CN112295015A - Preparation method of biological 3D printing composite ink for repairing cartilage defect - Google Patents
Preparation method of biological 3D printing composite ink for repairing cartilage defect Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y70/00—Materials specially adapted for additive manufacturing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y80/00—Products made by additive manufacturing
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
Abstract
The invention discloses a preparation method of biological 3D printing composite ink for repairing cartilage defects, which relates to the technical field of biological 3D printing and comprises the following steps: removing chondrocytes from fresh articular surface hyaline cartilage or costal cartilage of animals by physical, chemical and biological methods, sterilizing and freeze-drying; grinding into powder under the condition of liquid nitrogen low temperature; the extracellular matrix powder and the mixed material are uniformly mixed according to a certain proportion, and deionized water is added for dispersing and dissolving to form uniform and stable suspension, namely the target product. The invention provides a preparation method of biological 3D printing composite ink for repairing cartilage defects, which has the following beneficial effects: the obtained extracellular matrix powder is combined with different materials, so that biological 3D printing composite ink meeting different requirements can be prepared, can be quickly crosslinked and cured, has low cytotoxicity and high biocompatibility, can be co-printed with stem cells, meets the requirements of tissue engineering repair, and has good clinical application prospect and value.
Description
Technical Field
The invention relates to the technical field of biological 3D printing, in particular to a preparation method of biological 3D printing composite ink for repairing cartilage defects.
Background
The biological construction is a new research field, generally refers to a process of creating biological tissues containing specific hierarchical structures by applying bioengineering, the conventional biological construction technology is commonly known as particle leaching, freeze drying, electrostatic spinning and the like, although a series of biological materials with 3D structures can be generated by the technology, the biological materials are often poor in repeatability and functional expansibility, 3D biological printing becomes a new biological construction method, an automatic deposition technology based on digital control can provide precise control for the construction process of the tissue structures, the biological ink has high repeatability and functional expansibility, is an ink for a biological 3D printer, is an indispensable important component in the process of printing and constructing target tissue structures, meets the requirements of biological printing, and has high biocompatibility and biodegradability with different cells so as to simulate natural tissue microenvironment, a good bio-ink is a crucial step for successful printing.
However, the hydrogel prepared from alginate, chitosan, methacrylyl gelatin, hyaluronic acid or collagen serving as raw materials is not capable of simulating a natural three-dimensional microenvironment for cell growth due to biological inertia of the traditional biological 3D printing ink for cartilage repair process, so that the expression of cell adhesion, migration and directional induced differentiation is not satisfactory, and even part of the raw materials cannot be degraded or can generate components having toxic effects on cells after degradation. And to date, no material or ink capable of accurately simulating the complex three-dimensional structure of the natural extracellular matrix has been found, because the complex 3D structure and substance composition of the native ECM make accurate simulation very difficult, and for this reason, we propose a preparation method and application of a biological 3D printing composite ink containing cartilage extracellular matrix.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a preparation method and application of a biological 3D printing composite ink containing cartilage extracellular matrix, and solves the problems that the existing biological 3D printing ink provided in the background technology is hydrogel prepared by only taking alginate, chitosan, methacrylyl gelatin, hyaluronic acid or collagen and the like as raw materials, and because of biological inertia, the hydrogel cannot simulate a natural three-dimensional microenvironment for cell growth, the performance in the aspects of cell adhesion and directional differentiation is unsatisfactory, even a part of raw materials generate components with toxic action on cells after degradation, and the materials and the ink capable of accurately simulating the native extracellular matrix of the natural cells are not found so far, and the accurate simulation is very difficult due to the complex 3D structure and substance composition of the ECM.
Aiming at the defects of the prior art, the invention provides a preparation method of biological 3D printing composite ink for repairing cartilage defects, which comprises the following steps:
s1, preparing cartilage granules: collecting fresh hyaline cartilage or costal cartilage of animal joint surface, cutting hyaline cartilage from joint surface with sharp scalpel blade to obtain cartilage slice, cutting cartilage slice into granule with particle size of 1-2mm3；
S2, cell removal treatment: soaking cartilage particles in hypotonic Tris-HCl buffer solution, wherein the concentration of the hypotonic Tris-HCl buffer solution is 10mM, and the pH value is 8, and then circularly freezing and thawing for 6 cycles at the temperature of-80-37 ℃; then digesting with 0.25% pancreatin at 37 deg.C for 24 hr, and replacing pancreatin liquid every 4 hr; further washing away pancreatin with a hypertonic Tris-HCl buffer solution, wherein the hypertonic Tris-HCl buffer solution consists of NaCl 1.5M and Tris-HCl 50mM and has a pH of 7.6, and then carrying out digestion treatment with a nuclease at 37 ℃, wherein the nuclease consists of DNase 50U/mL, RNase A1U/mL and Tris-HCl 10mM, and wherein the pH of Tris-HCl is 7.5; removing nuclease, washing with hypotonic Tris-HCl for 20h, and soaking with 1% Triton-X-100 for 24 h;
s3, sterilization treatment: soaking the product obtained by the treatment of S2 in 0.5% PAA (PAA) prepared from peracetic acid and 0.1% PAA/4% ethanol for 24h, washing with sterilized deionized water for at least 72h, and changing the solution every 12 h;
s4, freeze-drying treatment: freezing the product obtained by the treatment of S3 at the temperature of minus 80 ℃, drying for 72h by a freeze dryer, and storing at the temperature of minus 20 ℃;
s5, grinding: grinding the product obtained by the processing of S4 into powder by using a liquid nitrogen ball mill under the condition of liquid nitrogen low temperature of-196 ℃, wherein the particle size of the powder is 3-5um, and obtaining extracellular matrix powder;
s6, preparing composite ink: uniformly mixing the extracellular matrix dECM powder with the mixed material according to a certain proportion, and then adding the mixture into deionized water for dispersing and dissolving to form uniform and stable suspension to prepare a target product;
s7, application: printing with 3D bioprinter or similar extrusion and spray forming system, and soaking the printed product in cross-linking agent CaCl2(the concentration is 2% -4%) to be cross-linked chemically or cross-linked by ultraviolet irradiation, and then the tissue engineering scaffold can be printed and prepared, and the scaffold can be freeze-dried under the temperature condition of minus 50 ℃ and stored under the temperature condition of minus 20 ℃ for standby.
Preferably, the mixed material in step S6 is alginate, and the mixing ratio is 1: 9.
Preferably, the mixed material in the step S6 is methacryl gelatin, and the mixing ratio is 1.2: 8.8.
Preferably, the mixed material in step S6 is polylactic acid, and the mixing ratio is 1.4: 8.6.
Preferably, the mixed material in the step S6 is chitosan, and the mixing ratio is 1.6: 8.4.
Preferably, the mixed material in the step S6 is hyaluronic acid, and the mixing ratio is 1.8: 8.2.
Preferably, the mixed material in the step S6 is collagen, and the mixing ratio is 2:8.
The invention provides a preparation method of biological 3D printing composite ink for repairing cartilage defects, which has the following beneficial effects:
according to the invention, extracellular matrix components (ECM) after removing cells from specific tissues and organs have natural three-dimensional structures of the specific tissues, natural superiority is shown for retaining specific functions and phenotypes of the cells, an optimized microenvironment can be provided, a natural three-dimensional microenvironment for cell growth is simulated, the growth of the three-dimensional structure tissues is facilitated, and inherent cell morphology and functions can be reconstructed;
by combining tissue engineering stem cells and a biological 3D printing technology, the three-dimensional stereospecific functional tissue modules with directional differentiation potential provide favorable environment for stem cell transplantation, survival, directional differentiation and long-term function, and provide theoretical basis and possibility for accurate repair in future tissue engineering repair technology;
according to the method for processing and obtaining the acellular extracellular matrix, disclosed by the invention, the obtained extracellular matrix powder is combined with different materials, so that the biological 3D printing composite ink meeting different requirements can be prepared, can be quickly crosslinked and solidified, has lower cytotoxicity and higher biocompatibility, can be co-printed with stem cells, meets the requirements of tissue engineering repair, and has better clinical application prospect and value.
Drawings
FIG. 1 is a diagram of fresh cartilage particles according to the present invention;
FIG. 2 is a diagram of decellularized cartilage particles of the invention;
FIG. 3 is a SEM image of a decellularized cartilage particle of the present invention;
FIG. 4 is a diagram of extracellular matrix-free lyophilized powder of the present invention;
FIG. 5 is a diagram of a composite bio-ink of the present invention;
FIG. 6 is an external view of a biological 3D printing support according to the present invention;
FIG. 7 is a SEM image of a biological 3D printing support according to the present invention;
fig. 8 is a laser confocal scanning image of the living and dead cells of the 3D printing scaffold containing cell organisms according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to specific 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.
s1, preparing cartilage granules: collecting fresh hyaline cartilage or costal cartilage of animal joint surface, cutting hyaline cartilage from joint surface with sharp scalpel blade to obtain cartilage slice, cutting cartilage slice into granule with particle size of 1-2mm3(as shown in FIG. 1);
s2, cell removal treatment: soaking cartilage particles in hypotonic Tris-HCl buffer solution, wherein the concentration of the hypotonic Tris-HCl buffer solution is 10mM, and the pH value is 8, and then circularly freezing and thawing for 6 cycles at the temperature of-80-37 ℃; then digesting with 0.25% pancreatin at 37 deg.C for 24 hr, and replacing pancreatin liquid every 4 hr; further washing away pancreatin with a hypertonic Tris-HCl buffer solution, wherein the hypertonic Tris-HCl buffer solution consists of NaCl 1.5M and Tris-HCl 50mM and has a pH of 7.6, and then carrying out digestion treatment with a nuclease at 37 ℃, wherein the nuclease consists of DNase 50U/mL, RNase A1U/mL and Tris-HCl 10mM, and wherein the pH of Tris-HCl is 7.5; removing nuclease, washing with hypotonic Tris-HCl for 20 hr, and soaking with 1% Triton-X-100 for 24 hr (as shown in FIGS. 2-3);
s3, sterilization treatment: soaking the product obtained by the treatment of S2 in 0.5% PAA (PAA) prepared from peracetic acid and 0.1% PAA/4% ethanol for 24h, washing with sterilized deionized water for at least 72h, and changing the solution every 12 h;
s4, freeze-drying treatment: freezing the product obtained by the treatment of S3 at the temperature of minus 80 ℃, drying for 72h by a freeze dryer, and storing at the temperature of minus 20 ℃;
s5, grinding: grinding the product obtained by processing S4 into powder with liquid nitrogen ball mill at liquid nitrogen low temperature of-196 deg.C, wherein the particle size of the powder is 3-5um, to obtain extracellular matrix powder (shown in figure 4);
s6, preparing composite ink: uniformly mixing extracellular matrix dECM powder with a mixed material according to a certain ratio, wherein the mixed material is alginate, the mixing ratio is 1:9, adding the alginate into deionized water, dispersing and dissolving to form uniform and stable suspension, and preparing a target product (shown in figure 5);
s7, application: printing with 3D bioprinter or similar extrusion and spray forming system, and soaking the printed product in cross-linking agent CaCl2(concentration is 2% -4%) to be cross-linked chemically or by ultraviolet irradiation, and then the tissue engineering scaffold can be printed and prepared, and the scaffold can be freeze-dried at-50 deg.C, and stored at-20 deg.C for use (as shown in fig. 6-8).
According to the method for processing and obtaining the acellular extracellular matrix, disclosed by the invention, the obtained extracellular matrix powder is combined with different materials, so that the biological 3D printing composite ink meeting different requirements can be prepared, can be quickly crosslinked and solidified, has lower cytotoxicity and higher biocompatibility, can be co-printed with stem cells, meets the requirements of tissue engineering repair, and has better clinical application prospect and value.
Embodiment 2, the present invention provides a technical solution: a preparation method of biological 3D printing composite ink for repairing cartilage defects comprises the following steps:
s1, preparing cartilage granules: collecting fresh hyaline cartilage or costal cartilage of animal joint surface, cutting hyaline cartilage from joint surface with sharp scalpel blade to obtain cartilage slice, cutting cartilage slice into granule with particle size of 1-2mm3(as shown in FIG. 1);
s2, cell removal treatment: soaking cartilage particles in hypotonic Tris-HCl buffer solution, wherein the concentration of the hypotonic Tris-HCl buffer solution is 10mM, and the pH value is 8, and then circularly freezing and thawing for 6 cycles at the temperature of-80-37 ℃; then digesting with 0.25% pancreatin at 37 deg.C for 24 hr, and replacing pancreatin liquid every 4 hr; further washing away pancreatin with a hypertonic Tris-HCl buffer solution, wherein the hypertonic Tris-HCl buffer solution consists of NaCl 1.5M and Tris-HCl 50mM and has a pH of 7.6, and then carrying out digestion treatment with a nuclease at 37 ℃, wherein the nuclease consists of DNase 50U/mL, RNase A1U/mL and Tris-HCl 10mM, and wherein the pH of Tris-HCl is 7.5; removing nuclease, washing with hypotonic Tris-HCl for 20 hr, and soaking with 1% Triton-X-100 for 24 hr (as shown in FIGS. 2-3);
s3, sterilization treatment: soaking the product obtained by the treatment of S2 in 0.5% PAA (PAA) prepared from peracetic acid and 0.1% PAA/4% ethanol for 24h, washing with sterilized deionized water for at least 72h, and changing the solution every 12 h;
s4, freeze-drying treatment: freezing the product obtained by the treatment of S3 at the temperature of minus 80 ℃, drying for 72h by a freeze dryer, and storing at the temperature of minus 20 ℃;
s5, grinding: grinding the product obtained by processing S4 into powder with liquid nitrogen ball mill at liquid nitrogen low temperature of-196 deg.C, wherein the particle size of the powder is 3-5um, to obtain extracellular matrix powder (shown in figure 4);
s6, preparing composite ink: uniformly mixing the extracellular matrix dECM powder with a mixed material according to a certain ratio, wherein the mixed material is methacryloyl gelatin, the mixing ratio is 1.2:8.8, and adding the mixed material into deionized water for dispersing and dissolving to form uniform and stable suspension to prepare a target product (shown in figure 5);
s7, application: printing with 3D bioprinter or similar extrusion and spray forming system, and soaking the printed product in cross-linking agent CaCl2(concentration is 2% -4%) to be cross-linked chemically or by ultraviolet irradiation, and then the tissue engineering scaffold can be printed and prepared, and the scaffold can be freeze-dried at-50 deg.C, and stored at-20 deg.C for use (as shown in fig. 6-8).
According to the method for processing and obtaining the acellular extracellular matrix, disclosed by the invention, the obtained extracellular matrix powder is combined with different materials, so that the biological 3D printing composite ink meeting different requirements can be prepared, can be quickly crosslinked and solidified, has lower cytotoxicity and higher biocompatibility, can be co-printed with stem cells, meets the requirements of tissue engineering repair, and has better clinical application prospect and value.
Embodiment 3, the present invention provides a technical solution: a preparation method of biological 3D printing composite ink for repairing cartilage defects comprises the following steps:
s1, preparing cartilage granules: collecting fresh hyaline cartilage or costal cartilage of animal joint surface, cutting hyaline cartilage from joint surface with sharp scalpel blade to obtain cartilage slice, cutting cartilage slice into granule with particle size of 1-2mm3(as shown in FIG. 1);
s2, cell removal treatment: soaking cartilage particles in hypotonic Tris-HCl buffer solution, wherein the concentration of the hypotonic Tris-HCl buffer solution is 10mM, and the pH value is 8, and then circularly freezing and thawing for 6 cycles at the temperature of-80-37 ℃; then digesting with 0.25% pancreatin at 37 deg.C for 24 hr, and replacing pancreatin liquid every 4 hr; further washing away pancreatin with a hypertonic Tris-HCl buffer solution, wherein the hypertonic Tris-HCl buffer solution consists of NaCl 1.5M and Tris-HCl 50mM and has a pH of 7.6, and then carrying out digestion treatment with a nuclease at 37 ℃, wherein the nuclease consists of DNase 50U/mL, RNase A1U/mL and Tris-HCl 10mM, and wherein the pH of Tris-HCl is 7.5; removing nuclease, washing with hypotonic Tris-HCl for 20 hr, and soaking with 1% Triton-X-100 for 24 hr (as shown in FIGS. 2-3);
s3, sterilization treatment: soaking the product obtained by the treatment of S2 in 0.5% PAA (PAA) prepared from peracetic acid and 0.1% PAA/4% ethanol for 24h, washing with sterilized deionized water for at least 72h, and changing the solution every 12 h;
s4, freeze-drying treatment: freezing the product obtained by the treatment of S3 at the temperature of minus 80 ℃, drying for 72h by a freeze dryer, and storing at the temperature of minus 20 ℃;
s5, grinding: grinding the product obtained by processing S4 into powder with liquid nitrogen ball mill at liquid nitrogen low temperature of-196 deg.C, wherein the particle size of the powder is 3-5um, to obtain extracellular matrix powder (shown in figure 4);
s6, preparing composite ink: uniformly mixing extracellular matrix dECM powder with a mixed material according to a certain ratio, wherein the mixed material is polylactic acid, and the mixing ratio is 1.4:8.6, adding the mixed material into deionized water for dispersing and dissolving to form uniform and stable suspension, and preparing a target product (shown in figure 5);
s7, application: printing with 3D bioprinter or similar extrusion and spray forming system, and soaking the printed product in cross-linking agent CaCl2(concentration is 2% -4%) to be cross-linked chemically or by ultraviolet irradiation, and then the tissue engineering scaffold can be printed and prepared, and the scaffold can be freeze-dried at-50 deg.C, and stored at-20 deg.C for use (as shown in fig. 6-8).
According to the method for processing and obtaining the acellular extracellular matrix, disclosed by the invention, the obtained extracellular matrix powder is combined with different materials, so that the biological 3D printing composite ink meeting different requirements can be prepared, can be quickly crosslinked and solidified, has lower cytotoxicity and higher biocompatibility, can be co-printed with stem cells, meets the requirements of tissue engineering repair, and has better clinical application prospect and value.
Embodiment 4, the present invention provides a technical solution: a preparation method of biological 3D printing composite ink for repairing cartilage defects comprises the following steps:
s1, preparing cartilage granules: collecting fresh hyaline cartilage or costal cartilage of animal joint surface, cutting hyaline cartilage from joint surface with sharp scalpel blade to obtain cartilage slice, cutting cartilage slice into granule with particle size of 1-2mm3(as shown in FIG. 1);
s2, cell removal treatment: soaking cartilage particles in hypotonic Tris-HCl buffer solution, wherein the concentration of the hypotonic Tris-HCl buffer solution is 10mM, and the pH value is 8, and then circularly freezing and thawing for 6 cycles at the temperature of-80-37 ℃; then digesting with 0.25% pancreatin at 37 deg.C for 24 hr, and replacing pancreatin liquid every 4 hr; further washing away pancreatin with a hypertonic Tris-HCl buffer solution, wherein the hypertonic Tris-HCl buffer solution consists of NaCl 1.5M and Tris-HCl 50mM and has a pH of 7.6, and then carrying out digestion treatment with a nuclease at 37 ℃, wherein the nuclease consists of DNase 50U/mL, RNase A1U/mL and Tris-HCl 10mM, and wherein the pH of Tris-HCl is 7.5; removing nuclease, washing with hypotonic Tris-HCl for 20 hr, and soaking with 1% Triton-X-100 for 24 hr (as shown in FIGS. 2-3);
s3, sterilization treatment: soaking the product obtained by the treatment of S2 in 0.5% PAA (PAA) prepared from peracetic acid and 0.1% PAA/4% ethanol for 24h, washing with sterilized deionized water for at least 72h, and changing the solution every 12 h;
s4, freeze-drying treatment: freezing the product obtained by the treatment of S3 at the temperature of minus 80 ℃, drying for 72h by a freeze dryer, and storing at the temperature of minus 20 ℃;
s5, grinding: grinding the product obtained by processing S4 into powder with liquid nitrogen ball mill at liquid nitrogen low temperature of-196 deg.C, wherein the particle size of the powder is 3-5um, to obtain extracellular matrix powder (shown in figure 4);
s6, preparing composite ink: uniformly mixing extracellular matrix dECM powder with a mixed material according to a certain ratio, wherein the mixed material is chitosan, the mixing ratio is 1.6:8.4, and adding the chitosan into deionized water for dispersing and dissolving to form uniform and stable suspension to prepare a target product (shown in figure 5);
s7, application: push buttonUsing a 3D bioprinter printing or similar extrusion, jet-forming system, the printed product is dipped into the cross-linking agent CaCl2(concentration is 2% -4%) to be cross-linked chemically or by ultraviolet irradiation, and then the tissue engineering scaffold can be printed and prepared, and the scaffold can be freeze-dried at-50 deg.C, and stored at-20 deg.C for use (as shown in fig. 6-8).
According to the method for processing and obtaining the acellular extracellular matrix, disclosed by the invention, the obtained extracellular matrix powder is combined with different materials, so that the biological 3D printing composite ink meeting different requirements can be prepared, can be quickly crosslinked and solidified, has lower cytotoxicity and higher biocompatibility, can be co-printed with stem cells, meets the requirements of tissue engineering repair, and has better clinical application prospect and value.
Embodiment 5, the present invention provides a technical solution: a preparation method of biological 3D printing composite ink for repairing cartilage defects comprises the following steps:
s1, preparing cartilage granules: collecting fresh hyaline cartilage or costal cartilage of animal joint surface, cutting hyaline cartilage from joint surface with sharp scalpel blade to obtain cartilage slice, cutting cartilage slice into granule with particle size of 1-2mm3(as shown in FIG. 1);
s2, cell removal treatment: soaking cartilage particles in hypotonic Tris-HCl buffer solution, wherein the concentration of the hypotonic Tris-HCl buffer solution is 10mM, and the pH value is 8, and then circularly freezing and thawing for 6 cycles at the temperature of-80-37 ℃; then digesting with 0.25% pancreatin at 37 deg.C for 24 hr, and replacing pancreatin liquid every 4 hr; further washing away pancreatin with a hypertonic Tris-HCl buffer solution, wherein the hypertonic Tris-HCl buffer solution consists of NaCl 1.5M and Tris-HCl 50mM and has a pH of 7.6, and then carrying out digestion treatment with a nuclease at 37 ℃, wherein the nuclease consists of DNase 50U/mL, RNase A1U/mL and Tris-HCl 10mM, and wherein the pH of Tris-HCl is 7.5; removing nuclease, washing with hypotonic Tris-HCl for 20 hr, and soaking with 1% Triton-X-100 for 24 hr (as shown in FIGS. 2-3);
s3, sterilization treatment: soaking the product obtained by the treatment of S2 in 0.5% PAA (PAA) prepared from peracetic acid and 0.1% PAA/4% ethanol for 24h, washing with sterilized deionized water for at least 72h, and changing the solution every 12 h;
s4, freeze-drying treatment: freezing the product obtained by the treatment of S3 at the temperature of minus 80 ℃, drying for 72h by a freeze dryer, and storing at the temperature of minus 20 ℃;
s5, grinding: grinding the product obtained by processing S4 into powder with liquid nitrogen ball mill at liquid nitrogen low temperature of-196 deg.C, wherein the particle size of the powder is 3-5um, to obtain extracellular matrix powder (shown in figure 4);
s6, preparing composite ink: uniformly mixing extracellular matrix dECM powder with a mixed material according to a certain ratio, wherein the mixed material is hyaluronic acid, the mixing ratio is 1.8:8.2, and adding the hyaluronic acid into deionized water for dispersing and dissolving to form uniform and stable suspension to prepare a target product (shown in figure 5);
s7, application: printing with 3D bioprinter or similar extrusion and spray forming system, and soaking the printed product in cross-linking agent CaCl2(concentration is 2% -4%) to be cross-linked chemically or by ultraviolet irradiation, and then the tissue engineering scaffold can be printed and prepared, and the scaffold can be freeze-dried at-50 deg.C, and stored at-20 deg.C for use (as shown in fig. 6-8).
According to the method for processing and obtaining the acellular extracellular matrix, disclosed by the invention, the obtained extracellular matrix powder is combined with different materials, so that the biological 3D printing composite ink meeting different requirements can be prepared, can be quickly crosslinked and solidified, has lower cytotoxicity and higher biocompatibility, can be co-printed with stem cells, meets the requirements of tissue engineering repair, and has better clinical application prospect and value.
Embodiment 6, the present invention provides a technical solution: a preparation method of biological 3D printing composite ink for repairing cartilage defects comprises the following steps:
s1, preparing cartilage granules: collecting animal fresh articular surface hyaline cartilage or costal cartilage, and cutting with sharp surgical bladeCutting the bright cartilage from the joint surface to obtain cartilage pieces, and cutting into granule with particle size of 1-2mm3(as shown in FIG. 1);
s2, cell removal treatment: soaking cartilage particles in hypotonic Tris-HCl buffer solution, wherein the concentration of the hypotonic Tris-HCl buffer solution is 10mM, and the pH value is 8, and then circularly freezing and thawing for 6 cycles at the temperature of-80-37 ℃; then digesting with 0.25% pancreatin at 37 deg.C for 24 hr, and replacing pancreatin liquid every 4 hr; further washing away pancreatin with a hypertonic Tris-HCl buffer solution, wherein the hypertonic Tris-HCl buffer solution consists of NaCl 1.5M and Tris-HCl 50mM and has a pH of 7.6, and then carrying out digestion treatment with a nuclease at 37 ℃, wherein the nuclease consists of DNase 50U/mL, RNase A1U/mL and Tris-HCl 10mM, and wherein the pH of Tris-HCl is 7.5; removing nuclease, washing with hypotonic Tris-HCl for 20 hr, and soaking with 1% Triton-X-100 for 24 hr (as shown in FIGS. 2-3);
s3, sterilization treatment: soaking the product obtained by the treatment of S2 in 0.5% PAA (PAA) prepared from peracetic acid and 0.1% PAA/4% ethanol for 24h, washing with sterilized deionized water for at least 72h, and changing the solution every 12 h;
s4, freeze-drying treatment: freezing the product obtained by the treatment of S3 at the temperature of minus 80 ℃, drying for 72h by a freeze dryer, and storing at the temperature of minus 20 ℃;
s5, grinding: grinding the product obtained by processing S4 into powder with liquid nitrogen ball mill at liquid nitrogen low temperature of-196 deg.C, wherein the particle size of the powder is 3-5um, to obtain extracellular matrix powder (shown in figure 4);
s6, preparing composite ink: uniformly mixing extracellular matrix dECM powder with a mixed material according to a certain ratio, wherein the mixed material is collagen, the mixing ratio is 2:8, and then adding the collagen into deionized water for dispersing and dissolving to form uniform and stable suspension to prepare a target product (shown in figure 5);
s7, application: printing with 3D bioprinter or similar extrusion and spray forming system, and soaking the printed product in cross-linking agent CaCl2(concentration 2% -4)%) is subjected to chemical crosslinking or ultraviolet irradiation crosslinking, and the tissue engineering scaffold can be prepared by printing, freeze-dried at the temperature of-50 ℃ and stored at the low temperature of-20 ℃ for later use (as shown in figures 6-8).
According to the method for processing and obtaining the acellular extracellular matrix, disclosed by the invention, the obtained extracellular matrix powder is combined with different materials, so that the biological 3D printing composite ink meeting different requirements can be prepared, can be quickly crosslinked and solidified, has lower cytotoxicity and higher biocompatibility, can be co-printed with stem cells, meets the requirements of tissue engineering repair, and has better clinical application prospect and value.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (7)
1. A preparation method of biological 3D printing composite ink for repairing cartilage defects is characterized by comprising the following steps:
s1, preparing cartilage granules: collecting fresh hyaline cartilage or costal cartilage of animal joint surface, cutting hyaline cartilage from joint surface with sharp scalpel blade to obtain cartilage slice, cutting cartilage slice into granule with particle size of 1-2mm3；
S2, cell removal treatment: soaking cartilage particles in hypotonic Tris-HCl buffer solution, wherein the concentration of the hypotonic Tris-HCl buffer solution is 10mM, and the pH value is 8, and then circularly freezing and thawing for 6 cycles at the temperature of-80-37 ℃; then digesting with 0.25% pancreatin at 37 deg.C for 24 hr, and replacing pancreatin liquid every 4 hr; further washing away pancreatin with a hypertonic Tris-HCl buffer solution, wherein the hypertonic Tris-HCl buffer solution consists of NaCl 1.5M and Tris-HCl 50mM and has a pH of 7.6, and then carrying out digestion treatment with a nuclease at 37 ℃, wherein the nuclease consists of DNase 50U/mL, RNase A1U/mL and Tris-HCl 10mM, and wherein the pH of Tris-HCl is 7.5; removing nuclease, washing with hypotonic Tris-HCl for 20h, and soaking with 1% Triton-X-100 for 24 h;
s3, sterilization treatment: soaking the product obtained by the treatment of S2 in 0.5% PAA (PAA) prepared from peracetic acid and 0.1% PAA/4% ethanol for 24h, washing with sterilized deionized water for at least 72h, and changing the solution every 12 h;
s4, freeze-drying treatment: freezing the product obtained by the treatment of S3 at the temperature of minus 80 ℃, drying for 72h by a freeze dryer, and storing at the temperature of minus 20 ℃;
s5, grinding: grinding the product obtained by the processing of S4 into powder by using a liquid nitrogen ball mill under the condition of liquid nitrogen low temperature of-196 ℃, wherein the particle size of the powder is 3-5um, and obtaining extracellular matrix powder;
s6, preparing composite ink: uniformly mixing the extracellular matrix dECM powder with the mixed material according to a certain proportion, and then adding the mixture into deionized water for dispersing and dissolving to form uniform and stable suspension to prepare a target product;
s7, application: printing with 3D bioprinter or similar extrusion and spray forming system, and soaking the printed product in cross-linking agent CaCl2(the concentration is 2% -4%) to be cross-linked chemically or cross-linked by ultraviolet irradiation, and then the tissue engineering scaffold can be printed and prepared, and the scaffold can be freeze-dried under the temperature condition of minus 50 ℃ and stored under the temperature condition of minus 20 ℃ for standby.
2. The preparation method and the application of the composite ink for biological 3D printing containing the cartilage extracellular matrix according to claim 1 are characterized in that: the mixed material in the step S6 is alginate, and the mixing ratio is 1: 9.
3. The preparation method and the application of the composite ink for biological 3D printing containing the cartilage extracellular matrix according to claim 1 are characterized in that: the mixed material in the step S6 is methacryl gelatin, and the mixing ratio is 1.2: 8.8.
4. The preparation method and the application of the composite ink for biological 3D printing containing the cartilage extracellular matrix according to claim 1 are characterized in that: the mixed material in the step S6 is polylactic acid, and the mixing ratio is 1.4: 8.6.
5. The preparation method and the application of the composite ink for biological 3D printing containing the cartilage extracellular matrix according to claim 1 are characterized in that: the mixed material in the step S6 is chitosan, and the mixing ratio is 1.6: 8.4.
6. The preparation method and the application of the composite ink for biological 3D printing containing the cartilage extracellular matrix according to claim 1 are characterized in that: the mixed material in the step S6 is hyaluronic acid, and the mixing ratio is 1.8: 8.2.
7. The preparation method and the application of the composite ink for biological 3D printing containing the cartilage extracellular matrix according to claim 1 are characterized in that: the mixed material in the step S6 is collagen, and the mixing ratio is 2:8.
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