursor
Sequentially overlapping and printing, wherein the printing thickness of the bionic subchondral bone is set to be 5-10% of the total thickness of the bionic cartilage, the printing thickness of the bionic calcified layer is set to be 2-5% of the total thickness of the bionic cartilage, the printing thickness of the bionic cartilage deep layer is set to be 15-30% of the total thickness of the bionic cartilage, the printing thickness of the bionic intermediate layer is set to be 40-55% of the total thickness of the bionic cartilage, the printing thickness of the bionic superficial layer is set to be 10-20% of the total thickness of the bionic cartilage, and the bionic cartilage based on 3D printing is obtained;
preferably, the type II collagen has a molecular weight of 1 × 106kDa；
Preferably, the molecular weight of the hyaluronic acid is 1.2-1.5 × 106kDa；
Preferably, the particle size distribution range of the nano hydroxyapatite is 150-250 nm;
preferably, the bionic printing of the bionic cartilage is performed by adopting a 3D printer of 3D systems ProX 800 model 3D stereolithography, the ultraviolet irradiation time of each bionic layer is set to be 30 s, and the ultraviolet irradiation intensity is set to be 10 mW/CM2；
The invention has the beneficial effects that:
(1) the bionic cartilage disclosed by the invention has the advantages of low immunogenicity, rich raw materials, good biocompatibility, good mechanical property and vascularization capacity.
(2) According to the invention, the 3D bionic printing material is prepared by taking the modified type II collagen, the modified hyaluronic acid, the nano-hydroxyapatite and the chondrocytes as matrixes, and then the bionic printing material is prepared by utilizing the three-dimensional printing technology to perform bionic printing, so that the problem of poor repairing effect caused by a single-phase structure of the traditional cartilage repairing material is solved, and the printed bionic cartilage can effectively promote the healing of damaged cartilage parts by compounding the chondrocytes and the printing material.
(3) Compared with the traditional preparation method of a tissue engineering cartilage material, the preparation method disclosed by the invention has the advantages that the scaffold microstructure is accurately formed, the adhesion of growth factors or cells is facilitated, the modeling can be carried out according to the actual condition of the damaged part of a patient, and the personalized treatment can be really achieved.
Drawings
FIG. 1 is a graph showing the results of mechanical property tests conducted in examples 1, 2 and 3 and a control group;
FIG. 2 is a graph showing the results of the cytotoxicity evaluation experiments conducted in examples 1, 2 and 3 in comparison with the control group;
FIG. 3 is a graph showing the results of the alkaline phosphatase assay (A L P) performed in examples 1, 2 and 3 in comparison with a control group;
FIG. 4 is a graph showing the results of experiments for measuring the secretion amount of glycosaminoglycan (GAG) in examples 1, 2 and 3 in comparison with a control group.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The bionic cartilage based on 3D printing is prepared according to the following steps:
(1) preparation of printing material:
① preparation of modified collagen type II (collagen type II) by weighing appropriate amount of collagen type II (molecular weight: 1 × 10)6kDa) was added to PBS phosphate at 50 ℃ to prepare a 10% solution, and then the volume ratio of type V II collagen PBS phosphate solution: adding methacrylic anhydride into V methacrylic anhydride =10:0.75, stirring and reacting for 4 hours at 50 ℃, after the reaction is finished, putting the solution obtained by the reaction into a dialysis bag with the molecular weight cutoff of 10000 for dialysis for 7 days, then putting the obtained dialysate into a freeze dryer with the temperature of minus 80 ℃ for freeze drying for 48 hours to obtain modified type II collagen, and storing the modified type II collagen at room temperature for later use;
② preparation of modified hyaluronic acid by weighing a certain amount of hyaluronic acid (molecular weight)1.2 to 1.5 × 106kDa) is dissolved in distilled water to prepare a hyaluronic acid solution with the mass concentration of 2 percent, then a sodium hydroxide solution with the molar concentration of 1 mol/L is added to adjust the pH of the solution to 8.5, then dimethylaminoethyl methacrylate is added according to the mass ratio of m sodium hyaluronate to m dimethylaminoethyl methacrylate =1:3.5, after the reaction at 10 ℃ for 24 hours, the obtained solution is added into a dialysis bag with the molecular weight cutoff of 14000 for flowing water dialysis for 7 days, and then the obtained dialysate is placed into a freeze dryer with the temperature of minus 80 ℃ for freeze drying for 48 hours, thus obtaining the modified hyaluronic acid which is stored at room temperature for later use;
③ chondrocytes were digested, and then added to a chondrocyte medium containing 10% by volume bovine serum to prepare a cell concentration of 1 × 106—5×106mL-1Placing the chondrocyte suspension in a cell culture box with the concentration of carbon dioxide of 5% and the temperature of 37 ℃ for standby; the chondrocytes are normally cultured chondrocytes after 3 generations;
(2) preparing precursors of all bionic layers:
① preparation of the bionic subchondral bone precursor, which is to add Irgacure2959 photoinitiator and the modified type II collagen prepared in the step (1) into distilled water in sequence, add the next substance after the substances added each time are completely dissolved, add nano-hydroxyapatite (particle size distribution range is 150-250 nm) into the obtained solution, stir until the nano-hydroxyapatite is completely and uniformly dispersed in the solution, obtain the bionic subchondral bone precursor solution for standby, wherein the concentration of the Irgacure2959 photoinitiator is 0.5wt%, the concentration of the nano-hydroxyapatite is 5-10wt%, and the concentration of the modified type II collagen is 5-10 wt%;
② preparation of a bionic calcification layer precursor, which is to add Irgacure2959 photoinitiator and the modified hyaluronic acid prepared in step (1) into distilled water in sequence, add the next substance after the substances added each time are completely dissolved, add nano-hydroxyapatite (particle size distribution is 150-250 nm) into the obtained solution, stir until the nano-hydroxyapatite is completely dispersed in the solution, obtain a bionic calcification layer precursor solution for standby, wherein the concentration of the Irgacure2959 photoinitiator is 0.5wt%, the concentration of the nano-hydroxyapatite is 1-5wt%, and the concentration of the modified hyaluronic acid is 1-3 wt%;
③ preparation of the bionic cartilage deep precursor, which comprises adding Irgacure2959 photoinitiator, the modified hyaluronic acid prepared in step (1) and the modified type II collagen prepared in step (1) into distilled water in sequence, adding the next substance after the substances added in each time are completely dissolved, adding the chondrocyte suspension prepared in step (1) with the same volume, and stirring gently until the mixture is uniform to obtain the bionic cartilage deep precursor solution for later use, wherein the concentration of the Irgacure2959 photoinitiator is 0.5wt%, the concentration of the modified hyaluronic acid is 1-3 wt%, the concentration of the modified type II collagen is 10-15wt%, and the concentration of the chondrocyte is 5 × 10wt%5—2.5×106mL-1；
④ preparation of a bionic intermediate layer precursor, which is to add Irgacure2959, the modified hyaluronic acid prepared in the step (1) and the modified type II collagen prepared in the step (1) into distilled water in sequence, and add the next substance after the substances added each time are completely dissolved to obtain a bionic intermediate layer precursor solution for later use, wherein the concentration of the Irgacure2959 is 0.5wt%, the concentration of the modified hyaluronic acid is 2-5 wt%, and the concentration of the modified type II collagen is 10-15 wt%;
⑤ preparation of a bionic shallow surface layer precursor, namely adding a photoinitiator Irgacure2959 and the modified type II collagen prepared in the step (1) into distilled water in sequence, and adding the next substance after the substances added each time are completely dissolved to obtain a bionic shallow surface layer precursor solution for later use, wherein the concentration of the photoinitiator Irgacure2959 is 0.5wt%, and the concentration of the modified type II collagen is 10-15 wt%;
(3) bionic printing of the bionic cartilage:
sequentially arranging the precursors of the bionic layers prepared in the step (2) according to the bionic subchondral bone precursor, the bionic calcification layer precursor, the bionic cartilage deep layer precursor, the bionic middle layer precursor and the bionic superficial layer precursor
Sequentially performing superposition printing, wherein the printing thickness of the bionic cartilage lower bone layer is 5-10% of the total thickness of the bionic cartilage, the printing thickness of the bionic cartilage lower bone layer is 2-5%, the printing thickness of the bionic cartilage lower bone layer is 15-30%, the printing thickness of the bionic cartilage deep layer is 40-55%, and the printing thickness of the bionic superficial layer is 10-20%, so that the bionic cartilage based on 3D printing is obtained;
example 1
The bionic cartilage based on 3D printing is prepared according to the method, wherein the molecular weight of the type II collagen used in the preparation of the printing material in the step (A) is 1 × 106kDa, molecular weight of hyaluronic acid used 1.2 × 106kDa; the particle size distribution range of the nano hydroxyapatite used in the preparation of the precursor of each bionic layer is 150-250 nm; in the preparation step of the bionic subchondral bone precursor, the dosage of the nano hydroxyapatite is 5 parts by mass, the dosage of the modified type II collagen is 5 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic calcified layer precursor, the dosage of the modified hyaluronic acid is 30 parts by mass, the dosage of the nano-hydroxyapatite is 5 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic cartilage deep precursor, the dosage of the modified hyaluronic acid is 25 parts by mass, the dosage of the modified type II collagen is 5 parts by mass, the dosage of the chondrocyte is 0.1 part by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic intermediate layer precursor, the dosage of the modified hyaluronic acid is 25 parts by mass, the dosage of the modified type II collagen is 5 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic superficial layer precursor, the dosage of the modified type II collagen is 5 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the bionic printing step of the bionic cartilage, the printing thickness of the bionic cartilage lower bone layer is 10 percent of the total thickness of the bionic cartilage, the bionic calcification layer is 2 percent, the bionic cartilage deep layer is 15 percent, the bionic intermediate layer is 55 percent, and the bionic shallow surface layer is 18 percent;
example 2
The bionic cartilage based on 3D printing is prepared according to the method, wherein the molecular weight of the type II collagen used in the preparation of the printing material in the step (A) is 1 × 106kDa, molecular weight of hyaluronic acid used 1.2 × 106kDa; the particle size distribution of the nano hydroxyapatite used in the preparation of the precursor of each bionic layer is 150-250 nm; in the preparation step of the bionic subchondral bone precursor, the dosage of the nano hydroxyapatite is 10 parts by mass, the dosage of the modified type II collagen is 10 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic calcified layer precursor, the dosage of the modified hyaluronic acid is 10 parts by mass, the dosage of the nano-hydroxyapatite is 5 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic cartilage deep precursor, the dosage of the modified hyaluronic acid is 15 parts by mass, the dosage of the modified type II collagen is 10 parts by mass, the dosage of the chondrocyte is 0.25 part by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic intermediate layer precursor, the dosage of the modified hyaluronic acid is 15 parts by mass, the dosage of the modified type II collagen is 10 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic superficial layer precursor, the dosage of modified type II collagen is 10 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the bionic printing step of the bionic cartilage, the printing thickness of the bionic subchondral bone layer is set to be 5% of the total thickness of the bionic cartilage, 5% of the bionic calcification layer, 30% of the deep layer of the bionic cartilage, 40% of the bionic intermediate layer and 20% of the bionic shallow surface layer;
example 3
The bionic cartilage based on 3D printing is prepared according to the method, wherein the molecular weight of the type II collagen used in the preparation of the printing material in the step (A) is 1 × 106kDa, molecular weight of hyaluronic acid used 1.2 × 106kDa; the particle size distribution of the nano hydroxyapatite used in the preparation of the precursor of each bionic layer is 150-250 nm; in the preparation step of the bionic subchondral bone precursor, the dosage of the nano hydroxyapatite is 10 parts by mass, the dosage of the modified type II collagen is 20 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic calcified layer precursor, the dosage of the modified hyaluronic acid is 10 parts by mass, the dosage of the nano-hydroxyapatite is 10 parts by mass, and the dosage of the photoinitiator Irgacure2959The using amount is 0.5 part by mass; in the preparation step of the bionic cartilage deep precursor, the dosage of the modified hyaluronic acid is 5 parts by mass, the dosage of the modified type II collagen is 20 parts by mass, the dosage of the chondrocyte is 0.5 part by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic intermediate layer precursor, the dosage of the modified hyaluronic acid is 5 parts by mass, the dosage of the modified type II collagen is 20 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the preparation step of the bionic superficial layer precursor, the dosage of the modified type II collagen is 20 parts by mass, and the dosage of the photoinitiator Irgacure2959 is 0.5 part by mass; in the bionic printing step of the bionic cartilage, the printing thickness of the bionic subchondral bone layer is set to be 5% of the total thickness of the bionic cartilage, 5% of the bionic calcification layer, 30% of the deep layer of the bionic cartilage, 50% of the bionic intermediate layer and 10% of the bionic shallow surface layer;
example 4
Comparative example: (a preparation process of a multilayer gradient bionic articular cartilage material based on 3D printing (prepared in example 1 with the reference of CN 201510752927.5).
Experiment groups 1-3: the bionic cartilage based on 3D printing is obtained in the embodiments 1-3.
(1) And (3) detection of mechanical properties: the 3D printed antibacterial hydrogel repair scaffold prepared in the above examples 1-3 and the comparative example were subjected to a mechanical evaluation experiment, an Instron 5575 mechanical testing machine was used to test the compression performance of the comparative example and the experimental group, the compression rate was set to 0.5mm/min, and the diameter and height of the sample used for the test were 6 mm and 4.5 mm. The instrument is zeroed before the test according to the instruction, and then the automatic reset function test is adopted in each test, 5 parallel samples are arranged in each group of samples, and the experimental result is shown in figure 1.
It can be seen that the compressive strength of the bionic cartilage prepared by the method disclosed by the invention in the examples 1-3 is obviously higher than that of the comparative example, the compressive strength of the example 3 is 2 times that of the comparative example, and the compressive strength reaches 5.16 MPa.
(2) Cytotoxicity evaluation experiment: cytotoxicity evaluation experiments (experiments according to national standard GB/T16886.5-2003) were carried out on the 3D printing-based bionic cartilage prepared in the above examples 1-3 and comparative examples, and the experimental results of the comparative examples 1-3 and comparative examples are shown in FIG. 2.
The cytotoxicity detection results show that the relative cell proliferation rates of the cells in the examples 1 to 3 after the cells are co-cultured with the bone marrow mesenchymal cells for 1 day and 7 days are more than 90%, the cytotoxicity rating is 0 grade, and the results prove that the cells have good cell compatibility, while the relative cell proliferation rates of the cells in the comparative examples and the bone marrow mesenchymal cells after the cells are co-cultured for 1 day and 7 days are 89.3% and 87.5%, respectively, the cytotoxicity rating is 1 grade, and the cells have slight cytotoxicity. In addition, the relative proliferation rates of the cells in the examples 1 to 3 are obviously improved after the co-culture time is prolonged, and the relative proliferation rates of the cells in the examples 2 and 3 are higher than that of the cells in the negative group after 7 days (both higher than 100%), so that the bionic cartilage prepared by the preparation method disclosed by the invention can promote the growth of bone marrow mesenchymal cells and has good biocompatibility.
(3) Detection of alkaline phosphatase (A L P)
After the 3D-printing-based bionic cartilage prepared in the above examples 1-3 and the comparative example are co-cultured with bone marrow mesenchymal cells for 7 days, the alkaline phosphatase value of the bionic cartilage and the alkaline phosphatase KIT (A L P assay KIT, purchased from Bio protocol KIT) are detected, and the bone induction capability of the experimental group and the comparative example is compared, and the experimental result is shown in FIG. 3.
The alkaline phosphatase (alkaline phosphate A L P) is a marker for differentiating osteoblasts and can promote mineralization of bone matrix, and experimental results show that compared with comparative examples, the A L P values of examples 1-3 are obviously higher than those of comparative examples.
(4) Detection of secretory amount of glycosaminoglycan (GAG)
After the 3D printing-based bionic cartilage prepared in the above examples 1-3 and the comparative example are co-cultured with the mesenchymal cells for 7 days and 14 days respectively, the secretion amount of glycosaminoglycan is detected by using a dimethyl methylene blue test KIT (DMMB assay KIT, purchased from Bio protocol KIT), and the specific operation is carried out according to the KIT instructions. The results of the experiment are shown in FIG. 4.
The secretion amount of glycosaminoglycan is somewhat related to the speed at which mesenchymal cells of bone marrow differentiate and form mature chondrocytes. The experimental results show that the secretion amounts of the glycosaminoglycans of examples 1 to 3 were significantly higher than that of the comparative example at both 7 days and 14 days, and the secretion amounts of the glycosaminoglycans of experimental groups 1 to 3 tended to increase with the increase of the culture time, and the secretion amount of the glycosaminoglycan of example 3 reached 430.15 at 14 days, which was 2 times that of the glycosaminoglycan of the comparative example. Therefore, the bionic cartilage prepared by the method based on 3D printing has higher glycosaminoglycan secretion amount, can promote bone marrow mesenchymal cells to be differentiated into chondrocytes, and is favorable for repairing damaged cartilage tissues.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention; it will be apparent to those skilled in the art that other variations and modifications may be made in the foregoing description, and it is not necessary or exhaustive for all embodiments to be present; any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (5)
1. A bionic cartilage based on 3D printing is characterized in that the bionic cartilage is prepared by gradient bionic 3D printing of modified type II collagen, modified hyaluronic acid, nano-hydroxyapatite and chondrocytes; the collagen; the bionic cartilage is formed by sequentially printing a bionic subchondral bone, a bionic calcification layer, a bionic cartilage deep layer, a bionic intermediate layer and a bionic superficial surface layer through a 3D printer from bottom to top, and the whole bionic cartilage is of a multi-layer interpenetrating network structure; the manufacturing method comprises the following specific steps:
(1) preparation of printing material:
① the modified type II collagen is prepared by adding type II collagen into PBS phosphate at 50 deg.C to prepare type II collagen PBS phosphate solution with mass concentration of 10%, adding methacrylic anhydride according to volume ratio V type II collagen PBS phosphate solution V methacrylic anhydride =10:0.75, stirring at 50 deg.C for reaction for 4 hr, dialyzing the obtained solution in a dialysis bag with molecular weight cutoff of 10000 for 7 days after the reaction is finished, freeze-drying the obtained dialysate in a freeze-drying machine at-80 deg.C for 48 hr to obtain modified type II collagen, storing at room temperature for use;
② preparing modified hyaluronic acid, dissolving hyaluronic acid in distilled water to obtain hyaluronic acid solution with mass concentration of 2%, adding sodium hydroxide solution with molar concentration of 1 mol/L to adjust pH of the solution to 8.5 to obtain sodium hyaluronate solution, adding dimethylaminoethyl methacrylate according to mass ratio of m sodium hyaluronate to m dimethylaminoethyl methacrylate =1:3.5, reacting at 10 ℃ for 24h, placing the obtained solution in a dialysis bag with molecular weight cutoff of 14000 for flowing water dialysis for 7 days, placing the dialysate in a freeze dryer at-80 ℃ for freeze drying for 48 hours to obtain modified hyaluronic acid, and storing at room temperature for later use;
③ the chondrocytes are digested and added to a chondrocyte medium containing 10% by volume bovine serum to prepare a cell concentration of 1 × 106—5×106mL-1Placing the chondrocyte suspension in a cell culture box with the concentration of carbon dioxide of 5% and the temperature of 37 ℃ for standby; the chondrocytes are normally cultured chondrocytes after 3 generations;
(2) preparing precursors of all bionic layers:
① preparation of the bionic subchondral bone precursor, which is to add Irgacure2959 photoinitiator and the modified type II collagen prepared in the step (1) into distilled water in sequence, add the next substance after the substances added each time are completely dissolved, add nano-hydroxyapatite, and stir until the nano-hydroxyapatite is uniformly dispersed in the solution to obtain a bionic subchondral bone precursor solution for later use, wherein the concentration of the Irgacure2959 is 0.5wt%, the concentration of the nano-hydroxyapatite is 5-10wt%, and the concentration of the modified type II collagen is 5-10 wt%;
② preparation of a bionic calcification layer precursor, namely adding Irgacure2959 photoinitiator and the modified hyaluronic acid prepared in the step (1) into distilled water in sequence, adding the next substance after the substances added each time are completely dissolved, then adding nano-hydroxyapatite, and stirring until the nano-hydroxyapatite is uniformly dispersed in the solution to obtain a bionic calcification layer precursor solution for later use, wherein the concentration of the Irgacure2959 photoinitiator is 0.5wt%, the concentration of the nano-hydroxyapatite is 1-5wt%, and the concentration of the modified hyaluronic acid is 1-3 wt%;
③ preparation of the bionic cartilage deep precursor, which comprises adding Irgacure2959 photoinitiator, the modified hyaluronic acid prepared in step (1) and the modified type II collagen prepared in step (1) into distilled water in sequence, adding the next substance after the substances added in each time are completely dissolved, adding the chondrocyte suspension prepared in step (1) with the same volume, and stirring gently until the mixture is uniform to obtain the bionic cartilage deep precursor solution for later use, wherein the concentration of the Irgacure2959 photoinitiator is 0.5wt%, the concentration of the modified hyaluronic acid is 1-3 wt%, the concentration of the modified type II collagen is 10-15wt%, and the concentration of the chondrocyte is 5 × 10wt%5—2.5×106mL-1；
④ preparation of a bionic intermediate layer precursor, which is to add Irgacure2959, the modified hyaluronic acid prepared in the step (1) and the modified type II collagen prepared in the step (1) into distilled water in sequence, and add the next substance after the substances added each time are completely dissolved to obtain a bionic intermediate layer precursor solution for later use, wherein the concentration of the Irgacure2959 is 0.5wt%, the concentration of the modified hyaluronic acid is 2-5 wt%, and the concentration of the modified type II collagen is 10-15 wt%;
⑤ preparation of a bionic shallow surface layer precursor, namely adding a photoinitiator Irgacure2959 and the modified type II collagen prepared in the step (1) into distilled water in sequence, and adding the next substance after the substances added each time are completely dissolved to obtain a bionic shallow surface layer precursor solution for later use, wherein the concentration of the photoinitiator Irgacure2959 is 0.5wt%, and the concentration of the modified type II collagen is 10-15 wt%;
(3) bionic printing of the bionic cartilage:
sequentially superposing and printing the precursors of the bionic layers prepared in the step (2) according to the sequence of the precursor of the bionic subchondral bone, the precursor of the bionic calcified layer, the precursor of the bionic cartilage deep layer, the precursor of the bionic intermediate layer and the precursor of the bionic superficial layer to obtain the bionic cartilage based on 3D printing; the bionic cartilage total thickness printing device comprises a bionic cartilage layer, a bionic cartilage layer and a printing layer, wherein the printing thickness of the bionic subchondral bone is set to be 5-10% of the total thickness of the bionic cartilage, the printing thickness of the bionic calcified layer is set to be 2-5% of the total thickness of the bionic cartilage, the printing thickness of the bionic cartilage layer is set to be 15-30% of the total thickness of the bionic cartilage, the printing thickness of the bionic intermediate layer is set to be 40-55% of the total thickness of the bionic cartilage, and.
2. The biomimetic cartilage based on 3D printing according to claim 1, wherein the type II collagen has a molecular weight of 1 × 106kDa。
3. The biomimetic cartilage based on 3D printing according to claim 1, wherein the molecular weight of the hyaluronic acid is 1.2-1.5 × 106kDa。
4. The biomimetic cartilage based on 3D printing according to claim 1, wherein the nano-hydroxyapatite has a particle size distribution range of 150-250 nm.
5. The bionic cartilage based on 3D printing according to claim 1, wherein the bionic cartilage is printed by a 3D printer of a three-dimensional photocuring forming method with the model number of 3D systems ProX 800, the ultraviolet irradiation time of each bionic layer is set to be 30 s, and the ultraviolet irradiation intensity is set to be 10 mW/CM2。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710695028.5A CN107537066B (en) | 2017-08-15 | 2017-08-15 | Bionic cartilage based on 3D printing and manufacturing method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710695028.5A CN107537066B (en) | 2017-08-15 | 2017-08-15 | Bionic cartilage based on 3D printing and manufacturing method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107537066A CN107537066A (en) | 2018-01-05 |
| CN107537066B true CN107537066B (en) | 2020-08-04 |
Family
ID=60970779
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710695028.5A Active CN107537066B (en) | 2017-08-15 | 2017-08-15 | Bionic cartilage based on 3D printing and manufacturing method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107537066B (en) |
Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108210072A (en) * | 2018-02-08 | 2018-06-29 | 扈玉华 | The preparation method of brain tissue and blood vessel entity composite model based on MRI and CTA |
| CN110171127B (en) * | 2018-06-29 | 2021-04-09 | 浙江大学 | 3D printing system |
| CN109481736B (en) * | 2019-01-18 | 2021-03-02 | 福州大学 | Cartilage-bone repair scaffold with bionic gradient and preparation method thereof |
| CN109646715A (en) * | 2019-01-25 | 2019-04-19 | 上海交通大学医学院附属第九人民医院 | Electrospinning 3D printing prepares the multilayer cartilage complex of factor-containing microballoon |
| CN109938885B (en) * | 2019-04-21 | 2021-02-02 | 西北工业大学 | Method for preparing osteochondral integrated bracket based on 3DP and laser cladding composite process |
| CN110302428B (en) * | 2019-07-30 | 2021-07-13 | 中国人民解放军陆军军医大学第一附属医院 | Cartilage-bone-marrow composite tissue structure and method based on living cell 3D printing |
| CN110790950A (en) * | 2019-10-21 | 2020-02-14 | 南京理工大学 | Photo-crosslinking recombinant collagen hydrogel, preparation method and application thereof in 3D bioprinting |
| CN111962210B (en) * | 2020-06-22 | 2022-03-25 | 华南理工大学 | Polycaprolactone/methacryloylated elastin nanofiber composite membrane and preparation method and application thereof |
| CN113440651A (en) * | 2020-08-06 | 2021-09-28 | 华夏司印(上海)生物技术有限公司 | Hydrogel composition, hydrogel, bionic cartilage and preparation method and application thereof |
| CN112354016B (en) * | 2020-11-24 | 2022-12-09 | 重庆电子工程职业学院 | Bionic artificial bone material and production method thereof |
| US20220298481A1 (en) * | 2021-03-19 | 2022-09-22 | Kaohsiung Medical University | Method for preparing zonal layered chondrocyte sheets and treating method thereof |
| CN115634311A (en) * | 2022-09-27 | 2023-01-24 | 润原生物科技(苏州)有限公司 | Multi-structure cartilage repair implant and preparation method thereof |
| CN115887775A (en) * | 2022-11-24 | 2023-04-04 | 吉林大学 | Gradient 3D printing polycaprolactone modified scaffold for growth plate repair and preparation method and application thereof |
| CN117887134A (en) * | 2024-03-14 | 2024-04-16 | 四川大学 | Hydrogel biomimetic mineralization stent and preparation method and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003022319A1 (en) * | 2001-09-11 | 2003-03-20 | Isis Innovation Limited | Tissue engineering scaffolds |
| CN1943801A (en) * | 2006-11-01 | 2007-04-11 | 华中科技大学 | A gradient laminated composite supporting frame material based on bionic structures and its preparation method |
| CN101020083A (en) * | 2007-03-08 | 2007-08-22 | 中国人民解放军第三军医大学第一附属医院 | Integral engineering rack of interface osteochondro tissue with bionic function |
| CN102274548A (en) * | 2011-08-26 | 2011-12-14 | 中国人民解放军军事医学科学院卫生装备研究所 | Bone-cartilage repair gradient active scaffold material and preparation method and use thereof |
| CN103877614A (en) * | 2014-02-26 | 2014-06-25 | 同济大学 | Dual-layer composite scaffold for repairing cartilage of tissue engineered bone and preparation method thereof |
| CN103920190A (en) * | 2013-01-14 | 2014-07-16 | 陕西博鸿生物科技有限公司 | Articular cartilage graft and preparation method thereof |
-
2017
- 2017-08-15 CN CN201710695028.5A patent/CN107537066B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003022319A1 (en) * | 2001-09-11 | 2003-03-20 | Isis Innovation Limited | Tissue engineering scaffolds |
| CN1943801A (en) * | 2006-11-01 | 2007-04-11 | 华中科技大学 | A gradient laminated composite supporting frame material based on bionic structures and its preparation method |
| CN101020083A (en) * | 2007-03-08 | 2007-08-22 | 中国人民解放军第三军医大学第一附属医院 | Integral engineering rack of interface osteochondro tissue with bionic function |
| CN102274548A (en) * | 2011-08-26 | 2011-12-14 | 中国人民解放军军事医学科学院卫生装备研究所 | Bone-cartilage repair gradient active scaffold material and preparation method and use thereof |
| CN103920190A (en) * | 2013-01-14 | 2014-07-16 | 陕西博鸿生物科技有限公司 | Articular cartilage graft and preparation method thereof |
| CN103877614A (en) * | 2014-02-26 | 2014-06-25 | 同济大学 | Dual-layer composite scaffold for repairing cartilage of tissue engineered bone and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《Design of a multiphase osteochondral scaffold III: Fabrication of layered scaffolds with continuous interfaces》;Harley BA et al;《JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A》;20100501;第92卷(第3期);第1078-1093页 * |
| 《骨软骨一体化仿生支架的研究现状与展望》;张学亮等;《中国医药生物技术》;20170810;第12卷(第4期);第350-355页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107537066A (en) | 2018-01-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107537066B (en) | Bionic cartilage based on 3D printing and manufacturing method thereof | |
| Zhu et al. | An injectable continuous stratified structurally and functionally biomimetic construct for enhancing osteochondral regeneration | |
| Gao et al. | Biomaterial–related cell microenvironment in tissue engineering and regenerative medicine | |
| Liu et al. | Facilitated vascularization and enhanced bone regeneration by manipulation hierarchical pore structure of scaffolds | |
| CN102526806B (en) | Tissue engineering cartilage and preparation method thereof | |
| CN112107731A (en) | Injectable double-layer drug-loaded osteochondral repair hydrogel scaffold and preparation method thereof | |
| Pan et al. | Selection of the optimum 3D‐printed pore and the surface modification techniques for tissue engineering tracheal scaffold in vivo reconstruction | |
| Chen et al. | A single integrated osteochondral in situ composite scaffold with a multi-layered functional structure | |
| CN110772668B (en) | Soft and hard tissue integrated regeneration and repair gradient three-dimensional fiber scaffold and preparation method thereof | |
| Jiang et al. | Effects of different aperture-sized type I collagen/silk fibroin scaffolds on the proliferation and differentiation of human dental pulp cells | |
| CN112791239B (en) | Preparation method of super-bionic soft and hard tissue composite scaffold | |
| CN112402701A (en) | Diagnosis and treatment integrated gradient osteochondral bionic scaffold and preparation method thereof | |
| CN113274553A (en) | Biomaterial-induced exosome three-dimensional scaffold and preparation method and application thereof | |
| CN107890585A (en) | A kind of composite ceramics support and preparation method thereof | |
| Abedin Dargoush et al. | A composite bilayer scaffold functionalized for osteochondral tissue regeneration in rat animal model | |
| Nogueira et al. | Three‐dimensional cell‐laden collagen scaffolds: from biochemistry to bone bioengineering | |
| Filová et al. | Composite hyaluronate-type I collagen-fibrin scaffold in the therapy of osteochondral defects in miniature pigs | |
| Gao et al. | Bi-lineage inducible and immunoregulatory electrospun fibers scaffolds for synchronous regeneration of tendon-to-bone interface | |
| Mitra et al. | Carboxymethyl Guar Gum nanoscaffold as matrix for cell growth | |
| CN111282021B (en) | Meniscus composite scaffold and preparation method thereof | |
| Yi et al. | Composite system of 3D-printed polymer and acellular matrix hydrogel to repair temporomandibular joint disc | |
| CN112354016B (en) | Bionic artificial bone material and production method thereof | |
| CN114848919B (en) | Composite hydrogel for TBI (Tunnel boring-induced plasticity) immune regulation and tissue repair and preparation method thereof | |
| WO2022156685A1 (en) | Method for realizing cartilage regeneration by means of inoculating gel cartilage into frame structure | |
| Tang et al. | A biomimetic in situ mineralization ECM composite scaffold to promote endogenous bone regeneration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |