CN108030958B - Formula and preparation method of 3D printing artificial bone composite fibrin scaffold - Google Patents

Formula and preparation method of 3D printing artificial bone composite fibrin scaffold Download PDF

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CN108030958B
CN108030958B CN201711384592.1A CN201711384592A CN108030958B CN 108030958 B CN108030958 B CN 108030958B CN 201711384592 A CN201711384592 A CN 201711384592A CN 108030958 B CN108030958 B CN 108030958B
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collagen
hydroxyapatite
bone
scaffold
artificial bone
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CN108030958A (en
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毕龙
宋岳
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Fourth Military Medical University FMMU
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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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/42—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having an inorganic matrix
    • A61L27/425—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having an inorganic matrix of phosphorus containing material, e.g. apatite
    • 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
    • A61L27/225—Fibrin; Fibrinogen
    • 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
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • B33Y10/00—Processes of additive manufacturing
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • 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
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

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Abstract

The invention discloses a formula and a preparation method of a 3D printing artificial bone composite fibrin scaffold, which comprises five components of medical collagen powder, 0.05mol/L acetic acid solution, medical hydroxyapatite powder, genipin solution with the mass fraction of 1 percent and platelet-rich fibrin: firstly, dissolving medical grade collagen powder in 0.05mol/L acetic acid solution to prepare collagen colloid, then uniformly stirring the medical grade hydroxyapatite powder and the collagen colloid to prepare hydroxyapatite and collagen printing raw materials, printing hollow individualized tissue engineering bone by a 3D bioprinter according to patient bone defect CT data, centrifuging patient blood to prepare platelet-rich fibrin in the operation process of transplanting an artificial bone scaffold, and then placing the platelet-rich fibrin in a hollow tube cavity of the tissue engineering bone to prepare the hydroxyapatite, collagen and platelet-rich fibrin artificial bone with the capability of promoting human bone regeneration. The artificial bone can slowly release autologous growth factors in platelet-rich fibrin of a patient, and promote the repair of human bone defects.

Description

Formula and preparation method of 3D printing artificial bone composite fibrin scaffold
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a formula of a 3D printing artificial bone composite fibrin scaffold and a preparation method thereof.
Background
The repair of large bone defects is a difficult problem which always troubles orthopedic surgeons, and the current repair strategies comprise autologous bone, allogeneic bone, artificial material transplantation and the like. Although autologous bone grafting is the gold standard for treatment, bleeding and infection of a bone taking area can be caused in the grafting process; the allograft bone transplantation has the risk of immunological rejection and disease transmission; the rapid development of bone tissue engineering provides a feasible method for repairing large bone defects. The internal structure of the traditional method for preparing the stent is often not controllable manually, and the 3D printing technology can tailor the shape of the stent for a patient according to the shape of the bone defect; meanwhile, the manufacturing process is strictly controlled by a computer, so that the pore diameter and the pore passage communication rate of the bracket can be finely controlled, and the bracket plays an important role in playing the functions.
In the 3D printing process, biological ceramic slurry is extruded from a printing spray head and is stacked and molded by a lamination manufacturing method, biological factors or specific medicines can be added into the slurry, the whole printing process is controlled at a low temperature, and the slurry containing the biological factors or the medicines is extruded from the printing spray head without damaging the activity of the biological factors and the medicines. Hydroxyapatite is a biological ceramic material, has excellent biocompatibility and biodegradability, and is currently and mostly applied to related researches of bone tissue engineering, bone cement and the like. Collagen is an important organic component in natural bone tissues, has good biocompatibility and degradability and the capacity of promoting osteogenesis, and a large number of experiments prove that the collagen can promote migration, adhesion, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. The platelet-rich fibrin is the second generation of platelet concentrate, the preparation method is simple, the three-dimensional network structure can be used for screening a large amount of biological factors activated and released by platelets, and the biological factors can be slowly released along with the degradation of the fibrin network structure, so that the tissue healing of the platelet-rich fibrin implantation part can be promoted. The artificial bone scaffold is prepared by mixing hydroxyapatite and collagen, performing low-temperature 3D printing, centrifuging blood of a patient to prepare platelet-rich fibrin, putting the platelet-rich fibrin into a hollow bone scaffold, promoting the repair of a bone defect part of the patient by using a cytokine generated by the patient, avoiding immunological rejection reaction, and simultaneously preparing the scaffold with any aperture and porosity by using a 3D printing technology in the preparation process, thereby really realizing individualized treatment.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the invention provides a formula of a 3D printing artificial bone composite fibrin scaffold and a preparation method thereof, and well solves the problem of individualized treatment of patients with large bone defects and nonunion.
In order to solve the technical problems, the technical scheme of the invention is as follows: the formula of the 3D printed artificial bone is characterized in that: the following formula is adopted: comprises 5g of medical collagen powder, 25mL of 0.05mol/L acetic acid solution, 10g of medical hydroxyapatite powder (average particle size of 100nm) and 20mL of genipin solution with the mass fraction of 1%.
The formula for preparing the 3D printing artificial bone composite fibrin scaffold of claim 1 and the preparation method thereof are characterized in that: the method comprises the following steps:
preparing a collagen colloid: dissolving 5g of medical grade collagen powder in 25mL of 0.05mol/L acetic acid solution to prepare collagen colloid;
(2) mixing: according to the mass ratio of collagen to hydroxyapatite of 1: 2 mixing colloid containing 5g collagen and 10g hydroxyapatite powder;
thirdly, stirring: and (3) uniformly stirring the mixed materials in the step (2) to prepare hydroxyapatite and collagen printing raw materials, namely the biological ink of a biological printer.
Fourthly, three-dimensional reconstruction: acquiring CT data of a bone defect part of a patient, segmenting a CT image by using a method combining threshold segmentation and region growth, obtaining a layered CT image Mask (Mask) through Boolean operation, then performing three-dimensional reconstruction, constructing a personalized hollow bone tissue engineering scaffold three-dimensional digital model by using RenetierHost software, and preparing a scaffold material by using a hydroxyapatite/collagen mixed tissue as a raw material and by using a 3D printing technology;
fifthly, 3D printing: and (3) loading the hydroxyapatite obtained in the step (3) and the collagen biological ink into a 3D biological printer (Bioprinter), and then printing the hollow individualized hydroxyapatite, namely the collagen artificial bone scaffold material, by the 3D biological printer (Bioprinter).
Sixthly, crosslinking: and (5) placing the hydroxyapatite/collagen artificial bone obtained in the step (5) into genipin solution with the mass fraction of 1% for crosslinking so as to increase the strength of the artificial bone scaffold.
Sirtuin, freeze-drying; and (4) drying the hydroxyapatite and the collagen artificial bone which are subjected to the crosslinking in the step (6) for 12 hours by using a freeze dryer.
And preparing and compounding platelet-rich fibrin: collecting 10mL of venous blood of a patient in the process of transplanting an engineering bone scaffold, immediately transferring the venous blood into a sterile glass centrifuge tube without anticoagulation measures, centrifuging for 10min at 400g, and taking out fibrin clots between red blood cells at the lowest layer and acellular plasma at the uppermost layer. And extruding serum from the fibrin clot by using sterile gauze to obtain an autologous fibrin membrane, and placing the autologous fibrin membrane into a hollow engineering bone scaffold tube cavity to prepare the hydroxyapatite/collagen/platelet-rich fibrin artificial bone scaffold with the osteogenesis inducing capability.
Further, in the step (8), the hydroxyapatite, the collagen colloid and the platelet-rich fibrin are compounded to form the hydroxyapatite, the collagen and the platelet-rich fibrin artificial bone scaffold.
According to the formula and the preparation method of the 3D printing artificial bone composite fibrin scaffold, bone defect parts of different patients are subjected to CT scanning and three-dimensional reconstruction, various parameters of the artificial bone can be customized in a 1:1 individuation mode, the 3D printing artificial bone with the functions of promoting bone regeneration, such as hydroxyapatite, collagen and platelet-rich fibrin, is prepared, the artificial bone is compounded with growth factors of the patients, and the effects of slowly releasing vascular endothelial growth factors, insulin growth factors, transforming growth factors and the like are achieved, the 3D printing artificial bone with the functions of promoting bone regeneration, inducing chemotaxis and osteogenic direction differentiation of mesenchymal stem cells, and having good bone conduction and bone induction functions. The invention can well solve the clinical problem of individualized treatment of patients with large bone defects and nonunion.
Drawings
FIG. 1 is a flow chart of the preparation of the present invention;
fig. 2 is a schematic view of mixing and stirring medical grade hydroxyapatite powder and medical grade collagen powder;
FIG. 3 is a schematic diagram of a 3D bioprinter;
FIG. 4 is a schematic illustration of a hydroxyapatite/collagen artificial bone;
FIG. 5 is a schematic representation of platelet rich fibroeggs in a test tube;
fig. 6 is a schematic view of an artificial bone made of hydroxyapatite, collagen and platelet-rich fibrin.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the concepts underlying the described embodiments. It will be apparent, however, to one skilled in the art, that the described embodiments may be practiced without some or all of these specific details. In other instances, well known process steps have not been described in detail.
The formula of the 3D printed artificial bone listed in this example is prepared from the following raw materials: medical collagen powder, 0.05mol/L acetic acid solution, medical hydroxyapatite powder and 1% genipin solution by mass fraction.
The formula and the preparation method of the 3D printing artificial bone composite fibrin scaffold according to claim 1 are characterized in that: the method comprises the following steps:
(1) preparing collagen colloid: dissolving 5g of medical grade collagen powder in 25mL of 0.05mol/L acetic acid solution to prepare collagen colloid;
(2) mixing: according to the mass ratio of collagen to hydroxyapatite of 1: 2 mixing colloid containing 5g collagen and 10g hydroxyapatite powder;
(3) stirring: and (3) uniformly stirring the materials prepared in the step (2) to obtain hydroxyapatite and collagen printing raw materials, namely the biological ink of a biological printer.
(4) Three-dimensional reconstruction: acquiring CT data of a bone defect part of a patient, segmenting a CT image by using a method combining threshold segmentation and region growth, obtaining a layered CT image Mask (Mask) through Boolean operation, then performing three-dimensional reconstruction, constructing a three-dimensional digital model of a personalized hollow bone tissue engineering scaffold by using RenetierHost software, and preparing an artificial bone by using a hydroxyapatite/collagen mixed tissue as a raw material and by using a 3D printing technology;
(5)3D printing: loading the hydroxyapatite/collagen bio-ink in (3) into a 3D bio-printer (Bioprinter), and then printing the hollow individualized hydroxyapatite/collagen artificial bone through the 3D bio-printer (Bioprinter).
(6) And (3) crosslinking: and (5) placing the hydroxyapatite and collagen artificial bone obtained in the step (5) into genipin solution with the mass fraction of 1% for crosslinking so as to increase the strength of the artificial bone scaffold.
(7) Freeze drying; and (4) drying the hydroxyapatite and the collagen artificial bone which are subjected to the crosslinking in the step (6) for 12 hours by using a freeze dryer.
(8) Preparation and composite of platelet-rich fibrin: collecting 10mL of venous blood of a patient in the process of transplanting an engineering bone scaffold, immediately transferring the venous blood into a sterile glass centrifuge tube without anticoagulation measures, centrifuging for 10min at 400g, and taking out fibrin clots between red blood cells at the lowest layer and acellular plasma at the uppermost layer. And extruding serum from the fibrin clot by using sterile gauze to obtain an autologous fibrin membrane, and placing the autologous fibrin membrane into a hollow artificial bone lumen to prepare the hydroxyapatite, collagen and platelet-rich fibrin artificial bone scaffold with the osteogenesis inducing capability.
Medical grade collagen powder: the natural organic matter has good biological safety, degradability and osteogenesis promoting capability.
0.05mol/L acetic acid solution: as a solvent for dissolving the collagen powder.
Medical grade hydroxyapatite powder: the commonly used bone tissue engineering scaffold raw material has the capability of promoting the mineralization of bone tissue.
1% by mass of genipin solution: as a cross-linking agent to increase the strength of the artificial bone scaffold.
And (8) compounding the hydroxyapatite, the collagen colloid and the platelet-rich fibrin to form the hydroxyapatite, collagen and platelet-rich fibrin artificial bone scaffold.
According to the formula and the preparation method of the 3D printing artificial bone composite fibrin scaffold, bone defect parts of different patients are subjected to CT scanning and three-dimensional reconstruction, various parameters of the artificial bone can be customized in a 1:1 individuation mode, the 3D printing artificial bone with the functions of promoting bone regeneration, such as hydroxyapatite, collagen and platelet-rich fibrin, is prepared, the artificial bone is compounded with growth factors of the patients, and the effects of slowly releasing vascular endothelial growth factors, insulin growth factors, transforming growth factors and the like are achieved, the 3D printing artificial bone with the functions of promoting bone regeneration, inducing chemotaxis and osteogenic direction differentiation of mesenchymal stem cells, and having good bone conduction and bone induction functions. The invention can well solve the clinical problem of individualized treatment of patients with large bone defects and nonunion.
The present invention is not limited to the above-described embodiments, and those skilled in the art will be able to make various modifications without creative efforts from the above-described conception, and fall within the scope of the present invention.

Claims (1)

  1. The preparation method of the 3D printing artificial bone composite fibrin scaffold is characterized by comprising the following steps: the following formula is adopted: comprises 5g of medical collagen powder, 25mL of 0.05mol/L acetic acid solution, 10g of medical hydroxyapatite powder and 20mL of genipin solution with the mass fraction of 1%;
    the method comprises the following steps:
    (1) preparing collagen colloid: dissolving 5g of medical grade collagen powder in 25mL of 0.05mol/L acetic acid solution to prepare collagen colloid;
    (2) mixing: according to the mass ratio of collagen to hydroxyapatite of 1: 2 mixing colloid containing 5g collagen and 10g hydroxyapatite powder;
    (3) stirring: uniformly stirring the mixed materials in the step (2) to prepare a hydroxyapatite/collagen printing raw material, namely biological ink of a biological printer;
    (4) three-dimensional reconstruction: acquiring CT data of a bone defect part of a patient, segmenting a CT image by using a method combining threshold segmentation and region growth, performing three-dimensional reconstruction after obtaining a layered C T image mask through Boolean operation, constructing a three-dimensional digital model of a personalized hollow bone tissue engineering scaffold by using RenetierHost software, and preparing a scaffold material by using a hydroxyapatite and collagen mixed tissue as a raw material and a 3D printing technology;
    (5)3D printing: loading the hydroxyapatite and the collagen biological ink obtained after the stirring in the step (3) into a 3D biological printer, and then printing the hollow individualized hydroxyapatite and collagen artificial bone scaffold material by the 3D biological printer;
    (6) and (3) crosslinking: placing the hydroxyapatite/collagen artificial bone obtained in the step (5) into genipin solution with the mass fraction of 1% for crosslinking so as to increase the strength of the artificial bone scaffold;
    (7) freeze drying; drying the hydroxyapatite and collagen artificial bone after the crosslinking in the step (6) for 12 hours by using a freeze dryer;
    (8) preparation and composite of platelet-rich fibrin: collecting 10mL of venous blood of a patient in the process of a bone scaffold transplantation engineering operation, immediately transferring the venous blood into a sterile glass centrifuge tube without anticoagulation measures, centrifuging for 10min at 400g, and taking out a fibrin clot between red blood cells at the lowermost layer and acellular plasma at the uppermost layer; and extruding serum from the fibrin clot by using sterile gauze to obtain an autologous fibrin membrane, and placing the autologous fibrin membrane into a hollow engineering bone scaffold tube cavity to prepare the hydroxyapatite, collagen and platelet-rich fibrin artificial bone scaffold with the osteogenesis inducing capability.
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CN110101914A (en) * 2019-05-04 2019-08-09 西北工业大学 A kind of Prevascularized two-phase artificial bone scaffold and preparation method thereof
CN110693724B (en) * 2019-11-05 2020-10-30 浙江大学 Tooth mineralizing liquid and mineralizing method thereof
WO2021123344A1 (en) * 2019-12-18 2021-06-24 Grifols Worldwide Operations Limited Bone composite and compositions for preparing same
CN113969066A (en) * 2020-07-23 2022-01-25 固安县朝阳生物科技有限公司 Bone density radiation image equivalent material
CN112704555B (en) * 2021-01-26 2022-06-07 山东建筑大学 Preparation method of degradable porous ulna middle-end fracture connector
CN112870450A (en) * 2021-01-28 2021-06-01 中南大学湘雅医院 Metformin-loaded artificial bone material and method for preparing artificial bone
CN114939226B (en) * 2022-07-25 2022-11-25 中南大学 Pancreatin inactivation stent drainage tube applied to pancreaticojejunostomy and preparation method thereof
CN116271222B (en) * 2023-03-28 2023-12-01 广东医科大学 Bone tissue engineering scaffold and preparation method and application thereof

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CN2889301Y (en) * 2005-04-14 2007-04-18 南方医院 An injection type bone reconstruction material
CN103990182A (en) * 2014-05-30 2014-08-20 东华大学 Three-dimensional scaffold material for bone tissue repair and preparation method thereof
CN104826171B (en) * 2015-04-23 2017-06-16 西安点云生物科技有限公司 A kind of increasing material manufacturing method of multiple dimensioned biomimetic artificial bone support
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